| Dairy cow mastitis is an inflammation of lacteal gland in dairy cow. It's one of the greatest disease to harm dairy cattle occupation. The problem especially subclinical mastitis hasn't been solved thoroughly. Lactoferrin(LF) is a kind of normal presence glucoprotein in cow's milk. The content of LF in milk has positive correlation with infected degree when lacteal gland of dairy cow get inflammation. And it's different along with different disease sources. Mouse anti-bovine LF monoclonal antibody(McAb) using hybridoma technique were prepared in the research to establish three detection methods of Lactoferrin by direct competition ELISA(c-ELISA),indirect competition ELISA(ci-ELISA) and double antibody sandwich ELISA. And the LF kit which is used to detect the quantitation of bovine Lactoferrin was been developmented using ci-ELISA.Three strains of Mouse anti-bovine LF monoclonal antibody(McAb) using hybridoma technique were prepared and 150mL of ascites of the monoclonal antibodie which were named 2E5, 3B8 and 2D3 were got by injection of BALB/c mice. These ascitie were purified by octanoic acid-ammonium sulfate and HiTrap Protein G HP affinity chromatography .These three McAbs were characterized for the cross reactivities, subtybes, relative affinity and antigen binding sites. The titer degree of ascites fluid of these three McAbs was above 1:5×105. Cross-reaction rates with casein,α-lactalbumin,β-lactoglobulin and serum-albumin which are four kinds of main proteins in milk were less than 0.1%. Relative affinity showed 2E5>3B8> 2D3.2E5-3B8 and 3B8-2D3 possiblly recognite different antigen binding site, with 2E5-2D3 the same antigen binding site. McAb 2E5 was successfully labeled with HRP . The labelling rate was 56.6%. And it could bind with LF specially.The experiment was operated by elementary procedure. With standard substance LF as a coated antigen, and HRP-labeled McAb 2E5(HRP-2E5) as a detection antibody, direct competition ELISA for LF was established. The concentration of coated antigen was 1.0μg/mL.And dilution of HRP-2E5 was1:40 000. The confining liquid was 2% bovine serum albumin(BSA). The TMB was chosen to be enzyme reaction substrate. The standard curve was obtained. Regression equation was got by a regression analysis. It was y=-1.8991x+5.044(R2=0.9873). The lowest detectable limit of the ELISA was 31.2ng/mL.The range of detection was 31.2~1 000ng/mL.This immunoassay provided a analytical method for the detection of LF. With standard substance LF and 2E5 as a coated antigen and a detection antibody , respectively. And with goat agaist mouse IgG labeled HRP as a second detection antibody, indirect competition ELISA for LF was established. The concentration of coated antigen was 1.5μg/mL.And dilution of 2E5 and HRP-labeled second detection antibody were 1:100 000,1:4 000, respectively. The confining liquid was 2% BSA. The TMB was chosen to be enzyme reaction substrate. The standard curve was obtained. Regression equation was got by a regression analysis. It was y=-1.6062x+4.8133(R2=0.9898).The lowest detectable limit of the ELISA was 125ng/mL.The range of detection was 125~8 000ng/mL.This immunoassay also provided a analytical method for the detection of LF. With purified McAb 3B8 against LF as a coated antibody, and HRP-2E5 as a second detection antibody, double antibody sandwich ELISA for LF was established. The concentration of coated antibody was 1:4 000. And dilution of HRP-2E5 was1:1 800. The confining liquid was 1% gelatinum. The TMB was chosen to be enzyme reaction substrate. The standard curve was obtained. Regression equation was got by a regression analysis. It was y =0.0981x -0.1387(R2=0.9380). The lowest detectable limit of the ELISA was 8.0ng/mL.The range of detection was 8~128ng/mL.And this immunoassay provided another analytical method for the detection of LF.Stabilizing agents of ELISA board with coated antiegen, standard antigen, McAb 2E5 and HRP-labeled second detection antibody were got by 37℃accelerating destroy experiments.And the ELISA kit for detecting LF was prepared to diagnose dairy cow mastitis mainly.The kit components were determined, including 1-16 No. reagents, one plate and one brochure. The sensitivity, specificity, repeatability, reproducibility, recovery rate and stability of the kit were detected. The results showed that Cross-reaction rates with casein,α-lactalbumin,β-lactoglobulin and serum-albumin which were four kinds of main proteins in milk were less than 0.1%. The lowest detectable limit of the kit was 105.6ng/mL . The variability of intar-assay and inter-assay of the kit were 4.35%,4.78%, respectively.The recovery rate for determining artificial addition Lactoferrin in dilution was 91.56%～109.20%, according with a standard of The Rule of China Biological Product.The preservation time got by 37℃accelerating destroy experiments of the kit was more than one year at 4℃and the detection time of the kit was about 3.5h.
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