A Novel Quality Control Slide for Quantitative Immunohistochemistry Testing

Abstract

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.

Accurate identification of paraprotein antigen targets by epitope reconstruction

“…Biopanning using MM paraproteins was performed essentially as previously described for other monoclonal antibodies, [6][7][8] with some modifications. Equal amounts of protein A-and protein G-coated paramagnetic beads (Invitrogen, Carlsbad, CA) were mixed and washed twice with IBBT (ImmunoPure [A/G] IgG Binding Buffer ϩ 0.05% Tween-20; Pierce Biotechnology, Rockford, IL).…”

Key Issues and Strategies of Standardization for Quantifiable Immunohistochemistry

“…Furthermore, it may be possible to establish a " barcode -like " quantitative standard reference material, based on different serial concentrations of the control protein, which can be read by computer simultaneously with the tested FFPE tissue section (Chapter 8 in detail). Recently, Sompuram et al documented synthetic peptides, identifi ed from phage -displayed combinatorial libraries, that could be used as positive standard controls for IHC 29,30 (Chapter 7 in detail). It is likely that synthetic peptide reference material may be further developed based on protein -embedding techniques to more closely mimic FFPE tissue sections.…”

Peptides as Immunohistochemistry Controls

“…Phagedisplayed peptide libraries have previously been used for epitope identifi cation in this context. 6,7 Choosing which method to use also depends on how the antibody was generated. Many widely used antibodies for clinical IHC laboratories bind to linear epitopes because the original immunogen was a peptide.…”

“…For each of the peptides described in this chapter, there was specifi c inhibition by soluble peptide but not by other, antigenically irrelevant peptides. 6,7 A drawback of using peptide controls is that they are useful only for antibodies that bind to a particular epitope. Other antibodies to the same protein, but which bind to different epitopes, will require their own peptides as positive control targets.…”

“…There is a difference in staining intensity that is detectable when the two images are held side by side, and quantifi able using image analysis (as per Figure 7.7 ). 6 However, the decrement might be missed if the 1:16 dilution image were examined by eye, independent of the other image. Subtle differences in staining intensity, such as twofold or fourfold dilutions of antibody, are often diffi cult to evaluate without the aid of image analysis.…”