Objective: To test serum NfL in patients with HSE. Samples of Thais Armengué Lab (Clinic Hospital). In some plates we add some IGLON5 patients from Lidia Sabeté, MOG patients from Thais Armengué and clinical outpatients from our hospital.
MATERIALS:
- Bead reagent
- Detector reagent
- SBG reagent (Streptavidin-ß-galactosidase)
- RGP reagent (Resorufin ß-D-galactopyranoside)
- Calibrators
- Controls
- Sample diluent
- Quanterix SR-X™ Instrument
- Simoa Microplate Washer
- Simoa Plate Shaker
- Simoa Wash Buffer A (#103078)
- Simoa Wash Buffer B (#103079)
- DI Water
- Simoa Sealing Oil (#102767)
- Simoa 96-well microplates (#101457)*
- Simoa disposable pipettor tips (#102919)*
- Simoa Discs (#100001)*
- Multi-channel pipettor and tips (for volumes 20 μL to 150 μL).
PREPARE SAMPLES:
- For optimal results, specimens should be free of fibrin, red blood cells, or other particulate matter. Specimens thawed after frozen storage must always be mixed THOROUGHLY by low-speed vortexing or inverting 10 times. Visually inspect the specimens. If layering or stratification is observed, continue mixing until specimens are visibly homogeneous.
- Centrifuge all specimens prior to assay. Centrifugation conditions should be sufficient to efficiently remove particulate matter and to clarify the sample, for example 5 minutes at 10,000 g for serum or plasma.
- Prior to starting sample preparation: Set the temperature on the shaker to allow equilibration – 30º.
- Dilute samples in the plate: Serum/plasma samples and controls should be diluted 4x with Sample Diluent in the plate by mixing 25 μL of sample with 75 μL Sample Diluent in each well.
BEADS: Vortex the beads for a minimum of 30 seconds. Pour beads into a clean reagent reservoir. Using a multi-channel pipettor dispense 25 μL of beads into each well, touching off to the samples and changing tips between each column.
ANTIBODY DETECTOR: Dispense 20 μL of detector into each well, touching off to the samples and changing tips between each column.
SHAKER – 1ST INCUBATION: Cover the plate with a black lid and place plate on orbital shaker to incubate at 30°C with shaking at 800 rpm for 30 minutes
2-STEP WASHER PROTOCOL:
- Place the plate on the washer magnet (remove lid!) and execute first wash of 2-step protocol.
- Keep the plate on the magnet after aspiration is complete. Pour SBG into a clean reagent reservoir. Use a multi-channel, multi-dispense pipettor to dispense 100 μL of SBG into each well.
- Cover the plate with a black lid and place plate on orbital shaker to incubate at 30°C with shaking at 800 rpm for 10 minutes (2ND INCUBATION)
- Place plate on the washer magnet (remove lid!) and continue the 2-step protocol to the addition of Buffer B.
- 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 (remove lid!) and press continue. Repeat the offline resuspension one more time, following washer prompts.
- 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.
RESULTS:
The results were here:
Plate 1: 19.05.2022 (ctrl inv: 207-3)
Plate 2: 21.05.2022 (ctrl inv: 207-3)
Plate 3: 12.07.2022 (ctrl inv: 207-3)
Plate 4: 15.07.2022 (+ IGLON5 patients) (ctrl inv: 207-3)
Plate 5: 05.08.2022 (ctrl inv: 201-9)
Plate 6: 03.10.2022 (ctrl inv: 201-9)
Plate 7: 10.12.2022 (ctrl inv: 201-9)
Plate 8: 06.02.2023 (+ MOG patients, +outpatients – 21-2-1323; 17-120 and 23-2-228) (ctrl inv: 201-9)
The intra-assay and inter-assay coefficients of variation at intermediate level (16.15 pg/mL) were 7.6% and 15%, respectively. The analysis can be found here.
