采用自身抗体建立氧化脂蛋白(a)水平的ELISA检测法  

Development of new ELISA for oxidized lipoprotein(a)by using purified human oxidized lipoProtein(a)autoantibodies as capture antibody

作　　者：汪俊军[1] 付丽[2] 顾振华[1] 张春妮[1] 

机构地区：[1]南京军区南京总医院全军临床检验医学研究所,210002 [2]湖北省肿瘤医院检验科

出　　处：《中华检验医学杂志》2007年第10期1101-1104,共4页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(30471649);江苏省自然科学基金青年科技创新人才项目(BK2006529)

摘　　要：目的提取和鉴定人血清中氧化脂蛋白(a)[ox-Lp(a)]自身抗体,建立 ox-Lp(a)ELISA 检测法并进行临床研究。方法采用溴化氰活化的 sepharose 4B 交联 ox-Lp(a)制备免疫吸附层析柱,从正常人血清中提取抗 ox-Lp(a)自身抗体,分析其免疫反应性。分别建立以抗 ox-Lp(a)的自身抗体、抗氧化 LDL(ox-LDL)多克隆抗体为包被抗体,酶标抗载脂蛋白(a)[apo(a)]为检测抗体的ox-Lp(a)ELISA 检测法,并对100例冠心病患者和100名健康体检人群进行分析。结果8份正常人血清中均存在抗 ox-Lp(a)自身抗体,与 ox-Lp(a),ox-LDL 均具有良好的反应性;再经亲和层析除出抗 ox-LDL 自身抗体后,2份标本同 ox-Lp(a)仍具有较高的反应性。同对照组相比,本组冠心病患者血浆 Lp(a)水平显著升高[(279.6±162.7)mg/L 比(206.3±126.4)mg/L,P<0.01];ox-Lp(a)水平亦明显增加(P<0.01),采用自身抗体和抗 ox-LDL 抗体建立的 ELISA 法测定结果分别为:(24.3±33.4)μg/ml 比(8.4±9.3)μg/ml 和(13.0±13.8)μg/ml 比(7.3±9.7)μg/ml;两种 ELISA 法间结果高度相关(r=0.78,P<0.01)。结论提取的人血清 ox-Lp(a)自身抗体可识别 apo(a)和 apoB 氧化位点,采用自身抗体建立的 ox-Lp(a)检测法能更真实、准确地反应体内 Lp(a)的氧化状态,冠心病患者 ox-Lp(a)水平显著升高。Objective To isolate and identify human autoantibodies against oxidized Lp（a） [ ox-Lp （a） ] and develop new ELISA method for ox-Lp （a） by using isolated human autoantibodies as capture antibody. Methods Ox-Lp（a） autoantibodies from healthy subjects were isolated and identified by affinity chromatography. Two ＂sandwich＂ ELISA methods for plasma ox-Lp（a） were developed by using either the human autoantibodies against ox-Lp（a） or rabbit antiserum against human ox-LDL as the capture antibodies. Enzyme-labeled monoclonal anti-ape（a） was used for the quantifications. Serum ox-Lp （a） levels in 100 coronary heart disease （CHD） patients and 100 control subjects were measured with the ELISA methods. Results Ox-Lp（a） autoantibodies were isolated from all 8 studied subjects. Autoantibodies from two of the 8 sebjects showed both reactivities to ox-LDL and ox-apo（a）. The Lp（a） levels in the patients with CHD were significantly different from those of control [ （279. 6 ± 162.7 ） mg/L vs （206. 3 ± 126. 4 ） mg/L, P 〈 0.0 1 ]. Plasma ox-Lp（a） levels in patients with CHD detected by two ELISAs were both significantly higher than those of control [ ELISA using human antibodies against ox-Lp（a） ： （24.3 ± 33.4） μg/ml vs （8.4 ± 9. 3 ） μg/ml, P 〈 0.01 ; ELISA using antibodies against ox-LDL： （ 13.0 ± 13.8 ） μg/ml vs （7. 3 ±9.7） μg/ml, P 〈 0. 01, respectively]. Results obtained with the two ELISA methods were closely correlated （r = 0. 78, P 〈 0. 01 ）. Conclusions Autoantibodies against ox-Lp （a） can recognize both ape （a） and apoB epitopes of ox-Lp （a）. A new ELISA for ox-Lp （a） by using human antibodies has been developed. Increased serum ox-Lp（a） has been observed in the CHD patients.

关 键 词：脂蛋白(a) 自身抗体 动脉硬化 

分 类 号：R446.6[医药卫生—诊断学]