[Preparation of colloidal gold test strips for the detection of antibodies to peste des petits ruminants based on monoclonal antibodies to N protein].

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.

Development of a sensitive immunochromatographic method using lanthanide fluorescent microsphere for rapid test for PPRV antibody.

Peste des petits ruminants virus (PPRV) causes a very devastating disease in sheep and goats. Rapid diagnosis and immunisation have been identified as key strategies for successful prevention of the disease. Therefore, a sensitive fluorescent microsphere immunochromatography test strips (FM-ICTS) was developed for rapid detection of special antibodies of PPRV in goats and sheep serum. The FM-ICTS were successfully prepared by fluorescent microspheres (FM) as tracer, which were covalently coupled to PPRV nucleocapsid protein (NP). The NP and monoclonal antibody of NP were separately dispensed onto a nitrocellulose membrane as test and quality control lines, respectively. The critical threshold for determining negative or positive through the ratio of the fluorescent signal of the test line and the control line (T/C) is 0.050. The repeatability of the FM-ICTS was excellent, with an overall average CV of 3.17 %. The detection limit of this assay was 1:5120. Additionally, the FM-ICTS no cross reaction with the sera of other related diseases was observed, only reacting with anti-PPRV serum. 70 serum samples were tested by FM-ICTS and commercial ELISA kit, and the results showed good agreement. Overall, a promising pen-side diagnostic tool was developed for the rapid qualitatively/semi-quantitatively detection of PPRV antibodies within 15 min.