Pyun J C, Cheong M Y, Park S H, Kim H Y, Park J S
Department of Chemistry, College of Natural Sciences, Seoul National University, South Korea.
J Immunol Methods. 1997 Oct 27;208(2):141-9. doi: 10.1016/s0022-1759(97)00136-1.
The hydrophobicity of short synthetic peptides of 5-10 residues was enhanced for high coating efficiency as antigens in indirect ELISA. To obtain enhanced hydrophobicity, coupling of epsilon-aminocaproic acids to the synthetic peptides was carried out during solid phase peptide synthesis. As a short peptide model, three analogues of a streptavidin binding peptide consisting of 5 amino acid residues were prepared with four epsilon-aminocaproic acid residues. HPLC analysis showed a dramatic increase in hydrophobicity after modification and the modified peptides showed a better adsorption ability than the unmodified peptides in indirect ELISA. The whole process from antigen coating to color development was carried out within 2.5 to 3 h by dissolving the peptide in methyl alcohol and evaporating the solvent in each well of the microplate. As an application of this method, a peptide assumed to function as one of the epitopes of the human 60 kDa Ro/SSA antigen was selected from hydrophilicity, acrophilicity and hydropathy plots. The peptide was synthesized having an epsilon-aminocaproic acid modification at both N and C terminal ends and was tested with 30 sera from patients with systemic lupus erythematosus (SLE), 20 normal sera and a standard anti-Ro/SSA serum. The ELISA results revealed that the method gave a high signal-to-background ratio without altering the specificity of the assay. Moreover, our process was far simpler and more rapid than conventional methods used in indirect ELISA. Thus this method could be useful in the development of techniques for the diagnosis of SLE.
为了在间接酶联免疫吸附测定（ELISA）中作为抗原获得高包被效率，对5 - 10个残基的短合成肽的疏水性进行了增强。为了获得增强的疏水性，在固相肽合成过程中进行了ε-氨基己酸与合成肽的偶联。作为一个短肽模型，制备了由5个氨基酸残基组成的链霉亲和素结合肽的三种类似物，每个类似物带有四个ε-氨基己酸残基。高效液相色谱（HPLC）分析表明修饰后疏水性显著增加，并且修饰后的肽在间接ELISA中比未修饰的肽表现出更好的吸附能力。通过将肽溶解在甲醇中并在微孔板的每个孔中蒸发溶剂，从抗原包被到显色的整个过程在2.5至3小时内完成。作为该方法的应用，从亲水性、嗜酸性和水合性图谱中选择了一种被认为是人60 kDa Ro/SSA抗原表位之一的肽。合成的该肽在N和C末端均具有ε-氨基己酸修饰，并使用来自系统性红斑狼疮（SLE）患者的30份血清、20份正常血清和一份标准抗Ro/SSA血清进行了测试。ELISA结果表明该方法在不改变测定特异性的情况下给出了高信噪比。此外，我们的方法比间接ELISA中使用的传统方法简单得多且快速得多。因此，该方法可能有助于SLE诊断技术的开发。
