Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

So the focus of our laboratory is to understand that the molecular basis of multiple sclerosis, immune disease of the central nervous system that is characterized by demyelination, neuroinflammation, and axonal injury. So this protocol represent an economic and flexible alternatives to commercial kits. Our x-ray can be easily adjusted to test B-cell antibody responses against different EAE peptides.

My lab is devoted to further understanding the connection between neuroinflammation and neurodegeneration in chronic neurological diseases such as multiple sclerosis. To begin, take a euthanized mouse and confirm the absence of vital signs by toe or tail pinch. Spray the mouse's fur with 70%ethanol.

Then place the mouse in a dorsal recumbency position on a dissection tray, securing the limbs with either pins or tape. Orient the mouse's body towards the LED light source to ensure proper illumination of the thorax during the dissection. Using dissector scissors, make a midline incision of three to four centimeters along the abdomen starting from the pelvis to the xiphoid.

Next, use spring scissors to cut through the diaphragm laterally. Then cut the rib cage anteriorly on both lateral edges and stop before reaching the sternum at the midline. Fold the created rib flap over the mouse's head to expose the heart.

Then while gently bracing the heart against a pair of forceps, insert a 25 gauge needle connected to a one milliliter syringe into the left ventricle. Gently pull back the plunger to collect the blood. Transfer the collected blood into a sterile 1.5 milliliter tube, and allow it to clot for 30 minutes at room temperature.

To remove the formed clot, centrifuge the tube at 2, 000 G for 20 minutes at four degrees Celsius. Collect the supernatant representing the serum fraction and carefully store single-use aliquots in cryogenic tubes at minus 80 degrees Celsius. Begin by re-suspending the lyophilized MOG 35-55 peptide in water.

Dilute the peptide stock to 10 micrograms per milliliter in coating buffer before the experiment. Now, pipette 100 microliters of the final solution into wells of a 96-well plate. Simultaneously, coat an equal number of wells with 100 microliters of BSA dissolved in coating buffer.

Seal the plate with an adhesive film to prevent evaporation and incubate the plate overnight at four degrees Celsius. The next day, perform three washes of the plate with 200 microliters of PBS supplemented with 0.5%tween 20. Subsequently, add 100 microliters per well of a blocking solution composed of 3%BSA in PBS.

Incubate the sealed plate for one hour at 37 degrees Celsius in a hybridization oven. After incubation, wash the plate three times with 200 microliters per well of PBST. Dilute each serum sample obtained from the blood of EAE immunized mice in blocking solution.

And add 100 microliters per well to both MOG 35-55 and BSA coated wells. Add the same volume of blocking solution to designated blank wells. Incubate the sealed plate for two hours at room temperature with constant shaking.

After incubation, remove the plate from the incubator and rinse the plate three times with 200 microliters per well of PBST. Dilute the HRP conjugated secondary antibody in a solution consisting of 0.2%BSA in PBST and add 100 microliters to each well. Incubate the sealed plate for one hour at room temperature, maintaining constant shaking.

Wash the plate three times with 200 microliters per well of PBST. Add 100 microliters of three 3-prime, five 5-prime tetraethyl benzine substrate, to each well. Incubate in the dark for one to five minutes monitoring the development of a blue color.

Add 100 microliters of stop solution to each well. Then use a plate reader set at a wavelength of 450 nanometers to measure the optical density in each well. The optical density values of the EAE samples were significantly higher than the control samples indicating a robust immunoglobulin G response against the MOG peptide.