Preparation And Preliminary Application Of Monoclonal Antibodies Against Bovine Haptoglobin

In many diseases of dairy cows, the subclinical inflammatory disease has become a bottleneckwhich restricts the healthy development of the dairy farming a lot, due to its clinical symptom isnot so obvious that it is not easy to be found in the early stage, thus bringing the seriouseconomic losses to the dairy farming. As a result, the research about early warning method of thesubclinical inflammatory disease of dairy cows has very important scientific significance andapplication value. Bovine haptoglobin(BoHp) is a kind of urgent phase plasma protein, which isalso a biomarker for a variety of infectious diseases of dairy cows, has potential applicatingvalue in the early diagnosis of inflammatory diseases.In this study, we used the purified pirBoHp recombinant protein as antigen to immuneBALB/c mice. By using the conventional lymphocyte hybridoma technology, we have preparedfive BoHp monoclonal antibodies, which are respectively named as1B3,6D6,6D3,6B7and1C1. According to the Western blotting test, among the five strains of prepared BoHpmonoclonal antibodies, only1B3,6D6,6D3and6B7can identify the alpha chains of naturalBoHp in bovine plasma. The further antibody subtype identification results has showed that allthe antibody subtypes of1B3,6D6,6D3and6B7are IgG1, the light chain types are kappa chain.Then using the affinity chromatography technology to purify the IgG protein of BoHpmonoclonal antibody1B3,6D6,6D3, and6B7, further using modified sodium periodate notationmethod to label HRP on the four strains of purified BoHp monoclonal antibodies. And applyingthe purified BoHp monoclonal antibodies and HRP labelled antibody to establish the quantitativeELISA detection method of BoHp. The sensitivity test showed that the quantitative ELISAminimum detectable concentration of BoHp was31.25ng/ml. According to the clinical samplestest, the coincidence rate between the established BoHp quantitative sandwich ELISA method inthis study and the commercialized ELISA method was75%. Using sodium citrate reductionmethod to prepare the colloidal gold particles, and to label gold on BoHp monoclonal antibody,further to apply the purified BoHp monoclonal antibody and sheep anti mouse IgG imprintingnitrocellulose membrane as test line and quality control line respectively to assemble into thecolloidal gold strip for BoHp detection. The experimental results of condition optimizationshowed that, the testing result of colloidal gold strip worked best when monoclonal antibody1B3 was used as the gold labeled antibody and6D3as the detection antibody. Sensitivity test showedthat the minimum detectable concentration of assembled BoHp colloidal gold strip was0.45μg/ml. The clinical testing results showed that, there were43samples appearing signal in48dairy cows’ serum samples with different degree of limp, of which there were14samplesappearing strong signal and the positive rate was89.58%. The results above can suggest that thecolloidal gold strip can specifically identify BoHp in bovine serum.This study has used the selected subclinical inflammatory disease warning marker of dairycows, BoHp, as the research target for preparation the monoclonal antibody of BoHp protein,futher to establish the diagnostic method of warning Marker BoHp in inflammatory diseases ofdairy cows. The study also can make sure that BoHp used as subclinical inflammatory diseasewarning Marker of dairy cows is feasible through clinical effect assessment, thus to provide aeffective tool for the early warning and treatment of subclinical inflammatory disease of dairycows.