DK150170B - PROCEDURE FOR DETERMINING AN ANTIGEN OR ITS SIMPLE ANTIBODY - Google Patents

Description

in 150170

The present invention relates to a method for determining an antigen or its corresponding antibody using the known binding affinity of such components.

5

For the detection and determination of substances that play a predominant role in biochemical processes, ie. low molecular weight substances such as vitamins and steroids or high molecular weight substances such as proteins and carbohydrates, it is often possible to use reactions between these substances and proteins with a specific binding affinity for these substances. Thus, it is possible to determine the concentration of a steroid using a protein capable of specifically binding that steroid. Examples of such combinations are cortisol and transcortin, 17/3 estradiol and the estradiol binding receptor protein in the uterus.

It is also possible to chemically link a low molecular weight substance to a protein and to inject this conjugate into an experimental 2Q animal which then reacts by forming antibodies against, among other things, the low molecular weight substance. The latter must in this case be regarded as a so-called hapten. The antibodies against the hapten can be considered as a specific case of specific binding proteins.

High molecular substances, such as simple and conjugated proteins and carbohydrates, are capable of causing antibody formation by injection into animals under the correct experimental conditions. Between these antibodies and the injected high-molecular-weight 3q coolant, the antigen, again there is specific binding affinity.

Detection methods using these specific binding affinities are often based on competition between the 35 substance to be determined in the sample and the known amount of the same substance that is radiolabelled with respect to a limited amount of its binding partner. . The amount of substance to be determined determines which part of the radiolabelled substance can be bound by its binding partner. It is also possible to determine an unknown amount of specific binding protein by reacting a sample thereof with a certain amount of radiolabeled specific binding substance. The terms used to describe these methods depend on the nature of the specific binding protein. These are "competing protein binding assays" using receptor or transport proteins, and "radioimmunological assays" using antibodies as specific binding protein.

Instead of labeling one of the components of the reactions described above with a radioactive isotope, it is also possible to use an enzyme as a labeling substance. When using the binding affinity between a low molecular weight substance and its specific binding protein, the low molecular weight substance can be coupled to an enzyme. The coupling product, ie.

2o low molecular weight substance / enzyme can then be used in experiments as described above. For example, when using the binding affinity between an antigen and its antibody, the antigen can be coupled to an enzyme instead of being labeled with a radioactive isotope, as is conventional in radioimmunological methods.

In all the methods in which such a labeled reaction component is used, a suitable method for separating the free labeled component associated with the binding partner is of paramount importance. Depending on the nature of the substances involved in the reaction, different separation methods can be used, e.g. electrophoresis, gel filtration, selective adsorption, use of one of the reaction components in an insolubilized form, and use of antibodies to one of the reaction components, also in an insolubilized form.

Despite the high sensitivity that can be achieved by these methods, it is not always possible to detect or determine substances that occur at extremely low concentrations in, for example, serum or urine. Thus, it is very difficult and often impossible to detect adrenocorticotropic hormone (ACTH) by a radioimmunological method unless a very rare antiserum containing antibodies with an extremely high binding affinity for ACTH is available.

10 Also, determinations of minor peptide hormones, such as oxytocin in unextracted serum, cause great difficulties because the required sensitivity cannot be achieved.

Although, in principle, the immunological cross-reaction between HCG and LH allows the determination of both hormones with a test system, most methods for the determination of human chorionic gonadotropin (HCG) by a coupling product between HCG and an enzyme cannot be used for detection and determination of luteinizing hormone (LH). This is due to the fact that 20 LH occurs at much lower concentrations in blood and urine than HCG during pregnancy, so that the sensitivity of the test systems is insufficient. This lack of sensitivity can be mitigated by extracting the substance to be determined from the medium in which it occurs or by concentrating the medium.

25 However, these methods are very labor intensive and the results are often unsatisfactory.

A method has now been found for determining a component of the reaction between specific binding proteins and the substances that can be specifically bound by these proteins, using the known binding affinity of such components to each other. Accordingly, the process of the invention is characterized in that the component to be determined is reacted with its binding partner, which is insolubilized or insolubilized, that the solid phase is separated from the liquid phase, that the solid phase is then reacted with a liquid phase. immunologic component capable of reacting with a solid phase component in which the immunological component is coupled to an enzyme and finally the enzyme activity of the liquid or solid phase is determined, which activity is a measure of the amount of substance to be determined.

The binding partner required for the determination of the component present in an unknown amount is used in an insoluble form. The assay can take place by putting the binding partner to the reaction mixture in an insoluble form and then reacting the mixture in a heterogeneous medium, but the binding partner can also be added in dissolved form, after which it can be insolubilized by the addition of antibodies to it.

15

The substance capable of reacting specifically with one of the reaction components used coupled to an enzyme may be the second reaction component, but may also be a third substance which must have a specific affinity for one of the reaction components.

The method can be used for the various reaction systems described above, ie. antigen / antibody, hapten / antibody, and a low molecular weight / specific binding protein. As a third substance, another antibody, i.e. an antibody to the γ-globulin fraction of the animal species in which the first antibodies have been formed. This situation can occur in the systems antigen / (first) antibody and hapten / (first) antibody. As a third substance, an antibody to a specific binding protein may also be used.

The core point of the present process is the two-stage design, between which a separation takes place between the liquid and the solid phase of the reaction mixture. The second step 35 is carried out with the solid phase. Only at this stage is the enzyme coupling product used and the enzyme activity determined.

5 150170

The main advantages of the method according to the invention are: 1) the first step can be carried out in a larger volume than is desirable or possible for the second step. This is of great importance when the substance to be determined occurs at too low concentrations to be determined by known methods.

2) In the liquid to be examined, substances may be present which adversely affect the reactions of the second step. In particular, the enzyme present in the coupling product as well as the enzymatically catalyzed reaction may be sensitive to disruptive substances in the sample liquid.

15

In particular, the present method can be used to determine substances contained in body fluids such as hormones, their antibodies and specific binding proteins, and enzymes.

Also factors for blood coagulation, fibrinolysis and complement systems, pathological proteins in body fluids, and antibodies to pathogenic microorganisms and iso-antibodies can be determined in this way.

The medium in which the first step of the assay takes place often consists of a large amount of test liquid, such as urine or serum.

If deemed necessary, the medium can be adjusted to the pH value required for the immunochemical reaction, namely between 5 and 9, by the addition of a dry buffer salt or a small volume of a concentrated buffer solution. The medium of the second step is also a buffer with a pH value needed for the immunochemical reaction. For this purpose, phosphate buffers, citrate buffers, tris (hydroxymethyl) aminomethane and imidazole buffers may be used. For the enzyme reaction in question, it may be necessary to use a buffer of a different composition and pH which depends on the enzyme used.

6 150170

The preparation of the insolubilized reaction component can take place in various ways, e.g. depending on the properties of this reaction component. Thus, antibodies, specific binding proteins, and protein-like antigen can be insolubilized by cross-linking, e.g. with glutaraldehyde and chloroformic acid ethyl ester, by physical adsorption or chemical coupling to an insoluble carrier, such as cellulose compounds, agarose, cross-linked dextran, polystyrene and the like, or by coupling to antibodies to that protein coupled to an insoluble carrier . Low molecular weight substances can be bonded by methods which depend on the structure of the low molecular weight substance and the carrier material. Some of the low molecular weight substances may already have groups capable of reacting with the reactive moieties on the carrier material. In other cases, such groups must be introduced by organic-chemical reactions.

The preparation of the enzyme coupling products can also be carried out by methods which depend on the properties of the molecule incorporated in the coupling product. A covalent binding of proteins to enzymes can be made by such reagents as carbodiimides, diisocyanates, glutaraldehyde and bis-diazobenzidine. The coupling of low molecular weight substances can take place in different ways. Some of these substances may already contain groups that can be crosslinked with reactive groups in the enzyme. In other cases, such groups must be introduced by organic-chemical reactions. It will be appreciated that in the preparation of these coupling products, the initial binding properties of the substances bound to the enzyme 30 must not be changed very much. Also, the activity of the enzyme must not be significantly reduced.

The choice of the enzyme to form part of the coupling product is determined by such properties as the specific binding activity (a high rate of conversion increases the sensitivity of the assay system) and the simplicity of the enzyme determination. The determination of an enzyme that catalyzes a conversion in which colored reaction components participate / is simple. Such colorimetric determinations can be automated in a simple way.

It is also possible to use enzymes that catalyze such transformations involving reaction components which can be determined spectrophotometrically or fluorimetrically.

These provisions can also be automated.

In the preparation of the coupling products, such enzymes as catalase, peroxidase, β-glucuronidase, β-β-glucosidase, β-D-galactosidase, urease, glucose oxidase and galactose oxidase are particularly preferred.

The determinations of the process according to the invention are broadly made as follows: A certain volume of a sample liquid, e.g. 10 ml, containing a very low concentration of the component to be determined, is mixed with a certain amount of an insolubilized reaction partner for the substance to be determined. The test liquid may be urine in which the component to be determined is present in low concentration, or serum which has been diluted to reduce the influence of interfering factors. To enable the reaction to be carried out in the heterogeneous system, the mixture is stirred. The reciting solid phase is separated from the sample liquid, e.g. by centrifugation, and washed if necessary. The first part of the assay can also be made by incubating the test liquid with a certain amount of the reaction partner for the substance to be determined in dissolved form, after which an amount of insolubilized antibody to this reaction partner is added and the mixture is stirred and finally centrifuged. Separation by filtration and decanting is also possible.

The second part of the assay is carried out by resuspending the insoluble substance which forms the solid phase, preferably in a buffer, and by reacting it with a certain amount of a coupling product between the substance to be determined, and enzyme, e.g. peroxidase. After a certain time, the solid phase is again separated, e.g. by centrifugation, upon which enzyme activity in the liquid phase is determined. This enzyme activity is a measure of the amount of substance to be determined in the sample liquid. The second part of the assay can also be performed by reacting the insoluble substance of the first part of the assay with a coupling product obtained by binding a third substance capable of specifically reacting with the reaction component to be determined. mes, to an enzyme. This will be the case when an antibody is determined by the insolubilized corresponding antigen in the first part of the assay and a second antibody to the first one coupled to an enzyme is used in the second part of the assay.

15

The enzyme activity in one phase of the reaction mixture is measured by incubating this phase with a substrate like other substances required for that enzyme reaction.

Preferably, a reaction is used in which a colored compound is formed or removed whose adsorption can be readily determined quantitatively.

The reagents can be used in a variety of forms. The component 25 of the reaction system coupled to an enzyme may be dissolved in a buffer or be lyophilized. Also a solid support can be used, e.g. a paper strip impregnated with the enzyme coupling product.

The insoluble component may be processed into particles of various sizes, such as grains, plates and rods, or into a strip of another carrier material.

For carrying out the process of the invention, a sample package is usually used, consisting mainly of: a) a certain amount of the enzyme coupling product, b) a corresponding amount of one of the components of the reaction system in insoluble form, and 5 c) a substrate for determining the amount of enzyme used.

The sample package, if required, may contain the desired auxiliaries for preparing a dilution series of the sample to be examined for a quantitative determination such as test tubes, pipettes and flasks containing a diluent. For the determination of antigens or haptenes or their antibodies, the sample package contains at least: a) a certain amount of the coupling product between the antigen hapten or an antibody relative thereto and an enzyme; b) a corresponding amount of an insolubilized component of the antigen / and c) a substrate for determining the enzyme activity.

The invention is further illustrated in the following Examples.

Example 1 150170

IQ

Determination of human chorionic gonadotropin (HCG) in serum.

a) Preparation of HCG-HEP.

5 mg of HCG · and 20 mg of horseradish peroxidase (HEP) were dissolved in 2 ml of 0.05 M phosphate buffer with pH 6.2. After adding 40 μ / 1 251 glutaraldehyde solution, the mixture was shaken at room temperature for two hours. Then the mixture was centrifuged for 5 min at 250 g, then fractionated over "Sephadex" G-2Q0 in 0.05 M phosphate buffer of pH 6.2. The fractions whose highest enzyme activity percentage was bound by antibodies to HCG were used in the assay system.

b) Preparation of antibodies against HCG ·.

Antibodies to HCG · were generated in rabbits, as described by Schuurs et. eel. in Acta Endocr. (Ebh.) 59, 120 (1968).

c) Preparation of the immunoadsorbent, anti-HCG cellulose.

The γ-Globulin fraction of the anti-HCG serum described under b) was prepared by precipitation with 18 $ (w / v) solid U 2 SO 2. The precipitate was washed and then taken up in so much 0.05 M borate buffer of pH 8.6 that the final protein concentration became 10 mg / ml.

M-aminobenzyloxymethylellulose (350 mg) prepared by G-urvich's method as described in Biokhimiya 26, 934 (1961) was suspended in 50 ml of distilled water and diazotized by the addition of 10 ml of 36 $ hydrochloric acid and drop by drop 10 ml of NaNO 2 solution. The suspension was centrifuged and washed and the precipitate was resuspended in 43 ml of 0.05 M sodium borate with pH 8.6. Of the γ-globulin solution prepared, 7 ml was added to this suspension. The mixture was stirred for 26 hours at 4 ° C, centrifuged and finally washed with 1 liter of 0.02 M phosphate buffer with pH 6.0.

d) Determination of HCG in serum.

A dilution series of HCG- (8-4-2-1-0.5-0.25-0 IU / ml) was prepared with a diluent of 1 part human serum and 11 parts 0.05 M phosphate buffer with pH 6.2. Of each of the HCG dilutions thus prepared, 0.5 ml was rotationally treated for 2 hours

Room temperature about 0.5 ml: of the inriad wadsorberite suspension (4 mg / ml), prepared according to c), and then centrifuged. The precipitate was washed twice, each time with 2 ml of 0.05 M phosphate buffer of pH 6.2. The precipitate was mixed with 1.0 ml of HCG-HEP solution in a suitable dilution as well as in 0.05 M phosphate buffer of pH 6.2 and again rotated at room temperature for 2 hours.

After centrifugation, the enzyme activity was determined in the supernatant by adding 0.5 ml thereof to 1.5 ml of a substrate solution containing 10 μΐ 30 * fo H₂O₂ and 20 mg of 5-aminosalicylic acid in 150 ml of 0. 02 M phosphal buffer with pH 6 , 0th After 30 minutes, the extinction was measured at 460 nm.

In the described test system, an HCG concentration of 0.25 I / ml caused a measurable increase in the enzyme activity in the supernatant liquid, while a concentration of 1 I.E./ml caused the maximum increase. In this test, the centrifugation step was found to be of crucial importance since omission of this step led to incorrect measurement results for the enzyme activity, i. due to the turbidity of the serum and its peroxidase-like activity.

The HCG content of pregnant women's serum could be determined by this experimental set-up, provided that the serum sample was diluted to at least 1: 3 ratio.

Example 2 3q Determination of HCG at low concentrations by an enzyme / antigen coupling product.

a) Preparation of antibodies to rabbit γ-globulin.

rabbit v-globulin was isolated from normal rabbit serum by precipitating it with 18 µ (w / v) solid NagSO Antibodies against it were formed in a sheep. The injection schedule was: 12 150170. Day - Amount of Freund * s Excipient Injection Method O 0.5 mg + Intramuscular 14 0.5 mg + ”5 28 1 mg +” 42 1 mg - Intravenous 56 1 mg - “10 On day # 70, the sheep were tapped for blood.

b) Preparation of / anti-(rabbit-γ-globulin) β-cellulose.

The γ-globulin fraction of the sheep compartment, described in (a), was readily prepared by precipitation thereof with 16% (w / v) solid sodium sulfate. This γ-globulin fraction was bound to m-aminobenzyloxymethyl cellulose, as described in Example 1 c).

c) Determination of human chorionic gonadotropin (HCG).

20

A dilution series of HCG was prepared in 0.01 M phosphate buffer of pH 6.0, which also contained 2 $ (v / v) normal sheep serum. The dilution range was in the range of 10 to 520 I.E./1, with the dilution factor being 2.

25

To 5 ml of an HCG solution was added 0.1 ml of a sheep (anti-HCG) serum (see Example 1b) diluted with the same buffer to the desired strength. However, the mixture was allowed to stand at room temperature for 30 minutes, after which 1.5 mg / year of anti-rabbit-γ-30-globulin) 7-cellulose was added and the mixture was rotated for 2 hours.

The cellulose was centrifuged for 10 minutes at 3000 rpm. and again suspended in 1 ml of a solution of HCG-HRP coupling product, prepared according to Example 1 a), diluted to the desired strength with 0.01 M phosphate buffer pH 6.0 with 2 $ normal sheep serum. The mixture was again stirred for 2 hours. After centrifugation, enzyme activity was determined in the supernatant by adding 0.5 ml of it to 1.5 ml of enzyme substrate consisting of 10 µl of 30 $ HgOg and 20 mg of 5-aminosalicylic acid in 150 ml of 0.01 M phosphate buffer with pH 6.0. The mixture was allowed to stand for 30 minutes at 25 ° C, after which the extinction was measured at 460 run. 100 $ of enzyme activity was measured in the supernatant provided no anti-HCG serum was added to the system.

5 A dilution series of HCGs, made with children's urine as a diluent liquid, showed an identical pattern, so that measurements in urine samples can also be performed.

The centrifugation step produces a ten times greater sensitivity than can be achieved in a system without centrifugation.

Example 3

Determination of HCG and EH at low concentrations by an enzyme / antibody coupling product.

A) Preparation of the immunoadsorbent HCG cellulose.

This immunoadsorbent was prepared by the method described in Example 1, but instead of rabbit γ-globulin, 100 mg of HCG was coupled to 500 mg of m-aminobenzyloxymethyl cellulose.

b) Purification of antibodies to HCG.

10 ml of rabbit anti-HCG serum, prepared according to Example 1b), was diluted with 90 ml of 0.05 M citrate of pH 5.0 and gently passed over the immunoadsorbent packed in a column and mixed with 10 parts. Sephadex "G-25. The immunoadsorbent was washed with the same buffer until no longer eluted protein. The antibodies were eluted with 0.05 M citrate at pH 2.0. The fractions were collected in 30 1/2 volumes of 0.25 M HaHCO 3, tested for their antibody and protein content, and the appropriate fractions were frozen.

c) Preparation of the Coupling Product (Anti-HCG) -HRP.

20 mg of horseradish peroxidase (HBP) 'was dissolved in 2 ml of an antibody solution with a protein content of 2 mg / ml. To this solution was added 8 μΐ $ 25 of glutaraldehyde solution, and the mixture was shaken for 2 hours at room temperature. Finally, the mixture was fractionated over a "sephadex" G-20Q column in 0.05 M phosphate buffer with pH

14 150170 6.5. The fractions, livi's highest enzyme activity percentage bound to the HCG cellulose, were used in the assay system.

d) Determination of HCG and IH.

10 ml of a HCG dilution series of HCG and 10 ml of a HCG dilution series of 1H were mixed with 5 mg of anti-HCG cellulose prepared according to Example 1 c) which was suspended in 1 ml of 0.15 M phos-pha tpuf with pH 6.0 and the mixture was rotationally treated at room temperature 10 12 hours. The immunoadsorbent was centrifuged and washed with 5 ml of 0.05 M phosphate buffer of pH 6.0.

The immunoadsorbent was added to 1 ml of the (anti-HCG) -HRP coupling product in the desired dilution, after which the mixture was again rationed for 2 hours. Finally, the enzyme activity in the supernatant was determined after centrifugation as described in the previous examples.

Ted using this method, a concentration of 5-10 2o I.E. HCG and 10-20 I.E. IH pr. liter is determined. If the centrifugation step is omitted, the optimum sample size is 0.5 ml and the sensitivity ea. 100 I.E. HCG per liter or approx. 200 I.E. IH pr. per liter, so that the process of the invention causes a 10-20 times increase in sensitivity.

25

Example 4

Determination of HCG and anti-HCG.

a) Rabbit serum was fractionated over DEAE cellulose according to the prescription of H.A. Sober and others in J. Am. Chejru Sog. 78f 756 (19561. The isolated γ-globulin fraction, which was immunoelectrophorically pure, was partly used to generate antibodies in a sheep in accordance with the scheme of Example 2 a), while additionally coupling 100 mg of rabbit γ-globulin to 500 mg of m-aminobenzyloxymethyl cellulose by the method described in Example .1 c).

From the prepared sheep / anti (rabbit Y-globulin) -7 serum, the specific antibodies were isolated by the method described in Example 3 b) with the immunoadsorbent prepared, · 15) b) The coupling product / anti- (rabbit γ -globulin)] - HEP was prepared analogously to the coupling product (anti-HCG ·) -HEP described in Example 3 c).

5 c) Determination of anti-HCG ·.

A dilution series of rabbit (anti-HCG ·) serum was prepared with 0.05 M phosphate buffer of pH 6.0.

Rabbit (anti-HCG ·) serum (0.5 ml was rotationally treated with 0.25 mg of HCG cellulose for 2 hours (see Example 3a)). The reaction mixture was centrifuged and the precipitate was washed four times with phosphate buffer each with 3 ml, then the precipitate was resuspended in 1 ml of a solution of the coupling product b. Bone obtained mixture was rotated for 2 hours. After centrifugation, the enzyme activity in the supernatant was determined as described in Example 1 d).

d) Determination of HCG.

With the reagents prepared, HCG was determined by incubating solutions thereof together with 0.5 ml of rabbit (anti-HCG) serum in a dilution which, in the system described under (c), precisely causes an almost maximum reduction in enzyme activity. the supernatant liquid, and by carrying out the procedure described under c) with the mixture obtained.

When using 5 ml of HCG solution, it was found possible to determine concentrations of approx. 10 X.E./l HCG.

Example 5 30. -

Determination of serum insulin, a) Preparation of insulin (glucose oxidase).

5 mg of swine insulin and 25 ml of glucose oxidase were dissolved in 2 ml of 0.05 M phosphate buffer with pH 6.5. To this solution was added July 5, $ 25 glutaraldehyde solution, and the mixture was shaken at room temperature for 90 minutes. The mixture was fractionated over "Sephadex" G-200 in 0.05 M phosphate buffer at pH 6.5 · The fractions, whose highest percent enzyme activity could be bound by 40 antibodies to insulin, were used in the assay system.

16 150170 b) Preparation of antibodies against insulin.

Ten guinea pigs were injected intramuscularly once a week with 1 mg of porcine insulin in Freund's complete excipient for a period of 4-8 weeks. After being left alone for 2 weeks, the animals were injected intravenously 5 with an additional 1 mg of insulin without adjuvant. Another 2 weeks later, the animals were tapped for blood. Occasional hypoglycaemia was combated by intraperitoneal glucose administration.

c) Preparation of antibodies to marsyin-γ-globulin.

10

Guinea pig γ-globulin was prepared by adding 1 volume of a saturated ammonium sulfate solution to 2 volumes of guinea pig serum. One resultant precipitate was washed 2 times with '33 # saturated (NH 2 gSO 4 solution and then taken up in a physiological saline solution. Sheep were immunized with increasing doses of the γ-globulin produced, namely 0.5, 1 and The injections were given every other week, with immunogenic t mixed with Freund's complete excipient, and 2 weeks after the last injection, an additional 2 mg of γ-globulin was administered.

1 a physiological saline solution. 1 Week later, the animal was tapped for 20 days d) Preparation of insoluble antibodies to guinea pig Y-globulin.

10 g of microcrystalline cellulose was activated by stirring, with stirring, to 400 ml of 2.5% (w / v) CNBr solution, then adjusting the pH to 10.5 with 1N NaOH solution and held on to this value for 2 minutes. ' The cellulose was washed with ice water and with 0.1 M NaHCO 3. To 10 ml of sheep anti-(guinea pig γ-globulin1 serum) was added 1.6 g Na-O 2. After stirring for 1 hour at room temperature, the precipitate was centrifuged, washed twice with 20 ml 16% (w / v). ) Na2 SO4 solution and finally taken up in 1Q ml of 0.1 M NaHCO3.

One activated cellulose was mixed with 40 ml of a 0.1 M NaHCO 5 solution and 10 ml of the γ-globulin solution. One suspension was rotated for 40 hours at 4 ° C and washed, 2 times with 5 ml Q, 5 M NaHCC, 2 times with 500 ml O, Q5 JA citrate with pH 1.1 and 2 times with 5QQ ml 0.05 M phosphate with pH 6.2.

E) Determination of serum insulin.

4 ml of an insulin dilution range in the range of 0 to 100 ng / ml was incubated at room temperature for 4 hours with 1.0 ml of anti-insulin serum (0.15 M phosphate buffer pH 6.0) to · The desired strength). Then, 5 mg of the irtimuno adsorbent prepared according to d) was added, suspended in 1 ml of pH 6.0 0.15 M phosphate buffer, and the resulting mixture was rotated overnight at 4 ° C. The immunoadsorbent was then centrifuged and washed 3 times with 5 ml of 0.05 M phosphate buffer of pH 6.0 (each time with 5 ml) to which 2 $ sheep serum had been added. Then 1.0 ml of insulin-glucose oxidase, diluted to the desired strength with the wash buffer, was added to the immunoadsorbent. The resulting mixture was again rotated overnight and centrifuged, ·· ·. after which the enzyme activity in the supernatant was measured by incubating 0.5 ml thereof with 2.5 ml of a substrate solution and the extinction was measured at 460 nm. The substrate solution contained 50 mg glucose, 10 µg peroxidase and 1 mg 5-aminosalicylic acid per ml. 2.5 ml of 0.05 M phosphate buffer with pH 6.0.

The centrifugation step, which in this case serves to both increase the maximum volume of sample liquid that can be used and to remove serum factors that interfere with the reaction, caused a 10-fold increase in sensitivity so that a concentration of few ng / ml insulin could detected.

25

Example 6

Determination of estradiol.

a) Preparation of estradiol-17-succinyl-HEP.

30 mg of estradiol-17-hemisucoinate and 0.08 ml of tri-n-butylamine were dissolved in 2.5 ml of dioxane. To the cold solution (2 ° C) was added 15 µX of isobutyl chlorocarbonate. After 30 minutes, this solution was mixed with 100 mg of horseradish peroxidase (HEP) in 7.5 ml of a mixture of dioxane and water (2: 3) which had been adjusted to pH 9.5 with caustic soda. This solution was stirred for 4 hours at 2 ° C, then dialyzed for 18 hours. The precipitate obtained after the pH of the dialysate was adjusted to 4.6 was centrifuged, washed and taken up in 5 ml of distilled water which had been adjusted to pH 8. The material was further purified to precipitate it. times with 10 ml of acetone. The synthetic product was taken up in 10 ml of 0.05 M phosphate buffer with pH 7.8.

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

The preparation was prepared by the mixed anhydride method as described in (a), the starting material was 100 mg of Trio 1-17 hemisuecinate and 150 mg of bovine rum albumin (BSA).

C) Preparation of antibodies for estradiol.

One sheep was injected every 4 weeks with 4 mg estradiol-17-succinyl-BSA in Freund's complete excipient. At regular intervals, clay was drawn from the sheep. The serum was adsorbed with 15 BSA.

d) Preparation of sheep γ-globulin antibodies.

Sheep V-globulin was prepared as described in Example 2b) but this time with 16 $ (w / v) sodium sulfate. Rabbits were immunized with all sheep γ-globulin according to the following scheme:

Behind Amount of Freund's Excipient Injection Method 0 200 pt> g + Intramuscular 25 14 400, ug + ”28 800 μβ +” 42 800 μβ - Intravenous 30 2 TJger After the last injection, the animals were tapped for blood.

e) Preparation of the immunoadsorbent / rabbit anti (sheep γ-globulin) J cellulose.

The γ-Globulin fraction of the antisera described below b1 was prepared by precipitation with 18 $ (w / v) Ha 2 SO 4. Bet obtained product was coupled with cellulose by Gurvich's method SOm described in Example 1 cl.

19 150170 (f) Determination of estradiol.

A dilution series of estradiol (0-0.5-1-2-4-8-16 ng / ml) was prepared in phosphate buffer with pH 6.0. 5 ml of sample was mixed with 5 0.5 ml of sheep anti-estradiol serum in the desired dilution. After incubating the mixture for 2 hours at room temperature, 1 ml of immunoadsorbent suspension (ee e) containing 60 mg / ml was added and the mixture was rotated for 4 hours at room temperature. The cellulose was centrifuged from / and washed with 5 ml of 0.05 M phosphate buffer of pH 6.0 containing 2 $ BSA. Then, 1 ml of estradiol-17-succinyl-HEP, diluted to the desired strength with the buffer with which the mixture was washed, was added to the cellulose. The mixture was rotated for 1 hour, then centrifuged again and the enzyme activity in the supernatant measured as described in Example 1.

By the method described, concentrations of 1 ng / ml estradiol could be determined, which means an increase in sensitivity by a factor of 10 compared to the sample without centrifugation 20 steps.

Example 7

Determination of cortisol.

A) Preparation of cortisol-21-galactose oxidase.

50 mg of Cortisol-21 hemisuccinate and 100 mg of galactose oxidase were coupled by the mixed anhydride method as described in Example 6 a).

B) preparation of insoluble transcortin.

100 mg of Transcortin purified by DEAE cellulose chromatography followed by hydroxylapatite chromatography was coupled to 5 g of epha-rose 4B by the CNBr method: Sepharose 4B suspension (5 g) was activated by mixing it. with 4 ml of 2.5 $ (w / v) CIBr solution in distilled water, after which the pH was adjusted with 1 U HaOH to between 10 and 11 at which value they were kept for 6 minutes. Then the sepharose was washed with ice water and with 0.1 M

40

Claims (3)

150170 NaHCO3, then 100 mg of transcortin was added in 20 ml of 0.1 M NaHCO3 and the suspension was shaken at 4 ° C for 24 hours. After being washed sequentially with 0.5 M IfaHCO 3, 0.05 M citrate buffer of pH 1.1 and 0.05 M phosphate buffer of pH 6.0, the sepharose 5 was kept in the last buffer added 0, 1% merthiolate. c) Determination of cortisol. A cortisol dilution range in the range of 0.25 to 16 ng / ml was prepared at 1 ° in 0.05 M phosphate buffer with pH 6.0); 5 ml of each cortisol solution (in 0.05 M phosphate buffer with jsH 6 0) was rotated overnight with 5 mg trans cort inisephar ose at 4 ° C. After centrifugation, tisatearl ml-coxtisol-21-galactose oxidase solution, diluted to a suitable concentration in the aforementioned buffer, then. The mixture was rotated for 2 hours at 4 ° C. After recent addition, enzyme activity in the supernatant is stormed by adding 0.5 ml thereof to 1.5 ml substrate containing 100 mg of D-galactose, 20 mg of 5-aminosalicylic acid and 10 µg of peroxidase in 150 ml of 0. 02 M phosphate buffer with pH 6.0. After 30 minutes, the extinction was finally measured at 460 nm. Using this method, approx. 1 ng / ml cortisol is determined, which increases the sensitivity by a factor of 10. 25 Patent claims. A method for determining an antigen or its corresponding antibody utilizing the known binding affinity of such components, characterized in that the component to be determined is reacted with its binding partner which is insolubilized or becomes insolubilized that the solid phase is separated from the liquid phase,. that the solid phase is then reacted with an immunological component capable of reacting with a component in the solid phase in which the immunological component is coupled to an enzyme and that the enzyme activity of the liquid or solid phase is finally, determine which activity is a measure of the amount of the substance to be determined.