Development of Two Murine Monoclonal Antibodies Recognizing Human nG1m(a)-Like Isoallotypic Markers

Abstract

Antigenic determinants of immunoglobulin molecules are categorized into three groups: idiotypes, isotypes, and allotypes. An isoallotype is defined as an isotypic determinant in one or more subclasses and an allotype in another subclass of a given isotype. The isoallotype nG1m(a), formerly called non-a, is an allotype located on human IgG1 molecules lacking the G1m(a) allotype. This marker, however, is also detectable on all human IgG2 and IgG3 molecules. Anti-isoallotypic antibodies are useful tools for structural studies of immunoglobulins, forensic science, and epidemiological demographic investigations. In this study, two murine monoclonal antibodies (MAbs) recognizing nG1m(a)-like epitope(s) were generated against a human IgG myeloma protein. These MAbs are produced by hybridoma clones (4F18B12 and 6F18D1) obtained by fusion of SP2/0 myeloma cells with splenocytes from BALB/c mice immunized with heavy chain of a human IgG3 myeloma protein. These MAbs reacted with Fc, but not Fab fragment of the immunizing IgG3 paraprotein. Specificity of the MAbs was further analyzed using a panel of purified myeloma proteins and polyclonal IgG3, including IgG1 (n = 9), IgG2 (n = 4), IgG3 (n = 8), and IgG4 (n = 6) subclasses. Our results demonstrated that these MAbs reacted with linear epitope(s) located on heavy chain of all IgG2 and IgG3 molecules tested and some paraproteins of IgG1 subclass, but none of the IgG4 molecules. So these MAbs seem to recognize nG1m(a)-like isoallotypic marker. These MAbs showed no cross-reactivity with serum of other species tested. Both MAbs belonged to IgG1 subclass with affinity constants of 4.5 x 10(9) mol(-1) (4F18B12) and 3.46 x 10(9) mol(-1) (6F18D1), respectively. These MAbs might be used as a tool to detect nG1m(a) + IgG molecules.

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