摘要
目的制备高效价的抗肠道病毒71型（enterovirus 71,EV71）兔血清。方法分别用RD细胞和Vero细胞培养EV71,纯化后,将经甲醛灭活和未灭活的EV71抗原分别加入佐剂或不加佐剂,经不同途径（肌肉、皮下、腹腔）免疫家兔,每周采血,分离血清,分别采用ELISA、免疫双扩散、免疫荧光和中和抗体试验检测兔血清抗体效价,并分析不同方法检测兔血清抗体结果的相关性。结果未灭活和灭活的纯化EV71抗原加弗氏佐剂后,经皮下或肌肉途径免疫家兔均可获得高效价的抗EV71兔血清,其中加弗氏佐剂的灭活疫苗经皮下途径免疫获得了最高滴度的中和抗体;当抗血清ELISA效价达到或接近1∶10^8,免疫双扩散效价达到或接近1∶512,抗血清1∶2 000稀释后荧光强度〉荻时,可进行免疫家兔的全血采集;不同方法免疫家兔全血采集的血清中和抗体效价为29 406～1 280 652 U/0.2 ml;免疫双扩散检测与中和抗体和免疫荧光检测结果的相关性较高。结论获得了高效价的抗EV71兔血清,为EV71疫苗抗原的相关检定创造了条件。
Objective To prepare high titer rabbit antiserum against enterovirus 71（EV71）. Methods EV71 was cultured in RD and Vero cells respectively and purified. The EV71 antigen inactivated with formaldehyde and that un-inactivated were mixed with Freund adjuvant respectively,and immunized to rabbits by various routes（intramuscular,subcutaneous and intraperitoneal injections）,using those containing no adjuvant as control. Serum samples of rabbits were collected weekly and determined for titers by ELISA,double immunodiffusion,IFA and neutralizing antibody assay,of which the relationship of results was analyzed. Results High titer antisera against EV71 were obtained by immunization of rabbits by subcutaneous and intramuscular routes with purified live and inactivated EV71 antigens mixed with Freund adjuvant. Neutralizing antibody at the highest titer was induced by subcutaneous injection with inactivated antigen containing Freund adjuvant. The total blood of rabbits were collected when the ELISA titer of antisera reached or nearly reached 1 ∶ 10^8,and double immunodiffusion titer reached or nearly reached 1 ∶ 512,and the fluorescence intensity of antisera after 1 ∶ 2 000 dilution was more than ＋＋. The neutralizing antibody titers in sera of rabbits immunized by various routes were 29 406 ～ 1 280 652 U / 0. 2 ml. The result of double immunodiffusion test showed high relationship to those of neutralizing antibody assay and IFA. Conclusion High titer rabbit antisrum against EV71 was obtained,which laid a foundation of relevant quality control of EV71 vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第5期664-670,共7页
Chinese Journal of Biologicals
