摘要
采用戊二醛法和物理方法,分别以BSA、HSA、OVA为载体蛋白合成了免疫抗原和包被抗原,并通过紫外扫描法鉴定了合成的抗原为载体蛋白和氨苄青霉素的复合物。将偶联抗原用弗氏佐剂乳化后免疫新西兰大白兔,制备出多克隆抗体,并对ELISA检测条件进行了优化。实验得到酶标二抗的最佳稀释倍数为1600。Ag5、Ag6、Ag7包被抗原的最佳稀释倍数分别为6400、800和400,且在ELISA试验中,选择与免疫抗原合成方法不同的合成抗原作为包被抗原为佳。底物的最佳配方为10mL底物缓冲液+7μLH2O2+200μLTMB。酶标板选用Corning酶标板。在以上条件下测得6只兔子抗血清的效价在1×103～128×103之间。亲和性试验表明以物理方法直接合成的免疫抗原比戊二醛法合成的免疫抗原亲和性好。交叉反应实验证实,抗血清与阿莫西林有极高的交叉反应率(78%),而与其它结构相似的5种抗生素的交叉反应都很小。本检测方法的检测极限是4.17ng/mL。
Bovine serum albumin （BSA）, human serum albumin （HSA） and ovalbumin （OVA） were used as three protein carries respectively to couple with semiantigen Ampicillin by glutaraldehyde method and physical method. The synthetic antigens Ampicillin-Glutaraldehyde-Carriers and Ampicillin-Carricrs were then prepared and acted as immunoantigens and coating antigens, The synthetic antigens were scanned by UV spectrum. Conditions of ELASA test were optimized in this paper. Working concentration of HRP linked goat anti-rabbit IgG antibody was 1 600x dilution. Working concentration of Ag5, Ag6 and Ag7 were 6 400x, 800x and 400x dilutions, Besides, coating antigen and immunogen should synthesize with different methods in ELISA. The best formula of reaction regent was 10 mL buffer＋7mL H2O2＋ 2004454μl TMB. New Coming microplate was chosed. Working concentration of six rabbit anti-sera were between 1×10^3 to 128×10^3 under these conditions, Competitive inhibitory indirect ELISA showed immunogens synthesized by physiological method had better affinities than immunogens synthesized by glutaraldehyde method. Amoxycillin had a high cross-reaction rate of 78% with anti-serum, Cross-reaction rate of other antibiotics with anti-serum were all very low. The detection limit of this method was 4.17ng/mL.
出处
《中国食品学报》
EI
CAS
CSCD
2006年第2期11-19,共9页
Journal of Chinese Institute Of Food Science and Technology
