肺炎支原体IgM和IgG抗体的时间分辨荧光免疫层析法的建立及初步应用

Time-resolved fluorescence immunochromatographic assay for the detection of mycoplasma pneumoniae IgM and IgG

目的构建可快速、灵敏、定量检测肺炎支原体(MP)免疫球蛋白(Ig)M和IgG抗体的时间分辨荧光免疫层析法(TFICA)。方法基于毛细管作用,应用铕微球示踪,通过对抗原/抗体微球偶联比、微球稀释度、划膜浓度和血清稀释度进行优化,建立检测MP-IgM和IgG抗体的TFICA,并考核其方法学性能(如灵敏度、特异性、稳定性)。通过对55名健康体格检查者进行检测获得TFICA的正常参考值;采用TFICA与市售化学发光法试剂盒分别检测88名受试者(33例患者,55名健康体格检查者)的血清样本,计算结果一致性(Kappa检验)。结果反应条件为:鼠抗人IgG抗体和MP抗原与微球分别按质量比1∶20和1∶100反应,微球工作稀释度分别为1∶200和1∶100;MP抗原和羊抗人IgM的划膜浓度分别为0.5和1.0 g/L;血清稀释度均为1∶300。建立的MP-IgM和IgG TFICA测量范围分别为(0.78~70.00)×10^(3)相对单位(RU)/L和(0.17~200.00)×10^(3) RU/L,检测灵敏度分别为0.78×10^(3)和0.17×10^(3) RU/L,与甲状腺过氧化物酶抗体、心磷脂酶抗体、甲状腺球蛋白抗体均无交叉反应;相对标准偏差分别为3.7%~14.8%和2.9%~14.0%。经37℃5 d快速老化,MP-IgM和IgG TFICA试剂的平均信号降低率分别为13.7%和14.2%,提示热稳定性好。该法正常参考值分别为3.33×10^(3)和2.61×10^(3) RU/L;与市售化学发光试剂盒检测结果的Kappa值分别为0.79和0.76。结论TFICA检测MP-IgM和IgG抗体具有操作简捷、灵敏度高、特异性好、可定量的优点,具备一定的临床应用价值。

Objective To establish time-resolved fluorescence immunochromatographic assay(TFICA)for rapid and quantitative detection of mycoplasma pneumoniae(MP)immunoglobulin(Ig)M and IgG.Methods Based on capillary effect and europium nanospheres,rapid TFICA for MP-IgM and IgG detections were developed with the optimized parameters(coupling rates of antigens or antibodies to microspheres,dilution of labeled nanospheres,fixture concentrations on test line and serum dilutions).The methodological performances were estimated such as sensitivity,specificity,stability.By testing 55 healthy control samples,the reference values of TFICA were obtained.The reliability was evaluated by Kappa test from detecting sera of 88 cases(33 patients and 55 healthy controls)using TFICA and commercial kits by chemiluminescence immunoassays(CLA).Results After screening the assay conditions,the mass ratios of mouse anti-human IgG and MP antigen with nanospheres were 1∶20 and 1∶100 respectively;the work dilutions of nanobeads conjugated with anti-human IgG and MP antigen were 1∶200 and 1∶100 respectively;the spraying concentrations of MP antigen and goat anti-human IgM were 0.5 and 1.0 g/L on the test line respectively,and the working dilutions of serum sample were both 1∶300.In the MP-IgM and IgG detections,the linear working ranges were(0.78-70.00)×10^(3) relative unit(RU)/L and(0.17-200.00)×10^(3) RU/L,while the sensitivities of the assays were 0.78×10^(3) and 0.17×10^(3) RU/L,respectively.No cross reactions were found with antithyroid peroxidase antibody,anticardiolipin antibody or thyroglobulin antibody.In these MP-IgM and IgG assays,the relative standard deviations were 3.7%-14.8%and 2.9%-14.0%,the average reduction rates of fluorescence were 13.7%and 14.2%respectively after incubation at 37℃for 5 d.The reference values of MP-IgM and IgG were 3.33×10^(3) and 2.61×10^(3) RU/L,while the Kappa values between TFICA and CLA were 0.79 and 0.76,respectively.Conclusion TFICA is a simple,sensitive,specific and quantitative method for detecting MP-IgM and IgG antibodies,and may show great promise for future clinical use.