Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.
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