Antibody Validation


As defined by the US Food and Drug Administration (FDA), Antibody Validation is “the process of demonstrating, through the use of specific laboratory investigations, that the performance characteristics of an analytical method are suitable for its intended analytical use.” Antibodies play a significant role as highly sought after research tools in multiple protocols including enzyme-linked immunosorbent assays (ELISA), immunohistochemistry (IHC), Western blot (WB), immunoprecipitation (IP), and quantitative immunofluorescence (QIF). In addition to these protocols, antibodies help facilitate critical processes in anatomic pathology, clinical management as well as laboratory medicine.

For antibody validation, it is necessary to confirm the selectivity, specificity and reproducibility of the antibodies selected. This process may also be referred to as standardization. In the case of certain protocols, such as IHC, the process of standardization can pose a particular challenge in the lab when it comes to staining of formalin-fixed paraffin embedded (FFPE) tissue which is utilized in cancer research. This is due to a myriad of pre and post analytical factors that may influence staining quality and the antigenicity of the chosen tissue sample.

There are three main points to consider for successful antibody validation: 1) Sample preparation 2) Protocol optimization and 3) Buffer selection. Let’s take a closer look at each of these categories to gain a better understanding of what specific factors should be considered throughout the antibody validation process.

Sample Preparation

Identification of true positive and negative controls plays an essential role in the process of selecting and preparing tissue samples or cell lines. This means that the target protein of interest must be expressed in the tissue sample. Conversely, cell lines will serve as the true negative control and thus will not express the protein of interest. To help determine which tissue samples and cell lines will be most appropriate toward your research goals, online protein databases and peer-reviewed literature are very useful references. Proper sample preparation also helps to ensure that the protein of interest will not take on a form which may render it unrecognizable to the protein of interest such as a denatured state. For example, if an antibody only recognizes the protein of interest in its native form, denatured conditions common for antibody validation by Western blotting, would not be appropriate for the test sample.

Protocol Optimization

In order to provide the antibody with best opportunity to successfully pass the antibody validation process, implementation of an optimized protocol is essential. There are a number of factors to consider such as the incubation period which varies widely depending upon the antibody, ranging from as little as one hour to overnight. Determining the period helps to ensure that challenges related to sensitivity or background staining are avoided if the incubation period is either too short or too long. Individual optimization of additional factors including but not limited to native vs denatured conditions, dilutions and blocking conditions must also be prioritized.

Buffer Selection

In preparation for the antibody validation protocol, Phosphate-buffered saline (PBS) and Tris-buffered saline (TBS) are the two most common buffers used in antibody assays. Based on the antibody selected it is important to determine the optimal corresponding buffer and pH among other key factors.

ProSci Inc has answers for all your antibody validation needs. Learn more about antibody validation today at ProSci Inc.