title: Development of a lateral flow immunochromatographic assay for rapid detection of Mycoplasma pneumoniae-specific IgM in human serum specimens.
abstract: 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. 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-specific IgM in serum specimen. Monoclonal mouse antibody against human IgM (μ-chain specific) and goat anti-rabbit IgG were labeled with Alexa Fluor® 647 (anti-IgM-AF647 and anti-IgG-AF647). A mixture of natural MP antigen and recombinant P1 antigen was coated as the test line (T line) and rabbit IgG was coated as the control line (C line) on a nitrocellulose (NC) membrane. The MP antigens captured IgM-anti-IgM-AF647 complex on the T line. Rabbit IgG captured anti-IgG-AF647 on the C line. The fluorescence intensity on the T line and C line was measured. Sartorius CN140 NC membrane showed higher sensitivity than CN95. The optimal reaction time for the LFIA was 10min. The area under the receiver operating characteristic curve based on 34 MP positive and 166 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 serum from patients infected with non-MP pathogens including influenza viruses and bacteria causing respiratory tract infection. The intra-assay and inter-assay coefficients of variation were between 3.28% and 10.14%. The shelf life was calculated to be 2years at room temperature. The LFIA strips and the commercial EUROIMMUN kit showed consistent results on 372 serum specimens. The overall consistency rate was 96.37% with a Kappa value of 0.842 (p<0.001). The LFIA in the current study may be a sensitive and specific approach to detect early MP infection rapidly and conveniently.
authors.affiliation: Anhui Medical University, Hefei 230032, China; State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Liming
authors.initials: L
authors.lastname: Ou
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Lv
authors.affiliation: Anhui Medical University, Hefei 230032, China; State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Canjun
authors.initials: C
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China; CAS Key Laboratory of Pathogen and Immunity, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
authors.forename: Huaijie
authors.initials: H
authors.lastname: Hao
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China. Electronic address: jiangyq@nic.bmi.ac.cn.
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Jiang
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood*; D051379:Mice; D009177:Mycoplasma pneumoniae / Q000276:immunology* / Q000302:isolation & purification; D011019:Pneumonia, Mycoplasma / Q000097:blood* / Q000382:microbiology; D011817:Rabbits; D012680:Sensitivity and Specificity
chemical_list: D000907:Antibodies, Bacterial; D007075:Immunoglobulin M
keywords: Alexa Fluor 647; Human IgM; Lateral flow immunochromatographic assay; Mycoplasma pneumoniae