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A new two-color Fab labeling method for autoantigen protein microarrays

Abstract

Antigen microarrays hold great promise for profiling the humoral immune response in the settings of autoimmunity, allergy and cancer. This approach involves immobilizing antigens on a slide surface and then exposing the array to biological fluids containing immunoglobulins. Although these arrays have proven extremely useful as research tools, they suffer from several sources of variability. To address these issues, we have developed a new two-color Fab labeling method that allows two samples to be applied simultaneously to the same array. This straightforward labeling approach improves reproducibility and reliably detects changes in autoantibody concentrations. Using this technique we profiled serum from a mouse model of systemic lupus erythematosus (SLE) and detected both expected and previously unrecognized reactivities. The improved labeling and detection method described here overcomes several problems that have hindered antigen microarrays and should facilitate translation to the clinical setting.

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Figure 1: Two-color Fab labeling for probing one array with different serum samples.
Figure 2: Signal intensity, sensitivity and dynamic range.
Figure 3: Autoantibody profiling of mouse serum and Ribo P autoreactivity in the pristane model of lupus.

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Acknowledgements

We thank S. Chan, J. Tenenbaum and R. Tibshirani and other members of our laboratory for helpful discussion and technical assistance. M.G.K. is funded by the Stanford Medical Scientist Training Program (MSTP). D.L.T. is funded by fellowships from the National Science Foundation and the PEO Sisterhood. I.B. is funded by the Arthritis Foundation Postdoctoral Fellowship. P.J.U. is the recipient of a Donald E. and Delia B. Baxter Foundation Career Development Award and was supported by the Dana Foundation, the Floren Family Trust, the Northern California Chapter of the Arthritis Foundation, National Institutes of Health grants DK61934, AI50854, AI50865 and AR49328, and National Heart, Lung, and Blood Institute (NHLBI) Proteomics contract N01-HV-28183.

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Correspondence to Paul J Utz.

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Competing interests

In the past 3 years P.J.U. has served as a consultant to Centocor (Horsham, Pennsylvania, USA), Biogen Idec (Cambridge, Massachusetts, USA), Genentech, Inc. (South San Francisco, California, USA), MedImmune (Gaithersburg, Maryland, USA) and Avanir, Inc. (La Jolla, California, USA), is a member of the Scientific Advisory Board of Monogram Biosciences (South San Francisco, California, USA) and XDx, Inc. (South San Francisco, California, USA), and is a cofounder and consultant at Bayhill Therapeutics (Palo Alto, California, USA). The authors are filing a patent on some of the information described in the article.

Supplementary information

Supplementary Fig. 1

Fold-changes from murine and human dye-swap experiments. (PDF 73 kb)

Supplementary Fig. 2

Cross-labeling of Fab fragments. (PDF 69 kb)

Supplementary Fig. 3

Correlation of single-color and two-color Fab methods with conventional ELISA. (PDF 66 kb)

Supplementary Fig. 4

Anti-ribosomal P reactivity by western-blot and immunoprecipitation in pristane-treated BALB/c mice. (PDF 186 kb)

Supplementary Table 1

Bias of Alexa and Cyanine dyes. (PDF 44 kb)

Supplementary Table 2

Measurement of artificial up- and down-regulation of antibody levels. (PDF 45 kb)

Supplementary Table 3

List of autoantigens and vendors. (PDF 55 kb)

Supplementary Methods (DOC 34 kb)

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Kattah, M., Alemi, G., Thibault, D. et al. A new two-color Fab labeling method for autoantigen protein microarrays. Nat Methods 3, 745–751 (2006). https://doi.org/10.1038/nmeth910

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