牛结合珠蛋白α链单克隆抗体制备及辣根过氧化物酶标记

Preparation of Monoclonal Antibodies against α Chains of Bovine Haptoglobin and Labeling of Horseradish Peroxidase Conjugates

为了制备牛结合珠蛋白主要抗原区重组蛋白(pir Bo Hp)单克隆抗体及对其进行辣根过氧化物标记。试验利用纯化的pir Bo Hp作为免疫原,免疫雌性BALB/c小鼠,然后利用淋巴细胞杂交瘤技术,制备Bo Hp单克隆抗体。通过Western blot试验分析制备的Bo Hp单克隆抗体的免疫反应性;利用抗体亚型鉴定试剂盒,分析鉴定Bo Hp单克隆抗体的亚型和轻链型。利用改良过碘酸钠标记法,对制备的Bo Hp单克隆抗体进行辣根过氧化物酶(HRP)标记。结果显示,试验成功制备了4株Bo Hp单克隆抗体,分别命名为1B3、6D6、6D3和6B7。Western blot结果显示,制备的4株Bo Hp单克隆抗体1B3、6D6、6D3和6B7能识别牛血浆中天然Bo Hp的α链。进一步抗体亚型鉴定结果表明,1B3、6D6、6D3和6B7抗体亚型均为Ig G1,轻链型为kappa链。利用改良过碘酸钠标记法,对纯化的Bo Hp单克隆抗体1B3、6D6、6D3和6B7成功进行了HRP标记。Bo Hpα链单克隆抗体的制备及其HRP标记为Bo Hp特异性诊断方法的建立奠定了基础。

In order to prepare the monoclonal antibodies （McAbs）against major antigen region recombinant protein of bovine conjugated globin （pirBoHp）and marker horseradish peroxidation,the study that bovine haptoglobin were prepared in BALB/c mice using recombinant protein of the predicted immunodominant region of bovine haptoglobin （pirBoHp）as immunized antigens by lymphocyte hybridoma technique. The immuno-response activity of the prepared monoclonal antibodies was evaluated by Western blot. The subtype of the McAbs was identified by using the Rapid ELISA Mouse mAb Isotyping Kit. Labeling of McAbs-horseradish peroxidase （HRP）was carried out by the modified sodium periodate labeling method. Results indicated that a total of four McAbs, named 1B3,6D6,6D3 and 6B7,were prepared by conventional B lymphocyte hybridoma technique. In Western blot,the McAbs 1B3,6D6,6D3 and 6B7 could reeognize or-chain of the native BoHp from plasma of dairy cows. The subtype of the McAbs 1B3,6D6,6D3 and 6B7 was IgG1K, The McAbs 1B3,6D6,6D3 and 6B7 were successfully labeled with HRP. Preparation and labeling of the McAbs against BoHp would have a potential use for developing diagnostic reagents of BoHp.