DMISA (DISSOCIATED MEMBRANE IMMUNOSORBENT-ASSAY), A NEW ELISA TECHNIQUE PERFORMED WITH BLOTTED SAMPLES

This study describes the use of electrophoretically purified antigens blotted onto nitrocellulose, as solid phase antigens for enzyme-linked immunosorbent assays. This procedure is called DMISA, for dissociated membrane immunosorbent assay. The method is illustrated using immunob lotted antigens of Dactylis glomerata grass pollen extract. The band o f interest was located on a print of nitrocellulose by light staining (India ink), then the corresponding strip of nitrocellulose was cut ou t. Immediately after its solubilization in ethyleneglycol monomethyl e ther, the antigen coated nitrocellulose was precipitated by the additi on of buffer. In this way the bulk of the antigen remained bound to th e membrane. The resulting suspension was carefully washed, and used as a solid phase antigen in an enzyme-linked immunosorbent assay. Two di fferent electrophoretic methods were used to separate the Dactylis glo merata antigens. We compared the results obtained with classical immun oblot and with DMISA, for IgG4 and IgE quantification using sera from patients allergic to D. glomerata and purified blotted antigens presen t at the nanogram level.