Establishment Of UPT Immunochromatography Test For Detection Of B.melitensis By Monoclonal Antibodies Against B.melitensis 16M

Brucella is an intracellular bacterial pathogen. Brucella invades the body, causing infection-allergic zoonosis called Brucellosis. Brucella causes abortions, infertility, testicular inflammation and arthritis in Mammals and every year incalculable economic losses to livestock. Human brucellosis mainly as a long-term fever, chronic granulomatous disease affecting person's various organ systems. In addition, Brucella has a stronger ability to survive in the environment and easily spread through the air of aerosols with high infectivity. Brucella can be used as biological warfare agents. The diagnosis of brucellosis traditionally relies on serological testing with a variety of agglutination tests and the well-standardised ELISA.These methods may be more suitable for confirmation of brucellosis in relapsing patients'diagnosis. Etiologic diagnosis depens on bacterial culture and identification. PCR has been a importent tool for the diagnosis of human brucellosis in laboratory, but utilized in practice is not much.Therefore, a rapid and effective method for detection of Brucella is necessary to diagnosis and in drug treatment. In this study, monoclonal antibodies were prepared by cell fusion, using in vivo whole-cell antigen and dead bacteria immune mice. Monoclonal antibodies that specific to Brucella melitensis and Brucella abortus were screened by ELISA.Brucella UPT immunochromatographic test strip was made by binding of light-emitting materials. At the same time the sensitivity, quantification, specificity, compatibility and stability were evaluated for the Brucella UPT immunochromatography test strip. At last, small-scale production, and white powder to simulate the experimental specimens were tested.The results showed that:(1) In vivo whole-cell Brucella antigen immunogenic than inactivated bacteria, the monoclonal anti-Brucella antibody titer were between 512000～1024000.(2)Developed Brucella monoclonal antibodies combined with the UCP particles prepared by UPT immunochromatographic strip; (3) Brucella UPT immunochromatographic test strip performance were evaluated. The results show that UPT immunochromatographic test strip has a good specificity(terrorist warfare agents for the common pathogens, Brucella near-source bacteria, and Brucella serological cross-reactions with pathogens can not be detected); Good stability(CV values were <15%); Good compatibility(several common Brucella can detected); good sensitivity (the detection limit is about 5.6×104 CFU, can quantitatively detected,105 to 109 CFU with a good linear relationship; (4) In external environmental, UPT test strip were not impacts the detection ability for simulation of white powder, detection limits can be maintained at 5.0×104 CFU Brucella.In the present study, we create a rapid, on-site testing method for quantitative detection of the whole cells of Brucella. The specificity and sensitivity of this assay was also satisfactory. And it has the potential to be optimized for greater sensitivity for on-site detection.