| Dairy cow mastitis is an inflammation of lacteal gland in dairy cow. It's one of the gravest 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 and mouse anti-rabbit LF polyclonal antibody(PcAb)were prepared in the research to establish a detection method of double antibody sandwich ELISA. And it can make a foundation for the development of LF kit which is used to diagnose dairy cow mastitis.Monoclonal antibodies against LF were achieved by injection of BALB/c mice with LF.Three strains of hybridoma cells were got by limiting dilution assay across three continuous clones. They were named 3C6,3F8 and 4D3. These three McAbs were characterized for the cross reactivities, specificities and relative affinity. The titer degree of ascites fluid of these three McAbs was above 1:2×105. There were no crossreactivities with casein,α-lactalbumin,β-lactoglobulin and serum-albumin which were four kinds of main proteins in milk. Relative affinity showed 3C6>4D3>3F8.PcAb against LF was achieved by injection of rabbits with LF. Getting blood from heart, dissociating serum, and the antiserum was last purified by caprylic acid- ammonium sulfate method. The titer degree of PcAb was above 1:64000. It's immunocompetence after purification didn't change. There were no cross reactivities with casein,α-lactalbumin,β-lactoglobulin and serum-albumin which were four kinds of main proteins in milk. PcAb was successfully labeled with HRP. The labelling rate was above 90%. And it could bind with LF specially.The experiment was operated by elementary procedure. With purified mouse anti-bovine McAb against LF as a coated antibody, and rabbit HRP-labeled PcAb as a second detection antibody, double antibody sandwich ELISA for LF was established. The concentration of coated antibody was 1.0μg/ml.And dilution of HRP-labeled PcAb was1:1000. The confining liquid was 1% gelatinum. The OPD was chosen to be enzyme reaction substrate. The standard curve was obtained. Regression equation was got by a regression analysis. It was y =1.174 log(cLF)-0.678. The lowest detectable limit of ELISA was 4.0ng/mL.The range of detection was 4~256ng/mL.This immunoassay provided a analytical method for the detection of LF.At present there are such detection methods as high efficiency liquid chromatography, immunodiffusion method, absorption spectrometry, electrophoresis method, radioactive immunoassay as well as enzyme linked immunosorbent assay(ELISA). Compared to other ones, ELISA method hashigher sensitivity and lower minimum detectability. And a number of samples can be handled one time. There are three ELISA methods to detect LF in our country. They were indirect ELISA, Competitive ELISA and double antibody sandwich ELISA. The minimum detectability in these ELISA methods can reach 0.5 ng/mL. And PcAbs are used in those detection methods. In our study double antibody sandwich ELISA was established. It's minimum detectability can reach 4.0 ng/mL, but the detection extent was bigger than other ELISA methods. And most important McAb was used to be coating antibody in this method. The specificity was improved. The influence of other composition to result could be avoided. So the accuracy of detection has been improved greatly. On this basis, the LF ELISA kit further can be prepared to diagnose dairy cow mastitis.
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