Laurent P, Magne L, De Palmas J, Bignon J, Jaurand M C
Unité de Recherche sur la Biopathologie, INSERM U 139, CHU Henri Mondor, Créteil, France.
J Immunol Methods. 1988 Feb 24;107(1):1-11. doi: 10.1016/0022-1759(88)90002-6.
A specific enzyme-linked immunosorbent assay (ELISA) was developed for the determination of desmosine, a cross-linked amino acid specific to fibrous elastin. Competition between solid phase-bound desmosine-protein conjugate and free desmosine for binding to monospecific anti-desmosine antiserum constituted the underlying principle of the assay. The conjugation of desmosine to different protein carriers was carried out with the 1-ethyl-3-(dimethylamino-propyl)carbodiimide (ECDI); rabbits were immunized with desmosine-bovine serum albumin and micro-titer plates were coated with desmosine-egg albumin. An avidin-biotin peroxidase system was used to reveal anti-desmosine antibodies bound to the desmosine-protein conjugate. As both conjugates revealed new non-specific common epitopes on the carrier proteins, prior absorption of the anti-desmosine antiserum on rabbit albumin polymerized with ECDI was required to remove the antibodies directed against these neo-antigens. The absorption procedure resulted in an increased specificity and sensitivity. Values ranging from 0.07 to 4 ng of desmosine/well could be detected and this sensitivity was greater than that obtained in previous immunoassays for desmosine. In order to assess the specificity of the test, samples containing aminoacids and urine hydrolysates were included in an assay. Some cross-reactivity was observed with the desmosine precursor lysinonorleucine and the desmosine isomer isodesmosine but, in contrast the very low cross-reactivity observed with collagen hydrolysate was similar to that exhibited by albumin hydrolysate. Analysis of urine samples from 118 normal male volunteers showed, firstly, that urinary creatinine measurement was a good indicator of the amount of urine which could be safely introduced in the assay without risk of non-specific interference by other organic compounds and, secondly, that the desmosine/creatinine ratio was a reliable index for an in vivo assessment of degraded elastin excretion. The assay also allowed quantitation of elastin fiber biosynthesis in the connective tissue matrix of cultured rat pleural mesothelial cells. This ELISA for demosine is a simple technique which should be useful for further in vivo or in vitro investigations of fibrous elastin tissue metabolism.
已开发出一种特定的酶联免疫吸附测定法（ELISA），用于测定去甲缬氨酸，这是一种纤维弹性蛋白特有的交联氨基酸。固相结合的去甲缬氨酸 - 蛋白质偶联物与游离去甲缬氨酸竞争结合单特异性抗去甲缬氨酸抗血清构成了该测定法的基本原理。使用1 - 乙基 - 3 - （二甲基氨基丙基）碳二亚胺（ECDI）将去甲缬氨酸与不同的蛋白质载体进行偶联；用去甲缬氨酸 - 牛血清白蛋白免疫兔子，并用去甲缬氨酸 - 卵白蛋白包被微量滴定板。使用抗生物素蛋白 - 生物素过氧化物酶系统来揭示与去甲缬氨酸 - 蛋白质偶联物结合的抗去甲缬氨酸抗体。由于两种偶联物都在载体蛋白上揭示了新的非特异性共同表位，因此需要用与ECDI聚合的兔白蛋白预先吸收抗去甲缬氨酸抗血清，以去除针对这些新抗原的抗体。吸收过程提高了特异性和灵敏度。每孔可检测到0.07至4 ng的去甲缬氨酸，这种灵敏度高于先前用于去甲缬氨酸的免疫测定法。为了评估该测试的特异性，在测定中纳入了含有氨基酸和尿液水解产物的样品。观察到与去甲缬氨酸前体赖氨酸正亮氨酸和去甲缬氨酸异构体异去甲缬氨酸有一些交叉反应，但相比之下，与胶原水解产物观察到的极低交叉反应与白蛋白水解产物相似。对118名正常男性志愿者的尿液样本分析表明，首先，尿肌酐测量是尿液量的良好指标，在测定中可安全引入尿液而无其他有机化合物非特异性干扰的风险；其次，去甲缬氨酸/肌酐比值是体内评估降解弹性蛋白排泄的可靠指标。该测定法还允许对培养的大鼠胸膜间皮细胞结缔组织基质中的弹性纤维生物合成进行定量。这种用于去甲缬氨酸的ELISA是一种简单的技术，应该有助于进一步在体内或体外研究纤维弹性蛋白组织代谢。
