新型氯霉素化学发光芯片免疫检测方法的建立及应用

Development and Application of A Novel Chemiluminescence Chip Immunoassay for Detection of Chloramphenicol

采用氯霉素(CAP)全抗原CAP-HS-BSA免疫新西兰兔,获得抗CAP多克隆抗体,测得效价为2×10~5,此抗体与CAP结构类似物的交叉反应率均小于0.01%,特异性良好。在此基础上,分别建立了特异性检测氯霉素残留的间接竞争酶联免疫吸附法(ic-ELISA)和基于iPDMS膜的化学发光芯片免疫检测法,并用于实际牛奶样品中CAP的检测。建立的ic-ELISA法的半数抑制浓度(IC_(50))为12.9 ng/mL,线性检测范围(IC_(20)~IC_(80))为0.8~250.0 ng/mL,检出限(IC_(10))为0.1 ng/mL,回归方程B/B_0=-0.2028lg C_(CAP)+0.7281(R^2=0.9926)。牛奶样品中加标回收率为81.8%~97.9%,相对标准偏差(RSD)小于10%。建立的化学发光芯片免疫检测方法的半数抑制浓度为263.0 ng/mL,线性范围(IC_(20)~IC_(80))为1~5000 ng/mL之间,检出限(IC_(10))为1 ng/mL,回归方程B/B_0=0.1781 lg C_(CAP)+0.9318(R^2=0.9891),牛奶中CAP的加标回收率为77.0%~100.5%,RSD<15%。

The rabbit anti-chloramphenicol(CAP)polyclonal antibodies were prepared by immunizing New Zealand rabbits by CAP antigen(CAP-HS-BSA),with antibody titer of 2×10^5.Then the indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)and chemiluminescence immunoassay based on iPDMS for detection of CAP were established.The proposed ic-ELISA showed a half-inhibition concentration(IC 50)of 12.9 ng/mL,with a linear detection range(IC 20-IC 80)of 0.8-250.0 ng/mL and limit of detection(LOD,IC 10)of 0.1 ng/mL.The standard curve was B/B 0=-0.2028lg C CAP+0.7281(R^2=0.9926).The cross-reactivity of ic-ELISA towards structural analogues of CAP was less than 0.01%.When applied to detection of CAP in real milk sample,the average recoveries of ic-ELISA were from 81.8%to 97.9%,with relative standard deviation(RSD)of less than 10%.However,for the proposed chemiluminescence immunoassay,the IC 50 was 263.0 ng/mL,the linear range(IC 20-IC 80)was 1-5000 ng/mL,and the LOD was 1 ng/mL.The standard curve was B/B 0=-0.1781lg C CAP+0.9318(R^2=0.9891).The average recoveries of iPDMS based chemiluminescence immunoassay were from 77.0%to 100.5%,with the RSD of less than 15%for spiked milk samples.