A sensitive method for the detection of specific antibody production in different isotypes from single lamina propria plasma cells

Abstract

A sensitive and reproducible method for the detection of specific antibody production (or total immunoglobulin secretion) at the single cell level from isolated lamina propria lymphocytes was developed. The cells were prepared from mouse intestinal mucosa by enzyme extraction with collagenase, and antibody secretion was demonstrated with a solid phase enzyme-linked immunospot (ELISPOT) assay. Oral immunizations with cholera toxin or keyhole limpet haemocyanin to mice gave high numbers of highly antigen-specific spot-forming cells (SFC) among isolated lamina propria lymphocytes. Spots were shown to result from active synthesis of immunoglobulin in vitro. The variation in SFC numbers between individual animals after a given protocol of oral immunizations was found to be 25% and between equal groups analysed on different occasions, 12%. Kinetics of primary as well as secondary immune responses after oral immunizations with cholera toxin were easily monitored. A single dose of cholera toxin gave rise to 230 antitoxin SFC/10(7) isolated lamina propria lymphocytes. Each additional dose stimulated to increasing numbers of specific SFC with roughly 7000 antitoxin SFC/10(7) cells after five immunizations. Monitoring of day-by-day responses after oral booster immunizations demonstrated peak SFC numbers on day 8 after antigen administration. The total number of immunoglobulin-secreting (Ig) cells and the isotype distribution of specific SFC could also be determined. In the peak antitoxin response, 8% of the isolated total Ig-secreting lamina propria cells were active against cholera toxin, and of these 80% were producing IgA. This method has also been successfully used in humans and rabbits to demonstrate specific antibody production by single lamina propria plasma cells.