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
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.
Polypeptides made by the solid phase method are ones in which the first amino acids in the sequence are attached to a polymer substrate. Anything attached to the polymer becomes insoluble, making it easier to separate them from soluble molecules that can be washed away. A chain of peptides that is anchored to a polymer also makes the desired chemical reactions between free-floating amino acids and the existing chain easier. The chain is held in place, which reduces the randomness within the aqueous system.
The free amino acids are modified by chemical groups that protect them from reacting with other molecules in the system. Different chemical groups are used, each of which can be removed by distinct chemical treatments. This allows the scientist to control what amino acids are made to be reactive first, which one second, and so on and so forth. This approach ensures that only the desired amino acid that is next in the sequence connects to the anchored chain.
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.
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