Generation and Characterization of a Mouse Monoclonal Antibody with Specificity Similar to Staphylococcal Protein A (SPA)

Abstract

Human IgG is comprised of four subclasses (IgG₁, IgG₂, IgG₃, and IgG₄). Each subclass possesses different biological properties. One of the differential specificities of human IgG subclasses is binding of Fc fragment of IgG₁, 2, and 4 but, not IgG₃ to staphylococcal protein A (SPA). This study was conducted to produce, select and characterize a monoclonal antibody (MAb) recognizing human IgG subclasses with specificity similar to SPA. Splenocytes from Balb/c mice immunized with Fc fraction of a human IgG₁ myeloma protein were fused with Sp2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAb was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. A murine hybridoma designated 6F11E1 was obtained that secretes an MAb specific for the Fc fragment of the immunizing protein. This MAb reacts with isotypic epitope common to IgG₁, 2 and 4 subclasses. An allelic epitope linked to IgG₃ molecules is also recognized by 6F11E1. This pattern of reactivity was found to be highly similar to that of SPA. Our findings imply that similar or overlapping epitopes are recognized by 6F11E1 and SPA.