During the process of developing herbal drugs with antimicrobial action, one of the most important phases is testing of antimicrobial activity in vitro. The disk-diffusion method is performed in Petri dish, on solid feeding surface. The disks with definite concentrations of antibiotics, pure substances or plant extracts are placed on the top of feeding plates previously inoculated with pure bacterial culture. Growth of the bacterial culture depends on its susceptibility to a tested substance; if the substance has antibacterial effect, a clear zone free of bacteria will form around the disk. Minimal inhibitory concentration (MIC) of the tested substance is determined by extrapolation of the regression line: concentration of the tested substance/radius or surface of area where growth of bacteria was inhibited. This method of testing microbial susceptibility to antibiotics, pure substances or plant extracts is highly sensitive and specific.

OBJECTIVE Anti-β2 GPI are a formal laboratory criterion for the antiphospholipid syndrome (APS). They were demonstrated to be a risk factor for thrombosis and fetal losses but can also be detected in patients with systemic autoimmune disease (SAD), in healthy adults individuals and pre-school children. It has been suggested that different subpopulations of anti-β2GPI may carry different pathogenetic potential: autoantibodies against Domain1 seem to be associated with thrombosis; autoantibodies against Domain4/5 have been identified in patients with non-thrombotic conditions. METHODS We studied 48 patients with SAD (32 systemic lupus erythematosus, 16 undifferentiated connettive tissue disease), 64 patients with APS, 57 one-year-old healthy children born to mother with SAD, 33 children with atopic dermatitis. All subjects were IgG anti-β2 GPI positive. The specificity of anti-β2 GPI was investigated using ELISA research products containing recombinant β2 GPI D1 and D4/5 antigens. Cut-off values are calculated as 95th percentile on 100 NHD. IgG anti-β2 GPI were tested at a validated home-made ELISA routinely performed in our laboratory. No thrombotic events were recordered in patients with SAD and in both groups of children. RESULTS Patients with SAD and APS showed prevalent reactivity for D1 while children in both groups preferentially recognize D4/5. CONCLUSIONS IgG anti-β2 GPI against D1 seem to cluster in patients with systemic autoimmune conditions. Their pathogenic potential in determine APS manifestations may be mitigated by adequate prophylaxis.