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

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 erythematosus^4-6. 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

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 groups of 9 when 9 pins are used for printing).
3. Cover source plate with foil and freeze at -80 °C until ready to print arrays.
4. Clean solid microarrayer pins by incubating them in a sonicating bath with deionized water for 3 x 1 min. Place pins in rack to dry and then arrange pins in microarray printhead. For 9 pins, use a 3 x 3 configuration.
5. Program microarrayer for print run by setting number pins on printhead, number of slides to print, number of pads on each slide, and number of replicate spots for each antigen. Typically, use 9 pins, 70 slides, 2 pads/slide, and 2 replicate spots for each antigen. Program microarrayer to sonicate pins in water between different groups of antigens.
6. Thaw source plate and then centrifuge plate for 1 min at 100 x g. Place source plate in designated spot in microarrayer. Remove the section of foil that is covering the first group of antigens to be printed.
7. Arrange unprinted slides on arrayer surface and run print program. Print slides at RT with humidity set on the arrayer at 55 - 60% with the build in humidifier of the array machine.
8. After each group of antigens is printed on all the slides, pause the microarrayer. Cover the antigens that were just printed with foil (to prevent evaporation) and uncover the next group of antigens to be printed. Continue print program.
9. After all antigens have been printed, cover source plate with new piece of foil and freeze at -80 °C. Place printed slides in slide box and vacuum seal. Slides may be used the next day or up to one month later.

2. Probing Antigen Microarrays with Diluted Sera

1. Place slides into frames using incubation chambers. Add 700 µl of blocking buffer (2.5% [vol/vol] Fetal calf serum (FCS), 0.1% [vol/vol] Tween 20 in PBS) to each array surface.
2. Place adhesive film over frame and place in a sealed container with a piece of wet tissue. Incubate O/N at 4 °C on a rocker.
3. Dilute serum samples 1:100 in blocking buffer. Aspirate blocking solution from arrays and add 500 µl of diluted sample to each array surface.
4. Cover with adhesive film and incubate for 1 hr at 4° C with rocking.
5. Dilute secondary antibodies in blocking buffer. For human studies, dilute a Cy3 labeled goat anti-human IgG antibody to 0.33 µg/ml and a Cy5 labeled goat anti-human IgM antibody to 0.25 µg/ml. For mouse studies, dilute a Cy3 labeled goat anti-mouse IgG antibody to 0.38 µg/ml and a Cy5 labeled goat anti-mouse IgM antibody to 0.7 µg/ml.
6. Aspirate samples from arrays and rinse array surfaces 4 times with rinse buffer (0.1% Tween 20 in PBS). Pour buffer onto to slides and flick off quickly.
7. Add 700 µl of blocking buffer to wash each array surface and incubate 10 min at RT with rocking. Repeat wash step two more times.
8. Add 500 µl of diluted secondary antibody to each slide surface and cover with adhesive film. Incubate 45 min at 4° C with rocking.
9. Aspirate secondary antibody mixture from slides and rinse 4 times as above. Wash slides 3 times with 700 µl of blocking/dilution buffer as above.
10. Remove slides from frames and place in metal slide rack that is immersed in PBS. Incubate 20 min at RT with orbital shaking. Place in new container of PBS and incubate another 20 min with shaking.
11. Place slide rack in container of deionized water 15 sec. Place rack in a new container of water and incubate another 15 sec.
12. To dry slides, place slide rack on an ELISA plate adapter in centrifuge and spin at 220 x g for 5 min at RT.
13. Place slides in light-tight box until ready for scanning.

3. Scanning Antigen Microarrays and Exporting Data

1. Scan slides using a microarray scanner that can detect Cy3 and Cy5 fluorescent signals. In order to adjust the photomultiplier tube (PMT) levels of the scanner, pre-scan a slide that was only probed with secondary antibodies.
   Note: This slide should have the full antigen library printed on it including positive (IgG and IgM) as well as negative (PBS) controls. The pre-scan is a low resolution scan that allows the user quickly to find the optimal PMT settings.
2. Set the PMT values so that the human IgG features (on the Cy3 channel) and the human IgM features (on the Cy5 channel) have a similar median fluorescence intensity minus background (MFI-B) (typically 40,000). The PMT levels are set by pressing the "hardware" button. Once set, keep these PMT settings constant.
3. Scan the experimental slides on the two channels by pressing the "scan" button. Save the slide images (both channels) after each scan.
4. Load the slide to be analyzed into the microarray software by using the "file" button.
5. Load the gene array list (GAL) file by using the "file" button. The GAL file has the layout of the array with the identities of the array features.
6. Place the array template over the scanned image so that the arrays features match the template as closely as possible.
7. Align features in all blocks with the template by pressing the "align" button. The program will generally find the outline of the circular features but some manual adjustments may be necessary. These adjustments can be made by entering the feature mode. The software will then calculate a MFI-B for each of the antigens.
8. Use the "file" button to export these results as a text file.

4. Analyzing Array Data with Significance Analysis of Microarrays (SAM)

1. Load individual text files into excel and identify the columns that have MFI-B for the Cy3 and Cy5 channels.
2. Calculate the average for the duplicate features.
3. For any negative MFI-B, replace the negative number with 10. Transform data by dividing raw MFI-B by 100 and then by calculating log base 2.
4. Analyze log transformed data with Significance Analysis of Microarrays (SAM) as described previously⁷.
5. For a study using two groups (e.g., healthy controls vs. patients), label one group "1" and the group "2." Use a two-class, unpaired analysis in SAM to identify antigens reactivities that are significantly different between the two groups (q value < 0.05).
6. Use clustering and heat-map generating software to make images for presentation⁸.

Results

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 ...

Discussion

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ribosomal P0	Diarect	14100	dilute to 0.2 mg/ml in PBS
human IgG	Jackson Immuno	009-000-003	dilute to 0.2 mg/ml in PBS
human IgM	Jackson Immuno	009-000-012	dilute to 0.2 mg/ml in PBS
mouse IgG	Sigma-Aldrich	I5381	dilute to 0.2 mg/ml in PBS
mouse IgM	Biolegend	401601	dilute to 0.2 mg/ml in PBS
double-stranded DNA	Sigma-Aldrich	D1626	dilute to 0.2 mg/ml in PBS
single-stranded DNA	Sigma-Aldrich	D8899	dilute to 0.2 mg/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)