Characteristics of immunoglobulin gene usage of the xenoantibody binding to Gal-alpha(1,3)Gal target antigens in the Gal knockout mouse

Background Natural antibodies that react with galactose-alpha (1,3)galactos e [gal alpha (1,3)gal] carbohydrate epitopes exist in humans and Old World primates because of the inactivation of the alpha1,3-galactosyltransferase (alpha1,3GT) gene in these species and the subsequent production of antibod ies to environmental microbes that express the gal alpha (1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-gal alpha (1,3)gal antibodies and can be used to study the genetic control of humoral immune responses to th is carbohydrate epitope. Methods. Six hybridomas that produce monoclonal antibodies (mAbs) to gal al pha (1,3)gal were generated in Gal o/o mice. The mAbs were tested to charac terize the binding activity with flow cytometry using pig aortic endothelia l cells and ELISA with gal alpha (1,3)gal carbohydrates, The V-H and V-K ge nes of these hybridomas were cloned, sequenced, and analyzed. Results. The mAbs showed distinct patterns of antibody binding to gal alpha (1,3)gal antigens, The V-H genes that encode the mAb binding activity were restricted to a small number of genes expressed in their germline configur ation. Four of six clones used closely related progeny of the same V-H germ line gene (V(H)441). Comparison of the mouse gene V(H)441 to the human gene IGWV3-11, a gene that encodes antibody activity to gal alpha (1,3)gal in h umans, demonstrates that these two genes share a nonrandom distribution of amino acids used at canonical binding sites within the variable regions (co mplimentary determining regions 1 and 2) of their immunoglobulin V, genes. Conclusions. These results demonstrate the similarity of the Gal o/o mice a nd humans in their immune response to gal alpha (1,3)gal epitopes. Gal o/o mouse can serve as a useful model for examining the genetic control of anti body/antigen interactions associated with the humoral response to pig xenog rafts in humans.