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Establishment of a quantitative bovine CXCL8 sandwich ELISA with newly developed monoclonal antibodies
Jiro Hirota1, Shinya Shimizu, Atsushi Watanabe
1The Research Team for Advanced Biologicals, National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
Three IgG class anti-bovine CXCL8 (bCXCL8) monoclonal antibody (mAb)-secreting hybridomas, SH8-8D7, SH8-12A5 and SH8-2A1, were developed. SH8-8D7 was IgG2a, and SH8-12A5 and SH8-2A1 were IgG1. All three mAbs detected recombinant bCXCL8 (rbCXCL8) by immunoprecipitation and Western blotting. SH8-2A1 could neutralise the chemotactic activity of rbCXCL8 towards neutrophils. The quantitative bCXCL8 ELISA was constituted by the combination of SH8-12A5 and biotin-SH8-2A1. The detection range was 20-1000 pg/mL. A sandwich ELISA was used to measure native bCXCL8 derived from the supernatant of cultured bovine peripheral blood mononuclear cells stimulated with ConA, LPS or PHA. Furthermore, SH8-2A1 could detect bCXCL8 in formalin-fixed, paraffin-embedded, pneumonic calf tissues. These findings indicate that the newly developed anti-bCXCL8 mAbs could contribute to research on bovine inflammatory responses and immunology.
Three IgG class anti-bovine CXCL8 (bCXCL8) monoclonal antibody (mAb)-secreting hybridomas, SH8-8D7, SH8-12A5 and SH8-2A1, were developed. SH8-8D7 was IgG2a, and SH8-12A5 and SH8-2A1 were IgG1. All three mAbs detected recombinant bCXCL8 (rbCXCL8) by immunoprecipitation and Western blotting. SH8-2A1 could neutralise the chemotactic activity of rbCXCL8 towards neutrophils. The quantitative bCXCL8 ELISA was constituted by the combination of SH8-12A5 and biotin-SH8-2A1. The detection range was 20-1000 pg/mL. A sandwich ELISA was used to measure native bCXCL8 derived from the supernatant of cultured bovine peripheral blood mononuclear cells stimulated with ConA, LPS or PHA. Furthermore, SH8-2A1 could detect bCXCL8 in formalin-fixed, paraffin-embedded, pneumonic calf tissues. These findings indicate that the newly developed anti-bCXCL8 mAbs could contribute to research on bovine inflammatory responses and immunology.
