A sensitive simple ELISA for quantitation of sensitizing IgG from dissolved erythrocytes.

A simple enzyme-linked immunosorbent assay has been developed for quantitating the rabbit IgG present on the surface of sensitized red blood cells. A suitable cell lysis was carried out by an alkaline buffer, which dissolved the erythrocytes without forming any precipitate and without disruption of IgG, and facilitated the dissociation of the immune complexes, i.e. the erythrocyte-anti-erythrocyte rabbit IgG. In this alkaline buffer of pH 11.4 the IgG adsorbed directly into wells of microtitration plates unprecoated with anti-rabbit IgG. The detection was performed with alkaline phosphatase-conjugated goat anti-rabbit IgG and p-nitrophenyl phosphate as substrate. Constructing a calibration curve from rabbit IgG made possible the calculation of molecules of IgG bound per red cell. The method was sensitive for the detection of fewer than 500 bound IgG molecules per erythrocyte.