摘要
建立检测疫苗中残余牛血清蛋白(CSP)含量的ELISA检测法。将混合的小牛血清分别免疫鸡和家兔,然后分别提取、纯化鸡卵黄免疫球蛋白(IgY)和兔抗小牛血清抗体IgG。IgY用作包被抗体,IgG用作酶标抗体,四甲基联苯胺(TMB)显色检测残余CSP含量。结果:包被用IgY的最适浓度为25—30μg/mL,用含0.4%明胶的0.01mol/LPBS(pH7.4)溶液作为包被缓冲液,HRPIgG的最适稀释度为1:20001:3000。该方法的检测灵敏度为2.5ng/mL,变异系数为6.3%—9.4%,回收率为90.4%—112.8%,与猪、猴、豚鼠血清皆无交叉反应。10批不含Al(OH)3的疫苗ELISA法与RPHA法(反相间接血凝法)检测结果无显著性差异(P>0.05);而10批含Al(OH)3的疫苗,两种方法检测结果相差很大(P<0.05)。建立的ELISA检测法不受Al(OH)3的影响,简便、特异和灵敏,更适用于检测生物制品中的残余CSP。
To develop a new DAS-ELISA (double antibody sandwich ELISA) kit for detecting residual calf serum protein (CSP) in vaccines, calf sera from different district were pooled and used to immunize rabbits and hens respectively. Then, the IgY from yolk and the anti-CSP IgG from rabbit were separated and purified. The purified IgY was used as the coating antibody, and purified rabbit anti-CSP IgG was labeled by HRP. The optimal concentration of IgY was 25-30μg/mL. The coating buffer was 0.01mol/L PBS(pH7.4) containing 0.4% glutin. The optimal dilutions of HRP-IgG were from 1:2000 to 1:3000. The sensitivity of this ELISA method was higher (up to 2.5ng/mL) than that of current RPHA, the variation coefficient was about 6.3%—9.4%, and the recovery rate was 90.4%—112.8%. Furthermore, there was no cross-reaction with sera of pig, monkey and guinea pig. Twenty lots of vaccines with Al(OH)_3 or without Al(OH)_3 were tested by ELISA and RPHA respectively. The results proved that the adjuvant of Al(OH)_3 had fewer influence on ELISA than on RPHA, the variation of PRHA among different lots of vaccines was more significant than ELISA. The ELISA method is a highly sensitive and useful method to detect CSP in vaccines.
出处
《标记免疫分析与临床》
CAS
2005年第2期107-109,共3页
Labeled Immunoassays and Clinical Medicine
