鸭血管紧张素转换酶2(ACE2)双抗体夹心ELISA检测方法的建立与应用

Establishment and application of double antibody sandwich ELISA for detection of duck angiotensin converting enzyme 2 (ACE2)

为建立鸭血管紧张素转换酶2 (angiotensin converting enzyme 2, ACE2)双抗体夹心酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)定量检测方法,首先利用纯化的鸭ACE2重组蛋白,成功制备了兔抗鸭ACE2多克隆抗体,间接ELISA法确定抗体效价为1∶800;然后利用制备的鼠抗鸭ACE2多克隆抗体作为捕获抗体,通过戊二醛二步法对兔抗鸭ACE2多克隆抗体进行HRP标记,并将HRP标记的兔抗鸭ACE2多克隆抗体作为检测抗体,纯化获得的鸭ACE2重组蛋白作为标准品,建立鸭ACE2双抗体夹心ELISA定量检测方法,并对抗体浓度、捕获抗体最适包被条件、孵育条件、封闭条件及显色条件等进行优化;通过Western blot法检测正常白鸭与患传染性浆膜炎的病鸭不同组织间ACE2表达的变化;利用RT-qPCR方法检测正常鸭与患传染性浆膜炎的病鸭不同组织中ACE2基因的相对表达量。结果表明:所建立的双抗体夹心ELISA方法最低检测浓度为5 ng·mL^(-1),最佳线性范围为25~1 600 ng·mL^(-1),批内变异系数为1.65%~5.62%,批间变异系数为3.66%~6.81%,可检测出麻鸭各组织中ACE2含量。传染性浆膜炎组白鸭以及健康鸭心脏、肝脏等组织样品ACE2含量或表达存在差异,心脏组织中显著升高或上调;鸭传染性浆膜炎组心脏中ACE2 mRNA水平显著上调,肝脏组织中ACE2 mRNA水平升高不明显。采用该ELISA方法对不同畜(大鼠、猪、羊)、禽(鸭、鸡、鹅)的不同组织中ACE2进行了定量测定,证实该方法在不同畜禽上存在较大种属差异,仅适用于家禽ACE2的定量检测。综上,该研究成功建立了鸭ACE2检测的双抗体夹心ELISA方法,可用于禽类样品中ACE2的快速定量检测。

To establish a method for quantitative detection of duck angiotensin converting enzyme 2(ACE2) by enzyme linked immunosorbent assay(ELISA). The rabbit polyclonal antibody against duck ACE2 was successfully prepared by using purified duck ACE2 recombinant protein. The titer of the antibody was determined to be 1∶800 by indirect ELISA. Then, the prepared mouse anti-duck ACE2 polyclonal antibody was used as the capture antibody, and the rabbit anti-duck ACE2 polyclonal antibody was labeled with HRP by glutaraldehyde two-step method, and the rabbit anti-duck ACE2 polyclonal antibody labeled with HRP was used as the detection antibody, and the recombinant duck ACE2 protein was purified as the standard product. A sandwich ELISA method for quantitative detection of duck ACE2 double antibody was established, and the antibody concentration, optimal encapsulation conditions, incubation conditions, sealing conditions and color conditions were optimized. Western blot was used to detect the expression of ACE2 in different tissues of normal white ducks and infected with infectious serositis ducks.RT-qPCR was used to detect the relative expression of ACE2 gene in different tissues of normal ducks and infected ducks with infectious serositis.The results showed that the minimum concentration of double-antibody sandwich ELISA was 5 ng· mL^(-1),the optimal linear range was 25-1 600ng·mL^(-1),the intra-batch coefficient of variation was 1.65%-5.62%,and the inter-batch coefficient of variation was 3.66%-6.81%,which could detect ACE2content in various tissues of ducks.The content or expression of ACE2 in heart and liver tissues of white ducks and healthy ducks in infectious serositis group was significantly increased or upregulated in heart tissues(P<0.05).The ACE2 mRNA level in the heart of infectious serositis group was significantly upregulated,but not in the liver.This ELISA method was used for the quantitative determination of ACE2 in different tissues of different livestock(rat,pig,sheep)and poultry(duck,chicken,goose).It was confirmed that this method had great species differences in different livestock and poultry,and was only suitable for the quantitative determination of ACE2 in poultry.This study suggested that a double-antibody sandwich ELISA method for ACE2 detection in duck was successfully established,which can be used for rapid and quantitative detection of ACE2 in poultry samples.