Referencing cross-reactivity of detection antibodies for protein array experiments

Abstract

Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires necessitates the use of extensively validated 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. Despite the identified non-specific binding, the tested antibodies are well suited for use in protein array experiments as the cross-reactive binding partners can be readily excluded from further analysis. The evident cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Furthermore, secondary antibody characterisation using protein arrays enables the generation of reference lists of cross-reactive proteins, which can be then marked as potential false positives in follow-up experiments. Providing such cross-reactivity reference lists accessible to the wider research community may help to interpret data generated with the same antibodies in applications not only related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

IgM and IgA augmented autoantibody signatures improve early‐stage detection of colorectal cancer prior to nodal and distant spread

“…Unique IgG, IgM and IgA antibody signatures were generated on the CRC protein arrays for each subject and were then compared between cancer and non‐cancer control groups. Non‐specific secondary antibody binding was identified in serum‐free experiments and excluded as previously described 88 …”

Don't blame it all on antibodies – The need for exhaustive characterisation, appropriate handling, and addressing the issues that affect specificity

“…The development of antibody-based-assays was a huge step forward in terms of the existing laborious analytical methods used to determine a variety of different targets. Initially polyclonal antibody preparations derived from serum from animal hosts immunised with the target antigen were used with the aim of generating high levels of specific antibody [8,[12][13][14]. The resulting serum was often used directly or subjected to relatively crude purification steps involving salt precipitation.…”

“…The secondary labelled antibody is used to demonstrate or quantitate the degree of binding of the primary antibody, and implementation of this strategy greatly increases the sensitivity of the assay and the secondary antibody can be used with many different mouse primary antibodies to target antigens. Many of the issues that may arise from immunoassay-based approaches are highlighted in Table 1 and all of these must be addressed if this type of assay is selected [13,14,17]. An ELISA begins with a coating step, in which the target analyte is adsorbed onto a 96-well polystyrene plate (e.g.…”

“…Antibodies are very widely used for a host of diagnostic, prognostic and therapeutic applications and represent a major focus for biotechnology and new pharmaceutical developments [1][2][3][4][5][6]. A number of very significant and thought-provoking articles highlighting the many issues identified with the overall quality of antibodies have been published indicating that many important clinical and other studies suffer from lack of reproducibility [7][8][9][10][11][12][13][14]. This was attributed, to a large degree, to the lack of specificity of the antibodies and the lack of adequate controls to ensure that non-specific binding to a plethora of irrelevant antigens was not occurring.…”

Automated semiquantitative analysis of protein macroarrays

“…An alternative to the microarray format are protein macroarrays produced by printing of annotated libraries of E.coli clones, expressing recombinant human protein, on large 22×22cm polyvinylidene fluoride (PVDF) membranes [2,3]. Since their introduction in the early 2000' the protein macroarrays have been frequently used with around 100 published studies in a wide range of applications from proteinprotein [4,5], peptide-protein [6,7], enzyme-substrate [8,9] and posttranslational modification interaction studies [10,11], to antibody specificity validation [12,13], antibody target discovery [14,15], antibody isotyping [16][17][18] and clinical autoantibody screening [11,16,19]. The extensive usage of the protein microarrays can be attributed to two innate characteristics unique to the platform: i) E.coli expression clones are spotted directly on PVDF membranes, ensuring consistent protein concentrations intrinsic to each individual expression clone, thereby avoiding the need for cumbersome large-scale protein purification and characterisation procedures essential for the generation of most protein microarray formats; and ii) each individual colony spot on the macroarray comprises of a single recombinant human protein and a collection of all E.coli proteins, thereby providing a natural blocking background consistent across the entire array and hence ensuring excellent experimental signal to noise ratios [20].…”