摘要
本研究将IBDV(CJ801)囊毒在鸡胚上盲传4代而后在鸡胚成纤维细胞上连传7代得到了CEF适应毒，大量繁殖细胞毒，经0.1％甲醛灭活，经蔗糖梯度密度离心法将其纯化。用纯化的IBDV作为包被抗原，建立了一种检测血清中IBDV抗体的间接ELISA方法。
通过对ELISA反应条件的系列摸索，确定了各组分的最适工作条件，并初步组装成试剂盒。试验结果表明，Costar公司生产的酶标板的变异系数为3.7％，达到ELISA的要求；IBD抗原最适包被浓度为0.0128微克／微升，最适包被条件为4℃过夜，血清稀释度为1：200，HRP-兔抗鸡IgG的最适工作浓度为1：800，待检血清和酶标二抗的反应时间均为37℃1小时，底物在室温显色20分钟。根据已经建立的ELISA方法及反应条件，确定阴阳性临界值为0.17。用本试剂盒检测已知的IBDV的BJ836、CJ801、2512、Cu和Harbin株单因子血清呈阳性反应。而与已知的SPF鸡血清呈阴性反应。用本试剂盒检测IBV，EDS’76，ND lasota，H9亚型AIV等单因子血清无交叉反应，说明建立的ELISA具有很好的特异性。本试剂盒与西班牙HIPRA公司IBDV抗体检测试剂盒的比较结果表明：两者共同检测60份血清，阴阳符合率达到96％；平行检测44份血清样品，两者检测结果呈线性关系，且相关系数为0.900。对包被好的反应板、酶标二抗、阴阳性血清和试剂盒的保存条件进行摸索，由于时间关系，只做到3个月。通过引入质控血清进行试剂盒的批内、批间重复性测试的方法，达到了试剂盒质量控制的目的。
本研究建立了一种快速、准确的检测方法，为我国IBD的免疫监测和血清学调查提供了行之有效的技术手段。
CJ801 strain of IBDV was cultured continuously for four times in chicken embryo, and then cultured for seven times in CEF. We obtained adapted virus of CEF. Then lots of these inactivitied virus was purified with methods of sucrose gradient density centrifugation.Using the inactivated CJ801 strain of IBDV as coating antigen,an indirect ELISA was successfully developed for detecting anti-IBDV antibody in serum and ELISA test kit was primarily prepared.
Conditions were optimized for using these preparations for an IBDV-specific ELISA. The result was shown that ELISA plates from Costar was suitable for ELISA kit,the optimal concentration of IBDV for coating of plate was 0.0128 u g/ul, the optimal coating condition of IBDV for ELISA was 4℃ overnight, the dilution of serum sample was 1:200 ,the working concentration of HRP-labeled rabbit anti-chicken IgG was 1:800, serum sample for detecting and HRP-labeled rabbit anti-chicken IgG should be incubated at 37℃ for 1h respectively, the substrate for ELISA was incubated at RT for 20 min before reading the OD655. Following the determination of conditions of ELISA, the threshold value of ELISA was 0.17.
Serum samples against BJ836,CJ801,2512 , Cu and Harbin were detected for the antibody to IBDV by using the developed ELISA. It was found that there were cross-reaction between the antibodies and CJ801 strain of IBDV. Meanwhile, it was confirmed that there were no cross-reaction between the antibodies against NDV, EDS'76, IBV, AIV and so on. The results revealed that the indirect ELISA by the purified IBDV had good specificity for the detection of IBDV antibody in serum.
60 serum samples from chicken were detected for the antibody to IBDV by using the developed ELISA and the HIPRA IBDV antibody test kit simultaneously. It was found that the coincidence of the developed ELISA were 96%. 44 serum samples from chicken were detected for the antibody to IBDV by using the developed ELISA and the HIPRA IBDV antibody test kit simultaneously. Both kits'result took on linary relation.The assay has met the standards of analogic method abroad.
Finally, the storage conditions of the plates coated by antigen of IBDV, the diluted HRP-rabbit anti-chicken conjugate, the control positive/negative serum and the kit were explored. So far the kit could be stored for 3 months at 4'C because of time. The study have developed an assay, which provides a available technique for immune detection and serological survey of IBD.
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