Genereren van tweekleurige Antigen Microarrays voor de simultane detectie van IgG en IgM Autoantistoffen
Summary
We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.
Abstract
Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.
Introduction
Autoantibodies are present in many disease states and can often have direct pathogenic activity1. Identification of autoantibodies is important for diagnosis of certain diseases, for prognosis of disease outcome, and for the classification of patients who may benefit from specific therapies2. Autoantibodies are typically identified in patient serum using the ELISA technique; however, screening for multiple antigens with this technique is laborious and consumes a large quantity of patient sample. New technologies are therefore needed to profile autoantibodies on a larger scale.
The antigen microarray technique is a proteomic technology that allows autoantibodies to be profiled in a multiplex fashion3. In the first step of this process, an antigen library is arrayed onto a slide surface using a robotic microarrayer. The slides are probed with diluted serum and then fluorescent-labeled secondary antibodies are added. Antibody reactivities are visualized by scanning the slides with a microarray scanner and quantified by fluorescent intensities. Antigen microarrays offer multiple advantages over the ELISA technique in screening for autoantibodies: 1) they require only microliters of serum to profile autoantibodies to multiple antigens simultaneously, 2) they use antigen sparingly, as only nanoliters of antigen are spotted onto the arrays, 3) they have enhanced sensitivity3 compared to ELISA and 4) they allow for the simultaneous yet, separate detection of more than one antibody isotype. Antigen microarrays have been used to profile autoantibodies in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus4-6. In all three of these diseases, new insight into disease pathogenesis was obtained from profiling autoantibodies on a large scale with the arrays.
Here we describe a protocol to generate antigen microarrays using nitrocellulose-coated slides. A variety of antigens including proteins, peptides, and cell lysates can be arrayed onto the slides using this technique. The arrays can be easily customized by including antigens of interest in the source plate that holds the antigen library. In addition, we show how a pair of secondary antibodies can be used to separate IgG and IgM reactivities on the same slide surface. We have now optimized this technique to measure autoantibodies in both humans and mice.
Protocol
Representative Results
Discussion
De hier beschreven protocol maakt de kwantificering van auto-antilichamen met het antigeen microarray techniek. Antigeen microarrays bieden verscheidene voordelen boven conventionele ELISA screening voor autoantilichamen. Allereerst een grote antigenen zoals nucleïnezuren, eiwitten, peptiden, en cellysaten worden opgesteld op de met nitrocellulose gecoate glaasjes, hetgeen aldus gemultiplexte screenen van autoantilichamen. Bovendien, slechts microgram antigeen nodig om de arrays genereren aangezien nanoliters antigeend…
Divulgations
The authors have nothing to disclose.
Acknowledgements
A.C. was supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada and the Training Program in Regenerative Medicine (Canadian Institutes of Health Research). F.Y.Y.H. was supported by the Training Program in Regenerative Medicine. This work was funded by a grant from Astellas Pharma Canada. We also would like to thank Dr. Mark Menenghini (University of Toronto) for use of his Axon microarray scanner.
Materials
| Ribosomal P0 | Diarect | 14100 | dilute to 0.2mg/ml in PBS |
| human IgG | Jackson Immuno | 009-000-003 | dilute to 0.2mg/ml in PBS |
| human IgM | Jackson Immuno | 009-000-012 | dilute to 0.2mg/ml in PBS |
| mouse IgG | Sigma-Aldrich | I5381 | dilute to 0.2mg/ml in PBS |
| mouse IgM | Biolegend | 401601 | dilute to 0.2mg/ml in PBS |
| double-stranded DNA | Sigma-Aldrich | D1626 | dilute to 0.2mg/ml in PBS |
| single-stranded DNA | Sigma-Aldrich | D8899 | dilute to 0.2mg/ml in PBS |
| microarrayer | Virtek | VersArray Chipwriter Pro | many types of arrayers are suitable |
| solid printing pins | Arrayit Corporation | SSP015 | |
| software for robotic microarrayer | Virtek | Chipwriter Pro | |
| FAST slides (2 Pad) | GVS Northa America | 10485317 | |
| FAST frame | GVS Northa America | 10486001 | |
| FAST incubation chambers (2 Pad) | GVS Northa America | 10486242 | |
| 384 well plates | Whatman | 7701-5101 | |
| plate sealers | VWR | 60941-062 | |
| foil plate covers | VWR | 60941-124 | |
| Tween-20 | Fisher Scientific | BP337-500 | |
| Fetal calf serum | Invitrogen | 12483020 | |
| Cy3 goat anti-human IgG | Jackson Immuno | 109-165-096 | use working stock in 50% glyercol |
| Cy5 goat anti-human IgM | Jackson Immuno | 109-175-129 | use working stock in 50% glyercol |
| Cy3 goat anti-mouse IgG | Jackson Immuno | 115-165-071 | use working stock in 50% glyercol |
| Cy5 goat anti-mouse IgM | Jackson Immuno | 115-175-075 | use working stock in 50% glyercol |
| Microarray Scanner | Molecular Devices | Axon 4200A | |
| Microarray software | Molecular Devices | Genepix 6.1 | |
| Clustering software | eisenlab.org | Cluster 3.0 | |
| Heatmap software | eisenlab.org | Treeview 1.60 | |
| Microarray statistical software | Stanford University | SAM 4.0 (Significance Analysis of Microarrays) | |
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