Production and characterization of anti-porcine CXCL10 monoclonal antibodies

Authors: HAILSTOCK, TAYLOR - Former ARS Employee; DAI, CHAOHUI - Orise Fellow; Aquino, Jovan; Walker, Kristen; CHICK, SHANNON - Former ARS Employee; MANIRARORA, JEAN - Former ARS Employee; SURESH, RAKSHA - The Ohio State University; PATIL, VEERUPAXAGOUDA - The Ohio State University; RENUKARADHYA, GOURAPURA - The Ohio State University; SULLIVAN, YVONNE - Kingfisher Biotech, Inc; LABRESH, JOANNA - Kingfisher Biotech, Inc; Lunney, Joan

Submitted to: Cytokine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2023
Publication Date: 12/22/2023
Citation: Hailstock, T., Dai, C., Aquino, J.F., Walker, K.E., Chick, S.T., Manirarora, J.N., Suresh, R., Patil, V., Renukaradhya, G.J., Sullivan, Y., Labresh, J., Lunney, J.K. 2023. Production and characterization of anti-porcine CXCL10 monoclonal antibodies. Cytokine. 174: Article e156449. https://doi.org/10.1016/j.cyto.2023.156449.
DOI: https://doi.org/10.1016/j.cyto.2023.156449

Interpretive Summary: Cellular immune responses are characterized as complex interactions of chemical and biological signals released from various cell types. One example of an important biological signal is the chemokine C-X-C motif ligand 10 (CXCL10). It is a cell signaling molecule that is secreted by various activated immune cells to control immune responses. This paper presents the characterization of our new panel of anti-porcine CXCL10 monoclonal antibodies (mAbs). These mAbs will enable pig disease and vaccine researchers to probe for important cellular interactions regulated by CXCL10 and provide targets for controlling protective as well as pathologic immune responses.

Technical Abstract: Research on C-X-C motif chemokine ligand 10 (CXCL10) has been widely reported for humans and select animal species, yet immune reagents are limited for pig chemokines. Our goal is to provide the veterinary community with new commercial immune reagents and standardized assays for swine research efforts. Recombinant porcine CXCL10 (rPoCXCL10) protein was produced by yeast expression and used to generate a panel of anti-CXCL10 monoclonal antibodies (mAbs). All mAbs were assessed for cross-inhibition and reactivity to orthologous yeast expressed CXCL10 proteins. Characterization of a panel of nine anti-PoCXCL10 mAbs identified six distinct antigenic determinants. Most of the mAbs did not cross-react with bovine and human recombinant orthologs. A quantitative sandwich ELISA was developed with anti-PoCXCL10-1.6 and -1.9 mAb as the most sensitive mAb pairs; reactivity was verified with both rPoCXCL10 as well as the native porcine CXCL10. The yeast expressed CXCL10 as well as native CXCL10 proteins were detected in supernatants of peripheral blood mononuclear cells (PBMC) stimulated with PMA/Ionomycin or IFNg. Selected anti-PoCXCL10 mAbs were screened for intracellular staining of stimulated pig spleen and blood cells as well as for staining of fixed tissues. Immunostaining assays verified CXCL10+ cells as CD3-CD4-CD172+, with occasional CD4+ subsets. Comparison studies determined that anti-PoCXCL10-1.4 mAb was the ideal mAb clone for intracellular staining, whereas immunohistochemistry analysis for CXCL10 expression on pig lymph nodes and spleen tissues indicated positive reactivity with anti-PoCXCL10-1.1 and -1.2 mAbs. These techniques and tools will be useful for evaluating swine immune development, responses to infectious diseases and vaccines, as well as for improving utility of pigs as an important biomedical model.