A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Preparation of buffers and reagents

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

1. Prepare citrate wash buffer by combining 0.941 g of sucrose (11 mM final concentration), 6.4 mL of 5 M sodium chloride (NaCl, 128 mM final), 5.4 mL of 0.2 M monosodium phosphate (NaH₂PO₄, 4.3 mM final), 9.4 mL of 0.2 M disodium phosphate (Na₂HPO₄, 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 to room temperature (RT) before use.
2. Prepare the rupture buffer by combining 250 mM sucrose, 1 mM ethylenediamine tetraacetic acid (EDTA), and 10 mM Tris (hydroxymethyl) aminomethane hydrochloride (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 a 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: 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 capillary electrophoresis immunoassay (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 the source.
   NOTE: When the required source volume is 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 required). Place them on ice in a 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. To reduce product loss, these should be spun down briefly using a mini centrifuge before opening. To open, the reagent tubes can be pierced by a pipette tip or pulled back from the corner.
6. To prepare the 400 mM dithiothreitol (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 the 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 the kit. Mix gently and transfer into a 0.2 mL polymerase chain reaction (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 the reverse pipetting technique; if not, pre-rinse the pipette tip before dispensing.
    NOTE: A-TDP-43 pan antibody and a-p(S409/410-2) TDP-43 antibody were used in this assay. Anti-ERK antibody was used for internal control to ensure assay components were working.
11. Perform the reverse pipetting for antibody dilution as described below. Alternatively, additional information can be found in the literature.
    NOTE: Reverse pipetting is preferred when dispensing small sequential volumes of solutions. 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 the pipet tip with the solution while the tip is still immersed. 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 to the 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 the 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 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, and then close immediately afterward. If volumes are identical, use a reverse pipetting technique. If not, pre-rinse the 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 the reverse pipetting technique. If not, pre-rinse the 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 a 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 must be optimized for the target protein.
    6. Repeat step 3.12.4.
    7. Return all PCR tubes to the tube rack and place them 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 the company website, as well.
       1. add 15 µL of luminol: peroxide mix to each well in row E.         
          NOTE: Ideally, prepare this reagent just before use and add it to each well. If this is inconvenient, this mixture may be prepared no more than 30 minutes before 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, add 10 µL of antibody diluent to each well.
       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.

Table 1: CEI assay plate loading template. 

Table 2: Interactive sample mixture preparation template. After entering the stock protein concentration from unknown samples, interactive cells will automatically calculate how much volume must be used to prepare the sample mix.