A non-recoverable hybridoma limits the production of monoclonal antibodies against bovine trophoblast non-classical <em>NC3*00101</em> protein

Abstract

The non-classical MHC class I (<em>MHC-Ib</em>) proteins are important modulators of immune system during pregnancy favoring survival of the fetus. Contrary to ubiquitously expressed classical <em>MHC-I </em> proteins, <em>MHC-Ib</em> proteins are oligomorphic, and expressed in specific cell/tissue types thus minimizing maternal immune-mediated rejection of fetal-allograft and a successful pregnancy. A unique characteristic of <em>MHC-Ib</em> glycoproteins is expression of surface and soluble isoforms due to alternative splicing phenomenon. Bovine fetal trophoblast cells, during the third trimester of pregnancy, express non-classical bovine leukocyte antigen class Ib (<em>BoLA-Ib</em>) antigens. <em>BoLA-NC3*00101</em>, is a non-classical class I allele from cattle AH11 haplotype and is expressed as cell surface isoform. However, lack of monoclonal antibody (mAb) hinders the development of specific assay to detect the soluble/secreted <em>BoLA</em>-I antigens released by fetal trophoblast cells. The objective of this study is to synthesize mAb specific to <em>NC3*00101</em> molecule to develop an isotype-matched ELISA to assess <em>BoLA-I</em> protein(s). We demonstrated that majority of <em>NC3*00101</em> hybridoma-supernatants were low-titer and showed inappreciable reactivity in ELISA and flow cytometry assays. We identified a clone of hybridoma (NC3-3*2) that secreted mAb specific to <em>BoLA</em>-<em>NC3*00101</em> as demonstrated with flow cytometry. NC3-3*2 clone secreted supernatant that captured <em>BoLA</em>-<em>NC3*00101</em> protein in ELISA. Unfortunately, despite several efforts we could not recover the cryopreserved hybridoma. Non-recoverability and instability, thus, limited production of <em>NC3*00101</em>-mAbs. This study provides the evidence that mice immunized with bovine class Ib proteins elicit a specific immunogenic response and warrants future studies into generation of stable hybridoma secreting antibodies against <em>BoLA-Ib</em> proteins using robust methods of fusion and cryopreservation of hybridomas.