Optimization of the intestinal lavage procedure for determination of intestinal immune responses

Optimal conditions to process, concentrate and store intestinal lavage fluid were studied in samples collected from volunteers before and after oral immunization with a prototype vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhoea. Total IgA and specific IgA antibody titres against enterotoxin and colonization factor antigen were determined in 22 lavage samples which were either enzyme-inhibited or heat-inactivated and then subjected to different long-term storage conditions. Samples were analysed within 1 month of collection and also after 3, 6 and 24 months of storage. Total IgA concentrations and specific IgA antibody levels were higher in lavage samples treated with enzyme inhibitors (soybean trypsin inhibitor and phenylmethylsulfonyl fluoride) than in those heat-inactivated. Similarily, concentration of the lavage fluid by freeze-drying was superior to concentration against polyethylene glycol. Specific antibody titres remained elevated after storage for at least 6 months but declined after 2 years in frozen compared with freeze-dried samples.