Use of human antibodies to identify antigens in cultured human tumor cells: Detection of discrete antigen molecules using electroblotting and enzyme-linked antibody probes

Abstract

To demonstrate antigens in cultured human tumor cells recognized by antibodies in human sera, extracts of cultured malignant melanoma cells were separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose paper (NCP). The resulting electroblots were incubated with human serum, and bound immunoglobulin (Ig) was visualized with rabbit antibodies specific for human IgG, IgA or IgM, followed by peroxidase-conjugated goat anti-rabbit IgG. Antigen-antibody reactions in the nitrocellulose paper were also detected using 125I-labeled anti-rabbit IgG. As little as 125 pg of bound antibody per band were detectable. The numbers of proteins recognized by antibodies in human sera depended both on the quantity of protein transferred and the concentration of Ig applied to the NCP. Whole serum could not be used at dilutions ≤ 1:20 without an unacceptable increase in background staining. Binding of Ig to tumor cell proteins transferred to NCP depended on interactions with the Fab′, not the Fc region of the Ig molecule. To determine the efficiency of transfer as a function of both time and molecular weight, tumor cell proteins were intrinsically labeled with 75Se-labeled methionine and transferred for up to 4 h after fractionation in gels containing acrylamide concentrations of 5%, 7.5%, 10% or 12%. Proteins <150 kDa were transferred with particularly high efficiency in ≥2 h. Different antigens were recognized by the IgA, the IgG and the IgM molecules from the same sera. The methods outlined herein are proving to be useful in monitoring the purification of specific antigens from whole tumor cell extracts.