Glutaraldehyde is an effective cross-linker for production of antibodies against advanced glycation end-products
Glutaraldehyde is an effective cross-linker for production of antibodies against advanced glycation end-products
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DOI：
10.1016/j.jim.2008.02.002
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发表时间：
2008-05-20
影响因子：
2.2
通讯作者：
Nagai, Ryoji
中科院分区：
文献类型：
--
作者：
Mera, Katsumi;Nagai, Mime;Nagai, Ryoji
免疫组织化学方法已广泛应用于晚期糖基化终产物（AGEs）的定位和定量。生产抗AGE抗体的传统方法使用交联剂如1-乙基-3-（3-二甲氨基丙基）盐酸碳二亚胺（EDC）将AGE抗原偶联到载体蛋白上。然而，这些方法通常不能产生针对特定AGE的抗体。本研究将主要抗原AGE结构N-epsilon-（羧甲基）赖氨酸（CML）与人血清白蛋白（HSA）结合，利用EDC、双亚基琥珀酰亚基（BS3）和戊二醛等交联剂，比较它们产生表位特异性抗体的效率。所有测试的交联剂都能够将CML与HSA偶联，并且每种CML偶联的HSA都被先前表征的抗CML抗体识别。然而，仅使用戊二醛作为交联剂，产生了CML特异性单克隆抗体2g11.2，该单克隆抗体能识别CML修饰的HSA和肽，而不识别N-epsilon-(carboxyethyl)lysine （CEL）修饰的HSA和肽，表明2G11对CML具有高度特异性，可以区分两个表位之间单个甲基的差异。为了进一步证明戊二醛的作用，通过与戊二醛偶联获得了针对CEL、S-（2-琥珀酰）半胱氨酸和S-（羧甲基）半胱氨酸的抗age抗体。这些研究证明了戊二醛作为交联剂对产生抗小分子抗体的功效。(C) 2008 Elsevier B.V.版权所有
Immunohistochemical approaches have been widely used in the localization and quantification of advanced glycation end-products (AGEs). Traditional approaches for production of anti-AGE antibodies use cross-linkers such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to conjugate the AGE antigen to the carrier protein. However, these approaches often fail to produce antibodies that are specific to the particular AGE of interest. In the present study, N-epsilon-(carboxymethyl)lysine (CML), a major antigenic AGE structure, was conjugated to human serum albumin (HSA) using various cross-linkers, including EDC, bis(sulfosuccinimidyl) suberate (BS3) and glutaraldehyde, to compare their efficiency for the production of epitope-specific antibodies. All of the cross-linkers tested were capable of conjugating CML to HSA, and each CML-conjugated HSA was recognized by previously characterized anti-CML antibody. However, only the use of glutaraldehyde as the cross-linker resulted in the production of a CML-specific monoclonal antibody, termed 2G11.2G11 significantly recognized CML-modified HSA and peptide, whereas it did not recognize N-epsilon-(carboxyethyl)lysine (CEL)-modified HSA and peptide, indicating that 2G11 is highly specific to CML, and can distinguish the difference of a single methyl group between the two epitopes. To further demonstrate the use of glutaraldehyde, anti-AGE antibodies against CEL, S-(2-succinyl) cysteine and S-(carboxymethyl)cysteine were obtained by conjugation with glutaraldehyde. These studies demonstrate the efficacy of glutaraldehyde as a cross-linker for the production of antibodies against small molecules. (C) 2008 Elsevier B.V. All rights reserved.