CA1302245C

CA1302245C - Process for the determination of a specifically bindable substance

Info

Publication number: CA1302245C
Application number: CA000551119A
Authority: CA
Inventors: Wilhelm Tischer; Josef Maier; Rolf Deeg
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1986-11-26
Filing date: 1987-11-05
Publication date: 1992-06-02
Anticipated expiration: 2009-06-02
Also published as: EP0269092B1; EP0269092A2; CN1032164C; DE3640412A1; ATE86743T1; DE3784638D1; KR890002227A; KR910002543B1; AU592910B2; GR3007910T3; ZA878824B; US5061640A; EP0269092A3; DD279741A5; JPH0731205B2; AU8169587A; CN87107982A; JPS63142268A; ES2054648T3

Abstract

ABSTRACT
A process for the preparation of a specifi-cally bindable protein substance bound to an insoluble carrier material, especially for use in a hetero-geneous analysis process, according to the immunoassay principle, wherein a soluble protein with a molecular weight above about 500,000, which is more hydrophobic than the specifically bindable substance, is coupled to the specifically bindable substance and then the conjugate of reaction component and protein is adsorbed on a hydrophobic solid phase; a carrier material for use in solid phase immunoassays prepared by this process consists of a hydrophobic solid phase which is adsorbed on a conjugate of a hydrophobic protein with a molecular weight above about 500,000 and of a specifically bindable protein substance.

Description

~L3~ 5 The present invention is concerned with a process for the determination of a specifically bindable protein substance according to the immunoassay principle, one of the components of the substance pair specifically bind-able with one another being present bound to a solidphase, and is also concerned with a carrier material suitable therefor.
For the determination of a specifically bindable substance, there are frequently used processes according to the immunoassay principle. One of the components of a substance pair specifically bindable with one another is thereby reacted with the receptor specific for it which is labelled in known manner. The conjugate of these two substances can then be reacted with a receptor which is specific for the conjugate or for one of the two parts of the conjugate. There are many variations for these immunological processes. It is thereby advantageous when one of the receptors is present bound to a solid phase. Thi9 makes easier the separation of reaction components present bound and non-bound~ For the determination of the specifically bindable sub-stance, there is then measured the amount of labelled reaction component bound to the solid phase or of labelled reaction component present in the solution and related in known manner to the amount of reaction com-ponent to be determined.

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~3~ 5 As solid phases, in the case of the immunological processes, there are usually used synthetic teqt tubes or microtitre plates on the inner surfaces of which is fi~ed the reaction component or spheroids on the outer surfaces of which is fixed the reaction component.
These synthetic resin test tubes, microtitre plate~ or spheroids usually consist of a relatively inert synthetic resin material so that the binding of the reaction component gives rise to difficulties. Furthermore, the binding of the specific reaction component to the sur-face in question mùst take place in ~uch a manner that it does not lose the ability of specific binding to the substance specifically bindable with it. For this reason, the binding of the reactive component to the solid phase mostly takes place adsorptively.
Therefore, it has already been suggested to bring about the fixing of the reaction component to the solid phase via a coupling agent which brings about the bind-ing. Care must thereby again be taken that the binding of the reaction component to the binding agent does not destroy the specifically reacting region of the molecule or that the reaction component is so bound that its reactive place is facing away from the solid phase to the binding component.
Furthenmore, in Federal Republic of Germany Patent Specification ~o. 25 33 701, it is suggested, in order to achieve a better binding, to cross-link the individual s immunologicaLly effective protein~ and then to absorb them on polystyrene spheroid~. A further posqibility given in this literature reference i~ ~imultaneously to cross-link an inert protein with the protein with immunological properties so that a cross-linked product results of inert and active protein which is then again adsorbed on polystyrene spheroids. However, depending upon chosen reaction conditions, this type of cross-linking leads to differing, non-reproducible cross-linkages with variable proportions of non-cross-linked protein, as well as of protein which has become insoluble.
Furthermore, due to the differing degree of cross-linking, products result with differing binding properties.
A similar process i3 described in European Patent Specification No. 0,122,209 and it also display~ the same disadvantages. Thus, all theqe known processes ( are still not satisfactory, still do not give an optimal adhesion of the specifically bindabLe substance and are of little suitability for the reproducible preparation of coated solid phases.

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Therefore, the present invention seeks to provide a process which reproducibly improves the adhesion of the specifically bindable substance to the solid phase and provides a carrier material suitable therefor. Since many immunological processes are carried out with the addition of detergents ~3~

in order to avoid turbidities, the present invention also seeks to improve the adhesion to such an extent that, even in the pxesence of detergents, the bound, specifically bindable substance is not dissolved S o~f.
'rhus, according to the present invention, there is provided a process for the preparation of a specific-ally bindable protein substance bound ~o an insoluble carrier material, especially for use in a heterogeneous lo analysis process, according to the immunoassay principle, wherein a soluble protein with a molecular weight above about 500,000, which is more hydrophobic than the specifically bindable substance, is coupled to the specifically bindable substance and then the conjugate lS of reaction component and protein is adsorbed on a hydrophobic solid phase.
The specifically bindable substance ~ixed in this way to a solid phase displays an improved adhesion. The binding is aIso stable with regard to detergents. In the case of the production of calibration curves which are necessary for the evaluation in the case of many immunological processes, the solid phase-bound, specifically bindable substances according to the present invention give steeper calibration curves, which results in an increase of the exactitude.
A further advantage of the process according to the present invention is that it is possible to control ,~

J'Z~:~s the bound amount more exactly. Since the adheqion is significantly better than in the case of the previously ~snown processes, the amount of specific protein which must be used is also smaller.
For the choice of soluble proteins which are suitable according to the present invention, there must be determined the molecular weight, as well aq the hydrophohicity, in comparison with the corresponding value for the specifically bindable substance. The molecular weight is determined according to known methods~
A comparison of the hydrophobicity between soluble protein and specifically bindable substance can also take place by conventional methods. Suitable methods are, for example, a comparison of the fluor-escent extinction after binding to coloured materials (Biochem. Biophys. Acta, 624, 13-20/1980), the elution behaviour in the case of hydrophobic chromatography (Biochem. Biophys. Acta, 576, 269-279/1979), the 20 surface tension (Biochem~ Biophys. Acta, 670, 64-73~
1981), and the retention times in the case of hydro-phobic interaction chromatography (HIC) (Angew. Chemie, 98, 530-548/1986, J. Chromat., 296, 107-114/1984, and Anal. Biochem., 137, 464-472/1984)o A comparison of the hydrophobicity of substances suitable according to the present invention is to be found in Sep. Sci. Technol., 14, 305-317/1979~ According ' ~a3~Z;~5 to that, the hydrophobicity increases, for example, in the following serie~: a2-macroglobulin (M.W. 820,000), bovine serum albumin/human serum albumin (M.W. about 70,0pO~, egg albumin, a2HS-glycoprotein (M.W. about 49,000), ~lc/~lA-globulin, immunoglobulin (M.W. about 150,0001 and transferrin (M.W. about 90,000).
Thus, if an immunoglobulin i5 used as specifically bindable æubstance, then, for example, human serum albumin or a2HS glycoprotein are not suitable as soluble proteins in the meaning of the present invention without further pre-treatment.
Both proteins must here be subjected not only to a hydrophobing but also to an increasing of the molecular weight. In the case of transferrin, in thi~
ca~e a cross-linking suffices and in the case of a2-macroglobulin a hydrophobing is sufficient.
Proteins which are suitable for the coupling wi~h imm~noglobulin as specifically bindable substance with-out pre-treatment include, for example, ~-lipoproteins (M.W. about 3.2 million) and ~2-lipoproteins (M.W. about 5 - 20 million).
The hydrophobing can take place, for example, by the use of heat, treatment with acids, denaturing agents and/or chaotropic ions and/or by chemical coupling with a hydrophobic compound, ; The increasing of the molecular weight can take place, for example, by the use of heat, treatment with ~3~ S

acids, denaturing agents and/or chaotropic ionq and/or by cross-linking with a bi- or polyfunctional protein reagent.
The treatment is carried out until a protein polymer is obtained with a molecular weight of 500,000 or more. It is e3pecially preferred to use a protein polymer with a molecular weight of from 500,000 to 20 million.
~hen a cross-linking is also to take place, the hydrophobing can be carried out before, during or after the cross-linking but not in the presence of the specifically bindable substance.
For hydrophobing by heating, there are usually used temperatures of from 40 to 95C. over a period of time of 1 minute to 10 hours, for example as described in Biochem. Biophys. Acta, 624, 13-20/1980.
As acids, there are used, for example, acetic acid, propionic acid, lactic acid or hydrochloric acid.
The usual concentrations are 1 to 100 mMole/litre with 20 a period of action of from 10 minute9 to 16 hoursO
Suitable chaotropic ions include, for example, thiocyanates, iodides, fluorides, bromides, perchlorates and sulphates. Suitable denaturing agents include, for example, guanidine hydrochloride and urea. Concent-rations of 10 mMole/litre to 6 mole/litre are u~ually here used.
For the derivatisation of hydrophobic compounds, g there are preferably used soluble fatty acids, lipoids in low and high molecular weight form, as well as synthetic polymers, such as polypropylene glycol, or soluble copolymers of polystyrene. The derivatisation takes place according to well known methods.
The cross-linking by way of bi- and polyfunctional compounds is carried out with known protein binding reagents. These are compounds which contain at least two functional groups, which can be the same or differ-ent and can react via these functional groups withfunctional groups of proteins. Compounds are prefer-ably used which consist of an alkyl chain on the ends of which are present, for example, succinimide, malein-imide and/or aldehyde groups.
The protein is cross-linked in the usual manner with the bi- or polyfunctional compounds by reacting together the soluble protein and the bi- or poly-functional compound.
For the hydrophobing and/or cross-linking, there are preferably used proteins with a molecular weight of from lO,000 to 700,000, bovine serum albumin, lipase and immune ~-globulin being especially preferred.
The specifically bindable protein substance to be bound is then coupled to the protein in known manner.
Suitable coupling methods are described, for example, by Ishikawa in J~ Immunoas~ay, , 209-327/1983. Proteins such as antibodies, antibody fragments, antigens and haptens can be used as specifically bindable substances.
The con]ugate obtained of specifically bindable protein substance and protein is then adsorbed adsorptively ~ion the synthetic resin surface serving as solid phase. The adsorptive binding to the solid phase takes place via strong and weak exchange actions, hydrophobic forces, dipole-dipole and ion-dipole inter-actions. As hydrophobic solid phases, there can be used carrier materials with a surface tension which is smaller than the surface tension of the hydrophobic soluble protein, i.e. æ e more hydrophobic than protein. Carrier materials with a surface tension of ~4O erg/cm2 æe preferably used. Poly-styrene, polymethacrylate, polytetrafluoroethylene (Teflon), poly-amlde, copolymers of styrene and acrylonitrile,~glass an~ cellulose product5 æ e especially preferred. m ey can be present in any desired form, for instance, in the ~orm of a film, plate, powder, ;~ granules or fikre fleece, preferably in the form of a glass fikre fleece or a fleece from cellulose-/cellulose ester fikr~s and poly~er fibres.
Hydrophoked proteins display an especially good adsorptive binding. pue to the hydrophobing, intramolecular bridge bonds of the protein are possibly opened so that hydrophobic parts of the protein reach the surface and there better a& ere to the hydrophobic synthetic resin surface than the hydrophilic parts which æ e particul æly to be found on the surface ~I the non-hydrophoked protein.
The process according to the present invention can ke used for the determination of a specifically bindable substance.
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~.3~22~5 As substance pairs, one reaction component of which is present bound to the solid phase, there can be used, for example, antigen-antibody, hapten-antibody and other proteins capable cf specific binding to one another, such as, in particular, the system streptavidin or avidin and biotin, which is preferred.
Before the conjugate of protein and specifically bindable substance is adsorbed on to the hydrophobic solid phase, it is also possible to pre-treat the solid phase physically or chemically. Thus, for example, a synthetic resin surface can be pre-swollen or activated in some other known way.
The carrier material according to the present invention for use in solid phase immunoassay is characterised in that it consists of a hydrophobic solid phase on which is adsorbed a protein with a molecular weight of above about 500,000 to which is bound a specifically bindable substance.
; This carrier material is outstandingly suitable for use in solid phase immunoassays since the specific-ally bindable substance adheres very well and is also not desorbed in the case of the addition of detergents.
The carrier material is present, for example, in the form of test tubes, microtitre plates or spheroids which are coated with a cross-linked protein to which is bound a specifically bindable substance.
On the solid base is adsorbed a conjugate con-sisting of a cross-linked protein and a specifically bindable substance. The protein is preferably bovine ~3~`ZZ~S

serum albumin, lipase or an immune ~-globulin which has been cross-linked in the described manner.
According to the present invention, there is provided a process and a carrier material in order to fix a specifically bindable substance with good adhesion and longlastingly to a hydrophobic solid phase. The adhesion could thus be improved to such an extent that even an addition of detergents does not lead to a dis-solviny off of the substance. The process according to the present invention is also simple to carry out.
The present invention will now be described in more detail in the following Examples, reference thereby ; being made to the accompanying drawings, in which~
Fig. l is a calibration curve for a TSH determination with the use of non-crosslinked Fab ~ TSH>
(curve l), crosslinked Fab < TSH> (curve 2), - conjugate of Fab ~ TSH~ Gal (curve 3) and conjugate of Fab ~ TSH ~ /thermo-BSA (curve 4) in Luran test tubes Fig. 2 is a calibration curve for a TSH determination with the use of immobilised streptavidin and biotinylated Ak ~ TSH ~ , non-crosslinked streptavidin (curve l), crosslinked streptavidin ~curve 2), streptavidin/~-gal conjugate (curve 3), streptavidin/BSA conjugate (curve 4), streptavidin/

thermo-BSA conjugate (curve 5) and streptavidin/
thermo-BS~ conjugate (curve 6), in the case of ;~a. 31~!2;;~:';iL S

curves 1 to 5, the solid phases are Luran test tubes and in the case of curve 6 the solid phases are polystyrene test tubes, Fig. 3 is a calibration curve ~or a T3 test, and Fig. 4 is a calibration curve for an HBs AG test~
Exam~le 1.

aqainst TSH to polystyrene test tubes.
a) Pre~aration of Fab fraqments (Fab ~ TSH ? ).
Monoclonal antibodie~ (~AB ~ TSH ? ~ are obtained by the method described by Gal~re and Millstein (Meth.
in Enzymology, ~ 1981). For further purification, the ascites li~uid is subjected to an ammonium sulphate precipitation and to a passage over DEAE-cellulose.
A papain fission is qubsequently carried out by tha method described in Biochem. J., 73, 119-126/1959 The Fab fragments hereby formed are separated from the non-digested IgG molecules and the Fc fragments by means of gel filtration over Sephadex G100 and ion exchanger chromatography over DEAE-cellulose according to Meth.
in Enzymology, 73, 418-459/1081.
b) sd~ti~ ~
50 mg. of Fab fragments are dissolved in 2 ml.
o.05 mole/litre potassium phosphate buffer (pH 7.5) and 0.4 ml. disuccinimidyl suberate (manufacturer Pierce) dissolved in dioxan (7.4 mg./ml.) is added thereto, with , ..
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~3~Z2~L5 _14-stirring. A~er incubating for 2 hours at 25C., the reaction is broken off by the addition of 0.2 ml. of 0.1 mole/litre lysine h~drochloride. The reaction batch is diluted with 0.2 ml. potassium phosphate buffer (v. supra) and centrifuged. The supernatant is desalinated over an Ultrogel AcA 202 column (LKB, Grafelfing, Federal Republic of Germany), 11.3 ml. being obtained with 45 mg. of protein. A part of this prepar-ation is fractionated on a Superose-6-column (Deutsche o Pharmacia GmbH) at a flowthrough rate of 0.5 ml./minute in 0~05 mole/litre potassium phosphate buffer (pH 7.0) and the fractions with a molecular weight of about 500,000 to 5 million are further used.
c) Cro ~ ated Y-alobulin (comparison), Fab fragments and bovine Y-globulin (Serva, Heidelberg, Federal Republic of Germany) are mixed in a weight ratio of 1:1. The crosslinking is carried out as described under b~.
d) Bind nq o _ b fraaments to ~re-crosslinked ~-qlobulin (accordinq to the ~r Qent inve tlO~
1.25 g. ~-globulin are dissolved in lo ml. of 0.05 mole/litre potassium phosphate buffer (pH 7.8) and centrifuged clear in a Sorvall cooled centrifuge for 10 minutes at 5000 r.p.m. 1.75 ml. Disuccinimidyl suberate are added to this solution which is then diluted with 2.5 ml. water. After stirring for 4 hours ..

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at 25C., 10 ml. of 0.1 mole/,itre lysine are added thereto and ~he pH value adjusted to 6.8 and centri~uged.
The supernatant is separated on a preparative gel ~iltration column ~TSK 3000, LI~B Grafelfing, Federal Republic of ~ermany~, concentxated by uLtrafiltration and stored at 4C.
50 mg. of this crosslinked ~-globulin are dissolved in 5 ml. of 0.05 mole/litre potassium phosphate buffer ( and the pH value adjusted to 9.5 by the addition of solid sodium carbonate. 50 mg. N-acetylhomocysteine thiolacekone (Serva, Heidelberg, Federal Republic of Germany) are then added thereto and stirred for 5 hours at 25C., while gassing with nitrogen. The batch is subsequently desalinated over an Ultragel AcA 202 column in a buffer of 0.1 mole/litre potassium phosphate (pH
7.0), 0.001 mole/litre magnesium chloride and 0.05 mole/
litre sodium chloride.
( Fab fragments prepared according to a) tlO mg. in 1 ml. of 0.01 mole/litre potassium phosphate buffer (pH
; 20 7~0)) are activated with 0O002 ml. maleinimidohexanoyl-N-hydroxysuccinimide ester (Boehringer Mannheim GmbH) in dimethyl sulphoxide t33 mg./ml.) for 2 hours at 25Co, subsequently centrifuged and desalinated over an AcA 202 column.
These fragments are subsequently combined with the ~-globulin (weight ratio of the proteins 1:1) and incub-ated for 1 hour at 25C. and at pH 7Ø Subsequently, * Trade Mark ~.3~;~2a~5 it is dialysed against desalinated water overnight at 4C. and at a protein concentration of 2.5 mg./ml.
This conjugate can be used directly for the adsorption on to a solid phase.
e) ~}~__tion of Fab_fra~ents coupled to thermo-BSA
(accordina to the present invention).
Preparation of thermo-BSA ~ m al umin¦:
1 g. BSA-I is dissolved in loo ml. of 50 mMolej litre potassium phosphate ~pH 7.0), heated to 70C. and kept at this temperature for 4 hours, with gentle stirring. The solution is cooled, filtered and adjusted in an ultrafiltration cell (exc~usion limit: 30,000 Dalton) to a concentration of 50 mg./ml. Subsequently, it is dialysed against a 30 fold volume of double distilled water and subsequently lyophilised. The product has a molecular weight of about 700,000.
Before coupling to the Fab fragments, the thermo-BSA is activated. For this purpose, 68 mg. thermo-BSA
are dissolved in 2 ml. 0.1 mole/litre potassium phosphate (pH 7.8) and slowly mixed with a solution of 3.8 mg.
S acetylmercaptosuccinic acid anhydride (SAMSA). After a reaction time of 3 hours, it is dialysed against 2 litres of 50 mMole/litre potassium phosphate (pH 6.5).
This thermo-BSA is incubated for 1 hour at 25C and pH 7.0 with the Fab fragments activated according to d) in a weight ratio of l:l, and subsequently dialysed against desalinated water overnight at 4C. and at a `2~91S

protein concentration of 2.5 mg./ml. This product is used directly for the coating.
f) Preparation of Fab fraqments cou~led t_ ~-~.
,~ 5 Fab fragments are activated as described in d) and coupled to the SH groups of ~-galactosidase accord-ing to J. Immunoassay, 4, 209-327/1983. The ~-galactos-idase used has a molecular weight of 500,000 to 2 million.
For another experiment, there is used crosslinked ~-galactosidase (M.W. about 5 million).
g) ~ ~''~
or conluq~es thereof.
50 mg. of a lyophilisate of the Fab fragment or of the conjugate are dissolved in 10 ml. double distilled 15 water. 1 ml. of this solution is diluted in 1000 ml.
of a loading buffer of 5.25 g./litre sodium dihydrogen phosphate and 1 g./litre sodium azide and stirred for 30 minutes at ambient temperature.
Test tubes of polystyrene or Luran (manufacturer BASF) are each filled with 1.5 ml. of the solution and loaded overnight (about 22 hours). Thereafter, the test tubes are com~letely emptied and the function test des-cribed hereinafter is carried out.
h) Function test Vl _ H determination.
The polystyrene and Luran test tubes loaded according to g) are used in a TSH determination reagent analogously to TSH-Enzymun test (Boehringer Mannheim GmbH, order No. 736 082) and a calibration curve ~3g~

_18-measured according to the test procedure. There are hereby obtained the measurement values shown in the following Table 1 and in Fig. 1 of the accompanying drawings.
It can be seen (Fig. 1) that with Fab fragments which have been immobilised without the addition of protein, only a very flat calibration can be obtained (curves 1 and 2~. By coupling to proteins with a molecular weight below 500,000 and low hydrophobicity, the calibration curve ~curve 3) is somewhat steeper but satisfactory results still cannot be achieved.
On the other hand, with the conjugates prepared accord-ing to the present invention, there can be achieved a sufficiently steep calibration curve (curve 4~.

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a) ~ .
Streptavidin (manufacturer Boehringer Mannheim GmbH) is crosslinked analogou~ly to Example lb.
b) A~.
The binding to non-crosslinked BSA or to ~-galactosidase takes place analogously to Example lf.
c) Pre~aration o ~ idin coupled to thermo-BSA.
Activation_g~ ptavidin:
60 mg. Streptavidin are dissolved in 6 ml~ of 50 mMole/litre potassium phosphate/100 mMole/litre sodium chloride ~pH 7.0) and stored at 4C. 6.16 mg.
maleinimidohexanoyl-N-hydroxysuccinimide ester are dissolved in 620 ~1. dio~an and stirred into the streptavidin solution. After a reaction timP of 2 hours at 4C., it is dialysed twice against 1 litre of 50 mMole/Litre potassium phosphate/100 mMole/litre -sodium chloride (pH 5) at 4Co Preparat ~ idin and thermo-BS~:
; 66 mg. Streptavidin are dissolved in 10 ml. of 50 mMole/litre potassium phosphate tpH 5.0) and 100 mMole/litre sodium chloride and 72 mg. of activated thermo-BSA-SAMBA (preparation according to Example le) in 5 ml. of 50 mMole/litre potassium phosphate/100 mMole/

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:. i 13~ 5 -21_ litre sodium chloride (pH 6.5) are added thereto.
After mixing, 50 ~1. of 1 mole/litre hydroxylamine (pH
7.0) are added thereto in order to stop the reaction.
After 3 hours, the reaction product is purified via gel chromatography (Superose 6, 50 mMole/litre potassium phosphate/100 mMole/litre sodium chloride; pH 7.5).
There is obtained a conjugate with a molecular weight of from 1 to 5 million.
d) 10 ~=_~.
The loading takes place in the manner described in Example lg) e~ Measurement of standard values via a TSH

determlnation .
The test tubes loaded according to d) are used in a TSH Enzymun reagent (4 fold conjugate activity).
However, in variation of the there-described procedure, instead of the immobilised antibody, there is used a biotinylated MAB ~ TSH > . The preparation of this biotinylated MAB takes place according to J.A.C.S., 100, 3585-3590/1978. The antibody is used in the test in a concentration of 400 ng. per test tube, together with the other reagents.
The results obtained are shown in Fig. 2. It can be seen therefrom that, with increasing molecular weight and with increasing hydrophobicity, the gradient of the calibration curve and thus of the achievable exacti-tude increases.

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A T3 test is carried out with the test tubes loaded according to the present invention. For this purpose, 200 ~1. of sample or of standard solution are pre-incubated together with 500 ~1. of a polyclonal antibody conjugate against T3, which is labelled with POD, in a test tube which had been coated with strepta-vidin coupled to thermo-BSA. After 5 minutes, 400 ng.
of polymerised T3 biotinylated in known manner are incubated for 30 minutes in 500 ~1. of buffer. As buffer, there is used a solution of sodium hydrogen phosphate with a pH of 8.65 which contains 0.20% BSA
and 0.04% 8-anilino-1-naphthalenesulphonic acid (ANA).
After incubation, washing is carried out three times and subsequently 1 ml. ABTS substrate solution is added thereto. After further incubation for 30 minutes, measurement is then carried out at 405 nm in a photo-meter. The measurement values obtained are given in the curve shown in Fig. 3. -Exam~le 4 .

An HBsAg test is carried out with test tubes coated according to the present invention~ For this purpose, in a test tube coated according to Example lg), r` there are simultaneously dissolved 400 ng. biotinylated monoclonal antibody against HBs-antigen and 50 mU of the same antibody which are labelled with POD and 1 ml, of the same composition as described in Example 3 added ~3~ 5 thereto, together with 200 ~1. of standard solution.
As standard solution, there is added, on the one hand, purified HBsAg sub-type ay and, on the other hand, purified HBsAg sub-type ad. Incubation is then carried out for 4 hours at ambient temperature. After washing the test tubes three times, 1 ml. ABTS substrate solution is added thereto. After 20 minutes, the reaction is substantially ended and the extinction is measured at 405 nm in a photometer. The measurement - 10 values are to be seen from Fig. 4, in which the unbro]cen curve gives the values for HBsAg sub-type ad and the broken curve gives the values for HBsAg sub-type ay.

The dissolving off of the test tubes coated according to the present invention is compared with test tuhes coated according to known processes. For this purpose, on the one hand, test tubes are loaded with 1.5 ml. of a streptavidin-thermo-BSA solution (4 ~g./ml.) in 40 mM sodium hydrogen phosphate buffer (pH 7.4) at 20 20C. for 18 to 24 hours. After sucking out the test tubes, there takes place an after-treatment with 1.8 ml.
of a 20/o saccharose solution which contains 0.9% sodium chloride and 0.3% BSA, the after-treatment being carried out for 30 minutes at 20C. Subsequently, the test 25 tubes are dried for 24 hours at 20C. and 40% relative humidity. These test tubes are ready for use for carry-ing out tests. Furthermore, test tubes are loaded in 13~ 45 -24~

known manner with streptavidin. The dissolving off behaviour in the case of the action of detergents is tested with these test tubes. The results obtained are given in the following Table 2:

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~26 The per cent dissolving off is determined according to the methods known to the skilled worker, for example, by 125J-labelling of streptavidin and streptavidin-thermo-BSA or by an enzymatic determination.
For the enz~matic determination of the pér cent dissolving off, the coated test tubes are incubated with 50 mMole/litre potassium phosphate buffer, pH 7.0, to which a detergent according to Table II was added, under the conditions according to Table II.
Subsequently, incubation is carried out at ambient for one hour, washing takes place with the above-mentioned buffer, and incubation is carried out with a conjugate of biotin POD (200 mU~ml.
PO3 activity) for one hour at ambient, washing takes place, and 2 ml ABTS ~ solution is added thereto ~Example 7). After substrate reaction for one hour, the extinction is measured at 405 nm, and from this the per cent dissolving off of streptavidin and streptavidin-thermo-BSA is determined via a standard calibration curve.
( 20 Example_6 Determination of the hydrophobicity of proteins with _ hydrophobic interaction chromatography (HIC).
The hydrophobicity of various compounds is invest-igated with a liquid chromatograph (Hewlett Packard 1090 ; 25 LUSI~. The pre-column is a BioRad Biogel TSK-phenyl-5PW
column (length 5 mm. x internal diameter 4.6 mm.).
Column: BioRad-Biogel TSK-phenyl-5PW (length 75 mm. x * Trade Mark .

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~3~ 5 internal diameter 7.5 mm., 10 ~m. 1000 ~). As detector, there is used a Hitachi F 1000 fluorimeter. Eluents/
gradient (see Table 3).
a) 1.5 mol/litre ammonium sulphate solution in 1/100 mole/litre monopotassium dihydrogen phosphate buffer 5IP~ 608) b) 1/100 mole/litre monopotassium dihydrogen phosphate buffer (pH 6.8).

Table 3 _ .... . _ _ I
10A% ¦ B,~ ¦ t/min.
._ ~
oo o o lOO O 5 O lOO 30 flow - 0.5 min.
0 loo 5 loo o s . ~ .... ..~
Working temperature: cold chamber ~7C.

.:

~3~ZZ4~

SampLe preparation:
The samples are used undiluted. The sample ~olume is lOf~l, The compounds to ~e investigated are dissol~Jed at a concentration of 0,2 to 1.4 mg./ml. in 10 mMole/
litre potassium phospha~e buffer (pH 6.8).
The follo~ing rrable 4 su~arises the, retention times for various proteins and specifically bindable substances. ~rhe longer is the retention tlme, the ~reater is the hydrophobicity.
Table 4 . . . .... ~
protein ¦ retention time tp ¦ (min.) . .
Fab rrSH 41.5 BSA 23.2 ~-globulin 32.0 ~-galactosidase 39.6 crosslinked,~-galactosidase 40.2 streptavidin 38.3 20 thermo-BSA (Example le) not elutable ~-globulin, crosslin~ed not elutable ,~ = . _ The proteins which are not elutable under these ,~ conditions are especially suitable and are preferably ~ 25 used, thermo-BSA being quite especially preferred.

' ' ` " , , ~Z2~5 ~,1_ Adsorption of thermo-BSA-streptavidin on ~lass fibre fleece.
~ glass fibre fleece (6 x 6 mm.) is soaked, in lts absorption volume (ca. 30 ~l.), with a solution of 30 ~g/ml. thermo-BSA-streptavidin (prepared accord-ing to Example 2c) in 50 n~lole/litre potassium phosphate buffer, p~ 7.0, and dried at 50C. in a circulating drier.
For determining the biotin binding capacity, the strip is soaked with 30 ~l. of a reagent consisting of a conjugate of peroxidase (POD) and biotin and having a POD activity of 50 mU/ml., biotin standard with concentrations of 0.5, 10, 20, 30, 40, 50, 100, 200, 1000 ng/ml. biotin, and incubated for 2 minutes.
Thereafter, 2 %o-Tweenk20 washing is carried out once with a surplus of 50 mMole/litre potassium phosphate buffer, pH 7.0, and subsequently 100 mMole/litre ; citrate buffer, pH 4.4; 3.2 mMole/litre perborate;
1.9 mole/litre ABTS ~ ( 2,2'-azino-di C-ethyl-benzthia-zolinesulphonic acid(6~ -diammonium salt are introduced in 2 ml. ABTS solution and agitated for 15 minutes.
-~ After substrate reaction for 1 hour, the extinction is measured at 405 nm., and from this the biotin binding capacity is determined via the semimaximum signal drop.

* Trade Mark : ~
. ,~

.

Claims

1. A process for the preparation of a specifically bindable protein substance bound to an insoluble carrier material comprising:
coupling a soluble protein with a molecular weight above about 500,000 to a specifically bindable substance, and adsorbing the resulting conjugate of said bindable substance and protein on a hydrophobic solid phase, said protein being more hydrophobic than the specifically bindable substance.

2. A process according to claim 1, wherein said soluble protein is a protein with a molecular weight of from 500,000 to 20 million.

3. A process according to claim 2, wherein said soluble protein is prepared from a protein with a molecular weight of 10,000 to 70,000 by increasing the molecular weight to above 500,000 to 20 million.

4. A process according to claim 1, 2 or 3, wherein, before coupling, the protein is cross linked with a bi- or polyfunctional protein reagent until the desired molecular weight is achieved.

5. A process according to claim 1, 2 or 3, wherein said protein is a hydrophobed protein.

6. A process according to claim 4, wherein said protein is a hydrophobed protein.

7. A process according to claim 5, wherein, before coupling, the protein is hydrophobed by the use of heat, treatment with acids, denaturing agents or chaotropic ions and/or by chemical coupling with a hydrophobic compound.

8. A process according to claim 6, wherein, before coupling, the protein is hydrophobed by the use of heat, treatment with acids, denaturing agents or chaotropic ions and/or by chemical coupling with a hydrophobic compound.

9. A process according to claim 1, 2 or 3, wherein, before coupling, the protein is cross-linked with disuccinimidyl suberate until the desired molecular weight is achieved.

10. A process according to claim 3, 6, 7 or 8, wherein said protein comprises at least one of bovine serum albumin, lipase and immune .gamma.-globulins.

11. A process according to claim 9, wherein said protein comprises at least one of bovine serum albumin, lipase and immune .gamma.-globulins.

12. A process according to claim 9, wherein said specifically bindable substance is selected from .beta.-lipoproteins and .alpha.2-lipoprotein.

13. A composition of a specifically bindable protein substance bound to an insoluble carrier material, comprising:
a conjugate in which a soluble protein with a molecular weight above about 500,000 is coupled to a specifically bindable substance, and a hydrophobic solid phase, said conjugate being adsorbed on said hydrophobic solid phase, said protein being more hydrophobic than said specifically bindable substance.

14. A composition according to claim 13, wherein said protein has a molecular weight of from 500,000 to 20 million.

15. A carrier material for use in solid phase immunoassays consisting of a hydrophobic solid phase on which is adsorbed a conjugate of a hydrophobic protein and a specifically bindable protein substance, said hydrophobic protein having a molecular weight above about 500,000.

16. A carrier material according to claim 15, wherein the hydrophobic solid phase consists of polystyrene, polymethacrylate, polyamide, polytetra-fluoroethylene or a copolymer of styrene and acrylo-nitrile.

17. A carrier material according to claim 15, wherein the protein is hydrophobed bovine serum albumin (thermo-BSA), hydrophobed lipase or hydrophobed immune .gamma.-globulin.

18. A carrier material according to claim 16, wherein the protein is hydrophobed bovine serum albumin (thermo-BSA), hydrophobed lipase or hydrophobed immune .gamma.-globulin.

19. A carrier material according to claim 15, wherein the specifically bindable substance is an antigen or an antibody.

20. A carrier material according to claim 16, 17 or 18, wherein the specifically bindable substance is an antigen or an antibody.

21. A carrier material according to claim 15, wherein the specifically bindable substance is strept-avidin or avidin.

22. A carrier material according to claim 16, 17 or 18, wherein the specifically bindable substance is an antigen or an antibody.

23. A carrier material according to claim 15, wherein the carrier is a glass fibre fleece or a fleece from cellulose-/cellulose ester fibre and polymer fibre.

24. A carrier material according to claim 16, 17, 18, 19 or 21, wherein the carrier is a glass fibre fleece or a fleece from cellulose-/cellulose ester fibre and polymer fibre.

25. A composition according to claim 13 or 14, wherein the specifically bindable substance is strept-avidin or avidin.

26. a composition according to claim 25, wherein the carrier material is a fleece of glass fibre.

27. Use of a composition of claim 13, 14 or 26, in a heterogeneous analysis process based on the immunoassay principle.

28. Use of a carrier material of claim 15, in a solid phase immunoassay.

#9/06/22/1990

29. A process for preparing an insoluble carrier material useful in binding assays comprising forming a cross-linked conjugate of a water-soluble first protein having a molecular weight of at least 500,000 daltons with a second protein substance which conjugates by cross-linking with the water-soluble first protein wherein the water-soluble first protein is more hydrophobic than said second protein substance, adsorbing the cross-linked conjugate on a hydro-phobic solid phase material to form said insoluble carrier material, wherein the second protein substance consists of one member of a specifically bindable pair of substances wherein the binding member is other than the water-soluble protein so that the cross-linked conjugate is specifically bindable.

30. The process according to claim 29 wherein said water-soluble protein has a molecular weight in the range of 500,000 to 20 million daltons.

31. The process according to claim 30 and further including the step of preparing said water-soluble protein prior to the forming of said cross-linked conjugate, the preparing of said water-soluble protein including linking a precursor protein having an original molecular weight in the range of 10,000 to 700,000 daltons with at least one other molecule to form a molecule having a molecular weight greater than the molecular weight of the precursor protein and in the range of 500,000 to 20 million daltons.

32. The process according to claim 31 and the linking of said precursor protein including cross-linking said precursor protein prior to conjugation with one or more reagents selected from the group consisting of bifunctional protein reagents and polyfunctional protein reagents.

33. The process according to claim 29 and further including the step of preparing said water-soluble protein from a precursor protein prior to forming the cross-linked conjugate, said preparing of said water-soluble protein including a step of causing the precursor protein to become more hydrophobic.

34. The process according to claim 33 and the step of causing the precursor protein to become more hydrophobic being accomplished by a method selected from the group con-sisting of heat treatment, treatment with acid, treat-ment with denaturing agents, treatment with chaotropic ions, and treatment to cause chemical coupling with a hydrophobic compound.

35. The process according to claim 32 and the cross-linking of the precursor protein including cross-linking with disuccinimidyl suberate.

36. The process according to claim 31 and said precursor protein being selected from the group consisting of bovine serum albumin, lipase, and immune gamma-globulin.

37. A specifically bindable complex adapted for adsorption onto a hydrophobic solid phase, said complex comprising:
a water-soluble first protein coupled with a second protein substance to form a cross-linked conjugate therewith wherein said second protein is one of a binding pair of substances which specifically bind with each other and which conjugate is specifically bindable with the other substance of the binding pair, said soluble first protein having a molecular weight of at least about 500,000 daltons and being more hydrophobic than said second protein substance.

38. A carrier material comprising:
a hydrophobic solid phase; and a cross-linked conjugate being absorbedly supported on said solid phase, said cross-linked conjugate comprising a water-soluble hydrophobic first protein having a molecular weight of at least about 500,000 daltons and being coupled with a second protein substance to form said cross-linked conjugate, wherein the second protein is one of a binding pair of substances which specifically bind with each other and said cross-linked conjugate is specifically bindable with the other substance of the binding pair of substances.

39. The carrier material according to claim 38 wherein said hydrophobic solid phase is of a material selected from the group consisting of polystyrene, polymethacrylate, polyamide, polytetrafluoroethylene, and copolymers of styrene and acrylonitrile.

40. The carrier material according to claim 38 and said hydrophobic protein being derived from a precursor protein selected from the group consisting of bovine serum albumin, lipase, and immune gamma-globulin, which precursor protein is treated to increase the molecular weight and the hydrophobic quality of said precursor protein.

41. The carrier material of claim 38 wherein the second protein substance is selected from the group consisting of antigens, antibodies and fragments thereof.

42. The carrier material of claim 38 wherein the second protein substance is selected from the group consisting of streptavidin and avidin.

43. The carrier material of claim 38 wherein said solid phase is a fleece containing material selected from the group consisting of glass fibers, and mixtures of polymer fibers and fibers of cellulose or cellulose ester.

44. The process of claim 29 wherein the binding assay is an immunoassay.