The presence of autoantibodies specific for NZB serum factors in adult NZB mice and the establishment of monoclonal autoantibodies against these humoral factors.
Humoral factors in serum of young NZB mice enhance maturation of B-lymphocyte precursors in vitro. A blot ELISA assay identified autoantibodies against the serum factors. NZB-SFs (designated NZB-SF alpha, pI 3.5-4.0, and NZB-SF beta, pI 7.8) were purified by sequential steps. Both had a molecular weight (MW) of approximately 23,000 in SDS-PAGE. NZB mice develop autoantibodies against NZB-SFs by 2 months of age; titers increased progressively with age. Non-autoimmune-prone mice did not produce autoantibodies against NZB-SFs. We then developed two hybridoma clones, IIC1C1 and IIC1M4, which produce monoclonal autoantibodies against NZB-SF alpha and NZB-SF beta, respectively. Both IgM autoantibodies could be affinity purified with a column of CNBr-Sepharose 4B gel conjugated with anti-mouse IgM antibody. Neither IIC1C1 nor IIC1M4 abolished bioactivity of recombinant mouse IL-1 alpha, human IL-1, mouse, rat, or human IL-2, mouse IL-3, or colony-stimulating factor. Neither antibody reacted to recombinant mouse IL-1 alpha, IL-4, TNF alpha, or IFN gamma in blot ELISA assays. Monoclonal autoantibodies IIC1C1 and IIC1M4 were used to purify NZB-SFs. SDS-PAGE of the affinity-purified NZB-SFs revealed bands of 23 and 60 kDa, and proteins extracted from the bands were reactive to our monoclonal autoantibodies.
Jyonouchi
H
H
Department of Pediatrics, University of South Florida, St. Petersburg 33701.
Voss
R M
RM
Good
R A
RA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D000918:Antibody Specificity; D001323:Autoantibodies / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D003429:Cross Reactions; D005260:Female; D006825:Hybridomas / Q000032:analysis; D007163:Immunosorbent Techniques; D008222:Lymphokines / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008811:Mice, Inbred DBA; D008814:Mice, Inbred NZB / Q000276:immunology*; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D008222:Lymphokines; C055837:NZB serum factor