| Brucellosis, also called undulant fever, or Malta fever, in humans is a highly contagious zoonosis (infectious disease transmitted from animals to humans) caused by bacteria of the genus Brucella. The most common clinical signs of animals infected with Brucella are high incidences of abortions, arthritic joints and retention of after-birth, known as retained placenta. Humans become infected by coming in contact with animals or animal products that are contaminated with these bacteria. In humans Brucellosis induces inconstant fevers, sweating, weakness, anemia, headaches, depression and muscular and bodily pain. It is found throughout the world,and cause great harm and loss to human and animal husbandry.Nowadays,Brucellosis is diagnosed in a laboratory by finding Brucella organisms in samples of blood or bone marrow. Also, blood tests can be done to detect antibodies against the bacteria.But these methods have low specificity and sensitivity,and also need a long time to get the results,so to establish a new simple method which have high specificity and sensitivity is more important in prevention and treatment of Brucellosis.LPS and O-chain of B.abortus 544A and B.melitensis 16M were extracted by hot phenol method and cold phenol method, and were purified by Sephadex G-50.The results of agarose gel diffusion,SDS-polyacrylamide gel electrophoresis and silver staining showed that the molecular weight of LPS and O-chain of B.abortus 544A was 70000Da and 7000Da respectively, and the molecular weight of LPS and O-chain of B.melitensis 16M was 100000Da and 6000Da, respectively.BaLb/C mouse were immunized by B.abortus and B.melitensis ,and hybridoma cell strain 4B8,1C9,1G9 and 1E2 of B.abortus and 3C6,2D10 and 1E10 of B.melitensis were screened while LPS and O-chain as antigen,and their subtype were identified.Monoclonal antibody 4B8,1C9,1G9 and 1E2 of B.abortus 544A belonged to IgG3,IgG2a, IgG3 and IgM respectively;and monoclonal antibody 3C6,2D10 and 1E10 of B.melitensis 16M belonged toIgG2b, IgG1 and IgM respectively.Cell strains 4B8 of B.abortus 544A and 2D10 of B.melitensis 16M were cultured,and ascites of monoclonal antibody was prepared by intraperitoneal injection the cell into Balb/C mice.Proteins in ascites of 4D8 and 2D10 were 9.70mg/ml and 8.67mg/ml respectively,and contents of antibody was 3.2mg/mL and 2.8mg/mL respectively.Monoclonal antibody was purified and identified by SDS-PAGE and the resulted showed that molecular weight of 4B8 and 2D10 was 150 000Da and 47 100Da respectively,affinity constant of 4B8 and 2D10 was 8.99×108M-1 and 5.30×107 M-1 respectively,antibody titer was more than 1×106.Polystyrene Latex Particles were allergized and carboxylated by chemical crosslinking,and the reaction condition was optimized by square matrix titration.The best fixation concentration, time and latex concentration of allergized monoclonal antibody 4B8 was 1.60mg/ml,4h and 1%,respectively.And the best fixation concentration,time and latex concentration of allergized monoclonal antibody 2D10 was 1.40mg/ml,4h and 1%,respectively. Thereby monoclonal antibody latex agglutination test for B.melitensis and B.abortus was established, and the results of sensitivity detection showed that the monoclonal antibody latex agglutination test of B.abortus 544A and B.melitensis 16M can not take place crossreaction with E.coli O:157,Yersinia O:9,Salmonella and Actinobacillus. The guarantee period of reagent was 10 months.The minimum detectable amount of monoclonal antibody 4B8 of B.abortus 544A was 1.0×106 CFU, and the limit quantitation of the method sampled from water,soil and milk was 1.0×106CFU, 3.0×106 CFU and 3.0×106 CFU.The minimum detectable amount of monoclonal antibody 2D10 of B.melitensis 16M was 3.0×106 CFU, and the limit of the method quantitation sampled from water,soil and milk was 3.0×106CFU, 5.0×106 CFU and5.0×106 CFU.
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