SU1285374A1 - Method of determining peroxydase - Google Patents

Abstract

The invention relates to analytical chemistry and can be used in medicine, agriculture, chemical, food, pharmaceutical industry. To quantify peroxidase, dilutions of the analyzed solution containing peroxidase were prepared by adding a solution of hydrogen peroxide in sodium acetate buffer. Using a microdosing device, dilute the iodide of a silver photographic plate in the dark on the surface. After incubation, the photographic plate is developed, fixed, dried. A qualitative assessment of peroxidase is performed by microphotometry of spots on a photographic plate and comparing the data with a calibration curve. The invention makes it possible to simplify the determination of peroxidase while maintaining the sensitivity and accuracy of the analysis. 2 tab. S Yu 00 SP 00 |

Description

112

The invention relates to analytical chemistry and can be used in medicine, agriculture, chemical, food and pharmaceutical industries, for example, to determine the enzymatic activity of gene-containing substances and biological preparations.

The purpose of the invention is to simplify the method for determining peroxidase.

The method is carried out as follows.

Prepare various peroxidase concentrations on a constant background of hydrogen peroxide in sodium acetate buffer pH 3.8. Peroxidase-containing solutions are applied to an emulsion surface moistened with distilled water or buffer iodide of silver photoplate in the dark using an automatic microdosing unit. After a certain time interval, depending on the expected peroxidase concentration, the photoplate is developed, fixed, and dried. Peroxidase quantification is carried out by microphotometry of spots on a photographic plate and comparison of data with a graduated graph.

Table 1

Example 1. To construct a calibration graph for determining the peroxidase, an emulsion surface of the IIford Q photoplate was applied using an automatic micropipette, 50 μl of peroxidase-containing solutions were prepared by serially diluting the outgoing horseradish peroxidase solution (RZ 0,6). , 1 M sodium acetate buffer (pH 3.8), containing 1 mM hydrogen peroxide. Range

the content of ieroxidase enzyme analysis using

second antibodies) is dissolved in saline. Protein concentration is determined on a spectrometer using nomograms at 260 nm and 280 nm. The peroxidase concentration is calculated using the formula C 0.44 mg / ml at L 403 nm. The resulting solutions are diluted with a 0.001 M solution of Hj Oj prepared in 0.1 M

(scale) contains 0.01 - 10.0 kg / mp of peroxidase.

The plate is left in the dark for 10 hours. Then, strewing solution drops, it develops. Obtained at the site of contact with the peroxidase-containing solution with a spot photometry was measured on a MF-4 microphotometer using a blackening scale. Build IgC calibration curve

SO

in coordinates 5 sodium acetate buffer pH 3.8. Different blackening. Coefficients of polysterification are carried out in the wells of variation in the determination of non-bulk microplates for carrying out an immunity of 10%. The results of the determination of the peri- nal (nological reactions, receiving 12 oxidases each) are given in Table 1. Dilutions (maximum - in 2048

0

EXAMPLE 2 Several crystals of a lyophilized immunoperoxidase conjugate for immunoenzymatic analysis: asparaginase — liep oxidase n immunoglobulin G donkey against rabbit immunoglobulin G labeled with peroxidase (respectively, conjugate for direct competitive immunoenzyme analysis for immunoglobulin G labeled with peroxidase (respectively, conjugate for direct competitive immunoenzyme analysis for immunoglobulin G labeled with peroxidase) the adjacent row of wells is Nl-standard concentrations of native peroxidase. The fitness of the conjugates was evaluated by comparing its activity with the activity of native peroxidase. on the effect on photographic plates. The results of the determination of peroxidase in conjugates for ELISA are presented in Table 2.

Asparaginase-peroxide conjugate Formula of the invention A method for determining peroxidase by carrying out an enzymatic oxide with hydrogen peroxide followed by taking into account the results obtained, characterized in that in order to simplify the process, in three samples, the conjugate has satisfactory activity, in one case it is unsatisfactory (12%) and cannot be used in an immuno-enzyme assay.

Thus, the best practices in terms of sensitivity and accuracy, proposed by

is a simpler way.

I

table 2

mental oxidation is carried out on a photographic material containing silver iodide, and the peroxidase concentration is determined by the intensity of illumination of the photographic material used after its appearance when compared with the calibration curve.

Claims (1)

SUMMARY OF THE INVENTION A method for determining peroxidase by conducting enzymatic oxidation of iodides with hydrogen peroxide, followed by the results obtained, characterized in that, in order to simplify the method, enzymatic oxidation ¹ is carried out on photographic material containing silver iodide, and the concentration of peroxidase is determined by the light intensity used photographic material after its manifestation when compared with calibration graphs. Compiled by E. Vorobiev Editor A. Szabo Tehred I.Popovich Corrector M. Samborskaya Order 7638/46 Circulation 776 Subscription VNIIIPI of the USSR State Committee for Inventions and Discoveries 113035, Moscow, Zh-35, Raushskaya nab., 4/5 Production and printing company, Uzhgorod, Project 4,