Immunohistochemistry

Interpreting positive and negative signals in immunohistochemistry (IHC) assays can be difficult. False negative signals may arise when antigens have been destroyed via the fixation process, or may result from the acknowledged difficulties with paraffin-embedded sections. False positive signals may be caused by an antibody binding to irrelevant epitopes or related epitopes in the wrong context; via the cross-reactivity of a secondary antibody with endogenous immunoglobulins or endogenous biotinylated proteins within the sample being bound by streptavidin-HRP.

Although it is common to validate an antibody in Western blotting before using it in IHC, the differential availability of antigens between formats also means that an antibody recognising a single band on a western blot may recognise multiple proteins in IHC.

There is no easy way around these problems. The usual approach is to compare samples to both positive and negative control sample staining and to demonstrate that similar signals can be obtained using multiple antibodies against the same target.

As Affimer technology has an inherent lack of partners in most samples, it is unlikely to generate the same level of concern – at the very least, the frequency of false positive and false negatives will be vastly reduced. Additionally, we are able to select against native and denatured samples and use FFPE material for positive or negative selection.

Detection of VEGF-R2 in prostate cancer sections

Figure 1: Detection of VEGF-R2 in prostate cancer sections. The left and middle images show two different Affimer binders from a screen for binders to VEGF-R2, which do not cross-react with any other VEGF-Rs. The right hand image shows a goat polyclonal anti-VEGF-R2 antibody. Affimer binders and antibodies were biotinylated and detected using streptavidin-HRP. All 3 probes give virtually identical signals.

When you receive an Affimer reagent our extensive validation process and selection of the best Affimer binder from at least 192 candidate clones, means that you can be confident that it recognises the correct target. For IHC – the high thermal and biophysical stability of Affimer proteins, combined with their lack of di-sulphide bonds or other post-translational modifications, means that it might be possible to add the Affimer reagent to your slide before or during the antigen retrieval protocol, which will at a minimum increase the speed of your assay but may also increase the rate of retrieval by stabilizing labile antigens.