EgM家族蛋白诱导犬免疫应答动态ELISA检测方法的建立

为建立用重组GST融合EgM家族(EgM9和EgM123)蛋白和GST蛋白免疫的犬血清中由靶蛋白(EgM家族)产生的特异性抗体的检测方法,用GST融合EgM9和EgM123蛋白及GST蛋白为抗原,辅以弗氏佐剂分别免疫试验犬3次,100μg/只,并设GST蛋白组和PBS组为对照。三免后第2周,用羊源原头蚴进行口服感染,25 000枚/只,每周采血直至感染后第45天,以GST融合EgM9和EgM123蛋白包板,用中和反应处理血清中由GST蛋白和大肠杆菌产生的抗体,之后用间接ELISA和Western-blot方法检测血清中IgG、IgM和IgA的水平。结果显示,预处理的犬血清能去除GST蛋白和大肠杆菌诱导的抗体。二免后IgG和IgM水平达到峰值,IgG应答水平随免疫和感染仍能保持高应答水平。三免后IgG应答水平未提高。第三次免疫后IgM应答水平逐渐下降。IgA产生低的免疫应答,随免疫刺激和感染没有显著的变化。结果表明,EgM9和EgM123靶蛋白免疫犬能产生高水平的免疫应答,说明用吸收非特异性抗体监测特异性抗体的方法是可行的。

Establishment of ELISA for detection of the specific antibodies against EgM family proteins in the vaccinated dogs

To establish an ELISA method for detecting specific antibodies against target vaccine proteins(EgM family) in sera from dogs vaccinated with GST fusion proteins,the dogs were vaccinated three times by GST fused EgM9 and EgM123 with GST as a control,and then orally challenged with protoscolices two weeks after the last vaccination.Blood sera were collected weekly until 45 days post-infection to measure immunoglobulin(IgG,IgM and IgA) by ELISA using the fusion EgM proteins coated onto the plates after absorbing procedure to remove antibodies against GST,and possible E.coli proteins.In result,the absorbing procedure removed all antibodies against tag GST and possible E.coli proteins were confirmed by both ELISA and Western-blot.The levels of IgG and IgM were boosted and reached to the peak immediately after the second vaccination.IgG was predominant and kept in high level.The third vaccination did not increase the levels of IgG antibody production.IgM antibodies gradually decreased after the third vaccination.IgA antibodies were in low level without significant change by boost vaccination and challenge infection. In conclusion,EgM9 and EgM123 induced high levels of antibody responses in the dogs indicating that an absorbing procedure to remove antibodies against tag GST can be used for monitoring specific antibodies against EgM proteins.