浙江省科技计划项目（编号：2015C37111）；浙江中医药大学比较医学创新团队（编号：XTD201301）。
目的 制备小鼠MHV、LCM、ECT和HANT液相芯片，建立转基因小鼠传染病抗体的xMAP多重检测方法。方法 将荧光微球分别与小鼠MHV、LCM、ECT和HANT蛋白进行偶联，并且对最佳蛋白偶联量、检测抗体最佳工作浓度和Streptavidin-PE最佳工作浓度进行优化，制备小鼠MHV、LCM、ECT和HANT液相芯片，对56个转基因小鼠血清样品、阴性血清、阳性血清和质控血清进行xMAP多重检测。并应用传统的ELISA方法对xMAP检测结果进行验证。结果 1）小鼠MHV、LCM、ECT和HANT蛋白的最佳偶联量分别为20 μg、20 μg、40 μg和20 μg，检测抗体最佳浓度分别为2 μg/mL、2 μg/mL、2 μg/mL和4 μg/mL；Streptavidin-PE抗体最佳浓度均为2 μg/mL；（2）xMAP检测结果显示，14号、23号、55号和56号转基因小鼠血清MHV MFI值和index值均高于质控血清，其余小鼠血清的MFI值和index值均低于质控血清，表明14号、23号、55号和56号转基因小鼠MHV抗体阳性，其余小鼠MHV、LCM、ECT和HANT抗体阴性；（3）ELISA检测结果显示，14号、23号、55号和56号转基因小鼠MHV A450值和index值均高于质控血清，其余小鼠血清的A450值和index值均低于质控血清，表明14号、23号、55号和56号转基因小鼠MHV抗体阳性，其余小鼠MHV、LCM、ECT和HANT抗体阴性，该ELISA检测结果与xMAP检测结果相一致。结论 建立的xMAP多重检测技术可应用于转基因小鼠的传染病检测。
Objective To establish a multiplex xMAP assay for detection of infectious disease antibodies in the serum of transgenic mice by liquid chips of mouse MHV, LCM, ECT and HANT proteins. Methods The fluorescent microspheres were conjugated with mouse MHV, LCM, ECT and HANT proteins, and the amount of protein-conjugates and the working concentrations of antibodies and Streptavidin-PE were optimized, respectively. The liquid chips of mouse MHV, LCM, ECT and HANT were prepared and used to detect the antibodies in the serum samples from 56 transgenic mice, together with the negative, positive and control serum, by the multiplex xMAP assay. The results of xMAP assay were verified by traditional ELISA. Results (1) The optimal conjugated amount of mouse MHV, LCM, ECT and HANT protein was 20 μg, 20 μg, 40 μg and 20 μg, respectively. The optimal concentration of their detection antibodies was 2 μg/mL, 2 μg/mL, 2 μg/mL and 4 μg/mL, and the optimal concentration of Streptavidin-PE for all of them was 2 μg/mL. (2) The results of xMAP assay showed that the MFI and index values of the serum samples from the No. 14, No. 23, No. 55 and No. 56 transgenic mice detected with the liquid chip of MHV were higher than those of the control serum, while other MFI and index values were lower than those of the control serum, indicating that the antibodies of MHV were positive in the No. 14, No. 23, No. 55 and No. 56 transgenic mice and negative in the other mice, and the antibodies of LCM, ECT and HANT were negative in all of the mice. (3) The results of ELISA showed that the A450 and index values of MHV in the No.14, No.23, No.55 and No.56 transgenic mice were higher than those of the control serum, while other A450 and index values were lower than those of the control serum, indicating that the antibodies of MHV were positive in the No.14, No.23, No.55 and No.56 transgenic mice and negative in the other mice, and the antibodies of LCM, ECT and HANT were negative in all of the mice. These ELISA results were consistent with the results of the xMAP assay. Conclusions The multiplex xMAP assay established in this study can be used in the detection of infectious disease antibodies in transgenic mice.
陈诚,朱科燕,褚燕青,齐月寒,蔡月琴.转基因小鼠常见传染病血清学液相芯片技术多重检测方法的建立[J].中国比较医学杂志,2017,27(9):30~35.
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