CAJ | 학술논문

目的选择理想的非特异性抗原封闭方法以适应基于免疫渗滤法的蛋白芯片制备.方法以杆状病毒-昆虫表达系统制备的人乳头瘤病毒16型(HPV16)L1蛋白抗原为例,采用脱脂奶粉、小牛血清、牛血清白蛋白及不同组合等5种方法对非特异性抗原进行封闭,检测其在免疫渗滤实验中对消除非特异性染色的效果,寻找最佳封闭非特异性染色的方法.结果经过多次重复试验,并以PBS代替血清作空白试验,结果显示,目前常用的先固相然后封闭的几种方法,其封闭效果的稳定性和重复性不佳,且空白对照易出现假阳性;同时发现使用脱脂奶粉做封闭剂封闭后,由于奶粉颗粒易堵塞NC膜的孔径,影响NC膜的渗滤速度;使用小牛血清做封闭剂封闭后,由于NC膜对小牛血清的吸附而产生背景,影响结果判读;而采用20g/L BSA先封闭后固相的方法,在实验中得到满意的结果.结论先封闭后固相的方法,在免疫渗滤实验中对非特异性抗原封闭的效果理想、结果稳定、重复性好,为一种理想的新的封闭方法.
목적 선택이상적비특이성항원봉폐방법이괄응기우면역삼려법적단백심편제비.방법 이간상병독-곤충표체계통제비적인유두류병독16형(HPV16)L1단백항원위례,채용탈지내분、소우혈청、우혈청백단백급불동조합등5충방법대비특이성항원진행봉폐,검측기재면역삼려실험중대소제비특이성염색적효과,심조최가봉폐비특이성염색적방법.결과 경과다차중복시험,병이PBS대체혈청작공백시험,결과현시,목전상용적선고상연후봉폐적궤충방법,기봉폐효과적은정성화중복성불가,차공백대조역출현가양성;동시발현사용탈지내분주봉폐제봉폐후,유우내분과립역도새NC막적공경,영향NC막적삼려속도;사용소우혈청주봉폐제봉폐후,유우NC막대소우혈청적흡부이산생배경,영향결과판독;이채용20g/L BSA선봉폐후고상적방법,재실험중득도만의적결과.결론 선봉폐후고상적방법,재면역삼려실험중대비특이성항원봉폐적효과이상、결과은정、중복성호,위일충이상적신적봉폐방법.
Objective To select an optimal non-specific antigen blocking method by using immuno-infiltration assay so as to suit protein chip preparation. Methods Human papillomavirus type 16 L1 protein expressed by insect-baculovirus espressin system was incubated with skimmed milk powder, calf serum, bovine serum albumin (BSA) combinations of five kinds of methods to block the non-specific antigen. PBS was used as control. The effect of eliminating non-specific stain was detected by immuno-infiltration assay. Results After repeated tests, the results showed that the stability and repeatability of blocking effects were poor for the fixing up antigen first and then blocking method, and the blank control was prone to false positive. The infiltration rate of NC membrane would be affected by using skimmed milk powder as a blocking agent because the pore of NC membrane was easily plugged by milk powder particles. The use of calf serum as a blocking agent made it very difficult to determine the result because the calf serum absorbed by NC membrane produced the background; however, when 20g/L BSA was used to blocking before fixing up antibody, the results became satisfactory. Conclusion Fixing up antibody after blocking in immuno-infiltration assay showed that the blocking effect against non-specific antigen was satisfactory, stable and repeatable, indicating this method is a novel optimal blocking method compared with others.