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)是一氧化氮合酶(NOS)的内源性竞争性抑制物。据报道，ADMA血浆水平升高与内皮精氨酸/NO途径受损的疾病和内皮功能障碍有关，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危险因素。因此，人们对测量受试者、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，应用最广泛的方法是高效液相色谱(HPLC)定量测定ADMA。我们设计了两种特定的检测系统，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)将ADMA降解成瓜氨酸的酶促反应方法，另一种是用抗ADMA的单抗进行酶联免疫吸附试验(EL ISA)。一般而言，首先通过评估其特异性、分析灵敏度和便利性来评估ELISA法的分析性能。因此，我们研究了一种新的检测ADMA的酶联免疫吸附试验的发展，重点是上述第二个系统。首先，我们尝试用ADMA-MBS-BSA结合物免疫BALB/c小鼠，制备抗ADMA的单抗。所获得的单抗3C12与多种氨基酸(包括精氨酸衍生物)中的ADMA发生特异性反应，并且对MBS酰化的ADMA比ADMA更敏感。由于mAb的抑制作用，我们构建了一套标准的检测体系。该分析系统的定量限为0.4 nm，平均回收率为95%。我们测定了人血浆中ADMA的水平，并发现用高效液相色谱法测定值之间有很好的相关性(r=0.98，p&lt；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).