抗乙型副伤寒沙门菌鞭毛抗原McAb的特性分析与初步应用

Characterization and primary application of monoclonal antibodies to Salmonella paratyphi B flagellin

目的:通过对抗乙型副伤寒沙门菌鞭毛抗原单克隆抗体的特性分析,研制检测乙型副伤寒沙门菌的ELISA法,对副伤寒传染病进行早期诊断。方法:用杂交瘤技术制备单克隆抗体,并进行鉴定,用双抗体夹心ELISA建立检测乙型副伤寒沙门菌鞭毛抗原ELISA方法,对临床标本进行检测。结果:7株抗乙型副伤寒沙门菌鞭毛抗原的单克隆抗体IgG15株、IgG2 a 1株、IgG2b 1株,均不与相关沙门菌、肠道菌反应,用菌体免疫制备的M cAb 3B5、3G12、7H1、2B5,与乙型副伤寒沙门菌、乙型副伤寒沙门菌鞭毛抗原反应,而用纯化的鞭毛抗原免疫制备的单克隆抗体1F4、5D3、3H7,只与乙型副伤寒沙门菌鞭毛抗原,3B5与7H1识别同一抗原表位,3B5、7H1与3G12、2B5相互识别不同抗原表位,1F4、5D3、3H7相互识别不同抗原表位。3B5包被、过氧化物酶(HRP)标记3G12建立双抗体夹心ELISA,鞭毛抗原、菌体的最低检出量分别为5 ng/m l、105个/m l。检测159份献血员血清及70份发热待查血清为阴性,1份发热待查血清、9份血培养阳性患者血清为阳性。结论:用纯化的鞭毛抗原免疫制备的单克隆抗体不适于检测乙型副伤寒沙门菌,菌体免疫制备的单克隆抗体特异性强,建立的双抗体夹心ELISA法,可对乙型副伤寒进行早期诊断。

Objective ：To analyze monoelonal antibodies （McAbs） against Salmonella paratyphi B flagellin protein and establish sandwich ELISA for early detection of Salmonella paratyphi B. Methods： McAbs against Salmonella paratyphi B flagellin protein were produced from hybridoma and their characterizations were performed. Sandwich ELISA assay was established with McAbs to Salmonella paratyphi B flagellin protein to detect Salmonella paratyphi B in clinical samples. Results： Seven hybridoma cell lines secreting monoclonal antibodies （McAbs） to Salmonella paratyphi B flagellin protein were established and five were IgG1, one IgG2a, the other one IgG2b. They did not react with related Salmonella bacteria and other related enterobacteria except Salmonella paratyphi B. McAbs 3B5,3G12,7H1 and 2B5 immunized with Salmonella paratyphi B reacted only with Salmonella paratyphi B and its flagellin protein. McAbs 1F4,5D3,3H7 immunized with Salmonella paratyphi B flagellin protein reacted with Salmonella paratyphi B flagellin protein only. 3B5,7H1 recognized the same immunogenic epitope on the flagellin protein. The results showed that 3B5,3G12,2B5 and 1 F4,5D3,3H7 recognized different immunogenic epitopes on the flagellin protein B. Double McAbs sandwich ELISA employing McAb 3B5 （ as coating） and 3G12 （ as enzyme labeled antibody） was used to detect Salmonella paratyphi B flagellin antigen. The established ELISA method could detect flagellin as low as 5 ng/ml or 105 cfu/ml of Salmonella paratyphi B. The result of ELISA detection of sera from 159 normal healthy blood donors and sera from 70 patients who had fever without known reason was negative,detection of serum from 1 patient who had fever without known reason and sera of 9 patients who were positive hemoculture for S. paratyphi B showed positive results. Conclusion： McAbs produced through immunization with Salmonella paratyphi B flagellin protein are unsuitable to detect Salmonella paratyphi B. McAbs produced through immunization with Salmonella paratyphi B with high specificity are used to develop the double McAKs sandwich ELISA. The method is of practical value in the early etiological diagnosis of paratyphoid fever.