Modification of short peptides using epsilon-aminocaproic acid for improved coating efficiency in indirect enzyme-linked immunosorbent assays (ELISA).
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.
Pyun
J C
JC
Department of Chemistry, College of Natural Sciences, Seoul National University, South Korea.
Cheong
M Y
MY
Park
S H
SH
Kim
H Y
HY
Park
J S
JS
D015119:Aminocaproic Acid / Q000737:chemistry* / Q000276:immunology; D001324:Autoantigens / Q000276:immunology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000175:diagnosis; D009842:Oligopeptides / Q000737:chemistry* / Q000276:immunology; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins / Q000276:immunology; D012680:Sensitivity and Specificity; D000099207:SS-A Antigen
D015119:Aminocaproic Acid; D001324:Autoantigens; D000939:Epitopes; D009842:Oligopeptides; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human