CA2008100A1

CA2008100A1 - Method for the determination of immunologically detectable substance

Info

Publication number: CA2008100A1
Application number: CA002008100A
Authority: CA
Inventors: Hubert Bayer; Peter Kirch; Erhard Kopetzki; Christian Klein
Original assignee: Individual
Current assignee: Roche Diagnostics GmbH
Priority date: 1989-01-20
Filing date: 1990-01-18
Publication date: 1990-07-20
Also published as: EP0379216B1; JPH02228561A; ES2055172T3; DK0379216T3; ATE107030T1; AU4858290A; DE59005973D1; JPH0827284B2; EP0379216A1; AU633041B2

Abstract

A b s t r a c t For the determination of immunologically detectable substances according to a heterogeneous immunoassay principle by incubation with at least two receptors R1 and R2, of which R1 mediates the binding to the solid phase and R2 is a conjugate of a ligand capable of specific binding to a substance to be determined and a label, in which a solid phase is used on which a specifically bindable reaction component is bound, separation of the solid from the liquid phase and determination of the label in one of the two phases a material is used as the solid phase on the surface of which specifically bindable partners are bound and the sample solution is incubated with several different receptors R1 whereby each receptor R1 has binding sites for the specifically bindable partner bound to the solid phase as well as binding sites for one of the substances to be determined.

Description

2(~(~8~

D e s c r i p t i o n The invention concerns a method for the determination of immunologically detectable substances according to a heterogeneous immunoassay principle by incubation with at least two receptors Rl and R2, of which Rl mediates the binding to the solid phase and R2 is a conjugate of a ligand capable of specific binding to the substance to be determined and a label, in which a solid phase is used on which a specifically bindable component is bound, separation of the solid from the liquid phase and determination of the label in one of the two phases as well as a suitable reagent therefor.

A multitude of clinically important parameters are determined with immunological detection methods. There are a large variety of homogeneous and heterogeneous variants of methods for immunoassays. Frequently a method according to the sandwich principle is used or a variant procedure derived from it. Usually the sample solution is incubated with a receptor which is capable of binding to the substance to be determined and which carries a label, and a receptor capable of specific binding to the substance to be determined which is bound to the solid phase or mediates the binding to the solid phase. In this process, bound complexes of substance to be detected and labelled receptor are formed on the solid phase via the receptor which mediàtes the binding to the solid phase. ~y separation of the solid from the liquid phase, the bound label can be easily isolated from the unbound label and can be detected in one of the two phases. The amount of label is a measure of the content of substance to be detected.

z~ o There are different situations in which not only one test but several different tests have to be carried out in parallel. This is, for example, the case in the diagnosis of antibodies directed towards particular viruses such as e.g. anti-HIV antibodies or antibodies directed towards hepatitis antigens. In general, a multitude of different antibodies are formed by the body to an antigen or hapten i.e. antibodies that are capable of binding to different epitopes. Some viruses have the property to continually change their surface so that it is very difficult either to find antibodies which enable a reliable detection of the antigen or to find model substances by which all antibodies formed to a particular virus can be detected. For this reason one tries to increase the accuracy of the detection in parallel tests by using different antibodies which are capable of binding to different viral antigens or by use of different antigenic determinants. Furthermore, for many disease conditions it is important to detect different parameters in parallel, for example, in tumour diagnosis in the search for tumour markers or in the investigation of allergy patients for the detection of allergens or of anti-allergen antibodies. It is also necessary to carry out different tests in parallel when investigating blood for transfusions in order to determine if the blood contains any risk factors such as HIV antibodies, hepatitis pathogens etc.

Up to now it has been very complicated to detect different antigens or antibodies in parallel, since a separate test has to be carried out for each individual substance which is time-consuming and laborious. It has also been suggested that several antibodies be determined simultaneously by immobilizing different antigens on the solid phase. However, as a rule, this method did not yield satisfactory results since, on the one hand, interfering reactions occurred and, on the other hand, the binding capacity of the solid phase was so low for such tests that the sensitivity was lower than that required in practice.

It was therefore the object of the present invention to provide a method with which several parameters can be determined simultaneously and with great accuracy and speed.
, This object is achieved by a method for the determination of immunologically detectable substances according to a heterogeneous immunoassay principle by incubation with at least two receptors R1 and R2, of which Rl mediates the binding to the solid phase and R2 is a conjugate of a ligand capable of specific binding to the substance to be determined and a label, in which a solid phase is used on which a specifically bindable reaction component is bound which is characterized in that a material is used as the solid phase on the surface of which specifically bindable partners are bound and that the sample solution is incubated with several different receptors Rl whereby each receptor R1 has binding sites for the specifically bindable partners bound to the solid phase as well as binding sites for one of the substances to be determined.

Surprisingly, different substances can be successfully determined simultaneously with the method according to the present invention. A whole spectrum of desired parameters can be covered simply and rapidly.

Z0~8~)0 The method according to the present invention is suitable for all variants of the heterogeneous immunoassay.
;
A solid phase is used for the method according to the present invention on the surface of which specific binding partners are bound. The well-known materials can be used for the solid phase such as plastic, glass, paper carriers, ceramics, latex, magnetic particles and others. The solid phase can be present e.g. in the form of reaction tubes, reagent carrier strips or beads. In a preferred embodiment the solid phase is present in the form of a reaction vessel and especially preferred in a cuvette form whose walls are at least partially coated on the inner surface with specific binding partners. The known materials are suitable as materials for the reaction vessel. For this polystyrene, copolymers of polystyrene, polycarbonates, polyacrylates and polymethacrylates are preferred.

The coating of the carrier material with the specific binding pairs can either be carried out directly or via a carrier material or a spacer. Binding to a soluble protein with a molecular weight above 500000 which is then adsorbed to the inner surface of the reaction vessel is, for example, suitable. Equally suitable is binding via a spacer which can be covalently or adsorptively bound to the surface of the reaction vessel via a functional group. Methods and agents for this are known. In a preferred embodiment a carr.ier material is used as the solid phase which is prepared according to the method described in DE-A-36 40 412.8. In a further preferred embodiment the solid phase is a reagent carrier which was obtained by activating a fibre pad made of a mixture of cellulose and synthetic fibres with 2(~ 0 periodate and coating it with the specific binding partner which had been previously treated with acid. A
method for producing such reagent carriers is described in DE-A-35 43 749.

A specific binding partner is bound to the surface of the solid phase. This partner must be capable of binding to a part of receptor R1. Since the binding between the partner bound to the solid phase and the receptor R
should not interfere with the ability of the receptor R
to bind to the substance to be determined, then, if receptor R1 is an antibody, either antibodies which are directed towards the Fc part of this antibody are used or protein A is used or partners Pl of a specific binding pair are imn~obilized. Antigen-antibody, hapten-antibody, lectin-carbohydrate, biotin-antibiotin-antibody, biotin-streptavidin as well as biotin-avidin are, for example, suitable as the specific binding pair.
Partners which are capable of binding to biotin, in particular streptavidin or avidin, are preferably immobilized on the solid phase. If protein A is used as P1 or P2 only receptor Rl should be a complete antibody in the immunoassay, while a Fab ar F(ab')2 fragment should be used as the labelled receptor in order not to cause non-specific binding of the labelled receptor to the solid phase which would result in a falsification of the results.

The sample solution is incubated with different xeceptors Rl. Receptors are used as the receptors Rl which have binding sites for the partner bound to the solid phase as well as binding sites for one of the substances to be detected. Complete antibodies of all subclasses which are capable of binding to one of the substances to be detected can be used as receptors Rl.

2~ n In this case either antibodies are bound to the solid phase which are directed towards the Fc part of the antibody used for Rl or protein A is bound. In the latter case, the labelled antibody should not be a complete antibody, in order not to cause non-specific binding to the solid phase which would result in a falsification of the result. In a further embodiment conjugates of partners P2 complementary to partner P1 which is bound to the solid phase as well as ligands capable of binding to one of the substances to be detected are used as receptors Rl. The immobilization is effected via the partner P2 which binds partner P1.
Preferably receptor Rl contains a hapten such as FITC, p-nitrophenol, saponin, digoxin, or, particularly preferably, biotin as partner Pl. The ligand binds to one of the substances to be detected. Depending on the substance to be detected the ligand can be an antibody, antibody fragment, antigen, hapten or binding protein.
Whole or purified parts of pathogens such as bacterial antigens, protozoal antigens, allergens or viral antigens can be used as antigens. The antigens can consist of native material, recombinant material or of chemically synthesized or modified peptides or carbohydrates. Thus, for example, in the detection of HIV antibodies, different antigenic determinants such as p24, gp41 and gp32 are used as ligands for the receptors Rl. If the type and course of a hepatitis illness is to be detected, then the different virus antigens HBcAg, HBsAg, HBeAg, HAV etc. can be used as ligands for receptor Rl. If the method is to be used for the detection of allergies, then the different allergens are used to detect the presence of corresponding antibodies.
For the detection of tumour markers, the corresponding different antibodies are used for receptor Rl, for example, the antibodies to CEA, AFP, CA15.3 can be used in parallel for the receptors Rl. For the determination 2~

of hormones, antibodies to TSH, LH, FSH, cortisol, prolactin etc. may be considered. Likewise for the detection of diseases, antibodies to the different viral, bacterial or protozoal antigens can be used.
Instead of the antibodies, their fragments such as Fab, Fab' or F(ab')2 fragments can, of course, also be used.
The antibodies can be polyclonal or monoclonal.

The second receptor R2 used in the method according to the present invention is a conjugate of a ligand capable of binding to one of the substances to be determined and a label. Many possibilities are known for the labelling and are suitable for the present method. As examples, radioactive isotopes such as 125I 131I 51Cr 35S and 3H, enzymes such as peroxidase, ~-galactosidase or alkaline phosphatase, fluorescent, chemiluminescent or substanc~s detectable by other means can be used. The ligand used for R2 can be a component capable of binding to all substances to be detected e.g. in the case in which different antigens are used for the detection of definite antibodies such as e.g. HIV. In this case the label for all receptors R2 is also the same. The simultaneous determination of IgE antibodies of different allergens is analogous. If different parameters are to be determined in parallel, different ligands can be used for each of the receptors R2 which are capable of specific binding to the individual substances to be detected. The determination of the individual parameters in parallel can be carried out in two variants. In a first variant the method is carried out in several phases. In this case the different receptors R2 which each carry the same label are added in succession. Since each receptor R2 can only react with one substance to be detected, the portion of bound or unbound label can be determined after each addition 2~

of the receptor and is a measure of the respective substance to be detected. In a second variant, the method is carried out in a s:ingle phase~ This variant is preferred because it can also be carried out in automated analysers. In this case, the different recept~rs R2 each carry different labels which enable a simultaneous determination e.y. different isotopes with distinguishable emissions or fluorescent compounds which fluoresce at different wavelengths. Equally suitable is labelling with different enzymes. In this case different substrates are added for the analysis which form colours under the action of the respective enzyme that has an adsorption maximum at different wavelengths. The same ligands as for receptor R1 are suitable as ligands for receptor R2. Depending of the determination to be carried out, the ligands used for receptor R2 can be identical with those ligands used for receptor Rl or different from them. For the detection of antigens, antibodies directed towards the antigens or their fragments, as well as binding proteins can be used as ligands.

Antibodies directed towards e.g. IgG, IgA, IgM, IgE or fragments of these antibodies or antigens capable of binding to the antibodies to be detected or haptens can for example be used for the detection of antibodies. The receptors R2 can be used in the form of mixtures or in the form of cross-linked products. A cross-linking can be carried out in a well-known way, for example, by use of bifunctional or polyfunctional linkers. Methods for this are known to the expert and do not need to be described in more detail.

The sample solution can either be incubated simultaneously with all receptors or first with receptor 20~
g R1 and then with the different receptors R2. Further variants of the method are possible and known to the expert. In this process, the reaction with both receptors Rl and R2 is carried out in a homogeneous phase.
:`
Using the method according to the present invention it is possible to carry out simultaneous determinations for many different parameters. The simple procedure enables the simultaneous determination in automated analysers.
`
By using a surface which is coated according to the present invention with a partner of a specific binding pair, sufficient binding capacity is available so that all parameters can be detected accurately and reproducibly.

The method is also suitable for a rapid diagnosis. By testing simultaneously for HIV antibodies and HBsAg blood samples which contain these risk factors can be eliminated immediately without the necessity for more accurate diagnostics. On the other hand, a rapid test can check at the outset of an examination if risk factors are present and, if this is the case, then a more detailed diagnosis can follow. It is thus possible according to the present invention to establish very quickly if particular indications for a disease are present which then simplifies the additional examinations.

20~81~0 -- 10 --`

The in~ention is elucidated in more detail by the following Figures and Examples.
ig. 1 shows a diagram for a simultaneous determination of HIV antibodies and HBsAg.
ig. 2 shows a diagram for a simultaneous determination of different tumour markers.

The simultaneous determination of HIV antibodies and HBsAg is shown diagrammatically in Fig. 1. Antibodies directed towards fluorescein isothiocyanate (FITC) are immobilized on a solid phase. The sample solution is incubated with different conjugates as receptors R1 which all contain FITC and either at least one epitope capable of binding to HIV antibodies or anti-HBsAg antibodies. After a wash step the sample is incubated with labelled anti-human-IgG antibody and anti-HBsAg antibody as receptors R2. The label is radioactive iodine. Complexes then form from the receptors Rl, HIV
antibodies or HBsAg and from receptors R2, which are bound to the solid phase by the binding of FITC to the immobilized anti-FITC antibodies.

Fig. 2 shows a diagram of a simultaneous determination of tumour antigens. In this case anti-Fc~ antibodies are immobilized on the solid phase. Antibodies directed towards tumour antigens are used for each of the receptors Rl. Cross-linked peroxidase which carries the Fab fragment of antibodies directed towards tumour antigens is used as receptor R2. During the incubation the anti-tumour-antigen antibodies bind the tumour antigen present in the sample~ In addition the cross-linked POD mol~cules also bind to the tumour antigens by zo~

; means of the corresponding Fab antibodies. The immobilization results from the binding via the anti-Fc antibodies.

E x a m p 1 e Anti-HIV test `:
HIV antibodies are determined in a two-step sandwich immunoassay. Reagents with the following compositions are used for the test.
.
Reaqent 1:
10 7 mol/l each of one or several biotinylated HIV
antigens, 40 mmol/l phosphate buffer, pH 7.0 0.9 % by weight sodium chloride 10 % by volume bovine serum albumin.

The following antigens were used for this: HIV antigens prepared by genetic engineering which correspond to HIVl-gp41 (gp41-rek., CentocorTM-pl21) and HIVl-p24 (p24-rek., CentocorTM-pg2), chemically synthesized peptides from HIV1-gp41 (gp41-pep, Wand et al., PNAS, 83, 6159, 1986) and HIV2-gp32 (gp32-pep, Gnann, J.W. et al., Science, 237, 1346, 1987). These antigens were labelled with biotin as described by Leary et al., PNAS, 80, 4045 (1983).

Reaqent 2.
20 mV/ml of a conjugate of sheep antibody to human immunoglobulin and POD
40 mmol/l phosphate buffer, pH 7.0 0.05 ~ by weight Tween 20 0.2 % bovine serum albumin 0.2 % bovine IgG.

Polystvrene tube Thermally aggregated BSA which is denoted below as thermo-BSA is prepared in the following manner: 1 g BSA
was dissolved in 100 ml 50 mmol potassium phosphate solution at a pH of 7.0, heated to 7CC and maintained for 4 hours at this temperature with gentle stirring.
The solution was cooled, filtered and adjusted to a concentration of 50 mg/ml. Subsequently it was dialyzed against the 30 fold volume of redistilled water.

Preparation of a conjugate of streptavidin with thermo-BSA: Streptavidin isolated from Streptomyces avidinii was reacted with maleimido-hexanoyl-N-hydroxy-succinimide and in this way streptavidin was obtained which carries ma]eimido groups. Thermo-BSA was reacted with S-acetylmercaptosuccinic acid-anhydride and afterwards the protected SH-groups were released by addition of hydroxylamine. The streptavidin containing maleimido groups was then mixed with the thermo-BSA
contain.ing SH-groups to form the desired conjugate.

Plastic tubes made of polystyrene were then coated with streptavidin-thermo-BSA conjugate. The coating of the tubes was carried out with 1.5 ml of a streptavidin-thermo-BSA solution in which the molar ratio of the two components was 1.8:1 (10 ~g/ml) in 40 mmol/l sodium phosphate buffer, pH 7.4 at 20C during 18 to 24 hours.

2~ )0 After aspirating the tubes they were re-coated for 30 minutes at 20'C with 1.8 ml of a solution of 2 %
saccharose, o.~ % sodium chloride and 0.3 % BSA. After drying (24 hours at 20C and 40 % relative humidity) the tubes were ready to use and stable.
,"
1 ml Reagent 1 and 10 ~1 human serum or plasma were incubated for one hour at room temperature in a polystyrene tube coated with streptavidin-thermo-BSA
conjugate. Afterwards it was washed three times with tap water and incubated for one hour at room temperature with 1 ml Reagent 2. It was washed again three times with tap water and 1 ml ABTS substrate solution was added for the test reaction. After 60 minutes the adsorbance at 422nm was measured photometrically.

The anti-HIV test was carried out using individual HIV
antigens and combinations of antigens. In doing so, it turned out that the test procedure in polystyrene tubes coated with streptavidin-thermo-BSA conjugate is suitable for the simultaneous determination of several antibodies or populations of antibodies (screening test;
Table 1) irrespective of whether these are directed towards the same virus or several viruses or antigens of interest.

2C~81~0 ,:.
H

H
C H ~ C~
:¢
.

C ~ ~ U~

.` ~

H ~ ~ ~ ) cn O r~l -1 ~ O ~ ~D ~ U~ ~
C H _ ,_, ,, a h C
Q) ~
Ul I H Q
L' ~ ~> O ~ ~1 a~
(~5 C HUl u~ N ~ ~D Ul a ~1 R I H ~ U~ _IO
C. C ~ ~ ~ _~
'¢ ;:
H O ~ ~ 1 O
,~ Ln ao ~ ~ o c~
:

Z U~ ~ I` ~ ~ U~

+
C) ~
h h ~ ~
~: h a) ~ ~ ` ` R.
~15 I h HQ ~ t~l tl~ CL 0 2~(381~() E x a m p 1 e 2 ., Hepatitis S antigen and HIV antibodies were determined in a sample solution using a two-step sandwich immunoassay. The reagents used had the following composition:

Reagent 1:
40 mmol/l phosphate buffer, pH 7.0 0.1 % Tween 20 0.9 % NaCl 0.01 % phenol 0.2 % bovine serum albumin 300 ng/ml of a FITC labelled monoclonal antibody to HBsAg as well as 600 ng/ml of FlTC-labelled p24 and of FITC-labelled gp41 antigen of HIV.
(FITC = fluorescein isothiocynate) Reaqent 2:
40 mmol/l phosphate buffer, pH 7.0 0.1 % Tween 20 0.2 mol/l sodium tartrate 0.01 % phenol 0.2 % bovine serum albumin 5000 cpm each of a 125I labelled polyclonal sheep antibody to human IgG antibody as well as of a monoclonal antibody to HBsAg.

Polystyrene tubes were coated for 18 hours at room temperature with 1 ml sheep antibody to FITC (50 ~g/ml in 0.1 mol/l carbonate buffer, pH 9.6) and coated with 1 ml bovine serum albumin (10 mg/ml in 0.1 mol/l phosphate buffer) for one hour at room temperature.

:~ 20(~81~(~
: - 16 -So ~l sample was incubated for 2 hours at room temperature with 1 ml Reagent l. After washing three times with water it was incubated for one hour at room temperature with l ml Reagent 2. After washing three .~ times the tube was measured in a gamma counter. The results are shown in Table 2.

T a b l e 2 sample HBsAg Anti-~IVmeasured value (ng/ml)(Western blot)in the simultaneous test 1 12 positive2125 cpm 2 <0.2 negative 52 cpm 3 <0.2 positive1159 cpm 4 <0.2 positive3041 cpm <0.2 negative 47 cpm 6 125 negative 830 cpm 7 203 negative1102 cpm 8 <0.2 negative 69 cpm 9 <0.2 negative 29 cpm <0.2 negative 58 cpm 11 <0.2 positive2552 cpm 12 <0.2 negative 54 cpm 13 52 negative 472 cpm 14 <0.2 negative 39 cpm ~0.2 negative 43 cpm E x a m p 1 e 3 The tumour mar~ers CEA and CAlS.3 were determined simultaneously in a sample using a one-step sandwich immunoassay. The reagent used had the following composition:

120 mU/ml of a conjugate of POD and Fab fragments each of a monoclonal antibody to the tumour marker CEA and CA15.3 ~0 mmol/l phosphate buffer, pH 7.0 TM

0.5 ~ Pluronic F6~
0.2 mol/l sodium tartrate 0.01 % phenol 0.2 % bovine serum albumin 300 ng/ml each of a biotinylated monoclonal antibody to CEA and CA15.3.

The requirements for the monoclonal antibodies to CEA
and CA15.3 are merely that the Fab fragments in the POD
conjugate are each directed towards another epltope than the biotinylated antibodies.

Polystyrene tubes were coated for 18 hours at room temperature with 1 ml sheep anti-mouse-Fc~ antibody (50 ~g/ml in 0.1 mol/l carbonate buffer, pH 9.6) and coated with 1 ml bovine serum albumin (10 mg/ml in 0.1 mol/i phosphate buffer) for one hour at room temperature. 1 ml of the reagent and 100 ~1 sample were incubated for 2 hours at room temperature. Afterwards they were washed three times with tap water. Then 1 ml ABT~ substrate solution was added for the test reaction. After one hour the absorbance at 405 nm was f~ `
.~ `
.

2~
~ 18 -measured photometrically. The results are shown in Table 3.

T a b l e 3 Simultaneous determination of tumour markers Content sample CEA CA15.3 measured value in the (ng/ml) (U/ml)simultaneous test 1 <0.5 1 76 mA
2 453 4 3580 mA
3 2 6 58 mA
4 212 5 2950 mA
4 7 76 mA
6 9 5 43 mA
793 mA
8 6 3 72 mA
9 50 4 250 mA
8 9 83 mA
11 3 7 59 mA
12 9 192 1483 mA
13 3 4 63 mA
14 5 4 51 mA

Claims

1. Method for the determination of immunologically detectable substances according to a heterogeneous immunoassay principle by incubation with at least two receptors R1 and R2, of which R1 mediates the binding to the solid phase and R2 is a conjugate of a ligand capable of specific binding to the substance to be determined and a label, in which a solid phase is used on which a specifically bindable reaction component is bound, separation of the solid and liquid phase and determination of the label in one of the two phases, w h e r e i n a material is used as the solid phase on the surface of which specifically bindable partners are bound and wherein the sample solution is incubated with several different receptors R1 whereby each receptor R1 has binding sites for the specifically bindable partner bound to the solid phase as well as binding sites for one of the substances to be determined.

2. Method as claimed in claim 1, w h e r e i n a material is used as the solid phase on the surface of which partners P1 of a specific binding pair are bound and wherein the sample solution is incubated with several different receptors R1 whereby each receptor R1 is a conjugate of the partner P2 of the specific binding pair and a ligand capable of binding to a substance to be detected.

3. Method as claimed in claim 1, wherein different receptors R1 are used, whereby different ligands capable of binding to the substance to be determined and different labels are used.

4. Method as claimed in claim 2, wherein different receptors R1 are used, whereby different ligands capable of binding to the substance to be determined and different labels are used.

5. Method as claimed in claim 1, wherein conjugate is used as receptor R2 consisting of one ligand capable of binding to all substances to be determined and a label.

6. Method as claimed in claim 2, wherein conjugate is used as receptor R2 consisting of one ligand capable of binding to all substances to be determined and a label.

7. Method as claimed in claim 3 or 4, wherein monoclonal antibodies are used for receptor R2.

8. A method for screening for the presence of any or all of two or more immunologically detectable substances in a sample by immunoassay resulting in a single signal for the presence of any or all said substances comprising:
a) providing a solid phase to which is bound a specific binding partner P1, b) providing two or more different receptors R1, each of which immunologically binds to a substance to be determined and specifically binds to the specific binding partner P1, c) incubating a sample containing two or more immunologically detectable substances to be determined with the different receptors R1 and the solid phase, to provide an incubation mixture, d) providing two or more different receptors R2, each of which immunologically binds to a substance to be determined and each conjugated to a same label, e) adding to the incubation mixture the different conjugated receptors R2 and determining the total amount of label bound or unbound to the solid phase as a measure of the sum of all immunologically detectable substances to be determined in the sample.

9. Method as claimed in claim 1, 2, 3, 4, 5, 6 or 8, wherein streptavidin-biotin, avidin-biotin, antibiotin-antibody-biotin, hapten-antibody, lectin-sugar or antigen-antibody is used as the specific binding pair P1-P2.

10. Method as claimed in claim 7, wherein streptavidin-biotin, avidin-biotin, antibiotin-antibody-biotin, hapten-antibody, lectin-sugar or antigen-antibody is used as the specific binding pair P1-P2.

11. Method as claimed in claim 1, 2, 3, 4, 5, 6, 8 or 10, wherein different antibodies are detected simultaneously in a sample solution.

12. Method as claimed in claim 7, wherein different antibodies are detected simultaneously in a sample solution.

13. Method as claimed in claim 9, wherein different antibodies are detected simultaneously in a sample solution.

14. Method as claimed in claim 1, 2, 3, 4, 5, 6, 8 or 10, wherein different tumour markers, hormones, allergens or pathogens are detected simultaneously in a sample solution.

15. Method as claimed in claim 7, wherein different tumour markers, hormones, allergens or pathogens are detected simultaneously in a sample solution.

16. Method as claimed in claim 9, wherein different tumour markers, hormones, allergens or pathogens are detected simultaneously in a sample solution.

17. Method as claimed in claim 1, 2, 3, 4, 5, 6, 8 or 10, wherein different antibodies and different antigens or haptens are detected simultaneously in a sample solution.

18. Method as claimed in claim 7, wherein different antibodies and different antigens or haptens are detected simultaneously in a sample solution.

19. Method as claimed in claim 9, wherein different antibodies and different antigens or haptens are detected simultaneously in a sample solution.

20. Method as claimed in claim 1, 2, 3, 4, 5, 6, 8, 10, 12, 13, 15, 16, 18 or 19, wherein a reaction vessel is used as the solid phase whose walls are coated on the inner surface with streptavidin or avidin.

21. Method as claimed in claim 7, wherein reaction vessel is used as the solid phase whose walls are coated on the inner surface with streptavidin or avidin.

22. Method as claimed in claim 9, wherein a reaction vessel is used as the solid phase whose walls are coated on the inner surface with streptavidin or avidin.

23. Method as claimed in claim 11, wherein a reaction vessel is used as the solid phase whose walls are coated on the inner surface with streptavidin or avidin.

24. Method as claimed in claim 14, wherein a reaction vessel is used as the solid phase whose walls are coated on the inner surface with streptavidin or avidin.

25. Method as claimed in claim 17, wherein a reaction vessel is used as the solid phase whose walls are coated on the inner surface with streptavidin or avidin.

26. Method as claimed in claim 1, 2, 3, 4, 5, 6, 8, 10, 12, 13, 15, 16, 18, 19, 21, 22, 23, 24 or 25, wherein the ligand for each receptor R1 is a monoclonal antibody.

27. Method as claimed in claim 7, wherein the ligand for each receptor R1 is a monoclonal antibody.

28. Method as claimed in claim 9, wherein the ligand for each receptor R1 is a monoclonal antibody.

29. Method as claimed in claim 11, wherein the ligand for each receptor R1 is a monoclonal antibody.

30. Method as claimed in claim 14, wherein the ligand for each receptor R1 is a monoclonal antibody.

31. Method as claimed in claim 17, wherein the ligand for each receptor R1 is a monoclonal antibody.

32. Method as claimed in claim 20, wherein the ligand for each receptor R1 is a monoclonal antibody.

33. Reagent for the determination of immunologically detectable substances comprising a solid phase on whose surface specific binding partners are bound, different receptors R1 which each have binding sites for one of the substances to be determined and binding sites for the specific binding partner bound to the solid phase, and receptors R2 which are conjugates of a ligand capable of specific binding to a substance to be determined and a label.

34. Reagent as claimed in claim 33, wherein the solid phase is a reaction vessel coated on the inside with streptavidin or avidin, the receptors R1 are each conjugates of biotin and a ligand capable of specific binding to a substance to be determined and R2 are conjugates of a ligand capable of specific binding to a substance to be determined and a label.

35. A reagent for screening for the presence of any and all of two or more immunologically detectable substances in a sample by immunoassay resulting in a single signal for the presence of any or all said substances comprising:
a) a solid phase to which is bound a specific binding partner P1, b) two different receptors R1 each of which immunologically binds to a substance to be determined and specifically binds to the specific binding partner P1, and c) two or more different receptors R2 each of which immunologically binds to a substance to be determined and each conjugated to the same label.