Establishment And Application Of Brucella Competitive ELISA

Obstract: At present,China is no based on lipopolysaccharide(LPS)antigen,commercial Brucella competitive ELISA(CELISA)kit for the serological diagnosis of clinical CELISA mostly foreign kits,because of the high cost,to the detriment of grassroots promotion.To this end,with independent intellectual property rights,based on the LPS CELISA kit is particularly important.Our aim is to LPS as antigen,a rapid,sensitive and specific CELISA kits.Method: This paper uses a modified hot phenol-water extraction of Brucella melitensis 16 M SLPS antigen,and SLPS antigen activity of the extract were identified by accredited SLPS antigen and reagents used in the preparation of monoclonal antibodies are PACKER antigen.Meanwhile,the monoclonal antibody rapid screening methods to explore,through the cross-screening,screening semisolid culture,screening S2 can react with Brucella melitensis 16 M,544 cattle Brucella species,breeds Brucella,not with E.coli O157,Yersinia O9 produce monoclonal antibodies cross react.And based on the monoclonal antibody establish CELISA kit to kit CELISA established a preliminary evaluation,such as sensitivity,specificity,consistent rate,repetition rate.Result:(1)The improved hot phenol-water extraction method,to obtain high purity and high antigenic activity of Brucella melitensis 16 M LPS antigen,and its standardization by the line experiments,extracted LPS can be used for research and development kits;(2)After crossing screening program to obtain five height-specific,high-affinity monoclonal antibody IgG subclasses,and select an optimal effect,6-7D4 methods were established;(3)The use of methyl cellulose semi-solid culture screening method for screening monoclonal antibodies,monoclonal antibody obtained improves the chances,while reducing the time required for the screening-specific monoclonal antibody;(4)Based on Brucella 16 M SLPS antigen-specific monoclonal antibody and anti 16 M LPS 6-7D4,initially established CELISA immunodiagnostic kits;(5)Detecting the established CELISA 240 bovine serum samples from clinical sensitivity of 95.65%,specificity of 91.07%,and compliance with AHVLA CELISA rate94.58%,showed good diagnostic performance and potential.Conclusion: This experiment can be used to smooth the initial establishment of the type Brucella clinical bovine serum samples were tested CELISA diagnostic kit for fast,high sensitivity,high specificity characteristics.