The overall goal of this protocol is to study the profile of circulating anti-glycan antibodies in the serum of small animals using a printed glycan array based technology. This approach has a potential in the early diagnosis and the right treatment in some pathological conditions where antibodies against glycan structures play an important role. The main advantage with this technique, is the possibility to measure in the same experimental setting hundreds of glycan targets with a very high sensitivity using a minimum amount of sample.
To prepare the micro array, use a non-contact robotic arrayer. Prints six replicates of 50 millimolar glycans and 10 micrograms per milliliter polysaccharides and 300 millimolar PBS, pH 8.5 onto n-hydroxy 6 cinnamic derivatized glass slides. Each slide contains four different blocks of subarrays repeated six times.
Every single subarray is formed by 112 different glycans spots including controls. Incubate the slides in a moisture box at room temperature for one hour. To block the micro arrays, incubate the slides for 1 1/2 hours with blocking buffer at room temperature.
Then wash the glycol chip with ultra pure water and dry it by air. Prepare buffer one through buffer four as described in the text protocol. To prepare the glycol chips and samples, put the storage box with the slides on the table until they reach room temperature.
Open the box and transfer the glycol chip 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 one in 1.5 millilitre tubes. Homogenize the serum solution for five 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 three minutes at 10, 000 times G and 25 degree Celsius. Collect the supernatant and discard any precipitated material.
Place the glycol chip carefully in the incubation chamber. Incubate it with one milliliter of buffer three to eliminate any residual material on the surface of the glycol chip. For 15 minutes, at 25 degree Celsius.
Hold the glycol chip in a vertical position, and rewash it with some drops of buffer three using a plastic pasteur pipette. Then, carefully remove the buffer from the glycol chip surface using filter paper. Place the glycol chip back into the incubation chamber.
Spread the diluted serum sample over the glycol chip surface using a micro pipette. Ensure that all the dry areas of the glycol chip surface are covered by the diluted serum sample using the tip of the pipette. Incubate with orbital agitation at 37 degrees Celsius for 1 1/2 hours.
Following incubation, remove any excess sample and immerse the glycol chip for five minutes in buffer three at 25 degrees Celsius. Pass the glycol chip to a container with buffer four and finally, wash the glycol chip with ultra pure water. Centrifuge the glycol chip for one minute at 175 times G and 25 degrees Celsius to remove the liquid.
After placing the glycol chip back into the incubation chamber, spread a solution of goat anti-mouse conjugated to biotin over the glycol chip 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 glycol chip in darkness at 25 degrees Celsius for 45 minutes with two micrograms per milliliter of the corresponding fluorochrome-labeled streptavidin solution in buffer two. After working in darkness to remove the unbound fraction and repeating the washing steps, dry the glycol chip by air. To scan the array, leave the glycol chip 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 micro array, slide the glycol chip into the slot until it touches the back. Scan the glycol chip by running EasyScan and save the scan as a tif file.
Quantify the array using a ScanArray analysis system. Open the previously scanned images by clicking file in the configure and file group on the main window. Load the corresponding array file template in GAL format which represents the disposition of printed glycans on the glass slide.
Adjust the GAL template by carefully aligning the array with the spots in the image and initiate quantification. Select the quantification perimeters as quantification type run easy quant and quantification method fixed circle. Unclick all options for the auto find spots and select lowess for the normalization method.
Save the quantified data as a csv file. Transfer this data into a common spreadsheet file using Microsoft Excel or another appropriate application. Genetically identical mice should not be considered as immunological equivalents because they have developed different patterns of natural anti carbohydrate antibodies.
As shown here, only 12 glycan specificities were conserved. Shown here, is a representative example of the images obtained from micro chips scanning using a florescent scanner. The grid is then aligned to the spots in every single sub array.
The florescent's is detected for each spot and results are transferred into a common spreadsheet file. Following incubation with mouse serum, the glyco chips were scanned using a scan array reader. Data were analyzed with a micro array analysis system and the results were expressed in RFU as median plus of minus the median absolute deviation.
Blue and white colors represent background binding signals of lower than 4000 RFU. Red color represents positive binding signals greater than or equal to 4000 RFU. Most of the glycan structures exposed in the glycol chips were not recognized by the repertoire of circulating anti glycan antibodies of balp C mice.
Once mastered this technique can be done in five hours and 45 minutes if it's performed properly. After watching this video, you should have a good understanding of how to use printer glycon array technology to investigate the repertoire and the levels of circulating anti carbohydrate antibodies in serum samples. While attempting this procedure, it's important to remember that the glyco chip must be manipulated by a bottom part of the glasses light.
Where the barcode is located. So as to avoid contact with the glycans printed. Also, ensure that all dry area of the glyco chip is covered by the diluted serum sample using the tip of the pipette.
The volume needed to totally cover a single surface of a glyco chip is approximately one milliliter. Following this procedure, other methods like microsphere base suspension array can be done in order to answer additional questions related to the flexible multiplex detection of anti glycan antibodies profile. After its introduction this technique paved the way for researchers in the field of biomacral molecules and immunology to explore a non protein glycol interaction in the set of small animals.
