SU1654750A1 - Method for immunoenzymatic analysis caring out - Google Patents

Abstract

The invention relates to medicine, in particular, to determination of the concentration of bio-LOGIC substances by an enzyme-linked immunosorbent assay. Objective of the invention — Increasing the sensitivity of the assay. Sensitize substrate is incubated with test samples. Then the substrate is washed and labeled conjugate is added. immediately before the measurement of iodine ions, the substrate is heated at 60–70 ° C. Compared to the prototype, the sensitivity of the analysis is increased by a factor of 100

Description

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The invention relates to medicine, veterinary medicine, agriculture, in particular, to methods for the immunoenzymatic determination of biologically active substances in various media and can be used in enterprises of the medical biotechnological and food industries.

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

The method is carried out as follows.

Electrochemical determination of the marker enzyme is carried out by molecular iodine — the product of peroxidase oxidation of iodide ions with hydrogen peroxide at pH 3.8–4.2 in the presence of 0.07–0.08% starch, followed by destruction of the starch-iodine complex by heating 60-70 ° C immediately before measurement.

Example An immunological reaction is poured into 200 µl y-globulin fraction of rabbit antiserum against L-acnapa-gichase containing 20 µg / ml protein in the wells of polystyrene plates, and 001 mol / l phosphate buffer (pH 7 4) is incubated for 2 hours at 37 ° C, the wells were washed 4 times with 0.01 mol / l phosphate buffer (pH 7.4) containing 0.15 mol / l sodium chloride and 0.05% Triton X-100. Then, 200 µl solutions with various the content of L-ac conjugate-paraginase-peroxidase on the background of the wash buffer is incubated for 1 hour at 37 ° C. Then repeat Washing is applied 5 times, 200 µl of substrate mixture containing 0.75 mmol / l of potassium iodide 0.3 mmol / l of hydrogen peroxide and 0.07% of starch on the background of O 1 mol / l acetate buffer, pH 4 1 are added. 50 min at room temperature. Results are recorded after injection. 100

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µl sample into the flow line of the amperometric sensor. The temperature of the flow line and the sensor is set to 70 ± 0.5 ° C.

EXAMPLE 2. The determination is carried out by a direct coupling method. For this purpose, 200 µl of the y-globulin fraction of rabbit antiserum against L-asparaginase containing 20 µg / ml of protein against a background of 0.01 mol / l phosphate buffer (pH 7.4) are added to the wells of polystyrene plates for immunological reactions. incubated for 2 hours at 37 ° C. The wells are washed 4 times with 0.01 mol / L phosphate buffer (pH 7.4). containing 0.15 mol / l sodium chloride and 0.05% Triton X-100. Then, 200 µl of solutions with different content of L-asparaginase-peroxidase conjugate are added to the wells against the background of wash buffer, and they are incubated for 1 hour at 37 ° C. The washing operation is repeated 5 times. Then, 200 µl of a substrate mixture containing 0.75 mmol / l of potassium iodide, 0.3 mmol / l of hydrogen peroxide and 0.08% of starch against a background of 0.1 mol / l of acetate buffer (pH 4.1) are added, incubate BOW at room temperature. The results are recorded after injection of 100 µl of the sample into the flow line of the amperometric sensor. The temperature of the flow line and the sensor is set to 60 ± 0.5 ° C.

It is statistically established (according to Student's t-criterion at P 0.95) that the lower limit of the definition of the conjugate is

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The recommended method is equal to 8 mol / l (1.0 ng / ml) in 1-asparaginase, by the known method 1 -10 mol / l (13.0 ng / ml).

Thus, the proposed method provides a significant increase in the sensitivity of the enzyme immunoassay with electrochemical detection of both the marker enzyme (from 1 10 to 6 mol / l) and the labeled antigen (from 1 10 10 to 8 10 12 mol / l), i.e. . 100 times compared with the known method.

Using the proposed method allows enzyme immunoassay of various physiologically active compounds, in the presence of immunoperoxidase diagnostics with high sensitivity. In this case, it becomes possible to expand the range of detectable objects, the content of which in the analyzed media is picomolar.

Claims (1)

Invention Formula An enzyme immunoassay method, including sensitization of the substrate, incubation with labeled conjugate, washing followed by the addition of the substrate and measuring ions, characterized in that, in order to increase the sensitivity of the method, after addition of the substrate, starch is added at a concentration of 0.07-0 , 08%, and before measuring the ions, the substrate is heated to 60-70 ° C. 35