DK150690B - PROCEDURE AND ANALYSIS PACKAGE FOR DETERMINING A COMPONENT IN THE REACTION BETWEEN A SPECIFIC BINDER PROTEIN AND THE SIMILAR BINDING SUBSTANCE OF ENZYM IMMUNO ANALYSIS - Google Patents

Description

150690 ø

It is known that a component of the reaction between a specific binding protein and the corresponding binding substance can be determined by incubating one of the subject components provided with a marker in a reaction mixture containing at least the other component, and then initiates a separation between the labeled component, which is and is not bound to its binding partner, respectively, and finally determines the marker in at least one of the two resulting fractions. The distribution of the labeled component over the two fractions is a measure of the amount of the searched component present in the sample. Three systems can be described in which the above method can be used, a) antibodies as specific binding proteins and the corresponding antigens as binding agents. Substances that can be determined 150690. 2 in this way are, inter alia, protein hormones and their antibodies or virus antigens and their antibodies, b) antibodies as specific binding proteins and haptenes as binding substance. Here, the haptenes are defined as protein-free substances that can react with antibodies without being able to induce them. Substances that can be determined in such a system include: steroid hormones and vitamins; c) proteins that act in the body as receptor or transport molecules as specific binding proteins and substances bound by them as binding substances. This system is e.g. suitable for the determination of steroid hormones but also of thyroxine and triiodothyronine, vitamin B12, the intrinsicole factor and the adrenocorticotropic hormone.

The main points of the described assay methods are the use of a marker and the separation of the labeled component into a bound fraction and a fraction which is not bound to the corresponding component.

From the specification of Danish Patent Application No. 5462/71 a method is known in which enzyme-labeled component is used in connection with a quantitative immunological assay. In this known method, the separation between liquid and solid phase occurs by gel filtration, but this method is impractical and cannot provide any reliable and easy determination. In the method, immunocomponents can only be determined at a concentration of 50-100 nanograms per minute. milliliter.

25

Further, from the specification to Danish Patent Application No. 2812/67, a method is used which utilizes insoluble antibodies to proteins or peptides which are antigens in a radioimmunoassay. Radiative atoms used as markers are e.g. 131j, 125 ^, 14 ^, 3 ^, 5? Co This process is characterized by a high sensitivity. However, this approach is limited to institutes where the necessary special equipment is available.

The separation methods can be divided as follows: a) Methods dependent on the difference in physical properties between the unbound, labeled component and its complex with the binding partner, such as gel filtration, electrophoresis, salt precipitation, and dextran-coated charcoal absorption.

40 b. The so-called solid phase method, in which one component is already pre-insolubilized by cross-linking or by covalent bonding or physical adsorption to a solid support, c) the so-called double antibody method according to which complex, antigen (or hapten) "antibody is precipitated by antibodies to the antibody in the complex, which method 5 is known only from systems in which antibodies in solute form are considered"

While the processes referred to in (a) and (c) are relatively complicated, those mentioned in (b) suffer from the disadvantage that a reaction component, when placed in insoluble form, will usually diminish its affinity for the reaction partner. However, a high affinity is important for providing a sensitive test system.

According to the invention, a method has been found for determining a component of the reaction between a specific binder protein and the corresponding binding substance using the known binding affinity of such components, using a known amount of a coupling product of the binding substance with an enzyme and optionally also a known amount of the specific binding protein (in the case that the binding substance is the component to be determined), which is characterized by further use. insolubilized antibodies or for the specific binder protein, and that after the reaction, enzyme activity is determined in the liquid or solid phase of the reaction mixture. This method allows the determination of components present at a concentration of 1-1C nanograms per minute. milliliter.

In the present description, the terms conjugate and enzyme-conjugate are used synonymously for the coupling product of the binding substance and the enzyme.

The binding substance can be detected and determined by comparing the unknown sample or dilution series of the same with a known amount of a conjugate of the substance to be determined and an enzyme and with an amount of specific binding protein depending on the amount enzyme conjugate which is added. Then an amount, preferably an excess of the antibodies rendered insoluble against the specific binder protein, is added so that the whole enzyme conjugate which has reacted with the binder protein is coupled to these insolubles with these insolubles. antibodies. The more binding substance ex 4 150690 present in the sample, the less enzyme conjugate will react with the specific binder protein and eventually enter the insoluble phase. The result is that more unbound enzyme conjugate remains in the liquid phase and can simply be determined there. The specific binder protein can be determined by incubating the sample or dilution series thereof with a known amount of enzyme conjugate and with an amount of the insolubilized antibodies to the specific binder protein. The enzyme activity can only take place in the insoluble phase if the conjugate has reacted with the specific binder protein, i.e. the more specifically the binding protein present in the sample, the less bound enzyme conjugate will remain in the liquid phase.

The sensitivity of the assay method described can be varied by changing the amounts of the reagents. However, the amount of enzyme conjugate that can be used is limited below the requirement that its enzyme activity can be reasonably measured, so the sensitivity of the assay method has its limitation. The minimally measurable enzyme activity is a. Depending on the nature of the enzyme used in the coupling and on the nature of the substrate and finally on the incubation time of the enzyme reaction. Still, the affinity of the specific binding protein strongly influences the sensitivity of the assay. A high sensitivity assay method requires high binding affinity specific binding proteins.

The quantities of reagents needed for a determination must be determined empirically.

For the determination of the binding substance, the amount of the enzyme conjugate must be determined by the enzyme activity. Then, this amount 30 is incubated with a dilution series of the specific binder protein to determine the required amount of this protein. Preferably, an amount of specific binding protein is selected which can bind 50-90% of the enzyme conjugate. Finally, one examines whether the desired sensitivity has been achieved, examining a dilution series of the substance to be tested by the method.

For the determination of the specific binding protein, the activity of the enzyme conjugate must be taken into account for its activity, which must be determined with reasonable accuracy.

s 150690

The antibodies to the specific binding protein that are made insoluble are preferably added in excess by the two assay methods. These dosages are determined by prior experiments.

The advantages of this method to the prior art are that the combination of the "Double Antibody" and the "Solid Phase" method, hereinafter referred to as the DASP method for convenience, offers several advantages over the known methods. Thus, the embodiment of the new method, i.e. delivery of insolubilized antibodies to the specific binder protein, incubation, centrifugation and measurement, very simple, the dosage is often easier than by the solid-phase methods because it is sufficient to add an excess of undissolved material, whereas the solid-phase methods must use an accurately measured amount of the solid material. Moreover, the affinity of the binding protein for the binding substance is not impaired by the binding to the carrier material, as may be the case with the solid phase method. A further advantage over the latter method is the rapid equilibrium adjustment of the reaction between the specific binder protein and the binding substance 20 (both in solution). A further advantage is that in the DASP method, the insolubilized antibody, hereinafter referred to as the immunoadsorbent, can be used in any system in which antibodies are used as specific binding proteins, provided these antibodies are prepared in the same animal species. On the other hand, for any antigen or hapten to be determined by the solid phase method, the antibodies must be rendered insoluble. The dual antibody method is very sensitive to relatively small changes in salt concentrations, pH and the like, which makes extensive control of the conditions necessary. Further, the method requires the addition of 30 "carrier" γ-globulin and, consequently, much other antibody to obtain an immune precipitate. In addition to being a simpler method, the DASP method, which does not require "carrier" γ-globulin, leads to the saving of materials. Finally, it should be mentioned that a double antibody-like separation used for transport or receptor proteins 35 is not possible as a "carrier" protein cannot be obtained for this purpose. The method according to the invention therefore offers a unique opportunity in this regard.

The process according to the invention can easily be done automatically. In principle, it is possible to bring the reaction components together immediately _ t t _. i _ j. _ -? However, preferably the insoluble antibodies to the specific binder protein for the reaction mixture as the last reagent have been found, since it has been found that the assay thereby obtains a higher sensitivity.

5

For the same reason, when using the method for determining the binding substance, it is convenient to proceed by first adding to the sample the specific binding protein and then the enzyme conjugate and finally the insoluble antibodies to the specific binding protein.

The reagent required for the invention, i.e. the coupling product of antigen, hapten, or binding agent with the enzyme may be prepared in known manner. These methods can also be used to bind a hapten or a low molecular weight binding substance to an enzyme, provided that one of the substances has one or more amino groups and the other one or more carboxyl groups. If the latter is not the case, it is possible to introduce the desired group into the molecule to be coupled by a known organochemical process. Methods are also known for linking amino and carboxyl groups, optionally by introducing a bridge. Finally, components such as glutaraldehyde, difluorodinodrodiphenylsulfone and di- and tri-chloro-s-triazine are often used for the present coupling. It may be necessary to separate the enzyme conjugates prepared from unchanged substances and from substances which have become inactive. For this purpose, known methods such as precipitation with organic solvents, gel filtration and centrifugation by a density gradient can be used.

The choice of the enzyme used in the coupling product is determined by a number of properties of the enzyme. Of course, it is important that the enzyme is resistant to the coupling with another molecule, i.e. to change one or more amino acid side chains. The specific activity of the enzyme is also of great importance. As a smaller amount of enzyme conjugate needs to be added to obtain a measurable enzyme effect, the study system becomes more sensitive. Still, the enzymes with which a determination of the activity can be made in a simple manner should be preferred, first of all, the enzymes for which the activity can be called colorime-40 should be taken into account. tric, eprophotometric or fluorimetric. Such provisions 7 150690

One can determine the activity of the colorimetric enzymes which catalyze a reaction at which. a colored substance arises or disappears, either by the primary or the secondary reaction.

As enzymes which "may be used as the enzymatically active component of conjugates, mention is made of catalase, peroxidase (3-glucoronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase, galactose oxidase and alkaline phosphatase.

At the end of the reaction between the components and the reagents, according to the invention, the enzyme activity in the liquid or solid phase is determined, or in both phases of the reaction mixture. The simplest, however, is to determine the enzyme activity in the liquid phase, 15

The antibodies rendered insoluble against the specific binding proteins, which are also important reagents for the method of the invention, may also be prepared in known manner. The antibodies can be prepared by taking a purified preparation of the specific binder protein or of those proteins which have at least partially the same antigenic properties as the specific binder protein, and then inject this into known way in a different animal species than the one from which one got it. The serum of the treated animal or the gamma globulin fraction thereof can be rendered insoluble by cross-linking with such substances as glutaraldehyde and chloroformic acid ethyl ester or by binding to solid carrier particles either physically or adsorptively or chemically by forming covalent bonds. As solid carriers such substances as cellulose (altered or not), agarose, cross-linked compound dextran, polystyrene and the like can be used. A covalent binding to these substances of 30 antibodies can be made by such substances as carbodiimides, di- and tri-chloro-s-triazines, glutaraldehyde, cyanogen bromide and e.g. by diazotizing.

The advantages of the method of the invention for the determination of binder protein are obtained if an excess of insoluble antibody is used, so that the specific binder protein goes completely into the solid phase.

The forms in which the reagents may be used are manifold. The 40 enzyme-conjugated component of the reaction system can be freeze-dried or dissolved in a buffer. You can also use a solid carrier, e.g. a strip of paper impregnated with the conjugate. This is also used for the necessary binder proteins.

The insoluble component may be in the form of particles of various sizes, such as granules, flakes, sticks or in the form of a strip of some carrier material.

The invention further relates to an assay package for determining * a specific binder protein or the corresponding binding substance by the disclosed method, characterized in that it mainly contains: a) a known amount of conjugate of the binding substance and an enzyme, b (c) one known amount of insoluble antibodies directed against the binding protein to be added or to be determined; (d) a substrate for determining the activity of the binding agent; enzyme used.

in

The assay package may still contain the necessary additional means for carrying out the assay, such as test tubes, pipettes and bottles of solutions.

The assay package can be used very frequently and with particular advantage for the detection and determination of an antigen or a hapten, and for this purpose it essentially contains: a) a known amount of a conjugate of the antigen or hapten and an enzyme, b) a corresponding amount of similar antibodies; c) a known amount of insolubilized antibodies directed against the antibodies used; d) a substrate for determining the activity of the enzyme used.

When using such assay packages for the detection and determination of antibodies, one does not need the antibodies mentioned in b).

150690 9

An important embodiment of an assay package according to the invention is one assay package for the determination of the gonadotropic hormones and in particular for the determination of HCG (Human Chorionic Gonadotropin) as a means of determining pregnancy 5 at a very early stage, which assay package is peculiar in that it consists of a container containing as essential constituents of lyophilized layers: a) a certain amount of an HCG conjugate and an enzyme, e.g. HCG peroxidase b) a certain amount of anti-HCG, c) a certain amount of insolubilized antibodies for anti-HCG together with a substrate to determine the enzyme activity of the residual HCG enzyme in the liquid phase.

By adding a certain amount of urine from a putative pregnant woman to this assay mixture and by incubating the urine with these ingredients, a mixture of insoluble matter is formed, the remainder containing the residual soluble HCG enzyme conjugate. The amount of the last depends on the amount of HCG in the urine examined. In determining the enzyme activity of this residual HCG enzyme conjugate, it can be determined whether or not the urine is due to a pregnant woman.

One method which is preferably used in the determination of enzyme activity consists in bringing an indicator paper impregnated with enzyme reagents, for example, if a peroxidase is used, an H₂O ^, with a color reagent such as o-tolidine.

By properly selecting the quantities of each of the reagents, it becomes possible to detect pregnancy already at a very early stage and in a simple, fast and very reliable way that can be performed by a non-trained person.

150690 10

Example 1.

Determination of the human chloride ion gonadotropin (HCG).

5 a) Preparation of HCG-HRP.

5 mg of HCG and 20 mg of horseradish peroxidase (HRP) are dissolved in 2 ml of 0.05 M phosphate buffer with the pH 6.2. After adding cow µ ko 25 $ glutaraldehyde solution, the mixture was shaken for 2 hours at room temperature. After 5 minutes centrifugation at 250 g, the liquid was fractionated over Sephadex G-200 in 0.05 M phosphate = buffer with pH 6.2. The fractions in which the highest enzyme activity was bound by antibodies to HCG were used during the assay.

B) Preparation of antibodies to HCG.

Antibodies to HCG were indicated in rabbits as indicated by Schuurs et al. Acta Endocr. (Kbh) 59j 120, (1968).

c) Preparation of antibodies to rabbit γ-globulin.

2q Rabbit Y-globulin was isolated from normal rabbit serum by precipitation with 1.8 $ w / v solid sodium sulfate. Antibodies to this were prepared by immunizing a sheep in accordance with the following 25 day schedule amount of Freud's adjuvant injection mode 0 o, 5 mg + intramuscular 1 5 · 5 mg + intramuscular 28 1 mg + intramuscular b2 1 mg - intravenous 3Q 56 1 mg - Intravenous on the 70th day, the sheep were released.

d) Preparation of immunoadsorbent cellulose / sheep anti (rabbit γ-globu = 35 lin) 7.

The γ-globulin fraction of the sheep serum described under c) was prepared by precipitation with 165¾ w / v solid sodium sulfate. After washing, the precipitate was taken up in so much 0.05 M borate buffer with pH 8.6 that the resulting protein concentration was 10 mg / ml.

4Q 350 mg of m-aminobenzyloxymethyl cellulose was slurried in 50 ml of distilled 11 15069¾ water and diazotized by addition of 10 ml of 30 $ hydrochloric acid and dropwise addition of 10 ml of 10 $ NaDiK 2 solution at 0 ° C. The slurry is centrifuged. It was washed and the precipitate was resuspended in * + 3 ml of 0.05 M sodium borate with pH 8.6, then 5 7 ml of the γ-globin solution prepared was added. The mixture was stirred at 26 ° C for 26 hours, then centrifuged and washed with 0.02 M phosphate buffer with pH 6.0.

e) Determination of HCG

A dilution series (32 -16-8 - 2-1-o, 5-IU / ml) of HCG was prepared in 0.02 M phosphate buffer with pH 6.0 containing 2 $ v / v v usually sheep serum.

0.5 ml of each of the HCG-containing samples was incubated with 0.1 ml of rabbit (anti-HCG) serum and 0.1 ml of HCG-KRP conjugate, both in appropriate dilution for half an hour. at room temperature. Then 0.3 ml of the immunoadsorbent (10 mg / ml) prepared according to d) was added and the resulting mixture was stirred for one hour at room temperature. After centrifugation, enzyme activity was determined: the residue by mixing 0.5 ml.

of this liquid with 1.5 ml of substrate (10µ1 30 $ ^. $ 2 ° & 20 mg 5-amino-20 salicylic acid in 150 ml 0.02 M phosphate buffer with pH.6.0) and after 30 minutes at At 25 ° C the extinction was measured at 1 + 60 mm.

In this way, it was demonstrated that it was possible to detect an HCG concentration of 0.5 to 10U / ml in the sample. This method could also examine urine samples. The study is therefore suitable for 25 a pregnancy test. The relationship with an existing study method, a hemaglutination inhibition test, was good. It was shown to increase the sensitivity of the system using a pre-incubation. Here, the sample is first incubated with the antiserum and then the HCG-HRP conjugate added.

30

Example II.

Determination of insulin and anti-insulin.

a) Preparation of insulin (glucose oxidase).

35 5 mg of pig insulin of 25 mg glucose oxidase was dissolved in 2 ml of 0.05 M phosphate buffer with pH 6.5. To this is added 5 μΐ of glutaraldehyde solution and the mixture is shaken for 90 minutes at room temperature. The mixture is fractionated over Sephadex 150690 12 G-200 in 0.05 M phosphate buffer with pH 6.5. The fractions of which the highest percent enzyme activity could be inhibited by antibodies to insulin were used in the assay 5 b) Preparation of antibodies to insulin.

10 guinea pigs &n; - weekly intramuscular injection with 1 mg of pig insulin in complete Freud's adjuvant over a period of +/- 8 weeks. After 10 weeks of rest, the animals were given an additional 1 mg of insulin by intravenous injection without adjuvant. Two weeks later, the 10 animals were released. Adjacent hypoglycemia was counteracted by intraperional glucose administration.

c) Preparation of antibodies against. maxsvin-γ-globulin.

There. guinea pig Y-globulin was prepared by adding 1 15 volume of saturated ammonium sulfate solution to 2 volume of guinea pig serum. The resulting precipitate was washed twice with 33 ° S saturated ammonium sulfate solution and then taken up in a physiological saline solution. Then, a sheep was immunized with increasing doses of the γ-globulin produced: 0.5-1 and 2 mg. The injections were given every two weeks, with the immunogen mixed completely

Freud's adjuvant. Two weeks after the last injection, an additional 2 mg of γ-globulin was added to a physiological saline solution, and a week later the animal was released yearly.

D) Preparation of insoluble antibodies to guinea pig Y-globulin.

10 g of microcrystalline cellulose was activated by adding, with stirring, to H00 ml of 2.5 $ w / v CWBr solution, after which the pH was increased to 10.5 ml of 1 N NaOH solution, keeping this value upright for 2 hours. minutes. Then the cellulose was washed with ice water and with 0.1 M NaHCO 3 1.6 g Na 2 SO 2 was added to 10 ml sheep anti (guinea pig γ-globulin) serum. After one hour of stirring at room temperature, the precipitate is centrifuged. Wash twice with 20 ml of 16 $ w / v Na 2 O 3 solution, then take up in 10 ml 0.1 M NaHCO 3 solution. The activated cellulose was mixed with * + 0 ml of 0.1 M NaKCO ^ solution and the 10 ml of γ-globulin solution. This suspension was stirred for 20 hours at 0 ° and was washed twice with 150 ml of 0.5 M NaHCO 2 twice with 500 ml of 0.05 M citrate of pH 1.1 and twice. with 500 ml of 0.05 M phosphate of pH 6? 5 5 e) Determination of antibodies to insulin.

0.1 ml of insulin (glucose oxidase) was incubated in appropriate dilution with 0.1 ml of a dilution series of a guinea pig anti-insulin serum for hours. Dilution s s are in a 0.05 M phosphate buffer with pH 6.0. Then, 0.3 ml of immunoadsorbent 10 (15 mg / ml) and 0.2 ml of buffer were added and the mixture was stirred overnight at * ° C. After centrifugation, the enzyme activity of the residual solution was determined by incubating 0.5 ml of it with 2.5 ml of substrate for 30 minutes, then measuring the extinction at * + 60 nm. The substrate contained 50 mg of glucose, 10 μg of peroxidase and 1 mg of 5-amino salicylic acid per ml. 2.5 ml of 0.05 M phosphate buffer with pH 6.0.

With this system, the antibody content of the different types of serum could be compared. As the reference point, the serum dilution was selected to which 5o5 of the total combinable enzyme activity is bound.

20 f) Determination of insulin.

0.2 ml of a dilution series of insulin was incubated for 2 hours with 0.1+ ml of anti-insulin serum in such a dilution that it could bind 60 $ of the added enzyme conjugate. Then 0.1 ml of insulin (glucose oxidase) was added for hours. Finally, 0.3 ml of immunoadsorbent (15 mg / ml) was added. The mixture was stirred overnight at H ° C. After centrifugation, enzyme activity in resto was measured. the solution as given under e). The sensitivity of the assay, which depends on the antiserum used, is within the order of nanograms: 20-100 mg / ml, i.e. * 0.5-2.5 mU / ml.

Example III.

Determination of oestradiol.

A) Preparation of oestradiol-17-succinyl-HRP.

50 mg of oestradiol-17-hemisuceinate and 0.08 ml of tri-n-butylamine were dissolved in 2.5 ml of dioxane. To the cold (2 ° C) solution was added 15 L of isobutyl chlorocarbonate. After 30 minutes, this solution was mixed

lif T5069Q

with 100 mg of horseradish peroxidase (HRP) in 7.5 ml of a dioxane / water mixture (2: 3) 5 adjusted to a pH of 9j5 with sodium hydroxide. The solution was stirred for * + hours at 2 ° C and then dialyzed for 18 hours. The precipitate formed after the pH of the dialysate was adjusted to * +, 6, was centrifuged, washed and taken up in 5 ml of distilled water which was adjusted to a pH of 8. The substance was further purified twice. precipitation with 10 ml of acetone. The finished product was taken up in 10 ml of 0.05 M pyospliat buffer with pH 738.

B) Preparation of estradiol-17-suecinyl-BSA.

The preparation was carried out according to the mixed anhydride method set forth in Example IIa. This preparation was made using 100 mg estradiol-17 hemisuccinate and 150 mg bovine bovine serum albumin (BSA).

c) Preparation of antibodies to estradiol.

One sheep was injected once for * + weeks with k mg estradiol-17-succinyl-BSA in complete Freund's adjuvant. With regular between 20 rooms, blood is drawn from the animal. The serum was absorbed with BSA, which was rendered insoluble.

d) Preparation of sheep Y-globulin antibodies.

Sheep Y-globulin was prepared as set forth in Example I, but now 25 with 16 $ w / v sodium sulfate. Rabbits were then immunized with this sheep-Y globulin according to the following scheme:

Day amount of Freund's adjuvant injection mode 0 200 µg + intramuscular 30 lb * + 00 µg + '"28 800 µg +" b2 800 µg - intravenous o c 2 Weeks after the last injection, the animal was released yearly.

e) Preparation of the immunoadsorbent / rabbit anti (sheep y-globulin) β-cellulose.

The γ-Globulin fraction from the antisera given in d) was prepared by precipitation with 18 $ w / v Wa 2 SO 2. The resulting product 5 was coupled with cellulose according to Gurvich's method, set forth in Example I.

f) Determination of estradiol.

The immune reaction was performed in 0.02 M phosphate buffer with pH 6.0 containing 2 $ BSA.

10 ml of the sample was mixed with 0.1 ml of the sheep anti-estradiol serum in the desired dilution. After 30 minutes of incubation at room temperature, 0.1 ml of estradiol-17-succinyl-HRP was added at an appropriate dilution, followed by another 30 minutes of incubation at room temperature. Then, qf3 ml of immunoadsorbent "su" suspension (30 mg / ml) was added and the mixture was stirred for 2 hours at room temperature. Then the liquid and solid phase were separated by centrifugation. The enzyme activity of the liquid component was measured as in Example I. could use the sheep anti-estradiol-20 serum in dilutions from 1: 1600 to 1: 12800 depending on the quality of estradiol-17-succinyl HRP used. At a dilution of 1: 12800 of the antiserum, an estradiol concentration could of 10 ng / ml is detected in the sample.Estradiol and estrone showed a cross reaction in this system.

25

Example IV

Determination of cortisol and corticoid-binding globulin.

a) Preparation of cortisol-21- (galactose oxidase).

30 mg of cortisol-21 hemisuccinate and 100 mg of galactose oxidase were coupled by the mixed anhydride technique set forth in Example IIa.

b) Corticoid-binding globulin (CBG) was isolated from human serum by succession chromatography over DEAE cellulose and hydroxyl apatite.

Antibodies to this were prepared by injecting rabbits into 1 µ day intervals with 500 µg CBG in complete Freund's adjuvant.

After 3 months, the animals were injected with 1 mg of CBG and two weeks later in the c) γ-Globulin fraction of anti-CBG serum coupled s to m-aminobenzyl = oxymethyl cellulose as set forth in Example III.

d) Determination of cortisol.

5 0.5 nil of a sample containing cortisol (standard solution) was extracted with 2 x 3 nil of methylene chloride. The combined extraction liquid was evaporated to dryness. The residue was taken up in 0.5 ml 0.05 M phosphate buffer with pH 6.2. Then, mixed with 0.1 ml of a solution of CBG in the same buffer at an appropriate concentration and incubated for 30 minutes at h ° C. Then, 0.1 ml of cortisol-21- (galactose oxidase), also in appropriate dilution, and 0.3 ml of the immunoadsorbent prepared under c) was added at a concentration of 5 mg / ml. The resulting mixture was stirred for 2 hours at 0 ° C and then centrifuged, after which the enzyme activity was measured in the residual liquid. To that end, 0.5 µl of the same was added to 1.5 ml of substrate consisting of 100 mg of D-galactose, 20 mg of 5-amino salicylic acid and 10 mg of peroxidase in 150 ml of 0.02 M phosphate buffer with pH 6.0 . After 30 minutes, the extinction was measured at * + 60 nm. Using a CBG concentration of 0.1 Mg / ml and 20 'cortisol-21- (galactose oxidase) so much that $ 80 of the enzyme conjugate was bound to the immunoadsorbent without the addition of steroids, it showed (c) Determination of CBG was also possible with the indicated reagents. From a transcortin 25 dilution series ranging from 0 to 1280 ng / ml, 0.5 ml was incubated for 15 minutes with 0.2 ml of cortisol-21- (galactose oxidase) in appropriate dilution. Then 0.3 ml of immunoadsorbent suspension (5 mg / ml) was added and the mixture was stirred for 15 minutes. The two incubations were held at Η- ° ϋ. Then, enzyme activity 30 in the residual fluid was measured as under d) c. The sensitivity of the assay method was found to be 50 ng / ml).

Example V

35 In a bottle, the following reagents were lyophilized in separate layers: 0.3 ml of the immunoadsorbent suspension (10 mg / ml) as set forth in Example 1a.

2) 0.1 ml of an Ifo mannitol solution.

Claims (2)

3) 0.1 nil of HCG-HRP as set forth in Example 1a. a second layer of 0.1 ml of a 1} mannitol solution. 5) 0.1 ml of rabbit (anti-HCG) serum as set forth in Example II. To this lyophilized Handing was added 0.5 ml of a urine sample and then 0.5 ml of distilled water. After 10 minutes, the enzyme activity in the residual fluid was determined by means of a paper strip impregnated with urea-hydrogen peroxide and o-tolidine. 10 If the urine sample came from a pregnant woman (> 2 IU HCG / ml), a blue color appeared within 5 minutes, whereas when the urine sample came from a non-pregnant woman, no color appeared within the same time. Patent Claims * A process for determining a component of the reaction 2Q between a specific binding protein and the corresponding binding substance using the known binding affinity of such components, using a known amount of a coupling product of the binding substance in the assay. with an enzyme and optionally also a known amount of the specific binding protein (in the case that the binding substance is the component to be determined), characterized in that further insolubilized antibodies to the specific binding protein are used, and after the reaction, the enzyme activity is determined in the liquid or solid phase of the 3Q reaction mixture. 1 Process according to claim 1, characterized in that the insoluble antibodies to the specific binding protein are added to the reaction mixture as the last reagent. A method according to claim 2 for determining the binding substance, characterized in that the specific binding protein and then the enzyme conjugate are first added to the sample and finally the insoluble antibodies to the specific binding protein.