Antibody validation of immunohistochemistry for biomarker discovery: Recommendations of a consortium of academic and pharmaceutical based histopathology researchers

Abstract

As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for “research use” continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers. We propose that antibodies are placed into a tier system, level 1–3, based on evidence of their usage in immunohistochemistry, and that the degree of validation required is proportionate to their place on that tier.

Claudin-2 Expression Levels in Ulcerative Colitis: Development and Validation of an In-Situ Hybridisation Assay for Therapeutic Studies

“…In agreement with a number of papers published, an antibody validation strategy was formulated to determine the selectivity, specificity and IHC compatibility of the selected antibodies [22],[23], [24]].…”

Evaluation of Cross-Sample Contamination in Tissue Microarrays by Polymerase Chain Reaction

“…(C) Representative spots of the tissue microarray stained with hematoxylin-eosin: control agarose empty core (1-a), cholecyst (2-e), lung (3-e), ileum (4-g), prostate (5-d), ovary (6-f), placenta (7-b), Panc1 (8-a), Hek293 (9-a), fibroblasts MGM18004E (10-a), and control agarose empty core (11-h). 6,7,8,9, and 10, containing cholecyst, lung, ileum, prostate, ovary, placenta, Panc1, Hek293, fibroblasts MGM18004E, respectively. Lack of cross-sample contamination is clearly demonstrated in the control lanes 1 and 11, showing no H&E staining.…”

“…[2][3][4][5][6][7] Proteins of potential interest can simultaneously be investigated by standard histological procedures on TMA slides containing hundreds of tissue samples and/or cells. 8,9 Thus, this technology significantly facilitates the analysis of fresh frozen or paraffin-embedded tissues. Analysis of protein markers using routine laboratory techniques is cumbersome, requiring a large amount of costly reagents, a great amount of time, and the results may not be reliable since they may be affected by person-to-person handling.…”

Generation and Validation of Monoclonal Antibodies Against the Maltose Binding Protein

“…(1)(2)(3)(4) In this study, we describe in detail the production of two mAbs against the maltose binding protein (MBP), as well as the production of the 6xhistidine-tagged protein and two 6xhistidine-tagged negative controls. MBP, which is a part of the maltose/maltodextrin transport system of Escherichia coli, 5 is a commonly used protein tag, as well as an affinity tag for the purification of recombinant proteins.…”