牛β-乳球蛋白胶体金免疫层析试纸条的研制及检测应用

Development and Application of Bovine β-lactoglobulin Gold Immunoassay Strip

摘要:

研制了一种可快速检测羊乳及制品中牛源β-乳球蛋白（β-lactoglobulin，β-lg）的胶体金免疫层析试纸条。通过杂交瘤技术制备牛β-lg特异性单克隆抗体（monoclonal antibody，mAb），利用柠檬酸钠还原氯金酸，形成30 nm胶体金颗粒，并用于标记牛β-lgmAb。采用竞争法研制免疫层析试纸条，将胶体金标记的牛β-lgmAb包被于金标垫，牛β-lg和羊抗鼠IgG标记于硝酸纤维素膜分别作为检测线（T线）和质控线（C线），牛β-lg和二抗的最佳包被浓度均为1.0 mg/mL。制得的单克隆抗体纯度都在90%以上，效价均在10000以上且特异性较好。该试纸条对牛β-lg的检测限（limit of detection，LOD）值为3.13 μg/mL，对牛源α-CN，牛源β-CN，牛源κ-CN，牛源α-LA，BSA均未产生交叉反应，对脱脂羊乳粉中掺杂脱脂牛奶粉的LOD值为5%，并用该方法对市售羊奶及配方羊奶粉进行分析，检测结果与商品化的ELISA试剂盒一致。该方法前处理快速简单，可在5 min内对牛β-lg进行检测，可用于羊乳制品的现场快速检测。

Abstract:

A colloidal gold immunochromatography method for the rapid determination of β-lactoglobulin (β-lg) in goat milk and its products were established. Bovine β-lg specific monoclonal antibody (monoclonal antibody, mAb) was prepared by hybridoma technique. Sodium citrate was used to reduce chloroauric acid. The 30 nm colloidal gold particles were formed and used to label bovine β-lgmAb. The competition method was used to develop immunochromatographic strips. A bovine β-lgmAb bag labeled with colloidal gold was placed on the gold label pad. Bovine β-lg and sheep anti-mouse IgG were labeled on nitrocellulose film as detection line (T line) and quality control line (C line), respectively. The optimal encapsulation concentration of bovine β-lg and secondary antibodies were both 1.0 mg/mL. The purity and titer of monoclonal antibody were all above 90% and above 10000 with high specificity. The limit of detection (LOD) of bovine β-lg was 3.13 μg/mL. There was no cross-reaction against other substrate components (bovine α-CN, β-CN, κ-CN, α-LA, BSA). The LOD of whole fat goat milk powder adulterated with skimmed cow milk powder was 0.5 %. The method was used to analyze the commercial goat milk and formula goat milk powder. The results of this method were consistent with that of the commercial ELISA kit. This method could be used to detect bovine β-lg in 5 min, and could be used to on-site rapid detection of sheep milk products.