The overall goal of this experiment is to identify Zika virus specific peptides for use in a diagnostic assay to detect Zika virus antibodies. This is critical, as the virus disappears from the blood in approximately 10 days, while antibodies persist for years. This method allows us to identify antigens and epitopes utilizing a microarray of the entire Zika proteome to identify peptides that capture either IgM or IgG antibodies in a sample of serum from an infected individual.
The main advantage in detecting both early antibodies, IgM, and later antibodies, IgG, is to provide the clinician a timeline of the infection. And this is particularly important in pregnant women. While this protocol provides insight into the cause of Zika virus infection, it can be applied to other pathogens, using a microarray specific for that pathogen if its protein sequence is known.
Handle the glass slide by the edges using powder-free gloves. The glass slide dimensions are 75.4 millimeters by 25.0 millimeters and 1 millimeter thick. Place the slide in the incubation tray with the microarray surface facing up.
Fill the empty slide holders with blank slides to prevent the microarray slide from breaking. Place the glossy side of the seal facing downwards onto the microarray printed surface. To ensure a good position, overlap the screw holes of the seal and the base plate.
Place the upper part of the tray onto the seal to create a microarray chamber. Secure the slide in the tray by equally tightening the thumb screws one after another by hand in an alternating pattern. Start from the upper left and proceed to the lower right.
Place the lid on the incubation tray. Careful handling of the microarray is essential to avoid scratching the microarray surface. Solutions are added to and then removed from the corner of the microarray chamber.
Drying the microarray is also important to provide a good scan image. Incubate the microarray with 2, 000 microliters of standard buffer per microarray for 15 minutes at room temperature on an orbital shaker at 140 rpm. Remove the standard buffer by aspirating with a pipette from the corner of the microarray chamber, then block the microarray with a blocking buffer for 60 minutes at room temperature on an orbital shaker at 140 rpm.
Remove the blocking buffer by aspirating from the corner of the microarray chamber with a pipette. Add anti-human IgM fluorochrome conjugated secondary antibody diluted 1:5, 000 in the staining buffer to the microarray. Then incubate the microarray for 30 minutes at room temperature in the dark on an orbital shaker at 140 rpm.
Use a pipette to remove the secondary antibody by aspirating from the corner of the microarray chamber. Wash the microarray for one minute with 2000 microliters with standard buffer per microarray at room temperature on an orbital shaker at 140 rpm. Remove the standard buffer using the demonstrated technique and repeat the wash two times.
Next, immerse the slide twice into 200 milliliters of a freshly prepared dipping buffer. Dry the microarray carefully for approximately one minute by aspirating excess fluid very carefully from the top to the bottom of the slide without touching the slide surface. Analyze the slide in a microarray scanner reader as demonstrated later in this video.
Inactivate the serum sample at 56 degrees Celsius for 30 minutes. Then centrifuge the sample at 16, 000 RCF for five minutes at four degrees Celsius. Keep the sample at four degrees Celsius until use.
Then incubate the microarray with the staining buffer for 15 minutes at room temperature on an orbital shaker at 140 rpm. Remove the staining buffer by aspirating from the corner of the microarray chamber with a pipette. Dilute the serum sample and staining buffer starting with a 1:1, 000 dilution.
Incubate the diluted sample with the microarray overnight at four degrees Celsius on an orbital shaker at 140 rpm. Remove the diluted serum sample using the demonstrated technique. Then wash the microarray three times as before.
Dilute the secondary antibody in the staining buffer. Use anti-human IgM fluorochrome conjugated antibody or anti-human IgG fluorochrome conjugated antibody at a dilution of 1:5, 000 in staining buffer. Then mix the control-labeled antibody at a dilution of 1:1000 in staining buffer with the diluted secondary antibody.
Incubate with the microarray for 30 minutes at room temperature in the dark and on an orbital shaker at 140 rpm. Remove the mix of diluted secondary and labeled antibodies by aspirating from the corner of the microarray chamber with a pipette. Then wash the microarray three times with standard buffer.
Each wash is for one minute on an orbital shaker at 140 rpm. Finally, remove the washing buffer from the microarray chamber as before. To perform microarray scanning, immerse the slide twice into 200 milliliters of freshly prepared dipping buffer.
Dry the microarray for around one minute by very carefully aspirating from the top to the bottom of the slide without touching the slide surface. Scan the microarray following the instructions of the scanner. Place the slide onto the scanner with the printed surface facing up.
Create a new project and select the scanning area. Set scanner parameters with a resolution of 21 microns, an intensity of 7.0 for both the 700 and 800 nanometer channels, a scanning quality of medium, and an offset of 0.8. Acquire and save the scan draw image as a 16-bit grayscale TIF file format.
To analyze the data, open the raw image. Then open the array grid file. Align the array grid to the scan image with the computer's mouse or keyboard arrow keys.
Select quantify selection in the specific software, which creates a readout file that contains the signal intensity for each spot, the background value, and the corresponding peptide sequence. Finally, store the microarray sealed in the dark at four degrees Celsius under oxygen-free nitrogen or argon gas. Shown here is the raw fluorescence image of staining the microarray with anti-human IgG after incubating with a specific Zika virus serum sample diluted 1:250.
HA control peptides frame the microarray. Peptides that react strongly to IgG are shown in the zoom panel. Similarly, shown here is a raw fluorescence image of staining the microarray with anti-human IgM after incubating with a specific Zika virus serum sample diluted 1:250.
Again, the HA control peptides frame the microarray. Peptides that react strongly to IgM are shown in the zoom panel. Shown here is a representative Zika virus epitope consensus sequence reacting with IgM in the host serum sample peptides.
Peptides from the epitope consensus sequence are highlighted in red, and the peptide with the highest fluorescence intensity is marked in bold. After watching this video, you should have a good understanding of how to process a high-density peptide microarray. Once mastered, this technique can be done in 24 hours if it is performed properly.
While attempting this procedure, it's important to remember to carefully handle the microarray to avoid scratching the microarray surface. Following this procedure, other methods like ELISA or a surface plasmon resonance binding assay can be performed to determine if the identified peptides bind specifically to Zika virus antibodies and not to other Flavivirus antibodies. After its development, this technique will pave the way for researchers in the field of diagnostics for the discovery of diagnostic peptides using serum and saliva specimens.
Don't forget that working with infectious agents as Zika virus can be extremely hazardous, and precautions such as heat-inactivating the serum sample and working in Biosafety Level 2 facilities should always be taken while performing this procedure.
