METHOD OF DETERMINING ANTIGEN STRESS LIMIT STIMULATING PRODUCTION OF HUMORAL ANTIBODIES Russian patent published in 2009 - IPC A61K39/00 C12N1/00 

FIELD: medicine.

SUBSTANCE: invention concerns medicine and development of conceptual immunisation procedure for experimental animals by antigens with unknown immunogenity parametres, related to efficient stimulation of humoral body production, allowing for determination of antigen stress limit which, if exceeded, results in reduction of antibody production function in microbe. Six animal groups are formed by 6-8 animals in each group. Each next group differs from the previous one in the number of injections of antigen under examination. Seventh group of intact animals from the same population, not injected with antigen forms reference group, yet they are used for obtainment of standard mice serum. For immunisation glycoprotein capsules of highly virulent Burkholderia pseudomallei 100 strain with molecular weight of 200 kDa and polysaccharide:protein ratio of 9:1-8:2 are applied. First injection is made to all test groups by the mix of equal 0.15 ml volumes of 25 mcg of antigen in saline solution and incomplete Freund's adjuvant subcutaneously in the back area. Over 7 days blood of first group animals is sampled. The other five groups undergo intraperitoneal injection of 0.5 ml of antigen solution in the dosage of 20 mcg per mouse. Over 7 days, when immunisation cycle is complete for respective group, blood sample is made, serum is separated, and solid-phase immunoenzyme method is applied for assessment of humoral body accumulation dynamics. For solid phase preparation, antigen in 10 mcg/ml concentration (for protein) is applied, polyvinylchloride plates are sensibilised at 4-8°C for 16-18 hours, individual serum samples are examined in solutions with twofold increment, beginning from 1:100 solution. When reaction is complete and hardware registration of reaction results is performed, statistic data processing for each animal group is performed, indicator differences between groups are verified, and animal group with highest indicators for antibody titre of 200 kDa Burkholderia pseudomallei antigen is the group obtaining optimal dosage stimulating humoral antibody production efficiently.

EFFECT: simple, unified multipurpose method of determining immunogenity of complex antigens for BALB/c line mice, allowing for relatively fast determination of antigen stress limit stimulating production of humoral bodies for this antigen.

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