An ELISA to Measure Mucosal IgA Specific forBacteroidesSurface Antigens in Whole Gut Lavage Fluid

Abstract

Whole gut lavage fluid (WGLF) is an ideal source of gut-derived immunoglobulin for the study of the immune response to the normal bacterial population of the gastrointestinal (GI) tract. An ELISA has been developed for measurement of IgA reacting with cell surface antigens (lipopolysaccharide LPS; and whole outer membrane, OM) of two species of Bacteroides : B. fragilis and B. vulgatus in WGLF. IgA to each species was detected at varying levels in specimens of WGLF from 80 patients attending a GI clinic. Specific IgA was compared with total IgA, and comparisons of the IgA antibody responses to the different antigens were made by plotting OD values for one antigen against values for the other. The resulting scattergrams were analysed by linear regression and correlation coefficients were calculated. There was poor correlation between any of the specific IgA levels and the total IgA in the lavage (r 0.7, P ≤ 0001). This indicated a possible response to a common epitope. This was confirmed by inhibition enzyme immunoassays where preincubation of the WGLF with LPS inhibited the reaction with OM and vice-versa . As the OM antigen contained LPS while the LPS was free of protein, it can be assumed that an epitope common to both species of Bacteroides is located in the LPS. No inhibition could be shown with enterobacterial LPS. When the 80 patients were divided into four diagnosis categories: normal, Crohn's disease, ulcerative colitis and other GI diseases; levels of IgA antibodies to B. fragilis LPS were significantly higher (P < 0.02) in the Crohn's disease patients than in the other groups. No significant differences in antibodies specific for other bacteroides antigens or total IgA were seen in the groups. Keywords: Bacteroides fragilis , Bacteroides vulgatus , LPS, outer membrane, ELISA, whole gut lavage, mucosal IgA, Crohn's disease