Antibody validation

P. Gagnon, 1996, Purification Tools for Monoclonal Antibodies, Validated Biosystems, p. 57, Tucson, AZ. 

A final caveat in the RPAIA field that limits biomarker discovery as well as other uses in drug development is the somewhat limited panel of antibodies that have been validated to date for the platform. Many antibodies that work on western blots have the potential to work with RPMAs as well, because in both platforms proteins are in a denatured state. However, because the molecular weight of analytes detected by RPMA cannot be known, it is imperative to demonstrate the specificity of antibodies, usually measured by the absence of nonspecific reactive bands on western blots developed under similar blocking conditions. A second requirement for antibody validation is proof of principle that an antibody detects quantitative changes in the expected target on an... 

Gagnon, P. (1996). Purification todsfor monoclonal antibodies. Validated Biosystems, Tucson, AZ. 

The fact that ceU culture-derived products are often injected into humans as therapeutic agents makes it imperative that there be no component in the final product that can pose a potential health risk to the patient. Health risks can be introduced into a product from many sources including the ceUs themselves raw materials, such as semm, media components, etc materials used in purification, eg, antibodies and external contamination. Eor a therapeutic product such risk factors are identified at the outset and ways of reducing them to acceptable levels are designed into the process. Before a product is released by the EDA the manufacturer has to demonstrate this risk reduction by rigorous validation of the process. 

Lutgens E, Faber B, Schapira K, et al. Gene profiling in atherosclerosis reveals a key role for small inducible cytokines validation using a novel monocyte chemoattractant protein monoclonal antibody. Circulation 2005 111(25) 3443-3452. 

G. Hunt, T. Hotaling, and A. Chen. Validation of a capillary isoelectric focusing method for the recombinant monoclonal antibody C2B8, J. Chromatogr. A, 800, 355 (1998). 

In order to conduct basic research and address the need for reagents and standards, several cell lines have been modified to express individual transporters. These can serve as a source of active protein to validate a chemical as a substrate or inhibitor, or as a source of protein to validate the specificity of an antibody. In order for this approach to be sufficiently robust to establish specificity (and to minimize false-negative findings), all of the key proteins need to be available and active in the system. However, as specific probes are being increasingly identified and developed, valuable mechanistic studies can be performed with the transporters and substrates/inhibitors that are currently available. 

In conclusion, based on the literature and our data, the traditional use of acetone-fixed frozen tissue sections as the gold standard for IHC is not justified for all antigen/antibody pairs. For validating any new antibody, it would be prudent to employ a combination of both acetone and NBF-fixed frozen sections. From our experience, FFPE tissue sections may serve as the standard for most antigens for IHC. 

Instruments). The antibody panel will be selected to include ubiquitous cytoplasmic, nuclear, and surface markers. Accurate biochemical quantification of proteins in the cell/tissue model will be undertaken for validation of the IHC findings. 

Carter, R, Smith, L., and Ryan, M. (2004) Identification and validation of cell surface antigens for antibody targeting in oncology. Endocr. Relat. Cancer 11, 659-687. 

Geng, D., Shankar, G., Schantz, A., Rajadhyaksha, M., Davis, H., and Wagner, C. 2005. Validation of immunoassays used to assess immunogenicity to therapeutic monoclonal antibodies. Journal of Pharmaceutical and Biomedical Analysis 39, 364-375. 

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