Objective: To test serum NfL in patients with MAG antibodies (@elba-pascual HNK project) and CNTN1 patients (@martacaballero project)
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:
- CNTN1 Samples and MAG 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.
LECTURE: ASSAY – NFL-LIGHT. Put the values of calibrators – Lot. 503836.
RESULTS:
The results were here:
- http://s811335031.mialojamiento.es/lab/wp-content/uploads/2023/12/simoa-11.12.xls.
- http://s811335031.mialojamiento.es/lab/wp-content/uploads/2023/12/simoa-12.12.23.xls
