Objective: To test NF-light, tau, GFAP, and UCH-L1 in serum of pediatric MOGAD patients, NMO, antibody negative ADS (15 MS, 3 CIS) 10 healthy controls.
SAMPLES: From Thais Armengué Lab – Clinic Hospital.
PROCEDURE:
Step 1: Remove kit reagents, calibrators, controls, and samples from storage and equilibrate to room temperature. Allow 60 minutes for frozen material and 30 minutes for refrigerated reagents.
Step 2: Set the temperature and speed on the shaker to the settings indicated in the table above.
Step 3: Prepare your assay plate using conical bottom plates provided by Quanterix. Pipette the appropriate volume of prepared calibrators, controls, and samples into the wells by replicate. Calibrator volume 100 uL; Serum/plasma samples and controls should be diluted 4x with Sample Diluent in the plate by mixing 25 uL of sample with 75 uL Sample Diluent in each well. CSF samples should be diluted 40x with Sample Diluent before distributing to individual wells.
Step 4: Vortex the beads for a minimum of 30 seconds. Pour beads into a clean reagent reservoir. Using a multi-channel pipettor dispense 20 uL of beads into each well, touching off to the samples and changing tips between each column. Ensure that beads are distributed across the plate within 2 minutes to avoid settling in the reagent reservoir.
Step 5: Pour detector into a clean reagent reservoir. Using a multi-channel pipettor dispense 20 uL of detector into each well, touching off to the samples and changing tips between each column. Pre-wet tips for accurate volume transfer.
Step 6: Cover the plate with a black lid and place plate on orbital shaker to incubate at 35°C with shaking at 800 rpm for 30 minutes.
Step 7: Start the 2-step protocol on the washer to prime the manifold. When first incubation is complete place plate on the washer magnet and execute first wash of 2-step protocol.
Step 8: Dilute the SβG Reagent. Transfer 7 mL of SR-X SβG Diluent and 7 mL of N4PB SβG Reagent to a conical bottom tube and mix by gentle vortexing.
Step 9: Keep the plate on the magnet after aspiration is complete. Pour diluted SβG into a clean reagent reservoir. Use a multi-channel, multi-dispense pipettor to dispense 100 uL of SβG into each well.
Step 10: Cover the plate with a black lid and place plate on orbital shaker to incubate at 35°C with shaking at 800 rpm for 10 minutes.
Step 11: Place plate on the washer magnet and continue the 2-step protocol to the addition of Buffer B.
Step 12: Remove the plate from the magnet, replace the lid, and resuspend the beads offline in the orbital shaker at 800 rpm for 1 minute. Return the plate to the washer magnet and press continue. Repeat the offline resuspension one more time, following washer prompts.
Step 13: After completion of two Buffer B resuspension steps, press continue on the washer for final aspiration. After aspiration, allow the plate to dry on the washer magnet for 10 minutes.
Step 14: If necessary, import the Neuro 4-Plex B analysis protocol into the Quanterix SR-X software. Refer to the lot-specific Certificate of Analysis (CoA) and update calibrator concentrations in the analysis protocol.
Step 15: After the 10-minute drying step is complete, transfer the plate with pelleted beads onto the SR-X instrument and start the run.
RESULTS:
We had a lot of problems with results and errors with one specific Lot in plate 1 and plate 3 (Lot 503270) but no problems in plate 2 performed with Lot 503327. After discussing the results with Quanterix, we performed a troubleshooting plate (plate 4) with them, without finding a response. Finally, Quanterix replace 2 kits from Lot 503327and we could provide the results:
Plate 1: 22/07/22 – Kit 503270
Plate 2: 04/10/22 – Kit 503327
Plate 3: 05/10/2022 – Kit 503270
Plate 4: Troubleshooting plate 08/11/2022 – Kit 503270
Plate 5: 30/11/2022 – Kit 503327
Plate 6: 02/12/2022 – Kit 503327
