奶牛孕酮半定量检测胶体金试纸条的研制
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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。
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.
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。
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.
引文
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[3]冯向辉,王建永,包永占,等.牛结核胶体金免疫层析快速诊断方法的建立及初步应用.动物医学进展,2010,31(2):32-35.
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[5]高成秀.免疫胶体金技术快速检测大肠杆菌O157和志贺毒素.[硕士学位论文].上海：上海交通大学,2008.
[6]巩月红,高晓黎,齐新伟,等.甾体激素人工抗原合成概况.西北医药杂志,2010,25(1):69-71.
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[21]沈民权,蒋静,陈鲁勇,等.抗孕酮Fab的制备和鉴定.上海交通大学学报(农业科学版),2003,21(1)：46-50.
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[2]冯梁.磺胺二甲嘧啶胶体金免疫层析试纸条的研制.[硕士学位论文].北京:中国农业大学,2005.
[3]冯向辉,王建永,包永占,等.牛结核胶体金免疫层析快速诊断方法的建立及初步应用.动物医学进展,2010,31(2):32-35.
[4]冯晓琼.抗奶牛孕酮单克隆抗体的制备及酶联免疫检测方法的建立.[硕士学位论文].长春:吉林大学,2007.
[5]高成秀.免疫胶体金技术快速检测大肠杆菌O157和志贺毒素.[硕士学位论文].上海：上海交通大学,2008.
[6]巩月红,高晓黎,齐新伟,等.甾体激素人工抗原合成概况.西北医药杂志,2010,25(1):69-71.
[7]赫丽娜,刘帅,孙瑾.纳米金溶胶的制备与应用.科技信息,2009,(13):30-31.
[8]侯吉波,丁再棣,何家惠.介绍一种简便的抗原乳化方法.上海免疫学杂志,1990(4):216.
[9]蒋韬,梁仲,陈涓,等.口蹄病毒O、A、Asia I型定型诊断胶体金免疫层析方法的建立.中国农业科学,2008,4(11):3801-3808.
[10]鞠莹,熊国华,曹远银.胶体金免疫层析法检测猪链球菌2型的研究.生物医学进展,2009,9(10):1881-1884.
[11]康熙雄.免疫胶体金技术临床应用.北京:军事医学科学出版社,2010,25-26.
[12]林彤,卲军军,丛国正,等.Asia I型口蹄疫病毒胶体金免疫层析检测方法的建立.生物工程学报,2009,25(5):767-772.
[13]刘洪贵,武瑞.免疫层析法检测金黄色葡萄球菌的研究.中国预防兽医学报,2009,31(4):283-287.
[14]刘显义,陈发菊,廖兴玉,等.应用乳汁孕酮的竞争性蛋白质结合分析法作乳牛早期妊娠诊断.原子能农业应用,1984(1):8-13.
[15]刘兆磊,马广鹏,姜艳平,等. A群猪轮状病毒胶体金免疫层析试纸条的研制.中国兽医科学,2008,38(11):952-956.
[16]吕申,王杉,常文保.胶体金免疫分析方法的进展.武汉大学学报,2000,46(4):393-399.
[17]吕世静.临床免疫学检测.北京:中国医药科技出版社,2004,282-285.
[18]庞观龙,何达崇,温和心.奶牛孕酮酶联免疫检测试剂盒的研制.广西农业科学,2010,41(4):386-389.
[19]彭伏虎.禽流感与新城疫胶体金免疫层析快速检测技术及初步应用研究.[博士学位论文].武汉:武汉华中农业大学,2008.
[20]秦银霞,崔晶,王中全.免疫层析试纸条诊断旋毛虫病的研究.中国人兽共患病学报,2007,23(9):866-868.
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