【作者】 李志源；

【导师】 杨百亮；

【作者基本信息】 天津农学院 ， 预防兽医学， 2012， 硕士

【摘要】 本课题所研制的竞争性胶体金试纸条以奶牛血清中的生殖激素孕酮为检测对象，通过测定血清中孕酮水平的变化来判断奶牛妊娠与否。为建立孕酮胶体金免疫层析快速检测技术，首先采用二环己基碳二亚胺法将孕酮的衍生物P4-11α-半琥珀酸酯与载体BSA、OVA相偶联分别制备出免疫原和检测原，通过紫外光谱扫描法证明偶联成功；并通过计算，孕酮与BSA、OVA的偶联比分别为18:1和10:1；再以获得的免疫原P4-BSA免疫Balb/c小鼠，进一步制备孕酮多克隆抗体，通过ELISA法确定免疫小鼠血清中的孕酮抗体效价高达1:25600。采用辛酸-硫酸铵法从小鼠血清获得了较高纯度的抗体，并通过自主设计方法（金标载体吸附法）进一步从纯化的多克隆抗体中有效地清除了大部分载体抗体，从而相对提高孕酮抗体的有效浓度(抗体终浓度为1.53mg/mL)，这为高质量金标抗体的制备奠定了基础。以柠檬酸三钠还原法制备胶体金颗粒平均粒径为17.4nm，标记抗体时的最佳pH为9.0，饱和1mL的胶体金溶液孕酮抗体用量为36μg；通过筛选，NC膜上检测线竞争抗原最适包被浓度为0.072mg/mL，金标垫上金标抗体复合物的稀释倍数为4×。试纸条的各组件分别为Whatman Prima90（NC膜）、Ahlstrom8964（金标垫）、GF-08（样品垫）和H-8（吸水垫）。组装成完整的试纸条后，对孕酮标准样品的检测极限为7.6ng/mL，与直肠法相比该方法的阳性符合率为85％（17/20），阴性符合率为93.3％。本课题所制备的试纸条具有较高的灵敏度，能够在5min内检测配种后22d～26d奶牛血清中孕酮的水平，从而实现提早判断奶牛妊娠状态。更多还原

【Abstract】 In this research, a colloidal gold lateral flow strip assay was prepared for detecting theprogesterone in blood serum of pregnant cows. The aim was to diagnose whether a cow waspregnant or not by the level of its progesterone.In order to establish a rapid assay for detection of progesterone, goldimmunochromatography assay (GICA) was studied. First, the derivant of progesteroneProgesterone-11alpha-hemisuccinate was conjugated with carrier protein Albumin Bovine V(BSA) and Albumin egg (OVA) using dicyclohexylcarbodiimide (DCC), and these two kindsof artificial antigen were used as immunogen and coating antigen respectively. This processwas proved successfully by ultraviolet scan and the results showed that the conjugationmolecular ratios of P4with BSA and OVA were18:1and10:1. Then the immunogen P4-BSAwas injected into10Balb/c mice to produce polyclone antibodies. The titer of anti-P4antibodies was as high as1:25600with the method of ELISA. Next, with the method caprylicacid-saturated ammonium sulfate, highly pured antibodies (IgG) were obtained from themice’s blood serum. In order to improve the proportion of anti-P4antibodies in total IgG, anew method was designed to get rid of anti-carrier antibodies and this method was proved tobe very eazy and effective. And this provided a possibility for preparing high-qualitygold-labeled antibodies. The average diameter of the colloidal gold made by trisodium citratemethod in the research was about17.4nm and the optimal conditions for preparing thecolloidal gold conjugates with the anti-P4antibodies were studied and found that pH9.0wasthe best condition and saturating colloidal gold solution of1mL needs the antibodies of36μg.By screening, the optimal concentration of coating antigen coated on NC membrane was0.072mg/mL and the colloidal gold-labelled anti-P4antibodies were dispensed on the glassfiber at4×dilution. The assemblies of the strip were as follows: NC membrane Prima90ofwhatman, glass fiber Ahlstrom8964, sample pad GF-08and absorbent pad H-8. The stripprepared in the research had a limit detection of7.6ng/mL. Compared with rectual assay, thepositive accordance of the method in this research was85%and the negative accordance was93.3%.The strip prepared in the research with a relative high sensitivity can give a result in5minby detecting the level of progesterone in blood serum of a cow which was fertilized after22dto26d, and by this way, whether the cow was pregnant could be known ahead of time.更多还原