How to read HuProtTM Antibody Validation Results

Due to the growing problem of non-specific and cross-reactive antibodies on the market, there has been a push for antibody validation and verification, using a variety of methods to confirm specificity. At NeoBiotechnologies, our antibodies are validated for specificity using the CDI HuProt™ Human Proteome Microarray. Each array contains 21,000 human proteins and protein isoforms. The recombinant proteins are expressed, purified, and printed on glass slides in duplicate, along with control proteins, covering more than 80% of the human proteome. Data are typically acquired and analyzed with GenePix Pro Image Acquisition and Analysis Software. Then, the “hits” are ranked according to the Z-score, which indicates the number of standard deviations above the background observed for the mean signal bound by the target antigen. The “hits” are identified by cross-referencing them to the array map. The antibody is considered monospecific if the expected target is ranked first and the Z-Score is >3 (i.e., the difference between #1 and #2). These Z-score versus ranking plots provide crucial information about the specificity and quality of the antibodies under evaluation. As shown in the figures below (in progress), antibodies can be classified as monospecific, high quality; monospecific, low quality; cross-reactive, high/low quality; or non-specific, high/low quality.