Modification, optimization and simplification of the spot ELISA technique for the enumeration of cells secreting anti-hapten antibodies

We describe here a modification of the spot ELISA assay for the detection and enumeration of antibody-secreting cells. The modification increases sensitivity, convenience and simplicity while optimizing parameters of time, temperature and reagents. Laboratory reagents and 96 well microtiter plates commonly used for the standard ELISA assay are adapted for use in the spot ELISA assay. Special emphasis is placed on the detection of anti-hapten IgM or IgG production by splenocytes in response to stimulation by a range of doses of antigen. In addition, two procedures are described whereby the spot ELISA assay is altered to yield a measurable optical density: (1) the blue precipitate obtained in the spot ELISA assay is dissolved and the optical density of the resultant blue color is measured, and (2) a standard ELISA substrate is substituted for the spot ELISA substrate to yield a colored solution rather than a precipitate. Both of these are shown to correlate with spot numbers and can therfore supplement or substitute for the enumeration of spots (produced by antibody-secreting cells), thus speeding and mechanizing the assay.