Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

All procedures involving mice were performed in compliance with experimental guidelines approved by the East Carolina University Institutional Animal Care and Use Committee (IACUC). Wildtype C57BL/6J female mice between 8-10 weeks of age were used in this study. The animals were obtained from a commercial source (see Table of Materials). EAE was induced following previously published reports^12,13,14.

1. Serum collection

1. Euthanize the experimental mouse by CO₂ asphyxiation or isoflurane overdose at the required time point (following institutionally approved protocols) after inducing EAE via MOG35-55 immunization.
   NOTE: The total number of mice and time points for serum collection can vary according to the specific experiment.
2. After the absence of vital signs is confirmed by toe or tail pinch, place the mouse in a dorsal recumbency position on a dissection tray and affix the limbs in position with pins or tape. Orient the mouse body to the LED light source (see Table of Materials) to illuminate the thorax of the animal.
3. Spray the mouse fur with 70% ethanol and make a midline incision of 3-4 cm along the abdomen from the pelvis to the xiphoid using the dissector scissors, taking care to avoid any organs and major vessels (Figure 1A-C). To facilitate the procedure, use the forceps to grab the skin over the xiphoid process.
4. Cut through the diaphragm laterally and then cut the rib cage anteriorly on both lateral edges using the spring scissors, stopping before reaching the sternum at the midline (Figure 1D). Fold the rib flap that was created over the mouse head to expose the heart (Figure 1E).
   NOTE: Cutting the sternum should be avoided as this will damage the main thoracic arteries laying adjacent to the bone and reduce the volume of blood that can be collected.
5. Insert a 25 G needle connected to a 1 mL syringe into the left ventricle and collect the blood by gently pulling back the plunger (Figure 1F). To facilitate the needle puncture, gently brace the heart against a pair of forceps.
   NOTE: If the blood does not appear immediately into the syringe, the needle should be slowly rotated, and a different angle should be tested. However, efforts should be made to place the needle properly on the first attempt to avoid creating unnecessary holes in the heart where blood can leak out.
6. Transfer the blood into a sterile 1.5 mL tube and allow it to clot for 30 min at room temperature. Remove the clot by centrifugation at 2000 × g for 20 min at 4 °C. Collect the supernatant, which represents the serum fraction, and store single-use aliquots into cryogenic tubes at -80 °C for future testing.

2. ELISA assay

1. Resuspend the lyophilized MOG35-55 peptide (see Table of Materials) in water to obtain a 10 mg/mL stock. Before starting the experiment, dilute the stock to 10 μg/mL in coating buffer (see Table of Materials) and pipette 100 μL/well of the final solution into a 96-well plate. Seal the plate with an adhesive film to avoid evaporation and incubate the plate overnight at 4 °C.
   NOTE: At least 2 wells should be calculated for each sample and 2 additional wells should be also included for a blank control.
2. In parallel, coat the same number of wells with 100 μL/well of bovine serum albumin (BSA) dissolved in coating buffer at 10 μg/mL concentration. These additional wells will serve as background controls.
   NOTE: It is recommended to coat with BSA the controls wells since the coating and blocking buffers have different compositions. Please note that other encephalitogenic peptides (such as PLP139-151 or MBP84-104) could be used as irrelevant antigens in alternative to BSA.
3. The day after, wash the plate 3 times with 200 μL/well of phosphate buffer saline supplemented with 0.05% Tween 20 (PBS-T). Subsequently, add 100 μL/well of a blocking solution made of 3% BSA in PBS (without Tween 20) and incubate the plate sealed for 1 h at 37 °C in a hybridization oven.
4. Wash the plate 3 times with 200 μL/well of PBS-T. Dilute each serum sample 1:100 in blocking solution and add 100 μL/well to both MOG35-55 and BSA coated wells. Add the same volume of blocking solution to the wells designated as blanks. Incubate the plate sealed for 2 h at room temperature, with constant shaking (250 rpm).
5. Wash the plate 3 times with 200 μL/well of PBS-T. Dilute the HRP-conjugated secondary antibody (1:2000, see Table of Materials) in a solution made of 0.2% BSA in PBS-T and add 100 μL/well to all wells. Incubate the plate sealed for 1 h at room temperature, with constant shaking (250 rpm).
6. Wash the plate 3 times with 200 μL/well of PBS-T. Add 100 µL/well of 3,3',5,5' tetramethylbenzidine (TMB) substrate (see Table of Materials) to all the wells and incubate in the dark for 1-5 min, monitoring the development of a blue color.
   1. Stop the reaction by adding 100 µL/well of stop solution (see Table of Materials) and measure the optical density (OD) in each well using a plate reader set at a wavelength of 450 nm.
      ​NOTE: The TMB substrate should be equilibrated at room temperature for 30-60 minutes before adding it to the wells.

3. Data analysis

1. Populate an Excel spreadsheet with the OD values read from the plate. Average the OD values obtained from duplicate wells (both MOG35-55 and BSA coated) for each sample.
2. For each sample, subtract the mean value of the BSA-coated wells from that of MOG35-55 coated wells. The resulting background corrected values will be proportional to the concentration of anti-MOG35-55 antibodies in the different serum samples.
   NOTE: Blank wells should result in corrected values around 0.
3. Apply a non-parametric statistical test such as the Mann-Whitney U test to compare the average OD values between two experimental conditions. Include at least 3 independent samples for each condition.