Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

All protocols concerning the processing of human platelets follow the guidelines of both the University of Kansas Medical Center and Kansas City University of Medicine and Biosciences IRB committees.

1. Preparation of buffers and reagents

NOTE: Prepare all samples as per the manufacturer's guidelines. Wear personal protection equipment (lab coats, gloves, and goggles) during this procedure.

1. Prepare citrate wash buffer by combining 0.941 g of sucrose (11 mM final concentration), 6.4 mL of 5 M NaCl (128 mM final), 5.4 mL of 0.2 M NaH₂PO₄ (4.3 mM final), 9.4 mL of 0.2 M Na₂PHO₄ (7.5 mM final), 0.352 g of sodium citrate (4.8 mM final), and 0.115 g of citric acid (2.4 mM final). Adjust the total volume to 250 mL with ddH₂O. Filter through a 0.45 µm filter disk and adjust the pH to 6.5. Store up to 1 year at 4 ˚C. Bring the solution room temperature (RT) before use²².
2. Prepare the rupture buffer by combining 250 mM sucrose, 1 mM EDTA, and 10 mM Tris-Cl (pH 7.4) in a 100 mL final volume. Store up to 1 year at 4 ˚C. Add 2 µL of phosphatase inhibitors cocktail (1:1000 final) and 1 µL of protease inhibitors cocktail (1:2000 final) into 2 mL of rupture buffer. Keep on ice until use. Discard the unused rupture buffer.

2. Platelet isolation

1. Collect 8–10 mL of human blood in yellow-cap blood collection tube containing acid-citrate-dextrose (ACD) solution (75 mM trisodium citrate, 124 mM dextrose, and 38 mM citric acid, pH = 7.4; ACD:blood = 1:9). Gently mix the tube content 5x–6x inverting by hand.
2. Centrifuge the tubes at 200 x g in a swinging bucket rotor for 20 min at RT.
3. Collect platelet-rich plasma (PRP) (~3–4 mL) into a 15 mL conical bottom tube and leave approximately 0.5 mL of the PRP from the buffy coat (hazy-looking fraction) to avoid contamination. If any red blood cell contamination occurs, repeat this step.
4. Centrifuge the PRP samples at 1,200 x g for 15 min at RT.
5. Wash the platelet pellets (P1) by gentle resuspension in 1 mL of citrate wash buffer and pellet by centrifugation at 1,200 x g for 15 min at RT.
6. Save the pure platelet pellet. Discard the supernatant.
7. Resuspend the platelet pellets in 600 μL of the rupture buffer containing inhibitor cocktails.
8. Sonicate the platelet suspension using a sonicator. Place the sample in a mini ice bucket. Set the sonicator at setting 3 for 20 s in continuous mode.
   NOTE: Make sure to clean the probe with 10% bleach followed by distilled water.
9. Centrifuge the sonicated samples at 20,000 x g for 30 min at 4 °C to remove membranous fractions. Aliquot supernatants in 60 μL and store at -80 ˚C. Avoid repeated thawing/freezing cycles for the platelet cytosolic fractions.

3. Preparation for CEI

NOTE: 100 µL of human platelet lysate was combined from ALS patients (n = 8–10), and healthy subjects (n = 8–10) were separately pooled and used for the assay optimization.

1. Fill out in-house generated templates for the CEI layout (Table 1) and sample preparation (Table 2). The sample mixture preparation table is dynamic and will automatically calculate how much volume needs to be removed from source.
   NOTE: When required source volume entered in dynamic Table-2, 0.1 X sample buffer volume will be automatically calculated.
2. Pre-label 25 0.2 mL PCR tubes with capillaries #1–#25 and place them in a PCR rack. Set on ice.
3. Pre-label 0.6 mL microcentrifuge tubes: one for each primary antibody and dilution (if needed) to be used, one for the 0.1x sample buffer, one for the luminol-S/peroxide, and one for each sample to be diluted (if needed). Place them on ice in tube rack.
4. Take out the sample buffer, wash buffer, one plate, and a cartridge provided in the CEI separation 12–230 kDa master kit separation module.
5. From the 4 °C refrigerator, take out the antibody dilution buffer, primary antibodies, secondary antibodies, luminol, hydrogen peroxide, and standard pack. Place all reagents on ice, except the standard pack, which remains at RT.
   NOTE: The reagents from the standard packs are lyophilized and sealed with a foil cover. These should be spun down briefly using a mini centrifuge before opening to reduce product loss. To open, the reagent tubes can either be pierced by a pipette tip or pulled back from the corner.
6. To prepare the 400 mM DTT, add 40 µL of deionized water to the clear tube containing the DTT.
7. To prepare 40 µL of fluorescent 5x master mix, add 20 µL of the 10x sample buffer and 20 µL of the prepared 400 mM DTT solution to the pink tube provided in kit.
8. To prepare the biotinylated ladder, add 16 µL of deionized water, 2 µL of 10x sample buffer, and 2 µL of the prepared 400 mM DTT solution to the white tube provided in kit. Mix gently and transfer into a 0.2 mL PCR tube for denaturing.
9. Prepare 0.1x sample buffer by adding 1.5 µL of 10x sample buffer and 148.5 µL of deionized water to a 0.6 mL micro-centrifuge tube. Vortex to mix and place on ice.
10. Prepare the desired antibody dilutions. Add antibody diluent in volumes designated to each pre-labeled micro-centrifuge tube. If volumes are identical, use reverse pipetting technique²³; if not, pre-rinse the pipette tip before dispensing.
    NOTE: In this assay, a-TDP-43 pan antibody and a-p(S409/410-2) TDP-43 antibody were used. Anti-ERK antibody was used for an internal control to make sure that assay components are working.
11. Perform the reverse pipetting for antibody dilution as described below. Alternatively, additional information can be found in the literature²⁴.
    NOTE: Reverse pipetting technique is preferred when dispensing small sequential volumes of solutions²³. This technique offers some advantages: (i) providing a precise volume, (ii) eliminating the reagent foaming in the tip orifice, and (iii) ideal for small volume (<5 µL) reagents, viscous solutions, surfactant solutions, and solutions with high vapor pressure.
    1. Put a proper tip in a pipette and press the plunger down to the second stop (Step-2). Immerse the pipet tip a few millimeters into the solution. Slowly release the plunger to fill up the pipet tip with the solution while the tip is still immersed in the solution. Remove the tip from the solution and gently touch against the edge of the reagent reservoir so that excess liquid remaining on the outside of the tip is removed.
    2. Dispense the solution by pressing the plunger down to first stop (Step-1). Do not dispense the remaining solution in the tip.
    3. Empty the remaining solution in the tip to the reagent reservoir by pressing the plunger to the second stop (Step-2). Release the plunger to the ready position for the next pipetting step.
    4. Add the required antibody in volumes designated to each pre-labeled microcentrifuge tube (Table 1) Do not pre-rinse the pipet tip: add it directly to the diluent and flush the tip multiple times to remove antibody. Place the tubes on ice.
12. To prepare the CEI sample-mix, perform the steps listed below for PCR tubes labeled cap#2 through cap#25: This is in the same order as it appears in Table 1.
    1. Open all tubes, add 1.6 µL aliquots of fluorescent 5x sample buffer to each tube using a reverse pipetting technique, then close each PCR tube upon the addition of the 5x buffer to minimize sample loss.
    2. Open all tubes, add 0.1x sample buffer in volumes designated in Table 2 to each tube, then close immediately afterward. If volumes are identical, use a reverse pipetting technique. If not, pre-rinse pipet tip before dispensing 0.1x sample buffer.
    3. Open all tubes, add protein sample in volumes designated in Table 2 to each tube, then close immediately afterward. If volumes are identical, use reverse pipetting technique. If not, pre-rinse pipet tip before dispensing 0.1x sample buffer.
    4. Briefly centrifuge all PCR tubes in a benchtop centrifuge (13,000 x g for 30 s), flick/vortex PCR tubes to mix, then repeat the centrifugation.
    5. Transfer all PCR tubes into thermocycler with a heated lid. Denature samples at defined temperature and duration (i.e., 95 ˚C for 5 min; 70 ˚C for 10 min).
       NOTE: The denaturation temperature and duration need to be optimized for target protein.
    6. Repeat step 3.12.4.
    7. Return all PCR tubes to tube rack and place on ice.
    8. During the denaturing step, prepare the development solution (1:1 luminol-S:peroxide solution), then add 200 µL of luminol-S and 200 µL of peroxide. Place on ice.
    9. To load a CEI pre-filled plate with the sample prepared above, dispense reagents and samples into the assay plate shown in the assay layout (Figure 1). Avoid introducing air bubbles.
       NOTE: If volumes and solution are identical, use a reverse pipetting technique. If not, pre-rinse the pipet tip before dispensing and do not expel the remainder into the plate well using the second tab stop on the pipette. A 12–230 kDa separation module may contain a color-coded plate-loading guide. Place this guide under the plate while adding reagents and samples to the well, which visually helps when sample loading. The plate-loading guide can be downloaded from company website, as well.
       1. In row E, add 15 µL of luminol:peroxide mix to each well.
          NOTE: Ideally, prepare this reagent just before use and add to each well. If this is not convenient, this mixture may be prepared no more than 30 min prior to plate loading.
       2. In row D, to well D1, add 10 µL of streptavidin-HRP.
       3. In row D, to wells D2–D25, add 10 µL of designated secondary antibody.
       4. In row B, to each well, add 10 µL of antibody diluent.
       5. In row C, to well C1, add 10 µL of antibody diluent.
       6. In row C, to wells C2–C25, add 10 µL of designated primary antibody.
       7. In row A, to well A1, add 5 µL of biotinylated ladder from PCR tube #1.
       8. In row A, to wells A2–A25, add 3 µL of the sample, PCR tubes #2–#25 into corresponding wells #2–#25.
       9. Add 500 µL of wash buffer to each designated wash buffer well.
       10. Centrifuge the plate for 5 min at 1,000 x g at RT.

Figure 1: Assay layout. Both primary antibody and target protein sample optimization can be performed in one assay. Capillaries 2–7, 8–13, 14–19, and 20–24 represent various protein concentration and primary antibody range. Capillary 25 represents positive control. Anti-ERK antibody was used; however, any appropriate positive control can be included. Please click here to view a larger version of this figure.

4. Performing the CEI on plate 1

1. First, turn on the CEI (Table of Materials) analyzer, then turn on the computer. Open the software (Table of Materials)
2. Connect the analyzer to the online system (Table of Materials). This is a necessary step to collect the run data for trouble shooting purposes and data recovery.
3. Click on Instrument from the top-left menu, then click Connect. Select the instrument serial number which appears as a pop-up menu. Click Connect.
4. Select the "Assay" tab and select New Assay or select a saved template.
5. Input assay parameters (Table 1) or modify template currently. Save the file name and location.
6. Make sure that the blinking blue color indicator in the analyzer remains solid blue.
7. Touch the silver metal button on top of the orange door to open.
8. Carefully remove the capillary cartridge from its packaging. Insert the capillary cartridge as described by manufacturer's protocol. If correctly installed, the inside light turns to "blue".
9. Remove the protective seal from the assay plate. Visually observe the pre-filled wells for air bubbles. If observed, pop them with a small pipet tip (Long-shaft P10 pipet tip works well).
10. Load the plate holder by placing the assay plate and close the door. In computer, click on Start button.
    1. Place ice tray containing temperature sensitive reagents and samples in the dark at 4 ˚C until ready to prepare second pre-filled plate.
    2. Leave reagents/supplies out at room temperature for the second plate.

5. Performing the CEI on plate 2

NOTE: This plate is set for analyzing phosphorylated TDP-43 levels.

1. Remove the ice tray being stored at 4 ˚C and place it on the bench, 1 h before the estimated completion time of the first plate. Retrieve a second plate and cartridge.
2. Prepare any antibody dilutions needed for the second run and store them on ice. Prepare fresh 1:1 luminol-S:peroxide solution (step 3.12.8 above).
3. Remix and briefly re-centrifuge the sample mix and reagents needed for loading the pre-filled plate. Load the second plate according to Figure 1. Load a-p(S409-410-2) TDP-43 antibody solution in row C wells.
4. When the first run is complete, discard the first plate and cartridge. Remove the cartridge and place in a sharp's container for disposal. Keep the stickers from the plate and cartridge for reference purposes.
5. Close the software file and reselect the same template. The software will remember the settings from the prior run. Make any changes to the annotation as needed (i.e., changing the primary antibody).
6. Repeat steps 4.8–4.10. Put away all 12–230 kDa master kit separation module reagents and supplies
7. Discard left over CEI sample-mix, antibody dilutions, 0.1x sample buffer and luminol-S:peroxide mixes in accordance with university regulations.

6. Data analysis

1. Once the run is completed, ensure that the following quality checks are performed.
   1. In software, select the Show Standards icon and Graph View tab. Check all 25 capillaries for the peak alignments to internal fluorescent marker sizes. Correct the misalignments by selecting Force Standard or right-clicking on the incorrect peak then selecting Not a Standard. Perform this check for each new capillary.
   2. Click on Samples and the Single View icon. Select capillary #1 (biotinylated ladder) in the experiment tab. Review the peak alignments to molecular weight markers. Click on the peak in Graph View and select Remove Peak, if an incorrect selection of the peak is done by the software.
      NOTE: As an example, the 12–230 KDa biotinylated ladder will show sizing peaks at 12 kDa, 40 kDa, 66 kDa, 116 kDa, 180 kDa, and 230 kDa. The sizing of the sample peaks will be incorrect if this step is not performed and will generating inaccurate results.
   3. View the electrophoretic movie and note if any abnormal migration occurred during the run.
   4. Derive data (e.g., peaks table, including molecular weight, peak area, peak height, and signal-to-noise [S/N]) as needed for further calculations. There are graph annotation tools located at the upper-right corner of Graph window for providing more information about the graph.