A method for titration of inhibiting antibodies to bacterial immunoglobulin A1 proteases in human serum and secretions.
Bacterial IgA1 proteases specifically cleave IgA1, including S-IgA1, molecules into Fab alpha and Fc alpha fragments. Hereby these enzymes interfere with the protective functions of antibodies belonging to this isotype. Antibodies inhibiting IgA1 proteases have been detected in humans, but the titration of such antibodies is a matter of methodological concern. Because human serum and secretions contain IgA1 substrate, it is impossible to provide uniform substrate conditions for samples of IgA1 protease incubated with inhibitors differing in their origin and state of dilution. This study demonstrates that such variations in substrate are not prohibitive for a reliable titration of inhibiting antibodies. This was evident from experiments demonstrating that the variations do not interfere with the quantification of residual IgA1 protease activity provided the activity is measured in terms of the proportion of IgA1 substrate cleaved during incubation. Proportions of cleaved IgA1 were measured by exploiting the differential reactivity of cleaved and intact IgA1 molecules in an ELISA using anti-Fc alpha and enzyme-conjugated anti-light chain antibodies for catching and development, respectively. A protocol for the titration of IgA1 protease-inhibiting antibodies based on this ELISA is described. By application of the protocol to chromatographic fractions of saliva, IgA1 protease-inhibiting activity was found to co-purify with salivary S-IgA.
Reinholdt
J
J
Royal Dental College, Faculty of Health Sciences, University of Aarhus, Denmark.
D000328:Adult; D000907:Antibodies, Bacterial / Q000097:blood / Q000737:chemistry / Q000494:pharmacology*; D001667:Binding, Competitive / Q000276:immunology; D005591:Chemical Fractionation; D003126:Colostrum / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D006801:Humans; D007070:Immunoglobulin A / Q000494:pharmacology*; D007071:Immunoglobulin A, Secretory / Q000494:pharmacology*; D015588:Observer Variation; D015203:Reproducibility of Results; D012463:Saliva / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D013379:Substrate Specificity; D017186:Titrimetry
D000907:Antibodies, Bacterial; D007070:Immunoglobulin A; D007071:Immunoglobulin A, Secretory