Open Access

ARTICLE

Establishment of a quantitative bovine CXCL8 sandwich ELISA with newly developed monoclonal antibodies

Jiro Hirota¹, Shinya Shimizu¹, Atsushi Watanabe², Fumiko Suzuta³, Kazue Yajima⁴, Kumiko Kimura⁵, Makoto Haritani⁵, Shigeki Inumaru¹, Yukio Yagi⁶

1 The Research Team for Advanced Biologicals, National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba,Ibaraki
2 The Research Team for Environmental/Enzootic Diseases, National Institute of Animal Health, National Agriculture and Food Research Organization, Sapporo, Hokkaido
3 Nagasaki South Livestock Hygiene Service Center, Shimabara, Nagasaki
4 Hyogo Himeji Livestock Hygiene Service Center, Himeji, Hyogo
5 The Research Team for Bacterial/Parasitic Diseases, National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba Ibaraki
6 National Institute of Animal Health, National Agriculture and Food Research Organization Tsukuba, Ibaraki, Japan

* Corresponding Author: S. Shimizu,

European Cytokine Network 2011, 22(1), 73-80. https://doi.org/10.1684/ecn.2011.0275

Accepted 12 October 2010;

Abstract

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 antibCXCL8 mAbs could contribute to research on bovine inflammatory responses and immunology.

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Keywords

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