This article describes the viral antigen microarray assay, a method for detecting antibodies in human serum against various viral antigens. The process involves the use of microarray slides containing multiple viral strains and employs quantum dot-conjugated secondary antibodies for quantification.
Begin the viral antigen microarray assay with a prepared viral antigen microarray slide.
The slide contains multiple pads, each with a single array containing hundreds of purified antigens of multiple viral strains printed onto spots arranged in a grid. Each spot contains a single antigen type adsorbed on the three-dimensional microporous nitrocellulose surface.
Secure the slide into a chamber for easy handling during subsequent steps. Add a blocking buffer containing proteins that bind to the slide's remaining surfaces to prevent the non-specific binding of antibodies to the surface.
Incubate the microarray slide with diluted human serum. The antibody isotypes in the serum, specific for the antigens on the slide, recognize and bind to the antigens' epitopes.
Remove the serum. Wash with a buffer to remove unbound antibodies and proteins on the slide.
Add quantum dot-conjugated secondary antibody mixtures, each with a different quantum dot. The isotype-specific secondary antibodies bind specifically to the respective serum antibody isotype bound to the antigens on the slide.
Use a microarray imager to quantify antibody binding to antigens within the microarray slide.
Upon excitation at appropriate wavelengths, the quantum dots bound to secondary antibodies return to the ground state and emit specific fluorescent signals that are detected by the photodetector.
The spot fluorescence intensity quantifies the serum antibody isotype within the serum bound to the individual antigen.
After programming the printing software with the desired protocol, start the print run. When completed, place un-probed microarray slides in a light-proof box in a desiccator cabinet at room temperature.
To probe sera for antibodies on the microarray, use clips to attach the microarray slides pad side up onto the probing chambers, and place the chambers in the frames.
Be careful when assembling the slides, as they are easily broken, and check for any leakage after rehydration. If necessary, disassemble and reassemble the slides to fix leaks.
Rehydrate the slides with 100 microliters of filtered blocking buffer per well, and dilute the sera at a 1 to 100 ratio in 100 microliters of blocking buffer per sample. Then, incubate the rehydrated microarray slides and the diluted sera for 30 minutes at room temperature and 100 to 250 rotations per minute on an orbital shaker.
At the end of the incubation, use pipette tips connected to a vacuum line with a secondary collection flask to carefully aspirate the blocking buffer from the corner of each chamber without touching the pads, and immediately add the diluted sera to the pads without allowing the pads to dry. Then, place the covered frames in trays containing moist paper towels sealed to maintain humidity, and incubate the frames overnight at 4 degrees Celsius on a rocking shaker.
For quantum-dot-conjugated secondary antibody labeling, carefully aspirate the sera, as just demonstrated, and rinse the slides with three washes in 100 microliters of fresh T-TBS buffer per well for 5 minutes each on an orbital shaker. The slides are then incubated with a mixture of quantum-dot-conjugated secondary antibodies, and then, wash three times as described in the protocol using the same technique as just demonstrated.
After the last wash, carefully remove the slides from the chambers, and gently rinse the slides with filtered double-distilled water, and then place each slide in a 50-milliliter tube. Then, dry the slides by centrifugation.
To acquire images of the microarray slides, first turn on the portable imager, and carefully place the slide to be imaged face-down into the slide holder in the imaging chamber. Open the imaging software, and under the "Configure Imager" tab, select the appropriate slide configuration.
Under the "Image Control" tab, select the appropriate fluorescent channel, and adjust the exposure and acquisition times, depending on the reactivity of the sera. Then, click 'Capture' to start the image acquisition. At the end of the acquisition, use the grids oriented on the fiducial markers to detect the array spots.
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