Quantification of natural antibody producing B cells in rats by an improved ELISPOT technique using the...

We describe here a new type of solid support for the ELISPOT assay, the 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 with the same combination of AP-labeled conjugates and substrate. We therefore used the PVDF membrane, coated with anti-rat IgM and IgG antibodies, ssDNA or bromelain-treated mouse erythrocytes (BrMRBC) (exposing phosphatidylcholine (PC) as major autoantigen) to develop ELISPOT assays for the quantification of isotype-specific natural antibody secreting cells (ASC) in rats. We confirmed the isotype specificity of the binding of the anti-rat IgM and anti-rat IgG coating antibodies and conjugates with the secreted rat antibodies in this assay, and, by inhibition of spot formation with soluble antigen, their specificity for ssDNA and BrMRBC. An in-house 18-well culture device for the easy manufacture of PVDF-lined culture wells greatly facilitated coating, blocking, and washing procedures, as compared to the original method in 24 well culture plates. This simple, fast, specific and sensitive ELISPOT assay was used to make an inventory of the numbers of natural splenic ASC in Wistar and Fischer rats.