Multiplexed immuno‐precipitation with 1725 commercially available antibodies to cellular proteins

Abstract

Antibody array analysis of complex samples requires capture reagents with exceptional specificity. The frequency of antibodies with label-based detection may be as low as 5%. Here, however, we show that as many as 25% of commercially available antibodies are useful when biotinylated cellular proteins are fractionated by size exclusion chromatography (SEC) first. A microsphere multiplex with 1725 antibodies to cellular proteins was added to 24 SEC fractions, labelled with streptavidin and analyzed by flow cytometry (microsphere-based affinity proteomics, MAP) The SEC-MAP approach resolved different targets captured by each antibody as reactivity peaks across the separation range of the SEC column (10-670kDa). Complex reactivity profiles demonstrated that most antibodies bound more than one target. However, specific binding was readily detected as reactivity peaks common for different antibodies to the same protein. We optimized sample preparation and found that amine-reactive biotin rarely inhibited antibody binding when the biotin to lysine ratio was kept below 1:1 during labelling. Moreover, several epitopes that were inaccessible to antibodies in native proteins were unmasked after heat denaturation with 0.1% of SDS. The SEC-MAP format should allow researchers to build multiplexed assays with antibodies purchased for use in e.g. Western blotting.

Reproducibility: Standardize antibodies used in research

“…This figure may be optimistic 4 . In fact, we believe that poorly characterized and ill-defined antibodies were in large part to blame for a study co-authored by C. Glenn Begley (a co-signatory to this article) being able to replicate the scientific results of only 6 of 53 landmark preclinical studies 5 .…”

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells

“…Reactivity of different antibody clones with the same target can be tested by clustering of the SEC-MAP results across samples (49). We track performance of all used antibodies across our projects (15,16,17,49). Where available, two or more antibody clones against particular target were used in this study (supplemental Table S1).…”

“…Unlike MS, affinity proteomics is a simple technology suitable for large-scale protein analysis in primary cancer samples in the clinical laboratories. Recently, a technique linking size exclusion chromatography (SEC) to microsphere-based antibody arrays (microsphere-based affinity proteomics (MAP)) has been developed (15,16). SEC-MAP enables the detection of hundreds of proteins in a single sample and provides essential information about protein size.…”

“…to classify acute leukemias or to find the marker with a prognostic relevance. We assembled MAP arrays to carry antibodies against proteins that are known to be important for leukemia diagnostics (18,9) and against components of intracellular signaling networks (16). Through extensive testing on leukemia samples, we have identified antibodies that are suitable for immunoprecipitation-based techniques.…”

Recombinant renewable polyclonal antibodies

“…1 This partly explains the variable quality [2][3][4][5] of most commercial polyclonal antibodies, only 20-50% of which actually recognize their targets specifically. [6][7][8] Although it may be possible to overcome this problem with affinity purification, 1,9 after which a majority of antibodies should recognize the target of interest, the finite and non-renewable nature of polyclonal antibodies limits the amount of specific antibody that can be isolated. Furthermore, as demand increases for highly specific antibodies that are able, for instance, to discriminate between closely related proteins, the potential amount of available antibody becomes substantially less.…”