Preparation Of The Colloidal Gold Immunochromatographic Dipstick For The Rapid Detection Of Plague F1 Capsule Antigen

PART I PREPARATION AND IDENTIFICATION OF MONOCLONAL ANTIBODIES AGAINST F1 CAPSULE ANTIGEN OF YERSINIA PESTISObjectives To prepare the monoclonal antibody (McAb) against F1 capsule antigen of Yersinia pestis, for the rapid detection of Yersinia pestis.Methods Immunize BALB/C mice with F1 capsule antigen which from EV76 (the attenuated Yersinia pestis).Establish hybridoma cell strains stably secreting McAb against F1 capsule antigen. The indirect ELISA was used to detect its titer in ascetic fluid and analyze relative affinity and antigen-binding epitope of McAbs. The indirect ELISA and Western Blot identified the speciality of McAb.Results Three hybridoma cell strains, H1,H2,H3,were obtained which could stably secret McAbs. The McAb secreted by strain H1 was IgM subclass,and the rest were IgG1 subclass. ELISA additivity test proved that two kinds of McAbs recognized the same antigen epitopes with difference from the third.The relative affinity of the three kinds of McAbs were as follows:H3, H2, H1. None of the three kinds of McAbs showed cross-reactions with other bacteria we used. The hybridoma cell strains grew well after continuous culture for more than 2 months or after being stored lyophilized for 4 months,and the titers of secreted McAbs were stable.Conclusion The McAbs against F1 Was obtained.It laid a foundation of the rapid detection of Yersinia pestis.PART II PREPARATION AND APPLICATION OF THE COLLOIDAL GOLD IMMUNOCHROMATOGRAPHIC DIPSTICK FOR THE RAPID DETECTION OF F1 ANTIGENObjectives To develop a rapid diagnostic dipstick kit basing on gold immunochromatographic assay(GICA). To detect clinical samples using F1 antigen colloidal gold immunochoratographic rapid detecting dipstick. Methods Sensitivity was determined with F1 antigen and Yersinia pestis EV76 and 141.To assess the specificity we used 51 bacteria including Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica. Samples from patients died from plague and 6 sputum samples from pneumonic plague patients were used. They were tested by rapid detecting dipstick.Results The lower detection threshold of the kit was 5ng/ml and its sensitivity was 2.5×105 Yersinia pestis EV76 or 141 per ml. Its specificity was 100% tested with above specimen.All the samples from dead bodies were tested positive. 5 out of 6 sputum samples gave positive results, and the negative one had been treated before detection.Conclusion The rapid detecting dipstick was proved to be a sensitive and specific kit for the detection of Yersinia pestis. It was a simple, quick and specific kit and was suitable for detecting Yersinia pestis in large scale and in field.