APPLICATION OF THE ILISPOT-IDIP SYSTEM FOR THE ENUMERATION OF DIFFERENT SIZES OF IGA SPOT FORMING CELLS IN THE MURINE SMALL AND LARGE-INTESTINE

The immunofluorescence-linked immunospot (ILISPOT) assay associated wi th the immunofluorescence digital image processing (IDIP) system was o riginally developed in our laboratory to allow enumeration of immunogl obulin (Ig) producing, spot forming cells (SFC) in a more objective an d quantitative manner. In this study, the ILISPOT-IDIP system was furt her advanced in order to analyze different sizes of SFC (e.g., IgA pro ducing cells) including large (L), medium (M), and small (S) cells whi ch correspond to high (> 2.4 pg), medium (1.2-2.4 pg) and low ( < 1.2 pg) IgA secreting cells by the adaptation of real time image processor and intensified video camera system. When the ILISPOT-IDIP system was used to characterize the frequency of IgA secreting cells among monon uclear cells isolated from different parts of the murine gastrointesti nal (GI) tract including the small (upper, middle and lower sections) and large (colon and rectum) intestine, the small intestine contained higher numbers of IgA SFC (approximately 8.4 x 10(6) SFC/10(6) cells) when compared with large intestine (approximately 1.3 x 10(-5) SFC/10( 6) cells). Among the 3 areas of small intestine, the upper (approximat ely 3.7 x 10(5) SFC/10(6) cells) and middle (approximately 2.4 x 10(5) SFC/10(6) cells) parts had higher numbers of IgA SFC when compared to the lower small (approximately 2.3 x 10(5) SFC/10(6) cells) intestine . When these IgA producing cells in different parts of the intestine w ere classified into three groups according to the size of individual s pots, the upper and middle intestine contained higher frequencies of l arge (approximately 20%) and medium (approximately 20%) SFC which corr esponded to high and medium IgA secretors in comparison to the lower s mall (approximately 9%) and large (approximately 6%) intestine. In con trast, the lower small and large intestine were dominated by small SFC since approximately 85% of IgA producing cells were categorized as lo w secretors. Using the advanced ILISPOT-IDIP system, a unique distribu tion of different sizes (or secretion rates) of IgA producing SFC was elucidated in the different regions of the mouse small and large intes tine.