Monoclonal autoantibodies against mouse red blood cells: a family of structurally restricted molecules.

Cultured mouse peritoneal cells from unstimulated mice developed plaque-forming activity against isologous bromelain-treated erythrocytes. Several IgM monoclonal autoantibodies obtained by fusion of peritoneal cells from NZB or CBA origin with BALB/c myeloma cells were purified by affinity chromatography on trimethyl ammonium (TMA) column on the basis of their cross-reactivity with TMA, phosphorylcholine (PC) or choline haptens. Binding affinity for PC hapten was of the order of 10(3) M-1. Idiotypic studies with a polyclonal rabbit anti-idiotypic reagent revealed strong cross-reactions with all hybridoma autoantibodies thus far tested. In addition, the rabbit anti-idiotypic serum detected idiotypes or cross-reactive idiotypes in the sera of NZB and CBA as well as BALB/c mice. N-terminal amino acid sequence analyses of three hybridoma autoantibodies from NZB mice and one from CBA mice were carried out. The sequences of the first 32 residues of the four heavy chains showed that three were identical while one had one amino acid interchange; they belong to the VHIII-subgroup. The light chains were identical in the first 35 residues with the exception of a substitution at position 3 in two light chains and are members of the VK-9-subgroup. These results entirely support the idiotypic data. These monoclonal autoantibodies from NZB and CBA mice although isolated and eluted from PC-related haptens do not have any apparent structural nor idiotypic relationship to PC-specific antibodies. Idiotypic and V-region N-terminal sequence data suggest that these autoantibodies constitute a highly restricted family of molecules likely to be encoded by unique germ-line genes which may be expressed as such or as somatic variants in different mouse strains.

Relationship between choline derivatives and mouse erythrocyte membrane antigens revealed by mouse monoclonal antibodies. I. Anticholine activity of anti-mouse erythrocyte monoclonal antibodies.

Different clones of mouse hybridomas, derived from the fusion of unstimulated mouse peritoneal cells with mouse myeloma cells, producing IgM monoclonal antibodies directed against the membrane of bromelain-treated mouse erythrocytes (MRBC(Br)) have been previously established. We have recently shown that one of these hybridomas produce, in ascites, antibodies cross-reacting with phosphorylcholine derivatives (trimethylammonium (TMA) derivatives). In this work the cross-reactivity for TMA derivatives of the monoclonal antibodies produced by 4 anti-MRBC(Br) hybridomas have been studied at the cell level (plaque-forming cells). Phosphorylcholine, choline bromide and p-aminophenyl-trimethylammonium were found to be potent specific inhibitors of plaque formation (anti MRBC(Br)). The hemolytic activities of ascites and tissue culture supernatants were studied and their inhibition by TMA derivatives was determined. Immunoglobulins from ascites purified on TMA immunoadsorbent column were analyzed by two-dimensional gel electrophoresis, their spectrotype was compared to the spectrotype of immunoglobulins from tissue culture supernatants from the same hybridoma radioactively tagged by internal incorporation of [14C]leucine. It could be shown without ambiguity that the PTMA column retained an IgM with the same characteristics as the IgM secreted in vitro.

The participation of trimethylammonium in the mouse erythrocyte epitope recognized by monoclonal autoantibodies.

Monoclonal IgM anti-erythrocyte autoantibody produced by a NZB-derived hybridoma has been found to react with trimethylammonium-containing compounds. Such compounds are able to prevent the lysis of bromelain-treated mouse erythrocytes (BrMRBC) by those autoantibodies. Using a column of insolubilized betaine hydrazine (BH) the monoclonal anti-erythrocyte antibody has been specifically retained. Elution of this antibody was accomplished by 0.15 M choline (Ch).