Antibody Validation Process

When you use an antibody for research, it is important to know that the antibody is reacting only with the target enzyme. In using antibodies for ELISA, the antibody must capture only the target enzyme or a complex containing that protein. Many antibody developers limit their validation to demonstrating that an antibody binds to or immunoprecipitates a peptide or an over-expressed protein. This limited level of analysis gives little, if any, confidence that the antibody can actually capture or label a native enzyme in a complex cell or tissue sample, or that its affinity is limited to the desired target.

MitoSciences takes a different, and much more thorough approach to validation. All of the antibodies used in our In-Cell ELISA, Sandwich ELISA, and Enzyme Activity ELISA Kits are IP+, and for all of these antibodies the protein that is immunoprecipitated by them is analyzed by mass spectrometry not just once, but twice during the development process. We also use cell and organ tissue samples for screening at every stage in development, ensuring that the antibodies will work with the samples that matter to your research.

The above figure provides an example of the mass spectrometry data that are generated for each of our ELISA antibodies. In this case, a group of hybridomas that secrete antibodies against catalase were tested with human liver at the clonal stage to choose, in conjunction with other pair testing data, the best candidate for final confirmation by mass spec. All of our antibodies are tested with organ tissue and cell samples at every screening stage to immunoprecipitate a target, which is then run on a gel. After confirmation that the antibody is capturing only one target, and that it runs to the expected molecular weight on the gel, the band is cut out of the gel, trypsinized, and analyzed by tandem mass spec. The resulting data are analyzed by Matrix Sciences' Mascot informatics system, which generates a MOWSE score based on the expected molecular weight of the trypsinized peptides. While a MOWSE score of 65 is considered sufficient to provide confidence in the accuracy of the identification of the target, MitoSciences' antibodies typically score many times that number (typical MOWSE scores for MitoSciences' antibodies are between 1000 and 3000). This level of analysis is undertaken at both the early development stage, where it is used to select the best hybridomas for cloning, and also after the cloning stage, when it is used to make final selection and to confirm that the antibodies are continuing to perform as expected. For sandwich ELISA pairs, this level of validation is conducted on both the capture and the detector antibodies, providing unparalleled confidence in the specificity and utility of the pair.

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