Despite the power and versatility of immunohistochemistry, limitations are imposed by the slow diffusion of antibodies through tissue and the need for secondary staining or signal amplification. It is shocking to think that with the millions of IHC antibodies lining the virtual shelves of the different suppliers that up to 76% of protein coding genes have no reliable antibody for IHC.
IHC plays an important role in both research and clinical settings, which includes the intraoperative environment where speed and accuracy are paramount. The lack of reliable antibodies in the identification of potential biomarkers in such situations hinders biological research and medical science.
Immunohistochemistry using an Affimer to Tenascin-C
Immunohistochemistry using an Affimer to Tenascin-C (A) in U251 sub-cut tumour in mouse shows equivalent performance to staining with an IHC grade anti-tenascin-C antibody (B). Antibody and Affimer were biotinylated and detected using strep-HRP. C & D show the corresponding control sections.
The main problem in IHC staining is non-specific background staining. The main source of this unwanted background staining was originally thought to be the binding of primary and secondary antibodies to endogenous FcRs in the tissue sample being probed. It was hypothesised that the FcRs would bind to the Fc portion of the IHC antibodies during immunostaining. To overcome this standard protocols generally advise blocking with 5-10% of serum from which the secondary antibody is derived. The blocking step is thought to remove the FcR, ionic and hydrophobic interactions thus preventing background staining.
More recent studies have brought into question this explanation of non-specific IHC staining, showing that omitting the blocking step from IHC protocols does not result in increased background staining. The authors concluded that the blocking step is largely unnecessary and derives from a time when antibodies were predominantly non-specific and scientists were looking for a reason to explain the lack of specificity of lab-grown antibodies. Though the causes for this non-specific background staining may differ, they all equally complicate the use of IHC antibodies in both the research and clinical settings.
One way of circumnavigating the issue of a lack of specificity in IHC antibodies is to use pre-adsorbed or cross-adsorbed antibodies, where an antibody is exposed to potential cross-reactive epitopes by passing through a column and only the antibodies in the mixture which do not bind to the homologous proteins are passed through the column. While this is theoretically promising unfortunately in practice such adsorbed antibodies have greatly reduced epitope recognition and may recognize some subclasses of IgG very weakly, especially those subclasses which are most closely homologous to the species they were adsorbed against.
While some of the commercially available IHC antibodies show improved specificity compared to the initial lab-developed antibodies, with up to 76% of the protein coding genes lacking reliable IHC antibodies, researchers need to look elsewhere for the reagents they require to remove the roadblocks from their IHC studies.
The use of aptamers for IHC applications has shown some promise, though take up of aptamers in this field has been generally low. While aptamers are smaller than antibodies thus offering the potential for better tissue penetration, the conformational limitations of aptamer chemistry mean that locating successful binders to specific proteins is not always possible.
As the development of custom antibodies can take months, halting research projects with their long lead times, Affimer technology offer an alternative to researchers hunting for IHC antibody answers. Affimer molecules are smaller than bulky antibodies allowing better tissue penetration of samples, they offer improved specificity so non-specific staining nightmares can become a distant memory and with our custom service taking just seven weeks, switching to Affimer technology for IHC allows you to continue with the production of publication quality data.