The method of obtaining colored 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 getting colored diagnosticum, including sensitization of latex - type core-shell, where the core - polystyrene latex sheath - copolymer of styrene and salts of zinc and copper unsaturated carboxylic acid at a mass ratio of links 1 : 0,3 - 3,0 : 0,3 - 3,0, moreover, the latex pre-incubated in buffer at pH 6.5 to 9.0 in the presence of 0.15 to 5.0 wt.h by weight of polymer of sodium dimethyldithiocarbamate within 1 to 3 months with subsequent sensitization at pH 7.0 - 9.0 and incubating at 37 - 56°C 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.

Closest to the proposed method is a method of obtaining diagnosticum for operosa polystyrene latex sensibiliser antibodies in 0.1 M glycine buffer at pH 7.0, and then carry out incubation at 37oC for 2 to 8 h [1].

Diagnosticum obtained by this method allows to detect HBsAg hepatitis B virus only at a concentration of 1 μg/ml of protein, it has a high enough frequency monopolistically reactions (30 - 40%). The specificity of such diagnosticum is expressed by the value 68,02 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 improving the sensitivity and specificity of diagnosticum to proteins and organic substances.

This objective is achieved in that in a method of producing colored diagnosticum for the determination of antigens and antibodies of infectious diseases, including sensitization of the modified polystyrene latex antibodies or antigens in a buffer solution at pH 7.0 and incubation sensitized latex, use a latex type core-shell, where the core - polystyrene latex sheath - copolymer of styrene and salts of copper and zinc unsaturated carboxylic acid at a mass ratio of 1 links : 0,3 - 3,0, with the specified latex pre-incubated in buffer pH of 6.5 - 9.0 in the presence of 0.15 to 5.0 wt.h. from the weight of the polymer is started when a 37 - 56oC within 24 - 72 hours Received diagnosticum can be used to determine HBsAg hepatitis B virus in a concentration of 1 PG/ml, hepatitis A concentration of 1 PG/MK, cancer antigens at a concentration of 10 PG/ml, protein doping control and alcoholism at a concentration of 50 PG/ml and serum of patients with AIDS, the virus-containing AIDS in known concentration. Diagnostics can be performed using serum, saliva and urine of the patient.

The specificity of diagnosticum is 97,0 2,2.

For diagnostic antigens and antibodies of infectious and other diseases used method of activated particles (MAH), namely, that the evaluation of the results of reactions performed according to the degree of activation of latex particles, visual or optical device. The degree of activation of the particles. Evaluation of the results of MAH,

+++ - large homogeneous flakes with a small amount of smaller to slightly turbid background, a positive result;

++++ big flakes in a fully transparent background, positive result;

++ - medium size particles in turbid background, equivocal result;

+ - fine grain particles in turbid background, negative result;

- - muteena latex type core-shell and diagnosticum is illustrated by the following examples.

Example 1. With the purpose of obtaining a polystyrene latex was charged to the reactor 10 wt.h. 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.

For part (A) of the obtained polystyrene latex was added 1 wt.h. styrene by weight of the total polymer (A), 1.0 wt.h. ethoxylated Nonylphenol OP-7, 0.3 wt.h. methacrylate zinc, 0.5.h. of potassium persulfate. The latex is stirred until dissolution of all components within 15 - 20 minutes and thermostatic at 70oC for 2 h Then enter 0.5.h. of sodium dimethyldithiocarbamate in the form of a 1% aqueous solution, the latex is washed by dialysis against 20 volumes of distillate from water-soluble impurities.

For part (B) of polystyrene latex was added 1 wt.h. styrene by weight of the total polymer (B), 0.3 wt.h. methacrylate copper (Cu2+), 1,0 wt.h. OP-7, 0.5. including potassium persulfate. The latex is stirred for 15 to 20 minutes and thermostatic at 70oC for 2 hours, the Latex is cooled and washed by dialysis. Part of the latexes (A and B) are mixed and used for diagnosticum.

Latex is characterized by a particle size of 0.5 μm and adsorption saturation of the surface Cham, glycerine-glycine buffer solution, pH of which 6,5 within 1 month.

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 37oC then 24 hours at 2oC.

The latex is also used for composite-based assays, for example, to different specific antigens of different cancers.

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

Example 2. The latex was prepared as in example 1, but using 3.0 mass.h. methacrylate copper, 3.0 mass.h. methacrylate zinc and 5.0 wt.h. dimethylthiocarbamate sodium.

The resulting latex incubated in 1 M glycerine-glycine buffer solution at pH 9.0 in 3 months. Then the concentration of the latex was adjusted with glycine buffer solution up to 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 incubated for 300 min at 56oC, and then at the 8oC for 72 hours.

The results of the diagnostic sleetgrout zinc and copper and 0.15 wt.h. of sodium dimethyldithiocarbamate.

The results are shown in table. 1.

Example 4. Get the latex of example 1, using 5 wt.h. the methacrylates of copper and zinc, sodium dimethyldithiocarbamate to enter failed, the latex was Smagulova. The results are shown in table. 1.

Example 5. Get the latex of example 1, using 0.3 wt.h. acrylate, copper and zinc. The results of diagnostic tests are given in table. 1.

Example 6. Get the latex of example 1, using 0.3 wt.h. itaconate copper and zinc. The results of diagnostic tests are given in table. 1.

The method of obtaining colored 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 - copolymer of styrene and salts of zinc and copper unsaturated carboxylic acid, in mass ratio units 1 : 0,3 - 3,0 : 0,3 - 3,0, paiticipate sodium for 1 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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