NO812397L - DETERMINATION OF ANTIGEN CONCENTRATION IN BODY LIQUIDS AND REAGENT USE BY DETERMINATION - Google Patents

Abstract

1. Process for binding, in an aqueous solution, an antigen which is specifically bound to a first antibody, and for separating this bound antigen from antigen which is not bound, for the determination of the antigen concentration in a body liquid with the aid of competitive radioimmunoassay, whereby the first antibody with the specifically bound antigen is separated from the free antigen which is dissolved in the aqueous solution, the separation being carried out by means of a second antibody, which is bound, on the one hand, to a Staphylococcus aureus cell, and, on the other hand, specifically binds the first antibody, and the process comprises adding a reaction accelerator to the aqueous solution, the reaction accelerator accelerating the specific binding of the first antibody by the second antibody, whereby 4-8 percent by weight polyethylene glycol, 5-10 percent by weight dextran, 5-10 percent by weight polyvinylpyrrolidone, 0,5-0,9 molar ammonium sulfate or 0,8-1,2 sodium sulfite are used as reaction accelerator.

Description

In competitive radioimmunoassay, a radioactively marked antigen is subjected to an unmarked antigen and an antigen-specific antibody for a competitive antigen-antibody reaction in an aqueous buffer solution. Thereby, a defined amount of radioactively marked antigen and unmarked antigen compete for

a defined amount of antibody, the antibodies being present in excess compared to the antigen. The part of the non-radioactively labeled antigen bound to the antibody is dependent

of the amount of unmarked antigen and decreases with increasing amount of unmarked antigen.

The principle of the radioimmunometric measuring method is based on

that the part of the radioactively labeled antigen bound to the antibody observed in a sample to be measured is compared with the result of adjustment samples with known antigen content and thereby the antigen content in the sample to be measured is determined.

For the measurement of the part of the radioactively labeled antigen bound to the antibody, it is necessary to separate the antigen-antibody complex from unbound antigen. The method of the competitive radioimmunoassay is discussed in detail in the literature (compare R.P. Ekins, Br.Med.Bull. 30.'3-11 (1974) and CM. Boyd, D.L. Herzberg; in: Practical radioimmuno-

assay pages 1-13 (A.J. Moss, G.V. Dalrymple, CM. Boyd, eds.) The CV. Mosby Company, Saint Louis 1976).

The accuracy of the competitive radioimmunoassay depends essentially on the separation of the radioactively labeled antigen bound to the antibody from free radioactively labeled anti-

gene after completion of the antigen-antibody reaction.

Methods and separation agents for separating antibody-bound and free antigen are known, e.g. 1. Precipitation of the antigen-antibody complex with protein-precipitation reagents compare B. Desbuquois, G.D. Aurbach J. Clin. Endocr. J33 732-738 (1971).

2. Adsorption of the free antigen on adsorbent

3. Binding of antibody (before the antigen-antibody reaction) to the surface of water-insoluble solids, compare DOS 2 322 533 4. Adsorption of the antigen-antibody complex on Staphylococcus aureus cells, compare S. Jonsson, G. Kronvall Eur. J. Immunol. 4, 29-33 (1974) 5. Precipitation of the antigen-antibody complex by means of another antibody (separating antibody), compare M.J. Martin, J. Landon Radioimmunoassay in Clinical Biochemistry pages 269-281 (Pasternak, editor) Heyden, London (1975). 6. Adsorption of the antigen-antibody complex on small water-insoluble particles (solids) coated with another antibody (separating antibody), compare F.C. Den Hollander, A.H.W.M Schuur's Radioimmunoassay Methods, pp. 419-422 (Kirkham and Hunter, eds.) Churchill Livingstone, London (1971); S. Jonsson, G. Kronvall IAEA Report SM 177/48, 287-298 (1974).

The separation of antibody-bound and free antigen' is carried out by means of a phase separation which mostly precedes a centrifugation step.

The above methods can be referred to as follows:

1. Precipitation of the antigen-antibody complex with protein precipitation reagents such as ethanol, ammonium sulfate or polyethylene glycol is easy to carry out, as separation by centrifugation is possible immediately after mixing with the precipitation reagent. However, this method assumes that the sample to be measured contains a sufficient amount of immunoglobulins. This is certainly the case in serum and plasma, but not in urine, liquor or gastric juice.

Furthermore, effective centrifugation is only possible with antigens

with a molecular weight that is significantly below the antibody's molecular weight. Consequently, this method is only applicable for special cases. Furthermore, it has the disadvantage that an often not insignificant part of the free antigen is co-precipitated. This co-precipitation fluctuates with the protein content and protein composition of the sample and can interfere with the measurement result. 2. Adsorption of the free antigen on adsorbents such as activated carbon, ion exchangers and silicates is only suitable for small antigens (eg steroids, peptides) and not generally applicable. Moreover, the reaction equilibrium can be disturbed by a longer reaction time with the adsorbent. 3. When using an antibody that is bound to the surface and water-insoluble solids (e.g. an inner wall of small test tubes, paper strips, polymer spheres of different sizes, glass beads, bacterial cells) the antigen-antibody complex formed during the incubation is also present

on solid bodies and is separated by the solid body from the free antigen contained in the reaction solution.

The each time the same amount of antibodies, with which such solid bodies are coated, is a prerequisite for the accuracy of the competitive radioimmunoassay and is only achievable with a great deal of work and care. In addition, in the meantime, unwanted disturbances occur during the adsorption of proteins from

the sample on the solid body and the associated blockage

of the antibody, which must be active in a defined quantity.

4. Due to the specific adsorption properties of staphylococcus aureus cells for some classes of immunoglobulins of different animal species - the antibodies used in radioimmunoassay belong to immunoglobulins - staphylococcus aureus cells can serve as adsorbent for antigen-antibody complexes. The method is only applicable when the antibodies belong to an immunoglobulin class of an animal species whose corresponding immunoglobulin is bound specifically by Staphylococcus aureus. Consequently, the method is not applicable to antibodies

of each desirable animal type.

Contains, in addition to the antibody used as a reagent

in addition also the sample to be measured (e.g. serum or plasma) larger amounts of Staphylococcus aureus-binding immunoglobulin, then a correspondingly larger

amount of Staphylococcus aureus cells. This it has

disadvantage is that in the case of non-specific adsorption of free antigen, separation is not complete. 5. Precipitation of the antigen-antibody complex by means of another antibody (separating antibody), which is directed against the antigen-specific first antibody, is time-consuming, because two antigen-antibody reactions must be carried out. The second antigen-antibody reaction can be shortened using a reaction accelerator. This method has the disadvantage that unwanted co-precipitation of free antigen occurs which is determined by the fluctuating protein content of the sample. Moreover, the precipitate is poorly visible and can be lost unnoticed when separating the above. 6. The adsorption of the antigen-antibody complex by means of another antibody (separating antibody) which is directed against the antigen-specific first antibody and is bound covalently to a water-insoluble solid (e.g. cellulose, staphylococcus aureus) has the disadvantage that a few hours are required for complete adsorption of antigen-antibody complexes during which, in addition to constant movement, the reaction vessel must produce a homogeneous suspension of the solid.

The invention's task is to provide a separation method and a separation reagent for the separation of free antigen from an antigen bound to a first antibody in the competitive radioimmunoassay, as the separation method and the separation reagent avoid the disadvantages of the above under items 1-6

said separation methods and separation agents.

The invention relates to a method for binding a

to a first antibody specifically bound antigen in an aqueous solution and to separate this bound antigen from unbound antigen for qualitative or quantitative determination of the antigen concentration in a body fluid by means of the competitive radioimmunoassay, the first antibody with the specifically bound antigen by with the help of another antibody which on the one hand is bound to a staphylococcus aureus cells and on the other hand binds the first antibody specifically, is separated from the free antigen dissolved in the aqueous solution, the method being characterized by the fact that to the aqueous solution a reaction accelerator is placed which accelerates the specific binding of the first antibody by means of the second antibody so that the binding reaction is completed within a few minutes.

The antigens used in the method according to the invention

are peptides, proteins or glycoproteins which correspond to their function may be hormones, enzymes, transport proteins or structural proteins and which upon immunization of different vertebrate species, preferably mammals, produce a formation of antigen-specific first antibodies.

Determination of antigens in body fluids such as blood plasma, blood serum, urine, liquor, gastric juice, duodenal juice, amniotic fluid or synovial fluid is carried out using the competitive radioimmunoassay, as the said body fluids can originate from humans or from an animal.

As a second antibody, such different classes of animals are used primarily from vertebrates, preferably from mammals, this second antibody being obtained by immunizing the animals with immunoglobulins of a foreign animal species from which the first antibody originates. Staphylococcus aureus cells bind gamma globulins from humans or from different animal species such as donkeys, shepherds, pigs, dogs, cats, guinea pigs or rabbits specifically over their Fc fragment, whereas the antigen-specific Fab fragment of gamma globulins remains unaffected by binding to the Staphylococcus aureus cells (compare J.W. Goding, J. Immunol.Me th. 20_, 241-253 (1978) .

Advantageous is the use of another antibody from larger animals such as goats or donkeys. The second antibody is allowed to bind with the serum of the immunized animal during incubation of Staphylococcus aureus cells, as the gamma globulin in particular binds specifically and the non-binding serum proteins and other serum components can be easily separated by washing the cells without again a significant amount of other antibody is desorbed.

For the determination of an antigen in body fluids containing large amounts of immunoglobulins, especially gamma globulins such as blood serum or blood plasma, it is advantageous to bind the other antibody in addition to the specific (adsorptive) binding mentioned covalently to the Staphylococcus aureus cells. Thereby, the second antibody is covalently bound after the specific adsorption to the staphylococcus aureus cells under mild conditions by means of a bifunctional reagent, the reactivity of the second antibody not significantly decreasing.

Due to the covalent bond, other antibodies cannot be released from the cell and cannot be displaced by gamma globulins from the sample.

By covalent bonding with a bifunctional reagent is connected

on the one hand amino groups of another antibody and on the other hand amino groups of the cell wall proteins of Staphylococcus aureus with each other. Dialdehydes, diimidates, diesters or diiso-

cyanates, e.g. glutardialdehyde, dimethyl adipindiimidate, suberic acid dimethyl ester or hexamethylene diisocyanate.

Before use in the method according to the invention, the Staphylococcus aureus cells are suitably killed by means of heat or formaldehyde treatment. Preferably, the cells are heated for 30 minutes at 80°C and incubated in a 1.5% formaldehyde solution, whereby the binding capacity of the cells for gamma-globulins does not significantly decrease.

Reaction accelerators according to the invention are substances that accelerate the specific binding reaction of a first antibody with the help of another antibody, as they do not significantly change the spatial polypeptide structure of immunoglobulins, especially gamma globulins, and thus do not destroy the antibodies' specific reactivity, e.g. hydrophilic polymers such as dextran or polyvinylpyrrolidone. Preferably substances are used which in higher concentrations preferably insolubilize immunoglobulins, especially gamma globulins or precipitate, e.g. hydrophilic polymers such as polyethylene glycol or salts such as ammonium sulfate or sodium sulfate. Hereby, a lower concentration of these substances has been used than would lead to precipitation of the antibody and the antigen.

The concentration of the reaction accelerator in the reaction mixture consisting of the first antibody and the antigen as well as the second antibody bound to staphylococcus aureus cells is chosen so that

the polyethylene glycol concentration generally amounts to 3 to

10% by weight, preferably 4 to 8% by weight, especially 5 to 6% by weight respectively, - the dextran concentration is generally 3 to 20% by weight, preferably 5 to 10% by weight, especially 7% by weight respectively,

the polyvinylpyrrolidone concentration is generally 3 to

15% by weight, preferably 5 to 10% by weight, especially 7% by weight respectively, the ammonium sulfate concentration is generally 0.3 to 1.2 molar, preferably 0.5 to 0.9 molar, especially 0.7 to 0.8 molar, respectively, - the sodium sulfite concentration is generally 0.5 to 1.5 molar, preferably 0.8 to 1.2 molar, especially 1.0 molar .

The molecular weight of the polymeric reaction accelerator in the case of polyethylene glycol is generally between 1,000 and 20,000, preferably between 2,000 and 10,000, especially at 6,000.

In the case of dextran gene.reit between 2,000 and 50,000, preferably between 4,000 and 20,0000, especially between 8,000 and 12,000.

In the case of polyvinylpyrrolidone generally between 2,000 and 25,000, preferably between 5,000 and 15,0000, especially at 10,000.

The reaction accelerator can already be added to the reaction mixture before the reaction of the first antibody with the antigen, before the second antibody bound to Staphylococcus aureus is added for separation. Preferably, however, a ready-to-use separation reagent is added to the reaction mixture consisting of antigen and first antibody.

The ready-to-use separation reagent contains in an aqueous buffer solution the second antibody bound to Staphylococcus aureus as well as the reaction accelerator, the concentration of which is chosen so that after adding the ready-to-use separation reagent to the reaction mixture consisting of antigen and first antibody, the reaction accelerator is present in excess

said concentration.

The amount of second antibody bound to Staphylococcus aureus (using 0.1 ml of body fluid to be determined) corresponds to the amount usually required for a precipitation of the antigen-antibody complex by means of an unbound second antibody.

The amount of staphylococcus is (using 0.1 ml of body fluid to be determined) usually 0.1 to 10 ml, preferably 1 to 5 mg, especially 2.5 mg.

To carry out the radioimmunometric determination, radioactively marked antigen, non-marked antigen and antigen-specific antibody are first incubated together. There-

by using as unmarked antigen either a solution of known antigen content for use as an adjustment sample or a body fluid of unknown antigen content as the sample to be determined.

Incubation is usually carried out at temperatures between

0 and 45°C, preferably between 17° and 27°C, and usually at room temperature.

The separation reagent is shaken before adding the reaction mixture so that a uniform cell suspension is obtained. The separation reagent's temperature is usually between 0° and +45°C, preferably adjusted to that temperature by the preceding incubation.

After mixing the separating agent with the reaction mixture

the binding of the first antibody by means of the second antibody is completed within a few minutes. During this time, the cells remain due to their small size is evenly distributed in the solution so that they are not lost to a bond by sedimentation. During subsequent centrifugation, the antigen bound to the first antibody is separated from those to the second

antibody charged Staphylococcus aureus cells as precipitation from the free antigen from the supernatant solution.

For the determination of the quantity of the marked antigen bound to the first antibody, the supernatant solution is decanted or aspirated, the precipitate being suitably then washed with a buffer solution. In the case of a larger volume of the liquid above, washing can be omitted.

The radioactivity is determined according to known procedures with radiation detectors.

A. The separation method according to the invention is suitable for the determination of antigens with low or. hqby molecular weight especially for peptides and proteins such as follicle-stimulating hormone, luteinizing hormone, mammary gland-stimulating hormone, chorionic gonadotropin, prolactin, placental lactogen, growth hormone, adrenocorticotropic hormone, gukagon, the g-chain of ;choriogonadotropin, parathormone, alpha-fetoprotein, carcino-embryonic, antigen , prostate-specific acid phosphates,

pregnancy-specific g^-glycoprotein as the absence of antigen-specific antibody does not bind to Staphylococcus aureus cells.

B. The separation method is generally applicable for separating free antigen from to a desirable first antibody bound antigen, the first antibody originating from a desirable first animal species and second antibody each time when immunizing another animal species with immunoglobin from the first animal species, the second animal species is chosen so that its gamma globulins are to a large extent specifically bound by

the staphylococcus aureus cells.

C. By the specific binding of the antigen-charged first antibody to the second antibody bound specifically to Staphylococcus aureus cells, the separation method provides a strong quantitative separation of the four antigen~of antigen bound to the first antibody, which is also not significantly disturbed by highly fluctuating protein - content of the sample to be determined. D. The reaction accelerator for binding of the antigen-charged first antibody by means of the Staphylococcus aureus-bound second antibody requires only a very short second incubation time, during which a constant movement of the reaction mixture for homogeneous distribution of the cells is superfluous so that after mixing with the separating agent, it can be centrifuged without a further laborious step and without major delay.

E. The cell precipitate after the centrifugation is clearly visible

and hangs tightly to the bottom of the centrifuge tube so that the separation of the above liquid by means of suction or decantation is problem-free and can easily be checked visually.

The invention will be explained with the help of some examples where all percentages refer to weight%.

Example 1

Determination of the human luteinizing hormone (hLH) in blood plasma or blood serum.

a) Preparation of separation reagent

16 ml of an antiserum from a goat containing antibodies against gamma-globulin from rabbits. The suspension was stirred for one hour. The antibody-charged cells are then separated from the above by centrifugation and decantation. The cells were resuspended in 45 ml of the above-mentioned buffer and for covalent binding of the antibody to the cells under constant stirring mixed with

■50 ml of a 0.2% glutardialdehyde solution in the above-mentioned buffer and after one hour stirring with 50 ml of a 5% sodium sulphite solution in the above-mentioned buffer to inactivate excess unreacted glutardialdehyde. After a further hour of stirring, the cells were separated by centrifugation and decantation from the supernatant. The cells were washed, being resuspended in 200 ml of 0.05 m aqueous sodium phosphate buffer of pH 7.4 and separated by centrifugation and decantation from the wash buffer.

The ready-to-use separation reagent was prepared, the thus treated cells being resuspended with the second antibody then in 500 ml of a 10% solution of polyethylene glycol with a molecular weight of 6000 in 0.05 m aqueous sodium phosphate buffer of pH 7.4 .

b) Implementation of the provision.

The analyzes were carried out in small centrifuge tubes of 3 to 5 ml

of polystyrene or polypropylene.

0.1 ml of the human serum or plasma sample to be tested was filled into a small tube. 0.1 ml of an adjustment sample with different hLH co-concentrations, e.g. 0, 4, 7, 14, 27, 54 and 100 m units of hLH/ml were each time held in a

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small pipe. 0.2 ml of a J-hLH containing solution (about 0.5 ng hLH/ml of a specific radioactivity of 100 nCi iodine-125/ng hLH) was kept in all small tubes. 0.2 ml of an antibody solution against hLH which, in the absence of unlabelled hLH, could bind 30

125

to .50% of J-hLH was held in all tubes. The incubation took place during 20 hours at room temperature.

0.5 ml of the ready-to-use separation reagent corresponding to example la) which was shaken before use to obtain a homogeneous cell suspension

pension was had in all pipes.

All tubes were shaken to mix the contents and after min

ten minutes was centrifuged approx. 2000 g ten minutes. The precipitate was separated by decantation or suction from the supernatant liquid and washed with 0.05 m aqueous sodium phosphate buffer solution of pH 7.4.

After separation of the supernatant liquid, gamma radiation of the marked hLH contained in the precipitate was measured in all tubes.

In a diagram, the impulse of the gamma radiation of the adjustment test per unit of time plotted linearly against the hLH concentration of the adjustment sample From this diagram, the hLH concentration of the unknown sample was then determined.

Example 2

Determination of human alpha-fetoprotein (AFP) in amniotic fluid,

a) Preparation of the separating agent

To 50 ml of a 10% suspension of staphylococcus aureus cells in a 0.9% aqueous sodium chloride was added 50 ml of an antiserum from a donkey containing antibodies against gamma-globulin from rabbits. The suspension was stirred for one hour. The cells were then separated by centrifugation and decantation from the supernatant liquid. The cells were washed, being resuspended with 200 ml of 0.9% aqueous sodium chloride solution and separated by centrifugation and decantation of the washing solution. The ready-to-use separation reagent was prepared,

the thus treated cells being then resuspended with another antibody in 500 ml of an aqueous buffer solution of pH 7.4 containing 1.6 m sodium sulphite and 0.05 m sodium phosphate.

b) Implementation of the provision.

The analyzes were carried out in small centrifuge tubes from 3 to 5 ml

of polystyrene or polypropylene.

0.1 ml of the amniotic fluid sample to be examined previously diluted 1:11 was held in a tube. 0.1 ml of an adjustment sample with different AFP concentrations, e.g. 0, 25, 50, 100, 200 and 400 units of AFP/ml were each time in a tube. 0.2 ml of a 12 5J-AFP-containing solution (approximately 1 unit AFP/ml of a specific radioactivity of 40 nCi iodine-125/units AFP) was placed in all tubes.

0.2 ml of an antibody solution against AFP which in the absence of

125

unlabeled AFP could bind 40 to 60% of J-AFP was present in all tubes. The incubation took place during 20 hours at room temperature.

0.5 ml of the ready-to-use separation reagent corresponding to example 2a) which was briefly shaken before use, to form a homogeneous cell suspension, was placed in all tubes.

All tubes were shaken briefly to mix the contents and 5 to 10 minutes later centrifuged at approx. 2000 g for 10 minutes.

The precipitate was separated by decantation or suction

from the supernatant liquid. After separation of the supernatant liquid, the gamma radiation of the marked AFP contained in the precipitate was measured in all tubes.

In a diagram, the impulse of the gamma radiation of the adjustment sample per time unit plotted linearly against the AFP concentration in the adjustment sample. From this diagram the AFP concentration in the unknown sample could then be determined.

Example 3

Determination of human alpha-fetoprotein (AFP) in amniotic fluid

a) Preparation of the separating agent.

The preparation of the separating agent, including the washing step, was carried out similarly to example la).

The ready-to-use separation reagent was prepared by resuspending the thus treated cells with another antibody in 500 ml of a 10% solution of polyvinylpyrrolidone with an average molecular weight of 10,000 in 0.05 m aqueous sodium phosphate buffer at pH 7.4.

b) Implementation of the provision.

The analyzes were carried out in small centrifuge tubes of 3 to 5 ml

of polystyrene or polypropylene.

0.1 ml of the amniotic fluid sample to be examined and previously diluted to 1:100 was placed in a tube. 0.1 ml of a calibration sample with different AFP concentrations, e.g. 0,

25, 50, 100, 200 and 400 units of AFP/ml were each time in a tube. 0.2 ml of a <125>J-AFP-containing solution (approx.

1 unit AFP/ml of a specific radioactivity of 40 nCi iodine-125/unit AFP/ was held in all tubes. 0.2 ml of an antibody solution against AFP which, in the absence of unlabelled AFP, could bind

125 40 to 60% of -J-AFP was present in all tubes. The incubation took place during 20 hours at room temperature.

1.0 ml of the ready-to-use separation reagent corresponding to example 3a) which was previously used and shaken to obtain a homogeneous cell suspension, was placed in all tubes.

All tubes were shaken briefly to mix the contents and centrifuged 10 to 30 minutes later at approx. 200 g for 10 minutes.

The precipitate was separated by decanting or aspirating the supernatant liquid.

After separation of the supernatant liquid, the gamma radiation of the marked AFP contained in the precipitate was measured in all tubes.

In a diagram, the impulse of -gamma's beam was listed in none of the adjustment test per time unit linearly against the AFP concentration in the adjustment sample. From this diagram, the AFP concentration in the unknown sample could then be determined.

Example 4

Determination of human thyroid-stimulating hormone (hTSH) in blood serum or blood plasma.

a) Preparation of the separating agent.

The production of the separating agent was carried out including the washing step corresponding to example la). The ready-to-use separation reagent was prepared by resuspending the thus treated cells with another antibody then in 500 ml of an aqueous buffer solution of pH 7.4 containing 1.3 m ammonium sulfate and 0.05 m sodium phosphate.

b) Implementation of the provision.

0.1 ml of the human serum or plasma sample that should

being examined was held in a tube. 0.1 ml of an adjustment sample with different hTSH concentrations, e.g. 0, 6, 12, 25,

50 and 100 y-units of hTSH/ml were each time held in a tube.

12 5

0.1 ml of a J-hTSH-containing solution (approx. 0.4 ng hTSH/

ml of a specific radioactivity of 80 nCi iodine-125/ng hTSH) was held in all tubes. 0.1 ml of an antibody solution against hTSH which, in the absence of unlabeled hTSH, could bind 30

125

to 50% of J-hTSH was retained in all tubes... The incubation took place during 20 hours at room temperature.

0.5 ml of the ready-to-use separation reagent corresponding to example 4a) which was briefly shaken before use to obtain a homogeneous cell suspension was placed in all tubes.

All tubes were briefly shaken to mix the contents

and 15 to 30 minutes later centrifuged at approx. 2000 g for 10 minutes.

The precipitate was separated by decanting or aspirating the supernatant and washed with a 0.05 molar aqueous sodium phosphate buffer solution of pH 7.4.

After separation of the supernatant liquid, the gamma radiation of the precipitate contained marked hTSH was measured.

in all pipes.

In a diagram, the impulse of the gamma radiation and the adjustment test per time unit linearly against the hTSH concentration in the adjustment sample. The hTSH concentration in the unknown sample could then be determined from this diagram.30.

Example 5

Determination of the human follicle-stimulating hormone

(hFSH) in blood serum or blood plasma

a) Preparation of the separating agent.

The preparation of the separating agent including a washing step

was carried out corresponding to example la).

The ready-to-use separation reagent was prepared by resuspending the thus treated cells with another antibody in 500 ml of a 15% solution of polyvinylpyrrolidone with an average molecular weight of 10,000 in 0.05 molar aqueous sodium phosphate buffer at pH 7.4.

b) Implementation of the provision.

The analyzes were carried out in small centrifuge tubes of 3 to 5 ml

polystyrene or polypropylene.

0.1 ml of the human serum or plasma sample to be tested was placed in a tube. 0.1 ml of an adjustment sample with different hFSH concentrations, e.g. 0, 5, 10, 20, 40 and 80 m units of hFSH/ml were each time held in a tube.

12 5

0.2 ml of a J-hFSH-containing solution (approximately 0.5 ng hFSH/ml of a specific radioactivity of 100 nCi iodine-125/ng hFSH) was kept in all tubes. 0.2 ml of an antibody solution against hFSH

125 which in the absence of unlabelled hFSH could bind 30 to 50% of J-hFSH was present in all tubes.

The incubation took place during 20 hours at room temperature.

0.5 ml of the ready-to-use separation reagent corresponding to example 5a) which was briefly shaken before use to obtain a homogeneous cell suspension was kept in all tubes.

Before mixing, all tubes were briefly shaken and 15 to 30 minutes later centrifuged at approx. 2000 g for 10 minutes.

The precipitate was separated by decantation or suction of the supernatant liquid and washed with 0.05 m aqueous sodium phosphate buffer solution of pH 7.4.

After the separation of the supernatant liquid, the gamma radiation of the marked hFSH contained in the precipitate was measured in all tubes. In a diagram, the impulses of the gamma radiation and the adjustment test per time unit linearly against the hFSH concentration of the adjustment samples. From this diagram, the hFSH concentration in the unknown sample could then be determined.

Claims (3)

1. Method for binding an antigen specifically bound to a first antibody in an aqueous solution and for separating this bound antigen from unbound antigen for determining the antigen concentration in a body fluid by means of a competitive radioimmunoassay, the first antibody being bound with the specifically bound antigen through a second antibody which on the one hand is bound to a Staphylococcus aureus cell and on the other hand specifically binds the first antibody, separates from the free antigen dissolved in the aqueous solution, characterized in that to the aqueous solution a reaction>accelerator is placed which accelerates the specific binding of the first antibody by means of the second antibody. 2. Method according to claim 1, characterized in that polyethylene glycol, dextran, polyvinylpyrrolidone, ammonium sulphate or sodium sulphite are used as reaction accelerators. 3. Reagent for separating free antigen from an antigen bound to a first antibody by determining an antigen by means of a competitive radioimmunoassay consisting of an aqueous buffer solution, a reaction accelerator and staphylococcus aureus cells, to which another antibody is specifically bound.