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 diagnosis 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 the core - polystyrene latex sheath - copolymer of styrene and a salt of iron and/or cobalt and/or Nickel unsaturated carboxylic acid at a mass ratio of 1 links : 0.3 to 0.5, and the latex pre-incubated in buffer at pH 6.5 to 9.0 in the presence of peroxide, sensitization is carried out at pH 7.0 - 9.0 and incubated first at 37 - 56oWith over 80 to 300 rpm, and then at 2 - 8oWith within 24 - 72 hours of 8 tablets.

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

Currently, for the diagnosis of viral infections has received widespread the so-called indirect reaction, based on the method of activated particles. As dia is elficiency carriers of organic or inorganic nature. Such carriers are often erythrocytes, coal, bentonite clay containing A protein, a 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.

The closest is the way to get diagnosticum for the determination of antigens and antibodies and other infectious diseases, such as antigens, HBsAg hepatitis B virus, according to which polystyrene latex sensibiliser antibodies in 0.1 M glycine buffer, pH which to 7.0, and then carry out incubation at a temperature of 37oC for 2 to 8 hours [2].

Diagnosticum received this and the above methods allows to detect HBsAg hepatitis B virus only in civicnet such diagnosticum is expressed by the value (68,01 + 0,88). In addition, the resulting diagnosticum allows you to get the results of the study in 20 minutes

The purpose of the invention is the increased sensitivity and specificity 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 diseases, including sensitization of the modified polystyrene latex at pH 7.0 and incubation sensitized latex, use a latex type core-shell, where the core - polystyrene latex sheath - copolymer of styrene and metal salts and unsaturated carboxylic acid at a mass ratio of 1 links : 0.3 to 0.5, as the metal salt of unsaturated carboxylic acid using iron salts or cobalt and/or Nickel. The shell is 0.2 - 20% by weight of the whole particle.

The specified latex pre-incubated in buffer at pH 6.5 to 9.0 in the presence of 0.5 - 0.7 wt.h. organic gidroperekisi within 1 to 3 months, followed by sensitization with pH 7.0 - 9.0 and incubating at 37 - 56oC for 80 to 300 rpm, and then at 2 - 8oC within 24 - 72 hours Received diagnosticum used to determine HBSAg hepatitis B virus in the concentration of the of thenov doping control and alcohol at a concentration of 50 PG/ml, as well as the AIDS virus in a known concentration contained in the serum of patients with AIDS. For diagnostic use serum, or urine, or saliva of the patient specificity of diagnosticum the 98.9 0,2. Received combined diagnosticum to different specific antigens of cancer, infectious and other diseases.

For diagnostic antigens and antibodies of infectious and other diseases of the method of activating particles (MAH), namely, that the evaluation of the results of reactions carried out according to the degree of activation of latex particles, visual or optical device. Assessment of the MAH on the degree of activation of the particles is as follows:

4+ - large flakes of activated particles in a fully transparent background, positive result;

3+ is a large homogeneous grains of activated particles with a small amount of smaller to slightly turbid background, a positive result;

2+ - medium grain sizes of activated particles in turbid background, equivocal result;

1+ - small-activated particles in turbid background, negative result;

- - murky background, active particles, a negative result.

The method of producing latex depoliticising latex was charged to the reactor 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. Part A received polystyrene latex was added 1 wt.h. methacrylate iron, 0.5.h. of potassium persulfate, 1.0 wt.h. ethoxylated Nonylphenol OP-7. The latex is stirred until dissolution of all components within 15 - 20 minutes and thermostatic at 70oC for 2 h and Then injected with 0.1 wt. including sodium sulfate, the latex is cooled and washed by dialysis against 20 volumes of distillate from water-soluble impurities. The latex particles part A consist of two parts: the inner core is polystyrene, the outer shell is a copolymer of styrene and methacrylate iron at a mass ratio of 1 : 0.3 to. The mass ratio between the links of iron methacrylate and styrene, which is part of the core and shell is 0,297 - 100.

Then the latex is diluted glycine buffer solution up to 2% concentration and mixed in a ratio of 1 : 1 with monoclonal antibodies in a buffer solution at pH 7.0. The resulting mixture was sequentially incubated in 0.01 M glycine solution at pH 7.0 for 80 min at 37oM, then 24 h at 2oC.

The results of the diagnostic issuedata iron, 0.5.h. sodium sulfite and 0.7 wt.h. the gidroperekisi GEPCPB.

10 ml of 6% aqueous latex incubated in 1 M glycerine-glycine buffer solution pH 9.0 in 3 months.

Then the concentration of the latex was adjusted with glycine solution to a value of 2%, then mix equal volumes of 2% latex and monoclonal antibodies in 1 M glycine buffer solution at a pH of 9.0. Sensitized latex is then incubated at a pH of 9.0 during the first 5 h at 56oC, and then at the 8oC for 72 h

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

To determine the sensitivity of diagnosticum use serum AIDS patients, the serum of pregnant women, the antigens of the virus of hepatitis A and B and cancer.

The sensitivity of the combined latex or antibody-based test-based assays (FRET) is shown in table. 7 and 5.

Examples 3 and 4. Latex and diagnosticum prepared as in example 1, but using 0.5 wt.h. the gidroperekisi p-Mentana and tertbutylphenol (HPCUPS). The results of diagnostic tests are given in table 1.

Examples 5 and 6. Latex and diagnosticum prepared as in example 1, but using methacrylate of cobalt elasticum prepared as in example 1, but using a 0.3 wt.h. acrylate iron and 0.3 wt.h. itaconate iron.

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

Example 9. The latexes A and B prepared as in example 1, the latexes A and B separately subjected to sensitization and incubation in example 1, and then mixed. The results of diagnostic tests are given in table. 1.

Example 10. Latex and diagnosticum prepared as in example 1, but using a 0.3 wt.h. and iron methacrylate and 0.2 wt.h. methacrylate cobalt. The results of diagnostic tests are given in table 1.

Example 11.

Latex and diagnosticum prepared as in example 1, but using 0.2 wt.h. the iron methacrylate and 0.3 wt.h. methacrylate Nickel. The results of diagnostic tests are given in table 1.

The method of producing 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, use a latex type core - shell, where the core - polystyrene latex, Obolon the ratio of parts of 1 : 0.3 to of 0.5, and the specified latex pre-incubated in buffer at 6.5 to 9.0 in the presence of 0.5 - 0.7 PM by weight of polymer organic gidroperekisi within 1 to 3 months with subsequent sensitization at pH 7 to 9 and incubated first at 37 - 56oWith over 80 to 300 rpm, and then at 2 - 8oWith within 24 - 72 hours

 

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