Time-resolved fluorescence immunochromatographic assay for the detection of mycoplasma pneumoniae IgM and IgG / 中华核医学与分子影像杂志

ABSTRACT

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.