Characterization of anti-porcine CXCL10 monoclonal antibodies

Abstract

Abstract C-X-C motif chemokine ligand 10 (CXCL10) facilitates chemoattraction of important immune cells to tissues. As part of the USDA-NIFA Swine Immune Toolkit Initiative, our goal is to provide the veterinary community with new commercial immune reagents and standardized assays for future research efforts. For the chemokine target CXCL10, we used yeast expressed, recombinant porcine CXCL10 (rPoCXCL10) to generate a panel of α-CXCL10 monoclonal antibodies (mAbs). Each of the α-PoCXCL10 mAbs was assessed by ELISA using cross-inhibition analyses of biotinylated mAbs, and direct binding to orthologous yeast expressed CXCL10 proteins. As a result, we assigned six distinct epitope groups for the nine generated α-CXCL10 mAbs. Subsequently, we screened AF647-tagged α-CXCL10 mAbs for intracellular staining of pig immune cells using different stimulation conditions. Of the nine mAbs only two detected intracellular CXCL10 expression in PMA/ionomycin or rPoIFNγ-stimulated porcine cells. Further, cell characterization assays verified CXCL10+ cells as CD3-CD4-CD172+, with occasional CD4+ subsets. Overall, we have determined that α-CXCL10-1.4 mAb is the best mAb clone for intracellular staining and recommend it for use in analysis of intracellular signaling to detect interactions that regulate cell migration. A sandwich ELISA was also developed to quantitate CXCL10 protein expression; it verified reactivity with native porcine CXCL10. Overall, these tools will be useful for evaluating immunity against swine infectious diseases and host-responses to vaccines. New reagents identified by the Swine Immune Toolkit Initiative will undoubtedly advance future swine research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815. Supported by USDA-ARS project # 8042?32000?102 and USDA-NIFA AFRI grant # 2019-67015-29815