QUANTIFICATION OF NATURAL ANTIBODY-PRODUCING B-CELLS IN RATS BY AN IMPROVED ELISPOT TECHNIQUE USING THE POLYVINYLIDENE DIFLUORIDE MEMBRANE AS THE SOLID SUPPORT

We describe here a new type of solid support for the ELISPOT assay, th e PVDF membrane. In parallel tests, spot yields on this membrane were superior to those obtained with the frequently used nitrocellulose (NC ) membrane, coated with the same rat anti-IgM and anti-IgG antibodies, incubated with the same rat spleen cell suspensions, and developed wi th the same combination of AP-labeled conjugates and substrate. We the refore used the PVDF membrane, coated with anti-rat IgM and IgG antibo dies, ssDNA or bromelain-treated mouse erythrocytes (BrMRBC) (exposing phosphatidylcholine (PC) as major autoantigen) to develop ELISPOT ass ays for the quantification of isotype-specific natural antibody secret ing cells (ASC) in rats. We confirmed the isotype specificity of the b inding of the anti-rat IgM and anti-rat IgG coating antibodies and con jugates with the secreted rat antibodies in this assay, and, by inhibi tion of spot formation with soluble antigen, their specificity for ssD NA and BrMRBC. An in-house 18-well culture device for the easy manufac ture of PVDF-lined culture wells greatly facilitated coating, blocking , and washing procedures, as compared to the original method in 24 wel l culture plates. This simple, fast, specific and sensitive ELISPOT as say was used to make an inventory of the numbers of natural splenic AS C in Wistar and Fischer rats.