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Immunology and Infection

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Published: September 15th, 2016



1Multi-Organ Transplant Program, University Health Network, 2Institute of Medical Science, University of Toronto, 3Divison of Neurosurgery, University Health Network, 4Division of Cardiac Surgery, University Health Network

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.

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.

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 prot....

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1. Diluting Antigens and Generating Antigen Microarrays

  1. Dilute antigens in PBS to a final concentration of 0.2 mg/ml. Print up to 162 unique antigens in duplicate with a microarrayer configuration with 9 pins. Include IgG and IgM antigens in the antigen library as positive controls. Include PBS only as a negative control.
  2. Add 20 µl of each antigen to the 384 well source plate. Add antigens to the source plate in groups that mirror the setup of the printhead (e.g., arrange antigens in g.......

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Antigens are arranged in a 384 well plate and printed onto slides by a robotic microarrayer as shown in Figure 1. Figure 2 shows slides placed in a frame with incubation chambers and a scanned slide after processing. Figure 3 shows positive and negative control slides. The negative slide is only probed with secondary antibodies, and the positive control slide is probed with serum from a patient with systemic lupus erythematosus. By using .......

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The protocol described here allows for the quantification of autoantibodies using the antigen microarray technique. Antigen microarrays offer several advantages over conventional ELISA in screening for autoantibodies. First of all, a variety of antigens including nucleic acids, proteins, peptides, and cell lysates can be arrayed onto the nitrocellulose-coated slides, thus allowing for multiplexed screening of autoantibodies. In addition, only micrograms of antigen are necessary to generate the arrays since nanoliters of .......

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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.


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Name Company Catalog Number Comments
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 Cluster 3.0
Heatmap software Treeview 1.60
Microarray statistical software Stanford University SAM 4.0 (Significance Analysis of Microarrays)

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