Vani Kodela | PubMed Health

PubMed2002

A novel quality control slide for quantitative immunohistochemistry testing.

We introduce a novel quality control technology that may improve intra- and interlaboratory immunohistochemistry (IHC) standardization. The technology involves the creation of standardized antibody targets that are attached to the same slides as the patient sample. After IHC staining, the targets turn the same color as the stained cells or tissue elements. Unlike current clinical practice, our proposed targets are neither cells nor tissue sections. To create reproducible standards that are available in unlimited supply, we use short constrained peptides as antibody targets. These peptides are attached directly to the glass slide. We show that these peptides simulate the portion of the native antigen to which the antibody binds. They are useful in detecting subtle changes in IHC staining efficacy. Moreover, the peptides do not degrade after deparaffinization or antigen retrieval treatments. This technology may be valuable in creating nationally standardized controls to quantify IHC analytical variability.

Animals↗

PubMed2002

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.

Amino Acid Sequence↗