A method of obtaining a suspension of diagnosticum (options)

 

(57) Abstract:

The invention relates to biotechnology. The methods of obtaining the suspension of diagnosticum by sensitization silica matrix immunoglobulins, as the carrier using a suspension of colored particles Aerosil, in the second case, particles of aluminosilicate. These methods allow to simplify and shorten the cooking time of diagnosticum and time studies. 2 S. p. f-crystals.

The invention relates to biotechnology and can be used in the production of suspension-based assays for antigen detection for rapid diagnosis of infectious diseases.

In recent years the tendency of the use of inert materials as carriers of ligands that have led to the development and application of the reaction suspension agglutination (PCA) to identify the causative agents of various diseases.

A method of obtaining the erythrocytic diagnosticum [1], including sensitization formalisation erythrocytes ligand in the presence kalugerovo agent with their subsequent laundering. Sensitization is carried out at a temperature of 54-S, and as conjugating agentel erythrocyte drugs limited first and foremost feature of the biological medium and the nature of the interaction between sensitiva and matrix. Of great importance is the quality of red blood cells, is dependent on many circumstances, such as gender, age, hormonal status of the animal producer, time of blood collection. In this regard, the reproducibility of the results can vary. The disadvantage is the length of the erythrocytic diagnosticums and that the final analysis of the reaction spend 16-18 hours.

Promising is the creation of diagnosticum synthetic based.

A method of obtaining the diagnosticum for serological reactions on synthetic particles due to the use as a carrier suspension painted polyacrolein particles treated with a solution of casein, activated cross-linking agent and sensitized by antibodies at pH 7,2-7,6 [2]. Use diagnosticum to identify antigens in the reaction suspension agglomeration (PCA) in the wells of a U-shaped microplate. Preparation of diagnosticum takes 7-8 hours, records of results performed in 1.5-2 hours

The aim of the invention is to simplify, reduce the time of Prigat is some sensitivity of diagnosticum.

This goal is achieved by a method of producing diagnosticum for serological indication of the causative agents of especially dangerous infections by binding immunoglobulin sensitive matrix on the basis of silicon compounds in a buffer solution at pH (7,2+0,2), in which the matrix is suggested to use Aerosil (fumed form of silicon dioxide or a silicate with the addition of the dye (auramine or Magenta), taken in a weight ratio of 2:1 as an activator of surface 1% secondary alkylsulfate sodium. Sensitin - immunoglobulin G immune serum. Sensitization is carried out at a temperature of (37+1)within 2 hours.

The choice of the matrix of Aerosil and aluminosilicate due to the fact that they are characterized by high reactivity, surface groups when interacting with many organic and inorganic compounds. Introduction to the matrix of the dye causes a color effect, resulting in increased visibility serological test.

Activating matrix secondary alkylsulfates sodium leads to prevention of approach of the particles, their aggregation, and is formed on the surface of monomolecular adsorption layer.structure and effective identification of causative agents of especially dangerous infections.

When increasing the pH buffer solution during sensitization reduced the percentage of binding sensity.

Within 2 hours is the maximum binding of immunoglobulins. Suspension of complex matrix-antibody should not give agglutination when interacting with heterologous antigens. To lock the free active sites of the matrix using bovine serum albumin (BSA) up to 1%.

Compared with the prototype [2] the proposed method reduces the cooking time of diagnosticum 2 times and scoring - 1-5 min, the proposed method there is no need for tablets, and the reaction is also evident.

The method is as follows.

Example 1. 100 mg of Aerosil, 50 mg auramine is ground in a porcelain mortar, sift. This powder to prepare a 2% suspension in 0.1 M phosphate-saline buffer solution (FSB), pH (7,2+0,2). Then hold the activation matrix, adding 0.075 ml secondary alkylsulfate sodium, inquira for 30 min at a temperature of (37+1)C. For sensitization using tularensis antibodies at a concentration of 0.5 mg/ml, 3 ml, incubated for 2 h at a temperature of (37+1), washed from unbound antibodies, BSA 1% suspension of diagnosticum and add 1% formalin.

The method of setting reaction.

To skim the glass, apply 2 drops of mist tularemia microbe in a concentration of from 21071104M. K./ml On glass negative control, apply 2 drops of 0.1 M FSB. On glass with experience and a negative control, add 1 drop of diagnosticum suspension tularemia immunoglobulin. Drops mix thoroughly and with a light rocking glass see within 1-5 min in comprobadas light of the changing structure of the suspension. Thanks to the intense color of the suspension (yellow) significantly increases the demonstrative reactions and facilitated its visual records.

In negative control agglutination is absent, while on the glass with suspensions of tularemia microbe observed full enlightenment liquid from coarse cereals, i.e. a clear agglutination. The sensitivity of diagnosticum was 2,5106M. K./ml.

Example 2. The process is carried out analogously to example No. 1, except as a matrix use the aluminosilicate instead of Aerosil. The results are similar.

Example 3. The process is carried out analogously to example No. 1, except as sensitive use immunoglobulins hall is but example No. 2, only as sensitive use immunoglobulins cholera. The sensitivity of diagnosticum amounted to 2107M. K./ml.

Example 5. The process is carried out analogously to example No. 1, except as the use dye Magenta instead of auramine. The sensitivity of diagnosticum was 2,5106M. K./ml.

Example 6. The process is carried out analogously to example No. 2, only use dye Magenta instead of auramine. The sensitivity of diagnosticum was 2,5106M. K./ml.

Tested on 3 series-based assays: tularemia and cholera in 5 replications. The diagnostics checked In the SAR on the glass with Homo - and heterologous particles. Used for comparison of erythrocyte tularemia diagnosticum in RNA, prepared from the same series of serums.

In the test drugs have proven themselves as highly specific, allowing you to conduct a rapid diagnosis within 1-5 min, the sensitivity is not inferior to the erythrocyte diagnosticum, and expressnet surpassing the last, as the end result of RNA after 16-18 h

This method is economically advantageous because it significantly reduces how about the. The method of obtaining erythrocytic diagnosticum. RF patent N 1774872, a 61 K 47/00, published 07.11.92, Bulletin No. 41.

2. E. P. Erokhin, S. C. Prozorovskii, I. S. Tartakovsky, O. C. Radchenko, Y. C. Lukin, V. P. Zubov, N. D. Temezhnikova, Century, Galtseva, C. M. Kostyukovsky, I. A. Bazikov, A. And Zaitsev, A. N.Avdeev and N. To.Mazurenko. The method of obtaining or antibody-based test diagnosticum. Copyright certificate №1596926 G 01 N 33/544, With 12 N 1/00, published 24.02.89.

1. A method of obtaining a suspension of diagnosticum by sensitization silica matrix immunoglobulins followed by washing of the target product, characterized in that as a matrix using a suspension of colored particles Aerosil activated secondary alkylsulfates sodium, the ratio of the matrix to the dye by weight is 2:1, sensitization is carried out at a temperature of 37 1C for 2 h, at pH of 7.2+0.2 and the ratio of 0.5 mg of immunoglobulins 50 mg matrix.

2. A method of obtaining a suspension of diagnosticum by sensitization silica matrix immunoglobulins followed by washing of the target product, characterized in that as a matrix using a suspension of colored particles of aluminosilicate, activated secondary Alki and a temperature of 37+1C, within 2 hours, at a pH of 7.2+0.2 and the ratio of 0.5 mg of immunoglobulins 50 mg matrix.

 

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