Production and immunoselection of IgM-IgA hybridomas: preparing immunoglobulins with dual binding specificity.
Fusion between the thioguanine resistant myeloma cell line MOPC-315 [which produces alpha, lambda-2 antibodies specific to the 2,4-dinitrophenyl (DNP) hapten] and a long term in vivo maintained hybridoma 6100.15 [which produces mu, lambda-1 antibodies specific to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten] resulted in the generation of 12 hybridomas. These hybridomas secrete a mixed family of immunoglobulins (Ig) that bind both DNP and NP and express both IgM and IgA serological determinants. Affinity purified molecules from NP, DNP, anti-mu, or anti-alpha immunosorbents react with both anti-mu and anti-alpha antisera, suggesting that these Ig represent IgM-IgA hybrid molecules. This conclusion was supported by idiotypic analyses. To determine the roles of individual immunoglobulin chains in determining antibody specificity this IgM-IgA hybridoma was used for immunoselection. Following lysis with specific anti-mu and anti-idiotype antibodies, an alpha+, mu- variant clone (A12) was identified, which secreted Ig that binds DNP but not NP. The DNP binding proteins were shown to express alpha, lambda-1 and lambda-2 chains. In contrast, the Ig which lack DNP binding activity only expressed alpha and lambda-1 determinants. The combined results demonstrate that the lambda-1 chain from 6100.15 hybridoma cannot replace lambda-2 of MOPC-315 for DNP binding activity. These data imply that these closely related lambda chains carry sites critical for antigen binding activity. An IgM-IgA hybridoma variant (MA2) which secretes Ig that binds to NP and DNP and expresses mu, alpha and lambda-2 chains was also characterized. This molecule lacked a lambda-1 chain. To determine if the Ig prepared with heterologous mu and lambda-2 chains had NP binding activity required immunoselection of a fourth clone (M2). M2 secretes homogeneous Ig bearing only mu and lambda-2 chains. In contrast to either parental Ig, the M2 antibody molecules express dual binding activity to both NP and DNP. Thus, critical amino acid substitutions in the MOPC-315 lambda-2 sequence are required for DNA binding specificity.
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Boston University School of Medicine, MA 02118.
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D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D002460:Cell Line; D004136:Dinitrobenzenes / Q000378:metabolism; D006825:Hybridomas; D007070:Immunoglobulin A / Q000378:metabolism*; D007075:Immunoglobulin M / Q000378:metabolism*; D007146:Immunoglobulin lambda-Chains / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009596:Nitrophenols / Q000378:metabolism; D010648:Phenylacetates; D014407:Tumor Cells, Cultured
D004136:Dinitrobenzenes; D007070:Immunoglobulin A; D007075:Immunoglobulin M; D007146:Immunoglobulin lambda-Chains; D009596:Nitrophenols; D010648:Phenylacetates; C026242:4-hydroxy-5-nitrophenyl acetic acid; C017906:3-dinitrobenzene