“全胚双氟碳法”制备禽脑脊髓炎琼扩抗原的研究

Preparation of the Agar Gel Precipitating Antigen for Avian Encephalomyelitis by Double Fluorcarban Extractions from Whole Embryo

用禽脑脊髓炎病毒 (AEV) Van Rockel毒株接种 6日龄 SPF鸡胚 ,感染 11d后 ,分别收集鸡胚、绒毛尿囊膜及尿囊液 ,鸡胚剔除羽毛、眼球、爪和喙后与绒毛尿囊膜一起 ,制成匀浆 ,反复冻融 3次。通过 2次氟碳 (三氯三氟乙烷 )从全胚匀浆中萃取制备 AE琼扩抗原 (AGP- Ag)。用进口标准 AE AGP试剂进行标化 ,结果发现 :制备的 AE AGP- Ag与标准阳性血清之间有清晰、致密的沉淀线 ,并与标准 AE AGP- Ag的沉淀线完全吻合 ,表明两者的沉淀反应一致 ,从而建立了 AE AGP- Ag制备新方法——“全胚双氟碳法”。其制备 AEAGP- Ag有如下特点 :1产量高 ,平均每 3个 SPF鸡胚可获得 1m L AE AGP- Ag;2特异 ,与类症阳性血清无交叉反应 ;3敏感 ,AGP试验中毋需重复加样 ,普通琼脂板即可出线 ;4稳定性好 ,保存期长 ;5与标准 AEAGP- Ag阳性检出符合率均达 10 0 %。本研究表明用“全胚双氟碳法”制备的 AEAGP- Ag达到了进口标准 AEAGP- Ag质量 ,可用于 SPF鸡 AE抗体的监测 ,且生产成本低 ,也可用于商品鸡

Six day old SPF chicken embryos were inoculated with Avian Encephalomylitis Virus (AEV) Van Rockel,and the whole embryos (without feathers,eyes,claws and beaks) were collected and homogenized at 11 days after inoculation.The homogenate was frozenthawed for 3 times,and extracted twice with Fluorocarban.The extracted sample was ultracentrifugated at 7×10 4g·min -1 for 3 h to obtain the concentrated AE AGP Ag .The Ag was evaluated with the standard AE AGP reagents.The results showed that there was clear precipitation line between the Ag and the standard positive serum,and the line matched with the standard Ag's line exactly,which suggested that the two kinds of Ag reacted in the same manner.The Ag output is higher and the Ag is specific and sensitive. 1 mL Ag could be producted with 3 SPF chicken embryos. The Ab detection rate of the Ag was identified with that of the standard Ag in monitoring SPF chickens and immunized or infected chickens.This revealed that the prepared Ag can be used to monitor AE Ab of SPF chickens,immunized breeder flocks and infected commercial chickens.