Developing a robust IHC method: Propath’s approach 

Our objective when developing a novel protocol for immunohistochemistry, is to identify a method which produces highly specific staining with good signal intensity, where positive cells are clearly distinguishable from any negative cells or background staining. In addition, to be considered robust, a method should produce staining which is consistent and reproducible.

The team at Propath is experienced at developing, optimising and validating methods for biomarkers and test articles with no prior IHC staining information.  

In addition to offering clients our own extensive portfolio of previously validated biomarker methods, we are able to develop and validate methods for most proprietary antibody structures, including antibody fragments, bi-specific antibodies, and antibody-drug conjugates (ADCs). 

Using the very versatile Roche Ventana Discovery Ultra platform, in most cases an entire IHC method can be developed and validated at Propath within two weeks, although more complex antibody structures may take longer.

Features assessed during method development 

Propath takes a detailed, systematic approach to method development, adjusting one single parameter at a time and evaluating the outcomes of that adjustment. 

Propath’s standard approach for IHC method development and validation includes assessment of:

Optimal positive and negative controls
Detection chemistry
Heat induced antigen retrieval parameters, including enzyme retrieval
Strategies to amplify the signal and reduce non-specific background staining
The precision, specificity, linearity and reproducibility of the final method

Method validation report

Following optimisation and validation, full details of the method are provided to sponsors within a comprehensive validation report, including representative images of staining where appropriate. Whole slide digital scans can also be provided, demonstrating optimum working conditions in positive control tissue.

Tissue Micro Arrays

Further testing of IHC protocols can be performed by Propath in TMAs, which provides additional insight into how an IHC protocol is likely to perform in a clinical trial involving multiple donors. In such cases, it is common for tissues to have undergone varying fixation and processing times, especially when they originate from multiple sites.   

Read more about how Propath successfully developed a method for a CD25 antibody in tissue microarrays here