Methods of determination and types of catalytic activity of polyclonal immunoglobulin A

Generalov I.I., Korotina O.L., Zherulik S.V., Generalova A.G., Volkova&⁸239;M.V.

Vitebsk State Medical University

The main goal of current work was to elaborate the methods of detection of oxidoreductase, proteolytic and nuclease IgA abzymes isolated from the sera of patients with systemic lupus erythemathosus (SLE) and oral fluids of healthy individuals and patients with chronic periodontitis with further assessment of catalytic parameters of abzyme IgAs.
Methods. Different methods of IgA purification from sera and oral fluids optimal for their next abzyme testing were studied. The best results were obtained with chromatography on immunoaffinity resin coupled with polyclonal goat antibodies against heavy chains of human IgA.
A group of methods was proposed to evaluate intrinsic catalytic activity of IgAs. DNAse abzymes were tested by original rivanol-DNA clot test, proteolytic elastase, cathepsin-like and amidase activities were determined with substrates Glp-Pro-Val-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, Gly-Phe-p- nitroanilide, Z-Arg-Arg-p-nitroanilide, and benzoyl-arginine-р-nitroanilide by plate colorimetric assay. Catalase activity was studied using hydrogen peroxide reaction with ammonium molybdate; peroxidase was tested in reaction with tetramethylbenzidine. Optimal pH of abzyme reactions and their kinetics were studied.
It has been found that IgA abzymes with peroxidase and elastase activities demonstrate variable reaction rates and different pH optima. Some abzyme IgA samples showed at least 2 pH optima in moderately acidic and alkaline zones.
Results. It was demonstrated also that IgAs isolated from oral fluids of patients with chronic periodontitis display the evident elastase, peroxidase and DNAse activities that is significantly (p<0,01-0,001) higher than activity of oral IgA abzymes of healthy persons and serum abzymes of SLE patients. The abzyme IgA activity mimicking the action of cathepsins B and C was not registered.