Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Begin with a functionalized glycochip featuring printed glycan sub-arrays organized into blocks of arrays.

Each spot in the sub-array represents a distinct glycan — a carbohydrate-based polymer with variations in monosaccharide composition, chain length, and branching pattern.

Treat the glycochip with a blocking buffer to block non-specific interaction sites.

Transfer the glycochip and introduce diluted mouse serum in a surfactant-supplemented buffer to minimize antibody aggregation. Incubate under agitation. 

Agitation promotes uniform serum distribution, facilitating selective binding of anti-glycan antibodies to specific glycans based on distinct molecular characteristics.

Wash with decreasing concentrations of a surfactant-containing buffer to disturb non-specific binding and rinse.

Overlay the glycochip with anti-mouse biotin-conjugated secondary antibodies, interacting with pre-bound anti-glycan antibodies and wash.

Introduce fluorophore-labeled streptavidin, binding to biotinylated secondary antibodies. Wash and remove excess solution.

Scan the glycochip and observe for distinct fluorescence spots at various positions suggesting glycan-specific circulating antibodies in serum.

Prepare buffer #1 through buffer #4 as described in the text protocol. To prepare the glycochips and samples, put the storage box with the slides on the table until they reach room temperature. Open the box and transfer the glycochip into the incubation chamber, which should be already conditioned with wet filter paper to keep the humidity constant inside the chamber.

Meanwhile, dilute the mice serum with buffer 1 in 1.5-milliliter tubes. Homogenize the serum solution for 5 seconds with a vortex mixer. After the homogenization, incubate the diluted serum at 37 degrees Celsius for 10 minutes in a water bath to avoid immunoglobulin aggregation. Then centrifuge the tubes for 3 minutes at 10,000 times g and 25 degrees Celsius.

Collect the supernatant and discard any precipitated material. Place the glycochip carefully in the incubation chamber. Incubate it with 1 milliliter of buffer #3 to eliminate any residual material on the surface of the glycochip. For 15 minutes, at 25 degrees Celsius, hold the glycochip in a vertical position, and rewash it with some drops of buffer #3 using a plastic Pasteur pipette.

Then, carefully remove the buffer from the glycochip surface using filter paper. Place the glycochip back into the incubation chamber. Spread the diluted serum sample over the glycochip surface using a micropipette. Ensure that all dry areas of the glycochip surface are covered by the diluted serum sample using the tip of the pipette.

Incubate with orbital agitation at 37 degrees Celsius for 1 and 1/2 hours. Following incubation, remove any excess sample and immerse the glycochip for 5 minutes in buffer #3 at 25 degrees Celsius. Pass the glycochip to a container with buffer #4, and finally, wash the glycochip with ultrapure water.

Centrifuge the glycochip for 1 minute at 175 times g and 25 degrees Celsius to remove the liquid. After placing the glycochip back into the incubation chamber, spread a solution of goat anti-mouse conjugated to biotin over the glycochip surface. Incubate with orbital agitation at 37 degrees Celsius for one hour. Remove the unbound fraction and repeat the washing steps.

Following centrifugation, incubate the glycochip in darkness at 25 degrees Celsius for 45 minutes with 2 micrograms per milliliter of the corresponding fluorochrome-labeled streptavidin solution in buffer #2. After working in darkness to remove the unbound fraction and repeating the washing steps, dry the glycochip by air.

To scan the array, leave the glycochip on the table until it reaches room temperature in the dark. At the same time, turn on the slide scanner and the laser. Holding the microarray, slide the glycochip into the slot until it touches the back. Scan the glycochip by running Easy Scan, and save the scan as a .tiff file.