CH617774A5 - - Google Patents

Description

617 774

2nd

PATENT CLAIMS

1. A method for the qualitative and quantitative determination of a hapten or a corresponding antibody, characterized in that a liquid sample containing the compounds to be determined, a predetermined, known amount of the coupling product of the hapten with an enzyme and a predetermined, known amount of Haptens or its antibody is added in the form of an insoluble preparation, a reaction of the hapten with the antibody taking place, whereupon the liquid phase is separated from the solid phase and the enzyme activity in one of these phases is qualitatively determined, which activity is a measure of the amount the connection to be determined.

2. The method according to claim 1, characterized in that the hapten is a vitamin or hormone.

3. The method according to claim 1 or 2, characterized in that an oxidoreductase is used as an enzyme.

4. Test pack for performing the method according to claim 1, characterized by a content of

(a) a predetermined amount of the coupling product of the hapten with an enzyme,

(b) a predetermined amount of components of the reaction system in insoluble form,

(c) a substrate for determining the activity of the enzyme used.

For the detection and determination of low-molecular substances, which are present in low concentrations, such as steroid hormones in body fluids, methods have been developed which use proteins which are capable of specifically binding the substance to be detected. These methods rely on competition between the substance to be detected in the sample and a known amount of the same substance, which is radiolabelled, with a limited amount of the specific binding protein. The unknown amount of the bindable substance determines which portion of the radioactively labeled substance is bound to the specific binding protein.

It is also possible to use these methods to determine an unknown amount of a specific binding protein by reacting a sample containing an unknown amount of the specific binding protein with a certain amount of a bindable radiolabeled substance.

It is common in the literature to distinguish these methods of determination depending on the type of specific binding protein used, although the basic principle of all of these determinations is the same. For example, spoken of “competitive protein binding assays” when using receptor or transport proteins that are found in the body and “radio-immunological determinations” when using anti-substances.

Radioactively labeled substances are required for both types of determinations. Working with these substances requires the presence of precise measuring devices (well-equipped laboratories and qualified personnel). These high requirements make it impossible to use these determination methods in general, especially in smaller laboratories.

A method has now been found for the qualitative and quantitative determination of a hapten or a corresponding antibody. Haptens can be viewed as a special group of small molecules.

Haptens and their antibodies mostly occur in low concentrations. According to the definition by K. Landsteiner, haptens are protein-free substances whose chemical configuration is such that they cannot react with specific antibodies, but also not so that they can lead to the formation of antibodies. However, in order to still be able to form antibodies to haptens, the haptens must be coupled to polypeptides before they are injected into the test animal. When determining a low molecular weight compound konkur-I »its the substance to be determined and its coupling product with an enzyme for a certain amount of the insoluble specific binding protein. The more of the low-molecular compound the sample contains, the less the chance for the coupling product from the soluble enzyme i * and the compound to combine with the insoluble specific binding protein and the more of the coupling product remains in the liquid phase, in which the enzyme activity can be measured easily.

The method is characterized in that a predetermined, known amount of the coupling product of the hapten with an enzyme and a predetermined, known amount of the hapten or its antibody in the form of an insoluble preparation are added to a liquid sample which contains the compounds to be determined, whereby a reaction of the hapten with the antibody takes place, whereupon the liquid phase is separated from the solid phase and the enzyme activity in one of these phases is determined qualitatively or quantitatively, which activity is a measure of the amount of the compound to be determined.

When determining a specific binding protein with the same reagents, the soluble protein to be determined and the insoluble protein compete to form a certain amount of the coupling product of the hapten with the enzyme. If the level of specific binding protein in the sample is higher, the insoluble protein will bind less of the enzyme coupling product and, consequently, more enzyme will remain in the liquid phase.

A specific binding protein with two or more binding sites can also be detected and determined by the method according to the invention, i.e. with the enzyme coupling product and the hapten preparation in insoluble form. The liquid phase of the reaction mixture can then contain the coupling product bound to the specific binding protein and the complex of the enzyme coupling product and specific binding protein and the water-insoluble hapten preparation can be contained in the solid phase. The more of the protein to be determined is contained in the sample, the more enzyme activity the liquid phase has.

With the aid of a determination curve for a specific system, in which the increasing content of the substance to be determined is plotted against the enzyme activity found, preferably in the liquid phase, the amount 55 of the substance to be determined contained in the sample can be determined for the value found for the enzyme activity can be read.

The most important reagent for this determination method is the coupling product from the hapten and an enzyme, hereinafter also called enzyme coupling product, which on the one hand can react with the specific binding protein via the hapten component and on the other hand has enzyme activity. This reagent can be prepared by a method described for similar products. The second reagent, the insoluble component in the reaction system, serves to facilitate the separation of the various enzyme-containing fractions of the reaction mixture. The addition of this reagent leads to the formation of a solid phase in addition to a liquid phase.

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617 774

The enzyme activity of a fraction of the reaction mixture is generally shown or measured by incubating that fraction with a substrate and other substances to carry out an enzyme reaction. A reaction is preferably used in which a colored compound is formed or removed, the absorption of which can also be easily measured quantitatively.

Low molecular weight substances that can be detected using the new method and have a molecular weight of up to approximately 1500 are, for example, steroids, vitamin B12, folinic acid, thyroxine and triiodothyronine, releasing factors, histamine, serotonin and other biogenic amines, digoxin, digitoxin , Prostaglandins, adrenaline, nor-adrenaline, herbal hormones such as auxin, kinetin, giberellic acid and antibiotics such as penicillin.

The method for the detection of specific binding proteins for low molecular weight substances can be used, e.g. for the determination of antigens against penicillin or for the determination of the intrinsic factor.

The production of coupling products of enzymes and haptens can be carried out in various ways. Some low molecular weight substances can already have groups which can be crosslinked with reactive groups on the surface of the enzyme, while other substances first have to obtain such groups through chemical reactions. It goes without saying that the original binding properties of the low molecular compound and the activity of the enzyme cannot be changed significantly during this process. The groups of the enzyme that are particularly suitable for coupling reactions are amino and carboxyl groups. If the modified or unmodified low molecular weight substance also has such groups, the coupling e.g. by reactions as are known from peptide synthesis. In addition, such substances as glu-taraldehyde, difluoronitrodiphenyl sulfone, tolui diisocyanate, di- and trichloro-s-triazine can be used for the coupling reaction.

Specific examples of the coupling of haptens with proteins are e.g. in Methods in Immunology and Immuno-chemistry, Volume 1. The methods described there are used for the production of coupling products for immunization, but they can also be used for the production of coupling products of the haptens and an enzyme which are important for the method according to the invention.

The choice of the enzyme, which is a component for the coupling system (hapten and enzyme), depends on properties such as the specific activity (a high conversion rate increases the sensitivity of the test system) and the ease of determination of the enzyme. Determining an enzyme that catalyzes a conversion in which colored products form or disappear is easy. Such colorimetric determinations can be automated easily.

It is also possible to use enzymes that catalyze transformations in which components occur or disappear, which can be determined spectrophotometrically or fluorimetrically. These provisions can also be automated.

For the production of the coupling products, enzymes such as catalase, peroxidase, β-glucuronidase, β-D-glocosidase, β-D-galactosidase, urease, glucose oxidase and galactose oxidase are preferred, especially the group of oxido-reductases.

The insoluble hapten preparation used in the process according to the invention can be used in a known manner, e.g.

by crosslinking with ethyl chloroformate, by covalent bonding with insoluble carriers, such as agarose, crosslinking with dextran or filter paper, or by physical coupling to insoluble carriers, such as plastics.

The form in which the reagents can be used is varied. The component of the reaction system that is coupled to an enzyme can be freeze-dried or dissolved in a buffer. In addition, a solid support, e.g. a paper strip impregnated with the coupling product can be used.

The insoluble preparation can be brought in the form of particles of various shapes, such as bodies, spheres and rods or in the form of a strip of one or the other carrier material.

A test pack, which is characterized by a content of, is preferably used to carry out the method according to the invention

(a) a predetermined amount of the coupling product of the hapten with an enzyme;

(b) a predetermined amount of one of the components of the reaction system in insoluble form;

(c) a substrate for determining the activity of the enzyme used.

If desired, the test pack can also contain the necessary aids for the preparation of a dilution series of the sample to be examined for a quantitative determination, such as test tubes, pipettes and flasks with diluent 111.

Example /

Determination of testosterone

A) Production of testosterone-3-HRP

100 mg testosterone-3- (0-carboxymethyl) oxime and 0.143 ml tri-n-butylamine were dissolved in 5 ml dioxane. The solution was cooled to 2 ° C and then 0.03 ml of isobutyl chlorocarbonate was added. After 30 minutes, the solution was added to 100 mg of HRP (horseradish peroxidase) in a mixture of 9, 4.1 ml of water and 6 ml of dioxane, and the pH was adjusted to 9 with 0.1N NaOH. This solution was stirred at 2 ° C for 4 h and dialyzed overnight. The precipitate, which was obtained after the dialysate had been adjusted to a pH of 4.6, was centrifuged after being left to stand overnight, suspended in 10 ml of water and dissolved with the aid of sodium hydroxide solution. The material was precipitated three times with 15 ml of acetone at a pH of 4.5, dissolved in 15 ml of water which had been adjusted to a pH of 7.8 with sodium hydroxide solution, dialyzed and finally lyophilized.

B) Preparation of Testosterone-3-BSA

This coupling product was prepared in the same manner as testosterone-3-HRP, except that 50 mg of testosterone-3- (0-carboxymethyl) oxime and 150 mg of BSA (bovine serum albumin) were used as the starting material.

C) Production of antibodies against testosterone-3-BSA

Five rabbits were injected with intramuscularly increasing doses of testosterone-3-BSA in Freund's complete adjuvant (0.5, 1 and 2 mg) at 3 week intervals. Two weeks after the last injection, the animals were intravenously injected with 2 mg of antigen in physiological saline. Blood was drawn from the animals a week later. The antibodies raised against BSA were removed by partially treating the serum with BSA-m-aminobenzyloxymethyl cellulose prepared by the Gurvich method (see D).

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D) Preparation of antitestosterone cellulose

This substance was prepared according to the method described by Gurvich in Biokhimiya 26, 934 (1961).

1. Production of «aminocellulose»:

50 g of Whatman cellulose, which had been washed and decanted several times, was suspended in 100 ml of a 0.7% sodium acetate solution containing 2 g of N (m-nitrobenzoxy) methyl pyridine. The mixture was dried at 60 to 80 ° C and heated to 125 ° C for 40 min! The resulting product was washed thoroughly with distilled water, dried at 80 ° C, washed with benzene and dried again. 50 g of the dried product were reduced by suspension in 300 ml of a 15 percent Na2S204 solution and stirred at 50 to 60 ° C. for 30 minutes. The product was filtered and washed successively with distilled water, 30 percent acetic acid and again with distilled water.

2. Treatment with ammoniacal copper solution:

40 ml of 10 percent sulfuric acid, 20 ml of 50 percent nitric acid and 140 ml of distilled water were heated to 90 ° C. with stirring. Then 5.9 g of CuO were added in small portions. The solution was heated to boiling for 2 hours and made up to 500 ml with distilled water. 80 ml of this solution were placed in an ice bath and added to 160 ml of cold 4N NaOH with stirring. After stirring for 30 minutes, the precipitate was washed twice with distilled water and dissolved in 80 ml of 25 percent ammonia. 1 g of "aminocellulose" was gradually added to this solution. The mixture was stirred for half an hour and then 40 ml of boiling water were added and the solution was rapidly cooled to 0.degree. The solution was neutralized with 10 percent sulfuric acid, whereupon the aminocellulose flocculated. It was washed with cold distilled water.

3. Production of y-globulin:

To rabbit antitestosterone serum 180 mg Na2S04 were added per ml serum. The mixture was stirred at room temperature for 1 h and the resulting precipitate was centrifuged, washed twice with an 18% Na2S04 solution and taken up in 0.05 M sodium borate with a pH of 8.6 such that the protein concentration was approximately 10 mg / ml was.

4. Binding of 7-globulin to aminocellulose:

350 mg of "aminocellulose" was suspended in 50 ml of distilled water. The suspension was cooled to 0 ° C. 10 ml of 36 percent hydrochloric acid were added and then 10 ml of 10 percent NaN02 solution was added dropwise. The suspension was centrifuged, washed with cold distilled water and then with 0.05 M sodium borate with a pH of 8.6. The cellulose was suspended in 43 ml of 0.05 M sodium borate with a pH of 8.6. 7 ml of the y-globulin solution prepared as above were added to this suspension. The mixture was stirred at 4 ° C for 26 h, centrifuged and washed with 0.02 M phosphate buffer with a pH of 6.0. A cellulose suspension was prepared from the antiserum of each of the 5 immunized rabbits (A to E).

E) Determination of testosterone using testosterone-3-HRP and antitestosterone cellulose

I) Immune response

0.5 ml of a sample containing testosterone, 0.2 ml of testosterone-3-HRP (100 mg / ml) and 0.3 ml of an antitestosterone cellulose suspension were rotated at room temperature for 2 h and then at 1000 for 5 min g centrifuged.

The immune reaction took place in 0.02 m phosphate buffer at a pH value of 6.0, containing 2% sheep serum.

II) Enzyme reaction

0.5 ml of the supernatant liquid was incubated at room temperature with 1.5 ml of substrate for 30 minutes. The absorbance was measured at 460 nm.

The enzyme substrate contained 10 ml, 30% hydrogen peroxide and 20 mg 5-aminosalicylic acid in 150 ml 0.02 M phosphate buffer with a pH of 6.2.

1 shows measured values in which testosterone-3-HRP is bound to the antitestosterone cellulose preparations in which it is. In this case only buffer was added as a sample in the test system. If cellulose is added instead of antitestosterone cellulose, more than 95% of the enzyme activity remains in the supernatant. Preparations B, D and E showed that almost no testosterone 3-1 s HRP had been bound, but with preparations A and C.

2 shows the results of the incubation of a testosterone dilution series with testosterone-3-HRP at four different concentrations of antitestosterone cellulose C.; ci 1 mg / ml (I), 2 mg / ml (II), 4 mg / ml (III ) and 16 mg / ml (IV). It is apparent that an amount of approximately 10ng testosterone can be shown with this system.

Example 2

Determination of estradiol

A) Oestradiol-17-succinyl-HRP was prepared by the mixed anhydride method described in Example 1A), using 50 mg of estradiol-17-hemisuccinate and 50 mg of HRP as starting materials. ,,, B) Oestradiol-17-succinyl-BSA was prepared according to the mixed anhydride method described in Example 1A), using 100 mg of oestradioI-17-hemisuccinate and 150 mg of BSA as starting materials.

C) To produce the antibodies against estradiol-17-is succinyl-BSA, 5 rabbits according to the method described in Example 1 C)

immunized scheme described. The sera were absorbed with BS A-m-aminobenzyloxymethyl cellulose.

D) Antiestradiol cellulose was prepared in the manner described in Example 1D) for antitestosterone cellulose. From

4ii Each of the immunized rabbits was made with a cellulose preparation numbered 16 to 20 inclusive.

E) The test was carried out analogously to that for testosterone in Example 1E).

4_, Figs. 3 and 4 show some results. Figure 3 shows that three useful antisera were obtained by immunization, 17 of which had the highest title. 4 shows the test system in which anti-oestradiol cellulose 17 was used in a concentration of 8 mg / ml. System 5 (, does not differentiate between estrone and 17a-estradiol. 17a estradiol, especially estriol, show a lower cross-reaction. Testosterone and progesterone only influence the system in very high concentrations

5; Example 3

Determination of antibodies against penicillin penicilloyl catalase

30 mg of benzylpenicillinic acid were dissolved in 5 ml of 96 percent ethanol and added dropwise to 200 mg of catalase in 45 ml of 0.1 M Phos-M1 phate buffer with a pH of 7.5. The reaction was continued for 2 hours, keeping the pH between 7.2 and 8.2 with 0.5N NaOH. The reaction mixture was dialyzed against 6X310.02 M phosphate buffer with a pH of 7.0.

h <In the same way, 250 mg of benzylpenicillic acid were coupled to 5 g of m-aminobenzyloxymethyl cellulose, which had been prepared by the method of Gurvich (Biokhimiya 26, 934 (1961)). The coupling product, however, was

not dialyzed, but washed on a glass filter.

Hypersensitivity to penicillin has been demonstrated in the following ways:

0.2 ml of a sample of non-hemolyzed serum was mixed with 0.5 ml of a solution of penicillol catalase (1: 800). After 30 minutes, 10 mg of penicilloyl-m-amino.benzyloxymethylcellulose were added. The mixture was rotated for 30 min and then the enzyme activity in the supernatant liquid was determined by adding 0.02 ml of this liquid to 2.8 ml of 0.05 m phosphate buffer with a pH of 6.8, the 1.2 | Contained 30 percent H202 and then the decrease in absorbance at 240 nm was measured. In the serum of patients who were hypersensitive to penicillin, less enzyme activity was found in the liquid than in rabbit serum. The values for people who were not hypersensitive did not differ significantly from those with rabbit serum.

Example 4 Determination of folic acid

A) Production of folate glucose oxidase

200 mg glucose oxidase (140 IU / mg) was dissolved in 10 mg PBS (with phosphate buffered saline, a phosphate-containing physiological saline) with a pH of 7.0. 30 mg of l-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (MCDI) were added and then 24 mg of folic acid. The reaction lasted 2 hours and then careful dialysis against PBS with a pH of 7.0 was carried out.

B) Preparation of Folate MBSA (Methylated Bovine Serum Albumin)

Folate MBSA was produced by the method described by Ricker and Stollar (Biochemistry 6, 2001 (1967)). 25 mg MCDI was added to 50 mg BSA in 50 ml water and then 20 mg folic acid. A yellow precipitate had formed 2 hours later. Finally the whole reaction mixture was dialyzed against physiological saline for a considerable time.

C) Preparation of antiserum against folate MBSA

On days 0, 21 and 42, 4 rabbits were injected intramuscularly with 2 mg of folate-MBSA in Freund's complete adjuvant and on day 35 with 3 mg of folant-MBSA intravenously in physiological saline. Blood was drawn from the animals on day 49.

D) Antifolate cellulose was prepared according to the procedure described in Example 1 D).

E) Determination of folic acid

100 ml of the sample to be examined and 700 ml of an anti-folate cellulose suspension were rotated for 3 hours. 200 ml of folate glucose oxidase (1: 1500) were added. The mixture was rotated again for 3 hours and centrifuged, and then the enzyme activity in the supernatant liquid This determination was carried out by mixing 0.5 ml of the supernatant liquid with a solution of 50 mg of glulose, 10 [ig HRP and 1 mg of 5-aminosalicylic acid in 2.5 ml of 0.05 N phosphate buffer with a pH of 6.0 and measurement of the absorbance after 30 min at 460 nm.

Figure 5 shows the percentage of bound enzyme against the concentration of antifolate cellulose.

Fig. 6 shows the sensitivity of the test system in an antifolate cellulose concentration of 2 mg / ml and the effect of glycine, asparagine, alanine, and glutamic acid.

Example 5

Determination of digoxin A) Production of digoxin -HRP

To 22 mg digoxin, in 1 ml abs. Suspended ethanol, 1 ml of 0.1 M sodium metaperiodate was added dropwise with stirring.

After 25 minutes, 0.3 ml of 0.1 M ethylene glycol was added. 5

617 774

Min later, this mixture was added dropwise with stirring to a solution of 32 mg of horseradish peroxidase (HRP) in 1 ml of distilled water, which was adjusted to a pH of 9.5 with 5% K2C03 solution. During the reaction, the pH was kept at 9 to 9.5 by adding 5% K2C03 solution. When the pH was stable, 15 mg NaBH4 in 1 ml distilled water was added. After 3 h the pH was adjusted to 6.5 with 1 M formic acid. 1 h later, 1 M NH4OH was added until a pH of 8.5 was reached. The mixture was dialyzed against cold running water overnight. Finally, the pH was adjusted to 4.5 with 0.1N hydrochloric acid. The mixture was left at room temperature for 1 hour and at 4 ° C. for 4 hours to obtain a precipitate, which was centrifuged at 1,000 g for 1 hour. The precipitate was dissolved in 5 ml of 0.1 M NaHC03, dialyzed thoroughly and freeze-dried.

B) Production of Digoxin-BSA Digoxin bovine serum albumin (BSA) was the same

Prepared as described above for Digoxin-HRP, but starting from 436 mg digoxin and 560 mg BSA and increasing the amounts of the other reagents in the same ratio as the dioxin.

C) Production of antibodies against digoxin

Five rabbits were injected with 400, 800 and 1600 [ig dioxin-BSA every 14 days. The immunogen was mixed with Freund's complete adjuvant and administered intramuscularly. 14 days after the last injection, the animals were intravenously injected with 800 [digoxin-BSA in physiological saline. Blood was drawn from the animals 10 days later. The serum was adsorbed with BSA-m-aminbenzyl-oxymethyl cellulose.

D) Production of Antidigoxin Cellulose Antidigoxin cellulose was prepared using the Gurvich process,

as described under 1 D).

E) Determination of digoxin

A dilution series was made with digoxin in 0.1 M phosphate buffer at pH 7.5 containing 0.9% NaCl, 0.5% Tween-20 and 1.0% BSA. The dilution range went from 0.1 to 100 ng / ml. 1 ml of a digoxin solution was mixed with 0.1 ml of digoxin HRP in a suitable dilution and then 2 mg of antidigoxin cellulose, which was suspended in 0.4 ml of buffer, were added. The mixture was rotated at room temperature for 6 h and then centrifuged and the enzyme activity in the supernatant liquid was determined.

The addition of 0.8 ng digoxin led to a measurable increase in the enzyme activity in the supernatant. Digoxin alone showed little cross reaction in the system while cholesterol, cortisol, estradiol, testosterone and progesterone showed no cross reaction in the system.

Example 6

Determination of cortisol

A) Preparation of cortisol-21-galactose oxidase

50 mg of cortisol-21-hemisuccinate and 100 mg of galactoseoxy-dase were prepared according to the mixed anhydride method described in Example 1A).

B) Preparation of Insoluble Transcortin

100 mg transcortin, which had been purified by chromatography with DEAE, cellulose or hydroxyapatite, were coupled to 3 g of Sepharose 4 B using the CNBr method as follows: 3 g of Sepharose 4 B suspension were activated by mixing with 4 ml of a 2nd , 5 percent (w / v) CNBr solution in distilled water and then the pH was adjusted to 10 to 11 with 1N NaOH and kept at this value for 6 min. The Sepharose was washed with ice water and 0.1 m NaHC03. Then

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100 mg of transcortin in 20 ml of 0.1 M NaHC03 were added and the suspension was shaken at 4 ° C. for 24 h. Then it was washed successively with 0.5 M NaHC03.0.05 M citrate buffer with a pH of 1.1 and 0.05 M phosphate buffer with a pH of 6 and the Sepharose was left in the last buffer to which 0 , 1% merthiolate had been added.

C) Determination of cortisol

0.5 ml of a sample containing cortisol (standard, plasma or urine) was extracted twice with methylene chloride (2X3 ml). The combined extracts were evaporated to dryness. The residue was taken up in 0.5 ml of physiological saline and mixed with 0.2 ml of cortisol-21-galactose oxidase in a suitable concentration and 0.3 ml of transcorcinol-Sepharose suspension (5 mg / ml). The mixture was rotated for 15 min at 4 ° C. and centrifuged. The enzyme activity in the supernatant liquid was then determined by adding 0.5 ml of this liquid to 1.5 ml of a substrate. The substrate consisted of 100 mg of D-galactose , 20 mg of 5-amino-salicylic acid and 10 (xg peroxidase in 150 ml of 0.02 m phosphate buffer with a pH of 6.0. 30 min later, the absorbance was measured at 460 nm.

5 ng / ml cortisol in the sample led to a measurable increase in enzyme activity in the supernatant. Corticosterone and progesterone only affect the system when larger amounts have been added. Testosterone and s aldosterone had little influence.

Example 7 Determination of transcortin

The reagents used to determine cortisol as described in Example 6 m were also used to determine transcortin.

From a dilution series of transcortin from 0 to 1280 ng / ml, 0.5 ml was incubated for 15 min at 4 ° C. with 0.2 ml cortisol-21-galactose oxidase in a corresponding dilution. 0.3 ml was added to this dilution series Trans-cortin-Sepharose (15 mg / ml) was added and the mixture was rotated at 4 "C for 15 min. The activity of the supernatant was measured as described in Example 6.

A sample containing 40 ng / ml transcortin showed a measurable increase in enzyme activity in the supernatant liquid, whereas in the presence of 320 ng / ml all enzyme activity was found in the supernatant liquid.

C.

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