A versatile synthetic peptide-based ELISA for identifying antibody epitopes.
A simple, versatile and very inexpensive procedure for cross-linking synthetic peptides to the polystyrene surfaces of micro-well assay plates for use in ELISA was developed. The method is based on the use of poly-L-lysine (PLL) as the anchor protein for synthetic peptides which were then easily and covalently linked to the PLL using glutaraldehyde. The synthetic peptides used for the study were based on the amino acid sequence of the equine infectious anemia virus (EIAV) envelope sequence and evaluated as antigens in an ELISA designed to detect antibodies in serum of EIAV-infected horses and ponies. The ELISA using cross-linked peptides proved to be significantly more sensitive when compared to assays where passively coated peptides were used. In one instance, a peptide was identified that was not recognized by any of our antisera and appeared not to bind to the assay plates. However, once this peptide was cross-linked to the assay plate it proved to be very useful for detecting EIAV-specific antibodies. This cross-linking approach functioned equally well with peptides of various charges and sizes and did not appear to alter epitopes contained in the peptides.
Ball
J M
JM
Division of Molecular Virology, Baylor College of Medicine, Houston, TX.
Henry
N L
NL
Montelaro
R C
RC
Newman
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D000941:Antigens; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003432:Cross-Linking Reagents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000032:analysis*; D005976:Glutaral; D006736:Horses; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides; D011107:Polylysine; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000906:Antibodies; D000941:Antigens; D003432:Cross-Linking Reagents; D000939:Epitopes; D010455:Peptides; D011107:Polylysine; D005976:Glutaral