A Non-Recoverable Hybridoma Limits the Production of Monoclonal Antibodies Against Bovine Trophoblast Non-Classical NC3*00101 Protein
Indian Journal of Experimental Biology
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The non-classical MHC class I (MHC-Ib) proteins are important modulators of immune system during pregnancy favoring survival of the fetus. Contrary to ubiquitously expressed classical MHC-I proteins, MHC-Ib 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 MHC-Ib 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 (BoLA-Ib) antigens. BoLA-NC3*00101, 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 BoLA-I antigens released by fetal trophoblast cells. The objective of this study is to synthesize mAb specific to NC3*00101 molecule to develop an isotype-matched ELISA to assess BoLA-I protein(s). We demonstrated that majority of NC3*00101 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 BoLA-NC3*00101 as demonstrated with flow cytometry. NC3-3*2 clone secreted supernatant that captured BoLA-NC3*00101 protein in ELISA. Unfortunately, despite several efforts we could not recover the cryopreserved hybridoma. Non-recoverability and instability, thus, limited production of NC3*00101-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 BoLA-Ib proteins using robust methods of fusion and cryopreservation of hybridomas.
Parasar, P., Wilhelm, A., Davis, W. C., White, K. L., & Davies, C. J. (2018). A non-recoverable hybridoma limits the production of monoclonal antibodies against bovine trophoblast non-classical NC3* 00101 protein. http://nopr.niscair.res.in/handle/123456789/44841