HU194284B - Process and in vitro reagent kit for the determination of primary glycol acid - Google Patents

Abstract

In a highly specific radio immunological assay method a hapten (1) is coupled with a protein, pref. BSA to obtain a new immunogen. The latter is used for immunisation and as a result an antiserum is obtd.

Description

Glycocols of the invention. storage kit, the in vitro reagent kit required for this, as well as the production of immunogen and kit components, high specificity antiserulin and tracem

It is a well-known fact that serum total acid concentration is elevated in patients with hepatobiliary disorders (liver, gall bladder and biliary tract). Biosynthesis and Conjugation of Primary Bile Acids, Cholic Acid (3a, 7α, 12a-Trichydroxy-50-Cholan-24-Carboxylic Acid) and Cenodoxoxy-Cholic Acid (3a, 7a-Dihydro-5-J-Colar-24-Carboxylic Acid) with Glycine and Taurine occurs in the liver, the conjugates are selected by the liver. bile, which is stored in the gallbladder. From there, they are transported to the dtiodennium via the bile / diet where they are emulsified and absorbed by the fat. Part of the bile acids in the intestine is deconjugated and is dehydroxylated by the intestinal bacterial flora and is called secondary bile acid, deoxycholic acid (3α, 12-xdihydroxy-5 / J-cholan-24-carboxylic acid) and htocholic acid (3α-hydroxy). 24-carboxylic acid). However, a small amount of bile acids is excreted in the faeces and absorbed from the intestine and returned to the liver by enterohepatic circulation. In healthy individuals, the liver filters bile acids from the potential circulation and recirculates artificially conjugated primary bile acids. If your hepatitis (liver cells) are damaged, inflammation, chemicals, etc. As a result, they will be unable to absorb bile acids, thereby increasing the concentration of primary bile acids in the peripheral circulation.

The increase in serum bile acid concentration is proportional to the degree of damage to the affected mast cells.

A number of methods have been developed for the measurement of bile acids, including gas chromatography (iGLC), high performance liquid chromatography (1HPLC) and gas chromatography (TLC). Their common disadvantage is that their sensitivity is inadequate, which requires a large volume of serum samples, labor, time and chemical preparation of the samples, and expensive measuring instruments.

For routine diagnostic purposes, only a test method that permits simultaneous measurement of a large number of serum samples with sufficient sensitivity is suitable. These requirements are met by the .adioimmune immunoassay (RIA) for the measurement of bile acids.

The first RIA method for the determination of conjugated derivatives of one of the primary bile acids, colic acid, was described by Simmonds et al. In 1973 (Gastroentero, ogy 65, 705, 1973). (Clin. Chim. Acta 54, 81 (1974), 72, 39 (1976); J. Steroid Biooem 13,440 () 980) The abolition of the radioimmunoassay enabled a large-scale clinical trial to be initiated. serous bile acids are a more sensitive indicator of hepatobiliary nerve disease than traditional liver function tests, such as BSP retention, prothrombin time, serum btlirubin, total serum protein, alkaline phosphase, and transaminase assay (N. Engl. (1975).

Determination of total acid levels may also be useful in the diagnosis of certain diseases of the heprtobiliary system, but it is generally accepted. the view is that by specifying serum bile acids, a more accurate diagnosis can be made. Japanese researchers include, for example, secondary wash acid conjugates such as glycolitolic acid (3a-hydroxy-5? -24-oxocolan-24-yl-glycine) and sulphoholcolitolic acid (3a-sulphonyloxy-5? - 24-o-o-cholan - 24-yl-glycine) using a 3- (0-carboxymethyl) oxime derivative as a hapten (Steroids 41.165 (1983).)

However, the method developed by most research teams mainly relies on the glycoconjugate of a single primary bile acid to determine glycolic acid (3a, 7a, 12 '-hydroxy-50-24-oxocholan-24-yl-glidn) as the liver is is able to extract to the greatest extent, about 80-9O70b.ni from the portal circulation. Therefore, changes in glycolic acid levels are considered to be the most sensitive indicator of hepatocyte injury (Clin. Chem / 10, 1698 (1981)).

The importance of the glycation of glycolic acid is further supported by the fact that glycolic acid is a bile acid that, unlike the free acid and its conjugates of monohydroxy and dihydroxy bile acids, is absorbed by active transport from the ternunae ileune. such as malabsorption, Chron disease, stagnant loop syndrome, etc. can be diagnosed on the basis of reduced serum glycolic acid (J. Pediatrics 96 / 3,582 (1980)).

It is known that radioimmunoassay requires a kit of reagents, one of the major components of which is antiserum, which can be obtained as a result of non-immunization. Anis / ions are characterized by three properties: titer, affinity, and specificity. The main disadvantage of antisera produced for the measurement of bile acids is that they are not specific enough, so that bile acids other than the bile acid to be measured are measured by the presence of other bile acids in the blood! also the antigen-annes complex formation, that is, the cross-reaction takes place. Undesirable cross-reactions are particularly problematic in the determination of bile acids, because in my stem the four major bile acids (kdic acid, canodeoxycholic acid, deoxycholic acid and lithol) are always present in at least three forms (free, glyco- or tauro-conjugated). Therefore, the numbers of bile acid measurement specialists have a particular task in eliminating or reducing cross-reactions.

A common disadvantage of RIA methods developed for the determination of glycolic acid is that the antisera used do not distinguish between glyco- or tauro-conjugated cholic acid, and to a lesser extent some other bile acids also rely on glycolic acid. This is because the antisera used are not specific enough (Gastroenterology, 1977, 72, 305).

Based on the foregoing, it is an object of the present invention to provide a process for the production of more specific antiserum than hitherto known which is suitable for primary bile conjugates. in particular the determination of glycolic acid.

The insufficient spedification of the antisera produced so far can be attributed to the structure of the immunogen used for immunization. Thus, there was a need for a novel fmmunogen capable of producing a more specific antiserum.

Bile acids are steroids and thus have a molecular weight of less than 1000, which makes them unsuitable for inducing an immune response

They are linked to 194,284 proteins. The researchers utilized the free carboxyl group of the glycine side chain of bile acids to produce a peptide bond with a protein, such as bovine (bovine) serum albumin (hereafter HSA), to produce immunogenesis of primary bile acids. It is common experience that anisera produced for steroid determination are most specific to the part of the steroid molecule that is "farthest" from the protein binding site and least specific to the part to which the protein is introduced. In the case of glycolic acid, the linkage to the protein has so far always been through the glycine side chain, so it was expected that. antiserum cannot distinguish between glycol and tauro-conjugates of cholic acid, and moreover, due to the inhibited spherical position of the 7a-hydroxyl group, interactions with the chenodeoxycholic acid and its conjugates are expected. The literature also confirms this (Clin. Cilim. Acta 54, 81 (1974); Gastroenetrology 65, 705 (1973)).

It has been discovered that if the protein binding site of the immunogen is not the glidn side chain, but the hydroxyl group furthest away from it, the aniserum produced can differentiate between glycol and tauro conjugate of cholic acid. In addition, the choice of the 3α-hydroxy group is advantageous because it is present in all bile acids, so that the antiserum need not be specific for this group. However, the 3 '-hydroxyl group of haptene, which is required for the production of immunogen, is not directly capable of forming a peptide bond, i.e. an acid amide, with the protein, and therefore the 3α-hydroxyl group has been selectively acylated with succinic acid. The free carboxyl group of the thus obtained succinate has already been used to form a peptide encoding. It was necessary to protect the carboxyl group of glycine so that BSA encoding would not occur at this site. The protection of the caboxyl group of glycine was resolved by ester formation.

The hapten of formula (I) was prepared by acylating the methyl ester of glycolic acid with succinic anhydride in acetonitrile at room temperature for 3 days. Spectroscopic data confirmed that additional hydroxyl groups of glycolic acid (7a and 12a) did not undergo acylation under these conditions.

The 3- (3-carboxypropionyl) ghkolic acid methyl ester thus prepared was used as a hapten to produce a new immunogen. Hapten is known to be coupled to a protein, preferably bovine serum albumin (BSA), according to the method known to Erlanger et al., J. Biol. Chem. 228 713 (1957), but the coupling is 1-ethyl-3- (3- diethylninopropyl) carbodinamide hydrochloride (J. Steroid Biochent. 13, 449 (1980)).

However, since hapten is a new structure, we had to modify the reaction conditions. Igv is the starting reaction mixture. The amount of steroid reactive with 1 mol of protein was raised from 40 mol to 100 mol, and during the course of the reaction the pH of the reaction mixture was maintained at 8.3 to 8.5. As a protein component, human serum albumin (HSA) or a peptide of appropriate molecular weight may also be used. The immunogen thus prepared was purified by dialysis (0.01 mol phosphate buffer, pL = 7.4) and the number of glycolic acid molecules bound to the protein molecules was determined from the ratio of tritiated gholic acid to the initial reaction and the dialyzed product in the final product. For example, 16 molecules of hapten (a novel glycolic acid dehydrogenase) were linked to a molecule of protein.

Aliquots of the above phosphate-buffered immunogen solution (containing 100 pg of immunogen) were frozen (at 20 ° C) until use.

As described above, the acid methyl ester of 3a- (3-carboxypropionyl) glycols was coupled to BSA by the mixed anhydride method.

SUMMARY OF THE INVENTION The present invention relates to a process for the production of a residual-novel immunogen for the production of an alpha X NH of formula II, an immunogen comprising the carboxyl group of a hapten of formula (1) and a suitable protein of formula X NH ₂ Acid bonding is performed between the amino group of the molecule in a manner known per se in peptide chemistry while adjusting the pH of the reaction mixture to between 8.3 and 8.5.

A further basis of the invention is the discovery that a new immunogen produced as described above is immunized to produce a specific anlis / ene.

An aliquot of the novel immunogen (3α- (3-carboxypropionyl) -glucocollic acid methyl) ester-BSA conjugate prepared according to the method of the invention was diluted to 0.5 m with saline, and a 1: 1 emulsion was prepared with Ereund's complete adjuvant. . Ai immunization was performed as known in the literature (Gastroenterology 65, 705 (1973); Clin. Cilim. Acta 72, 39 (1976)) as follows: The animal to be immunized, e.g., rabbit, sheep, sheep, pig, horse, etc. The immunogen was administered intradermally to 10 different sites of the spinal cord, once a week for 4 weeks, followed by monthly booster immunization. The titre and specificity of the antiserum produced was periodically checked with a glycollic acid tracer. At the end of 4 months from the start of immunization, the animal was bled by cardiac puncture and the antiserum was separated by centrifugation, and the specificity of the antiserum obtained was characterized by cross-reactivity compared to the best known literature (C) and commercially available reagent kits. B) with trap reactions antiserum lből characteristics obtained are shown in. Table 1 below. It can be seen that according to the invention practically antiszérunt glycocholic monospecific. With 100% cross-reactions with glycolic acid, Abraham, G.E. (J. Clin. Endocrinol. 29, 866 (1969)), the rate of aphid reaction with other bile acids is 2 to 3 orders of magnitude lower. Because of the rabbit biological dispersion, the specificity of the antisera obtained is not the same for the same immunization. It is a requirement that the cross-reactivity value for each bile acid should be at most 10% of that of glycolic acid, and this requirement is also satisfied by the results obtained with iodinated tracerr.

Another major component of the kit for radioimmunoassay is the tracer. Tracers are conjugates of haptens with amino acids or anins; which may be indicative of p ⁽¹³ⁱ í song or song Λ tracerekben ¹⁵¹ 1 - Ck is then established between the hapten and the amino acids or peptide bonds most commonly h.isznált amines for this purpose are the following amino acids and amines - like the immunogens: tyrosine-32...

194,284 Zin, Thiramine, Phenylglycine, Tryptophan, 5-Hydroxytryptophan, Witness and Histidine.

Table 1

Cross-reactions (%)

Bile acids (trivial name) Immunization data of the invention Literature data THE B i c glycocholic 100 85 100 100 taurocholic 0.5 100 130 16.5 cholic <0.1 3.1 50 7.8 Glikokenode- zoxikólsav 3.1 62 4.2 14.5 Taurokenode- zoxikólsav <0.1 210 1.3 3.2 Kenodezoxi- cholic <0.1 8.1 1.5 3.3 Glikodezoxi cholic 0.8 3.3 1.2 2.8 Taurodezoxi- cholic <0.1 4.0 3.7 1.7 deoxycholate <0.1 0.2 0.9 1.0 Glikohto- cholic <0.1 3.3 1.4 0.4 Taurolitokól acid <0.1 not ¹ 0.3 0.3 lithocholic <0.1 Exam. 0.7 0.1 0.1

A: Gastroenterology 72, 305 (1977).

B: International Atomic Energy Agency (ΙΛΕΑ)

-SM 200/87 (1977)

C: Clin. Chem. 27/10, 1698 (1981) and U.S. Patent No. 4,264,514. U.S. Pat

Tracer preferably used 3a- (3-carboxy-propionyl) ^{-glikokólsav-methylester-12} 'l-histamine conjugate of the invention is a reagent kit. The coupling of histamine with the haptene of formula (I), methyl 3- (3-carboxypropionyl) -glycolic acid, was carried out in a known manner by a mixed anhydride method (Brit. J. Sports. Med. 9, 89 (1975) but with a carbodiimide method). (IAEASM 220/87 (1977). Radiolabelling of iodine, for example with ¹¹⁵ I, can occur prior to or after coupling. ^{13 5} Idal labeling using the Chloranun-T technique (J. Endocrinol. 57, XVII. 1973) both before and after coupling.The resulting tracer was purified by column chromatography and thin layer chromatography.

Accordingly, the present invention further relates to a process for the preparation of a novel tracer comprising the step of forming a peptide coding between the free carboxyl group of a hapten of formula (I) and an amino acid or amine suitable for radiolabeling in the usual manner in peptide chemistry. before or after coupling is denoted by ^{12 5} 1 in known manner.

The high specificity antiserum and tracer produced by the process of the present invention enables accurate determination of glycolic acid. Therefore, another object of the present invention is to provide an in vitro reagent kit for the determination of glycolic acid comprising the following components a) to f):

a) The tracer produced by the process of the present invention is provided in a suitable buffer solution having a p1 = 7.4. The activity of the tracer solution is preferably max. 0.25 pCi / ml (9.25 kBq / ml).

b) The antiserum produced by the process of the invention is lyophilized, which can be used at a final dilution of 5 to 50,000.

(c) Lyophilized glycolic acid standards to be determined.

(d) Lyophilized control serum samples containing glycolic acid to be determined at known concentrations.

(e) A suitable buffer solution providing component (b) and a pH of 7,4 to dilute the samples to be tested.

(f) Reagent solution to separate the free and bound fractions.

The components a) to f) of the in vitro reagent kit for the determination of glycolic acid may be as follows for a 100 tube kit:

a) Tracer: 3 '- (3-carboxy propionyl) -glikokólsavmetil ester ^{and 13} l histamine konjugpt reagent solution 20 ni 0,9'Aos sodium chloride and phosphate buffer (PBS), pH

- 7.4). The activity of the tracer solution is n, 25 µCi / ml (9.25 kBq / ml) or less. The tracer solution also contains 0/2% bovin gamma globulin, 0.3% BSA and 0.1% sodium azide. To be measured: 0.2 ml per tube

b) Glycolic acid antiserum: the rabbit antiserum produced by the process of the invention in a lyophilized state. For reconstitution, 20 ml of sodium chloride phosphate buffer (pH 7.4) containing 0.2% bovin gamma globulin, 0.3% BSA and 0.1% sodium azide were used. To be measured: 0.1 per 0.2 ml tube.

c) Glycolic acid standards: 0 - 0.50 1.5 - 5.0 - 15.0

- solutions at a concentration of 50.0 µmol / l glycolic acid in the lyophilized state. For reconstitution, 6x0.5 ml of buffer solution is used. Quantity to record the standard curve: 0.1 ml per tube.

(d) Known low (0.50-1.5 pmol / l) and high concentration (15.0-50.0 μηιόΐ / l) glycollic acid control serum when lyophilized. For reconstitution, 2x0.5 ml distilled water was used.

e) Phosphate buffer solution (pH 7.4) containing 0.2% bovin gammaglobulin, 0.3% BSA and 0.1% sodium azide in a volume of 50 ml and containing antiserum (b) and used to dilute the serum samples to be tested.

f) Polyethylene glycol 6000 reagent: 60 ml of a 30% aqueous solution containing 0.9% sodium chloride and 0.1% sodium azide, which is used to separate the free and bound fractions. To be measured: 0.5 ml per tube.

With the above reagent kit, biological fluid samples (blood serum, bile, urine, thymus extract, etc.) are preferably used to determine the amount of glycolic acid in blood serum. The determination of glycolic acid by radioimmunoassay is carried out in a manner known per se, by allowing the above listed components to be kept at room temperature up to 4 ° C and dissolving the lyophilisates in distilled water or buffer. 0.2 ml of tracer solution is added to the first two RIA tubes (labeled Complete Activity). In tubes 3 and 4 (NSK = non-specific bond

194,284) 0.2 ml of tracer solution and 03 ml of gamma-globin-PBS solution were measured. Under tube 5. to 0.1 ml of standard solutions of glycolic acid (measuring 2 to 2 replicates) at 0; 0.5; 1.5; 5.0; 15.0 and 50.0 pmol / l glass bottles. 0.2 ml of tracer solution and 0.2 ml of antiserum are then added to the tubes and vortexed well. Starting from tube 17, weigh 0.1 ml (previously diluted 1: 10 in gamma globulin phosphate buffer) of the serum samples to be tested using duplicate samples. Similarly, 0.1 ml of properly dissolved control sera (low and high) are weighed. Subsequently, 0.2 ml of tracer and 0.2 ml of antiserum are added to each tube and shaken well. The tubes are incubated at room temperature for 1 hour, then 0.5 ml of polyethylene glycol solution (except 1-2) is added to each tube, fine! shake the samples with a vortex mixer and centrifuge at 1500 g for 15 minutes at room temperature. The supernatants were removed with a water air piston. The radioactivity remaining in the tubes is measured with a device capable of measuring gamma emitting isotopes for at least 1 minute. Divide the score of the standard solution tubes by the score of the standard 0 and multiply by 100 to give the binding (K)% to the standards, plotted against concentration to give a standard curve.

Dividing the tube of unknown serum samples by plotting standard 0 gives the cause of K% for the samples to be tested. Plotting these values against the standard curve, the bile acid concentration of the unknown serum sample can be read. The non-specific binding values are considered when calculating K% values.

The advantage of the method according to the invention over the known methods is that the most specific, very sensitive method for measuring the content of glycocholic acid in blood serum has been obtained so far, which allows more accurate diagnosis of diseases of the hepatobiliary system and the intestinal tract.

The invention is illustrated by the following non-limiting examples.

Example 1

Preparation of Intmunogen (Π)

17.4 mg (0.03 mmol) of 3- (3-carboxypropionyl) glycollic acid methyl ester in 0.2 mL of dioxane was dissolved with gentle heating. The solution was cooled to 8 ° C and radioactive ('H) -glycolic acid (0.04 pCi) was added. Thereafter, 7.1 μΐ (0.03 mmol) of tri-n-butylamine and 3.8 μΐ (0.03 mmol) of isobutyl ester of chlorocarbon were mixed. The reaction mixture was stirred for 30 minutes at 8 ° C. Bovine serum albumin (BSA) (21 mg, 0.30 μιηόΐ) was dissolved in 0.8 mid distilled water, 0.05 mL of 0.5 M sodium hydroxide solution was added and mixed with 0.8 mL of dioxane. The solution was cooled to 4 ° C, and the mixed anhydride prepared above was added dropwise to the BSA solution with constant stirring. The reaction mixture was stirred at 4 ° C for 3 hours under constant pH control, making sure the pH was between 8.3 and 8.5.

The pH was maintained with 0.5 M sodium hydroxide solution. After the reaction time, the radioactivity was determined from 1% of the sample volume, and the remainder of the sample was dialyzed for 3 days against 0.01 M sodium phosphate buffer, pH 7.4. 5% of the volume purified by dialysis was assayed for activity. Based on the two activity measurements, the protein / steroid molar ratio of BSA conjugate was 1:16. Anquots of the purified fraction (equivalent to 100 μβ protein) were frozen at -20 ° C until use.

Example 2

Preparation of Tracer: 3oe- (3-Carboxypropionyl) -Glycol Chloric Acid Methyl- ¹ ? ⁵ 1-histamine conjugate.

(a) Mixed anhydride preparation:

3.7 mg (6.4 μmol) of methyl 3- (3-carboxypropionyl) glycolic acid was dissolved in 0.1 ml dioxane and 10 μΐ tri-n-butylamine 1: 5 solution of dioxane and 10 μ 1: of a 1:10 solution of isobutyl chlorobenzyl ester in dioxane. The reaction mixture was stirred at 8 ° C for 20 minutes and then diluted with dioxane to 3.5 ml.

(b) ¹²⁵ liters of histamine:

37-74 MBq ^Na122I (MTA Isotope Institute: I-RB-4 1-125 solution in 0.1% NaHCO3, 7-11 GBq / cm ³ ) was added to the well, 222 ng of hyalamine was added. , 10 μΐ 0.5 μl / L phosphate buffer (pH 8.0) 10 μ μ Aqueous solution of 50 μβ chloramine-T. The reaction mixture was stirred for 30 seconds and then mixed with 300 μg of a 10 μΐ aqueous solution of sodium metabisulfite.

(c) Switching and cleaning:

50 μΐ of the mixed anhydride solution prepared in a) was added to the iodinated histamine solution prepared in b) at 0 ° C. Subsequently, ΙΟμΙΟ, ΙN sodium hydroxide was added to the reaction mixture and allowed to stand for 1 hour, then another 10 μ további of 0.1N sodium hydroxide was added and the solution was allowed to stand again for 1 hour, followed by 1 ml of 0.01 M using phosphate buffer (pH 7.4), the reaction mixture was applied to an Amberlite XAD-2 column (Ig) for purification. The column was washed with the above buffer and then with distilled water until the free ¹²⁵ L was completely removed. The column was then abs. was washed with alcohol and activity measurements were performed on 0.2 mL fractions of eluate. The active fractions (about 2 mL) were combined and concentrated to a quarter. For further purification, the evaporated fraction was applied to a thin layer of DC-Alufolien Kieselgel 60 (Merck). The starting mixture was composed of ethyl acetate-methanol-benzene triethylamine = 50:25:25:10. Detection was performed by autoradiography. The area corresponding to the spot at Rf 0.5 on the radiochromatogram was plated off the layer with methanol. Aliquots of this methanolic cldat (stored at 18 ° C) (20,000 cpm / 0.1 mL) were used as a tracer in PBS.

Example 3

Preparation of Tracer: 3- (3-Carboxypropionyl) -Glycolic Acid Methyl ¹² -I-Histamine Conjugate

s) Preparation of methyl methyl ester histaphthalate of .beta.-p-carboxyl- ^1- europionyl) -glycolic acid:

7.0 ng (35 pmoles) of histamine hydrochloride salt in 50 μl distilled water, 12.0 mg (60 μηύόΐ) of 1-ethyl-3- (3-dietH-51)

194.284 Amino-propyl) -carbodiimide HCl was dissolved in 50 µl of 1: 1 pyridine-water and then 5.8 mg (from 10 ppm) of 3- (3-carboxyppropionyl) glycolic acid methyl ester in 100 µl of pyridine. The reaction mixture also contained the tritiated derivative of the bile acid derivative (0.01 pCl). The solutions were combined, the ρΗ was adjusted to 6.0-6.3, and the mixture was stirred at room temperature for 72 hours. The solution was then diluted with 400 µl of 0.1 M phosphate buffer (pH 74) and extracted with 800 µl of butanol. The conjugate appeared at Rf 0.46 in ethyl acetate-methanol-pyridine-55:35:10 in a thin layer chromatogram. According to the activity measurements, the concentration of the conjugate in the butanol solution was 2400 nM.

(b) Marking ¹¹⁵ 1 and cleaning of the labeled material:

A 1 µl solution of the above conjugate in butanol was added to a labeled vessel and evaporated under a stream of nitrogen, then reconstituted with 10 µl of 0.5 M phosphate buffer, pH 7.5, followed by 37 MBq (1 inCi) of Na ¹² fl, 20 µg. Chloramine-T was added in 10 µl of 0.05 M phosphate buffer. The reaction mixture was stirred for 30 seconds and then mixed with 10 µl of a solution of 40 µg of sodium metabisulph and 10 µl of potassium iodide in buffer. It was diluted to 200 µl with buffer and extracted with 0.5 mL of butanol. The butanol phase was evaporated and the residue was dissolved in 100 µl of methanol and applied to a thin layer. The starting mixture was first mixed with ethyl acetate-methanol-pyridine ~ 58: 37: 5 and then purified as described in Example 2. Methanol aliquots of 40,000 cpm / 0.1 µl were used for the assays.

Example 4 Reagent kit:

A set of 100 tubes for the determination of glycolic acid contains the following components (a) to (f):

a) Tracer: 20 ml of 3a- (3-carboxy-propionyl) -glikokólsav methyl Aromatic plants ^12s I-histamine conjugate in PBS (e). The activity of the tracer solution is max. 0.25 pCi / ml.

b) Glycolic acid antiserum: lyophilized rabbit antiserum produced by the process of the invention, reconstituted with PBS solution (e) (20 ml).

c) Glycolic acid standards: 0-0.50-1.5-5.0-15.050.0 pmol / L solutions of glycolic acid lyophilized and redissolved in buffer (6x0.5 ml).

(d) Control serum samples: known low (0.5-1.5 pnol / l) and high concentration (15.0-50.0 pmol / l) glycolic acid sera, lyophilized using redistilled water (2x0.5 ml) .

e) PBS solution, pH 7.4, containing 0.2% bovine gamma globulin, 0.3% BSA and 0.1% sodium azide, 50 ml.

f) Reagent solution for separating the bound fraction: polyethylene glycol 6000, 30% aqueous solution, volume 60 ml containing sodium chloride and 0.1% sodium azide.

Claims (7)

PATENT CLAIMS A process for the preparation of a novel immunogen of formula II, wherein X-NH5 is a protein moiety for the production of immunogens, comprising coupling 70-120 moles of the hapten of formula I in a manner known per se in peptide chemistry. ₂ while maintaining the pH of the tea mixture between 8.3 and 8.5. 10 2. The method of claim 1, wherein the bovine serum albumin (BSA) is used as the X-NH ₂ . 3. A method for producing highly specific antiserum by immunizing warm-blooded animals, preferably rabbits. j. and by isolating the antiserum with 11 je ⁰ e comprises using as imintinogénnel formula I prepared according to the method of claim 1 (II) were immunized, and the antiserum kinyeijük known manner. 4. A process for the preparation of a new tracer labeled with ^{12 5} 1, comprising the step of forming a peptide bond between the free carboxyl group of the haptene of formula I and the free amino group of a radiolabeled antinoic acid or amine. labeled amino acid or before or after coupling ^{12 5} 1-dioxide. The process of claim 4, wherein the amine is histamine. 6. A reagent kit for the determination of glycolic acid in biological fluid samples, preferably in blood serum, by radioimmunoassay, characterized by a) the max. 30 tracer with activity of 0.25 pCi / ml in buffer solution pH 7.4, (b) the antiserum prepared by the process of claim 3 in a lyophilized state which can be used in a final dilution of 5 to 50 thousand times; (d) control serum samples containing known concentrations of bile acid to be determined, (e) reconstitution of component (b) and dilution of strain samples to be examined with pH 7,4 phosphate buffer solution, f) a solution of polyethylene glycol reagent separating the free and bound fractions. The reagent kit for the determination of glycocholic acid in blood serum according to claim 6, characterized in that (a) methyl 3- (3-carboxypropionyl) -glycolic acid ¹⁵ I-histane conjugate in PBS containing 0,2% bovine gamma globulin, 0,3% BSA and 0,1% sodium azide, __ (b) lyophilized rabbit antiserum, c) 0; 0.50; 1.5; 5.0; 15.0; Lyophilized glycolic acid standard solutions at 50.0 ppm, d) known low (0.5-1.5) pmole / l and high (15.0-50.0) pmole / l lyophilized glycolic acid serum samples, cg e) PBS solution (pH 7.4), containing 0.2% bovine gammaglobulin, 03% BSA and 0.1% sodium azide to dissolve component (b) and dilute the serum samples to be tested, f) a 30% aqueous solution of a polyethylene glycol reagent containing sodium chloride and 0.1% sodium azide, 60 contains.