Viral Antigen Microarray Assay to Detect Antibody Isotypes in Serum Against Viral Antigen
DEŞİFRE METNİ
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.
