|
|
|
马立克病肿瘤相关抗原间接ELISA检测方法的建立
|
|
|
|
穆杨1，王玉东2，张彦明1，李崴2，李玉清2，郑增忍2，高敬1，童德文1
|
|
|
|
（1.西北农林科技大学 动物科技学院，陕西 杨凌712100；2.农业部动物检疫所，山东 青岛266032）
|
|
|
|
摘要：
|
|
用自制并纯化的马立克病肿瘤相关表面抗原（MATSA）免疫SPF级BALB/c小鼠制备阳性对照抗体，以SPF级BALB/c小鼠血清为阴性对照抗体，筛选间接ELISA检测MATSA抗体的最佳反应条件。结果，包被液为0.1mol/L pH 9.6的碳酸盐缓冲液，抗原包被浓度为4μg/mL，封闭液为含10g/L BSA的PBST,抗血清稀释比例为1∶800，酶标抗体稀释比例为1∶1200，酶标抗体作用时间为37℃温育45min，底物作用时间为37℃温育15min，终止液为2mol/L H2SO4，洗涤4次，每次1min为最佳反应条件。将建立的方法应用于MATSA单克隆抗体研制过程中阳性克隆的筛选，成功获得了MATSA的单克隆抗体，表明该优化条件为间接ELISA检测MATSA抗体的最佳反应条件。 |
|
关键词：
|
|
马立克病肿瘤相关表面抗原；间接ELISA；条件；筛选 |
|
中图分类号：S 852.43：R 446.61 文献标识码：
A 文章编号：1673-4696(2006)07-0538-05 |
|
|
Development of an indirect ELISA for the detection of MATSA |
|
|
|
MU Yang1， WANG Yu-dong2，ZHANG
Yan-ming1， LI Wei2，LI Yu-qing2，
ZHENG Zeng-ren2, GAO Jing1，TONG De-wen1 |
|
|
|
( 1.College of Animal Science and Technology, Northwest Sci-Tech University of Agriculture and Forestry,Yangling
712100, China; 2. National Animal Quarantine Institute,Ministry of Agriculture,Qingdao 266032,China) |
|
|
Abstract: BALB/c mice were immunized with Marek's disease tumor-associated surface antigen(MATSA) and the serum was used as positive control. Serum of SPF grade BALB/c mice without immunization was used as negative control. Detection conditions of MATSA antibody by indirect ELISA were optimized as following: coating liquid was buffer bicarbonate of 0.1mol/L(pH 9.6), optimal coating concentration of antigen was 4μg/mL, confining liquid was PBST containing 1% of BSA, optimal dilution of antiserum was 1∶800, optimal dilution of enzyme labelled antibody was 1∶1200, action time of enzyme labelled antibody was 45min at 37℃, action time of substrate was 15min in 37℃, stop buffer was 2mol/L of sulfuric acid, and washing protocol was 1min per wash for four times. The developed assay was used in the screening of positive clones of monoclonal antibodies against MATSA and the monoclonal antibodies were obtained successfully. |
|
|
|
Key words: MATSA； indirect ELISA；condition；screening |
|
|
|
|
|
《中国兽医科学》 ©
版权所有
Chinese Veterinary Science
zgsykx@zgsykx.com |
