禽呼肠孤病毒S1733株单克隆抗体的制备及特性鉴定

摘要

The reovirus S1733 harvested from chicken embryos allantoic fluid was purified by sucrose gradient purification. The concentration was tested by protein testing kit. Then BALB/c mice were regularly immunized with purified reovirus S1733 contain 100 μg protein. After injecting four times, the spleen cells were collected and infused with myeloma cell SP2/0 follow ratio 5: 1. After selection in time and 3 times clone by limiting dilution. The two monoclonal antibodies were detected by indirect ELISA, Dot-ELISA, and immunofluorescence methods. The results showed that the purification protein was 43 mg/mL. Two hybridoma cells lines against reovirus named SF6-3K3 and SB2-1K3 were obtained. The titers were all above 10° by indirect ELISA detection. It was not cross with others virus strains in this test by Dot-ELISA. The subtype was IgG1. It indicated that two hybridoma cell lines possessed the favorable specificity. Both of them could be used to detect reovirus in the future.%将增殖的禽呼肠孤病毒S1733毒株进行浓缩,然后进行蔗糖梯度纯化,测定纯化后病毒的浓度,按每只鼠100 μg的病毒用量免疫BALB/c小鼠,免疫5次后取其脾细胞与骨髓瘤细胞SP2/0按5:1进行融合.对融合后的杂交瘤细胞进行筛选,阳性孔经3次有限稀释法克隆.通过间接ELISA方法测定抗体效价,并通过Western blotting、Dot-ELISA、直接免疫荧光和中和反应等方法对2株单克隆抗体特性进行检测.试验结果表明,纯化的病毒含量为43 mg/mL,用纯化的病毒免疫BALB/c小鼠后与骨髓瘤细胞融合,通过克隆筛选成功获得2株能稳定传代并分泌抗禽呼肠孤病毒单克隆抗体的杂交瘤细胞株SF6 3K3和SB2 1K3.2株单克隆抗体的腹水经间接ELISA测定效价达105以上,其抗体为IgG1亚类,特异性试验表明其与其他病毒株没有交叉反应,具有良好的特异性,并且这2株单克隆抗体没有中和禽呼肠孤病毒的能力,具有识别禽呼肠孤病毒的能力,可以用于禽呼肠孤病毒的特异性检测.