双抗体夹心酶联免疫吸附试验在肺炎支原体抗原检测中的建立与应用  

Establishment and application of a double-antibody sandwich enzyme linked immunosorbent assay method for the detection of Mycoplasma pneumoniae antigens

作　　者：张凤莲 钏鸿云 童菲 袁广波 李诚伟 廖国阳[1] ZHANG Fenglian;CHUAN Hongyun;TONG Fei;YUAN Guangbo;LI Chengwei;LIAO Guoyang(Peking Union Medical CollegeChinese Academy of Medical SciencesInstitute of Medical Biology,Yunnan Province,Kunming650000,China)

机构地区：[1]北京协和医学院中国医学科学院医学生物学研究所,云南昆明650000

出　　处：《中国医药导报》2025年第6期165-170,共6页China Medical Herald

基　　金：中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-043)。

摘　　要：目的构建用于检测肺炎支原体抗原的双抗体夹心酶联免疫吸附试验(ELISA)法,并应用其检测不同耐药表型临床分离株抗原。方法鉴定抗肺炎支原体单克隆抗体4G4、8H6的抗体结合位点竞争性及结合活性;以4G4为包被抗体,8H6为酶标抗体建立双抗体夹心ELISA法,优化其线性范围、稳定性、特异性、广泛适用性与准确度。结果单克隆抗体4G4、8H6具有不同的抗原结合位点,半数最大效应浓度(EC50)分别为0.4113、0.1881μg/ml;最佳反应体系为包被抗体浓度4μg/ml,酶标抗体1∶4000稀释,封闭液为含2%BSA、2%蔗糖的0.01 mol/L PBS。该方法线性范围为1~32 U,线性回归方程Y=0.0742X+0.073,R2=0.9969;批间变异系数为0.4784%~7.9613%,批内变异系数为1.6158%~13.1754%;特异性与广泛适用性佳,不与新型冠状病毒感染、流感、猴痘病毒及多种细菌交叉反应,可检测不同耐药株抗原;与肺炎支原体抗原检测商用试剂盒(ELISA法与胶体金法)检测结果符合率96.7%~103.3%。结论该双抗体夹心ELISA法线性范围优、稳定性强、特异性好、适用广、准确度高,可用于肺炎支原体疫苗生产中的抗原定量。Objective To construct a double-antibody sandwich enzyme linked immunosorbent assay(ELISA)method for the detection of Mycoplasma pneumoniae antigens and apply it to detect antigens of clinical isolates with different drug resistance phenotypes.Methods The antibody binding site competitiveness and binding activities of anti-Mycoplasma pneumoniae monoclonal antibodies 4G4 and 8H6 were identified.A double-antibody sandwich ELISA method was established using 4G4 as the coating antibody and 8H6 as the enzyme-labeled antibody,and its linear range,stability,specificity,broad applicability,and accuracy were optimized.Results Monoclonal antibodies 4G4 and 8H6 have different antigen-binding sites,with the half-maximal effective concentration(EC50)of 0.4113μg/ml and 0.1881μg/ml,respectively.The optimal reaction system was a coating antibody concentration of 4μg/ml,an enzyme-labeled antibody dilution of 1∶4000,and a blocking buffer of 0.01 mol/L PBS containing 2%BSA and 2%sucrose.The linear range of this method was 1-32 U,with a linear regression equation of Y=0.0742X+0.073 and R2=0.9969.The inter-batch coefficient of variation was between 0.4784%-7.9613%,and the intra-batch coefficient of variation was between 1.6158%-13.1754%.It had excellent specificity and broad applicability,without cross-reaction with corona virus disease 2019,influenza,monkeypox viruses,and various bacteria,and could detect antigens of different drug-resistant strains.The coincidence rate of the detection results with commercial Mycoplasma pneumoniae antigen detection kits(ELISA method and colloidal gold method)was between 96.7%-103.3%.Conclusion This double-antibody sandwich ELISA method has an excellent linear range,strong stability,good specificity,wide applicability,and high accuracy,and can be used for antigen quantification in Mycoplasma pneumoniae vaccine production.

关 键 词：肺炎支原体 双抗体夹心酶联免疫吸附试验法 抗原检测 单克隆抗体 

分 类 号：R375.2[医药卫生—病原生物学]