SU1205913A1 - Method of making immunofermental analysis - Google Patents

Description

The invention relates to medicine and biological chemistry, in particular to enzyme immunoassay methods for the determination of biologically active substances in blood serum or other biologic material, and can be used in clinical diagnostic laboratories and enterprises of the Ministry of Medical Industry.

The purpose of the invention is to increase the sensitivity, simplify and reduce the time of the study when detecting the results of the ELISA analysis.

The method is carried out as follows.

I immunoassay determination of asparaginase.

Quantitative determination of asparaginase was carried out using a competitive method. 100 μl of a -globulin fraction of antibodies against asparaginase containing 0.1 mg / ml protein are added to the wells of the plates for immunological reactions. The plates are incubated for 18 hours at C, then they are washed five times with buffered saline at pH 7.4. In the wells containing immo. bilized antibodies. make 150 µl each of the sample to be analyzed and asparaginase conjugate with peroxidase corresponding to 10 µg / ml of native peroxidase. After a three-hour incubation at 37 ° C, the wells are again washed with physiological saline at pH 7 containing non-ionic detergent (0.05% Triton X-100). 150 µl of substrate mixture containing 0.2 mmol of hydrogen peroxide and 0.75 mmol of potassium iodide prepared in 0.1 M sodium acetate buffer at pH 3.8 with 0.1 M potassium chloride is added to the washed wells. After a 15-minute incubation at room temperature, 100 μl of the sample using; an automatic microdosing device; enter into the flow amperometric sensor

In the sensor, the product of the enzymatic reaction — pn – iodine molecules, electrically recovering on the surface of the indicator electrode, causes the appearance of a current in the external circuit of the sensor, the value of which is proportional to the concentration associated with the solid phase peroxidase label. It has been established that the linear portion of the dependence of the cathode current on the end

five

0

0

peroxidase concentration (marker enzyme) is observed in the concentration range 1-10 - 1 10 M. - Sensitivity factor is about I. IO μA / mmol. The number of samples analyzed with the aid of a flow ampere sensor per hour is 60-80.

The proposed method allows to determine up to 25 ng / ml of asparaginase (210 M) in serum with a volume of 50 µl, makes it possible to work in the kinetic mode and reduces the measurement time by 3-5 times.

The results of the determination of asparaginase in model solutions are given in Table I.

PRI im p Immunoenzyme determination of rabbit antibodies against X-potato virus.

The method is based on the sandwich method, which is implemented according to the following scheme. 0.2 ml samples of purified 1PO virus (100 µg / ml) are added to the moon and polystyrene plates, incubated with 5 at 4 ° C for 18 hours. Washed three times with 0.01 M potassium phosphate buffer at pH 7, 4, containing 0.05% of detey5

0

five

0

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Genta Twin-20. The immobilized preparation is sequentially injected with various dilutions of the test serum at 37 ° C for 1 h, and then, with the JgG peroxidase-labeled donkey anti-rabbit fraction (5000 ng / ml). During the analysis, the wells were washed with 0.01 M potassium phosphate buffer containing 0.1 M sodium chloride and 0.05% Tween-20 detergent. After washing, the amount of the sorbed immunoassay complex is determined ampermetrically. 150 μl of a substitute mixture containing 0.4 mmol of hydrogen peroxide and 1.5 mmol of potassium iodide prepared in O, 1 M sodium acetate buffer at 4.2 s O, GM with potassium chloride is added to the wells.

A 15-minute incubation at room temperature. 1 OOmcl of the sample is introduced into the flow-through amperometric sensor using an automatic microbatcher. The results obtained by the proposed method are comparable in sensitivity with the data: trophotometric recording and much simpler.

A comparative description of the electrochemical methods for enzyme-linked immunosorbent assay (ELISA) is presented in Table. 2

J + 2e - “2 J (recovery)

Two-electrode system with galvanic mode

The range of detectable concentrations of the product of the enzymatic reaction

or 0.5 ng / ml peroxidase

Sc I-IO

Lower Limit of Determination: 0.025 μg / ml of antigen laziness (antibodies) (L asparaginase)

Measurement time

40 s

Editor A. Kozoriz

Compiled by V. Drozdov

Tehred M.Nad Corrector S. Shekmar

Order 8583/7 Circulation Subscription

VNISH1I USSR State Committee

for inventions and discoveries 113035, Moscow, D-35, Raushsk nab., d. A / 5

Lilial PPP Patent, Uzhgorod, st. Project, 4

Table 1

NADH-NAD + 2e + H (oxidation)

Three-electrode system with a superimposed potential of + 0.75V. Ag / AgCl

8.7- Yu - 1.75-1b mol / l (according to NADH)

CH 2.5 µg / ml (phenytoy)

180 s.

Claims (3)

1. METHOD FOR IMMUNO-ENZYME ANALYSIS, including incubation of a detectable and labeled antigen or antibody with an immunosorbent and electrochemical determination of the concentration of the enzyme label, characterized in that, in order to increase sensitivity, simplify and shorten the study time, electrochemical determination is carried out by the product of peroxidase oxidation hydrogen peroxide at pH 2. The method according to p. Sh and th with the fact that, root in concentration, Iodide ions hydrogen peroxide 3.8-4.2. I distinguish iodide ions Bemmol: 0.75-1.5 0.2-0.4 3 cl I £ g. n * to SP CD 00 f 1205913 2