CA2188920A1 - Method for detecting hemoglobin advanced glycosylation endproducts - Google Patents
Method for detecting hemoglobin advanced glycosylation endproductsInfo
- Publication number
- CA2188920A1 CA2188920A1 CA002188920A CA2188920A CA2188920A1 CA 2188920 A1 CA2188920 A1 CA 2188920A1 CA 002188920 A CA002188920 A CA 002188920A CA 2188920 A CA2188920 A CA 2188920A CA 2188920 A1 CA2188920 A1 CA 2188920A1
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- CA
- Canada
- Prior art keywords
- age
- hemoglobin
- sample
- concentration
- level
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
Abstract
The present invention relates to methods for the diagnosis and monitoring of diseases and disorders associate with advanced glycosylation endproducts (AGE) formation, such as diabetes and the ageing process. In particular, the invention is directed to detecting AGE-modified hemoglobin (Hb-AGE) for the foregoing purposes, and in improved assay therefore. The method involves diluting the sample in a dilution buffer, which dilution buffer comprises an anionic protein denaturing detergent at a concentration sufficient to denature hemoglobin-AGE without interfering in binding of reagents with hemoglobin-AGE the dilution buffer may also include a non-ionic surfactant at a concentration sufficient to facilitate detection of hemoglobin-AGE; and a denaturing agent at a concentration sufficient to denature hemoglobin-AGE and increase assay sensitivity, without denaturing binding of reagents to hemoglobin-AGE. After diluting the sample in the dilution buffer, the sample is contacted with means for detecting the presence of hemoglobin-AGE in the sample, and the presence of hemoglobin-AGE in the sample is detected with the detection means. Dilution buffers and kits for practicing the invention are also provided. In specific examples, the level of AGE in hemoglobin in samples from human and rat normal subjects and diabetic subjects is detected. The results obtained from human samples show a high degree of correlation between the level of hemoglobin-AGE in a sample and the level of hemoglobin-A1c in a sample. Most importantly, the invention is used to detect the "aminoguanidine effect", which is the decrease in the level of hemoglobin-AGE in a sample from a subject undergoing therapy with the AGE-inhibitor aminoguanidine.
Description
2 ~ 8 8 9 2 0 r~ J~
METEIOD FOR ~ gTN
ADV~NCED Gl.YCO8YLaTION .~.)Du~;~o FIELD OF THE INVENTION
The present invention relates to method for ~ gnnciC and monitoring of 1; ce~coc and disorders associated with advanced glycosylation e~ CJdaULs (AGE) formation.
Accordingly, the invention relates to ~;agnnsin~ and 10 monitoring diabetes and the ageing process. In particular, the invention is directed to detecting AGE-modified hemoglobin (Hb-AGE~ for the foregoing ~uL~o~es, and in; uved assay therefore.
BA~ uNL~ OF T~F INVENTION
The reaction between glucose and proteins has been known for some time. Its earliest manifestation was in the appearance of brown pigments during the cooking of food.
20 In 1912, Naillard observed that glucose or other reducing sugars react with amino acids to form adducts that undergo a series of dehydrations and reaLLc.l.y Ls to form stable brown pigments (Naillard, 1912, C.R. Acad.
Sci . 154: 66-68 ) .
Thus, the nonenzymatic reaction between glucose and the free amino groups on proteins to form a stable amino, 1-deoxy ketosyl adduct, known as the Amadori product, has been shown to occur with hemoglobin, wherein the reaction 30 of glucose with the amino t~nm;n-lc of the B-chain of hemoglobin forms the adduct known as hemoglobin A1C. The reaction has also been found to occur with a variety of other body proteins, such as lens crystallin, collagen and nerve proteins (see Bunn et al., 1975, Biochem.
35 Biophys. Res. Commun. 67:103-109: Koenig et al., 1975, J.
Biol. Chem. 252:2g92-2997; Nonnier and Cerami, in Naillard Reaction in Food ~nfl Nutrition, ed. Waller, G.A., American Chemical Society 1983 , pp. 431-448 ; and W095130153 2~8~;2~ IIU.,.- I ~
Nonnier and Cerami, 1982, Clinics in Enaocrinology and Metabolism 11:431-452).
IloleuveL, brown pigments with spectral and fluorescent 5 properties similar to those of late-stage Naillard products have also been observed in vivo in association with several long-lived proteins, such as lens proteins and collagen from aged individuals. An age-related linear increase in pigment was vbseL ved in human dura 10 collagen between the ages of 20 to 90 years (see Monnier and Cerami, 1981, Science 211:491-493; Monnier and Cerami, 1983, Biochem. Biophys. Acta 760:97-103; and Monnier et al., 1984, Proc. Natl. Acad. Sci. USA 81:583-587) .
Glucose and other reducing sugars attach non-enzymatically to the amino groups of proteins in a cu..ct~ L~tion-dor~ndPnt manner. Over time, these initial Amadori adducts can undergo further reaLLc.l,y Ls, 20 dehydrations and cross-linking with other proteins to Ar 1 ~te a family of complex nLLu.;LuLes referred to as Advanced Glycosylation El~d~LVdUULS tAGEs). Substantial p~ UyLèSS has been made toward the elucidation of the role and rlinirAl cignifjcA"re of advanced glycosylation 25 ell~Lvdu~Ls, so that it is now acknowledged that many of the conditions heretofore attributed to the aging process or to the pathological effects of d i CP:~cc-c 6uch as ~1; Ahet ~PC, are attributable at least in part to the formation of AGEs in vivo.
AGE ~r~ l Ation can be indicative of protein half-life, sugar Cvll~ell~L~tiOn~ or both. These factors have important c~m~P~lonrPs. N~lr-~uus studies have indicated that AGEs play an important role in the nLLU~LUL~ll and 35 functional alterations which occur during aging and in chronic disease. Additionally, advancêd glycosylation WO 95/3~153 2 1 ~ ~ 9 2 0 P~
Loducts are noted to form more rapidly in diabetic and other ~i c~AcO~l tis6ue than in normal tissue.
Hb-AGE has been found to be predictive of aging or 5 disease ~Luu,L~assion over the long term (International Patent Publication No. W0 93/13421 by Bucala, published July 8, 1993; Makita et al., 1992, Science 258:651-653).
Hb-AGE measurements provide an d~Lu~iate index of long-term tissue --A;fic~tion by AGEs and are useful in 10 AC5.ocC; nrj the contribution of advanced glycosylation to a variety of diabetic and age-related l; cations. While hemoglobin A1C (HbA1C) has been reported as predictive of the extent of glycation on the hemoglobin B chain, HbA~C
is only a reversible int~ a; Ate in the advanced 15 glycosylation pathway and -- UUS other int~ Ates are believed to exist. Hb-AGE, as an irreversible adduct, is a superior measure of disease ~LoyL~s6ion~
drug effectiveness, etc.
20 Hb-AGEs are used to more readily correlate the progression of disease and longer term control of blood sugar levels. HbA~C ls a reversible inf - "; ate, and reaches ~qll~lihrium with glucose over a 3-4 week period.
Hence, levels of HbA~C only reflect blood glucose only 25 during this short time period. Hb-AGE, in contrast, is an irreversible adduct and reflects blood sugar over the lifespan of hemoglobin. Thus, the effectiveness of t Leai L for AGE-related ~ Ar-c or disorders can also be det~rm;nr~ from the level of Hb-AGE in samples. Thus, 30 the reduction in Hb-AGE levels as a result of aminogll ~ni~lin~ therapy is a primary example of the s~lcc~ccful rhArr--ological inhibition of advanced glycosylation in human subjects.
35 Prior to the instant invention, detection of Hb-AGEs required a complex assay format, that included TCA
precipitation of the hemoglobin from the hemolysate, wo 9S/301~3 2 1 8 3 ~ 2 0 F~~
followed by centrifugation to separate the precipitated protein from the ,,u~eL.Id~ lL liquid fraction. Resolution of the precipitate was accomplished by adding sodium hydroxide at high pH (pH>ll), followed by pH adjustment 5 with 0.3 M KH2P04, pH ~.4 buffer (Makita et al., l99l, Diabetologia 34:40-45). After pH adjustment to about 7 . 8, some of the hemoglobin precipitates out of solution, thus requiring a separation step prior to assay of the liquid fraction. In short, the current protocol complex, 1~ is , -, time Çcmcllmin~, subject to and generally not ~rP1 ic~hle to a çl inic~l laboratory setting.
Accordingly, there is a need in the art for a simpler, faster, and milder sample treatment protocol to 15 facilitate testing for Hb-AGE in clinical laboratory settings.
There is a further need in the art for kits containing the reagents neC~cs~ry to perform such assays.
20 The citation of references herein shall not be construed as an ~llmiccinn that such is prior art to the present invention .
STTMMARY OF TTlF~ INvENTIoN
The present invention is directed broadly to a method for detecting the presence of ~ l nhin-AGE in a sample.
The method involves diluting the sample in a dilution buffer, which dilution buffer comprises an anionic 3 0 protein denaturing detergent at a concentration sufficient to denature hemoglobin-AGE without interfering in binding of reagents with hemoglobin-AGE. Preferably, the dilution buffer further comprises a non-ionic surfactant at a cu"c~l,LLcltion sufficient to facilitate 35 detection of hemoglobin-AGE; and a polar denaturing agent at a cu"c~1,LL~tion sufficient to denature hemoglobin-AGE
and increase assay sensitivity, without denaturing .
binding of reagents to hemoglobin-AGE. After diluting the sample in the dilution buffer, the sample is contacted with means for detecting the presence of hemoglobin-AGE in the sample, and the presence of 5 hemoglobin-AGE in the sample is detected with the detection means. The sample can be assayed directly after dilution.
In specific aspects, the Cu--c~ Lation of anionic protein 10 denaturing detergent in the dilution buffer ranges from about 0 . 04% to about 0.16% (w/v) . The ~u..c~l-LLation of non-ionic surfactant, if present, ranges from about O . 005% to about 0 . 1% (w/v); and the cc,~ ..LLation of the denaturing agent, if present, is between about 0. 5 M to 15 about 3 M.
In a specific pre~erred 'i L, the anionic protein denaturing detergent is sodium dodecyl sulfate. In a further specific preferred: '_'ir L, the non-ionic 20 surfactant is a polyoxyethylene ester, in particular Triton X-100 polyoxyethylene ester, and the denaturing agent is urea.
The best mode contemplated by the inventor for practicing 25 the invention uses a dilution bu~fer in which the .~ "c~ ation of sodium dodecyl sulfate is about 0. 08%, the CullC~ Llat.ion of Triton X-100 polyu,Ly-:L~lylene ester is s~ cted from the group consisting of about 0 . 01% and about 0. 04%, and the CUllC~:llLLat.iOn of urea is about 2 M.
Preferably, the dilution buffer is buffered to between about pH 7 to about pH 8, and contains salts at a con~-~ntration approximating physiological ionic strength.
35 In a further -~;r L, the invention provides a method for quantitating the amount of hemoglobin-AGE in a sample. Quantitation can be accomplished by detecting _ _ _ _ _ _ _ WO 95130153 2 1 8 ~ 9 2 0 r~llu~ Jaul the ~les~.1ce of hemoglobin-AGE in the sample as described above. The extent of binding of a binding partner to hemoglobin-AGE, or other means for detecting the presence of hemoglobin-AGE, can be quantitated. The extent of S detection CULL~ VIIdS to the amount of hemoglobin-AGE in the sample.
The invention further provides a method for detecting or nncin~ the pLt s~11ce of a disease associated with lO elevated h~ -~lnhin-AGE levels in a li;ln subject by comparing the amount or level of h~ Jl nhin-AGE detected in a sample to a level of hemoglobin-AGE normally present in the 1 i ~n subject. An increase in the level of hemoglobin-AGE as compared to normal levels indicates a 15 disease associated with elevated levels of hemoglobin-AGE .
In another aspect, the invention provides a method for monitoring the course of a disease associated with 2~ elevated hemoglobin-AGE levels in a 1 i ~n 5ubject.
The level or amount of hemoglobin-AGE in a series of samples obtained at different time points from a r-r~ n subject is detc~rminpcll as described above. An increase in the level of hemoglobin-AGE over time 25 indicates progression of the disease; a decrease in the level of hemoglobin-AGE over time indicates regression of the disease.
In yet a further aspect, the invention-provides a method 30 for monitoring a theLcl~t uLic LLaa, 1 of a disease associated with elevated hemoglobin-AGE levels in a r-mr-l i~n 5ubject. The level or amount of hemoglobin-AGE
in samples obtained at different time points from a 1 i 1n subject undergoing a therapeutic treatment for 35 a disease associated with elevated hemoglobin-AGE levels is ~ot~rmin~-d. A decrease in the level of AGEs over time indicates an effective therapeutic outcome.
W095130153 21 8~920 r~
In particular, the invention provides a preferred method for titrating a dosage of an inhibitor of AGE formation to determine the optimum dosage. The level or amount of hemoglobin-AGE in a series of samples obtained at 5 different time points from a 1; ~n subject receiving aL~ively larger doses of an inhibitor of AGE
formation over the time points is evaluated. An optimum dosage of the inhibitor is a dosage above which no further decrease in the level of hemoglobin-AGE is lO observed.
In still another aspect, the invention provides a method for monitoring the long term glucose level in a r-m~ n subj ect . The level or amount of hemoglobin-AGE in a 15 sample from a 1 ;:~n subject is de~;n~ The level or amount of hemoglobin-AGE i5 indicative of the long term glucose level in the 6ubject.
The methods of the present invention are achieved by 20 dilution of the sample in the dilution buffer of the invention. Accordingly, the present invention further provides a dilution buffer. The dilution buffer of the invention comprises an anionic protein denaturing detergent; a non-ionic~surfactant; and a polar denaturing 2~ agent. These l~:ayt~ can be provided in a cu~ 1LLG~e, for ~ t j nn such that the anionic protein denaturing detergent is present at a co1~ct:111 LGLiOn sufficient to denature hemoglobin-AGE without interfering in binding of reagents with hemoglobin-AGE; the non-ionic surfactant is 30 present at a ~ ullcc~ La~ion sufficient to facilitate detection of hemoglobin-AGE; and the denaturing agent is present at a cu11c~1--L~tion sufficient to denature hemoglobin-AGE and increase assay sensitivity, without denaturing binding of reagents to hemoglobin-AGE.
35 Preferably, the dilution buffer i8 pll:,ua~ cd such that the cu~ La~lon of anionic protein d~ aLuLing detergent in the dilution buffer ranges from about 0. 04% to about WO95/30153 2 1 3~9~0 ..I~U~,~ J~ul 0 . 16% (w/v); the ~u-.c~:..LL.ltion of non-ionic surfactant ranges from about û. 00596 to about 0 . 1% (w/v); and the . ul.c~:..LLation of the denaturing agent is between about 0.5 M to about 3 M. In another preferred aspect, the 5 anionic protein denaturing detergent is sodium dodecyl sulfate, the non-ionic surfactant is Triton X-100 polyoxyethylene ester, and the denaturing agent is urea.
In the most preferred aspect of the invention, which is the best mode contemplated by the inventors for 10 practicing the invention, the Cu-.c~:..LLation of sodium dodecyl sulfate is about 0.08%, the c~ -tion of Triton X-lO0 polyoxyethylene ester is selected from the group consisting of about 0. 01% and about 0. 04%, and the cu.,~m:-.LLation of urea is about 2 M.
In a further _'i L, the dilution buffer is buffered to between about pH 7 to about pH 8, and contains salts at a cu.lc~-lLLation approximating physiological ionic strength .
In a further aspect, the present invention relates to a kit for detertin~ the presence of hemoglobin-AGE in a sample. The kit of the invention comprises a dilution buf f er as described above, in ,_u..~ LL at.ed cr ready-to-25 use form; means for detecting the presence cf hemoglobin-AGE; other reagents; and directicns for use of said kit.
In an Example, infra, the present invention provides for 3 o detection of the level of AGE in hemoglobin in samples from normal subjects and diabetic subjects. Samples from both humans and rats were successfully tested using the method of the invention. Furthermore, the results obtained from human samples show a high degree of 35 correlation between the level of hemoglobin-AGE in a sample and the level of hemoglobin-A1c in a sample. Most importantly, the assay of the instant invention can be ~ W095/30153 21 88920 P~
used to detect the "aminog~1An;~1;ne effect," which is the decrease in the level of hemoglobin-AGE in a sample from a subject undergoing therapy with the AGE-inhibitor aminog~An;~l;nP. Thi6 effect cannot be detected by 5 measuring the level of hemoglobin-A~c, since aminoguAn;~l;nP, or other inhibitors of AGEs, do not affect hemoglobin-A1c levels. The so called "aminoguAn;~l;nP effect" applies to other AGE-inhibitors as well.
Thus, the present invention a~v~..Lly~u,-sly provides for monitoring the efficacy of a therapy for preventing advanced glycosylation ~ duct formation, as well as a more sensitive test for diagnosis of an AGE-associated 15 disease or disorder.
Accordingly, it is an object of the present invention to provide an improved assay Por hemoglobin-AGE.
20 It is a further object of the invention to provide a dilution buffer that increases the speed, ease, and simplicity of assays for hemoglobin-AGE.
It is a yet another object Or the invention to provide a 25 buffer that ~nhAnl-PC immuno-detection of hemoglobin-AGE.
Yet a further obj ect of the invention is to provide kits for detecting the ~L~se1lce of hemoglobin-AGE in a sample.
30 These and other objects of the invention are addL~_sed and will be better understood by reference to the - following drawings and detailed description of the invention .
~ ,",~Vt!,~ 33~
21 88~20 ~RIEF DESt'RTPTTON OP TT~R D~WINGS
FIC-~JRE 1 preaents the level of AGE modification of hemoglobin. The samples were pretreated with O 08~ SDS, 5 0.049~ or 0.01~ Triton ~-LOQ, and 2 M urea in the sample diluent prior to testing in an antibody ELISA assay for the level of AGE modification. The data are reported as units of AGE per mg of hemoglobin in samples from diabetic and normal subj ects ~ standard error of the mean 10 (SEM) . The amount of AGE was determined- using an EI,ISA
assay developed earlier (Makita et al., 1992, J. Biol.
Chem. 267:5133-38; Int~rn~t;on~l Patent Publication WO
93/13g21) i the concentration of protein (which consists mostly of hemoglobin) was determined with the ~owry reagent using purified bovine serum albumin as a standard --(I,owry et al., 1951, J. ~iol. Chem 193 :265) . (A) Samples were obtained from 11 normal and 11 diabetic humans. The hemoglobin Al~ levels for the normals were 4.9:t0.52, and for diabetics were 8.3~1.49. A
20 concentration of 0.04~ Triton was used to pre-treat human samples. (B) Samples were obtained from 10 normal and 10 diabetic rats. Diabetes was induced in rats by treatment with streptozocin, and hyperglycemia was confirmed by assaying blood glucose.
FIGURE 2 presents data which demonstrate the correlation between measurement of hemoglobin-AGE (units o~ AGE per ~--mg of he~oglobin) according to the present invention versus the level of hemoglobin Alc (percentage o~ total 30 hemoglobin). The correlation was performed with 22 human samples (the same samples as sho~n in Figure L~
FIGURE 3 presents data that demonstrate the sensitivity of the present assay to detecting the hemoglobin effect.
35 The level of AGE-hemoglobin (units of AGEs per mg o~
hemoglobin) was measured after pretreatment of samples according to the present invention. Samples were ~ =
WO 95B0153 2 1 g g 9 2 0 . ~1I~J...5, ~ul obtained from normal rats, induced diabetic rats, and diabetic rats that received 50 mg per kg of aminog~An i tl i nP: each sample group included 10 members .
Statistically significant differences in the level of Hb-5 AGE were observed between normal and diabetic rats (p<O.Ol), and between aminog~lAnitlin-~-treated and diabetic rats (p<O . 05) .
n~T~ DES~l?TPTION OF ~ NV~LlUN
The present invention relates to an improved assay for hemoglobin-AGE. In particular, the invention discloses a dilution buffer containing an anionic protein denaturing detergent, such as sodium dodecyl sulfate. Preferably, 15 the dilution buffer further comprises a non-ionic surfactant, such as polyoxyethylene esters like Triton X-100, and a polar denaturant, such as urea. Dilution of hemolysate in this dilution buffer ~i~nifirAntly increases the simplicity and speed of assays for 2 O hemoglobin-AGE .
As used herein, the term "AGE-" refers to the _ ' which it modifies as the reaction product of either an advanced glycosylation ~l~d~-u-lu~:L or a . ' which 25 forms AGEs and the ~ d so - 'ified, such as the bovine serum albumin (BSA). Thus, AGEs include, but are not limited to, AGE-proteins (such as BSA-AGE), AGE-lipids, AGE-peptides, and AGE-DNA. AGE polypeptides or AGE proteins can be formed in vitro or-~in vivo by 30 reacting a polypeptide or protein with an AGE, such as AGE-peptide, or with a ~ ' such as a reducing sugar, e.a., glucose, until the polypeptide or protein is r ~'ifiocl to form the AGE-polypeptide or protein.
35 The term "glycosylation" is used herein to refer to the non-enzymatic reaction of reducing sugars with a nucleophile, in particular an amine group, on a polypeptide or protein, such a~ hemoglobin, a lipid, or DNA, which leads to formation of AGEs. These processes are well known in the art, as described above. Recently, the term "glycation" has become more favo~ed to refer to 5 non-enzymatic glycosylation processes. Thus, the term "glycosylation, " as specifically defined herein, and "glycation" are equivalent.
As stated above, according to the present invention, a 10 sample ~on~in;n~ red blood cells or hemoglobin from a subject is treated by dilution in a diluti~n buffer, e.g., buffer l-,.n~A;nin~ sodium dodecyl sulfate, Triton X-100, and urea. The sample may be blood, or red blood ceils isolated from blood, that has been treated to 15 hemolyze the red blood cells. Alternatively, the sample may be hemoglobin isolated from red blood cells.
Although not intending to be limited by any particular . theory or hypothesis, it is believed that moderate 20 ~pn~tl~r~ion of hemog~obin exposes additional AGE
epitopes to antibody binding, thus significantly increasing the sensitivity of an; ~csay for ~b-AG~
compared to untreated samples.
25 According to the invention, samples can be obtained from any source, lncluding in v1tro or ~n vlvo sources. In particular, the instant invention contemplates assays on a sam~ole from an animal, and more preferably, from a mammal, :including humans, as well as mammals such as 30 dogs, cats, horses, cows, pigs, guinea pigs, mice, and rats. Thus, the present invention provides for monitori~g ~G~ levels in human and veteri~y medicine.
The concentration of anionic protein denaturiny 35 deteryent, such as SDS, in the dilution buffer is sufficient to denature hemoglobin-AGE without inter~ering in immunological binding or causing detachment or elution Wo 951301~3 of a solid phase reagent in a solid phase; :~esay.
Accordingly, the concentration of anionic protein denaturing detergent can range from about 0. 04% to about 0.1696 (w/v), ~lPrPn~lin~ on the stability of the detection 5 assay, e.q., antibody binding. Preferably, the anionic protein denaturing detergent is SDS, and the Cu~ La~ion of SDS i5 about 0 . 0896 .
In addition to SDS, the invention further contemplates lO use of analogous detergents that interact with and effectively denature proteins. Although SDS has been found to be the most useful protein denaturing detergent, other such detergents, although le6s effective than SDS
f or denaturing proteins, can be used according to the 15 present invention. Obvious choices include C8 to C20 hydrocarbon acyl sulfate salts, i.e., SDS analogs.
Substitution of a different anionic protein denaturing may require retesting to ~PtP~n; ne an optimal l U~ LLCl~iOn, which merely requires straightforward 2 0 experimentation .
The cu~ lL~clLion of non-ionic surfactant, if any, such as Triton X-lOO, in the dilution buffer is sufficient to facilitate detection of hemoglobin-AGE in a sample. The 25 non-ionic detergent may function, in part, to buffer harsh effects of the anionic detergent. In a sperifir~
, the non-ionic surfactant is the polyoxyethylene ester Triton X-lO0, and the c;ullcel.LL~-t.ion of Triton X-l00 ranges from about 0 . 00596 to about O .1%
30 (w/v). In a preferred aspect of the invention, the ~U~ LClLiOn of Triton X-l00 is approximately 0. 0l96 for - samples from rats, and 0.049~ for samples from humans.
Optimal cc,1~c~,1LL~Lion for samples from other animals, for different AGE-specific antibodies, and for the choice of 35 anionic detergent can be readily de~PrminPd using standard experimental techniques.
21 8~q20 The invention further contemplates use of an analogous non-ionic surfactant in place of Triton X-100. A non-ionic surfactant to be substituted ~or Triton X-100 may be tested to ~lP~Pr;ll; nP an optimal conceI:Ltration; such 5 testing involves straightforward experimentation.
..
The concentration of a polar denaturing agent in the dilution buffer is sufficient to denature the Xb-AGE or stabili~e and maintain detergent-denatured Hb-AGE in a 10 denatured state, and increase assay sensitivity, without denaturing immunological reagents used to ~etect the - --presence of Hb-AGE in a sample. In a specific aspect, the denaturant is urea, in a concentration of between about 0.5 M to about 3 M. Preferably, the rnnrPntri~t;r,n 15 of urea is 2 M. Furthermore, the invention contemplates use of other polar denaturing reagents, such as guanidine-XCl, at a suitable concentration in place of urea .
20 Preferably, the dilution buffer is pH-buffered and ionic strength controlled. For example, the ~ t; nn bu~fer --may contain a physiological concentration of sodium chloride, or other salts, as well as buffers to maintain pH of the ~ t inn solution between about pH 5 and about 25pH 9. Preferably, the pX of the buffer is about pH 7 to about pX 8; more preferably, the pH is about pH 7 4. In a specific embodiment, ill~ra, the dilution bu~fer is a sodium phosphate bu~fered solution cnnt;~in;ng sodium chloride: It is preferable not to use potassium salts in 30the ~ilution buffer, since it is known that potassium ion can induce precipitation of SDS. An.other suitable buffer salt is Tris-HCl.
A sample rrnt~ining hemoglobin can be diluted from about 3510-fold to about 100-fold with dilution buffer prior to conducting an immunoassay to detect Hb-AGEs.
Alternatively, the sample can be diluted so that the ---~ WO 95/30153 2 1 8 ~3 9 2 ~ r~
protein col~cerlLL~-tion (mo5t of the protein being hemoglobin) ranges from about 0.1 mg/ml to about 10 mg/ml. In a spP~ ;f1c ~ho~;- L, inf~, a hemolysate sample is diluted 40-fold in the dilution buffer of the 5 invention prior to cn~ t;n~ the i - cSay. In the example, ~, the con~n~ration of protein in the sample is approximately 1-2 mg/ml, as ~ tprminpd by the Lowry assay, using BSA as a protein standard (Lowry et al., 1951, J. Biol. Chem. 193:265).
The present invention can enhance many of the; - - CRay or ; ~ C~-c y-type formats that can be used to detect the presence of, and measure the quantity of, hemoglobin-AGE, by increasing the accessibility of AGE-epitopes 15 present on hemoglobin-AGE. As used herein, the terms "means for ~lPte~;n~ and "means for quantitating"
hemoglobin-AGE refer to detection or quantitation of hemoglobin-AGE in an any such; ccay-type format.
Generally, such means comprise contacting a sample with 20 one or more binding partners of hemoglobin-AGE, followed by detection of binding of the one or more binding partners to hemoglobin-AGE in the sample, and, if desired, quantitation (measuring t~e quantity) of binding that occurs. Binding partners include, but are not 25 limited to, ant;ho~;~C to hemoglobin, antibodies to AGEs, Il:ctl~LuLa for AGEs, small molecules that bind either hcmoglobin or AGEs, and the like. At least one such binding partner must be spPc;f;c for an AGE. Detection of specific binding of a binding partner of hemoglobin-30 AGE with Hb-AGE in the sample is indicative of the presence of Hb-AGE in the sample.
Accordingly, once a sample containing hemoglobin has been diluted in the dilution buffer of the invention, the 35 presence of Hb-AGE can be det~rm;n~cl using many formats of immunological assay means, e.q., using well known techniques such as but not limited to radio; -- y, _ _ _ _ _ _ _ , . .
Wo9S/30153 2~ 8g9~1~ r ~
ELISA (enzyme-linked immunosorbant assay), "sandwich"
- -ys (in particular ELISA assays), competitive assays (in particular competitive ELISA assays), LL1C assays, precipitation r~prtionc~
5 immunofluoL~s~:ence assays, protein A assays, etc. In one ;- ~, antibody binding is detected by detecting a label on the primary antibody. In another ~mho~; , the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
10 In a further ~mho~ , the secondary antibody is labeled .
In a spDc; f; r ~mho~ , a sandwich assay format is used, in which an anti-AGE antibody is attached to the 15 solid phase, and an anti-hemoglobin antibody (either labeled directly or detected with a s~rnnrl~ry labeled antibody) is used to detect binding of Hb-AGE with the anti-AGE antibody. In another . ; ~, the anti-hemoglobin antibody can be attached to the solid phase, 20 and an anti-AGE antibody used to bind any AGE epitopes present on the hemoglobin molecules bound to the ant~-hemoglobin antibody.
As used herein, the term "antibody" refers to polyclonal, 25 monoclonal, chimeric, and single chain antibodies, as well as Fab fragments (F(ab' )z, F(ab), Fv, etc. ), and an Fab expression library.
The amount of hemoglobin-AGE in a sampl:e can be 30 det~rm;n~rl quantitatively. For example, the extent of binding can be ~uantitated, and the amount of Hb-AGE
t~rm; n~d by extrapolation from a standard curve. In a specific aspect, a standard amount of hemoglobin-AGE can be provided in a kit of the invention for use as a 35 standard. In another ;- ~, the amount of AGE
(e. a ., units of AGE) can be ~l~t~rm; n~d by comparison with WO 95/30153 l7 / ~~
a standard amount, such as a BSA-AGE sample as described in the examples, ~.
In a 5pc~c;f;r-: ' ~ ';~ L, the assay described in ~akita 5 et al. (1992, J. Biol. Chem. 267:5133-38; see also International Patent Publication No. W0 93/13421) can be used to detect the presence of Hb-AGEs in a sample prepared according to the present invention.
In a further ~ of this invention, commercial test kits suitable for use by medical or clinical te~-hnnl o~ists or speciAl; cts may be prepared to determine 15 the presence or absence of hemoglobin-AGEs. Such kits will include at least a dilution buffer of the invention, and means for detecting the ~sc.~.~e of hemoglobin-AGE as described suPra. The dilution buffer can be provided in cu..~-~..LLated form, which reguires the addition of water 20 or buffer to bring the -- L~ of the buffer to their appropriate cu..c~..LLa~ion. Providing the dilution buffer in cu..L:e..LLated rorm alvallLa~uusly reduces the weight and size of the kit, and thus is more cost effective.
25 ~he contents of the kit are preferably in~uLuuLal ed in a package, such as cardboard or plastic packaging material, which is ~ n~ to hold all of the --tc of the kit. Each ~ L can be held in separate containers, such as glass or plastic vials, for use or dilution prior 3 0 to use .
- In~ accordance with the testing techniques ~; CcllcF^1 above, one class of such kits will contain at least a binding partner of an AGE epitope and dilution buffer of 35 the invention comprising an anionic protein denaturing detergent, a non-ionic detergent, and a denaturant, as described above, and means for detecting binding of the _ _ _ _ _ _ wo 95/30153 2 1 8 8 ~ 2 0 . ~
binding partner of an AGE epitope to an AGE epitope present in the sample diluted with the dilution buffer according to the invention.
5 The kit may also contain directions for conducting an assay in A~ror~nne with the method selected, e.g., "competitive", "sandwich", "DASP" and the like. The kits may also contain peripheral reagents 6uch as buffers, stabilizers, etc.
Accordingly, a test kit may be prepared for the cl LL~ltion of the ~L~:St:llCe, S~uantity or activity of AGEs, comprising:
(a) a labeled specifically reactive component 15 obtained by the direct or indirect att~ L of a binding partner of an AGE epitope or a specific binding partner thereto, to a detectable label;
(b) a dilution buffer, in ~u..cc~ c.Led or ready-to-use form, compri6ing an anionic protein denaturing detergent, a non-ionic detergent, and a polar denaturant:
(c) other ~ay~.lLs; and (d) directions for use of said kit.
More specifically, the diagnûstic test kit may comprise:
(a) a known amount of a first binding partner to an AGE epitope as described above, generally bound to a solid phase to form an ;r-~nnsorh~nt;
(b) a second binding partner to an AGE epitope or to hemoglobin, which second binding partner is labelled:
(c) a tl;ll1ti n buffer, in ~;u.,-~l.LLc.ted or ready-to-use form, comprising an anionic protein denaturing detergent, a non-ionic surfactant, and a polar denaturant;
(d) if n~c-~RRIry, other reagents; and (e) directions for use of said test kit.
~ WO95130153 21 ~8920 1 "- 1 In another specific embodiment, a diagnostic test kit may comprise:
(a) a known amount of a first binding partner to hemoglobin as described above, generally bound to a solid 5 phase to form an i --orh~nt;
(b) a second binding partner to an AGE epitope, which second binding partner is labelled;
(c) a dilution buffer, in CU~ LC-ted or ready-to-use form, comprising an anionic protein denaturing lO detergent, a non-ionic surfactant, and a polar denaturant;
(d) if nPc~q~-ry, other reagents; and (e) directions for use of said test kit.
15 In another ^ ~'i-- L, a diagnostic test kit may comprise:
(a) a binding partner of an AGE epitope, generally associated with a solid phase to form an i ~..L1.~
(b) a known quantity of a labelled AGE that i8 2 0 capable of binding to the binding partner of an AGE
epitope;
(c) a dilution buffer, in .:u,.~ Lc~ted or ready-to-use form, comprising an anionic protein d~,lla-uLing detergent, a non-ionic surfactant, and a polar 2 5 denaturant;
(d) if nPl.F-qqAry, other reagents; and (e) directions for use of said test kit.
In another P~-~Or~ a diagnostic test kit may 3 0 comprise:
(a) an AGE, generally associated with a solid phase to f orm an i - _ l;
(b) a known quantity of a labelled binding partner of an AGE epitope that is capable of binding to the AGE
35 i -~_L~e~t;
(c) a dilution buffer, in concentrated or ready-to-use form, comprising an anion~c protein denaturing W0 95130153 21 8 8 9 2 0 F~
detergent, a non-ionic surfactant, and a polar denaturant;
(d) if ne~cs~ry, other reagents; and (e) directions for use o~ said test kit.
The test kits of the invention can comprise an AGE
standard for quantitating the amount of AGE in a sample.
In a specific embodiment, BSA-AGE is used as an AGE
standard .
Preferably, in a test kit of the invention, the binding partner of an AGE epitope is an antibody to an AGE , e . q ., as described above (see Makita et al., 1992, sup~a;
International Patent Publication No. W0 93/13421).
Preferably, in a test kit of the invention that comprises an AGE, the AGE is BSA-AGE as described above and in the examples, infra.
20 In a preferred ~ t, the dilution buffer ~Pc SDS, Triton X-100, and urea in the C~ r~LtiOnS that are ~ i ccl oced g~.
MOnit:Or;n~l IL~a~ lent to Prevent ~:P Formation The hemoglobin-AGE assay of the present invention is particularly well suited to monitor the aminog~l~n;ti;n~, or other AGE-inhibitor, effect in a large scale study or trial, since the assay in fast and simple. Si~ilarly, 3 0 the assay of the present invention can provide ror monitoring the course of therapy of any product ~ ect~d to inhibit AGE formation. In particular, the present method for detQct~ n~ hemoglobin-AGE provides for carefully titrating the ~h.or~r~--tic dose of ~n AGE-35 inhibitor.
WO 95130153 2 ~ 8 8 9 2 0 P~
In a particular aspect, the invention provides fortitrating the optimum dosage of an agent that inhibits AGE formation. Titration of the optimum dosage allows a physician to select a dosage with maximum benefit for a 5 patient, while m;n;m;7;r~ the risk of side effects.
Since t.-hol;c p~ y~ and rates of clearance dirfer from one individual to another, the optimum dose o a therapeutic agent can vary from one person to another.
The term "optimum" when used to modify "dose" should not 10 be cu.lrur~ed with the term "effective"; a dosage of a therapeutic agent can be effective without being the optimum dose.
Thus, the invention provides for monitoring the effect of 15 administration of agents that block the post-glycosylation step, e., the formation of fl.lores~;el-t or cros61inking .:}-~ nres whose presence is associated with, and leads to, the adverse sequ~lae Or glycosylation. An ideal agent would prevent the 2 O formation of a chromophore and its associated cross-links of proteins to proteins and trapping of proteins on the other proteins. The ideal agent would prevent or inhibit the long-term, post-glycosylation steps that lead to the formation of the ultimate advanced glycosylation end 25 products that are a direct cause of AGE-associated pathology .
An inhibitor of the formation of AGEs includes that react with a carbonyl moiety of an early 30 glycosylation product. R~ s~ c.tive Or such advanced glycosylation inhibitors are aminog~lAn i ~; n~, lysine and - ~-hydrA~;nnh;~tidine. In a specific embodiment, the inhibitor is aminog~lAn;-l;n~ (AG) and derivatives thereof.
- Pharmaceutical compositions and methods involving AG and 35 derivatives thereof are well known, as described in U.S.
Patents No. 4, 758, 583, issued July 19, 1988; No.
4, 9O8, 446, issued March 13, 199o; No. 4, 983, 604, issued '1 88~20 January 8, 1991; No. 5,100,919, issued March 31, 1992;
No. 5,106,877, issued April 21, 1992; No. 5,114,943, issued May 19, 1992; ~o. 5,128,360, issued July 7, 1992;
No. 5,1~0,324, issued July 14, 1992; No. 5,130,337, 5 issued July 14, 1992; No. 5,137,916, issued August 11, 1992; No 5,140,048, issued August 18, 1992; No.
5,175,192, issued December 29, 1992; No. 5,218,001, issued June 8, 1993; No. 5,221,683, issued June 22, 1993;
No. 5,238,963, issued August 24, 1993; No. 5,243,071, 10 issued September 7, 1993; and No. 5,254,593, issued October 19, 1993 Other inhibitors of AGE formation are ~
described in U.S. Patents No. 5,258,381, issued November ~=:
2, 1993; No. 5,356,895, issued October 18, l99g; No.
5,272,116 issued December 21, 1993; No. 5,358,960, issued :
15 October 25, 1994; and Serial No. 08/095,095, filed July 20, 1993. ~ach of the foregoing patents and patent applications is specifically incorporated herein by ref erence in its entirety .
20 The invention may be more completely understood by reference to the following non-limiting example, which is provided solely as exemplary of a specif ic embodiment o~
the invention.
.
F;I~AI~qpJ,~ rlob; n At~.~ AssaY
M~ter;; l R j3rll~ hnrlR
-Pre~rea~ment Buf~er. SDS/~riton/Urea Hemolysate 30 pretreatment buf~er (1 liter) was prepared as follows:
A sodium phosphate (0.02 M) bu~er solution was prepared by adding 0.91 g of sodium monobasic (rr~onohydrate); 1.902 g of sodium dibasic (anhydrous); 3 g of sodium chloride, 35 and 0.2 g of sodium azide to 800 ml of distilled water.
~ WO95/30153 ? 1 8 ~ 92 0 r_"~
To the above buffer (800 ml) were added 120.12 gm of urea (2 M final .,v..~:..LLaLion); 0.8 g of SDS (0.08%) sa~ples, and 0 . 4 or 0.1 g of Triton X-100 (yielding concentrations of 0.04%, for human samples, or 0.01% for rat samples).
5 The pH of the solution was adjusted to 7.4 and the volume was brought to 1 liter with distilled water.
Sodium phosphate buffer was also prepared as described above, without inrlllAlng urea, SDS and Triton X-100.
Pre~aration of the Hemolvsate. Red blood cells were prepared using a distilled water/toluene extraction uceduLa. Briefly, red blood cells (RBCs) were separated from whole blood collected in a heparinized 15 tube by centrifugation at 2-3000 rpm for 10 min. The RBCs were washed by rD~ p~n~ion in sterile isotonic saline (0 . 8596) in a volume approximately equal to the plasma, and repacked by centrifugation as described above . RBCs were stored for up to one week at 4 C in 20 buffered saline after two washing steps that included 30 min soaks in the wash buf f er . RBCs could be stored ror more than 1 week prior to use in an assay by freezing the pellet to -20C after incubation in isotonic saline at 4 C overnight to dialyze out glucose contained in the 25 cells.
Hemolysate was prepared by pelleting enough fresh cells in a SC;L~ a~ed tube to give 1 ml of packed RBCs.
(This step was omitted with frozen RBCs, however, as 30 freezing hemolyses the cells. Instead, the thawed packed cells are used directly for the next step. ) To 1 ml of - packed R3Cs was added 3 ml of distilled water to lyse the cells. Following addition of water, 2 ml of toluene were added to delipidate the suspension. The cells were 35 shaken vigorously for a few minutes, or vortexed intermittently six times to ensure complete lipid extraction. The RBC preparation was then centrifuged at _ wo95/30153 21 88920 1~ s~ul ~
3000 rpm for 10 min to separate the two phases and pellet the c~ l Ar debris. An interface of white insoluble material is founa between the toluene (top) phace and the aaueous (bottom) phase. The toluene phase, above the 5 insoluble material layer overlaying the aaueous hemolysate, was removed using a glass Pasteur pipette with a ~uction flask. After removing the toluene, the tuhe was tipped to expose the red aqueous phase underlying the layer of insoluble material. The aqueous 10 phase was removed with a Pasteur pipette, taking care not to disturb either the layer of; ncol l~hle material or the pellet at the bottom of the tube. The hemolysate (aaueous phzse) was stored overnight at 4-C if the assay was not to be peL L~ - ' immediately . Long term storage 15 of the hemolysate requires use of a preservative or sterile filtration.
Coatina Antiaen. BSA-AGE was ~ aLtd at 30 ~Lg/ml in a 0.1 M bicarbonate buffer, pE~ 9.6, with azide. One 20 hundred ,ul of coating solution were incubated in microtiter wells for 1 hr. at 37-C, or overnight st 4-C.
Coating solution was washed away and a blorkin~ buffer containing PBS with 0.1 g BSA, 1 ml goat serum and azide was added; the plates were incubated for 1 hr. at 37-C.
25 The plates were washed with a PBS, Tween solution prior to addition of sample.
Microtiter Plate AGE AssaY. The hemolysate was diluted in the SDS/Triton/Urea Buffer to give approximately a 1-2 30 mg/ml concentration of protein. Usually, the hemolysate was diluted 1:40-1:60 to achieve the desired protein concentration range. The protein c~ ,l r, Lion of the hemolysate was detPrmin~d using the Lowry procedure (Lowry et al., 1951, J. Biol. Chem. 193:265), with 35 "; fi~ations.
~ Wogs/3ols3 21 88q20 P~
Briefly, 3-10 ,ul of hemolysate preparation were added using an Eppendorf positive displ P~ - L pipetter (0. 5-10 ~1) to a 2-5 ml glass test tube (12 x 75 mm). To this tube was added 1 ml of Lowry reagent. The sample was 5 mixed well and incubated at room t~ .UL~ (RT) for 15-30 min. To each tUbe were added 100 ~Ll of Folin &
Ciocalteu's phenol reagent, and the mixture was vortexed immediately in two short intervals. Aggressive or excessive vortexing was avoided to eliminate or reduce 10 Ahnt~ results. The sample was incubated for 30 min at RT, and optical density read in a dual-beam ~ue~ LL~pl.otometer at 750 nm against a blank consisting of a mixture of 1 ml of Lowry reagent with 100 ~1 of the Folin reagent.
A 1 mg/ml BSA (Sigma RIA grade A7888 or A7030) solution in 300 mM KE12P04 with 3 mM sodium azide was used as the standard for the protein determination assay. The standard samples were prepared by pipetting 3 .12, 6 . 25, 20 12 and 25 ~1 of standard solution into test tubes and performing the assay as described above.
The Lowry reagent, pH 12.8, was prepared by adding 0.5 ml of 2% potassium sodium~ tartrate (KNaC4H406 H20) and 0.5 ml 25 of 1% CUS04 to 50 ml of 2% Na2CO3 in 0.1 N NaOH. The Lowry reagent is stable at RT and should not be stored in the refrigerator. Fresh solution was pLt:~aL~d for each assay.
30 Folin & Ciocalteu's phenol reagent was made by dilution of the stock (Sigma F9252) 1:1 with distilled water in an amber glass bottle to yield a 1 N solution. This solution was stored at RT.
35 All samples were either diluted by the same amount, and concentration calculated from the protein cu.~c~ Lc-tion, or all WO gS/30153 2 ~ ~ ~ 9 2 0 hemolysates were adjusted to the same protein cu.,ce--LLcl~ion tapprox. 1 mg/ml) by using an eYact ratio of buffer to hemolysate.
5 To each well of the assay plate were added 50 ~Ll of solution of diluted hemolysate. An additional 50 ~Ll of the SDS/Triton/Urea Buffer was added to each well to nnrrql i 7e the sample.
10 PreParation of AGE Standard. BSA-AGE for use as a standard was prepared by incubating BSA with glucose in a molar ration of 500:1 glucose:BSA in a phosphate buffer, pH 7.4, for six weeks at 37-C. After the incubation period, the solution was dialyzed against PBS/0 . 02%
15 azide, and stored at -80-C.
Pr~nqration of SamPle Standards. BSA-AGE standards (0.1-3.75 ~Lg/well) were made up in sodium phosphate buffer lacking SDS/Triton/Urea. Fifty ~l of each standard was 20 added to assay wells.
Primarv Antibodv Dilution. Anti-AGE antisera was diluted to give a B50 of 1 ~Lg BSA-AGE. The BSA-AGE was prepared as described above for preparation of the BSA-AGE
2 5 standard .
Assav Procedure. After coating the microtiter wells with BSA-AGE, 50,u1 of sample or standards, diluting buffers, and 50 ~l of primary antibody were added to each well.
30 The microtiter plate was incubated for 2 hrs. at room t ~ULC:. The microtiter plate was washed 3X with Tris buffered saline (TBS)/Tween. To each well were added 100 ~Ll of A l kq l i n q phosphatase labeled secnn~ ry antibody at the appropriate titer, and the wells 35 incubated at 37 C for 1 hr. The microtiter plate was washed 3X with TBS/Tween. Para-niLLuuht ..yl phosphate WO 95/30153 2 ~ 8 ~ q ~ 0 P~
tPNPP) aUll~LL~te was added to all wells and incubated for 1-2 hrs until the 0. D. of the Bo wells reached 1. 7 O . D.
B is the 0. D. of binding of lAh~ d antibody in the 5 ~les~ ce of a sample that contains a competitor. Bo is the signal (O.D. ) from control samples without competitor .
Norlr~lization of Values. T nACSay results (O.D. of 10 sample wells) were e~ cased as % of B/Bo. The AGE
concentration was det~rmi ni d from 0 . D. by extrapolation from a standard curve (see Makita et al., 1992, J. Biol.
Chem. 267:5133-38; International Patent Publication No.
W0 93/13421).
All sample readings were normalized to the protein ~ ullcc:~lLLGtion~ which was det~rm;nPcl in the Lowry assay.
Thus, the final data are expressed as units of AGE per mg of hemoglobin.
Reslll ts Anrl D~ crllCcion A much simpler and reproducible Hb-AGE ELISA assay, with greater sensitivity due to what is believed to be maximal 25 ~uoauL~: of AGE epitopes, has been developed. The new assay includes incubation of samples containing h ~~lnhin with SDS, Triton X-100, and urea.
In pr-ol ;m;n~ry experiments, it was found that incubation 30 with SDS alone ~nhAnrl~ detection of hemoglobin-AGE.
However, SDS alone altered the standard assay conditions, and affected the binding of anti-AGE antibody to the AGE-model dipeptide, Gly-Lys browned with ribose, compared to - binding in the absence of SDS.
After further experimentation, it was found that the assays of samples ~L~:LLe:ated only with SDS could be wo 9~/30153 P~~ ul 21 88S~ --_uved by ;nr~ in~ an optimal combination of ~riton X-100 and urea in the dilution buffer. The optimal ~ullc~llLLaLions of SDS/Triton/urea were empirically found to be 0 . 08~6/0 . 01%/2M in rat samples and 0 . 08%/0 . 04%/2M in 5 human 6amples, respectively. Neither of the above samples diluents, 0.08% SDS/0.01% Triton/2M Urea used for rat samples or 0.08% SDS/0.04% Triton/2 M urea used for human samples, induced ~PtA~ ~ or elution o~ the coated BSA-AGE antigen from the well (data not shown).
Using the newly developed assay pL~LL~ a; L method, significant differences in the level of hemoglobin-AGE
were obselvt:d between normal patients and ~l;Ahetics (Figure lA). Similar data are obst:Lvtd in samples from 15 normal rats and rats with induced diabetes (Figure lB).
The present assay method for Hb-AGE shows good correlation with Hb-Alc in normal and diabetic humans (Figure 2). Thus, Hb-AGE is as effective a marker as Hb-20 A1C for diagnosis of AGE-associated ~lic~Acr~c~ such as 1l i Ahet-,c .
With the newly modified method, the inhibitory effect of aminog~l~n;-linP on the Hb-AGE content in vivo from samples 25 in a rat assay was clearly d LLated (Figure 3).
Hemoglobin-AGE is a much more relevant marker for the Aminn~lAnid;n-~ effect than Hb-A1C, since the latter is not affected by the presence of Aminn~lAni~lin~, or other AGE-inhibitors, at their rhArr-^~ntically relevant 30 cullc~ LLation.
Using the method of the present invention, delipidated hemolysates can be diluted down to around 1-2 mg/ml of protein, which consists mostly of hemoglobin. For 35 example, the samples shown in Figures 1, 2, and 3 were diluted 40 fold befûre addition to the 2ssays. One possible explanation for this increased sensitivity is ~ WO95130153 21 8~920 Y~
that the SDS/Triton/Urea pretreatment eYposes ID~-AGE
epitopes that would otherwise be in~cc~cc;hle to antibody binding in the ELISA or other i csay.
5 This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of 10 the invention being indicated by the ~rp~n~ Claims, and all changes which come within the meaning and range of es~uivalency are intended to be ~ c~d therein.
Various references are cited throughout this 15 speci~ication, each of which is incoL~uLated herein by rerere=ce ln 1t~ ~ntirety.
-
METEIOD FOR ~ gTN
ADV~NCED Gl.YCO8YLaTION .~.)Du~;~o FIELD OF THE INVENTION
The present invention relates to method for ~ gnnciC and monitoring of 1; ce~coc and disorders associated with advanced glycosylation e~ CJdaULs (AGE) formation.
Accordingly, the invention relates to ~;agnnsin~ and 10 monitoring diabetes and the ageing process. In particular, the invention is directed to detecting AGE-modified hemoglobin (Hb-AGE~ for the foregoing ~uL~o~es, and in; uved assay therefore.
BA~ uNL~ OF T~F INVENTION
The reaction between glucose and proteins has been known for some time. Its earliest manifestation was in the appearance of brown pigments during the cooking of food.
20 In 1912, Naillard observed that glucose or other reducing sugars react with amino acids to form adducts that undergo a series of dehydrations and reaLLc.l.y Ls to form stable brown pigments (Naillard, 1912, C.R. Acad.
Sci . 154: 66-68 ) .
Thus, the nonenzymatic reaction between glucose and the free amino groups on proteins to form a stable amino, 1-deoxy ketosyl adduct, known as the Amadori product, has been shown to occur with hemoglobin, wherein the reaction 30 of glucose with the amino t~nm;n-lc of the B-chain of hemoglobin forms the adduct known as hemoglobin A1C. The reaction has also been found to occur with a variety of other body proteins, such as lens crystallin, collagen and nerve proteins (see Bunn et al., 1975, Biochem.
35 Biophys. Res. Commun. 67:103-109: Koenig et al., 1975, J.
Biol. Chem. 252:2g92-2997; Nonnier and Cerami, in Naillard Reaction in Food ~nfl Nutrition, ed. Waller, G.A., American Chemical Society 1983 , pp. 431-448 ; and W095130153 2~8~;2~ IIU.,.- I ~
Nonnier and Cerami, 1982, Clinics in Enaocrinology and Metabolism 11:431-452).
IloleuveL, brown pigments with spectral and fluorescent 5 properties similar to those of late-stage Naillard products have also been observed in vivo in association with several long-lived proteins, such as lens proteins and collagen from aged individuals. An age-related linear increase in pigment was vbseL ved in human dura 10 collagen between the ages of 20 to 90 years (see Monnier and Cerami, 1981, Science 211:491-493; Monnier and Cerami, 1983, Biochem. Biophys. Acta 760:97-103; and Monnier et al., 1984, Proc. Natl. Acad. Sci. USA 81:583-587) .
Glucose and other reducing sugars attach non-enzymatically to the amino groups of proteins in a cu..ct~ L~tion-dor~ndPnt manner. Over time, these initial Amadori adducts can undergo further reaLLc.l,y Ls, 20 dehydrations and cross-linking with other proteins to Ar 1 ~te a family of complex nLLu.;LuLes referred to as Advanced Glycosylation El~d~LVdUULS tAGEs). Substantial p~ UyLèSS has been made toward the elucidation of the role and rlinirAl cignifjcA"re of advanced glycosylation 25 ell~Lvdu~Ls, so that it is now acknowledged that many of the conditions heretofore attributed to the aging process or to the pathological effects of d i CP:~cc-c 6uch as ~1; Ahet ~PC, are attributable at least in part to the formation of AGEs in vivo.
AGE ~r~ l Ation can be indicative of protein half-life, sugar Cvll~ell~L~tiOn~ or both. These factors have important c~m~P~lonrPs. N~lr-~uus studies have indicated that AGEs play an important role in the nLLU~LUL~ll and 35 functional alterations which occur during aging and in chronic disease. Additionally, advancêd glycosylation WO 95/3~153 2 1 ~ ~ 9 2 0 P~
Loducts are noted to form more rapidly in diabetic and other ~i c~AcO~l tis6ue than in normal tissue.
Hb-AGE has been found to be predictive of aging or 5 disease ~Luu,L~assion over the long term (International Patent Publication No. W0 93/13421 by Bucala, published July 8, 1993; Makita et al., 1992, Science 258:651-653).
Hb-AGE measurements provide an d~Lu~iate index of long-term tissue --A;fic~tion by AGEs and are useful in 10 AC5.ocC; nrj the contribution of advanced glycosylation to a variety of diabetic and age-related l; cations. While hemoglobin A1C (HbA1C) has been reported as predictive of the extent of glycation on the hemoglobin B chain, HbA~C
is only a reversible int~ a; Ate in the advanced 15 glycosylation pathway and -- UUS other int~ Ates are believed to exist. Hb-AGE, as an irreversible adduct, is a superior measure of disease ~LoyL~s6ion~
drug effectiveness, etc.
20 Hb-AGEs are used to more readily correlate the progression of disease and longer term control of blood sugar levels. HbA~C ls a reversible inf - "; ate, and reaches ~qll~lihrium with glucose over a 3-4 week period.
Hence, levels of HbA~C only reflect blood glucose only 25 during this short time period. Hb-AGE, in contrast, is an irreversible adduct and reflects blood sugar over the lifespan of hemoglobin. Thus, the effectiveness of t Leai L for AGE-related ~ Ar-c or disorders can also be det~rm;nr~ from the level of Hb-AGE in samples. Thus, 30 the reduction in Hb-AGE levels as a result of aminogll ~ni~lin~ therapy is a primary example of the s~lcc~ccful rhArr--ological inhibition of advanced glycosylation in human subjects.
35 Prior to the instant invention, detection of Hb-AGEs required a complex assay format, that included TCA
precipitation of the hemoglobin from the hemolysate, wo 9S/301~3 2 1 8 3 ~ 2 0 F~~
followed by centrifugation to separate the precipitated protein from the ,,u~eL.Id~ lL liquid fraction. Resolution of the precipitate was accomplished by adding sodium hydroxide at high pH (pH>ll), followed by pH adjustment 5 with 0.3 M KH2P04, pH ~.4 buffer (Makita et al., l99l, Diabetologia 34:40-45). After pH adjustment to about 7 . 8, some of the hemoglobin precipitates out of solution, thus requiring a separation step prior to assay of the liquid fraction. In short, the current protocol complex, 1~ is , -, time Çcmcllmin~, subject to and generally not ~rP1 ic~hle to a çl inic~l laboratory setting.
Accordingly, there is a need in the art for a simpler, faster, and milder sample treatment protocol to 15 facilitate testing for Hb-AGE in clinical laboratory settings.
There is a further need in the art for kits containing the reagents neC~cs~ry to perform such assays.
20 The citation of references herein shall not be construed as an ~llmiccinn that such is prior art to the present invention .
STTMMARY OF TTlF~ INvENTIoN
The present invention is directed broadly to a method for detecting the presence of ~ l nhin-AGE in a sample.
The method involves diluting the sample in a dilution buffer, which dilution buffer comprises an anionic 3 0 protein denaturing detergent at a concentration sufficient to denature hemoglobin-AGE without interfering in binding of reagents with hemoglobin-AGE. Preferably, the dilution buffer further comprises a non-ionic surfactant at a cu"c~l,LLcltion sufficient to facilitate 35 detection of hemoglobin-AGE; and a polar denaturing agent at a cu"c~1,LL~tion sufficient to denature hemoglobin-AGE
and increase assay sensitivity, without denaturing .
binding of reagents to hemoglobin-AGE. After diluting the sample in the dilution buffer, the sample is contacted with means for detecting the presence of hemoglobin-AGE in the sample, and the presence of 5 hemoglobin-AGE in the sample is detected with the detection means. The sample can be assayed directly after dilution.
In specific aspects, the Cu--c~ Lation of anionic protein 10 denaturing detergent in the dilution buffer ranges from about 0 . 04% to about 0.16% (w/v) . The ~u..c~l-LLation of non-ionic surfactant, if present, ranges from about O . 005% to about 0 . 1% (w/v); and the cc,~ ..LLation of the denaturing agent, if present, is between about 0. 5 M to 15 about 3 M.
In a specific pre~erred 'i L, the anionic protein denaturing detergent is sodium dodecyl sulfate. In a further specific preferred: '_'ir L, the non-ionic 20 surfactant is a polyoxyethylene ester, in particular Triton X-100 polyoxyethylene ester, and the denaturing agent is urea.
The best mode contemplated by the inventor for practicing 25 the invention uses a dilution bu~fer in which the .~ "c~ ation of sodium dodecyl sulfate is about 0. 08%, the CullC~ Llat.ion of Triton X-100 polyu,Ly-:L~lylene ester is s~ cted from the group consisting of about 0 . 01% and about 0. 04%, and the CUllC~:llLLat.iOn of urea is about 2 M.
Preferably, the dilution buffer is buffered to between about pH 7 to about pH 8, and contains salts at a con~-~ntration approximating physiological ionic strength.
35 In a further -~;r L, the invention provides a method for quantitating the amount of hemoglobin-AGE in a sample. Quantitation can be accomplished by detecting _ _ _ _ _ _ _ WO 95130153 2 1 8 ~ 9 2 0 r~llu~ Jaul the ~les~.1ce of hemoglobin-AGE in the sample as described above. The extent of binding of a binding partner to hemoglobin-AGE, or other means for detecting the presence of hemoglobin-AGE, can be quantitated. The extent of S detection CULL~ VIIdS to the amount of hemoglobin-AGE in the sample.
The invention further provides a method for detecting or nncin~ the pLt s~11ce of a disease associated with lO elevated h~ -~lnhin-AGE levels in a li;ln subject by comparing the amount or level of h~ Jl nhin-AGE detected in a sample to a level of hemoglobin-AGE normally present in the 1 i ~n subject. An increase in the level of hemoglobin-AGE as compared to normal levels indicates a 15 disease associated with elevated levels of hemoglobin-AGE .
In another aspect, the invention provides a method for monitoring the course of a disease associated with 2~ elevated hemoglobin-AGE levels in a 1 i ~n 5ubject.
The level or amount of hemoglobin-AGE in a series of samples obtained at different time points from a r-r~ n subject is detc~rminpcll as described above. An increase in the level of hemoglobin-AGE over time 25 indicates progression of the disease; a decrease in the level of hemoglobin-AGE over time indicates regression of the disease.
In yet a further aspect, the invention-provides a method 30 for monitoring a theLcl~t uLic LLaa, 1 of a disease associated with elevated hemoglobin-AGE levels in a r-mr-l i~n 5ubject. The level or amount of hemoglobin-AGE
in samples obtained at different time points from a 1 i 1n subject undergoing a therapeutic treatment for 35 a disease associated with elevated hemoglobin-AGE levels is ~ot~rmin~-d. A decrease in the level of AGEs over time indicates an effective therapeutic outcome.
W095130153 21 8~920 r~
In particular, the invention provides a preferred method for titrating a dosage of an inhibitor of AGE formation to determine the optimum dosage. The level or amount of hemoglobin-AGE in a series of samples obtained at 5 different time points from a 1; ~n subject receiving aL~ively larger doses of an inhibitor of AGE
formation over the time points is evaluated. An optimum dosage of the inhibitor is a dosage above which no further decrease in the level of hemoglobin-AGE is lO observed.
In still another aspect, the invention provides a method for monitoring the long term glucose level in a r-m~ n subj ect . The level or amount of hemoglobin-AGE in a 15 sample from a 1 ;:~n subject is de~;n~ The level or amount of hemoglobin-AGE i5 indicative of the long term glucose level in the 6ubject.
The methods of the present invention are achieved by 20 dilution of the sample in the dilution buffer of the invention. Accordingly, the present invention further provides a dilution buffer. The dilution buffer of the invention comprises an anionic protein denaturing detergent; a non-ionic~surfactant; and a polar denaturing 2~ agent. These l~:ayt~ can be provided in a cu~ 1LLG~e, for ~ t j nn such that the anionic protein denaturing detergent is present at a co1~ct:111 LGLiOn sufficient to denature hemoglobin-AGE without interfering in binding of reagents with hemoglobin-AGE; the non-ionic surfactant is 30 present at a ~ ullcc~ La~ion sufficient to facilitate detection of hemoglobin-AGE; and the denaturing agent is present at a cu11c~1--L~tion sufficient to denature hemoglobin-AGE and increase assay sensitivity, without denaturing binding of reagents to hemoglobin-AGE.
35 Preferably, the dilution buffer i8 pll:,ua~ cd such that the cu~ La~lon of anionic protein d~ aLuLing detergent in the dilution buffer ranges from about 0. 04% to about WO95/30153 2 1 3~9~0 ..I~U~,~ J~ul 0 . 16% (w/v); the ~u-.c~:..LL.ltion of non-ionic surfactant ranges from about û. 00596 to about 0 . 1% (w/v); and the . ul.c~:..LLation of the denaturing agent is between about 0.5 M to about 3 M. In another preferred aspect, the 5 anionic protein denaturing detergent is sodium dodecyl sulfate, the non-ionic surfactant is Triton X-100 polyoxyethylene ester, and the denaturing agent is urea.
In the most preferred aspect of the invention, which is the best mode contemplated by the inventors for 10 practicing the invention, the Cu-.c~:..LLation of sodium dodecyl sulfate is about 0.08%, the c~ -tion of Triton X-lO0 polyoxyethylene ester is selected from the group consisting of about 0. 01% and about 0. 04%, and the cu.,~m:-.LLation of urea is about 2 M.
In a further _'i L, the dilution buffer is buffered to between about pH 7 to about pH 8, and contains salts at a cu.lc~-lLLation approximating physiological ionic strength .
In a further aspect, the present invention relates to a kit for detertin~ the presence of hemoglobin-AGE in a sample. The kit of the invention comprises a dilution buf f er as described above, in ,_u..~ LL at.ed cr ready-to-25 use form; means for detecting the presence cf hemoglobin-AGE; other reagents; and directicns for use of said kit.
In an Example, infra, the present invention provides for 3 o detection of the level of AGE in hemoglobin in samples from normal subjects and diabetic subjects. Samples from both humans and rats were successfully tested using the method of the invention. Furthermore, the results obtained from human samples show a high degree of 35 correlation between the level of hemoglobin-AGE in a sample and the level of hemoglobin-A1c in a sample. Most importantly, the assay of the instant invention can be ~ W095/30153 21 88920 P~
used to detect the "aminog~1An;~1;ne effect," which is the decrease in the level of hemoglobin-AGE in a sample from a subject undergoing therapy with the AGE-inhibitor aminog~An;~l;nP. Thi6 effect cannot be detected by 5 measuring the level of hemoglobin-A~c, since aminoguAn;~l;nP, or other inhibitors of AGEs, do not affect hemoglobin-A1c levels. The so called "aminoguAn;~l;nP effect" applies to other AGE-inhibitors as well.
Thus, the present invention a~v~..Lly~u,-sly provides for monitoring the efficacy of a therapy for preventing advanced glycosylation ~ duct formation, as well as a more sensitive test for diagnosis of an AGE-associated 15 disease or disorder.
Accordingly, it is an object of the present invention to provide an improved assay Por hemoglobin-AGE.
20 It is a further object of the invention to provide a dilution buffer that increases the speed, ease, and simplicity of assays for hemoglobin-AGE.
It is a yet another object Or the invention to provide a 25 buffer that ~nhAnl-PC immuno-detection of hemoglobin-AGE.
Yet a further obj ect of the invention is to provide kits for detecting the ~L~se1lce of hemoglobin-AGE in a sample.
30 These and other objects of the invention are addL~_sed and will be better understood by reference to the - following drawings and detailed description of the invention .
~ ,",~Vt!,~ 33~
21 88~20 ~RIEF DESt'RTPTTON OP TT~R D~WINGS
FIC-~JRE 1 preaents the level of AGE modification of hemoglobin. The samples were pretreated with O 08~ SDS, 5 0.049~ or 0.01~ Triton ~-LOQ, and 2 M urea in the sample diluent prior to testing in an antibody ELISA assay for the level of AGE modification. The data are reported as units of AGE per mg of hemoglobin in samples from diabetic and normal subj ects ~ standard error of the mean 10 (SEM) . The amount of AGE was determined- using an EI,ISA
assay developed earlier (Makita et al., 1992, J. Biol.
Chem. 267:5133-38; Int~rn~t;on~l Patent Publication WO
93/13g21) i the concentration of protein (which consists mostly of hemoglobin) was determined with the ~owry reagent using purified bovine serum albumin as a standard --(I,owry et al., 1951, J. ~iol. Chem 193 :265) . (A) Samples were obtained from 11 normal and 11 diabetic humans. The hemoglobin Al~ levels for the normals were 4.9:t0.52, and for diabetics were 8.3~1.49. A
20 concentration of 0.04~ Triton was used to pre-treat human samples. (B) Samples were obtained from 10 normal and 10 diabetic rats. Diabetes was induced in rats by treatment with streptozocin, and hyperglycemia was confirmed by assaying blood glucose.
FIGURE 2 presents data which demonstrate the correlation between measurement of hemoglobin-AGE (units o~ AGE per ~--mg of he~oglobin) according to the present invention versus the level of hemoglobin Alc (percentage o~ total 30 hemoglobin). The correlation was performed with 22 human samples (the same samples as sho~n in Figure L~
FIGURE 3 presents data that demonstrate the sensitivity of the present assay to detecting the hemoglobin effect.
35 The level of AGE-hemoglobin (units of AGEs per mg o~
hemoglobin) was measured after pretreatment of samples according to the present invention. Samples were ~ =
WO 95B0153 2 1 g g 9 2 0 . ~1I~J...5, ~ul obtained from normal rats, induced diabetic rats, and diabetic rats that received 50 mg per kg of aminog~An i tl i nP: each sample group included 10 members .
Statistically significant differences in the level of Hb-5 AGE were observed between normal and diabetic rats (p<O.Ol), and between aminog~lAnitlin-~-treated and diabetic rats (p<O . 05) .
n~T~ DES~l?TPTION OF ~ NV~LlUN
The present invention relates to an improved assay for hemoglobin-AGE. In particular, the invention discloses a dilution buffer containing an anionic protein denaturing detergent, such as sodium dodecyl sulfate. Preferably, 15 the dilution buffer further comprises a non-ionic surfactant, such as polyoxyethylene esters like Triton X-100, and a polar denaturant, such as urea. Dilution of hemolysate in this dilution buffer ~i~nifirAntly increases the simplicity and speed of assays for 2 O hemoglobin-AGE .
As used herein, the term "AGE-" refers to the _ ' which it modifies as the reaction product of either an advanced glycosylation ~l~d~-u-lu~:L or a . ' which 25 forms AGEs and the ~ d so - 'ified, such as the bovine serum albumin (BSA). Thus, AGEs include, but are not limited to, AGE-proteins (such as BSA-AGE), AGE-lipids, AGE-peptides, and AGE-DNA. AGE polypeptides or AGE proteins can be formed in vitro or-~in vivo by 30 reacting a polypeptide or protein with an AGE, such as AGE-peptide, or with a ~ ' such as a reducing sugar, e.a., glucose, until the polypeptide or protein is r ~'ifiocl to form the AGE-polypeptide or protein.
35 The term "glycosylation" is used herein to refer to the non-enzymatic reaction of reducing sugars with a nucleophile, in particular an amine group, on a polypeptide or protein, such a~ hemoglobin, a lipid, or DNA, which leads to formation of AGEs. These processes are well known in the art, as described above. Recently, the term "glycation" has become more favo~ed to refer to 5 non-enzymatic glycosylation processes. Thus, the term "glycosylation, " as specifically defined herein, and "glycation" are equivalent.
As stated above, according to the present invention, a 10 sample ~on~in;n~ red blood cells or hemoglobin from a subject is treated by dilution in a diluti~n buffer, e.g., buffer l-,.n~A;nin~ sodium dodecyl sulfate, Triton X-100, and urea. The sample may be blood, or red blood ceils isolated from blood, that has been treated to 15 hemolyze the red blood cells. Alternatively, the sample may be hemoglobin isolated from red blood cells.
Although not intending to be limited by any particular . theory or hypothesis, it is believed that moderate 20 ~pn~tl~r~ion of hemog~obin exposes additional AGE
epitopes to antibody binding, thus significantly increasing the sensitivity of an; ~csay for ~b-AG~
compared to untreated samples.
25 According to the invention, samples can be obtained from any source, lncluding in v1tro or ~n vlvo sources. In particular, the instant invention contemplates assays on a sam~ole from an animal, and more preferably, from a mammal, :including humans, as well as mammals such as 30 dogs, cats, horses, cows, pigs, guinea pigs, mice, and rats. Thus, the present invention provides for monitori~g ~G~ levels in human and veteri~y medicine.
The concentration of anionic protein denaturiny 35 deteryent, such as SDS, in the dilution buffer is sufficient to denature hemoglobin-AGE without inter~ering in immunological binding or causing detachment or elution Wo 951301~3 of a solid phase reagent in a solid phase; :~esay.
Accordingly, the concentration of anionic protein denaturing detergent can range from about 0. 04% to about 0.1696 (w/v), ~lPrPn~lin~ on the stability of the detection 5 assay, e.q., antibody binding. Preferably, the anionic protein denaturing detergent is SDS, and the Cu~ La~ion of SDS i5 about 0 . 0896 .
In addition to SDS, the invention further contemplates lO use of analogous detergents that interact with and effectively denature proteins. Although SDS has been found to be the most useful protein denaturing detergent, other such detergents, although le6s effective than SDS
f or denaturing proteins, can be used according to the 15 present invention. Obvious choices include C8 to C20 hydrocarbon acyl sulfate salts, i.e., SDS analogs.
Substitution of a different anionic protein denaturing may require retesting to ~PtP~n; ne an optimal l U~ LLCl~iOn, which merely requires straightforward 2 0 experimentation .
The cu~ lL~clLion of non-ionic surfactant, if any, such as Triton X-lOO, in the dilution buffer is sufficient to facilitate detection of hemoglobin-AGE in a sample. The 25 non-ionic detergent may function, in part, to buffer harsh effects of the anionic detergent. In a sperifir~
, the non-ionic surfactant is the polyoxyethylene ester Triton X-lO0, and the c;ullcel.LL~-t.ion of Triton X-l00 ranges from about 0 . 00596 to about O .1%
30 (w/v). In a preferred aspect of the invention, the ~U~ LClLiOn of Triton X-l00 is approximately 0. 0l96 for - samples from rats, and 0.049~ for samples from humans.
Optimal cc,1~c~,1LL~Lion for samples from other animals, for different AGE-specific antibodies, and for the choice of 35 anionic detergent can be readily de~PrminPd using standard experimental techniques.
21 8~q20 The invention further contemplates use of an analogous non-ionic surfactant in place of Triton X-100. A non-ionic surfactant to be substituted ~or Triton X-100 may be tested to ~lP~Pr;ll; nP an optimal conceI:Ltration; such 5 testing involves straightforward experimentation.
..
The concentration of a polar denaturing agent in the dilution buffer is sufficient to denature the Xb-AGE or stabili~e and maintain detergent-denatured Hb-AGE in a 10 denatured state, and increase assay sensitivity, without denaturing immunological reagents used to ~etect the - --presence of Hb-AGE in a sample. In a specific aspect, the denaturant is urea, in a concentration of between about 0.5 M to about 3 M. Preferably, the rnnrPntri~t;r,n 15 of urea is 2 M. Furthermore, the invention contemplates use of other polar denaturing reagents, such as guanidine-XCl, at a suitable concentration in place of urea .
20 Preferably, the dilution buffer is pH-buffered and ionic strength controlled. For example, the ~ t; nn bu~fer --may contain a physiological concentration of sodium chloride, or other salts, as well as buffers to maintain pH of the ~ t inn solution between about pH 5 and about 25pH 9. Preferably, the pX of the buffer is about pH 7 to about pX 8; more preferably, the pH is about pH 7 4. In a specific embodiment, ill~ra, the dilution bu~fer is a sodium phosphate bu~fered solution cnnt;~in;ng sodium chloride: It is preferable not to use potassium salts in 30the ~ilution buffer, since it is known that potassium ion can induce precipitation of SDS. An.other suitable buffer salt is Tris-HCl.
A sample rrnt~ining hemoglobin can be diluted from about 3510-fold to about 100-fold with dilution buffer prior to conducting an immunoassay to detect Hb-AGEs.
Alternatively, the sample can be diluted so that the ---~ WO 95/30153 2 1 8 ~3 9 2 ~ r~
protein col~cerlLL~-tion (mo5t of the protein being hemoglobin) ranges from about 0.1 mg/ml to about 10 mg/ml. In a spP~ ;f1c ~ho~;- L, inf~, a hemolysate sample is diluted 40-fold in the dilution buffer of the 5 invention prior to cn~ t;n~ the i - cSay. In the example, ~, the con~n~ration of protein in the sample is approximately 1-2 mg/ml, as ~ tprminpd by the Lowry assay, using BSA as a protein standard (Lowry et al., 1951, J. Biol. Chem. 193:265).
The present invention can enhance many of the; - - CRay or ; ~ C~-c y-type formats that can be used to detect the presence of, and measure the quantity of, hemoglobin-AGE, by increasing the accessibility of AGE-epitopes 15 present on hemoglobin-AGE. As used herein, the terms "means for ~lPte~;n~ and "means for quantitating"
hemoglobin-AGE refer to detection or quantitation of hemoglobin-AGE in an any such; ccay-type format.
Generally, such means comprise contacting a sample with 20 one or more binding partners of hemoglobin-AGE, followed by detection of binding of the one or more binding partners to hemoglobin-AGE in the sample, and, if desired, quantitation (measuring t~e quantity) of binding that occurs. Binding partners include, but are not 25 limited to, ant;ho~;~C to hemoglobin, antibodies to AGEs, Il:ctl~LuLa for AGEs, small molecules that bind either hcmoglobin or AGEs, and the like. At least one such binding partner must be spPc;f;c for an AGE. Detection of specific binding of a binding partner of hemoglobin-30 AGE with Hb-AGE in the sample is indicative of the presence of Hb-AGE in the sample.
Accordingly, once a sample containing hemoglobin has been diluted in the dilution buffer of the invention, the 35 presence of Hb-AGE can be det~rm;n~cl using many formats of immunological assay means, e.q., using well known techniques such as but not limited to radio; -- y, _ _ _ _ _ _ _ , . .
Wo9S/30153 2~ 8g9~1~ r ~
ELISA (enzyme-linked immunosorbant assay), "sandwich"
- -ys (in particular ELISA assays), competitive assays (in particular competitive ELISA assays), LL1C assays, precipitation r~prtionc~
5 immunofluoL~s~:ence assays, protein A assays, etc. In one ;- ~, antibody binding is detected by detecting a label on the primary antibody. In another ~mho~; , the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
10 In a further ~mho~ , the secondary antibody is labeled .
In a spDc; f; r ~mho~ , a sandwich assay format is used, in which an anti-AGE antibody is attached to the 15 solid phase, and an anti-hemoglobin antibody (either labeled directly or detected with a s~rnnrl~ry labeled antibody) is used to detect binding of Hb-AGE with the anti-AGE antibody. In another . ; ~, the anti-hemoglobin antibody can be attached to the solid phase, 20 and an anti-AGE antibody used to bind any AGE epitopes present on the hemoglobin molecules bound to the ant~-hemoglobin antibody.
As used herein, the term "antibody" refers to polyclonal, 25 monoclonal, chimeric, and single chain antibodies, as well as Fab fragments (F(ab' )z, F(ab), Fv, etc. ), and an Fab expression library.
The amount of hemoglobin-AGE in a sampl:e can be 30 det~rm;n~rl quantitatively. For example, the extent of binding can be ~uantitated, and the amount of Hb-AGE
t~rm; n~d by extrapolation from a standard curve. In a specific aspect, a standard amount of hemoglobin-AGE can be provided in a kit of the invention for use as a 35 standard. In another ;- ~, the amount of AGE
(e. a ., units of AGE) can be ~l~t~rm; n~d by comparison with WO 95/30153 l7 / ~~
a standard amount, such as a BSA-AGE sample as described in the examples, ~.
In a 5pc~c;f;r-: ' ~ ';~ L, the assay described in ~akita 5 et al. (1992, J. Biol. Chem. 267:5133-38; see also International Patent Publication No. W0 93/13421) can be used to detect the presence of Hb-AGEs in a sample prepared according to the present invention.
In a further ~ of this invention, commercial test kits suitable for use by medical or clinical te~-hnnl o~ists or speciAl; cts may be prepared to determine 15 the presence or absence of hemoglobin-AGEs. Such kits will include at least a dilution buffer of the invention, and means for detecting the ~sc.~.~e of hemoglobin-AGE as described suPra. The dilution buffer can be provided in cu..~-~..LLated form, which reguires the addition of water 20 or buffer to bring the -- L~ of the buffer to their appropriate cu..c~..LLa~ion. Providing the dilution buffer in cu..L:e..LLated rorm alvallLa~uusly reduces the weight and size of the kit, and thus is more cost effective.
25 ~he contents of the kit are preferably in~uLuuLal ed in a package, such as cardboard or plastic packaging material, which is ~ n~ to hold all of the --tc of the kit. Each ~ L can be held in separate containers, such as glass or plastic vials, for use or dilution prior 3 0 to use .
- In~ accordance with the testing techniques ~; CcllcF^1 above, one class of such kits will contain at least a binding partner of an AGE epitope and dilution buffer of 35 the invention comprising an anionic protein denaturing detergent, a non-ionic detergent, and a denaturant, as described above, and means for detecting binding of the _ _ _ _ _ _ wo 95/30153 2 1 8 8 ~ 2 0 . ~
binding partner of an AGE epitope to an AGE epitope present in the sample diluted with the dilution buffer according to the invention.
5 The kit may also contain directions for conducting an assay in A~ror~nne with the method selected, e.g., "competitive", "sandwich", "DASP" and the like. The kits may also contain peripheral reagents 6uch as buffers, stabilizers, etc.
Accordingly, a test kit may be prepared for the cl LL~ltion of the ~L~:St:llCe, S~uantity or activity of AGEs, comprising:
(a) a labeled specifically reactive component 15 obtained by the direct or indirect att~ L of a binding partner of an AGE epitope or a specific binding partner thereto, to a detectable label;
(b) a dilution buffer, in ~u..cc~ c.Led or ready-to-use form, compri6ing an anionic protein denaturing detergent, a non-ionic detergent, and a polar denaturant:
(c) other ~ay~.lLs; and (d) directions for use of said kit.
More specifically, the diagnûstic test kit may comprise:
(a) a known amount of a first binding partner to an AGE epitope as described above, generally bound to a solid phase to form an ;r-~nnsorh~nt;
(b) a second binding partner to an AGE epitope or to hemoglobin, which second binding partner is labelled:
(c) a tl;ll1ti n buffer, in ~;u.,-~l.LLc.ted or ready-to-use form, comprising an anionic protein denaturing detergent, a non-ionic surfactant, and a polar denaturant;
(d) if n~c-~RRIry, other reagents; and (e) directions for use of said test kit.
~ WO95130153 21 ~8920 1 "- 1 In another specific embodiment, a diagnostic test kit may comprise:
(a) a known amount of a first binding partner to hemoglobin as described above, generally bound to a solid 5 phase to form an i --orh~nt;
(b) a second binding partner to an AGE epitope, which second binding partner is labelled;
(c) a dilution buffer, in CU~ LC-ted or ready-to-use form, comprising an anionic protein denaturing lO detergent, a non-ionic surfactant, and a polar denaturant;
(d) if nPc~q~-ry, other reagents; and (e) directions for use of said test kit.
15 In another ^ ~'i-- L, a diagnostic test kit may comprise:
(a) a binding partner of an AGE epitope, generally associated with a solid phase to form an i ~..L1.~
(b) a known quantity of a labelled AGE that i8 2 0 capable of binding to the binding partner of an AGE
epitope;
(c) a dilution buffer, in .:u,.~ Lc~ted or ready-to-use form, comprising an anionic protein d~,lla-uLing detergent, a non-ionic surfactant, and a polar 2 5 denaturant;
(d) if nPl.F-qqAry, other reagents; and (e) directions for use of said test kit.
In another P~-~Or~ a diagnostic test kit may 3 0 comprise:
(a) an AGE, generally associated with a solid phase to f orm an i - _ l;
(b) a known quantity of a labelled binding partner of an AGE epitope that is capable of binding to the AGE
35 i -~_L~e~t;
(c) a dilution buffer, in concentrated or ready-to-use form, comprising an anion~c protein denaturing W0 95130153 21 8 8 9 2 0 F~
detergent, a non-ionic surfactant, and a polar denaturant;
(d) if ne~cs~ry, other reagents; and (e) directions for use o~ said test kit.
The test kits of the invention can comprise an AGE
standard for quantitating the amount of AGE in a sample.
In a specific embodiment, BSA-AGE is used as an AGE
standard .
Preferably, in a test kit of the invention, the binding partner of an AGE epitope is an antibody to an AGE , e . q ., as described above (see Makita et al., 1992, sup~a;
International Patent Publication No. W0 93/13421).
Preferably, in a test kit of the invention that comprises an AGE, the AGE is BSA-AGE as described above and in the examples, infra.
20 In a preferred ~ t, the dilution buffer ~Pc SDS, Triton X-100, and urea in the C~ r~LtiOnS that are ~ i ccl oced g~.
MOnit:Or;n~l IL~a~ lent to Prevent ~:P Formation The hemoglobin-AGE assay of the present invention is particularly well suited to monitor the aminog~l~n;ti;n~, or other AGE-inhibitor, effect in a large scale study or trial, since the assay in fast and simple. Si~ilarly, 3 0 the assay of the present invention can provide ror monitoring the course of therapy of any product ~ ect~d to inhibit AGE formation. In particular, the present method for detQct~ n~ hemoglobin-AGE provides for carefully titrating the ~h.or~r~--tic dose of ~n AGE-35 inhibitor.
WO 95130153 2 ~ 8 8 9 2 0 P~
In a particular aspect, the invention provides fortitrating the optimum dosage of an agent that inhibits AGE formation. Titration of the optimum dosage allows a physician to select a dosage with maximum benefit for a 5 patient, while m;n;m;7;r~ the risk of side effects.
Since t.-hol;c p~ y~ and rates of clearance dirfer from one individual to another, the optimum dose o a therapeutic agent can vary from one person to another.
The term "optimum" when used to modify "dose" should not 10 be cu.lrur~ed with the term "effective"; a dosage of a therapeutic agent can be effective without being the optimum dose.
Thus, the invention provides for monitoring the effect of 15 administration of agents that block the post-glycosylation step, e., the formation of fl.lores~;el-t or cros61inking .:}-~ nres whose presence is associated with, and leads to, the adverse sequ~lae Or glycosylation. An ideal agent would prevent the 2 O formation of a chromophore and its associated cross-links of proteins to proteins and trapping of proteins on the other proteins. The ideal agent would prevent or inhibit the long-term, post-glycosylation steps that lead to the formation of the ultimate advanced glycosylation end 25 products that are a direct cause of AGE-associated pathology .
An inhibitor of the formation of AGEs includes that react with a carbonyl moiety of an early 30 glycosylation product. R~ s~ c.tive Or such advanced glycosylation inhibitors are aminog~lAn i ~; n~, lysine and - ~-hydrA~;nnh;~tidine. In a specific embodiment, the inhibitor is aminog~lAn;-l;n~ (AG) and derivatives thereof.
- Pharmaceutical compositions and methods involving AG and 35 derivatives thereof are well known, as described in U.S.
Patents No. 4, 758, 583, issued July 19, 1988; No.
4, 9O8, 446, issued March 13, 199o; No. 4, 983, 604, issued '1 88~20 January 8, 1991; No. 5,100,919, issued March 31, 1992;
No. 5,106,877, issued April 21, 1992; No. 5,114,943, issued May 19, 1992; ~o. 5,128,360, issued July 7, 1992;
No. 5,1~0,324, issued July 14, 1992; No. 5,130,337, 5 issued July 14, 1992; No. 5,137,916, issued August 11, 1992; No 5,140,048, issued August 18, 1992; No.
5,175,192, issued December 29, 1992; No. 5,218,001, issued June 8, 1993; No. 5,221,683, issued June 22, 1993;
No. 5,238,963, issued August 24, 1993; No. 5,243,071, 10 issued September 7, 1993; and No. 5,254,593, issued October 19, 1993 Other inhibitors of AGE formation are ~
described in U.S. Patents No. 5,258,381, issued November ~=:
2, 1993; No. 5,356,895, issued October 18, l99g; No.
5,272,116 issued December 21, 1993; No. 5,358,960, issued :
15 October 25, 1994; and Serial No. 08/095,095, filed July 20, 1993. ~ach of the foregoing patents and patent applications is specifically incorporated herein by ref erence in its entirety .
20 The invention may be more completely understood by reference to the following non-limiting example, which is provided solely as exemplary of a specif ic embodiment o~
the invention.
.
F;I~AI~qpJ,~ rlob; n At~.~ AssaY
M~ter;; l R j3rll~ hnrlR
-Pre~rea~ment Buf~er. SDS/~riton/Urea Hemolysate 30 pretreatment buf~er (1 liter) was prepared as follows:
A sodium phosphate (0.02 M) bu~er solution was prepared by adding 0.91 g of sodium monobasic (rr~onohydrate); 1.902 g of sodium dibasic (anhydrous); 3 g of sodium chloride, 35 and 0.2 g of sodium azide to 800 ml of distilled water.
~ WO95/30153 ? 1 8 ~ 92 0 r_"~
To the above buffer (800 ml) were added 120.12 gm of urea (2 M final .,v..~:..LLaLion); 0.8 g of SDS (0.08%) sa~ples, and 0 . 4 or 0.1 g of Triton X-100 (yielding concentrations of 0.04%, for human samples, or 0.01% for rat samples).
5 The pH of the solution was adjusted to 7.4 and the volume was brought to 1 liter with distilled water.
Sodium phosphate buffer was also prepared as described above, without inrlllAlng urea, SDS and Triton X-100.
Pre~aration of the Hemolvsate. Red blood cells were prepared using a distilled water/toluene extraction uceduLa. Briefly, red blood cells (RBCs) were separated from whole blood collected in a heparinized 15 tube by centrifugation at 2-3000 rpm for 10 min. The RBCs were washed by rD~ p~n~ion in sterile isotonic saline (0 . 8596) in a volume approximately equal to the plasma, and repacked by centrifugation as described above . RBCs were stored for up to one week at 4 C in 20 buffered saline after two washing steps that included 30 min soaks in the wash buf f er . RBCs could be stored ror more than 1 week prior to use in an assay by freezing the pellet to -20C after incubation in isotonic saline at 4 C overnight to dialyze out glucose contained in the 25 cells.
Hemolysate was prepared by pelleting enough fresh cells in a SC;L~ a~ed tube to give 1 ml of packed RBCs.
(This step was omitted with frozen RBCs, however, as 30 freezing hemolyses the cells. Instead, the thawed packed cells are used directly for the next step. ) To 1 ml of - packed R3Cs was added 3 ml of distilled water to lyse the cells. Following addition of water, 2 ml of toluene were added to delipidate the suspension. The cells were 35 shaken vigorously for a few minutes, or vortexed intermittently six times to ensure complete lipid extraction. The RBC preparation was then centrifuged at _ wo95/30153 21 88920 1~ s~ul ~
3000 rpm for 10 min to separate the two phases and pellet the c~ l Ar debris. An interface of white insoluble material is founa between the toluene (top) phace and the aaueous (bottom) phase. The toluene phase, above the 5 insoluble material layer overlaying the aaueous hemolysate, was removed using a glass Pasteur pipette with a ~uction flask. After removing the toluene, the tuhe was tipped to expose the red aqueous phase underlying the layer of insoluble material. The aqueous 10 phase was removed with a Pasteur pipette, taking care not to disturb either the layer of; ncol l~hle material or the pellet at the bottom of the tube. The hemolysate (aaueous phzse) was stored overnight at 4-C if the assay was not to be peL L~ - ' immediately . Long term storage 15 of the hemolysate requires use of a preservative or sterile filtration.
Coatina Antiaen. BSA-AGE was ~ aLtd at 30 ~Lg/ml in a 0.1 M bicarbonate buffer, pE~ 9.6, with azide. One 20 hundred ,ul of coating solution were incubated in microtiter wells for 1 hr. at 37-C, or overnight st 4-C.
Coating solution was washed away and a blorkin~ buffer containing PBS with 0.1 g BSA, 1 ml goat serum and azide was added; the plates were incubated for 1 hr. at 37-C.
25 The plates were washed with a PBS, Tween solution prior to addition of sample.
Microtiter Plate AGE AssaY. The hemolysate was diluted in the SDS/Triton/Urea Buffer to give approximately a 1-2 30 mg/ml concentration of protein. Usually, the hemolysate was diluted 1:40-1:60 to achieve the desired protein concentration range. The protein c~ ,l r, Lion of the hemolysate was detPrmin~d using the Lowry procedure (Lowry et al., 1951, J. Biol. Chem. 193:265), with 35 "; fi~ations.
~ Wogs/3ols3 21 88q20 P~
Briefly, 3-10 ,ul of hemolysate preparation were added using an Eppendorf positive displ P~ - L pipetter (0. 5-10 ~1) to a 2-5 ml glass test tube (12 x 75 mm). To this tube was added 1 ml of Lowry reagent. The sample was 5 mixed well and incubated at room t~ .UL~ (RT) for 15-30 min. To each tUbe were added 100 ~Ll of Folin &
Ciocalteu's phenol reagent, and the mixture was vortexed immediately in two short intervals. Aggressive or excessive vortexing was avoided to eliminate or reduce 10 Ahnt~ results. The sample was incubated for 30 min at RT, and optical density read in a dual-beam ~ue~ LL~pl.otometer at 750 nm against a blank consisting of a mixture of 1 ml of Lowry reagent with 100 ~1 of the Folin reagent.
A 1 mg/ml BSA (Sigma RIA grade A7888 or A7030) solution in 300 mM KE12P04 with 3 mM sodium azide was used as the standard for the protein determination assay. The standard samples were prepared by pipetting 3 .12, 6 . 25, 20 12 and 25 ~1 of standard solution into test tubes and performing the assay as described above.
The Lowry reagent, pH 12.8, was prepared by adding 0.5 ml of 2% potassium sodium~ tartrate (KNaC4H406 H20) and 0.5 ml 25 of 1% CUS04 to 50 ml of 2% Na2CO3 in 0.1 N NaOH. The Lowry reagent is stable at RT and should not be stored in the refrigerator. Fresh solution was pLt:~aL~d for each assay.
30 Folin & Ciocalteu's phenol reagent was made by dilution of the stock (Sigma F9252) 1:1 with distilled water in an amber glass bottle to yield a 1 N solution. This solution was stored at RT.
35 All samples were either diluted by the same amount, and concentration calculated from the protein cu.~c~ Lc-tion, or all WO gS/30153 2 ~ ~ ~ 9 2 0 hemolysates were adjusted to the same protein cu.,ce--LLcl~ion tapprox. 1 mg/ml) by using an eYact ratio of buffer to hemolysate.
5 To each well of the assay plate were added 50 ~Ll of solution of diluted hemolysate. An additional 50 ~Ll of the SDS/Triton/Urea Buffer was added to each well to nnrrql i 7e the sample.
10 PreParation of AGE Standard. BSA-AGE for use as a standard was prepared by incubating BSA with glucose in a molar ration of 500:1 glucose:BSA in a phosphate buffer, pH 7.4, for six weeks at 37-C. After the incubation period, the solution was dialyzed against PBS/0 . 02%
15 azide, and stored at -80-C.
Pr~nqration of SamPle Standards. BSA-AGE standards (0.1-3.75 ~Lg/well) were made up in sodium phosphate buffer lacking SDS/Triton/Urea. Fifty ~l of each standard was 20 added to assay wells.
Primarv Antibodv Dilution. Anti-AGE antisera was diluted to give a B50 of 1 ~Lg BSA-AGE. The BSA-AGE was prepared as described above for preparation of the BSA-AGE
2 5 standard .
Assav Procedure. After coating the microtiter wells with BSA-AGE, 50,u1 of sample or standards, diluting buffers, and 50 ~l of primary antibody were added to each well.
30 The microtiter plate was incubated for 2 hrs. at room t ~ULC:. The microtiter plate was washed 3X with Tris buffered saline (TBS)/Tween. To each well were added 100 ~Ll of A l kq l i n q phosphatase labeled secnn~ ry antibody at the appropriate titer, and the wells 35 incubated at 37 C for 1 hr. The microtiter plate was washed 3X with TBS/Tween. Para-niLLuuht ..yl phosphate WO 95/30153 2 ~ 8 ~ q ~ 0 P~
tPNPP) aUll~LL~te was added to all wells and incubated for 1-2 hrs until the 0. D. of the Bo wells reached 1. 7 O . D.
B is the 0. D. of binding of lAh~ d antibody in the 5 ~les~ ce of a sample that contains a competitor. Bo is the signal (O.D. ) from control samples without competitor .
Norlr~lization of Values. T nACSay results (O.D. of 10 sample wells) were e~ cased as % of B/Bo. The AGE
concentration was det~rmi ni d from 0 . D. by extrapolation from a standard curve (see Makita et al., 1992, J. Biol.
Chem. 267:5133-38; International Patent Publication No.
W0 93/13421).
All sample readings were normalized to the protein ~ ullcc:~lLLGtion~ which was det~rm;nPcl in the Lowry assay.
Thus, the final data are expressed as units of AGE per mg of hemoglobin.
Reslll ts Anrl D~ crllCcion A much simpler and reproducible Hb-AGE ELISA assay, with greater sensitivity due to what is believed to be maximal 25 ~uoauL~: of AGE epitopes, has been developed. The new assay includes incubation of samples containing h ~~lnhin with SDS, Triton X-100, and urea.
In pr-ol ;m;n~ry experiments, it was found that incubation 30 with SDS alone ~nhAnrl~ detection of hemoglobin-AGE.
However, SDS alone altered the standard assay conditions, and affected the binding of anti-AGE antibody to the AGE-model dipeptide, Gly-Lys browned with ribose, compared to - binding in the absence of SDS.
After further experimentation, it was found that the assays of samples ~L~:LLe:ated only with SDS could be wo 9~/30153 P~~ ul 21 88S~ --_uved by ;nr~ in~ an optimal combination of ~riton X-100 and urea in the dilution buffer. The optimal ~ullc~llLLaLions of SDS/Triton/urea were empirically found to be 0 . 08~6/0 . 01%/2M in rat samples and 0 . 08%/0 . 04%/2M in 5 human 6amples, respectively. Neither of the above samples diluents, 0.08% SDS/0.01% Triton/2M Urea used for rat samples or 0.08% SDS/0.04% Triton/2 M urea used for human samples, induced ~PtA~ ~ or elution o~ the coated BSA-AGE antigen from the well (data not shown).
Using the newly developed assay pL~LL~ a; L method, significant differences in the level of hemoglobin-AGE
were obselvt:d between normal patients and ~l;Ahetics (Figure lA). Similar data are obst:Lvtd in samples from 15 normal rats and rats with induced diabetes (Figure lB).
The present assay method for Hb-AGE shows good correlation with Hb-Alc in normal and diabetic humans (Figure 2). Thus, Hb-AGE is as effective a marker as Hb-20 A1C for diagnosis of AGE-associated ~lic~Acr~c~ such as 1l i Ahet-,c .
With the newly modified method, the inhibitory effect of aminog~l~n;-linP on the Hb-AGE content in vivo from samples 25 in a rat assay was clearly d LLated (Figure 3).
Hemoglobin-AGE is a much more relevant marker for the Aminn~lAnid;n-~ effect than Hb-A1C, since the latter is not affected by the presence of Aminn~lAni~lin~, or other AGE-inhibitors, at their rhArr-^~ntically relevant 30 cullc~ LLation.
Using the method of the present invention, delipidated hemolysates can be diluted down to around 1-2 mg/ml of protein, which consists mostly of hemoglobin. For 35 example, the samples shown in Figures 1, 2, and 3 were diluted 40 fold befûre addition to the 2ssays. One possible explanation for this increased sensitivity is ~ WO95130153 21 8~920 Y~
that the SDS/Triton/Urea pretreatment eYposes ID~-AGE
epitopes that would otherwise be in~cc~cc;hle to antibody binding in the ELISA or other i csay.
5 This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of 10 the invention being indicated by the ~rp~n~ Claims, and all changes which come within the meaning and range of es~uivalency are intended to be ~ c~d therein.
Various references are cited throughout this 15 speci~ication, each of which is incoL~uLated herein by rerere=ce ln 1t~ ~ntirety.
-
Claims (27)
1. A method for detecting the presence of hemoglobin-AGE in a sample comprising:
a) diluting the sample in a dilution buffer, which dilution buffer comprises an anionic protein denaturing detergent at a concentration sufficient to denature hemoglobin-AGE;
b) contacting the diluted sample with means for detecting the presence of hemoglobin-AGE in the sample; and c) detecting the presence of hemoglobin-AGE in the sample with the detection means.
a) diluting the sample in a dilution buffer, which dilution buffer comprises an anionic protein denaturing detergent at a concentration sufficient to denature hemoglobin-AGE;
b) contacting the diluted sample with means for detecting the presence of hemoglobin-AGE in the sample; and c) detecting the presence of hemoglobin-AGE in the sample with the detection means.
2. The method according to Claim 1, wherein the dilution buffer further comprises:
i) a non-ionic surfactant at a concentration sufficient to facilitate detection of hemoglobin-AGE; and ii) a denaturing agent at a concentration sufficient to denature hemoglobin-AGE and increase assay sensitivity, without interfering with the binding of reagents to hemoglobin-AGE.
i) a non-ionic surfactant at a concentration sufficient to facilitate detection of hemoglobin-AGE; and ii) a denaturing agent at a concentration sufficient to denature hemoglobin-AGE and increase assay sensitivity, without interfering with the binding of reagents to hemoglobin-AGE.
3, The method according to Claim 1 wherein concentration of anionic protein denaturing detergent ranges from about 0.04% to about 0.16% (w/v).
4. The method according to Claim 3 wherein the anionic protein denaturing detergent is sodium dodecyl sulfate.
5. The method according to Claim 2 wherein the concentration of non-ionic surfactant ranges from about 0.005% to about 0.1% (w/v).
6. The method according to Claim 5 wherein the non-ionic surfactant is a polyoxyethylene ester.
7. The method according to Claim 6 wherein the non-ionic surfactant is Triton X-100.
8. The method according to Claim 2 wherein the concentration of the denaturing agent is between about 0.5 M to about 3 M.
9. The method according to Claim 8 wherein the denaturing agent is urea.
10. The method according to Claim 1 wherein the dilution buffer is buffered to between about pH 7 to about pH 8, and contains salts at a concentration approximating physiological ionic strength.
11. A method for detecting the presence of hemoglobin-AGE in a sample comprising:
a) diluting the sample in a diluent buffer, which diluent buffer comprises i) sodium dodecyl sulfate at a concentration of from about 0.04% to about 0.16% (w/v);
ii) Triton X-100 polyoxyethylene ester at a concentration of from about 0.005% to about 0.1% (w/v); and iii) urea at a concentration of between about 0.5 M to about 3 M;
b) contacting the diluted sample with means for detecting the presence of hemoglobin-AGE in the sample; and c) detecting the presence of hemoglobin-AGE in the sample with the detection means.
a) diluting the sample in a diluent buffer, which diluent buffer comprises i) sodium dodecyl sulfate at a concentration of from about 0.04% to about 0.16% (w/v);
ii) Triton X-100 polyoxyethylene ester at a concentration of from about 0.005% to about 0.1% (w/v); and iii) urea at a concentration of between about 0.5 M to about 3 M;
b) contacting the diluted sample with means for detecting the presence of hemoglobin-AGE in the sample; and c) detecting the presence of hemoglobin-AGE in the sample with the detection means.
12. The method according to Claim 11 wherein the concentration of sodium dodecyl sulfate is about 0.08%, the concentration of Triton X-100 polyoxyethylene ester is selected from the group consisting of about 0.01% and about 0.04%, and the concentration of urea is about 2 M.
13. The method according to Claim 11 wherein the dilution buffer is buffered to between about pH 7 to about pH 8, and contains salts at a concentration approximating physiological ionic strength.
14. A method for quantitating the amount or level of hemoglobin-AGE in a sample comprising:
a) detecting the presence of hemoglobin-AGE in the sample according to the method of Claim 1, 2 or 11;
and b) quantitating the extent of detection of hemoglobin-AGE in the sample;
in which the extent of detection corresponds to the amount of hemoglobin-AGE in the sample.
a) detecting the presence of hemoglobin-AGE in the sample according to the method of Claim 1, 2 or 11;
and b) quantitating the extent of detection of hemoglobin-AGE in the sample;
in which the extent of detection corresponds to the amount of hemoglobin-AGE in the sample.
15. A method for detecting or diagnosing the presence of a disease associated with elevated hemoglobin-AGE levels in a mammalian subject comprising:
(a) quantitating the amount of hemoglobin-AGE in a sample from a mammalian subject according to Claim 14; and (b) comparing the level detected in step (a) to a level of hemoglobin-AGE normally present in the mammalian subject;
in which an increase in the level of hemoglobin-AGE as compared to normal levels indicates a disease associated with elevated levels of hemoglobin-AGE.
(a) quantitating the amount of hemoglobin-AGE in a sample from a mammalian subject according to Claim 14; and (b) comparing the level detected in step (a) to a level of hemoglobin-AGE normally present in the mammalian subject;
in which an increase in the level of hemoglobin-AGE as compared to normal levels indicates a disease associated with elevated levels of hemoglobin-AGE.
16. A method for monitoring the course of a disease associated with elevated hemoglobin-AGE levels in a mammalian subject comprising evaluating an amount of hemoglobin-AGE in a series of samples obtained at different time points from a mammalian subject, wherein the amount of hemoglobin-AGE is quantitated according to the method of Claim 14, in which an increase in the level of hemoglobin-AGE over time indicates progression of the disease, and in which a decrease in the level of hemoglobin-AGE over time indicates regression of the disease.
17. A method for monitoring a therapeutic treatment of a disease associated with elevated hemoglobin-AGE levels in a mammalian subject comprising evaluating the level of hemoglobin-AGE in a series of samples obtained at different time points from a mammalian subject undergoing a therapeutic treatment for a disease associated with elevated hemoglobin-AGE levels, wherein the level of hemoglobin-AGE is quantitated according to the method of Claim 14, in which a decrease in the level of hemoglobin-AGE over time indicates an effective therapeutic outcome.
18. A method for determining the optimum dosage of an inhibitor of advanced glycosylation endproduct formation in a mammalian subject with elevated levels of hemoglobin-AGEs comprising evaluating the level of hemoglobin-AGE in a series of samples obtained at different time points from a mammalian subject receiving progressively larger doses of an inhibitor of AGE
formation over the time points, wherein the level of hemoglobin-AGE is quantitated according to the method of Claim 14, and wherein an optimum dosage of the inhibitor is a dosage above which no further decrease in the level of hemoglobin-AGE is observed.
formation over the time points, wherein the level of hemoglobin-AGE is quantitated according to the method of Claim 14, and wherein an optimum dosage of the inhibitor is a dosage above which no further decrease in the level of hemoglobin-AGE is observed.
19. A method for monitoring the long term glucose level in a mammalian subject comprising evaluating the level of hemoglobin-AGE in a sample from a mammalian subject, wherein the level of hemoglobin-AGE is quantitated according to the method of Claim 14, and wherein the level of hemoglobin-AGE is indicative of the long term glucose level.
20. A dilution buffer comprising:
a) an anionic protein denaturing detergent at a concentration sufficient to denature hemoglobin-AGE
without interfering in binding of reagents with hemoglobin-AGE;
b) a non-ionic surfactant at a concentration sufficient to facilitate detection of hemoglobin-AGE; and c) a denaturing agent at a concentration sufficient to denature hemoglobin-AGE and increase assay sensitivity, without denaturing binding of reagents to hemoglobin-AGE, wherein the concentration of anionic protein denaturing detergent in the dilution buffer ranges from about 0.04%
to about 0.16% (w/v); the concentration of non-ionic surfactant ranges from about 0.005% to about 0.1% (w/v);
and the concentration of the denaturing agent is between about 0.5 M to about 3 M.
a) an anionic protein denaturing detergent at a concentration sufficient to denature hemoglobin-AGE
without interfering in binding of reagents with hemoglobin-AGE;
b) a non-ionic surfactant at a concentration sufficient to facilitate detection of hemoglobin-AGE; and c) a denaturing agent at a concentration sufficient to denature hemoglobin-AGE and increase assay sensitivity, without denaturing binding of reagents to hemoglobin-AGE, wherein the concentration of anionic protein denaturing detergent in the dilution buffer ranges from about 0.04%
to about 0.16% (w/v); the concentration of non-ionic surfactant ranges from about 0.005% to about 0.1% (w/v);
and the concentration of the denaturing agent is between about 0.5 M to about 3 M.
21. The dilution buffer of Claim 20 wherein the anionic protein denaturing detergent is sodium dodecyl sulfate, the non-ionic surfactant is Triton X-100 polyoxyethylene ester, and the denaturing agent is urea.
22. The dilution buffer of Claim 21 wherein the concentration of sodium dodecyl sulfate is about 0.08%, the concentration of Triton X-100 polyoxyethylene ester is selected from the group consisting of about 0.01% and about 0.04%, and the concentration of urea is about 2 M.
23. The dilution buffer of Claim 22 which is buffered to between about pH 7 to about pH 3, and contains salts at a concentration approximating physiological ionic strength.
24. A kit for detecting the presence of hemoglobin-AGE
in a sample comprising:
a) the dilution buffer of any one of Claims 20 to 23, in concentrated or ready-to-use form;
b) means for detecting the presence of hemoglobin-AGE;
c) other reagents; and d) directions for use of said kit.
in a sample comprising:
a) the dilution buffer of any one of Claims 20 to 23, in concentrated or ready-to-use form;
b) means for detecting the presence of hemoglobin-AGE;
c) other reagents; and d) directions for use of said kit.
25. A kit for detecting the presence of hemoglobin-AGE
in a sample comprising:
a) a dilution buffer which comprises i) an anionic protein denaturing detergent at a concentration sufficient to denature hemoglobin-AGE without interfering in binding of reagents with hemoglobin-AGE;
ii) a non-ionic surfactant at a concentration sufficient to facilitate detection of hemoglobin-AGE; and iii) a denaturing agent at a concentration sufficient to denature hemoglobin-AGE and increase assay sensitivity, without denaturing binding of reagents to hemoglobin-AGE;
b) means for detecting the presence of hemoglobin-AGE;
c) other reagents; and d) directions for use of said kit.
in a sample comprising:
a) a dilution buffer which comprises i) an anionic protein denaturing detergent at a concentration sufficient to denature hemoglobin-AGE without interfering in binding of reagents with hemoglobin-AGE;
ii) a non-ionic surfactant at a concentration sufficient to facilitate detection of hemoglobin-AGE; and iii) a denaturing agent at a concentration sufficient to denature hemoglobin-AGE and increase assay sensitivity, without denaturing binding of reagents to hemoglobin-AGE;
b) means for detecting the presence of hemoglobin-AGE;
c) other reagents; and d) directions for use of said kit.
26. The method according to Claim 1 wherein the means for detecting the presence of hemoglobin-AGE is an immunoassay.
27. The kit of claim 24 or 25 wherein the means for detecting the presence of hemoglobin-AGE is an immunoassay.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/236,416 | 1994-04-29 | ||
| US08/236,416 US5610076A (en) | 1994-04-29 | 1994-04-29 | Method for detecting hemoglobin advanced glycosylation endproducts |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2188920A1 true CA2188920A1 (en) | 1995-11-09 |
Family
ID=22889409
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002188920A Abandoned CA2188920A1 (en) | 1994-04-29 | 1995-04-28 | Method for detecting hemoglobin advanced glycosylation endproducts |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US5610076A (en) |
| EP (1) | EP0757795B1 (en) |
| JP (1) | JP3490715B2 (en) |
| AT (1) | ATE182008T1 (en) |
| CA (1) | CA2188920A1 (en) |
| DE (1) | DE69510668T2 (en) |
| WO (1) | WO1995030153A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5811242A (en) * | 1995-10-24 | 1998-09-22 | Tokuyama Corporation | Marker and reagent for diabetes mellitus and diabetes mellitus complication |
| US6033862A (en) * | 1996-10-30 | 2000-03-07 | Tokuyama Corporation | Marker and immunological reagent for dialysis-related amyloidosis, diabetes mellitus and diabetes mellitus complications |
| EP1217378A1 (en) * | 2000-12-21 | 2002-06-26 | Roche Diagnostics GmbH | Detection of advanced glycation endproducts in a cerebrospinal fluid sample |
| WO2002050548A1 (en) * | 2000-12-21 | 2002-06-27 | Roche Diagnostics Gmbh | Detection of advanced glycation endproducts in a cerebrospinal fluid sample |
| DE60202008T2 (en) * | 2001-03-22 | 2005-12-01 | Roche Diagnostics Gmbh | A method of finding reagents and solid phase components in specific binding assays free of advanced glycosylation endproducts |
| WO2006044728A2 (en) * | 2004-10-18 | 2006-04-27 | U.S.Genomics, Inc. | Methods for isolation of nucleic acids from prokaryotic spores |
| JP2013054029A (en) * | 2011-08-08 | 2013-03-21 | Arkray Inc | Immunoassay method of carboxymethyl arginine |
| KR102443804B1 (en) * | 2018-01-11 | 2022-09-19 | 도요보 가부시키가이샤 | Measurement sample dilutions, kits and measurement methods |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5254593A (en) * | 1984-03-19 | 1993-10-19 | The Rockefeller University | Compositions containing biguanides and derivatives thereof as inhibitors of nonenzymatic cross-linking |
| US4658022A (en) * | 1985-08-08 | 1987-04-14 | Molecular Diagnostics, Inc. | Binding of antibody reagents to denatured protein analytes |
| JP2619900B2 (en) * | 1988-01-27 | 1997-06-11 | 東亜医用電子株式会社 | Reagent and method for measuring leukocytes and hemoglobin in blood |
| IL94724A (en) * | 1989-07-13 | 1994-02-27 | Miles Inc | Lysis of red blood cells and hemoglobin denaturing by the use of lithium salts |
| US5624804A (en) * | 1991-12-20 | 1997-04-29 | The Rockefeller University | Immunochemical detection of In vivo advanced glycosylation end products |
| DE4206932A1 (en) * | 1992-03-05 | 1993-09-09 | Boehringer Mannheim Gmbh | IMMUNOLOGICAL METHOD FOR DETERMINING A HAEMOGLOBIN DERIVATIVE |
-
1994
- 1994-04-29 US US08/236,416 patent/US5610076A/en not_active Expired - Lifetime
-
1995
- 1995-04-28 WO PCT/US1995/005301 patent/WO1995030153A1/en active IP Right Grant
- 1995-04-28 DE DE69510668T patent/DE69510668T2/en not_active Expired - Fee Related
- 1995-04-28 JP JP52841895A patent/JP3490715B2/en not_active Expired - Fee Related
- 1995-04-28 AT AT95917751T patent/ATE182008T1/en not_active IP Right Cessation
- 1995-04-28 CA CA002188920A patent/CA2188920A1/en not_active Abandoned
- 1995-04-28 EP EP95917751A patent/EP0757795B1/en not_active Expired - Lifetime
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| Publication number | Publication date |
|---|---|
| DE69510668D1 (en) | 1999-08-12 |
| WO1995030153A1 (en) | 1995-11-09 |
| ATE182008T1 (en) | 1999-07-15 |
| US5610076A (en) | 1997-03-11 |
| JPH10504640A (en) | 1998-05-06 |
| EP0757795A1 (en) | 1997-02-12 |
| EP0757795B1 (en) | 1999-07-07 |
| DE69510668T2 (en) | 1999-10-28 |
| JP3490715B2 (en) | 2004-01-26 |
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