METHOD FOR ACCELERATED LINE IMMUNOELECTROPHORESIS FOR BURKHOLDERIA DIFFERENTIATION Russian patent published in 2013 - IPC G01N33/561 

FIELD: medicine.

SUBSTANCE: invention concerns a method for accelerated line immunoelectrophoresis (LIEP) for burkholderia differentiation. The method involves preparing an antigenic preparation followed by conducing line immunoelectrophoresis of the examined microorganism antigens and analysing the results; for the purpose of detecting the malleus and melioidosis pathogens quickly using rabbit or goat antiserums with an antibody titre in an agar gel immunodiffusion (AGID) assay not less than 1:16, prepared of VSE immunisation from acetone-dried cells of the strains B. pseudomallei 100 or B. mallei 10230 sonically pre-processed twice for 2 min at 50 Hz and cooled, containing the specific high-molecular antigen complex antibodies of molecular weight not less than 336 kDa; the line electrophoresis is conducted in the following assembly: GelBond plate 8.5×10 cm is filled with 1% Calbiochem agarose 10 ml prepared in a tris-veronal buffer containing calcium lactate 0.1 g and sodium azide 0.7 g in the buffer 1 l, pH 8.6 dissolved in 5 times (the same buffer is used in electrode trays); a gel strip 4-5 cm wide is cut out at 1 cm from a plate edge from the anode side; the gel is melted, cooled to 48°C, added with serum 5-10 vol. % containing the examined antigen antibodies, the plate is filled once more; a contact gel strip 1-2 mm wide is left between the serum gel and the analysed antigen samples; after serum gel setting-up, rectangular gel samples with the antigens to be examined are placed on the border of the contact strip; gel samples are prepared in the same way as the serum by introducing the examined antigens 5-10 vol. % into the cooled gel; 5 filter-paper bridges impregnated in the electrode buffer are attached to the gel borders; the electrophoresis is conducted at electric field intensity - 4-5 V/cm for 3-5 h at room temperature; after electrophoresis termination, the plates are surveyed; if observing any precipitate lines on the border of the gel sample and the rabbit or goat antiserum gel containing the antibodies to the examined antigen complex under the given LIEP conditions testifies to the presence of the pathogenic B. pseudomallei and B. mallei antigens; the negative result shows the absence of the pathogenic microorganism antigens that enables differentiating the malleus and melioidosis agents caused by non-pathogenic closely related burkholderia.

EFFECT: method has simplicity and speed of execution, visibility, possibility to determine the general and specific Burkholderia antigens, selection of the antiserums containing antibodies to the respective antigens, possibility to quantify the results, no complex equipment required.

5 dwg, 4 ex