高效价腮腺炎病毒抗血清的制备及检定

Preparation and identification of high titer antiserum against mumps virus

目的制备高效价腮腺炎病毒抗血清,用于麻腮、麻腮风、麻腮风水痘联合疫苗的病毒滴定及鉴别试验等指标的检定。方法将腮腺炎病毒ME株接种于SPF鸡胚尿囊腔中,优化病毒制备工艺条件,收获高滴度病毒原液,制备免疫抗原;采用皮下多点注射法免疫SPF豚鼠,制备抗血清并对其进行中和效价、中和能力以及特异性干扰试验的检定。结果 ME株腮腺炎病毒以102倍稀释接种鸡胚尿囊腔,培养5 d经-20℃预冷1 h后,收获的尿囊液病毒滴度最高;以其免疫豚鼠所制备的抗血清平均中和效价达1∶3 276,高于鸡抗腮腺炎病毒血清;当豚鼠抗腮腺炎病毒血清稀释度为1∶320时,可完全中和1 000 CCID50/m L的腮腺炎病毒;豚鼠抗腮腺炎病毒血清对Vero细胞、RK-13细胞及2BS细胞的生长,均未见干扰及细胞毒性作用;豚鼠抗腮腺炎病毒血清对异种病毒(麻疹病毒、风疹病毒和水痘病毒)滴度的干扰试验显示,各病毒滴度其试验组与对照组的差值均<0.50 lg CCID50/m L,表明豚鼠抗腮腺炎病毒血清对麻疹病毒、风疹病毒和水痘病毒的滴度均无干扰;豚鼠抗腮腺炎病毒血清及鸡抗腮腺炎病毒血清对麻腮风水痘联合疫苗各病毒滴度均无干扰,且两种抗血清之间差异无统计学意义(P>0.05)。结论采用优化后的病毒制备工艺条件及免疫方法,可获得较高效价的抗腮腺炎病毒血清,经检定和验证,均符合含腮腺炎成分疫苗检定抗血清使用要求。

Objective To prepare a high titer antiserum against mumps virus,which can be used in titration and identification of the vaccines including measles-mumps,measles-mumps-rubella,and measles-mumps-rubella-varicella combined vaccines,etc. Methods The mumps virus strain ME was injected into SPF chicken embryos,in order to obtain a high titer virus fluid,and the related preparation process was optimized. The male guinea pigs were immunized by using the allantoic fluid as a antigen and a multiple subcutaneous injection,then the antiserum was obtained. The detection of neutralization titer,neutralizing capacity and specific interference experiment for the antiserum were carried out. Results The ME strain was inoculated in102 dilution,cultured in the SPF chick embryo for 5 days,then stored at-20 ℃ for 1 h for pre-cooling before harvest,the obtained allantoic fluid had a high virus titer by such a treatment. The neutralization titer for the obtained antiserum was 1 ∶ 3276,which was a significantly higher than antiserum of chicken against mumps virus. The serum in 1 ∶ 320 dilution can completely neutralize 1 000 CCID50/ m L of mumps virus,and it had no interference and cytotoxic effect on growth of Vero cells,RK-13 cells and 2BS cells. The interference test showed that the differences were less than 0. 50 lg CCID50/ m L between the experimental groups and the control groups,and indicated that the antiserum had no interference with the titer of heterologous virus,such as measles,rubella,and varicella virus. The results showed there were no interference to the virus titer of MMRV by using the guinea pig antiserum and the chicken antiserum（ P0. 05）. Conclusion We obtained a high titer of antiserum against mumps virus by optimizing the preparation condition of the virus and the immune method with a multipie subcutaneous injection.,The prepared antiserum was in accordance with the requirements in detection of mumps containing vaccines by related detection and validation tests.