抗氨甲酰化蛋白抗体的制备及其ELISA检测方法的建立

Preparation of Antibodies against Carbamylated/Homocitrullinated Proteins and Development of ELISA Method

为了制备抗氨甲酰化蛋白抗体,探讨了氨甲酰化与瓜氨酸化之间的关系,开发氨甲酰化蛋白检测试剂盒。以牛血清白蛋白(BSA)和氰酸钾(KCNO)为原料合成人工完全抗原(Hcit-BSA),然后免疫新西兰大白兔和BALB/c小鼠,获得抗血清,间接ELISA测定抗体效价,用方阵实验确定抗体最佳稀释倍数和包被抗原的最佳包被浓度,间接竞争ELISA检测抗体特异性。结果表明:抗体效价大于1∶8 000,抗体最佳稀释倍数为1∶4 000,包被原最佳包被浓度为0.25μg/mL,抗体能特异性识别氨甲酰化产物(carbamylation-derived products,CDPs)。Hcit-BSA具有较强免疫原性,使BALB/c小鼠产生抗氨甲酰化蛋白抗体,也为建立一种简便、快速、特异性强的免疫分析方法奠定了基础,为一些疾病的早期快速检测提供了可能。

In order to prepare antibodies specific for carbamylated/homocitrullinated proteins,called as anticarbamylated protein antibody(Anti-CarP),this paper studied the relationships between carbamylation/homocitrullination and citrullination,and developed ELISA kit.Albumin from bovine serum(BSA) was homocitrullinated by potassium cyanate.Complete antigen(Hcit-BSA) was prepared,New Zealand white rabbits and BALB/c mice were immunized,and specific antibodies were developed.Antiserum titer,checkerboard and specificity determination were done by indirect ELISA.Antiserum titers were up to 1∶8 000.The optimum conditions were that antiserum was diluted at 1∶4 000 and coater was coated at 0.25 μg/mL.Anti-CarP recognized carbamylation-derived products(CDPs) specifically,such as Hcit-BSA,Hcit-KLH & Hcit-OVA.Hcit-BSA was with strong immunogenicity.Anti-CarP were inducted in BALB/c mouse.Simple,rapid and specific ELISA method was established.Anti-CarP may be used in early,rapid detection of some disease.