Characterization of a polyreactive monoclonal antibody to dsDNA, F x 1A, and heparan sulfate generated from BALB/c mice immunized with rat renal homogenates.
Monoclonal antibodies (MoAb) were prepared by fusing spleen cells from BALB/c mice immunized with rat renal cortical homogenates to the mouse myeloma cell. One of them, designated MoAb26-3, revealed a positive antinuclear activity by screening an indirect immunofluorescence test on kidney cryostat sections. The reactivity of MoAb26-3 with double-stranded deoxyribonucleic acid (dsDNA) was confirmed by the Crithidia luciliae assay. The isotype of MoAb26-3 was determined to be IgM-kappa. To test the nephritogenicity of MoAb26-3, the hybridomas were grafted intraperitoneally into BALB/c mice. A deposition of IgM was observed along the base of the epithelial foot processes and on the luminal surface of the endothelium by immunoelectron microscopy. By direct enzyme-linked immunosorbent assay (ELISA), MoAb26-3 was shown to react not only with dsDNA but also with fraction 1A of renal cortical supernatant (F x 1A) and heparan sulfate. On the basis of inhibition ELISA, the dsDNA inhibited F x 1A and heparan sulfate binding of MoAb26-3 and F x 1A blocked the reactivity of Mo26-3 with dsDNA and heparan sulfate, while heparan sulfate showed a less inhibition on the binding of MoAb26-3 with F x 1A, dsDNA, and even with heparan sulfate. Using immunoprecipitation with radiolabeled F x 1A, MoAb26-3 was shown to react with MW 330,000, 440,000, and 700,000 bands which were the same with those which polyclonal Heymann nephritis serum could react. An intravenous injection of MoAb26-3 to rats resulted in the deposition of IgM along the glomerular capillary wall, but resulted in an only transient appearance of proteinuria.
Chen
X M
XM
Department of Medicine, Kitasato University School of Medicine, Sagamihara, Japan.
Aozuka
S
S
Kamata
K
K
Okumura
K
K
Kobayashi
Y
Y
D000818:Animals; D000974:Antibodies, Antinuclear / Q000008:administration & dosage / Q000032:analysis / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000032:analysis / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001324:Autoantigens / Q000302:isolation & purification; D002474:Cell-Free System; D003421:Crithidia; D004247:DNA / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005260:Female; D005629:Frozen Sections; D006025:Glycosaminoglycans / Q000276:immunology*; D006497:Heparan Sulfate / Q000276:immunology*; D006825:Hybridomas / Q000637:transplantation; D007132:Immunoglobulin Isotypes / Q000032:analysis; D007275:Injections, Intravenous; D007672:Kidney Cortex / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011919:Rats, Inbred Strains; D011933:Reagent Kits, Diagnostic
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D001324:Autoantigens; D004247:DNA; D000939:Epitopes; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D007132:Immunoglobulin Isotypes; D011933:Reagent Kits, Diagnostic