Preparation and characteristics analysis of blockers for in vitro diagnostic test kits
He Ruqin, Lei Peng, Deng Longzhi, et al
(1. School of Environmental Ecology and Bioengineering, Wuhan Institute of Technology, Hubei, Wuhan 430205,China; 2. Wuhan Yesda Biotechnology Co., Ltd, Hubei, Wuhan 430200, China)
Abstract: 【Abstract】 Objective To explore the preparation methods and characteristics of blockers for in vitro diagnostic test kits. Method Six-week-old BALB/c mice were used for antigen immunization. Hybridoma cells were constructed and cloned. The method of inducing monoclonal antibodies in animal bodies was used to prepare a large quantity of monoclonal antibody, which was purified by two methods (G-column affinity chromatography, and blue gel + Q-column ion-exchange chromatography). The optical density (OD) was recorded at a wavelength of 280 nm using a nucleic acid protein detector. Finished product concentration was detected using a ultraviolet spectrophotometer. The purity of the antibody was detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Enzyme-linked immunosorbent assay (ELISA) double antibody sandwich method was used to detect the blocking ability of this blocker (CN302) on false positive samples. Result The yields of the blocker extracted by G-column affinity chromatography (6.49, 6.12, and 6.27 g/L for three batches, respectively) were higher than those by blue gum + Q-column ion-exchange chromatography (1.78, 1.68, and 1.61 g/L for three batches, respectively). The purity of both purification methods was up to more than 95%. The activities of the blockers obtained by G-column affinity chromatography and blue gum + Q-column ion-exchange chromatography were 106.54% and 92.45%, respectively. Compared with the blank group, the detection of false-positive samples was reduced by blockers extracted using both purification methods, and the OD values of the blocker CN302 were less than 0.2, which were about the same as that of the reference blocker. The activity of the blocker CN302 obtained by G-column affinity chromatography was 99.86% without freezing and thawing, and 99.83% after 5 times of freezing and thawing. Without blocker, the OD values of false-positive samples were about 2.5 and up to 4.0, that decreased significantly after the addition of the blocker. The OD values of the blocker CN302 were lower than those of the other blockers for the detection of false-positive samples. Conclusion The blocker obtained in this study significantly eliminates the endogenous interference in the samples, and the activity was almost completely retained. The yield and blocking ability of the antibody extracted by G-column affinity chromatography were higher than those of the blue gel + Q-column ion-exchange chromatography.
何汝琴 雷鹏 邓龙芝 朱文武 朱雄伟 熊梦瑶. 体外检测试剂盒阻断剂的制备方法及特性分析[J]. 发育医学电子杂志, 2024, 12(1): 1-6,19.
He Ruqin, Lei Peng, Deng Longzhi, et al. Preparation and characteristics analysis of blockers for in vitro diagnostic test kits. Journal of Developmental Medicine(Electronic Version), 2024, 12(1): 1-6,19.
2024, Vol. 12 
