Summary

דור שני צבעים Antigen Microarrays לאיתור סימולטני של IgG ו- IgM נוגדנים עצמיים

Published: September 15, 2016
doi:

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

אנטיגנים מדללים 1. יצירת Microarrays Antigen לדלל אנטיגנים ב PBS לריכוז סופי של 0.2 מ"ג / מ"ל. הדפס עד 162 אנטיגנים ייחודיים כפולים עם תצורת microarrayer עם 9 פינים. כלול אנטיגנים IgG ו IgM בספריית אנטיגן בקרות חיוביות כמו. כלול PBS רק כביקו?…

Representative Results

אנטיגנים מסודרים צלחת 384 היטב מודפסים על גבי שקופיות על ידי microarrayer רובוטית כפי שמוצג באיור 1. איור 2 מראה שקופיות להציב מסגרת עם תאי הדגירה שקופית סרק לאחר עיבוד. איור 3 מציג שקופיות בקרה חיובית ושלילית. השקופית השלילית חקר?…

Discussion

הפרוטוקול המתואר כאן מאפשר כימות של נוגדנים עצמיים באמצעות טכניקת microarray אנטיגן. microarrays Antigen להציע מספר יתרונות על פני קונבנציונאלי ELISA ב בדיקות סקר לאיתור נוגדנים עצמיים. קודם כל, מגוון רחב של אנטיגנים כולל חומצות גרעין, חלבונים, פפטידים, ו lysates תא ניתן ערוך על השקופיו?…

Disclosures

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)

References

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Cite This Article
Chruscinski, A., Huang, F. Y. Y., Ulndreaj, A., Chua, C., Fehlings, M., Rao, V., Ross, H. J., Levy, G. A. Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies. J. Vis. Exp. (115), e54543, doi:10.3791/54543 (2016).

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