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

The overall goal of this technique is to rapidly screen for autoantibodies in a multiplex fashion. This method can help identify key questions in the field of autoimmunity, such as which autoantibodies are correlated with different disease states. The main advantages of this technique are that autoantibodies can be screened in a parallel fashion, only microliters of serum are needed, and only micrograms of antigen are needed.

To generate antigen microarrays, begin by diluting antigens in PBS to a final concentration of 2 milligrams per milliliter. Set up a micro arrayer configuration with nine pins to print up to 162 unique antigens in duplicate. Include IgG and IgM antigens in the antigen library as positive controls.

Include PBS only as a negative control. Add 20 microliters of each antigen to the 384 well source plate in groups that mirror the setup of the print head. Use foil to cover the source plate, and freeze the plate at minus 80 degrees Celsius, until ready to print arrays.

Clean the solid microarray pins by incubating them in a sonicating bath with deionized water three times for one minute each. Place the pins in a rack to dry. Then arrange the pins in the microarray print head.

For nine pins, use a three by three configuration. Next, program the microarrayer for the print run by setting the number of pins on the print head, the number of slides to print, the number of pads on each slide, and the number of replicate spots for each antigen. In addition, program the microarrayer to sonicate the pins in water between different groups of antigens.

Next, after thawing the source plate, centrifuge it for one minute at 100 times G.Place the source plate in the designated spot in the microarrayer, then remove the section of foil that is covering the first group of antigens to be printed. Arrange the unprinted slides on the arrayer surface and run the print program. Print slides at room temperature with the humidity set on the arrayer at to 55 to 60%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. Then, continue the print program. After all the antigens have been printed, cover the source plate with a new piece of foil, and freeze it at minus 80 degrees Celsius.

Place the printed slides in a slide box, and vacuum-seal it. Slides may be use the next day, or up to one month later. To probe microarrays with diluted sera using incubation chambers, place the slides into frames.

Then add 700 microliters of blocking buffer to each array surface. Place the adhesive film over the frame and transfer it into a sealed container with a piece of wet tissue. Then, incubate the slides at four degrees Celsius on a rocker overnight.

The following day, dilute the serum samples one to 100 in blocking buffer. Then aspirate the blocking solution from the arrays, and add 500 microliters of diluted sample to each array surface. Next, cover the arrays with adhesive film, and incubate at four degrees Celsius, with rocking for one hour.

Then, aspirate the serum samples from the arrays, and use rinse buffer to rinse the array surfaces four times by pouring the buffer onto the slides and quickly flicking it off. Use 700 microliters of blocking buffer to wash each array surface, and incubate 10 minutes at room temperature with rocking. Then, add 500 microliters of diluted secondary antibody to each slide surface, and use adhesive film to cover it.

Incubate the slides at four degrees Celsius with rocking for 45 minutes. After the incubation, aspirate the secondary antibody mixture from the slides and rinse four times, as just demonstrated. Then, wash the slides with 700 microliters of blocking dilution buffer, three times for 10 minutes each.

Remove the slides from the frames, then place them in a metal slide rack, and immerse them in PBS. Incubate at room temperature with orbital shaking for 20 minutes. Place a slide rack in a container of deionized water for 15 seconds.

Then place the rack in a new container of water, and incubate for another 15 seconds. To dry the slides, place the slide rack on an ELISA plate adapter in the centrifuge, and spin at 220 times G and room temperature for five minutes. Place the slides in a light tight box until ready for scanning.

To scan the slides, use a microarray scanner that can detect SI three and SI five fluorescent signals. Determine the optimal multiplier tube, or PMT settings, by pre-scanning a full antigen library slide that was only probed with secondary antibodies. Set the PMT values used by pressing the hardware button so that the human IgG features on the SI three channel, and the human IGM features on the SI five channel, have a similar median fluorescence intensity minus background, or MFIB, which is typically 40, 000.

Next, scan the experimental slides on the two channels by pressing the scan button. Save the slide images in both channels after each scan. Using the file button, load the slide to be analyzed into the microarray software.

Then, use the file button to load the gene array list, or GAL file, which has the layout of the array, with the identities of the array features. Place the array template over the scanned image, so that the arrays features match the template as closely as possible. Press the align button to align features in all the blocks with the template.

The software with then calculate a MFI minus background for each of the antigens. Finally, use the file button to export the results as a text file, and analyze the data according to the text protocol. In this figure, showing 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.

As indicated here, the positive control serum, with known reactivity to riboP is shown to have IgM antibodies against single-stranded DNA, and IgG antibodies against riboP. Linear responses were observed with IgM over all the serum dilutions, and with IgG up to an MFI minus background of approximately 30, 000. In this experiment, the array is probed with human serum from a patient with cryoglobulinemic vasculitis, with a documented rheumatoid factor, which is an IgM antibody against IgG.

The IgG feature is yellow-green due to the binding of IgM in the patient's serum to the immobilized IgG on the array. Using secondary antibodies alone, no IgM reactivity is seen against IgG that is spotted on the array. Shown here, mouse IgM and IgG spotted onto the array, are only detected with anti-mouse secondary antibodies to IgM and IgG respectively.

This figure shows the alignment of a template grid on a scanned array image, using microarray analysis software. Once aligned, the array features can be analyzed to obtain median fluorescence intensity minus background for each feature. Once mastered, the probing of antigen microarrays can be done in four hours if it is done properly.

Following this procedure, other techniques such as ELISA can be performed in order to validate the results obtained from the antigen microarrays. After watching this video, you should have a good understanding of how to print antigen microarrays, how to probe antigen microarrays with serum, and how to scan antigen microarrays with a scanner. Don't forget that working with human serum can be hazardous, and universal precautions should be taken while performing this procedure.