[The production of monoclonal anti-human spectrin antibody cross-reactive with single-stranded DNA].
Five monoclonal anti-human red blood cell (RBC) antibodies (all IgM kappa type) were produced by immunizing BALB/c mouse with human O-type RBC. Solid phase enzyme linked immunosorbent assays (ELISAs) were used for studying the reactivities of monoclonal antibodies against RBC, DNA, cardiolipin, dermatan sulfate and spectrin. Because two monoclonal antibodies (1B1A2E5, 1B1B2G7) of these antibodies also reacted with heat-denatured DNA (ssDNA) derived from calf thymus, salmon sperm and human white blood cell, the cross-reactivities of two monoclonal antibodies were analyzed. They reacted with spectrin, one of the RBC cytoskeleton proteins, by western blotting using RBC ghost protein and ELISA using spectrin, but not react with other anionic antigens such as cardiolipin and dermatan sulfate. The anti-RBC activities of the two monoclonal antibodies were not changed by DNase I treatment, but were decreased by pronase E treatment of RBC ghost protein. And, the anti-ssDNA activities of these antibodies were abolished by DNase I treatment, but were not changed by pronase E treatment of DNA. The reactivities of these antibodies to RBC and ssDNA were inhibited by ssDNA, The reactivities of these antibodies to RBC and ssDNA were inhibited by ssDNA, RBC ghost protein and rabbit anti-mouse IgM antiserum, but were not inhibited by dsDNA, RBC, rabbit anti-mouse IgG antiserum, bovine serum albumin and spectrin in liquid phase. In inhibition test, spectrin in liquid phase did not inhibit the reaction for RBC or ssDNA of these antibodies. Complements were fixed to these antibodies in RBC, ssDNA and spectrin ELISA. In the view of the above results, it was concluded that the RBC antigen of the 2 monoclonal antibodies was directed against spectrin in solid phase, and that these antibodies were cross-reactive with both RBC (spectrin) and ssDNA (derived from calf thymus, salmon sperm and human white blood cell).
Mukai
M
M
Second Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis*; D003429:Cross Reactions; D004277:DNA, Single-Stranded / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D013047:Specific Pathogen-Free Organisms; D013049:Spectrin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D004277:DNA, Single-Stranded; D013049:Spectrin