Résumé
A biosensor was constructed to detect the endotoxin.The amine-terminated aptamer was immobilized on the gold electrode surface covered in advance with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA).The modification of the gold electrode was confirmed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).It was found that MPA assembly time at 6 h in the gold electrode surface to form a stable self-assembled monolayer.The biosensor has a good linear relationship with concentration of endotoxin in the range of 0.001 ～0.1 EU/ml,it is possible to be applied for quality control in the actual biological sample.
Résumé
Purpose To prepare monoclonal antibody against hCG. Methods Balb/c mice were immunized with hCG, and spleen cells from the mice were fused with myeloma cells SP2/0 at ratio of 5:1. Positive clones were screened by indirect enzyme-linked-immunoadsorbent assay (ELISA) ,then the clones were subcloned by limiting dilution and amplified further. After intraperitoneal injection of hybridoma cells, mouse ascites antibody was prepared. Titres and specificity of the ascites antibody were identified and determined by indirect ELISA. The ascites were purified by protein A affinity chromatography. Results Two strains of hybridoma cell lines obtained could secrete MAb stably. The titre of MAb of cell culture supernate is more than 10~(-3), and the titre of prepared ascites MAb is more than 10~(-7).The purity of the obtained monoclonal antibody was more than 98% and recovery reached 75 % after purification. Conclusion The obtained two strains of hybridoma cell lines can secrete MAb stably. The monoclonal antibodies can be used in the research of early pregnancy and tumor diagnosis .
Résumé
Anti-HBcAg monoclonal antibodies from mouse ascites were purified by using immobilized metal ion affinity chromatography. We optimized the conditions of sample loading and elution. The results showed that when the pH stepwise elution was used, the best solution for sample loading was 20 mmol/L phosphate buffer containing 0.5 mol/L sodium chloride at pH 8.0 and the mAb was eluted at pH 5.0. The purity of obtained mAb was more than 85% and recovery reached 80%. When the adsorbed proteins were eluted by using gradient elution of an imidazole, the best solution for loading condition was 20 mmolL phosphate buffer containing 5 mmol/L imidazole at pH 8.0. The purity and recovery of antibody were up to 95%.