Preparation and preliminary application of monoclonal antibodies against chicken Toll-like receptor 21

Abstract:

[Objective] To develop monoclonal antibodies (MAbs) against chicken Toll-like receptor 21 (chTLR21) and determine the expression of chTLR21 in chicken tissues challenged by avian pathogenic Escherichia coli (APEC) or highly pathogenic avian influenza virus (HPAIV). [Methods] The 6-week-old BALB/c mice were immunized with the synthetic polypeptide chTLR21 (203-225 aa) conjugated with keyhole limpet hemocyanin (KLH). The chTLR21 (203-225 aa) conjugated with bovine serum albumin (BSA) was employed as the coating protein in indirect enzyme-linked immunosorbent assay (ELISA). The positive hybridoma cells were screened by indirect ELISA. The MAbs selected by ELISA were detected by indirect immunofluorescence assay (IFA) and then used to trace the localization of chTLR21 in chicken macrophages (HD11) and determine the expression of chTLR21 in tissues of the chickens infected with APEC O1 serotype E516 strain and HPAIV H5N6 strain. [Results] An indirect ELISA method was established, with the optimal antigen coating concentration of 2.5 μg/mL and serum dilution of 1:6 400. Four positive hybridoma cell strains were obtained and named 1G3, 2C10, 3B6, and 4F11, which belonged to the IgG2b, IgG1, IgG2b, and IgG2a subclasses, respectively. The MAb 3B6 had a robust fluorescence reactivity. The results of IFA demonstrated that chTLR21 was localized in the cytoplasm of HD11 cells. Compared with that in mock birds, the expression of chTLR21 was up-regulated in liver, spleen, lung, kidney, and bursa of Fabricius while down-regulated in pancreas of the 35-day-old specific pathogen-free (SPF) chickens infected with APEC E516 strain while exhibited no changes in the tissues of the chickens challenged with HPAIV H5N6 strain. [Conclusion] We developed the MAbs against chTLR21, among which the MAb 3B6 can be used to probe the localization and expression of chTLR21 in both chickens and macrophages, providing a tool for studying the effect of chTLR21 on the pathogenesis of bacterial or viral infections in chicken.

Key words:chTLR21;monoclonal antibody;subcellular localization;Western blotting;immunohistochemistry