The method of producing drug for indication of the toxin of diphtheria pathogens in a liquid nutrient medium

 

(57) Abstract:

A new method of producing drug for indication of diphtheria toxin in liquid nutrient medium. The method is carried out in 0.05 M glycine-saline buffer solution with a pH of 8.2 - 8,25 by immobilization of gammaglobulin, immune to the toxin of diphtheria pathogens on the surface of particles of polystyrene latexes activated methacrylic acid and methacrylate zinc, in the presence of bovine serum albumin, polyethylene glycol and preservatives. Used latex is a mixture of modified polystyrene latexes with particle size of 0.20 - 0.25 μm and 1.1 to 1.2 μm. Components for the production of drug taking in a certain weight ratio.

The inventive method relates to the field of Microbiology, and in particular to a drug necessary for carrying out immunological analysis to indicate the toxin of diphtheria pathogens.

Specified drug is needed to identify toxinogenesis the causative agent of diphtheria patients and carriers, to monitor the effectiveness of treatment of patients with diphtheria and sanitation carriers of the causative agent of diphtheria, for assessing the efficacy of preventive and protivoradarnyh articles Mazurova I. K., Kombarov C. Yu, L. Spring M, Grigorian, K. Reaction of coagglutination for the detection of diphtheria toxin. Journal of Microbiology, epidemiology and Immunobiology". - 1989 - N 3 C. 68-74; Osipova, F., Barashkova L. N. Determination of toxigenic properties corynebacteria diphtheria in the reaction of indirect hemagglutination with the use or antibody-based test diagnostic drug. Abstr. Novgorod, 1989. Topical issues of infectious diseases and its prevention in the Novgorod region. 8th conference of the epidemiology, Microbiology, Parasitology. - S. 54-56; Mazurov, I. K., Potemkin, E. E. , Sviridov centuries, Zaitsev, E. M. Detection of toxin and the assessment of the degree of coccinobaphi Corynebacterium diphitheriae using enzyme immunoassay. "The journal of Microbiology, epidemiology and Immunobiology", - 1989 N 6 C. 66-70).

In the first drug as a carrier of antibodies used protein And Staphylococcus, which complicates the technology of preparation of the drug and does not provide receive high quality product. The second drug used in the reaction of indirect haemagglutination, is a sheep erythrocytes loaded with antibodies to diphtheria toxin. This drug provides good sensitivity of the method, but obtaining the purified antibodies to diphtheria toxin, adsorbed on the surface of special tablets. Immunoassay method has high sensitivity, but costly, complex staging, requires special equipment and trained personnel.

The prototype of the proposed method is latex preparation, previously proposed by the authors for the identification of streptococcal groups A, B, C, D and described in applications NN 92008090, 92008091, 92008092 of 26 November 1993, the Essence of the prototype is immobilization gamma globulin, immune to Streptococcus, on the surface of latex particles, which are particles of polystyrene of different dimensions and activated methacrylate zinc.

The technical essence of the proposed method is that the target of the drug is produced by sorption of highly purified, immune to diphtheria toxin antibodies on the latex particles produced from polystyrene modified with methacrylic acid and methacrylate zinc. The process includes the following operations.

1. The mixture of antibody-gammaglobulin, immune to diphtheria toxin diluted in 0.05 M glycine-saline buffer solution with a pH of 8.2, latex, diluted with the same buffer solution to the latex content in the buffer solution of 1%m zinc with surround enabled dispersion 1,1 - 1.2 µm and latex polystyrene carboxylating with a dispersion of 0.2 - 0.25 μm. The volumetric ratio of these latexes in the mixture is 1:2.

2. Received on p. 1 mixture incubated at 56oC for 45 minutes

3. The mixture obtained under item 2, cool to 4 - 6oC.

4. The cooled mixture is mixed with bovine serum albumin, pre-diluted with 0.05 M glycine-saline buffer solution with a pH of 8.2 to a concentration of 0.1%.

5. To the mixture obtained under item 4, enter the polyethylene glycol-6000 and preservative.

6. The components necessary to prepare the target of the drug, taken in the following ratio in g/l:

gammaglobulin, immune to diphtheria toxin - 0,05 - 0,06

the latex mixture - 2,50 - 2,55

bovine serum albumin - 0,5 - 0,55

the polyethylene glycol-6000 - 55,0 - 60,0

preservative - 0,1 - 1,0

0.05 M glycinato-salt buffer solution with a pH of 8.2 - 8,25 - Rest

The inventive method is simple and provides highly sensitive drug.

The authors argue that the claimed method meets the criteria of the invention "inventive step", since on the basis of acquaintance with scientific, technical and patent information, and twitcam from the prior art, related to the declared object.

The inventive method is carried out as follows.

1. Gammaglobulin, immune to diphtheria toxin, plant 0.05 M glycine-saline buffer solution with a pH of 8.2 to a concentration of 0.25 mg/kg

2. Polystyrene latex, activated methacrylate zinc, with a particle size of 1.2 μm bred 0.05 M glycine-saline buffer solution with a pH of 8.2 to a concentration of 1% (based on the polymer.

3. Polystyrene carboxypropanoyl latex with a particle size of 0.2 μm bred 0.05 M glycine-saline buffer solution with a pH of 8.2 to a concentration of 1% (based on the polymer.

4. Prepared by PP. 2 and 3 polystyrene latexes take in a volume ratio of 1:2.

5. 10 ml of the composition obtained under item 1, incubated with 10 ml of latex suspensions, obtained under item 4, in a water bath at 56oC for 45 minutes

6. The mixture obtained under item 5, cooled to 4 to 6oC for 18 - 20 h, add 20 ml of 0.1% solution of bovine serum albumin in 0.05 m glycine-saline buffer solution with a pH of 8.2, containing 12% peg-6000. In the resulting mixture was added 0.004 g of thimerosal (preservative).

The results of operations item is manufactured in the above way, it has a high specificity, which is manifested in the absence of cross-reactions against other antigens toxins secreted by pathogens other diseases.

The results of sensitivity studies of the drug received in response latex agglutination (RLA) presents test RLA on glass with diphtheria toxoid - positive at a concentration of 10-3- 10-4Lf/ml

Note: Lf - unit flocculation toxin causative agent of diphtheria.

The method of producing drug for indication of the toxin of diphtheria pathogens in a liquid nutrient medium under conditions of glycine-saline buffer solution with a pH of 8.2 to 8.25 by immobilization of gammaglobulin, immune to diphtheria toxin, particles of polystyrene latex, including stabilization in the presence of bovine serum albumin, polyethylene glycol-6000 and preservative, characterized in that the process is carried out in glycine-saline buffer solution with both molarity of 0.05, as polystyrene latex, a mixture of polystyrene latex with a particle size of 1.1 to 1.2 μm, activated methacrylate zinc when surround is enabled, and carboxylating polystyrene latex with a particle size of 0.2 to 0.25 m
The latex mixture 2,5 2,55

Bovine serum albumin 0,5 0,55

The polyethylene glycol-6000 55 60

Preservative 0,1 1,0

0.05 M glycine-saline buffer solution with a pH of 8.2 to 8.25 Ostalnoe

 

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