The overall goal of this miniaturized glycan array is to analyze the specificity and avidity of influenza A virus hemagglutinins. This assay can help to answer key questions regarding influenza A virus receptor binding and avidity. The main advantage to this technique is that within a single assay we can address specificity as well as relative binding avidity, normally not achieved within typical glycan array and plate-based assays.
This protocol begins with preparations of the glycan samples. The base solution for the glycans is a stock printing buffer which, when made, should be slowly titrated to a pH of 8.5 and then given an addition of surfactant. Using the prepared printing buffer, dilute the PAA-conjugated glycans to 100 micrograms per milliliter.
Then, dilute the monovalent glycans to 100 micromolar also in the printing buffer. Finally, make working stocks of the amine-functionalized dyes at one micromolar. Now load ten microliters of each glycan sample into a well of a 384-well microtiter plate.
This is enough solution to print five slides. Also transfer ten microliters of the dye marker to one well of the printing plate. Prepare to print the arrays by first positioning the arrayer pins and printing head according to the layout.
In this case, one pin can print all the samples. The arrays of glycans are printed in blocks of six surrounded by an empty spot on all sides. Put on gloves to handle the slides.
Unpack them and blow any debris off their surfaces with ultra high purity nitrogen gas. Next, position the slides coated-side up on the arrayer stage. Then, program the arrayer.
In this case, program 27 spots per visit to the source plate. Three pre spots placed on the non-array area of a poly-L-lysine coated slide are used to remove excess liquid at the tip of the pin. The other 24 spots per pin visit are used to place six spots in four replicate arrays.
Details are provided in the text protocol. Now, load the multi-well plates into the printer and start printing the arrays. While printing, make sure that the humidity stays between 55 and 65%Pay attention to the appearance of the pre spots on the poly-L-lysine coated slides.
After printing, visually inspect each slide. Check for correct alignment of the spots within the teflon borders, which is vital, and check for the correct number of spotted features. To help homogenize the attachment of the dots, put the printed slides in a 100%humidity chamber with their printed side up immediately after they are made.
Use wet paper towels to line a container and a makeshift rack. Wrap the chamber in plastic to trap in the moisture. After half an hour, remove the slides.
Then, number the slides with a solvent-resistant marker and store them in a desiccation box overnight. The next day, prepare a fresh stock of blocking buffer with vigorous stirring. Adjust the pH of 9.2 using concentrated sodium hydroxide.
Then, incubate the slides in the buffer for an hour. To remove the block, rinse the slides in double-distilled water. Then, transfer the slides to glass staining holders and load the holders into a swinging plate holder to spin the slides dry at 10 G's for five minutes.
Once dry, if needed store the slides at minus 20 degrees celsius in sealed plastic bags. In preparation, make another humidification chamber as before. To detect the immobilized glycans, prepare SNA and ECA biotinylated lectins at 10 micrograms per milliliter in probing buffer, and add two micrograms per milliliter of streptavidin 555 to each dilution.
To stain with the hemagglutinins, make a 20 microgram per milliliter solution of pre complex antibody in blocking buffer at a four-to-two-to-one molar ratio as described in the text protocol. Transfer 20 microliters of the prepared solutions into wells of a 384-well plate. Next, serially dilute the lectins and hemagglutinins one-to-one in their appropriate buffers.
Make eight dilutions, filling each well with 10 microliters of sample and 10 microliters of buffer. Now, incubate the plate on ice for 30 minutes. Now add eight microliters of each dilution to one of the 48 microwells on a printed slide.
Then, incubate the prepared slide in the humidification chamber for 90 minutes. After 90 minutes, wash the slide while holding it from the edge;dip it in PBS-T four times, then dip it in PBS four times and finally, dip it in deionized water four times. Now, spin-dry the slide as before.
After spin-drying, immediately scan the slide. Be sure to carefully load the slide into the scanner in the correct orientation. Using a low laser power of five milliwatts iteratively scan the slides, gradually increasing the gain from 25 to 75%While optimizing the scan settings keep in mind that the fluorophores will bleach out with repeated scans.
Analyze the saved image for binding signals using the associated software. An array list can be loaded which has the spotting pattern. Overlay it on the scan to help navigate the placement of the grid markers.
Then, use the software to analyze the spot intensities and save the results as a tab delimited text file to export into a spreadsheet or other statistical package. Analyze the data by calculating the mean signal intensity minus the background. Take an average of four of the six replicate spots for each sample, leaving out the highest signal and the lowest signal.
Then, produce a line graph of the average mean signal minus the background against the eight protein concentrations. Smooth the resulting line using nonlinear regression. The PAA array was used to assess receptor binding specificity of influenza A virus hemagglutinins.
The array included non-sialylated controls in the same microwells. Plant lectins with known specificities for the printed compounds were used as positive controls. The ECA lectin bound to terminal galactose beta 1-4 linked to anacetylglucosamine and did not bind to the same compound if the terminal galactose was capped with sialic acid.
To test alpha 2-6 linked sialic acid, the SNA lectin was used. As expected, SNA only bound to alpha 2-6 linked sialic acids, but with notable differences in affinity for PAA linked versus non-PAA linked dye lacNAc repeats. Next, an H5 hemagglutinin derived from a Vietnam/1203/04 virus which only recognizes alpha 2-3 linked sialic acids was tested.
The binding profile of the H5 hemagglutinin showed the expected specificity with a higher avidity for the PAA linked structures. Finally, the H1 hemagglutinin from a human seasonal H1N1 strain was tested. As expected, this hemagglutinin only bound to alpha 2-6 linked sialic acid-containing structures.
Once mastered, this technique can reveal the binding specificities and relative avidities of influenza viruses within a single assay. This technique will greatly enhance the efficiency of influenza binding assays as well as reducing the consumption of precious biologicals.
