摘要
目的制备耐甲氧西林金黄色葡萄球菌(MRSA)青霉素结合蛋白2a(PBP2a)抗原的卵黄抗体IgY,建立检测MRSA的酶联寡聚核苷酸吸附试验(ELONA)方法。方法用大肠埃希菌原核表达并纯化PBP2a,采用胸部肌肉多点注射方式免疫产蛋母鸡,水稀释法粗提IgY,BCA法测定蛋白浓度,Western blot分析其特异性;以IgY抗体为捕获分子,以生物素标记的PBP2a特异性适配体为检测分子,建立检测MRSA的ELONA方法;对IgY包被量、生物素标记适配体用量及HRP标记链霉亲和素稀释度进行优化,测定该方法的灵敏度、特异性和重复性。结果成功表达和纯化PBP2a蛋白,Western blot显示IgY抗体能特异识别PBP2a蛋白。建立了检测MRSA的ELONA方法,其最佳包被用IgY稀释度为1∶1 600,生物素标记适配体用量为400nmol/L,HRP标记链霉亲和素稀释度为1∶2 000。优化的ELO-NA对MRSA的检测限为2.87×10~2 CFU/ml;重复性试验的变异系数为4.7%。用该方法检测34株临床分离的金黄色葡萄球菌,与梅里埃VITEK2Compact全自动细菌鉴定及药敏分析系统分析结果一致性为100%。结论制备的抗MRSA PBP2a卵黄抗体具有较高特异性,基于该抗体建立的ELONA方法灵敏、特异、重复性好,可用于MRSA PBP2a的检测。
Objectives To prepare yolk antibody(IgY)against penicillin binding protein 2 a(PBP2 a)of methicillin-resistant Staphylococcus aureus(MRSA)and to create an enzyme-linked oligonucleotide assay(ELONA)for detection of MRSA. Methods PBP2 awas expressed in Escherichia coli and purified using Ni-NTA agarose.Chickens were immunized via multiple intramuscular injection of the recombinant PBP2 a,and IgY antibodies were extracted using the water dilution method.The concentration and specificity of IgY antibodies were analyzed using the bicinchoninic acid(BCA)method and Western blotting.An ELONA for MRSA detection was created by using IgY antibodies to capture PBP2 a from the MRSA lysate.Biotin-labeled aptamer was used to detect the captured PBP2 a,horseradish peroxidase(HRP)-labeled streptavidin was added to bind to the biotin,and MRSA was detected based on colorimetric signals generated by the catalytic reaction between HRP and the substrate 3,3’,5,5’-tetramethylbenzidine(TMB).Different parameters(for instance,the IgY titer,biotin-labeled aptamers,and HRP-labeled streptavidin)were optimized.The sensitivity,specificity,and repeatability of this method were determined under optimized conditions. Results PBP2 awas successfully expressed and purified.Western blotting indicated that the IgY antibody specifically recognized PBP2 a.Optimal conditions were IgY at a dilution of 1:2,000,400 nmol/L of biotin-labeled aptamers,and HRP-labeled streptavidin at a dilution of1:1,600.The limit of detection(LOD)for the assay was 2.87×102 CFU/ml,and the assay was highly specific for MRSA.The coefficient of variation(CV)in a repeatability test was 4.7%.Thirty-four clinical isolates of Staphylococcus aureus were tested using this method,and results agreed 100% with results from the automated Vitek 2 system. Conclusion IgY was highly specific for MRSA PBP2 a,and an ELONA technique with a high level of sensitivity,specificity,and repeatability was developed to detect MRSA.
作者
孙誉芳
李宁
孟祥英
王彰骄
张羽飞
乔晋娟
SUN Yu-fang;LI Ning;MENG Xiang-ying;WANG Zhang-jiao;ZHANG Yu-fei;QIAO Jin-juan(Department of Medical Laboratory Science,Weifang Medical University,Weifang 261053,Shandong,China 261053)
出处
《中国病原生物学杂志》
CSCD
北大核心
2019年第1期55-59,共5页
Journal of Pathogen Biology
基金
山东省自然科学基金项目(No.ZR2017LH057
ZR2016BL19)
潍坊医学院大学生科技创新项目(No.KX2017029)
