Referencing cross-reactivity of detection antibodies for protein array experiments

Introduction

Secondary label-conjugated and non-conjugated detection antibodies are frequently used in a wide range of research applications. However, they are often affinity-isolated, polyclonal reagents that may lack the highest standard of antibody validation. 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). Although the use of the rabbit anti-IgY antibody in the literature is limited, the goat anti-rabbit IgG AP has been extensively utilised for over 15 years^1,2.

The research conducted in this laboratory examines complex antibody repertoires in humans and animals by means of protein arrays. Protein arrays are frequently used to profile antibody binding to human proteins in autoimmune disease³, cancer⁴ and in healthy individuals⁵. Other protein array applications include recombinant⁶ and hybridoma-derived⁷ antibody characterisation studies. 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 protein array technology applied here, developed by Büssow and colleagues⁸, is comprised in its current version of a fully annotated set of 7,390 distinct human proteins, that may serve as potential antigens. The aim of this study is to define a cross-reactivity reference list for the two described secondary antibodies, which can then be used to eliminate non-specific binders from ongoing chicken IgY profiling studies. Furthermore, publication of the cross-reactivity reference list provides a valuable resource of potential false-positive binders to researchers using the same antibodies. may support other researchers using these antibodies in the evaluation of their experiments.

Materials and methods

Results

Probing protein arrays with antibodies allows the assessment of their specificity and cross-reactivity across a large numbers of potential antigens in parallel^12,13. Here we investigated the cross-reactivity of a secondary rabbit anti-chicken IgY and a goat anti-rabbit IgG labelled with AP, using a single set of human protein arrays in the absence of chicken serum. We identified a total of 63 binding events, of which 61 corresponded to unique proteins (Table 4). The identified positive signals varied in strength, as shown in Figure 1, with intensity 3 being the strongest and 1 the weakest. Five of the identified signals were scored as intensity 3, twelve signals were scored as intensity 2 and remainder scored as intensity 1. The original protein array images are shown in Figure S1 and Figure S2 (Supplementary material) and protein array images with highlighted positive signals, which correspond the cross-reactive proteins listed in Table 4, are shown in Figure S3 and Figure S4 (Supplementary material).

Figure 1. Cross-reactivity of rabbit anti-chicken IgY and goat anti-rabbit IgG identified by protein array screening.

(A) Image of a whole protein array and a representative section illustrating antibody-antigen binding at three different signal intensities; 3 = strong, 2 = intermediate and 1 = weak. (B) The proteins are arranged in a 3×3 pattern on the array and all proteins are arrayed twice and appear as duplicate spots in a particular pattern within a block after a successful hybridization. (C) Description of proteins chosen as examples provided on the representative array image above; signal intensities, patterns, Unigene IDs and protein names are listed.

The 61 identified proteins comprised of a wide range of human proteins, including immunoglobulins, as well as a variety of nuclear, cytoplasmic and cell-membrane proteins with a diverse range of functions (Table 4). In order to identify shared epitopes that could explain the observed antibody cross-reactivity, and to deduce the origin of the non-specific binding to either of the two tested antibodies, we investigated sequence similarities between the human proteins and the immunogens used to produce the antibodies. We conducted a linear (BLAST) and a segmented (SIM) in silico sequence analysis of chicken IgY and rabbit IgG immunoglobulins against 61 array-identified human proteins as detailed in supplementary Table 1. In total, 5 proteins met the BLAST threshold criteria of E-values below 1 × 10^-10, as well as the SIM threshold criteria of scores above 50. A further 9 proteins met the SIM threshold, but they did not meet the BLAST criteria (Table 5).

All 5 proteins that met both threshold criteria belong to the immunoglobulin class of proteins, four being variable regions of Ig heavy chains and one a heavy constant gamma chain. The in silico sequence analysis revealed the highest sequence similarity to Ig heavy variable and constant chains, respectively, of both, the chicken IgY and rabbit IgG in all cases (Supplementary Table 1) making it impossible with this approach to deduce the origin of the cross-reactivity.

The 9 proteins that met only the SIM threshold criteria belong to a wide range of protein classes, however, none of those proteins belongs to the immunoglobulin class of proteins. In silico sequence analysis revealed that 8 of those proteins have a high local sequence similarity to the chicken immunoglobulin Y heavy chain constant region, but not to any other chicken and rabbit Ig regions. The analysis revealed further, that Inverted Formin, FH2 and WH2 Domain Containing (INF2) showed high local similarity exclusively to the rabbit Ig gamma chain constant region (Supplementary Table 1).

Conclusion

This work illustrates the cross-reactivity of an antibody-based detection system for IgY binding. The polyclonal anti-IgY rabbit antibody in combination with an anti-rabbit IgG alkaline phosphatase-conjugated antibody was shown to bind to 61 human proteins present on Unipex protein arrays comprising of 7,390 human proteins. Characterisation of this cross-reactivity provides a ‘false-positive’ database for future chicken antisera characterisation on protein array systems not limited to the Unipex protein array used here. These results, in combination with ‘false-positives’ from earlier research investigating antibody cross-reactivity by this group¹² and others¹³ may provide valuable information for future protein array-based experiments. Reference lists provided by such experiments would be further strengthened by arrays that include additional portions of the human proteome and/or post-translational modifications. Using antibodies that have been extensively characterised on protein arrays will reduce the risk of identifying irrelevant cross-reactive secondary antibody binding to the array as a host-antigen response.

It is important to note that the current study was a one-off experiment and repeat experiments may increase the reliability of the data. The reproducibility of the binding events identified in this study was further warranted by evaluating each protein in two discrete positions on the array. Of the 63 binding events, five were scored as intensity 3, twelve were scored as intensity 2 and the remainder were intensity 1. While the assay is unable to conclusively distinguish the precise cause of the differences in signal intensities, it can be assumed to be due to variations in antibody affinity and avidity, the availability of the epitope for binding, and protein concentrations on the array. A follow-up quantitative Western blot analyses and titration experiments would help further to shed more light into differences in antigen-binding kinetics.

The secondary antibodies utilized in this study are polyclonal, isolated by immunoaffinity chromatography. The presented cross-reactivity reference list may, therefore, show some variation when a different lot of the antibody is used. We have previously shown that conditions applied during affinity chromatography may affect specificity¹⁴. When assessing protein array images, we found a considerable discrepancy in background intensity of array part 1 and 2. It is important to highlight that the part 1 and part 2 of the array are generated from distinct clone libraries of different tissue origin. Part 1 of the array was generated from human brain tissue using a pQE30NST vector, whereas part 2 of the array was generated from different sources of tissue, including T cells and lung tissue, using a pQE80LSN vector. The tissue origin and the utilised bacterial vector are potential contributing factors for the variances in background noise.

Since both antibodies were used as a pair in this study, it was not possible to directly deduce the exact cross-reactivity profile for each individual antibody. We have therefore taken an in silico sequence analysis approach and we found that five of the identified proteins were of the immunoglobulin class of proteins with very high sequence similarities to both, the chicken IgY and the rabbit IgG immunoglobulins. Such cross-reactivity is not surprising considering that the antibodies are polyclonal and the immunogens were immunoglobulins of both hosts. In addition, the data sheet provided with the anti-chicken IgY antibody produced in rabbit (31104, Thermo Fisher) has specified that this antibody may cross-react with immunoglobulins from other species. The data sheet for the goat anti-rabbit IgG AP antibody (A3687, Sigma-Aldrich) has specified binding to all rabbit immunoglobulins. The in silico sequence analysis revealed furthermore 8 proteins with high sequence similarity to chicken IgY heavy chain constant region and one protein with high sequence similarity to rabbit Ig gamma chain constant region. In order to tackle this issue experimentally, a single labelled antibody should be tested on its own in future experiments. Furthermore, if a non-labelled antibody is to be tested, two experiments should be performed, one with a labelled and non-labelled antibody pair such as demonstrated in this study, and one additional experiment with the labelled antibody alone, thereby allowing allocation of exact cross-reactivates by simply subtracting ‘false-positives’ from both sets of results.

In conclusion, the antibodies tested in this study showed cross-reactivity to unrelated human proteins as well as to human immunoglobulin proteins, which are homologous to the original immunogens. Despite the identified non-specific binding, the tested antibodies are suitable for use in protein array experiments as the cross-reactive binding partners can be readily excluded from further analysis. As both antibodies were used as a pair in this study, the possibility to deduce the exact cross-reactivity profile for each individual antibody may be limited. However, the cross-reactivity reference list provided in this paper can be further utilised to validate research using those antibodies in applications other than protein arrays.