Antibody produced against isolated Rh(D) polypeptide reacts with other Rh-related antigens

Rh(D) antigen-containing polypeptide was prepared by immune precipitation of intact cDE/cDE erythrocytes by using a high-titer preparation of polyclonal anti-D. When isolated Rh(D) polypeptide was administered to rabbits, antibody was produced that was unresponsive toward Rh-positive and -negative cells but reacted strongly with the immunogen in enzyme-linked immunosorbent assay-type immunobinding and Western blot immunostaining assays. Rabbit antibody also immunostains isolated Rh(c) polypeptide as well as the Rh antigen-containing components of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated membrane proteins from Rh(D)-positive (cDE/cDE,CDe/CDe), Rh(D)-negative (cde/cde,Cde/Cde), and -D-/-D- cells. It does not react with any membrane protein from Rh-null regulator type cells, thus indicating a specificity for Rh-related proteins. We have also been able to demonstrate that polyclonal and monoclonal anti-D preparations that do not immunostain isolated Rh(D) polypeptide will react with it in our immunobinding assay.