抗广州管圆线虫Ⅴ期幼虫55ku抗原单克隆抗体的制备、鉴定及初步应用

Production and characterization of monoclonal antibodies against 55 ku antigen from the fifth-stage larvae of Angiostrongylus cantonensis

目的制备抗广州管圆线虫Ⅴ期幼虫55 ku抗原单克隆抗体,初步应用于大鼠广州管圆线虫感染检测。方法利用Western blot鉴定能被感染广州管圆线虫Ⅴ期幼虫大鼠血清识别的特异性抗原,经电渗法获得抗原后免疫BALB/c小鼠;取脾细胞,采用常规细胞融合方法建立筛选分泌高滴度、高特异性单克隆抗体杂交瘤细胞株,用ELISA、Western blot对获得的杂交瘤细胞株进行鉴定,用生产的单克隆抗体以抗体夹心ELISA法检测感染广州管圆线虫Ⅴ期幼虫大鼠血清循环抗原水平。结果经Western blot鉴定,得到能被感染广州管圆线虫Ⅴ期幼虫大鼠血清识别的广州管圆线虫Ⅴ期幼虫55 ku抗原;获得2株分泌高滴度抗广州管圆线虫Ⅴ期幼虫55 ku抗原的单克隆抗体杂交瘤细胞株,命名为ACV1和ACV2,其分泌的抗体均为IgG2b亚型,均能识别广州管圆线虫Ⅴ期幼虫55 ku抗原,与日本血吸虫抗原等其他寄生虫抗原无交叉反应。用该单抗以ELISA方法检测感染广州管圆线虫Ⅴ期幼虫大鼠血清循环抗原,阳性率为73.3%(22/30)。结论制备的抗广州管圆线虫Ⅴ期幼虫55 ku抗原杂交瘤细胞株能分泌高滴度、高特异性的单克隆抗体,该单抗可应用于广州管圆线虫Ⅴ期幼虫循环抗原的检测。

Objective To develop and identify monoclonal antibodies（McAbs） against the 55 ku antigen from the fifth-stage larvae of Angiostrongylus cantonensis and to use McAbs to detect A.cantonensis infection in rats.Methods Western blot of the sera of infected rats identified specific antigens from the fifth-stage larvae of A.cantonensis,and the obtained antigens were purified by PAGE and electroendosmosis.Hybridomas secreting high titers of McAbs with a high level of specificity were established and screened after immunization and cell fusion.Then,McAbs were characterized using ELISA and Western blot.Finally,an antibody sandwich ELISA method based on McAbs was established to detect the level of circulating antigens in the sera of rats infected with A.cantonensis.Results Western blot revealed that the 55 ku antigen from the fifth-stage larvae of A.cantonensis has the ability to react with the sera of infected rats.Two hybridomas,designated ACV1 and ACV2,secreting high titers of McAbs with a high level of specificity were obtained.The isotype of both McAbs was IgG2b.Western blot demonstrated that McAbs are able to specifically recognize the 55 ku antigen from the fifth-stage larvae of A.cantonensis and show no cross-reaction with other antigens from Schistosoma japonicum,Trichinella spiralis,Paragonimus westermani,or Cysticercus cellulosae.Antibody sandwich ELISA detection resulted in a positive rate of 73.3 %（22/30）.Conclusion Two hybridoma cell lines against the 55 ku antigen from the fifth-stage larvae of A.cantonensis were able to secrete high titers of McAbs with a high level of specificity and could be used to detect the circulating antigens of the fifth-stage larvae of A.cantonensis.