Development of a novel assay system of asymmetric dimethylarginine (ADMA), a risk factor for atherosclerosis.

不对称二甲基精氨酸（ADMA）是一种内源性竞争性一氧化氮合酶（NOS）抑制剂。据报道，ADMA血浆水平升高与内皮精氨酸/NO通路受损和内皮功能障碍相关的疾病有关，如动脉粥样硬化、高胆固醇血症、慢性心力衰竭、糖尿病和高血压。此外，ADMA最近被证明是心血管疾病的一个危险因素。因此，人们对测量人类受试者、实验动物和细胞培养中的ADMA水平越来越感兴趣。到目前为止，高效液相色谱法（HPLC）是应用最广泛的测定ADMA的方法。我们设计了两种特异性的检测系统，第一种是用N^G，N^G-二甲基精氨酸二甲氨基水解酶（E.C. 3.5.3.18: DDAH）酶促反应将ADMA降解为瓜氨酸，第二种是用ADMA单克隆抗体酶联免疫吸附法（ELISA）。一般来说，ELISA的分析性能首先是通过评估其特异性、分析灵敏度和方便性来评估的。因此，我们研究了一种针对上述第二种系统的新型ADMA ELISA检测方法的开发。首先，我们尝试用ADMA- mbs - bsa偶联物免疫BALB/c小鼠制备抗ADMA的单克隆抗体（mAb）。获得的mAb 3C12可在多种氨基酸（包括精氨酸衍生物）中特异反应ADMA，对mbs酰化ADMA比ADMA更敏感。由于单克隆抗体的抑制作用，我们构建了一个标准的ELISA检测系统。定量限为0.4nM，各处理平均回收率为95%。我们测定了人血浆中ADMA的水平，发现与HPLC测定值有良好的相关性（r=0.98,p<0.01）。

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Elevated ADMA plasma levels have been reported in connection with diseases associated with an impaired endothelial arginine/NO pathway and endothelial dysfunction, such as atherosclerosis, hypercholesterolemia, chronic heart failure, diabetes mellitus, and hypertension. Moreover, ADMA has recently been shown to be a risk factor for cardiovascular diseases. Therefore, there is increasing interest in measuring ADMA levels in human subjects, experimental animals, and cell culture. So far, the quantitative determination of ADMA by high-performance liquid chromatography (HPLC) analysis has been the most widely applied method. We designed specific two assay systems, the first is a method using enzymatic reaction with N^G,N^G-dimethylarginine dimethylaminohydrolase (E.C. 3.5.3.18 : DDAH) that degrades ADMA to citrulline, and the second an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against ADMA. In general, the analytical performance of ELISA is assessed above all by evaluating its specificity, analytical sensitivity, and facility. Therefore, we investigated the development of a novel ELISA assay for ADMA focusing on the second system described above. First we attempted to prepare a monoclonal antibody (mAb) against ADMA using BALB/c mouse immunized with ADMA-MBS-BSA conjugate. The mAb 3C12 obtained reacted specifically ADMA within a variety of amino acids including arginine derivatives and more sensitively to MBS-acylated ADMA than ADMA. We constructed a standard assay system due to the inhibition ELISA using the mAb. The limit of quantitation was 0.4nM, and the mean recovery was 95% after all treatments for the assay system. We determined ADMA levels in human plasma and found good correlation of the values measured by HPLC (r=0.98,p<0.01).