Validating the selectivity of antibodies used in multiplexed serum profiling via parallel immunocapture analysis
(English)Manuscript (preprint) (Other academic)
The increasing availability of antibodies towards human proteins drives the generation of new and exploratory data, which can be generated by profiling e.g. plasma samples using multiplexed arrays. However, antibody assays can lead to erroneous results due to cross-‐reactivity to off-‐targets. Here, we describe an approach in which an antibody-‐based suspension bead array is combined with subsequent validation of on-‐ target binding using a coupled affinity purification assay. Based on differential heat treatment of samples, antibody performance was investigated and the results for antibodies directed towards several complement factors provide insights on the detection of proteins in sera from patients with complement deficiency. In conclusion, the combined parallel flow and blot based immunocapture analysis serves an important, first line tool for resolving differently detected serum or plasma protein profiles proposed by antibody arrays.
Suspension bead array, protein profiling, antibody, immunocapture, Western blot
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
IdentifiersURN: urn:nbn:se:kth:diva-117959OAI: oai:DiVA.org:kth-117959DiVA: diva2:604021
QS 20132013-02-072013-02-072013-02-08Bibliographically approved