The method of producing diagnosticum for the determination of antigens and antibodies of infectious and other diseases

 

(57) Abstract:

Usage: in medicine, veterinary medicine, food industry for the determination of antigens and antibodies of infectious diseases and cancer, alcoholism, drug abuse, drug testing, AIDS. The inventive method of producing diagnosticum, including sensitization of latex type "core-shell", where "core" is a polystyrene latex, the shell - sodium silicate at a mass ratio of 1 : 0.5 to 2.0, and the latex pre-incubated in buffer at pH 6.5 to 9.0 in the presence of 0.3 wt.h. by weight of polymer of zinc acetate or cobalt, or Nickel within 1 to 3 months with subsequent sensitization at pH 7 to 9 and a first incubation at 37 - 56oC for 80 to 300 rpm, and then at 2 - 8oWith within 24 - 72 hours of 8 tablets.

The invention relates to medicine, veterinary medicine and food industry, namely the method of producing diagnosticum for the determination of antigens and antibodies of infectious diseases and cancer, alcoholism, drug abuse, doping control and AIDS.

Currently, for the diagnosis of viral infections has received widespread the so-called indirect reaction, based on the method of activated particles. surface nonspecific carriers of organic or inorganic nature. Such carriers are often erythrocytes, coal, betonite clay containing protein And synthetic latex.

A method of obtaining the diagnosticum for determination of viral antigens and meningococcal infections based latex media [1]. In accordance with this method, 2% synthetic latex containing functional groups such as carboxyl, aldehyde or epoxypropyl, incubated in 0.1 M glycine buffer at pH 7.0 - 8.2 for 2 h at 37oC to obtain the target product.

Get diagnosticum more stable, has a high activity and specificity and allows results in a shorter time.

Closest to the invention is a method of obtaining diagnosticum for determining antigen HBs Ag hepatitis B virus, according to which polystyrene latex sensibiliser antibodies in 0.1 M glycine buffer, pH 7, and then carry out incubation at a temperature of 37oC for 2 to 8 h [2].

Diagnosticum received these and the above-mentioned methods, allows to detect HBs Ag hepatitis B virus only at a concentration of 1 μg/ml of protein, it has sufficient 68,02 0,88. In addition, the resulting diagnosticum allows you to get the results of the study in 20 minutes

The aim of the invention is to increase the sensitivity and specificity of diagnosticum to proteins and organic substances.

This objective is achieved in that in the method of producing diagnosticum for the determination of antigens and antibodies of infectious and other diseases, including sensitization of the modified polystyrene latex antibodies or antigens in a buffer solution, pH 7, and incubation sensitized latex, use a latex type "core-shell", where "core" is a polystyrene latex, a "shell" is sodium silicate, at a mass ratio of 1 : 0.5 to 2.

The specified latex in the presence of zinc acetate or cobalt, or Nickel incubated in buffer at pH 6,5 - 9,0 within 1 to 3 months with subsequent sensitization.

Sensitization is carried out in buffer solution, pH 7 to 9, and incubation are initially at a temperature of 37 - 56oC for 80 to 300 rpm, and then within 24 - 72 hours at a temperature of 2 - 8oC. the Obtained diagnosticum used to determine HBs Ag hepatitis B virus in a concentration of 1 PG/ml, hepatitis A concentration of 1 PC/ml, intercentral 50 PG/ml, as well as the AIDS virus in a known concentration contained in the serum of patients with AIDS. Diagnostics can be performed using serum, saliva and urine of the patient. The specificity of diagnosticum 98,0 - 0,2.

For diagnostic antigens and antibodies of infectious and other diseases (painted diagnosticum) the method of activated particles (colored) (MAH), namely, that the evaluation of the results of reactions carried out according to the degree of activation of colored latex particles visually or optical device. Assessment of the MAH the following:

4+ - large flakes painted activated particles in a fully transparent background, positive,

3+ is a large homogeneous grain painted activated particles with a small amount of smaller to slightly turbid background, positive,

2+ - medium size colored grains of activated particles in turbid background, equivocal result.

1+ - painted activated particles in turbid background, a negative result,

"-" - muddy background, colored particles is not activated, a negative result.

The method of producing latex type "core-shell" and diagnosticum illustr 10 wt. including styrene, 0.1 wt.h. of potassium persulfate, to 89.9 wt.h. water, and the reaction mixture thermostatic under stirring at 70oC until complete conversion of the monomer. Latex add 5% solution of sodium hydroxide to bring the pH of the latex to 11 and add 5 wt.h. by weight of polymer of sodium silicate in the form of a 5% solution. Then under stirring injected 0,3 wt.h. by weight of polymer of zinc acetate in a 1% solution.

Latex is acidified with 1% solution of acetic acid to pH 7. The resulting latex is washed by dialysis and is used for diagnosticum.

Latex before sensitization is kept in a buffer solution, such as 0.01 M glycerine-glycine buffer solution, pH of which 6,5 within 1 month.

The next stage of this method is the accession of the antigen or antibody to latex particles (sensitization). According to the invention sensitization is carried out in a buffer at pH 7, using monoclonal antibodies or antigens. Sensitization is carried out until complete bonding, that is, education for sustainable relationships between functional groups of the latex with protein and organic components of the monoclonal antibody or antigen.

The resulting mixture was sequentially To determine the sensitivity of diagnosticum use the serum of patients with AIDS, specific antigens of different cancers, the serum of pregnant women, the antigens of the virus of hepatitis B and A. the Sensitivity of the combined latex or antibody-based test of diagnostical (LAD) is shown in table. 7.

The results of diagnostic tests are presented in table. 1 - 8.

Example 2. The latex was prepared as in example 1, but using 20 wt.h. on the weight of the polymer of sodium silicate.

The resulting latex incubated in 1 M glycerine-glycine buffer solution at pH 9 for 3 months.

Then the concentration of the latex was adjusted with glycine buffer solution to a value of 2%, then mix equal volumes of 2% latex and monoclonal antibodies in 0.1 M glycine buffer solution, pH 9. Received sensitized latex incubated at pH 9 during the first 300 min at 56oC, and then for 72 h at 8oC.

The results of diagnostic tests are given in table. 1-8.

Example 3. Latex and diagnosticum prepared as in example 1, but using a 0.3 wt.h. by weight of polymer acetate cobalt.

The results of diagnostic tests are presented in table. 1.

Example 4. Latex and diagnosticum get primerov get diagnosticum for the determination of antigens and antibodies of infectious and other diseases, including sensitization of polystyrene latex antibodies and antigens in a buffer solution at pH 7 and incubation sensitized latex, characterized in that, in order to increase the sensitivity and specificity to proteins and organic substances, as latex uses latex type core - shell, where the core - polystyrene latex sheath - sodium silicate, at a mass ratio of 1 : 0.5 to 2.0, and the latex pre-incubated in buffer at pH 6.5 to 9.0 in the presence of 0.3 hours from the weight of the polymer of zinc acetate, or cobalt, or Nickel within 1 to 3 months with subsequent sensitization at pH 7 to 9 and a first incubation at 37 - 56oWith over 80 to 300 rpm, and then at 2 - 8oWith within 24 - 72 hours

 

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SUBSTANCE: invention relates to preparing aqueous cationic latexes with hollow polymeric particles serving as multifunctional additives used when preparing polymer compositions, paintwork materials, coatings (including paper coatings), and in other applications as white pigment and filler reducing density of material and internal stresses arising during formation of coatings or polymer materials. Process comprises at least three following stages: (A) preparing functional core copolymer, (B) preparing particles with core-shell morphology, and (C) ionization of functional groups of core copolymer. All process stages are carried out as a series of consecutive transformations in the same reactor without discharge of intermediate products. As monomer containing functional groups in stage A, vinylbenzyl chloride is used, and stage C represents amination of vinylbenzyl chloride units with tertiary aliphatic amines, pyridine, or derivatives of the latter.

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