IE850007L - Process and reagent for the determination of a polyvalent¹antigen - Google Patents

Abstract

A polyvalent antigen is determined by using three different receptors, of which the first and the third are dissolved in liquid phase and are able to bind to the antigen, the second receptor is in solid phase and is able to bind with only part of the first receptor, and the third receptor carries a label and does not cross-react with the first and second receptor, and incubating the antigen with the first and the third receptor, then contacting the incubation solution with the second receptor, then separating the phases and measuring the label in one of the phases. <IMAGE> [EP0147848A2]

Description

3 25 4 -2- The invention concerns an immune chemical process for the determination of a polyvalent, i.e. at least bifunctional, ligand (antigen) in a liquid sample. 5 The sensitive determination of polyvalent antigens (peptides, proteins) with the use of two antibodies which are directed against different antigen determinants, is known as 2-site immunoradiometric or immuno-enzymometric assay, e.g. from J. Clin. Chem. Clin. 10 Biochem., JJ3 (1980), 197-208. The most common is the carrying out of this known method of determination in that the antigen to be determined is first incubated with a first antibody which is present in solid phase by binding to a suitable carrier material, such as 15 Sepharose, agarose, synthetic material test tubes or the like. During this first incubation, the first antibody, which, as a rule, must be present in large excess, binds with an antigen determinant of the antigen to be determined. The sample liquid is then usually separated 20 from the solid phase in order to exclude disturbances due to non-specific substances, such as human plasma protein, or due to cross-reacting antigens in the subsequent second incubation. A definite amount of a second labelled antibody is then incubated in liquid 25 phase with the solid phase. The specificity of the second antibody is thereby preferably so chosen that it is directed against a determinant of the antigen to be determined different from that of the first antibody in order to exclude a competition between the two anti-30 bodies for the same binding position on the antigen to be determined since the sensitivity of the test would hereby be impaired.

During this second incubation, the labelled second antibody, which is also usually present in excess, 35 reacts with all binding positions on molecules of the antigen to be determined. After the second incubation, the activity of the labelling substance can be measured either on the solid phase or in the supernatant. The measurement thereby usually takes place in the solid phase after washing out of the liquid phase. In the most favourable case, the activity determined is almost proportional to the amount of the antigen to be determined.

This 2-stage sandwich process has the advantage that possibly cross-reacting antigens are already removed in the case of the first phase separation.

This is of particular importance in the case of test systems in which the two antibodies must indeed possess differing specificity because of the sensitivity requirements. In this case, the high requirement of the specificity only exists for one of the two antibodies, the other one must then not be absolutely specific towards cross-reacting antigens.

However, this process has three important disadvantages: 1. In the first incubation step, one reactant is present in solid phase, the other in solution. The velocity constant of the reaction is thus smaller than if both reaction components (antigen and first antibody) were present in solution. However, one must incubate until all antigen molecules are present bound to the solid phase since non-bound antigen would be removed in the case of the phase separation and this would result in a falsification of the result. 2. The binding of a specifically bindable antibody for the antigen on to a solid phase requires, because of the low binding yields, relatively large amounts of first antibody, which is usually a very expensive substance, especially when the antiserum must be obtained, for reasons of specificity, in small animals, such as -4- rabbits, guinea pigs and the like. Furthermore, in the case of the binding reaction to the solid phase, affinity deteriorations arise on the antibody which acts disadvantageously on the sensitivity. 5 3. As a rule, the obtaining of two antibodies of different specificity against the same antigen is laborious and frequently only of limited possibility, for example, in that one immunises two different animal species or in that one attempts to separate the anti-10 body populations from one animal into suitable fragments by immune adsorption on the antigen to be determined.

Various attempts are already known to overcome the above deficiencies. Thus, according to US-PS 4,098,876, the first incubation of the antigen is 15 carried out with isotope-labelled, dissolved antibody. Only thereafter is the solid phase-bound antibody added thereto and the second incubation carried out. Here, only one phase separation is necessary, which results in a certain increase of the sensitivity and 20 practicability. However, the specificity problem remains unsolved since boch antibodies must be directed highly specifically against the antigen since otherwise, in the presence of comparatively large amounts of cross-reacting antigens, the apparent concentration of 25 the antigen to be determined is increased in the case of a possible non-specificity of the second antibody and is reduced in the case of non-specificity of the first antibody.

In DE-OS 29 25 565 is described a process in 30 which the first antibody present in solid phase and the labelled, dissolved second antibody are incubated simultaneously with the antigen. The same problems here exist as in the case of the previously mentioned process. Furthermore, disadvantages 2 and 3 described 35 above for the basic process also remain unsolved. -5- Finally, from published Japanese Patent Application 123,802 of the 06.10.1978, a process is known in which a solid phase antibody is used which is specifically directed against the second antibody 5 which is bindable with the antigen to be measured. The binding of an antibody directed against the antigen to the solid phase is here avoided. However, the sensitivity is lowered since only a single antibody specifically bindable with the antigen is used. 10 The concentration determination of the antigen to be determined takes place via the competitive reaction between the antigen and a definite amount of isotope-labelled antigen with the specific antibody.

From EP-A 0,105,714, a process is known in the 15 case of which there is used a solid phase antibody, together with two further antibodies, at least one of which must be a monoclonal antibody, according to the sandwich principle for antigen determination. The antigen is thereby first incubated with the two anti-20 bodies present in liquid phase and thereafter with the solid phase antibody. The process has the disadvantage of requiring two incubation steps, which hinders automation, of resulting in too long incubation times and of not permitting the use of undiluted samples. A 25 similar process is known from Clin. Chem., 22/6 (1981), 823-827. There is there described a radiometric method of determination for creatine kinase MB isoenzyme in which, in a first incubation step, the antigen is reacted simultaneously with an unlabelled and with a 30 labelled antibody of differing specificity and from different animal species. In the second step, an antibody present in solid phase, with specificity against the unlabelled antibody of the first incubation step, is then added thereto. The disadvantage of this 35 process is again the fact that there are needed two different antibodies specific for the antigen and three different animal species.

Therefore, the task forming the basis of the invention is better to avoid one or more of the above-5 described disadvantages of the basic process than the previously known processes. In particular, such a process is to be provided which only requires one incubation, none of the antibodies has to be used in' liquid phase, is suitable for automation and which 10 displays high sensitivity and exactitude. Furthermore, the process is to make it possible to manage possibly with a single animal species for obtaining the necessary antibodies or to be able to use large animals for obtaining the antiserum. 15 According to the invention, this task is solved by a process for the determination of a polyvalent antigen by incubation with three different receptors, the first and third of which are present dissolved in liquid phase and bindable with the antigen, the second 20 receptor is present in solid phase and is only bindable with a part of the first receptor and the third receptor carries a labelling and does not cross-react with the first and second receptor, separation of the solid from the liquid phase and measurement of the 25 labelling in one of the phases, which is characterised in that one brings together a solution containing the antigen with a first solid carrier material, which contains, impregnated or lyophilised, the first and the third receptor in removable form and, after removal 30 of the receptors, immediately contacts the solution obtained with a second solid carrier material which contains the second receptor in non-removable form, thereafter separates the liquid from the second solid carrier material and measures the labelling, whereby, 35 as third receptor, one uses either a Fab antibody fragment which originates from the same animal species as the antibody component of the first receptor or uses a receptor which originates from the same animal species as the second receptor, whereby the first receptor either originates from a different animal species and/or is labelled with a hapten or antigen.

As receptors in the scope of the invention, there are used either specifically bindable complete antibodies (polyclonal or monoclonal), antibody fragments thereof or conjugates of antibodies or antibody fragments with haptens or antigens. As first receptor, there is preferably used a complete antibody or an antibody or antibody fragment bound or labelled with a hapten or antigen, in each case especially preferably a monoclonal antibody.

As second receptor, there is used an antibody which is only bindable with a part of the first receptor, especially preferably an antibody which is only bindable with the Fc part of the first receptor. Alternatively, as second receptor, there can also be used an antibody which is only specifically bindable with the hapten part or antigen part of the first receptor. The second receptor can also be generally directed against the Fc part of immunoglobulin.

The third receptor, which must not cross-react with the second receptor, can be a labelled antibody, bindable with the antigen, which is obtained from an animal species different from that from which the first receptor is obtained.

However, one preferably uses a third receptor which is a labelled Fab fragment. Within the scope of the invention, by Fab fragment are thereby also understood (Fab>2 and Fab' fragments. In this case, receptor 3 can be obtained from the same animal species as the antibody component of the first receptor. This also applies when the first receptor consists of an antibody which carries a hapten or antigen and the second receptor is only bindable with this hapten or antigen. Especially preferably, the third receptor 5 is a part, especially a Fab fragment, of the first receptor. Alternatively, as third receptor there can also be used an antibody which originates from the same animal species as the second receptor. Both alternatives have the advantage that only two animal species are 10 required for the three receptors. If the second receptor is only bindable with the hapten or antigen of the first receptor, only one animal species is needed for all three receptors. Especially preferably, the third and the first receptor contain monoclonal anti-15 bodies or fragments thereof which are directed against different and/or repetitive determinants of the antigen.

The third receptor is preferably used in an amount which is greater than the amount of the antigen to be determined. In this case, thus in the case of excess, 20 an exact dosing or a precisely known amount is not necessary. The third receptor is labelled in the manner known to the expert. The labelling preferably takes place by coupling with an enzyme, a fluorescing, chemiluminescing and radioactive substance. Processes 25 for the labelling of such receptors are known to the expert, e.g. from Clin. Chim. Acta, jU (1977), 1-40, and do not require further explanation here.

The 1st and the 3rd receptors are applied dissolved to a suitable carrier, e.g. to absorbent paper, 30 and, by drying or lyophilising, converted into a form which can easily be removed therefrom.

The binding of the second receptor present in solid phase to an insoluble carrier material can be carried out according to the usual methods known to 35 the expert for the fixing of biologically active -9- proteins to solid carrier substances. Not only a covalent but also an adsorptive binding is suitable. However, because of the hereby achievable higher yields and of the simplified method of working, there 5 is preferred solely an adsorptive binding, for example on a synthetic material or on to a fleece, such as e.g. paper. There have proved to be suitable e.g. reagent vessels made of polystyrene and similar synthetic materials which are adsorptively coated on the inner 10 surface with the second receptor. The incubation can then be carried out in these test tubes or in containers of some other shape and, for the phase separation, it suffices to remove the liquid phase from the test tube, for example, by sucking out, shaking out and the like. 15 After washing of the test tube, the measurement of the labelling can be carried out directly therein, especially when the labelling has taken place with an enzyme. It then suffices simply to measure the activity of the labelling enzyme, for example peroxidase 20 or 0-galactosidase, in the manner generally known here-for in that one adds a known detection reagent for this labelling enzyme, e.g. a colour reagent, and measures either in the presence of the solid phase or after separation. The measured enzyme activity is then a 25 measure for the amount of polyfunctional antigen to be determined. However, as solid phase carrier for the second receptor, there also come into consideration particulate substances, e.g. ion exchangers, molecular sieve materials, glass particles, synthetic material 30 tubes and the like. Especially preferred as carrier for the second receptor is a porous, laminar carrier, such as paper.

If the labelling takes place not with an enzyme but rather with a radioactive substance, an isotope, 35 a fluorescing or chemiluminescing substance, here, too, -10- the measurement is carried out according to one of the methods well known to the expert. Therefore, a description of these measurement methods is here not necessary. 5 For the carrying out of the process, the sample solution, which according to an especially advantageous characteristic of the invention can even be used in undiluted form, e.g. as blood, plasma or serum, is placed together with the first carrier material for a 10 sufficient length of time for the removal of the two receptors, then separated off and incubated with the second carrier material. In general, the contact time with the first carrier material lies between about 10 seconds and 10 minutes. Advantageously, the process 15 for this purpose is carried out in a device such as is described in DE-A 34 25 008.

Surprisingly, it has been found that, according to the invention, there is achieved a clearly higher sensitivity than in the case of the conventional 2-step 20 sandwich method and of the 1-step sandwich method.

If, according to the preferred embodimental form of the invention, there is used a second receptor directed against the Fc part of the first receptor or against the hapten or antigen which is coupled with 25 the first receptor, then there can hereby be achieved a further increase of the sensitivity since a possible steric hindrance of the binding reaction of the antigen with the binding position of the first receptor is avoided. This is an especial advantage when antigens 30 with especially low concentration must be determined, such as e.g. the case in the determination of thyrotropin in serum.

A further subject of the invention is a dry reagent for the determination of a polyvalent antigen 35 according to the process of the invention, characterised by -11- a) a first solid carrier material impregnated or lyophilised with the antigen-bindable receptors 1 and 3, of which receptor 3 carries a labelling, and b) a second solid carrier material, which is present 5 physically separated from the first carrier material, which contains an insoluble receptor 2 only bindable with a part of the non-labelled soluble receptor 1, with the proviso that the labelled third receptor is a Fab antibody fragment. 10 The physical separation can e.g. take place in that the insoluble second receptor and the soluble first and third receptors are applied to separate carrier materials. This can be achieved in that receptor 1 and receptor 3 are lyophilised or impregnated into or 15 on to a suitable carrier simultaneously or successively (for example synthetic material test tubes or absorbent carriers, such as paper or the like) and the receptors 2 are bound adsorptively or covalently on to a suitable carrier (for example synthetic material test tubes, 20 paper or particles). The sample with the polyvalent antigen to be determined is first brought into contact with the carrier 1, which contains the soluble first and third receptors, and thereafter with the carrier 2, on which is fixed the insoluble receptor 2. Altern-25 atively, the first and third receptor, on the one hand, and the second receptor, on the other hand, can be present on carriers connected with one another which consist e.g. of a paper strip which contains the receptors at different places separated from one 30 another.

According to a first preferred embodimental form, a reagent according to the invention is characterised in that it contains a first receptor, which is an antibody, and a third receptor, which is an enzyme-35 labelled Fab fragment of an antibody. Preferably, it -12- also contains a buffer and a system for the determination of the enzyme labelling.

According to a further preferred embodimental form of the reagent according to the invention, this 5 is characterised in that the receptors 1 and 3 are monoclonal and are directed against different determinants of the antigen and originate from the same animal species.

According to a further preferred embodimental 10 form of the reagent according to the invention, this is characterised in that the second receptor is an antibody against the Fc part of the first receptor and the third receptor originates from the same animal species as the second receptor, the first receptor, on 15 the other hand, from another animal species.

According to a further preferred embodimental form of the reagent according to the invention, this is characterised in that all three receptors contain antibodies from the same animal species, the first 20 receptor is a hapten-labelled monoclonal antibody, and the second receptor, on the other hand, is directed against the hapten of the first receptor.

The receptors 1 and 3 of the reagent according to the invention are present impregnated or lyophilised 25 on a solid carrier material.

The invention is suitable for the determination of all antigens with at least two antigen determinants. Examples herefor include thyreotropin (TSH), alpha-1-foetoprotein (AFP), hCG, carcinoembryonal antigen, 30 luteinising hormone (LH), follicle-stimulating hormone (FSH), 02-microgl°bulin» acidic prostate phosphatase, prolactin, ferritin and insulin.

In the case of the invention, receptor 1 can react homogeneously with the antigen. Consequently, 35 this reaction possesses a higher velocity constant than -13- in the case of the use of a solid phase-bound insoluble first receptor. Since the antibody losses in the case of the fixing (making insoluble) on to the solid phase are avoided, the amount of first receptor can be 5 further decreased.

In contradistinction to the process described in Clin. Chem., 27/6 (1981), it is also not necessary to obtain the first and the third receptor from two different animal species which, in addition, have to be 10 so chosen that receptor 2 only reacts with receptor 1 but not with receptor 3.

Finally, the obtaining of the antibody for the receptor 1 and for the receptor 3 from the same animal species also makes it possible to use large animals 15 for obtaining the antiserum.

Finally, according to the invention, the sensitivity is also increased. Thus, for TSH there is found a sensitivity of less than 40 pg./ml. (1 pU = 170 pg./ml.), whereas in the case of the 2 site assay 20 (sandwich process) it lies above 100 pg./ml.

The following Examples further explain the invention in combination with the drawings. In these are illustrated: Fig. 1 a calibration curve according to Example 2, 25 Fig. 2 a calibration curve according to Example 3, Fig. 3 a calibration curve according to Example 4, Fig. 4 an insert element for a centrifugal aytomatic analysis apparatus which is used according to Example 2. 30 Example 1.

Determination of thyreotropin (TSH).

Receptor 1 and 3 originate from the same animal species (mouse); receptor 3 is a labelled Fab fragment. Receptor 1 is a monoclonal antibody, the Fab fragment 35 in receptor 3 also originates from a monoclonal -14- antibody which is directed against a determinant of the TSH other than that against which the monoclonal antibody of receptor 1 is directed. Receptor 2 originates from sheep and is directed against the Fc part of the monoclonal mouse antibody.

The development of monoclonal antibodies takes place according to the method of Kohler and Milstein, Eur. J. Immunol., 292 (1976). a) Carrying out of the test according to the invention: Reagents: 1. Incubation buffer (IB): 15 mM sodium phosphate buffer, pH 7.4; 154 mM NaCl; 5 mM EDTA; 0.2% BSA; pH 7.4. 2. Receptor 1: Mouse anti-TSH antiserum (receptor 1): The ascites liquid from mice containing monoclonal anti-TSH-antibodies is mixed ad 1.8M with NH^SO^. The precipitate is taken up in a buffer of 15 mM sodium phosphate, pH 7.0, and 50 mM NaCl and the solution so obtained is subjected to a passage over DEAE-cellulose. 3. Receptor 3: Anti-TSH antibody (monoclonal) which recognises a determinant different from that recognised by receptor 1, Peroxidase conjugate (Receptor 3): mouse anti-TSH antiserum is purified like Receptor 1. The subsequent obtaining of (Fab)2 from the complete antibody molecule takes place by the method described in Example 2. The coupling with horseradish peroxidase takes place according to the method of Nakane (M.B. Wilson, P.K. Nakane, "Recent developments in the periodate method of conjugating horseradish peroxidase to antibodies, 1978, Elsevier, North Holland Biomedical Press, pp. 215-224 in "Immuno- -15- fluorescence and related staining techniques"). 4. Receptor 2: Adsorber material with fixed sheep anti-mouse-FcY-antibody (Receptor 2 adsorber): 5 Sheep anti-mouse FcY antiserum is mixed ad 1.8M with NH^SO^. The precipitate is taken up in a buffer of 15 mM sodium phosphate, pH 7.0, and 50 mM NaCl and the solution so obtained is subjected to a passage over DEAE-cellulose. The IgG-containing fraction 10 (Receptor 2) is coupled with the "affinity adsorbent, activated glutardialdehyde" of Boehringer Mannheim (order No. 665 525) according to the manufacturer's working instructions. 5. Indicator reagent: * 15 1.8 mM ABTS 2,2*-azino-di-[3-ethylbenzthiazoline- sulphonate (6)], 3.3 mM sodium perborate in 100 mM phosphate-citrate buffer, pH 4.4.

Carrying out: 50 ng. Receptor 1 and 170 mU Receptor 3, 20 dissolved in incubation buffer, are together applied dropwise to a fleece consisting of commercial polyester paper with a size of 6 x 6 mm., 'thickness 1.0 mm., and dried at room temperature. The reagent-containing paper fleece is called reagent carrier 1. 25 On to reagent carrier 1 are pipetted 40 yl. of the sample to be determined or known amounts of antigen-containing standard solution, subsequently the fleece is immediately centrifuged off in an Eppendorf centrifuge, whereby reagent carrier 1 is placed on an 30 Eppendorf cup and the eluted liquid in the cup impinges during the centrifuging on to the receptor 2, which is coupled on an adsorber, and reacts with this. Of receptor 2, 2.5 mg. of adsorber material are used per test.

♦Trade Mark -16- The reaction mixture is incubated on a shaking apparatus for 15 minutes at 37°C.

After precisely 15 minutes, the reaction vessels are removed from the apparatus and centrifuged. 5 An aliquot of the supernatant is removed with a pipette and the coloured material formed is determined via its extinction at 405 nm.

The following extinction values were determined with 2 different TSH samples: 10 TSH E405 nm/cm* (yU/ml.) 0 0.74 50 7.54 * The extinction values were determined at 0.2 cm. 15 layer thickness and recalculated to 1 cm. layer thickness. b) Simple sandwich (comparison): The carrying out takes place with the same reagents and in the same manner as described under a) 20 but with the following changes: 1. 2.5 mg. adsorber-receptor 2 is pre-incubated for 1 hour with 20 pg. receptor 1 in 0.2 ml., with shaking. Subsequently, the supernatant is sucked off and the residue washed three times with, in 25 each case, 1 ml. IB. 2. Adsorber receptor 2 pre-treated in this manner with receptor 1 is incubated at 37°C. for 4 hours with sample and receptor 3 on a shaking apparatus, the addition of soluble receptor 1 is omitted in this 30 variant. -17- Subsequently, the supernatant is sucked off as usual, the residue washed and the fixed enzyme activity detected with indicator reagent.

The following extinction values were determined 5 with 2 different TSH samples: TSH . (yU/ml.) E405 nm/cm* 0 50 0.20 2.87 10 * The extinction values were determined at 0.2 cm. layer thickness and recalculated to 1 cm. layer thickness.

The sensitivity is clearly smaller than according to a) although in the case of b) more receptor 1 is 15 used than in the case of a) (ratio 1:400).

Example 2.

Determination of human choriogonadotropin (HCG). Reagents: Buffer A: 20 100 mmol sodium phosphate, pH 7.3 (37°C.) 2 mmol magnesium chloride 0.9% NaCl 0.5% BSA (bovine serum albumin) 120 pg./ml. anti-HCG monoclonal antibodies from 25 mice (as ascites) (receptor 1) 100 mU/ml. anti-HCG antibody (sheep) Fab fragment-0-galactosidase conjugate Receptor 3 The preparation of the Fab fragment takes place 30 according to T. Kitagawa in "Enzyme immunoassay, E. Ishikawa, T. Kawai, K. Miyai eds. Igaku-Shoin, Tokyo/New York, 1981, pp. 81-89". The covalent coupling of 0-galactosidase to antibodies or AB -18- fragments takes place according to the method of R.R. Porter, Biochem. J., 7_3 (1959), 119.

Substrate solution: As buffer A, additionally 5 mmol (ONPG) ortho-5 nitrophenyl-galactoside. 1. The production of reagent carrier 1 takes place analogously to Example 1 in such a manner that 40 pi. of solution A is applied dropwise to a fleece consisting of commercial polyester paper and subsequently dried at 10 room temperature. Until its use, this fleece is stored at 4°C. and relative humidity <£ 20%. 2. Production of reagent carrier 2: On to a cellulose fleece are fixed, according to the cyanogen bromide activation process (DE-OS 17 68 512), 15 antibodies from sheep (IgG fraction), whereby 10 mg. of antibodies are provided for fixing per gram of fibre material. Non-coupled antibody is removed by washing and the fleece gently dried at room temperature. The storage of this fleece takes place analogously to 20 reagent carrier 1.

The determination of HCG with the help of these two reagent carriers 1 and 2 takes place with the help of the device for the carrying out of analytical determinations described in German Patent Application 25 24 25 008.5. This discloses a rotor insert element for centrifugal automatic analysers consisting of a formed body, which has a sample application chamber which stands in connection with a plurality of reagent zones, each of which contains an absorbent carrier 30 material impregnated with a particular reagent, at least one mixing valve chamber and a measurement chamber which together form a sample liquid transport path which passes from radially inwardly to radially further outwardly when the insert element is fixed on to the 35 rotor and, in addition, at least one further chamber -19- for the reception of a liquid and a transport path which leads from this chamber to the measurement guide and is at least partly identical to the sample liquid transport path. The sample liquid transport path 5 thereby passes from a sample application chamber (P) via a chamber (a) filled with absorbent material containing buffer, a chamber (c) and a first valve chamber (VK1) arranged between the chambers (a) and (c) to a second valve chamber (VK2) and from this, via the 10 chamber (d) and via a collection chamber (AK) to a measurement chamber (K). For the reception of a further liquid, there is provided a substrate chamber (PK) formed as a pump chamber which is connected with the second valve chamber (VK2) via a dosing device, 15 consisting of a dosing chamber (DK) and capillary (Kap), and an overflow chamber (UK). Fig. 4 shows schematically the rotor insert element used. Reagent carrier 1 is hereby placed on field c of the insert element and the reagent carrier 2 on field d. The carrying out of 20 the reaction corresponds essentially to that of Example 1 of this Patent Application. 40 pi. of sample are thereby pipetted through an opening on the upper edge directly on to the field a. The sample is not diluted. 25 By means of a suitable programme, in which high speeds of rotation alternate with stopping, sample and substrate are then transported in the direction of the separating matrix and cuvette. In the following, centrifuging stands for high speed of rotation, inter-30 mediate steps with lower speeds of rotation serve for the more sensitive control of the liquid transport. The substrate/wash solution is divided up by the dosage capillary (DK) into equally large portions. 1st centrifuging 35 Sample and sample buffer are centrifuged in VK1, the first portion is in the dosage chamber DK. -20- lst stopping Sample runs to field c and dissolves the receptors 1 and 3. The 1st portion of substrate solution passes into the overflow chamber UK 5 2nd centrifuging HCG and receptors 1 and 3 pass into the VK2, it is centrifuged in order to achieve homogeneous mixing of the reagents. The 1st portion of substrate solution is held back in UK, the 2nd 10 portion of substrate solution passes into the dosage chamber DK. 2nd stopping On the sample side, the liquid is transported to field d, there follows a stopping for 5 minutes 15 during which time the complex binds to the carrier. Non-complexed anti-HCG is also bound. At the end of the reaction, not yet complexed Fab conjugate is present in the solution. The 2nd portion of substrate solution passes into the UK. 20 3rd centrifuging The liquid coming from the sample canal is centrifuged in the reception chamber (AK), with it the excess of Fab conjugate. The 2nd substrate portion is retained in UK. The 3rd substrate 25 portion is present in dosage chamber DK. 3rd stopping The 3rd substrate portion is transported to UK. 4th centrifuging Portion 4 to DK 30 Portion 3 to VK2 4th stopping Portion 4 to UK Portion 3 to field d The 1st wash portion is present on reagent carrier 2. -21- 5th centrifuging Portion 5 to DK Portion 4 to VK2 Portion 3 to AK 5 5th stopping Portion 5 to UK Portion 4 to d 2nd wash portion on reagent carrier 2 6th centrifuging 10 Portion 6 to DK Portion 5 to VK2 Portion 4 to AK 6th stopping Portion 6 to UK 15 Portion 5 to d 3rd wash portion on reagent carrier 2. 7th centrifuging Portion 7 to DK Portion 6 to VK2 20 Portion 5 to AK 7th stopping Portion 7 to UK Portion 6 to d 4th wash portion on reagent carrier 2 25 8th centrifuging Portion 8 to DK Portion 7 to VK2 Portion 6 to AK 8th stopping 30 Portion 8 to UK Portion 7 to d Detection portion on reagent carrier 2. In 5 minutes reaction, the substrate is cleaved by the enzyme bound to reagent carrier 2, i.e. an -22- amount of enzyme which, due to the complex formation, is proportional to the amount of HCG introduced, and the colour to be measured is formed 5 9th centrifuging The liquid coming from the reagent carrier 2 completely fills the AK with a first aliquot and the remainder is passed into the cuvette. The measurement of the colour formed takes place in 10 the cuvette at 578 nm.

The transfer of the sample from field c to d takes place within a very short time (4 60 seconds). The final substrate volume leading to the detection also amounts to 40 pi. so that no sample silution takes 15 place up to the signal measurement.

With the use of calibration serum, there is obtained the calibration curve of Fig. 1 which includes the range of 0 to 100 mU HCG/ml. (standardised according to the 1st IRP standard for HCG) and makes possible 20 a sufficiently sensitive measurement of HCG in serum and plasma.

Example 3.

Determination of alpha-1-foetoprotein (AFP).

Use of a hapten-labelled 1st receptor, whereby all three 25 antibodies originate from the same animal species.

The carrying out and the buffers used correspond to Example 2.

As receptor 1 are used anti-AFP antibodies from sheep (100 ng./ml.) which are labelled with digoxin 30 (according to DE Patent Specification 25 37 129), as receptor 3 are employed anti-AFP antibodies from sheep labelled with 0-galactosidase according to the method mentioned in the case of HCG (Example 2) and as solid phase-coupled receptor 2 are used small paper discs of 35 polyester paper on to which antibodies against digoxin -23- from sheep are coupled adsorptively. The coating process corresponds to the process known for plastics test tubes. Fig. 2 of the drawings shows the calibration curve obtained. 5 Example 4.

Determination of thyreotropin (TSH).

Reagents: 1. Incubation buffer (IB): 15 mM sodium phosphate buffer, pH 7.4, 154 mM NaCl, 5 mM EDTA, 0.2% BSA, 10 pH 7.4. 2. Receptor 1: sheep anti-TSH antiserum: The crude serum is mixed ad 1.8M with NH^SO^. The precipitate is taken up in a buffer of 15 mM sodium phosphate, pH 7.0, and 50 mM NaCl and the solution so obtained is subjected 15 to a passage over DEAE-cellulose. The IgG-containing fraction is subsequently freed from the portions reacting with the alpha chain of TSH by passage over an { immune adsorber loaded with HCG. The eluate is applied to an immune adsorber loaded with TSH. After washing 20 of the adsorber, there takes place the elution of the antibodies immune-reactive against TSH with 1M propionic acid. The eluate is subsequently dialysed against IB, possibly concentrated by ultrafiltration. 3. Receptor 3: antibody-peroxidase conjugate: A 25 further sheep anti-TSH antiserum is purified like receptor 1 and subsequently (Fab)2 fragments obtained therefrom. The preparation of the (Fab^ fragments takes place according to E. Lamoye et al^., J. Immunol. Methods, 5JS, 235-243 (1983). The coupling. with horse-30 radish peroxidase takes place according to the method of Nakane (M.B. Wilson, P.K. Nakane, "Recent developments in the periodate method of conjugating horseradish peroxidase to antibodies", 1978, Elsevier, North Holland Biomedical Press, pp. 215-224, in "Immuno-35 fluorescence and relating staining techniques"). -24- 4. Receptor 2: Adsorber material with fixed donkey anti-sheep-FcY antibody: Donkey anti-sheep-FcY - antiserum is mixed ad 1.8 M with NH.SO,. The 4 4 precipitate is taken up in a buffer of 15 mM sodium 5 phosphate, pH 7.0, and 50 mM NaCl and the solution so obtained is subjected to a passage over DEAE-cellulose. The IgG-containing fraction (receptor 2) is coupled with the "affinity adsorbent, glutardialdehyde activated" of Boehringer Mannheim (order No. 665 525) 10 according to the manufacturer's working instructions. 5. Indicator reagent: 1.8 mM ABTS (2,2'-azino-di-3-ethylbenzthiazoline-sulphonate-(6), 3.3 mM sodium perborate in 100 mM phosphate-citrate buffer, pH 4.4).

Carrying out: 15 The carrying out corresponds to the process described in Example 1, whereby, in this case, 100 ng. of receptor 1 and 100 mU of receptor 3, dissolved in incubation buffer (40 pi.) are applied dropwise on to a cellulose fleece of the size 6x6 mm. After drying, 20 the storage of the fleece up to its use again takes place at 4°C.

The further carrying out corresponds to the manner of working described in Example 1. As sample, 40 pi. are used. 25 Fig. 3 shows the calibration curve so obtained.

Claims (22)

-25-Patent Claims

1. Process for the determination of a polyvalent antigen by incubation with three different receptors, the first and third of which are present dissolved in 5 liquid phase and are bindable with the antigen, the second receptor is present in solid phase and is only bindable with a part of the first receptor and the third receptor carries a labelling and does not cross-react with the first and second receptor, separation 10 of the solid from the liquid phase and measurement of the labelling in one of the phases, characterised in that one brings a solution containing the antigen together with a first solid carrier material, which contains, impregnated or lyophilised, the first and 15 the third receptor in removable form and, after removal of the receptors, immediately contacts the solution obtained with a second solid carrier material which contains the second receptor in non-removable form, thereafter separates the liquid from the second solid 20 carrier material and measures the labelling, whereby, as third receptor, one uses either a Fab antibody fragment which originates from the same animal species as the antibody component of the first receptor or uses a receptor which originates from the same animal species 25 as the second receptor, whereby the first receptor originates either from another animal species and/or is labelled with a hapten or antigen.

2. Process according to claim 1, characterised in that one uses an antibody as first receptor. 30

3. Process according to claim 1, characterised in that one uses an antibody labelled with a hapten or antigen as first receptor.

4. Process according to claim 1, characterised in that, as the first receptor, one uses an antibody 35 fragment which is labelled with a hapten or antigen. -26-

5. Process according to claim 2, characterised in that one uses a second receptor which is only bindable with the Fc part of the first receptor.

6. Process according to claim 3 or 4, characterised 5 in that one uses a second receptor which is only bindable with the hapten part or the antigen part of the first receptor.

7. Process according to claim 5, characterised in that one uses a second receptor which is directed 10 against the Fc part of immunoglobulin.

8. Process according to claim 1, characterised in that one uses a part of the first receptor as third receptor.

9. Process according to claim 3 or 4, characterised 15 in that one uses a third receptor which consists of a labelled complete antibody.

10. Process according to claim 3 or 4, characterised in ,that all three receptors contain antibodies or fragments thereof which originate from the same animal 20 species.

11. Process according to one of the preceding claims, characterised in that one uses a third receptor which is labelled with an enzyme, a fluorescing, chemi-luminescing or radioactive substance. 25

12. Process according to one of the preceding claims, characterised in that one uses an undiluted sample solution.

13. Dry reagent for the determination of a polyvalent antigen according to a process according to one of the 30 preceding claims, characterised by a) a first solid carrier material impregnated or lyophilised with two different soluble receptors 1 and 3, bindable with the antigen, of which receptor 3 carries a labelling, and -27- b)m a second solid carrier material, which is present physically separated from the first carrier material, which contains an insoluble recep-.or 2 only bindable with a part of the non-labelled soluble receptor 1, with the proviso that the labelled third receptor is a Fab antibody fragment.

14. Reagent according to claim 13, characterised in that the first receptor is an antibody and the third receptor is an enzyme-labelled Fab fragment of an antibody.

15. Reagent according to claim 14, characterised in that the receptors 1 and 3 are monoclonal and are directed against different and/or repetitive determinants of the antigen and originate from the same animal species.

16. Reagent according to claim 14, characterised in that the second receptor is an antibody against the Fc part of the first receptor and the third receptor originates from the same animal species as the second receptor, whereas the first receptor originates from another animal species.

17. Reagent according to claim 15, characterised in that all three receptors contain antibodies from the same animal species, the first receptor is a hapten-or antigen-labelled monoclonal antibody and the second receptor is directed against the hapten or antigen first receptor.

18. Reagent according to claim 14, characterised in that the first receptor is a complete antibody, the third receptor is from the same animal species as the first receptor and the second receptor is an antibody directed against the Fc part of the first receptor.

19. Reagent according to one of claims 13 to 18, characterised in that it contains a buffer and a system for the determination of the enzyme labelling. -28-

20. Reagent according to one of claims 13 to 19, characterised in that the third receptor is labelled with peroxidase or alpha- or 0-galactosidnse.

21. Process according to claim 1 for determining a 5 polyvalent antigen, substantially as hereinbefore described and exemplified.

22. Reagent according to claim 13 for determining a polyvalent antigen, substantially as hereinbefore described and exemplified,. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS. f