Epitope mapping and serodiagnosis using Ata- and (Lys)7 peptides.
The general applicability of the new peptide immobilization strategy in which the peptide of interest is N-terminally extended with an acetyl-thio-acetyl group or (poly)-Lys extension during synthesis, has been demonstrated in epitope-mapping experiments and serodiagnosis. Ala-scanning experiments and minimal epitope determination showed that the antigenicity of Ata-extended peptides derived from the human chorionic gonadotropin (hCG) and hepatitis B virus (HBV) amino acid sequence, was superior to the free parent peptides. Further, it could be shown that the choice of the epitope-mapping procedure (peptide in solution or immobilized on a solid support) may lead to a different perception of which residues constitute the epitope. In addition, a time-consuming conjugation process could be circumvented since the ELISA reactivity of BSA-conjugates was comparable to that of Ata-extended peptides. In the serodiagnosis using sera from various HIV-positive individuals, the lysyl-peptide showed a signal/noise ratio 10 times higher than the parent peptide, indicating that sensitivity increased as a result of this N-terminal lysyl tail. In all experiments we observed that antibody detection could be performed at roughly 10 times lower amounts of peptide when N-terminally linked to an Ata-group or lysyl-extension compared to the free parent peptide or the BSA-conjugated equivalent.
Loomans
E E
EE
Chemistry Research Unit, Organon Teknika B.V., Akzo Nobel, Boxtel, The Netherlands.
Nijholt
L J
LJ
Cremers
A J
AJ
Paulij
W P
WP
Schielen
W J
WJ
D015492:AIDS Serodiagnosis / Q000379:methods; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping / Q000379:methods*; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006513:Hepatitis B e Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006515:Hepatitis B virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011107:Polylysine / Q000737:chemistry*; D012698:Serologic Tests / Q000379:methods*
D006063:Chorionic Gonadotropin; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D006513:Hepatitis B e Antigens; D010446:Peptide Fragments; D011107:Polylysine; D000409:Alanine