摘要
为了建立一个更为简单和敏感的方法,以作为不使用试剂红细胞检测犬DEA1.1血型抗体的替代方法,从犬DEA1.1阳性红细胞中分离出细胞膜。将携带DEA1.1阳性抗原的膜置于硝酸纤维素膜上,加入初级抗体和辣根过氧化物酶标记的二级抗体,采用TMB显色试剂盒检测。对照试验采用凝聚胺抗球蛋白试验检测DEA1.1抗体。用DEA1.1阳性和阴性血型抗原检测Dot-ELISA的灵敏度、稳定性和特异性。用158只犬的血清样本检测Dot-ELISA的准确性。结果显示,Dot-ELISA方法比凝聚胺抗球蛋白试验灵敏8倍以上。即使细胞膜反复冻融20次,其抗原稳定性也不受影响,并且可在-20℃时长期稳定保存。在对158只犬血清检测的过程中,该方法展现了与凝聚胺抗球蛋白试验较高的相关性。表明本研究成功建立了犬DEA1.1血型抗体Dot-ELISA检测方法,并且方法稳定、灵敏、无需试剂红细胞,可以作为输血前传统抗体检测的一种有效替代方法。
To establish a simpler and more sensitive method for the detection of canine DEA1. 1 blood group antibodies without the use of reagent red blood cells. Cell membranes were isolated from canine DEA 1. 1 positive red blood cells. The membranes with canine DEA1. 1 blood group antigens were placed on nitrocellulose membranes, followed with primary antibodies and horseradish per- oxidase-conjugated secondary antibodies. The reaction was detected by using TMB colorimetric kit. The polybrene antiglobulin assay was performed to detect DEA1. 1 antibodies. The sensitivity, stability and specificity of the Dot-ELISA were detected by using the DEA 1. 1 positive and negative blood group antigens. Serum samples from 158 dogs were used to determine the accuracy of the Dot- ELISA. The sensitivity of the Dot-ELISA was 8-fold greater than the polybrene antiglobulin assay. The plasma membrane antigens were not affected by repeated freezing and thawing for 20 times. The antigens were stable at long-term when stored at -20 ℃. The assay presented higher correlation with polybrene antiglobulin assay on testing of serum samples from 158 dogs. Conclusion Dot- ELISA is established in this experiment and it is a stable and sensitive method for detecting anti-erythrocyte antibody without using re- agent red blood cells.
作者
王娟
姜忠玲
丰艳妮
丛霞
曹荣峰
田文儒
李华涛
WANG Juan;JIANG Zhong-ling;FENG Yan-ni;CONG Xia;CAO Rong-feng;TIAN Wen-ru;LI Hua-tao(College of Veterinary Medicine , Qingdao Agricultural University , Qingdao 266109 , China)
出处
《中国兽医杂志》
CAS
北大核心
2019年第4期85-89,I0005,共6页
Chinese Journal of Veterinary Medicine
基金
国家自然科学基金(31502138,31572590,31772815)
山东省重点研发计划(2018GNC110005)
