US 3867518 A
A rapid radioimmunoassay for detecting immunoreactive insulin in body fluids, e.g., blood plasma, utilizing zirconyl phosphate gel at a pH at which free insulin will not bind to it, i.e., pH of 6.0 to 6.5, preferably 6.25, to adsorb antibody-bound insulin while leaving free insulin in solution, is disclosed.
Description (OCR text may contain errors)
United States Patent Coffey et al.
[451 Feb. 18,1975
RADIOIMMUNOASSAY FOR INSULIN Inventors: John William Coffey, West Caldwell; Hans John Hansen, Allendale, both of NJ.
Assignee: Hoffmann-La Roche Inc., Nutley,
\ NJ. Filed: Mar. 9, 1973 Appl. No.: 339,803
U.S. Cl. 424/1, 23/230 B Int. Cl A6lk 27/04 Field of Search 424/1; 23/230 B References Cited UNITED STATES PATENTS 1/1971 Axen et a1. 424/1 3,646,346 2/1972 Catt 23/2308 3,697,638 10/1972 Hanson 424/1 OTHER PUBLICATIONS Moody et a1., Nuclear Science Abstracts, Vol. 20. No. 8, April 30, 1966, pp. 1564-1565, Item No. 12754.
Primary Examiner-Benjamin R. Padgett Attorney, Agent, or FirmSamuel L. Well  ABSTRACT 4 Claims, 2 Drawing Figures PAIENIEB FEB I 81975 CPM X10" IN Z-GEL PELLET CPM x 10- IN z-sa. PELLET DILUTION 0F ANTISERUM FIG. 1
4 I I I I I I I l MICROUNITS 0F INSULIN FIG 2 RADIOIMMUNOASSAY FOR INSULIN BACKGROUND OF THE INVENTION Various radioimmunoassays have been developed for the measurement of the level of immunoreactive insulin in the plasma of animals and humans. All the procedures depend on the competition between the endogenous insulin in body fluids, e.g., blood plasma, and exogenous radioactive insulin for the binding sites on anti-insulin antibodies.
Thus, Yalow et al., Journal of Clinical Investigation, 35, 1157 (1960) disclose a laborous process wherein paper electrophoresis is used to separate antibodybound insulin from free insulin. Morgan et al., Diabetes 12, 115 (1963), on the other hand, utilize the double antibody technique wherein anti-guinea pig y-globulin is used to precipitate the soluble complex formed between insulin and guinea pig anti-insulin antibody.
These known radioimmunoassays for measuring the level of immunoreactive insulin in body fluids, particularly in blood plasma are sufficiently accurate for the desired measurement but are relatively impractical since they require extended periods of time to complete and in some processes require the use of carriers for the antibody.
There is thus a need for a radioimmunoassay for measuring the level of immunoreactive insulin in the body fluids, e.g., blood plasma, spinal fluid, urine and the like, of animals and humans which is accurate, relatively rapid, economical and commercially practical.
The invention as described hereinafter provides such a radioimmunoassay.
SUMMARY OF THE INVENTION This invention relates to an improved radioimmunoassay for measuring the level of immunoreactive insulin in the body fluids, particularly blood plasma of animals and humans. More particularly, this invention relates to a radioimmunoassay for measuring immunoreactive insulin which can be completed within about three hours by utilizing zirconyl phosphate gel at a pH at which free insulin will not bind to it, i.e., at a pH of from 6.0.to 6.5, preferably 6.25, as the adsorbent for the insulin-antibody complex.
DETAILED DESCRIPTION OF THE INVENTION This invention is based upon the discovery that a radioimmunoassay for immunoreactive insulin in body fluids, preferably blood plasma can be completed within three hours when zirconyl phosphate gel (Z-gel) at a pH at which free insulin will not bind to it, i.e., at a pH of from 6.0 to 6.5, preferably pH 6.25 is used to adsorb antibody-bound insulin while leaving free insulin in solution.
The method is accurate and reproducible and eliminates the need for utilizing carriers to which the antibody is coupled. For example, the accuracy is shown by a comparison to a solid phase commercial assay using carriers having antibody coupled thereto. In the comparison thirty-two samples of human blood plasma were assayed for immunoreactive insulin using the solid phase commercial assay and using the assay of this invention. The results showed a statistically significant (p 0.01) coefficient of correlation between the two assays (0.998) indicating the assay of this invention is accurate. The reproducibility of the assay of this invention is within 10 percent when duplicate samples are run.
In conducting radioimmunoassays, procedures based on both the techniques of isotope dilution and sequential competitive-inhibition can be used. The preferred procedure for the detection of immunoreactive insulin according to this invention is the sequential competitive-inhibition method.
In this method a titration curve shown in FIG. 1 then a standard inhibition curve shown in FIG. 2 are developed.
The titration curve, used to determine the proper dilution of anti-insulin antiserum for used in the radioimmunoassay. This is necessary in order to avoid having excess antibody present, causing readings which are too high. The ideal is to have a dilution at which about half the labelled insulin is complexed with antiserum. The curve is prepared by adding increasing dilutions of the antiserum, diluted with a buffer composition at pH 7.4. A typical suitable buffer is composed of 0.04 M phosphate (prepared from NaH PO, and NaOH) containing 0.6 mM thimersal and 0.5 percent wt./vol. bovine serum albumin (BSA) to a constant amount of radioiodinated insulin I-insulin). A typical suitable buffer composition utilized in the incubation mixtures for preparing the titration curve is 0.2 ml. of a 0.04 M phosphate buffer at pH 7.4, containing 0.6 mM thimersal, 0.5 percent wt./vol. BSA and 0.15 M NaCl. The incubation buffer is mixed with 0.1 ml. of varying dilutions of the antiserum and 0.1 ml. l-insulin in an incubation test tube and the mixture is incubated at 37C. for 45 mins. The reaction is stopped by the addition of 5 ml. of an ammonium acetate slurry of Z-gel at pH 6.0 to 6.5, preferably 6.25, to each tube followed by 10 ml. of 0.1 M ammonium acetate, pH 6.25. The Z-gel slurry contains, on a volume basis, about 40-45 percent solids. The contents of the tubes are mixed, then centrifuged to separate the Z-gel-antibody-insulin precipitate and assayed for radioactivity. The proper dilution of antiserum is determined from the curve and utilized in subsequent assays utilizing the antiserum.
The standard inhibition curve is used to determine the concentration of insulin in the test fluid as follows:
Standard inhibition curves are prepared using mixtures containing 0.1 ml. of a buffer at pH 7.4. A typical buffer is composed of 0.04 M phosphate, containing 0.6 mM thimersal, 0.5 percent wt./vol. BSA and 0.15 M NaCl; 0.1 ml. of the appropriate dilution of antiserum and 0.1 ml. of a standard solution of bovine insulin, e.g., 1.0 to 10.0 uU in the same buffer. The mixture is incubated for 2 hours at 37C. then 0.1 ml. of I- insulin is added to each tube and incubated 45 minutes at 37C. The incubation is terminated by the addition of 5 ml. of an ammonium acetate slurry of Z-gel at a pH 6.0-6.5, preferably 6.25, solids content about 40-45 percent v/v, and 10 ml. of ammonium acetate at pH 6.25. The radioactivity is determined by centrifuging and assaying the resulting pellet for radioactivity.
0.1 MI. of blood plasma containing unknown amounts of insulin are treated in the same manner as the standards used to prepare the inhibition curve. The amount of insulin in the plasma samples is determined by comparison to the standard inhibition curve. If the plasma samples contain very high levels of insulin, they are diluted with appropriate amounts of a buffer at pH 7.4, preferably composed of 0.04 M phosphate, 0.6
mM thimersal and 0.5 percent wt./vol. BSA and then assayed.
In conducting the assay according to this invention, any inorganic buffer at pH 7.4 can be used provided its ionic strength is similar to that of the test fluid, e.g., blood plasma. Phosphate buffers are preferred because of their ready accessibility.
The following Examples illustrate the invention.
EXAMPLE 1 Preparation of Radioiodinated Insulin Twenty-five ug. of insulin (25 ,ul) in 0.4 M phosphate buffer (pH 7.4) was mixed with a mixture of 5 mCi of Na l in 50 pl. of dilute NaOH at pH 8-11 and 10 1.1.1. of 0.3 M phosphate buffer, pH 7.4.
100 pg. (10 pl.) of chloramine T (sodium-ptoluenesulfochloramine) in 0.05 M phosphate buffer, pH 7.4, was added to the reaction mixture to initiate the iodination reaction which was allowed to proceed at room temperature for 2 minutes, with constant shakmg.
The reaction was stopped by adding 200 pg. ,ul.) of sodium metabisulfite in 0.05 M phosphate buffer pH 7.4. Then 0.2 ml. of 1 percent aqueous bovine serum albumin was added. The mixture was chromatographed on a 2.5 X 20 cm. column of Sephadex G-25, a hydrophilic water-insoluble cross-linked dextran polymer gel commercially available from AB Pharmacia, Uppsala, Sweden, which had been equilibrated at 4C. with 0.1 M Tris.HCl buffer, pH 7.4, containing 0.9 percent NaCl.
The column was eluted with the same Tris buffer at a flow rate of 0.5 ml./min. and 1.0 ml. fractions were collected into tubes containing 1 ml. of 1 percent bovine serum albumin. The amount of radioactivity in a ul. aliquot of each fraction was determined and the fraction near the void volume of the column containing the largest amount of radioactivity was used as the source of l-insulin for the assay after dilution with a 0.04 M phosphate buffer at pH 7.4 containing 0.6 mM thimersal and 0.5 percent wt./vol. BSA until 1 ml of the diluted solution contains about-450,000 cpm/ml. of
EXAMPLE 2 Preparation of Zirconyl Phosphate Gel 100 Gm. of Zirconyl chloride SH O was dissolved in 15 liters of 0.1 M hydrochloric acid and the solution was vigorously stirred while 200 ml. of concentrated phosphoric acid was slowly added. The resulting gel was allowed to settle to about one-third of the original volume. The supernatant fluid was removed by aspiration. The gel was washed by adding distilled water to the gel to a volume of 15 liters; the gel was allowed to settle, and the supernatant fluid again removed by aspiration. This washing procedure was repeated five times. After the last washing, distilled water was added to a volume of 15 liters and enough concentrated acetic acid was added to make the gel slurry 0.1 M with respect to acetic acid. The gel was allowed to settle for 2 days in this solution after which the supernatant fluid was removed as completely as possible by aspiration. The Z-gel slurry remaining had a volume of approximately 3 to 4 liters and was adjusted to pH 6.25 with concentrated ammonium hydroxide. The resulting slurry contained, on a volume basis, about 40 to 45 percent solids.
EXAMPLE 3 Preparation of Titration Curve lnto five incubation test tubes 0.2 ml. of a 0.04 M phosphate buffer at pH 7.4 containing 0.6 mM thimersal, 0.5 percent wt./vol. BSA and 0.15 M NaCl. then each of five separate 0.] ml. aliquots of antiserum diluted respectively 1 to 5,000, l to 10,000, 1 to 20.000, 1 to 40,000 and l to 80,000 with a 0.04 M phosphate buffer at pH 7.4 containing 0.6 mM thimersal and 0.5 percent wt./vol. BSA and finally 0.] ml. of l-insulin were added.
The mixtures were then incubated at 37C. for 45 mins. The reaction was stopped by the addition of 5 ml. of a slurry of Z-gel, as prepared in Example 2, to each tube followed by the addition of 10 ml. of 0.1 M ammonium acetate at pH 6.25.
The contents of the tubes were then mixed by inversion and the Z-gel fraction was separated by centrifugation at 1,000 X g for 5 minutes at room temperature. The supernatant was decanted and the pellet washed by resuspension in 10 ml. of ammonium acetate followed by recentrifugation. The supernatant was again decanted and the amount of l in the Z-gel pellet was measured.
The results are shown in FIG. 1.
EXAMPLE 4 V H Preparation of Standard Inhibition Curve this time 0.1 ml. of l-insulin containing 43,000 cpm. I
were added and the mixtures incubated for an additional 45 minutes at 37C. The reaction was stopped by the addition of 5 ml. ofa Z-gel slurry as prepared in Example 2 and 10 ml. of ammonium acetate, pH 6.25. The'Z-gel fraction was separated and assayed for radioactivity as in Example 3. The results are shown in FIG. 2
EXAMPLE 5 g In order to test the accuracy of the assay, 2.5 uU of bovine insulin in 0.1 ml of an 0.04 M phosphate buffer at pH 7.4 containing 0.6 mM thimerosal, 0.5 percent wt./vol. BSA and 0.15 M NaCl were added to plasma samples in which the levels of immuno reactive insulin had been determined, using the assay of this invention, by the use of the standard inhibition curve. The assay was then repeated. The percent recovery was calculated by comparing the level ofimmunoreactive insulin in the samples before and after the addition of exogenous bovine insulin.
TABLE Patplasma sample +2.5 [.LU bovine insulin ient p.U insulin/ amount expected amount recovered No 0.1 ml. sample (pU) (uU) recovery I 1.45 3.95 4.15 108 2 1.75 4.25 3.95 88 3 1.95 4.25 4.35 96 4 1.20 3.70 3.35 86 5 1.35 3.85 4.25 116 6 2.35 4.85 4.80 98 7 1.60 4.10 3.85 90 8 3.70 6.20 5.50 72 9 4.40 6.90 6.00 64 I0 l.45(1/5)* 3.95 4.60 126 11 3.20 5.70 5.50 92 12 3.90 6.20 6.40 92 13 2.15( H40) 465 4.40 90 14 2.50( l/50) 5.00 4.20 68 15 2.45(1/50) 4.95 4.65 88 16 2.05 4.55 4.50 98 17 l.45(%) 3.95 4.40 118 18 3.90 6.40 6.85 H8 19 3.35 5.85 6.55 I28 20 1.40 3.90 3.80 96 21 1.85016) 4.35 4.90 122 22 3.50(%) 6.00 6.15 106 23 2.3506) 4.85 5.10 110 24 2.35 4.85 4.85 100 25 1.75 4.25 3.95 88 26 1.70(%) 4.20 4.55 114 27 2.0006) 4.50 5.35 134 28 2.35( /a) 4.85 4.90 102 29 2.30 4.80 4.60 92 30 2.75(1/10) 5.25 5.40 106 31 2.05( 4.55 4.70 106 The values in parentheses indicate the extent to which the plasma samples were diluted with an 0.04 M
phosphate buffer at pH 7.4 before the assay was done. "The mean percentage recovery was l00.4 17.0 (5.1).)
antibodyinsulin comple cand measuring the radioactivity thereof.
2. The radioimmunoassay of claim 1 wherein the pH of the zirconyl phosphate gel slurry is 6.25.
3. The radioimmunoassay of claim 1 wherein the zirconyl phosphate gel slurry contains on a volume basis about 40 percent to 45 percent solids.
4. The radioimmunoassay of claim 1 wherein the test fluid is blood plasma.