| Background and Objective:Mycoplasma pneumoniae(MP) is a common respiratory which is the main cause of primary atypical pneumonia. 20% of community-acquired pneumonia is associated with MP. For human, especially for young child and the aged, the increasing highly morbidity has aroused more concern. Early diagnosis of Mycoplasma pneumoniae(MP) infection is crucial for prompt treatment and good patient outcome. However, serological tests to detect MP rapidly and conveniently are still lacking. The common enzyme-linked immunosorbent assay is laborious and time-consuming and colloidal gold method is unable to quantify, low sensitivity, as well as possible in diagnosis. This study aimed to use the fluorescent dye Alexa Fluor? 647 as the detection marker to develop a lateral flow immunochromatographic assay(LFIA) for detection of MP quantitatively and quickly in serum specimen.Method: Monoclonal mouse antibody against human Ig M(μ-chain specific) and goat anti-rabbit Ig G were labeled with Alexa Fluor? 647(anti-Ig M-AF647 and anti-Ig G-AF647).A mixture of natural MP antigen and recombinant P1 antigen was coated as the test line(T line) and rabbit Ig G was coated as the control line(C line) on a nitrocellulose(NC)membrane. The MP antigens captured Ig M-anti-Ig M-AF647 complex on the T line. Rabbit Ig G captured anti-Ig G-AF647 on the C line. The fluorescence intensity on the T line and C line was measure. Meanwhile, we optimize our method.The 34 MP positive and 166 MP negative serum specimens confirmed by particle agglutination test were tested on the LFIA strips that were developed in this current study. A receiver operating characteristic(ROC) curve of the LFIA strip was plotted using the software SPSS 16.0(SPSS Inc., Chicago, IL, USA). The cutoff value was determined based on the ROC curve. Samples with T/C area ratio ≥ 0.383 are considered MP positive, while samples with T/C area ratio < 0.383 are considered MP negative. Then we assess performance of specificity, sensitive accuracy and reproducibility.A total of 386 serum specimens were tested on the commercial EUROIMMUN kit and the LFIA strips that were developed in the current study. Sensitivity, specificity, the consistency of the 2 tests, and Kappa coefficient were determined and compared using the software SPSS 16.0.Result: We have developed a lateral flow immunochromatographic assay for rapid detection of Mycoplasma pneumoniae-specific Ig M in human serum specimens. The Sartorius CN140 NC membrane showed higher sensitivity than CN95. The optimal reaction time for the LFIA was 10 minutes. It was confirmed that trehalose and polyvinylpyrrolidoneplay played an important role in the stability of the strips.The area under the receiver operating characteristic curve based on 34 MP positive and166 MP negative serum samples was 0.986(p<0.001). The cutoff value of T/C area ratio was 0.3830. The LFIA strips did not react with non-MP respiratory pathogens including influenza viruses and bacteria causing respiratory tract infection. The LFIA strips show higher sensitivity than colloidal gold immunochromatographic assay for detection of MP-specific Ig M.The intra-assay and inter-assay coefficient of variation were between 3.28% and 10.14%. The shelf life was approximately 2 years at room temperature.The LFIA strips and the commercial EUROIMMUN kit showed consistent results on 368 serum specimens. The overall consistency rate was 96.37% with a Kappa value of 0.842(p<0.001).The LFIA in this current study may be a sensitive and specific approach to detect early MP-specific Ig M infection rapidly and convenient. |
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