仿刺参体腔液补体类似物化学发光免疫检测

首次应用酶联化学发光免疫检测(Chemiluminesent Immunoassay,CLIA)技术测定仿刺参体腔液补体类似物AjC3和AjC4。羊抗人C3、C4抗体吸附到经过紫外线处理的聚苯乙烯管内,采用辣根过氧化物酶(HRP)标记抗体。过氧化氢和鲁米诺为辣根过氧化物酶的底物。捕获抗体包被最适浓度为1μg/ml,免疫反应20℃孵育2h达到平衡。HRP-IgC3、IgC4抗体复合物适宜稀释度为1:2000,HRP-IgC3I、gC4抗体复合物4℃下保存8d性能稳定,室温下5d内性能稳定。标准品浓度在0.1～10ng/ml范围内时与化学发光值之间具有良好的线性相关性,检测灵敏度为0.1ng/ml。结果表明应用酶联化学发光免疫检测技术能够检测到仿刺参体腔液中含有补体类似物,AjC3含量为6.58±1.4μg/ml,AjC4含量为0.67±0.3μg/ml。

DETECTION METHOD OF COMPLEMENT ANALOGUES OF COELOMIC FLUID IN Apostichopus japonicus BY CHEMILUMINESCENT IMMUNOASSAY

A highly sensitive and specific chemiluminescent immunoassay(CLIA) method was developed for quantitative detection of complement analogues,AjC3 and AjC4 in Apostichopus japonicus.Sheep anti-human C3,C4 antibodies were absorbed inside of polystrene tubes which were treated with ultraviolet.The antibodies were labeled with horseradish peroxidase(HRP).Luminol solution and H_2O_2 were used as the substrate of HRP.The most optimum concentration of antibody coated in the polystyrene tubes was 1μg/ml.The optimum dilution level of HRP-anti-IgC3 and HRP-anti-IgC4 was 1∶2000.The good stabilities of HRP-anti-IgC3 and HRP-anti-IgC4 were monitored at 4℃ during 8 days and at room temperature during 5 days.The immunoreaction reaches equilibrium after 2h of incubation at 20℃.The detection range of the method was 0.1～10ng/ml and it displayed good linearity.The sensitivity of the method can be used to detect AjC3 and AjC4 concentrations as low as 0.1ng/ml.The results showed that the complement analogues,AjC3 and AjC4 in Apostichopus japonicus could be detected by the method,and the content of AjC3 was 6.58±1.4 μg/ml,and AjC4 was 0.67±0.3 μg/ml.