Abstract
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.
Highlights
Secondary label-conjugated and non-conjugated detection antibodies are frequently used in a wide range of research applications
The antibodies characterised in this study are a polyclonal anti-chicken IgY antibody produced in rabbit (31104, Thermo Fisher) and a polyclonal goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (AP) (A3687, Sigma-Aldrich)
This article investigates the cross-reactivity of a rabbit anti-chicken IgY and an alkaline phosphatase-conjugated goat anti-rabbit IgG, which were used for the profiling of IgY antibody responses to human antigens in chickens immunised with human cancer cells
Summary
Secondary label-conjugated and non-conjugated detection antibodies are frequently used in a wide range of research applications. The antibodies characterised in this study are a polyclonal anti-chicken IgY antibody produced in rabbit (31104, Thermo Fisher) and a polyclonal goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (AP) (A3687, Sigma-Aldrich). This article investigates the cross-reactivity of a rabbit anti-chicken IgY and an alkaline phosphatase-conjugated goat anti-rabbit IgG, which were used for the profiling of IgY antibody responses to human antigens in chickens immunised with human cancer cells. The aim of this study is to define a cross-reactivity reference list for the two described secondary antibodies, which can be used to eliminate non-specific binders from ongoing chicken IgY profiling studies. Publication of the cross-reactivity reference list may support other researchers using these antibodies in the evaluation of their experiments
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