In other fields of laboratory testing, assay performance specifications are established before a test is commercialized. It is usually part of an FDA regulatory submission. However, until now, that was impossible for IHC tests – there was no method to measure limit of detection (LOD), an important measure of test sensitivity.
IHCalibrators provide the first tools for quantitative measurement of analytic sensitivity. Verifying analytic sensitivity will harmonize laboratory practice and lower test error rates.
Boston Cell Standards is the only diagnostic product manufacturer to overcome the technical challenges that, until now, precluded the development of calibrators.
With the creation of IHControls®, our team pioneered the use of peptide epitopes for use as immunohistochemistry controls and calibrators. The native proteins of these cellular protein biomarkers are otherwise generally unavailable, unstable, or excessively expensive to purify or manufacture. Peptides, on the other hand, are stable, inexpensive, reproducible to high purity, and available in any desired quantity. Through the development of IHControls, we learned that all clinically relevant IHC stains are associated with linear epitopes derived directly from the native protein sequence. This is why these epitopes recover immunoreactivity after formalin fixation and antigen retrieval.
To create traceability in the measurement of peptide epitopes, we incorporate one fluorescein per peptide during peptide synthesis. Rather than calculate the peptide concentration (for which there is no traceable standard), we measure the fluorescein concentration. The fluorescein concentration equals the peptide concentration. This facilitates analytic traceability to a NIST standard reference material for fluorescein.
The same concept can be used for any IHC test by incorporating a fluorescein into a peptide epitope. The fluorescein is positioned outside the epitope so that it does not interfere with primary antibody binding. Peptide epitopes incorporating a fluorescein have a built-in system of measurement traceability.
Figure 1. Schematic illustration of a peptide analyte with an attached fluorescein, represented as the small yellow glowing sphere attached to a lysine (K). Each sphere represents a single amino acid using the single letter amino acid code. The peptide is covalently linked to a glass microbead(left). The fluorescein facilitates calculation of the peptide concentration per bead as the microbead’s fluorescein concentration equals the peptide concentration.