Preparation Of Brucella 16M LPS Monoclonal Antibody And Establishment Of Fluorescent Polarized Antibody Detection Method

Lipopolysaccharide(LPS)is the main component of the cell wall of most Gram-negative bacteria,also known as endotoxin or pyrogen.The structure of the LPS of different bacteria is very different.Because the LPS on the surface of Brucella is atypical LPS,once the defect will seriously affect the proliferation and viability of Brucella in vivo and in vitro,it is also the most important surface antigen of Brucella,becoming the main marker in its diagnostic.At present,the the coating antigens of Rose Bengal Plate Test and the standard-tube agglutination test for diagnosis of brucellosis are bacteria and LPS,but the specificity of these two detection methods is poor,the false positive is high,and subjective human factors have a great influence on the judgment.Therefore,the establishment of rapid,sensitive and specific Brucellosis detection methods is essential for the purification of brucellosis.By subcutaneous multi-point injection,using 60?g and 30?g extracted and purified Brucella standard strain 16M LPS as immunogen to immunize Balb/c mice for the first immunization and booster immunization.After cell fusion,selection of positive hybridoma cell lines,preparation and purification of ascites,two monoclonal antibodies were obtained,and titer analysis,subclass determination,purity detection and specificity test were performed.Preparation of fluorescently labeled antigens by using fluorescein isothiocyanate(FITC)and quantum dots(QDs)to label Brucella 16M LPS.To determine the optimal reaction volume ratio for labeling,the optimal reaction concentration of LPS and its influencing factors,and to detect the labeling effect of the two fluorescently labeled antigens prepared by fluorescence spectroscopy.Using the prepared two fluorescently labeled antigens as tracers to establish two Brucella fluorescence polarization detection methods,and to determine their optimal tracer concentration,serum optimal dilution,optimal reaction time,critical value.Using the method established in this experiment and imported fluorescent polarization(FPIA)antibody detection kit to detect 623 bovine serum samples and 30 sheep serum samples,compare the specificity,rapid accuracy and coincidence rate of the methods.In this experiment,we obtained two cell lines secreting monoclonal antibodies against Brucella 16M LPS.Their antibody titers were 1:6 400 and 1:12 800;the subclasses were IgG3 and IgGl,and the light chain types were both ? type;purity greater than 90%;both can recognize Brucella A and M antigen sites;can form obvious agglutination with standard test tube agglutination antigens,and name them Anti-A-LPS:5 and Anti-A-LPS:10.The maximum fluorescence emission wavelengths of the two fluorescently labeled antigens FITC-LPS and QDs-LPS were 524nm and 624nm,which were shifted 4nm compared to the maximum fluorescence emission wavelengths of FITC and QDs alone;when QDs labeled LPS,the optimal volume ratio is 1:5,the optimal concentration of LPS is 300ng/mL;FITC-LPS and QDs-LPS react with Brucella standard positive sera and the obtained two monoclonal antibodies,the FP value occurs obvious changes.Using FITC-LPS and QDs-LPS as fluorescent labeled antigens,the optimal tracer concentrations were determined to be 1:1500 and 1:2000,the optimal reaction time is 5min and 4min;the critical value is 101mP and 84mP;the sensitivity is 88.9%and 93.4%;the specificity is 96.6%and 89.8%;compared with the test results of the imported FPIA antibody test kit,the total coincidence rates for bovine serum samples were 94%and 89%;for sheep serum samples,the total coincidence rates were 93%and 90%.In this experiment,we obtained two monoclonal antibodies against Brucella ovis 16M LPS with high titer,good purity,high sensitivity and strong reactivity;We have established two Brucella fluorescent polarized antibody detection methods,which are fast,sensitive,convenient,highly repeatable,can achieve high-throughput detection,and can be used for qualitative and quantitative analysis of the serum samples to be tested by specific fluorescence polarization values,and suitable for clinical sample testing.Compared with the test results of imported FPIA antibody test kits,the positive detection rate of sheep serum samples is higher than that of imported FPIA antibody test kits.The research results can lay the experimental foundation for the futher establishment of Brucella fluorescence polarization pathogen detection method.