Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation

Abstract

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

Antibody anarchy: A call to order

“…Aled Edwards at the University of Toronto, Canada, is director of the international Structural Genomics Consortium (SGC). He and his SGC colleagues used mass spectrometry to detect and compare the sets of proteins pulled down by immunoprecipitation with more than 1,000 antibodies 6 . The collaboration ran across 5 reference laboratories, took 4 years and cost US$3 million, not counting in-kind donations.…”

A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes

“…First, conventional antibodies do not exist for most Drosophila RNP complex proteins (or, for that matter, for most proteins encoded by any organism's genome). Second, IP-MS is a quantitative method for determining the ability of synthetic antibodies to immunoprecipitate their target proteins, as has been confirmed recently for a panel of 1124 synthetic antibodies against 152 chromatin-associated proteins (Marcon et al 2015). Finally, IP-MS will be useful, not just for validation, but also for identification of protein-protein interactions that occur in each RNP complex.…”

“…antibody selections (Hornsby et al 2015;Marcon et al 2015), annotated domains or full-length proteins have previously been the most commonly used antigens for synthetic antibody production (Schofield et al 2007;Colwill and Graslund 2011;Hornsby et al 2015;Huang et al 2015). However, although this provides a straightforward approach to choosing antigens, for our purposes, we sought to exclude annotated RNA-binding domains as antigens in order to prevent the production of antibodies that would disrupt RBP-RNA interactions and, for 75% of the RNA-binding RNP complex proteins, were left with no other annotated domains from which to choose.…”

“…This was determined using IP-westerns with available conventional antibodies against the same RNP complex proteins. The use of conventional antibodies for Western blots was necessitated as synthetic antibodies typically recognize native rather than denatured antigen and thus may not work well on westerns when detecting their endogenous target protein (Marcon et al 2015).…”

“…An analysis of 381 synthetic antibodies that recognize human proteins revealed that 37% worked for IF of the endogenous protein on tissue microarrays (Schofield et al 2007). The abovementioned study of synthetic antibodies for chromatin-associated proteins found that 50 of 66 (76%) of the "gold-standard" antibodies were useful for IF of the endogenous protein in HEK293 cells (Marcon et al 2015).…”