基于上转发光免疫层析检测鼠疫抗体方法的初步优化与评价  

Optimization and evaluation of an up-converting phosphor technology-based lateral flow assay for detection of antibodies against Yersinia pestis

作　　者：周裕贵 孙竹林 宋亚军 牟荣 张平平 杨瑞馥 ZHOU Yu-gui;SUN Zhu-lin;SONG Ya-jun;MOU Rong;ZHANG Ping-ping;YANG Rui-fu(School of Basic Medical Science,Guizhou Medical University,Guiyang 550025,China;Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Beijing 100071,China;College of Veterinary Medicine,South China Agricultural University,Guangzhou 510630,China)

机构地区：[1]贵州医科大学基础医学院,贵阳550025 [2]军事科学院军事医学研究院微生物流行病研究所,北京100071 [3]华南农业大学兽医学院,广州510630

出　　处：《中国人兽共患病学报》2022年第7期582-588,共7页Chinese Journal of Zoonoses

基　　金：国家重点研发计划(No.2021YFC1200202);病原微生物生物安全国家重点实验室自主资金(No.SKLPBS2121)。

摘　　要：目的优化基于上转发光免疫层析检测鼠疫抗体的方法(YPAb-UPT-LF)并对其进行评价。方法依据《中国生物制品规程》收录方法改进制备的F1抗原免疫家兔,制备兔多抗作为质控品和质控带;通过筛选试纸上的最适F1抗原对YPAb-UPT-LF进行优化,再用健康人血清稀释的多种血清和多抗进行评价。结果质控品兔抗F1抗体的ELISA效价为31.25~62.5μg/L。优化后的YPAb-UPT-LF对质控品的检测灵敏度为1 mg/L,比之前提升了10倍;已被ELISA方法滴定的F1疫苗接种者血清和多种鼠疫菌株兔免血清稀释100倍后均可被YPAb-UPT-LF检出,覆盖度良好;对F1免疫兔血清、兔抗EV76和羊抗EV76的检测灵敏度分别为1∶40960、1.25 mg/L、0.625 mg/L,考虑到免疫层析的样本需经10倍稀释处理,判定YPAb-UPT-LF的检测能力与ELISA基本持平。结论本研究成功优化了YPAb-UPT-LF,为鼠疫免疫诊断和F1疫苗评估等提供了备选方法。An up-converting phosphor technology based-lateral flow assay for detection of antibodies against Yersinia pestis(Y.pestis)was optimized and further evaluated.The F1 antigen,obtained through an improved method based on the approach recorded in the Chinese Biological Product Regulation,was used to prepare rabbit anti-F1 antibody as a control.YPAb-UPT-LF was optimized by screening of F1 antigens applicable to the strip,and then evaluated in various types of sera and antibodies diluted with human serum.The ELISA titer of the control rabbit anti-F1 antibody was 31.25-62.5μg/L.The sensitivity of the optimized YPAb-UPT-LF for the control was 1 mg/L,indicating a 10-fold improvement.Serum from F1 vaccine vaccinator,as well as sera from rabbits immunized with different strains of Y.pestis,which had been titrated by ELISA,were successfully detected with YPAb-UPT-LF after 100 fold dilution,thus demonstrating the method’s excellent coverage.The detection sensitivity of YPAb-UPT-LF was 1∶40960,1.25 mg/L and 0.625 mg/L for anti-F1 rabbit serum,rabbit anti-EV76 antibodies and goat anti-EV76 antibodies,respectively.The method is as sensitive as ELISA,given that the sample required 10 fold dilution for immunochromatographic assays.In conclusion,YPAb-UPT-LF was optimized successfully and may serve as a candidate method for immunodiagnosis of plague and the evaluation of F1 vaccines.

关 键 词：鼠疫抗体 上转发光 免疫层析 F1抗原 

分 类 号：R37[医药卫生—病原生物学]