Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza antigen microarray allows the simultaneous measurement of multiple antibody isotypes against more than 300 strains of influenza from a small blood sample volume of just one microliter. The microarray facilitates a high-throughput measurement of the breadth of the influenza antibodies across the antigenic landscape of influenza strains. By characterizing influenza antibodies in more detail than had been previously possible, this microarray can help determine why certain people develop an influenza infection and others do not.

The antigen microarray technique has been applied to 35 different pathogens in our laboratory, allowing the measurement of antibodies against 60, 000 antigen targets and from 50, 000 serum specimens collected from infectious disease cases and healthy controls worldwide. Having taught this technique through in-person workshops, we have found that being able to visually demonstrate the technique is crucial to its understanding and performance. Demonstrating the procedure will be Al Jasinskas and Rafael De Assis, project scientists from our laboratory.

To print antigens using a microarray printer, select a printer with low volume microarray spotting pins that can aspirate the antigens from a 384-well source plate into the sample channel and deposit the antigens via direct contact and capillary action onto 16 pad nitrocellulose coated glass slides. In the printing software, enter the number of sample plates that will be used in the number of plates text box and select the plate type that will be used for the analysis. Select a pin configuration and set the origin offsets to the distances between the slide origin and the location where the printing pins will start printing on the slides.

Under the array design tab, define the size and shape of the arrays and select the parameters for how the printing pins will pick up and dispense the samples. Configure the pin cleaning and blotting protocols and define the sequence in which the sample blocks are printed onto the slides. The array software will construct an annotated grid index file to describe the arrangement of antigens within each microarray.

After programming the printing software with the desired protocol, start the print run. When completed, place unprobed 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 one 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 four 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 TTBS buffer per well for five minutes each on an orbital shaker. The slides are then incubated with a mixture of quantum dot conjugated secondary antibodies and then washed 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.

To measure the spot intensity, in the file info panel, upload the gal file and specify the folder where the analysis output files are to be saved in the analysis options section. Under the image control tab, open one of the acquired images to be quantified and select auto. In the array analysis section, create a fiducial template as instructed by the software.

Click batch analysis to select the folder that contains the images to be quantified and select the fiducial template that was just created. The software will analyze each image and quantify the spot intensity. Then, analyze the raw data to compare the antibody binding across antigens and across serum specimens.

In this representative study, the secondary antibodies used were Goat Anti-Human IgG conjugated to quantum dot emitting at 800 nanometers and Goat Anti-Human IgA conjugated to quantum dot emitting at 585 nanometers for multiplex detection of IgG and IgA antibodies respectively. In the resulting heat map, only the clinically relevant subtypes with high representation were labeled to save space with the plus sign denoting all of the remaining subtypes of a higher number. Serum IgA and IgG were grouped by their hemagglutinin and neuraminidase molecular forms and subtypes.

To demonstrate the high specificity of the hemagglutinin head group antibodies for the clinical subtypes and the high cross-reactivity of whole hemagglutinin and triamerized whole hemagglutinin antibodies with the inclusion of the stock region. Influenza antibody responses measured on the microarray can be confirmed by traditional techniques, including hemagglutination inhibition and microneutralization assays. This technique has produced insights into the subtype specificity of antibodies to the influenza head group and the relative importance of IgA and IgG antibodies to influenza susceptibility.

While none of the materials are extremely hazardous, all human serum samples should be handled in accordance to institutional biosafety protocols.