Study on the establishment of immune side flow chromatography method for detecting human Mycoplasma pneumoniae lgM antibody

Abstract

In this study, in response to the clinical demand for the early diagnosis of Mycoplasma pneumoniae infection, a novel rapid detection system for IgM antibodies based on immunochromatography technology was constructed. By optimizing the antigen-antibody reaction system, using the C-terminal domain of recombinant P1 protein as the capture antigen and colloidal gold-labeled murine anti-human IgM monoclonal antibody as the signal probe, the chromatographic membrane treatment process of the test strip was innovatively improved. The experiment systematically investigated key parameters such as the particle size of the marker, the antigen coating concentration (1.2 mg/mL), and the sample dilution ratio (1:10), and determined the optimal reaction conditions through orthogonal experiments. The checkerboard titration method was adopted to optimize the spatial arrangement of the detection line/quality control line, and a four-module detection device including the sample pad, bonding pad, nitrocellulose membrane and water-absorbing pad was constructed. The performance evaluation shows that this method can complete the detection within 15 minutes, with the minimum detection limit reaching 3.12 IU/mL, and the coincidence rate with the ELISA detection results is 95%. Cross-experiments confirmed that there was no cross-reaction with 12 common respiratory pathogens such as influenza virus and adenovirus, and the intra-batch/inter-batch coefficient of variation was low. In clinical verification, the detection sensitivity of 602 suspected case samples was 100% and the specificity reached 95%. The stability test indicates that the reagent can be stored for 12 months at 4 ℃, and the accelerated test at 37 ℃ shows that the validity period is 6 months. Studies have shown that this detection system is characterized by simple operation, rapidity and sensitivity. Its detection performance meets the requirements of the "Guidelines for Stability Evaluation of In Vitro Diagnostic Reagents", providing primary medical institutions with an immediate detection solution that does not require professional equipment. It has important application value for the early screening and epidemiological surveillance of Mycoplasma pneumoniae infection.