DD202073A5 - PROCESS FOR BINDING AND DISCONNECTING ANTIGEN - Google Patents

Abstract

Described is a method for binding an antibody specifically bound to an antibody in an aqueous solution and separating that bound antigen to determine the antigen concentration in a fluid, wherein the antibody having the specifically bound antigen is free from the antigen dissolved in the aqueous solution is separated, wherein the aqueous solution, a reaction accelerator is added. The reaction accelerators are polyethylene glycol, dextran, polyvinyl pyrrolidone, ammonium sulfate or sodium sulfite.

Description

«C« J i WU \ J % - · / -

Methods for Binding and Separation of Antigen Field of the Invention

The invention relates to a "method for binding and separating for the competitive radioimmunoassay and reagent for this purpose"

Characteristic of the known technical solutions

In competitive radioimmunoassay, a radiolabelled antigen is subjected to an unlabelled antigen and antigen specific to a competitive antigen-antibody reaction in an aqueous buffer solution. In this case, a defined amount of radioactively labeled antigen and unlabeled antigen compete for a defined amount of antibody _s . Antibody is deficient compared to the antigen. "The amount of radiolabeled antigen bound to the antibody is dependent on the amount of unlabelled antigen and decreases with increasing amount of unlabeled antigen.

The principle of the radioimmunometric measuring method based on the fact that the bound to the antibody portion of the radioactively labeled antigen, the wirdj observed in a sample to be measured is compared with the result of calibration samples with known antigen content and thereby the antigen content of the sample to be measured is determined _o I ¹ For the measurement of the portion of radiolabelled antigen bound to the antibody, it is necessary to separate the antigen-antibody complex from the unbound antigen. The method of competitive radioimmunoassay is described in detail in the literature (see R, P, Ekins , Dr, Med _e BuIl ₆ 22 * 3-11 (1974), as well as C. M _e Boyd, D _e L ₃ Herzberg _s in: Practical radio immunoassay pp. 1-13 (AJ Moss »G, Y. Dalrymple ₃ CM Boyd, eds. _E. The G. Y. ₀ Mosby Company, Saint Louis 1976),

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The accuracy of the competitive radioimmunoassay depends essentially on the separation of the radioactively labeled antigen bound to the antibody from the radiolabeled free antigen upon termination of the antigen-antibody reaction,

Methods and separation reagents for the separation of antibody-bound and free antigens are known, for example

1. Precipitation of the antigen-antibody complex with protein precipitation reagents, cf., B _e _Desbuquois _f Q ₉ D, Aurbach J. din. Endocr. 33, 732-738 (1971).

2. Adsorption of the free antigen on adsorbent

3. Binding of the antibody (before the antigen-antibody reaction) to surfaces of water-insoluble plague bodies, cf. DE-OS 2 322 533 "

Adsorption of the antigen-antibody complex on Staphylococcus aureus cells, see S, Jonsson, G, Kronvall Eur. J ₀ Immunol, 4, 29-33 (1974)

-5. Precipitation of the antigen-antibody complex by a second antibody (separating antibody), see, IL J. Martin, J ₉ Landon Radio Immunoassay in Clinical Biochemistry pp. 269-281 (Pasternak, editor) Heyden, London (1975)

6. Adsorption of Ant antigen-antibody complex on small, water-insoluble particles (plague bodies), which are coated with a second antibody (separating antibody), see, P. 0, Denander. A, H * W * M. Schnur radio immunoassay Methods S _e 419-422 (Kirkham and Hunter, eds ")

231

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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 performed by a phase separation, which usually precedes a centrifugation step _#

The above methods can be described as follows;

1. The precipitation of antigen-antibody complexes by protein precipitation reagents, such as ethanol, ammonium sulfate or polyethylene glycol, is easy to carry out, since separation by centrifugation is possible immediately after mixing with the precipitating reagent. However, this method assumes that the sample to be measured contains a sufficient amount of immunoglobulins. "This is true in serum or plasma, but not in urine, cerebrospinal fluid or gastric juice of the pail.

Effective separations are also only possible with antigens with a molecular weight that is substantially below the molecular weight of the antibodies. Accordingly, this method is only applicable for special cases, Furthermore, it has the disadvantage that an often not insignificant proportion of the free antigen is mitfällällt »This co-precipitation varies with the protein content and the protein composition of the sample and can interfere with the measurement result«

2. The adsorption of the free antigen on adsorbents such as activated carbon _? Ion exchangers and silicates are only suitable for small antigens (ζ _β Β »steroids, peptides) and.

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not generally applicable. In addition, the reaction equilibrium can be disturbed with a longer reaction time with the adsorbent.

When using an antibody bound to surfaces of water-insoluble solids (eg, test tube inner wall, paper strips, polymer beads of various sizes, glass beads, bacterial cells), the antigen-antibody complex formed during the incubation is also bound to the solid and is separated with the solid from the free antigen contained in the reaction solution.

Each same amount of antibody that are coated with such a solid state is a prerequisite for the accuracy of the competitive radioimmunoassay and is accessible only with great effort and care _β occasionally Additionally, undesirable interference on by adsorption of proteins from the sample to the solid and the associated blocking of the antibodies, which must be effective in a defined amount.

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

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Contains in addition to the used as the reagent antibody in addition, the sample to be measured (e.g., B * serum or plasma) larger amounts τοπ Staphylococcus aureus binding immunoglobulin _s so a correspondingly greater amount of Staphylococcus aureus ~ cells * are used must This has the laughing part that due to non-specific adsorption of friem antigen the separation is not complete.

5 · The precipitation of the antigen-antibody complex with a second antibody (separating antibodies) directed against the antigen-specific first antibody is zeitraubendj because zwei.Antigen-antibody reactions must be carried out "The second antigen-antibody reaction can be abbreviated by reaction accelerator. This method has the disadvantage that undesirable precipitations of foreign antigen occur, which are determined by the fluctuating protein content of the sample. In addition, the precipitate is poorly visible after the precipitation and can be lost unnoticed when the supernatant is separated off,

6 * The adsorption of the antigen-antibody complex by a second antibody (separation antibody) directed against the antigen-specific first antibody and covalently bound to a water-insoluble solid (eg B _s cellulose, Staphylococcus aureus) the Uaehteil that for complete adsorption of the antigen-antibody complex several hours are needed, 'where in addition by constant movement of the reaction vessel, a homogeneous suspension of the solid must be generated »

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Object of the invention

The aim of the invention is to provide a separation process. and a separation reagent for separating free antigen from an antigen bound to a first antibody in the competitive radioimmunoassay, the separation method and the separation reagent avoiding the disadvantages of the separation methods and separation reagents mentioned above under 1 to 6,

Explanation of the essence of the invention

The invention has for its object to find a suitable reaction accelerator for the binding of the antibody.

The invention therefore relates to a method for binding an antigen specifically bound to a first antibody in an aqueous solution and separating this bound antigen from unbound antigen for the 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 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 is separated from the free, dissolved in the aqueous solution antigen, characterized in that the aqueous solution Reaction accelerator is added, which accelerates the specific binding of the first antibody by the second antibody ^ so that the binding reaction is completed in a few minutes.

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The antigens used in the method according to the invention are peptides, proteins or glycoproteins which according to their function can be hormones, enzymes, transport proteins or structural proteins and which, in the immunization of various vertebrate species, preferably mammals, cause formation of antigen-specific first antibodies.

The determination of the antigen in body fluids such as blood plasma, blood serum, urine, cerebrospinal fluid, gastric juice, duodenal juice, amniotic fluid or synovial fluid is determined with the help of. made of competitive radioimmunoassays, said body fluids may be derived from humans or from an animal.

As second antibodies, such different animal classes ₉ are primarily used by vertebrates, preferably mammals, whereby this second antibody was obtained by immunizing the animals with immunoglobulins of a foreign species from which the first antibody originated. Staphylococcus aureus cells specifically bind human gamma globulins or various animal species such as donkey, goat, pig, dog, cat, guinea pig or rabbit over their Fc pragment, with the antigen-specific Fab fragment of the gamma globulins of the binding to the Staphylococcus aureus cell remains unaffected (see J. Y / Goding, J, Immunol, Meth. _, 20, 241-253 (1978)),

The use of a second antibody from larger animals such as goat or donkey is advantageous. The second antibody can be bound to the serum of the immunized animal by incubation of the Staphylococcus aureus cell

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In particular, the gamma-globulins are specifically bound and the non-binding serum proteins and other serum components can be readily separated by washing the cells without resorbing a substantial amount of the second antibody.

I ¹ for the determination of an antigen in body fluids which contain relatively large amounts of immunoglobulins, especially gamma-globulins, such as blood serum or blood plasma, it is advantageous for the second antibody in addition to the above specific (adsorptive) binding covalently to the Staphylococcus aureus cell to bind. For this purpose, the second antibody after specific adsorption to the Staphylococcus aureus cell is covalently bound under mild conditions by means of a bifunctional reagent, wherein the reactivity of the second antibody does not decrease significantly. Due to covalent binding, the second antibody can not detach from the cell and be displaced by gamma globulins of the sample,

By covalent bonding with a bifunctional reagent, on the one hand, amino groups of the second antibody and, on the other hand, amino groups of cell wall proteins of Staphylococcus aureus are bound together. As bifunktioneile reagents dialdehydes, diimidates, diesters or diisocyanates, z, B, glutaric dialdehyde, Dimethyladipindiimidat, suberic acid or hexamethylene diisocyanate are used.

The Staphylococcus aureus cells are advantageously killed before use in the method according to the invention by heat or Pormaldehydbehandlung. Advantageously, the cells are heated to 80 ^° C. for thirty minutes and incubated in a 1.5% strength formaldehyde solution, whereby the binding

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The ability of the cells for gamma globulins does not substantially decrease.

*** "" The expression enhancers according to the invention are substances which accelerate the specific binding reaction of a first antibody by a second antibody, while not substantially altering the spatial polypeptide structure of the immunoglobulins, in particular the gamma globulins, and thus do not destroy the specific reactivity of the antibodies, e.g. Hydrophilic polymers such as dextran or polyvinylpyrrolidone Advantageously, substances are used which, in higher concentrations, preferentially insolubilize or precipitate immunoglobulins, in particular gamma-globulins, e.g. As hydrophilic polymers such as polyethylene glycol, or salts such as ammonium sulfate and sodium sulfite. In this case, a lower concentration of these substances should be used than would lead to the fall of the first antibody and the antigen.

The concentration of the Seaktionsbeschleuniger in the reaction mixture consisting of the first antibody and the antigen and the second antibody bound to the Staphylococcus aureus cell is chosen so that

the polyethylene glycol concentration is generally from 3 to 10% by weight, preferably from 4 to 8% by weight, in particular from 5 to 6% by weight, or

the dextran concentrate ion is generally from three to twenty percent by weight, preferably from five to ten percent by weight, in particular seven percent by weight or

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the polyvinylpyrrolidone concentration is in general from 3 to 15% by weight, preferably from 5 to 10% by weight, in particular 7% by weight, or

- the ammonium sulfate concentration is generally 0.3 to 1.2 Molar, vorzugsweise.0,5 - O ₉ 9 molar, in particular from 0.7 to 0.8 molar is or

the sodium sulphite concentration is generally 0.5-1.5 molar, preferably 0.8-1.2 molar, in particular 1.0 molar.

The molecular weight of the polymeric reaction accelerators in the case of polyethylene glycol is generally between 1000 and 20,000, preferably between 2000 and 10,000, in particular 600 O,

in the case of dextran generally between 2,000 and 50,000, preferably between 4,000 and 20,000, in particular between 8,000 and 12,000,

in the case of polyvinyl pyrrolidones, generally between 2,000 and 25,000, preferably between 5,000 and 15,000, in particular 10,000.

The reaction accelerator can 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. However, a ready-to-use separation reagent is advantageously added to the reaction mixture consisting of antigen and first antibody.

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The ready-to-use separation reagent contains in an aqueous buffer solution bound to Staphylococcus aureus second antibody and the reaction accelerator, the concentration of which nach.der the addition of the ready, ready separating reagent to the reaction mixture consisting of antigen and the first antibody of the reaction accelerator in the above Concentrations.

The amount of second antibody bound to Staphylococcus aureus (using 0.1 ml of body fluid to be determined) is the amount usually required for precipitation of the antigen-antibody complex by an unbound second antibody. The amount of Staphylococcus is (when using 0.1 x ml to be determined body fluid) is generally 0 _s 5 to 10 mg, preferably 1 to 5 mg, in particular 2.5 mg.

To carry out the radioimmunometric determination, radioactively labeled antigen, unlabelled antigen and antigen-specific first antibody are first incubated together. As unmarked antigen, either a solution of known antigen content for use as a calibration sample or a body fluid of unknown antigen content is used as the sample to be determined ,

The incubation is generally carried out at temperatures between 0 and 45 ° ö, preferably between 17 ⁰ C and 27 ⁰ C and usually carried out at room temperature

The separation reagent is shaken before addition to the reaction mixture, so that a uniform cell suspension is obtained. The temperature of the separation reagent is generally

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between 0 ° and + 45 ⁰ C, preferably it is the temperature of the preceding incubation adapted "

After mixing the separation reagent with the reaction mixture, the binding of the first antibody by the second antibody is completed in a few minutes. During this time, because of their small size, the cells remain evenly distributed in the solution so that they are not lost by sedimentation for binding By subsequent centrifugation, this is the first antibody. bound antigen with the Staphylococcus aureus cell loaded with the second antibody precipitate from the free antigen in the supernatant solution.

To determine the amount of labeled antigen bound to the first antibody, the supernatant solution is decanted or aspirated, the precipitate being then conveniently washed with a buffer solution. "Washing may be dispensed with for a larger volume of the supernatant fluid.

The determination of the radioactivity is carried out by known methods by radiation detectors,

A ₀ The separation method according to the invention is in the determination of antigens with low or high molecular. especially suitable for peptides and proteins such as pollinator-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, chorionic gonadotropin, prolactin, placentalactogen * growth hormone, adrenocorticotropic hormone, guccagon, β-chorionic gonadotropin chain, parathyroid hormone alphafetoprotein, carcinoembryonic antigen, prostate gland specific acid phosphates,

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Pregnancy-specific ß ^ -glycoprotein, the absence of antigen-specific antibodies are not bound to Staphylococcus aureus cells,

B, The separation method is generally applicable to the separation of free antigen and antigen bound to any first antibody, wherein the first antibody is from any one animal species and the second antibody is each obtained by immunizing a second animal species with immunoglobulin of the first animal species, wherein the second animal species is selected so that its gamma globulins are mostly bound by the Staphylococcus aureus cell.

C. By the specific binding of the antigen-loaded first antibody to the second antibody specifically bound to the Staphylococcus aureus cell, the separation process yields a largely quantitative separation of the free antigen from the antigen bound to the first antibody, which is also due to greatly varying protein content loading. sounding sample is not significantly disturbed »

The reaction accelerator for the binding of the antigen-loaded first antibody by the Staphylococcus aureus-bound second antibody requires only a very short second incubation period, during which a constant movement of the reaction mixture for homogeneous distribution of the cells is superfluous, so that after mixing with the separation reagent without a further step and can be centrifuged without major delay »

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The cell pellet after centrifugation is clearly visible and adheres well to the bottom of the centrifuge tube, so that the separation of the supernatant liquid by suction or decantation is easy and visually easy to control.

embodiment

The invention is explained in more detail by the following examples. All details given there relate to percent by weight.

example 1

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

a) Preparation of the separation reagent

To 50 ml of a 10% suspension of Staphylococcus aureus cells in a 0.9 % sodium chloride 0.05m aqueous sodium phosphate buffer solution of pH 7.4 was added 16 ml of a goats antiserum containing antibodies to rabbit gamma globulin , given. The suspension was stirred for one hour. Subsequently, the antibody-loaded cells were separated from the supernatant by centrifugation and decantation. The cells were resuspended in 45 ml of the above buffer and for covalent binding of the antibody to the cells with constant stirring with 50 ml of a 0.2 followed glutardialdehyde solution in the above buffer and after stirring for 1 hour with 50 ml of a 5% sodium sulfite solution added the above buffer to remove the excess unreacted glutardial

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In order to inactivate the dehydrogenation, stirring the cells for an additional hour, the cells were separated from the supernatant by centrifugation and decantation. The cells were washed by resuspending in 200 ml of 0.05 M pH 7.4 aqueous sodium phosphate buffer and separating from the washing buffer by centrifuging and decanting.

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

b) Implementation of the provision

The analyzes were carried out in centrifuge tubes of 3 to 5 ml of polystyrene or polypropylene »

0.1 ml of the human serum or plasna sample to be tested was placed in a tube *

0.1 ml of a calibration sample with various hIH concentrations, eg 0, 4, 7, 14, 27, 54 * and 100 m units hLH / ml, were each placed in a tube.

"IPK

0.2 ml of a solution containing J-hLH (about 0.5 ng hLH / ml of a specific radioactivity of 100 nCi iodine-125 / ng hlH) was added to all tubes.

0.2 ml of an antibody solution against hlH, which in the absence of unlabelled hLH 30 to 50% of the ¹² . J-hLH was allowed to bind to all tubes. The incubation took place for twenty hours at room temperature »

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0.5 ml of the ready-to-use separation reagent according to Example 1a), which was shaken before use to obtain a homogeneous cell suspension, was added to all the tubes.

All tubes were shaken to mix contents and centrifuged for at least ten minutes at about 200 g for ten minutes.

The precipitate was separated by decanting or aspirating the supernatant liquid and washing with a 0.05M aqueous pH 7.4 sodium phosphate buffer solution.

Each time the supernatant was separated, the gamma radiation of the labeled hite contained in the precipitate was measured in all the tubes.

In a diagram, the pulses of the gamma radiation of the calibration samples per unit time were plotted linearly against the hlH concentration of the calibration samples. From this diagram, the hEH concentration of the unknown sample was then determined »

Example 2

Determination of Human Alphafetoprotein (APP) in Scarewater

a) Preparation of the separation reagent

To 50 ml of a 10% suspension of Staphylococcus aureus cells in a 0.9% aqueous sodium chloride were added

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The suspension was stirred for one hour, then the cells were separated from the supernatant by centrifugation and decantation, and the cells were washed by adding 200 ml of 0 ml of 50 ml of donkey anti-serum containing rabbit anti-Gainma globulin antibody resuspended 9% aqueous Hatriumchloridlösung and ^centrifugal: fugieren and decanting were separated from the washing solution. "

The ready-to-use separation reagent was prepared by resuspending the cells thus treated with the second antibody in 500 ml of pH 7.4 aqueous buffer solution containing 1.6 M sodium sulfate and 0.05 M sodium phosphate.

b) Implementation of the provision

The analyzes were carried out in centrifuge tubes of 3 to 5 ml of polystyrene or polypropylene.

0.1 ml of the foul water sample, previously diluted 1: 100, was placed in a tube «

0.1 ml of a calibration sample with different APP concentrations, e.g. B. 0, 25, 50, 100, 200 and 400 units of AFP / ml were each added to a tube.

1 hp

0.2 ml of a ^ J-AFT- containing solution (about I.U. unit APP / ml of a specific radioactivity of 40 nOi iodine-125 / unit APP) was added to all tubes »

0.2 ml of an antibody solution to APP capable of binding 40-60% of the ^2- J-APP in the absence of unlabelled APP,

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was added to all tubes. The incubation took place for twenty hours at room temperature,

0.5 ml of the ready-to-use separation reagent according to Example 2a) which was shaken briefly before use to obtain a homogeneous cell suspension was added to all the tubes.

All tubes were shaken briefly to mix the contents and centrifuged five to ten minutes later at about 2000 g for ten minutes *. ,

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

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

In a diagram, the pulses of the gamma radiation of the calibration samples per unit time were plotted linearly against the AFP concentration of the calibration samples. AFP concentration of the unknown sample can be determined «. ,

Example 3 ^:

Determination of Human Alpha-Feptide Protein (AFP) in Scarce Waters "

a) Preparation of the separation reagent

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The preparation of the separation reagent was carried out including the washing step according to Example 1a). The ready-to-use separation reagent was prepared by subsequently resuspending the cells thus treated with the second antibody in 500 ml of a 10% solution of polyvinyl pyrrolidone having a mean molecular weight of 10,000 in 0.05M aqueous sodium phosphate buffer of pH 7.4.

b) Carrying out the determination

The analyzes were carried out in centrifuge tubes of 3 to 5 ml of polystyrene or polypropylene,

0.1 ml of the foul water sample, previously diluted 1: 100, was placed in a tube.

0.1 ml of a calibration sample with different AFP concentrations, eg "B.O, 25, 50, 100, 200 and 400 units of AFP / ml, were each placed in a tube"

0.2 ml of a ^ J-AFP-containing solution (approximately 1 unit AFP / ml of a specific radioactivity of 40 nCi iodine i25 / unit AFP) was added to all the tubes,

0.2 ml of antibody solution against AFP capable of binding 40 % to ⁹ % of ^9- J-AFP in the absence of unlabelled AFP was added to all tubes. The incubation took place for twenty hours at room temperature,

1.0 ml of the ready-to-use separation reagent according to example 3a), which was briefly shaken before use to obtain a homogeneous cell suspension, was placed in all tubes.

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All tubes were shaken briefly for venning the contents and centrifuged for ten to thirty minutes later at about 2000 g for ten minutes. The IT deposit. was separated by decanting or 'purging the supernatant. Separating the supernatant liquid, the gamma radiation of the labeled AFP contained in the IT precipitate in all tubes was measured.

In a diagram were the pulses of gamma radiation ^; the calibration samples per unit time linearly plotted against the AFP concentration of the calibration samples. From this diagram, the AFP concentration of 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 separation reagent

The preparation of the separation reagent was carried out including the washing step according to Example 1a). The ready-to-use separation reagent was prepared by resuspending the cells thus treated with the second antibody, then in 500 ml of pH 7 »4 aqueous buffer solution containing 1.3m ammonium sulfate and 0.05m sodium phosphate,

b) Implementation of the provision

0.1 ml of the human serum or plasma sample to be tested was placed in a tube.

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0.1 ml of a sample with different hTSH concentrations, eg 0, 6, 12, "25, 50 and 100, u units hTSH / ml were in each case placed in a tube"

125 ml of a solution containing .J-IaTSH (about 0-4 ng hTSH / ml of a specific radioactivity of 80 nCi iodine-125 / ng hTSH) were added to all tubes.

0.1 ml of an antibody solution to hTSH capable of binding 30 to 50 % of ¹² ^ J-hTSH in the absence of unlabeled hTSH was added to all tubes »

Incubation took place at room temperature for twenty hours.

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

All tubes were shaken briefly to mix the contents and centrifuged fifteen to thirty minutes later at about 2000 g for ten hours.

The precipitate was separated by decanting or aspirating the supernatant liquid and washed with a 0 _s 05 m aqueous sodium phosphate buffer solution of pH 7.4,

After removal of the supernatant liquid, the gamma radiation of the labeled hTSH contained in the precipitate was measured in all tubes *

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In a diagram, the pulses of the gamma radiation of the calibration samples per unit time were plotted linearly against the hTSH concentration of the calibration samples. The hTSH concentration of the unknown sample could then be determined from this diagram.

Example 5

Determination of human follicle-stimulating hormone (hPSH) in blood serum or blood plasma

a) Preparation of the separation reagent

The preparation of the separation reagent was carried out including the washing step according to Example 1a).

The ready-to-use separation reagent was prepared by using the. cells treated with the second antibody were then resuspended in 500 ml of a 15% solution of polyvinylpyrrolidone having an average molecular weight of 10,000 in 0.05M pH 7j4 aqueous sodium phosphate buffer.

b) Implementation of the provision

The analyzes were carried out in 3 to 5 ml centrifuge tubes made of 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 a calibration sample with different hPSH concentrations, e.g. B. 0, 5, 10, 20, 40 and 80 m units of hFSH / ml were each added to a tube.

0.2 ml of a ¹² ^ -J-IiFSH-containing solution (about 0.5 ng

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hPSH / ml of a specific radioactivity of 100 nCi iodine-125 / ng KB ⁵ SH) were added to all tubes.

0.2 ml of an antibody solution against hPSH capable of binding 30 to 50% of the ^ J-KE ¹ SH in the absence of unlabeled KE ¹ SH was added to all tubes.

The incubation took place for twenty hours at room temperature.

0.5 ml of the ready-to-use Irenn reagent according to Example 5a) which was shaken briefly before use to obtain a homogeneous cell suspension was added to all the tubes.

All tubes were shaken briefly to mix the contents and fifteen to thirty minutes later at about 2000g. Centrifuged for ten minutes.

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

After separating the supernatant liquid, the gamma radiation of the labeled hFSH contained in the precipitate was measured in all the tubes.

In a diagram, the pulses of the gamma radiation of the calibration samples per unit of time were plotted linearly against the hFSH concentration of the calibration samples. The KFSH concentration of the unknown sample could then be determined from this diagram.

Claims (3)

- 24 - 59 349 11 28.4.8.2 Claim for invention A method for binding an antibody specifically bound to a first antibody in an aqueous solution and separating that bound unbound antigen antigen to determine the antigen concentration in a body fluid by competitive radioimmunoassay, wherein the first antibody is specifically bound to the specific antibody Antigen by a second antibody which is bound on the one hand to a Staphylococcus aureus cell and on the other hand, the first antibody specifically binds separated from the free, dissolved in the aqueous solution antigen, characterized in that the aqueous solution is added to a Reaktionsbescheluniger, the accelerates specific binding of the first antibody by the second antibody. 2. The method according to item 1, characterized in that is used as a reaction accelerator polyethylene glycol, dextran, polyvinylpyrrolidone, ammonium sulfate or sodium sulfite. ^j _K 3. A reagent for the separation of free antigen from an antigen bound to a first antibody in the determination of an antigen by means of the competitive radioimmunoassay, characterized in that it consists of an aqueous buffer solution, a reaction accelerator and Staphylococcus aureus cellsj to the second antibody is specifically bound