AU609301B2 - Process for the determination of an immunologically detectable substance and a suitable reaction vessel therefor - Google Patents

Abstract

To determine an immunologically detectable substance on the principle of the heterogeneous immunoassay using a solid phase to which one of the immunologically active reactants is bound, the solid phase used is a reaction vessel to whose inner surface streptavidin or avidin is bound in an amount such that 0.1 to 2.5 mu g are present per ml of reaction volume. A reaction vessel suitable for this purpose has opposite optically transparent wall regions and, moreover, is coated, at least partially in the region of the inner wall provided to receive liquid, with streptavidin or avidin, where the interior of the container intended to receive liquid, and the streptavidin or avidin content of the coating are matched with one another so that 0.1 to 2.5 mu g of streptavidin or avidin are present per ml of reaction volume. <IMAGE>

Description

'1 301 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 CQWLMSPECICAflQ NAME ADDRESS OF APPLICANT: Boehringer Mannheim GmbH Sandhofer Strasse 112-132 D-6800 Mannheim-Waldhof Federal Republic of Germany NAME(S) OF INVENTOR(S): This d0'"met contains the section 49and is correct f Or printing, 0 Urban SCHMIT Eberhard MAURER Wolfgang RUDINGER Rolf DEEG ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneys I Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: Process for the determination of an immunologically detectable substance and a suitable reaction vessel therefor 44 4 0 49 4 I 0 The following statement. is a full description of this performing it kniown to rue/us;invention, including the best method of 4 4 4 4 of the application.

Insert place and date of signature. Declared at Mannheim this 3rd day of April, 1989 Boehringer Mannheim GmbH Signature of diclarant(s) (no attestation required) pp-aa Note: initial all alterations. Dr- Fou q. r DAVIES COLLISON, MELBOURNE and CANBERRA, I 1% 4 -2- Description The invention concerns a process for the determination of an immunologically detectable substance based on a heterogeneous immunassay by use of a solid phase on which one of the immunologically active reaction components is bound, a suitable reaction vessel therefor as well as use of a standard reaction vessel.

Immunoassay procedures are often used to determine substances which can be specifically bound. In this o process one partner of a binding pair, capable of specific binding to one another, is reacted with its specific receptor which is labelled in a known way. The conjugate of both these substances can then still react with a receptor which is specific for the conjugate or for one of the two parts of the conjugate. There are o omany variants of these immunological procedures. In such Smethods it is advantageous if one of the receptors is present bound to a solid phase. This facilitates the So separation of the bound and non-bound reaction partners.

For the determination of the substance capable of being specifically bound, the amount of labelled reaction partner bound to the solid phase or of labelled reaction partner present in the solution is measured and related to the amount of the reaction partner to be determined according to known procedures.

Plastic tubes or microtitre plates on the inner surface of which the reaction partner is bound, or spheres on the outer surface of which the reaction partner is bound, are usually used as the solid phase for immunological methods.

-3- In these methods the binding of the specific reaction partner to the respective surface must be carried out in such a way that it does not lose its ability to specifically bind to the substance which is capable of specific binding to it. For this reason the reaction partner is usually bound adsorptively to the solid phase. It was therefore already suggested that the binding of the reaction partner to the solid phase should be achieved via a coupling agent which mediates the binding. In this process one should take care that the binding of the reaction partner to the binding agent does not destroy the region of the molecule which specifically reacts or alternatively that the reaction partner is bound in such a way that its reactive site is facing away from the solid phase and towards its binding partner.

A disadvantage of all these known processes is that a special solid phase has to be provided for each specific reaction. This means that for each individual test another solid phase has to be produced, stored and then used for the determination which is tedious.

It was therefore the object of the present invention to provide a process and a reaction Vessel which are suitable for many different methods of detection. For routine clinical diagnosis many different parameters such as hormones, tumour markers, as well as parameters for infection, allergy or fertility are determined from a single blood sample. The concentration of the parameters to be determined covers a wide range.

Parameters in low concentrations such as for example TSH and prolactin are in the range of 10-10 to 1i- 12 mol/l, whereas parameters in high concentrations like T 3 and T are at a concentration of about 10-7 to 10-8 mol/l. For -4this purpose it would be desirable if a different tube did not have to be used in every case for the individual determinations but rather if a standard reaction vessel could be used for all determinations.

This object was achieved by a process for the determination of an immunologically detectable substance based on a heterogeneous immunoassay by use of a solid phase on which one of the immunologically active reaction components is bound ,wherein a reaction vessel oC is used as the solid phase on the inner surface of which streptavidin or avidin is bound in such an amount that 0.1 to 2.5 Ag are present per ml reaction volume. The amount quoted refers to streptavidin or avidin which is 00O accessible to the binding partner in the form of a biotinylated protein or hapten during the course of the immunological reaction.

o00 0o0 The process according to the present invention is applicable to all methods of determination in which the Poeo a" receptor, which can be an antibody or an antigen, which is to be immobilized on the solid phase is conjugated bound, to the solid phase according to the present invention, adhere well to all tube materials and the binding is stable against detergents. In the process of establishing calibration curves which are necessary for the evaluation of many immunological methods, the streptavidin or avidin bound to the solid phase yield steep calibration curves with small blank readings which lead to an increase in the accuracy. A further advantage is that lot to lot variations in the amount of coated strepta,idin had only negligible influences on the results of measurements. The particular advantage of the process according to the present invention is that the

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amount of streptavidin or avidin bound can be so adjusted that the solid phase can be used for all known processes based on heterogeneous immunoassays. A further advantage is that several substances, especially antibodies, but also antigens, can be determined simultaneously with the process according to the present invention. This will be elucidated in more detail in Example 7.

In a particularly preferred embodiment the reaction t r vessel used as the solid phase is at the same time used as a cuvette for the photometric determination of the label. This is particularly advantageous since only a single vessel is necessary to carry out the entire S' reaction. In this case the reaction vessel is so constructed that it has two optically transparent wall areas facing one another. The entire wall area can be coated with streptavidin or avidin.

According to the present invention a reaction vessel is used as the solid phase on the inner surface of which streptavidin or avidin is bound. The binding of streptavidin or avidin is carried out by methods known to the expert (EP 12 22 09). The streptavidin or avidin can either be bound directly to the solid phase or alternatively bound via a spacer or substances which mediate the binding.

In a preferred embodiment of the present invention streptavidin or avidin is coupled to a soluble protein with a molecular weight above about 500 000 and then this conjugate is adsorbed to a hydrophobic solid phase as described for example in the patent application P 36 40 412.8. It is preferable to use a protein which has been made hydrophobic.

-6- This increase in hydrophobicity can result from the application of heat, treatment with acid, denaturing agents and/or chaotropic ions and/or by chemical coupling to a hydrophobic compound. Treatment with these agents also leads to an increase in the molecular weight. The increase in molecular weight can be also achieved by cross-linking with a bi-or polyfunctional protein reagent.

Acetic acid, propionic acid, lactic acid or hydrochloric Sacid can for example be used as the acid. The usual *on, concentrations are 1 to 100 mmol/l with reaction times from 10 min to 16 hours.

a Q '"no Suitable chaotropic ions are ior example thiocyanates, iodides, fluorides, bromides, perchlorates and sulphates. Suitable denaturing agents are for example guanidine hydrochloride or urea. The concentrations of these are usually from 10 mmol/l to 6 mol/l.

o 1 «For the derivatisation with hydrophobic compounds soluble fatty acids, lipoids in a low or high molecular weight form as well as synthetic polymers such as polypropylene glycol or soluble copolymers of polystyrene are preferably used. The derivatisation is carried out by methods familiar to the expert.

The cross-linking by means of bi- or polyfunctional compounds is carried out with known protein binding reagents. These are compounds which have at least two functional groups which can be either the same or different and which can react with functional groups of proteins by means of these functional groups. It is preferred to use compounds which consist of an alkyl -7chain at the end of which succinimide, maleimide, and/or aldehyde groups are located.

The protein is cross-linked with the bi- or polyfunctional compound in a known way by reacting the soluble protein and the bi- or polyfunctional compc, In order to increase the hydrophobicity and/or to crosslink, it is preferable to use proteins with a moleculax weight from 10 000 to 700 000. Particularly preferred is bovine serum albumin, lipase or immuno-e-globulin.

Streptavidin or avidin is then coupled to the protein using a known method. Suitable coupling methods are o4 described for example in Ishikawa, Immunoassay 4.

(1983), 209-327.

44The conjugate obtaired from streptavidin or avidin and S protein is then adsorbed on to the plastic surface which serves as the solid phase. Before the conjugate of S 4protein and streptavidin or avidin is adsorbed onto the hydrophobic solid phase it is also possible to cherically or physically pretreat the solid phase. Thus for example a plastic surface can be swollen in advance or activrated in another known manner. The adsorptive binding to the solid phase results from strong and weak inte-actions, hydrophobic forces, dipole-dipole or ion-dipole interactions.

Reaction vessels made of supporting materials with a surface tension which is smaller than the surface tension of the soluble hydrophobic protein i.e. which are more hydrophobic than protein are particularly suitable as the hydrophobic solid phase. Use of -8supporting materials with a surface tension of erg/cm 2 is preferred. Particularly suitable are polystyrene, polymethacrylate, teflon, polyamide, copolymers of styrene and acrylonitrile, glass and cellulose products.

The solid phase material produced according to the present invention is used in the determination of many different parameters. In this process one of the substances capable of binding to the parameter to be determined is conjugated with biotin which in turn binds «o to the streptavidin or avidin respectively bound to the solid phase. By this means the specific complexes which S form as a result of the inuunoassay process can be immobilized and then determined. Therefore in accordance 0 with the present invention as much streptavidin or avidin respectively is bound to the solid phase that per ml reaction volume 0.1 to 2.5 pg are available for binding to the biotinylated substance capable of o specific binding.

0 00O The said reaction volume denotes the sum of sample volume and test reagent volume.

ft It is preferred to coat the solid phase material such that 1 to 2 pg streptavidin or avidin respectively are available for binding to the biotinylated substance capable of specific binding. The amounts of the biotinylated substance capable of specific binding is in the range of from 10 16 to 10 8 mol/reaction volume.

A further embodiment of the present invention is a reaction vessel with optically transparent wall areas which face one another and with avidin or streptavidin Nk-- -9coated walls which are at least partially within the inner wall region intended as a receptacle for liquid, wherein the inner space of the container intended as a receptacle for liquid and the respective streptavidin or avidin content of the coating are so matched that 0.1 to gg streptavidin or avidin are present per ml reaction volume.

This reaction vessel is particularly suitable for carrying out the process according to the present invention. Since it has optically transparent wall areas which face one another and also a coating of streptavidin or avidin it can be used simultaneously to carry out the reaction and for the photometric determination. As a result of the coating with 0.1 to 2.5 Ag streptavidin or avidin respectively per ml reactionl volume, this reaction vessel can be used for the determination of different parameters on the basis S' r of a heterogeneous immunoassay.

The reaction vessel constitutes the solid phase. It consists of materials which are usually used for this purpose such as plastics, glass etc. The optically transparent wall areas also consist of known materials.

Particularly preferred in this case are polystyrene, copolymers of polystyrene, polycarbonates, polyacrylates and polymethacrylates.

Streptavidin or avidin can either be coated directly or via a carrier material or a spacer. For example the binding of streptavidin or avidin to a soluble protein with a molecular weight above 500 000 which is then adsorbed to the inner surface of the reaction vessel is suitable.

i An embodiment of the invention is also the use of a standard reaction vessel for the determination of different parameters using processes based on immunoassays, which consists of a vessel on the inner surface of which streptavidin or avidin is bound in such an amount that for the test reaction 0.1 to 2.5 pg streptavidin or avidin are available per ml reaction volume.

According to the present invention a process and a tt* reaction vessel are provided in which streptavidin or avidin are bound permanently and with good adhesion to the inner surface of a reaction vessel which serves as a solid phase. The ahesion is so good that even the t addition of detergents does not lead to the detachment 41 a of the substance. The reaction vessel provided according to the present invention can be universally employed and is suitable for carrying out methods of determination S° for very many parameters and therefore facilitates the performance of routine determinations.

i The invention is elucidated by the following figures and examples. The figures show Fig. 1 a family of calibration curves for a TSH determination using immobilized streptavidin in various amounts and biotinylated anti-TSHantibody; Fig. 2 a calibration curve for a prolactin test; Fig. 3 a calibration curve for a CEA-test and Fig. 4 a calibration curve for a T4-test.

-11- Fig. 5 a family of calibration curves for an anti-HBs determination using immobilized streptavidin in different amounts.

The monoclonal antibodies against TSH used in the examples originate from hybridoma cell lines which were deposited at the European Collection of Animal Cell Cultures, Porton Down, GB, under the numbers ECACC 87 122 201 and ECACC 87 122 202.

Ex amp e 1 The binding to the solid phase of the conjugate o according to the present invention and of the hydrophobic protein and streptavidin was investigated: Thermally aggregated BSA, from now on referred to as thermo-BSA, was prepared as follows: 1 g BSA was dissolved in 100 ml of 50 mmol/l potassium phosphate solution with a pH of 7.0, and was heated to 701C and kept at this temperature for 4 hours during which it was gently stirred. The solution was cooled down, filtered s and adjusted to a concentration of 50 mg/ml. Afterwards it was dialyzed against a thirtyfold volume of redistilled water.

Preparation of a conjugate of streptavidin with thermo-BSA: streptavidin was isolated from Streptomyces avidinii and was converted by maleimido-hexanoyl-Nhydroxy-succinimide to a streptavidin containing maleimido groups. Thermo-BSA was converted with s-acetyl-mercapto-succinic acid anhydride and the protected SH-groups were afterwards released by the addition of hydroxylamine, The desired conjugat6 was

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-12then obtained by mixing the streptavidin containing maleimido groups and the thermo-BSA containing SH-groups.

Plastic tubes made of polystyrene were then coated with the streptavidin-thermo-BSA-conjugate. The coating of the tubes was carried out with 1.5 ml of a streptavidin-thermo-BSA-solution for 18 to 24 hours, whereby the molar ratio of both components was 1.8 1 in 40 mmol/1 sodium phosphate buffer, pH 7.4 at 20 0

C.

After aspiration of the tubes they were recoated with 1.8 ml of a solution containing 2% sucrose, 0.9% sodium chloride and 0.3% BSA for 30 min at 20°C. After drying .o (24 hours at 20°C and 40% relative humidity) the tubes were ready for use and stable.

E x a m p 1 e 2 One-step test for TSH Biotinylated monoclonal anti-TSH-antibody was prepared, For this purpose the anti-TSH-antibody, ECACC 87 122 201, was converted with biotin in the usual way, whereby the coupling took place via amino groups.

Antibody and biotin were used in the proportion of 1 16. 400 ng of the biotinylated antibody thus obtained were dissolved in 1 ml buffer (40 mmol/1 sodium phosphate, 0.5% Pluronic F68, 0,2 mol/1 sodium tartrate as well as 0.01% phenol) and then incubated for two hours in a coated tube as described in Example 1 with 200 p1 sample or standard solution respectively an amount that 0.1 to 2.5 pg are present per ml reaction volume.

/2 I I -13containing a known amount of TSH and 75 mU of an anti- TSH-antibody conjugated with peroxidase (ECACC 87 122 202). Afterwards it was washed three times with tap water and 1 ml ABTS-substrate solution was added. After one hour the absorption was measured at 405 nm. The results are shown in Fig. 1. It turns out that a satisfactory result is obtained with immobilized streptavidin when an amount of more than 0.1 Ag/ml test volume is used.

The streptavidin required for binding of the biotinylated antibody used in the test was determined.

For this the amount of freely available biotin was determined according to the method of Bayer, Edward A., Meir-Wilchek, Analytical Biochemistry 154 (1986), 367- 370. Assuming that 1 Ag antibody was biotinylated in an 2 antibody to biotin ratio of 1 15 (Guesdon, I. J.

Histochem, Cytochem. 27 (1979) 1131-1139), it follows that 1 to 2 mol biotin per mol antibody are available for binding, i.e. 1.5 to 3 ng biotin are freely accessible per Ag antibody.

A tube coated with 10 Cg/ml streptavidin-thermo-BSAconjugate (Example 1) has a binding capacity for biotin of 15 to 20 ng per ml test volume, determined with radioactive 14 C-biotin.

The binding capacity of the streptavidin tubes for biotinylated immuno-Z-globulin is 1.7 to 2 pg/ml test volume. The determination was carried out with 1 2 5 1-labelled -globulin (labelled according to the method of McConahey and Dixon 1966, Int. Arch. Allergy 29/185).

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-14- Exa m 1 e 3 Prolactin test Prolactin is determined by a one-step-sandwichimmunoassay. A reagent of the following composition is used for the detection: 50 mU/ml of a conjugate of POD and a monoclonal antibody specific for prolactin, o P Son n a 9 9 0 e o a 9 0 0 40 mmol/l 0.5% 0.2 mol/1 0.01% 0.2% 400 ng/ml phosphate buffer, pH Pluronic F65 (polyoxyethylene polypropylene) sodium tartrate phenol bovine serum albumin of a biotinylated monoclonal antibody against prolactin.

Both monoclonal antibodies are only required to detect prolactin and to be directed against the various epitopes of prolactin. 1 ml of this reagent and 50 Al sample were incubated for 30 min at room temperature in a polystyrene tube coated with streptavidin-thermo-BSAconjugate, obtained according to Example 1. Afterwards it was washed three times with tap water. 1 ml ABT substrate solution was added for the test reaction. The absorbance was measured photometrically after 30 min at 405 nm. The results are shown in Fig.2.

ABTS@ 2.2'-azino-di-[3-ethylbenzthiazoline-6sUlphonate] -di-ammonium salt W, a- -ii Examp le 4 CEA test CEA was determined in a sample solution by a one-stepsandwich-immunoassay. The reagent used contained the following: 120 mU/ml of a conjugate of POD and a monoclonal antibody against CEA, 4 t #4(w I I 4 4a 44 4 os 4 4444 4 4 4 44 4 4c 4c 44p 4 40 mmol/1 0.5% 0.2 mol/l 0.01% 0.2% 500 ng/ml phosphate buffer, pH Pluronic F68 sodium tartrate phenol bovine serum albumin of a biotinylated monoclonal antibody against CEA.

The only requirements for both monoclonal antibodies against CEA are that they detect CEA and that each is directed against a different epitope of CEA.

1 ml reagent and 100 il sample were incubated for 2 hours at room -Cmperature in a polystyrene tube coated with streptavidin-thermo-BSA, obtained according to Example 1. Afterwards it was washed three times with tap water. I ml ABTS-substrate solution was added for the test reaction. After one hour the absorbance was measured photometrically at 405 nm. The results are shown in Fig. 3.

Y 1 111 'i I~r~rrr~

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-16- Examp 1 e T4 test T4 was determined in a competitive immunoassay using biotinylated anti-T4-antibodies. For this purpose 500 ji of a reagent consisting of: 100 mU/ml 120 mmol/l 18.2 mmol/1 0.04% by weight 0.2% by weight thyroxine-POD-conjugate (prepared according to EP 209 155) barbiturate phosphate buffer, pH 8.6 ANS (8-anilino-l-naphthaline -sulphonic acid) bovine serum albumin, 44 4 4 O 4 *9 9 as well as 20 pl sample were incubated for 10 min at room temperature in a polystyrene tube coated with streptavidin-thermo-BSA-conjugate, obtained according to Example 1. Afterwards 500 ji of a second reagent consisting of: 1 Ag/ml biotinylated polyclonal antibody against T4, 120 mmol/l phosphate buffer, pH 8.6, 0.04% by weight ANS (8-anilino-l-naphthaline -sulphonic acid), 0.2% by weight bovine serum albumin, were added and incubated for a further 30 min at room temperature. After washing it three times with tap water 1 ml ABTA-substrate solution was then added and incubated for 30 min at room temperature. Afterwards the v. -vla aLre in tne range of 1 0 to 10- L mol/l, whereas parameters in high concentrations like T 3 and T 4 are at a concentration of about 10 7 to 10 8 mol/1. For I III- 4i m A

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-17absorbance was measured photometrically at 405 nm. The results are shown in Fig. 4.

Ex a mp 1 e 6 Anti-HB s test HBs-antibodies were determined in a cne-step-sandwichimmunoassay. A reagent with the following composition was used for the determination: 4444 4 4 4 4n 4 OU 0 4444 4O 4 44 0( 60 mU/ml mmol/l 200 mmol/1 by weight 0.01% by weight 0.2% 0.1% by weight 150 ng/ml of a conjugate of POD and HBs-antigen phosphate buffer, pH sodium tartrate Pluronic F68 phenol bovine serum albumin bovine-IgG biotinylated HBsAg (prepared according to Immunol. Letters 8 (1984), 273).

0 44*4 44 4 4 44 a *4 a 1 ml of this reagent and 200 Al sample were incubated for 4 hours at room temperature in a polystyrene tube coated with a conjugate of streptavidin-thermo-BSA.

After washing it three times with tap water 1 ml of ABTS-substrate solution was added for the test reaction.

After 60 min the absorbance at 422 nm was measured photometrically.

The anti-HBg-test was performed in tubes using different amounts of immobilized streptavidin. The results are shown in Fig. 5. It can be seen that when streptavidin is used in amounts which are more than those recommended results of measurements. The particular advantage of the process according to the present invention is that the 4 0900 4 4 4 t o 4 4 44 44 0; 4li 4 *L 40 04Q 0 64 4440 g4 4 4 4 4 44 -18in the present invention then the test becomes significantly less sensitive.

Examp e 7 Anti-HIV test HIV-antibodies are determined in a two-step-sandwichimmunoassay. A reagent with the following composition was used for the determination.

Reagent 1: 10 7 mol/l each of one or several biotinylated HIVantigens 40 mmol/l phosphate buffer, pH 0.9 by weight sodium chloride 10% by volume bovine serum The following were used as antigens: HIVl-antigen produced by genetic engineering corresponding to HIV1gp41 (gp41-rek., CentocorTM-pl21) and HIVl-p24 (p24rek., CentocorTM-pg2), chemically synthesized peptide from HIVl-gp41 (gp41-pep, Wang et al., PNAS, 83, 6159, 1986) and HIV2-gp32 (gp32-pep, Gnknn, J.W. et al., Science, 237, 1346, 1987). These antigens were labelled with biotin as described by Leary et al., PNAS, A0, 4045 (1983).

L11u padienr application P 36 40 412.8. It is preferable to use a protein which has been made hydrophobic.

L L -19- Reagent 2: mU/ml of a conjugate of sheep antibodies against human immunoglobulin and POD mmol/1 phosphate buffer, pH 0.05% by weight Tween 0.2% bovine serum albumin 0.2% bovine-IgG 1 ml of reagent 1 and 10 Al human serum or plasma were incubated for 1 hour at room temperature in a polystyrene tube coated with a conjugate of streptavidin-thermo-BSA. Afterwards the tube was washed three times with tap water and incubated for 1 hour at room temperature with 1 ml reagent 2. The tube was again washed three times with tap water and 1 m" ABTS 1 ^substrate solution was added for the test reaction.

0 After 60 mmin the absorbance at'422 nm was measured photometrically.

o° 0 The anti-HIV-test was carried out using individual HIVantigens and combinati..ns of antigens. As a result it a. was demonstrated that the test procedure in polystyrene ti'bes coated with a conjugate of streptavidin-thermo-BSA is suitable for the determination of individual antigenspecific antibodies as well as for the simultaneous A4 4 determination of several antibodies or antibody populations (screening test table irrespective of whether these antibodies are directed against the same virus or against several viruses or against the antigens of interest.

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A A A 00 *0 0 0 0 a .0 00 A A a A P pA Ta bl1e 1 Human serum sample Anti- Anti- IAnti- Anti- I Anti- Anti- HIV-Antigens Neg. HIV I HIV I HV I HIV II HIV I HIV II Amount of HIV-Antigen gp4l-rek. 52 1250 812 521 102 223 75 p24-rek. 43 786 1515 212 491 1531 263 gp4l-pep 71 831 301 295 63 52 59 gp32-pep 38 41 50 62 1819 45 810 gp4l-,p24-rek. 45 1402 1818 618 550 1559 266 gp4l-,p24-rek.+ 55, 1380 1753 599 1723 1529 878 gp3 2-pep

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Claims (7)

1. Process for the determination of an immunologically detectable substanze based on a heterogeneous immunoassay by use of a solid phase on which one of the immunologically active reaction components is bound ,wherein a reaction vessel is used as the solid phase on the inner surface of whic'i streptavidin or avidin is permanently bound in such an amount that 0.1 to 2.5 pg are present per ml reaction volume.

2. Process as claimed in claim 1, wherein a cuvette is used as the reaction vessel whose walls are coated on the inner surface with streptavidin or avidin.

3. Process as claimed in one of the previous claims, wherein streptavidin or avidin is coupled to a soluble protein with a molecular weight above 500 000 and then the conjugate of streptavidin or avidin and protein is adsorbed to a hydrophobic solid phase.

4. Process as claimed in one of the previous claims, wherein several substances in particular several antibodies are determined simultaneously. adsorbed to the inner surface of the reaction vessel is suitable. c -22- 0 S ##ff <r Reaction vessel with optically transparent wall areas which face one another and with avidin or streptavidin coated walls which are at least partially within the inner wall region intended as a receptacle for liquid, wherein the inner space of the container intended as a receptacle for liquid and the respective streptavidin or avidin content of the coating are so matched that 0.1 to 2.5 ig streptavidin or avidin are present per ml reaction volume.

6. Reaction vessel as claimed in claim 5, wherein streptavidin or avidin is bound to the solid phase via a soluble protein with a molecular weight above 500 000.

7. Reaction vessel as claimed in claim 5 or 6, wherein the reaction vessel is a cuvette.

8. A process as claimed in claim 1, or a vessel as claimed in claim 5, substantially as hereinbefore described with reference to the accompanying drawings. DATED this 11th day of December, 1990 BOEHRINGER MANNHEIM GmbH By Its Patent Attorneys DAVIES COLLISON I i i i i,