Adsorption studies of tritium-labeled peptides on polystyrene surfaces.
In this study, three presentation formats of an epitope peptide (hepta-peptide), derived from the human chorionic gonadotropin amino acid sequence, were compared for adsorption to the polystyrene wells of a microELISA plate. The peptides had either a free N-terminus, an Ata-group or a linear (Lys)7-extension at the N-terminal. In order to measure the adsorption properties, all peptides were tritiated by synthesizing an additional 3H-labeled glycyl residue to the N-terminus of their peptide sequence. Over a broad range of peptide concentrations used as coat solution, extension of the peptide by an Ata-group consistently increased adsorption by a factor of 1.5 to 3 compared to the free parent peptide. Of the three peptides studied, the Ata-peptide showed the highest surface coverage of 0.6 mg/m2 when 1.0 mmol/l was offered as the concentration of peptide in the coating solution. The highest surface coverage observed for the parent peptide was 0.4 mg/m2 (at 1.5 mmol/l). The lysyl (K7) peptide showed a maximum plateau value of 0.2 mg/m2, and therefore the lysyl (K7) extension reduced the peptide surface coverage at relatively high coat concentrations (above 0.1 mmol/l) compared to the parent peptide. At lower input concentrations (below 0.1 micromol/l), however, the packing density of the lysyl (K7) peptide was up to 25 times higher when compared to the other two peptide analogs. We conclude that better adsorption as well as improved antibody binding activity and (functional) affinity could explain the higher reactivity observed in ELISA procedures when peptides are N-terminally extended by an Ata-group or lysyl (K7) extension.
Loomans
E E
EE
ATO-DLO, Department of Industrial Proteins, Wageningen, The Netherlands.
Gribnau
T C
TC
Bloemers
H P
HP
Schielen
W J
WJ
D000327:Adsorption; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011137:Polystyrenes / Q000737:chemistry*; D013499:Surface Properties; D014316:Tritium
D010455:Peptides; D011137:Polystyrenes; D014316:Tritium