SU1022712A1 - Method of quantitative determining of biologically active substance - Google Patents

Abstract

METHOD OF CATERING DEFINITION. BIOLOGICALLY ACTIVE BE-, CRUSHES by incubating 1 (and the studied material with specific serum in the presence of the labeled biologically active substance, taken in a certain concentration, followed by taking into account unprotected labeled substance, o t and h and so that, p. the purpose of gaining sensitivity of the method, the incubation is carried out in the presence of a biologically active substance, labeled with nicotine-midadene dinucleotide or adenosine triphosphate.

Description

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This invention relates to medicine and relates to a method for the quantitative determination of a biologically active substance.

A known method for the quantitative determination of antigen using fluorescent dyes.

There is also known a method for the quantitative determination of a biologically active substance (VLV) by incubating the test material with a specific serum in the presence of the labyrinth of the test BAS, taken at a certain concentration and then taking into account the amount of unreacted labeled matter. Sensitivity for

steroid hormones 10 m, at 2.

for insulin

However, the known method does not provide a high sensitivity of the method.

The aim of the invention is to increase the sensitivity of the method.

The goal is achieved by the method of quantitative determination of a biologically active substance by incubating the test material with a specific serum in the presence of a labeled test biologically active substance, taken at a certain concentration, followed by taking into account the amount of unreacted labeled substance, and incubating it in the presence of a biologically active substance labeled with nicotinamide adenine dinucleotide or adenosine phosphate.

Labeled NAD BAS is obtained by modifying NAD of gto Cg - amino group of the adenine ring, followed by binding the modified coenzyme to the BAS with carbodimide.

Example G. Definition of insulin.

Construction of a calibration curve for determining insulin in solution.

1 ml of a solution of insulin in phosphate buffer pH 7.8, containing standard amounts (0.05; 0.1; 0.5; 1.5; Yu; 20; kG; 60; 100 and 200 nm of insulin, is incubated with 200 μl guinea pig immune serum against pig insulin diluted 1000 times and 1 ml of a solution of the NAD-insulin complex (nicotinamide-denindinucleotide-insulin) for 2-25 minutes at.

After incubation, 0.1 ml of the incubation solution is added to a spectrophotometer cuvette containing 1.9 ml of 0.05 M potassium phosphate buffer pH 7, 0.1 ml of NDMA (para-nitroso-N N-dimethylaniline) and cyclohexanol / NDMA dissolved in cyclohexanol to a concentration of 40 mM, and then diluted with 0.05 M potassium phosphate buffer to a concentration of 300 µM / ml and 0.1 ml of alcohol hydrogenase. The decrease in the reduced form of NDMA is measured by the change in optical density at kkO nm. Calibration curve is presented in figure 1. Insulin determination is carried out as follows.

1 ml of human blood serum diluted 10 times with potassium phosphate buffer is incubated with 200 μl of guinea pig serum containing antibodies to insulin 100 times diluted and 1 ml of 10 M solution of NAD 5 insulin, after which the spectrophotometer is applied to the cuvette measuring the recovery rate of NDMA. All other reagents are added in the same ratios as when constructing the calibration curve. The insulin concentration is determined from a calibration curve.

Example 2. The definition of the 17th extradiol.

c To construct a calibration curve, 1 ml of a solution of 17-) b-extradiol in 0.05 M potassium phosphate buffer (pH 7.8), containing standard quantities of 10; 0.8; one; 1.5; 2.10; 20;

0 50 and 100 ng) are incubated with 1 ml of a M solution of the NAD-17-fb-3KCTradi complex with bovine serum albumin (BSA) diluted 100 times for 20 minutes at. Dose and dilution of antisera are selected to inhibit the recovery rate of NDMA by 60.

The definition of 17 - (- extradiol is carried out analogously to example 1.

The sensitivity of the determination of biologically active substances reaches 10,. n, especially for insulin M

In order to obtain complexes, adenosine triphosphate (ATP) with BAS ATP is pre-modified to ATPN -N - (6-aminohexyl) -acetamide and then covalently bound to the corresponding biologically active substance in the presence of soluble carbodiimide. Example 3. Insulin determination. Construction of a calibration curve. 1 ml of a solution of insulin in O, 1 M trisacetate buffer (pH 7.8, containing standard amounts (5; 10; 20; 60; LLP and 20 ng) of insulin is incubated with 100 µl of guinea pig immune serum, diluted in 1000 Pa and 1 ml of ATP-insulin complex solution for 20-30 minutes. The volume and dilution of initial antisera causing inhibition of bioluminescence with 1 ml of 10 M ATP-insulin complex solution per 80 are used for work. After incubation 0, 5 ml of the reaction mixture are added to a bioluminometer cuvette containing 1 ml of 0.1 M trisacetate o Buffer, pH 7.8; 0.2 ml 0.1 M MgSO solution, 0.2 ml O, m solution of luceferin and 0.2 ml solution of luciferase in the same buffer measure the bioluminescence intensity. The calibration curve is shown in FIG. .2 Upon receipt of the calibration curve, the content of in 1 1 4 suline in a sample of biological fluid, 1 ml of human serum divided by a factor of 10 can be determined. (P "1 ml of serum and 0.9 ml of tris-acetated buffer, pH 7, 3) incubated with 100 l of immune serum of guinea pig, diluted 1000 times, and 1 ml of 10 M solution of ATP-insulin complex for 2 0 min at, after which 0.5 ml of the reaction mixture is introduced into a cuvette to measure the bioluminescence intensity. All other reagents are added to the cuvette in the same ratios as the calibration curve. Based on the obtained intensity value, the insulin concentration determines the calibration curve. The detection sensitivity is about 10 N. Example 4. Determination of ampicillin concentration. For the immune response, antiserum obtained for the ampicillin-bovine serum albumin (BSA) conjugate is used, the dose and dilution of which are selected experimentally depending on the activity and antibody titer. A 10 M solution of the ATP-ampicillin complex is used as the labeled standard. The use of the method allows to increase the sensitivity (X1, to reduce the time of analysis.

Insulin concentrations, mim

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Claims (1)

METHOD OF QUANTITATIVE DETERMINATION. BIOLOGICALLY ACTIVE BE-, SOCIETY by incubating the test material with specific serum in the presence of a labeled test biologically active substance, taken in a certain concentration, followed by inconsistency. Aging labeled substance, with the exception of the fact that, with ·. in order to increase the sensitivity of the method, incubation is carried out in the presence of a biologically active substance labeled with nicotine midadenine dinucleotide or adenosine triphosphate. J ---------------------------- 1- 'f i J insulin concentration, t / n FnL __l>