Establishment of a high-affinity anti-mouse CXCR5 monoclonal antibody for flow cytometry

Abstract

The CXC chemokine receptor 5 (CXCR5) is a member of the G protein-coupled receptor family that is highly expressed in B cells and a subset of T cells, such as T follicular helper cells. Various types of cancers, including non-small cell lung cancer, breast cancer, and prostate cancer, also express CXCR5. Therefore, antibodies that specifically bind to CXCR5 could be useful for clarification of the mechanisms of cancer progression. In this study, we aimed to develop high-affinity monoclonal antibodies targeting mouse CXCR5 (mCXCR5) for flow cytometry. The established anti-mCXCR5 mAb (Cx₅Mab-3; rat IgG_2b, kappa), demonstrated reactivity with mCXCR5-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/mCXCR5) in flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (K_D) of Cx₅Mab-3 for CHO/mCXCR5 cell is 7.2 × 10⁻¹⁰ M. Furthermore, Cx₅Mab-3 did not cross-react with other mouse CC, CXC, CX3C, and XC chemokine receptors. These results indicate that Cx₅Mab-3 is useful for detecting mCXCR5 in flow cytometry with high affinity and specificity.

Keywords

Mouse CXC chemokine receptor 5 / Monoclonal antibody / Peptide immunization / Flow cytometry

Funding

This research was supported in part by Japan Agency for Medical Research and Development (AMED) under Grant Numbers: JP24am0521010 (to Y.K.), JP24ama121008 (to Y.K.), JP24ama221339 (to Y.K.), JP23am0401013 (to Y.K.), JP24bm1123027 (to Y.K.), and JP24ck0106730 (to Y.K.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant nos. 22K06995 (to H.S.) and 22K07224 (to Y.K).

Conflict of interest

The authors declare that they have no competing interests.

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