Nagatsu T, Sawada M, Yamaguchi T, Sugimoto T, Matsuura S, Akino M, Nakazawa N, Ogawa H
Anal Biochem. 1984 Sep;141(2):472-80. doi: 10.1016/0003-2697(84)90073-3.
Specific antibodies against D-erythroneopterin have been prepared in rabbits using a conjugate of D-erythroneopterin to bovine serum albumin (D-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak.(ABSTRACT TRUNCATED AT 250 WORDS)
利用D - 赤藓糖型蝶呤与牛血清白蛋白的偶联物（D - 赤藓糖型蝶呤己酰 - 牛血清白蛋白）在兔体内制备了抗D - 赤藓糖型蝶呤的特异性抗体。该抗血清能够区分D - 赤藓糖型蝶呤与其他蝶啶，即新蝶呤的三种立体异构体、L - 赤藓糖型生物蝶呤、叶酸、黄蝶呤以及其他四种合成蝶啶。利用这种特异性抗血清，开发了一种用于D - 赤藓糖型蝶呤的放射免疫测定法，以测量尿液和组织中的新蝶呤浓度。合成了D - 赤藓糖型蝶呤与酪胺的偶联物（NP - Tyra）并用125I进行标记，作为放射免疫测定的标记配体NP - [125I]tyra。新蝶呤的最小可检测量约为0.1皮摩尔。在放射免疫测定之前，通过酸性条件下的碘氧化获得生物样品中总新蝶呤（新蝶呤、7,8 - 二氢新蝶呤、醌型二氢新蝶呤和四氢新蝶呤）的浓度，通过碱性条件下的氧化获得新蝶呤加7,8 - 二氢新蝶呤的浓度。通过这种新的放射免疫测定法获得的人尿中总新蝶呤值与通过荧光检测的高效液相色谱法获得的值显示出良好的一致性。对于与生物蝶呤相比新蝶呤浓度非常低的大鼠组织样品，生物蝶呤通过我们先前报道的放射免疫测定法同时测定，新蝶呤值针对交叉反应性（0.1%）进行校正。通过该方法获得的新蝶呤浓度与通过高效液相色谱分离后新蝶呤和生物蝶呤的放射免疫测定法获得的值一致。由于大的生物蝶呤峰掩盖了新蝶呤峰，仅靠高效液相色谱 - 荧光法无法测定大鼠组织中与生物蝶呤相比含量极少的新蝶呤。（摘要截短于250字）
