Synthetic peptides identified from phage-displayed combinatorial libraries as immunodiagnostic assay surrogate quality-control targets.
BACKGROUND
Quantitative immunohistochemical (IHC) assays currently lack optimal reference quality-control material for cellular protein targets. To address this problem, we identified peptides that mimic the site on the native analyte to which the primary (monoclonal) antibody binds and used them as surrogate peptide controls.


METHODS
We identified peptide candidates from a combinatorial peptide phage-display library that mimic the epitope for the 1D5 estrogen receptor (ER) monoclonal antibody (mAb). The peptide inserts of the phage clones were sequenced. Several phage-encoded peptides were then synthesized and analyzed for affinity and specificity.


RESULTS
We identified phage clones that specifically bound to the ER 1D5 mAb. The binding was specific, in that the phage clones did not bind to two other isotype-matched mAbs. Their ability to bind the ER 1D5 mAb was related to the presence of a consensus sequence. Binding analysis revealed a K(d) of 8.3 x 10(-8) mol/L. The peptide was not recognized by any of 15 other mAbs commonly used for clinical IHC testing. Moreover, the peptide was able to inhibit the binding of ER 1D5 mAb to native ER, indicating that the peptide bound to ER 1D5 mAb at or close to the antigen-binding site.


CONCLUSIONS
Surrogate peptide controls behave like the native analyte in terms of affinity and specificity. This technology may be especially useful when the native analyte is in short supply.
Sompuram
Seshi R
SR
CytoLogix Corporation, 99 Erie St., Cambridge, MA 02139, USA. ssompuram@cytologix.com
Kodela
Vani
V
Ramanathan
Halasya
H
Wescott
Charles
C
Radcliffe
Gail
G
Bogen
Steven A
SA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001943:Breast Neoplasms / Q000737:chemistry; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods / Q000592:standards; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000592:standards; D011485:Protein Binding; D011960:Receptors, Estrogen / Q000737:chemistry; D012015:Reference Standards; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019151:Peptide Library; D010455:Peptides; D011960:Receptors, Estrogen