Methods

SOP of V-PEX

Reagent Preparation

Prepare Calibrator Dilutions

Prepare the highest calibrator in diluent A then Perform a series of 4-fold dilution steps to generate 7 calibrators. Use Diluent A as the zero calibrator

Dilute Samples

Dilute samples in Diluent A based on stimulation and analyte concentrations in the sample.

Prepare Detection Antibody Solution

MSD provides each detection antibody separately as a 50X stock solution.

Prepare 1x detection antibody solution in Diluent B immediately prior to use.

Prepare Wash Buffer

Make 1x Phosphate-buffered saline (PBS) plus 0.05% Tween-20 for plate washing

 

Prepare Read Buffer T

MSD provides Read Buffer T as a 4X stock solution. Dilute to 2x or 1x with deionized water

as working solution

Assay Protocol

STEP 1: Wash and Add Sample

Wash the plate 3 times with 1x Wash Buffer then add prepared samples, calibrators into well.

Seal the plate and incubate at RT with shaking for 2 hours.

STEP 2: Wash and Add Detection Antibody Solution

Wash the plate 3 times then add detection antibody solution to each well.

Seal the plate and incubate at RT with shaking for 2 hours.

STEP 3: Wash and Read

Wash the plate 3 times then add 150μL of Read Buffer to each well.

STEP 4: Analyze the plate on an MSD instrument.

SOP of U-PEX

Prepare U-PLEX Plate

  1. Create Individual U-PLEX Linker-Coupled Antibody Solutions by adding 200μL of each biotinylated antibody to 300μL of the assigned Linker in the Linker vial. Mix by vortexing then Incubate at RT for 30 minutes.
  2. Add 200μL of Stop Solution. Mix by vortexing. Incubate at room temperature for 30 minutes.
  3. Prepare the Multiplex Coating Solution by combining 600μL of each U-PLEX Linker-coupled antibody solution into a single tube and bring the solution up to 6 mL with Stop Solution for 1 plate.

4.Coat the U-PLEX Plate by add 50μL of multiplex coating solution to each well. Seal the plate and incubate with shaking at RT for 1 hour or overnight at 2–8 °C.

  1. Wash the plate 3 times and the plate is ready for use.

Reagent Preparation

Prepare Calibrator Dilutions

Reconstitute each vial of Calibrator by adding 250 μL of Diluent A to the glass vial. This will result in a 5X concentrated stock of each Calibrator.

Dilute the 5X Calibrator stock solution in Diluent A to 1X as the highest calibrator then Perform a series of 4-fold dilution steps to generate 7 calibrators. Use Diluent A as the zero calibrator

Dilute Samples

Dilute samples in Diluent A based on stimulation and analyte concentrations in the sample.

Prepare Detection Antibody Solution

MSD provides each detection antibody separately as a 100X stock solution.

Prepare 1x detection antibody solution in Diluent B immediately prior to use.

Prepare Wash Buffer

Make 1x Phosphate-buffered saline (PBS) plus 0.05% Tween-20 for plate washing

 

Prepare Read Buffer

MSD provides MSD GOLD Read Buffer solution ready to use.

 

Assay Protocol

STEP 1: Add Diluent A then Sample

Add 25μL of Diluent A to each well then add 25μL of the prepared Calibrators or sample to each well. Seal the plate and incubate at RT with shaking for 1 hours.

STEP 2: Wash and Add Detection Antibody Solution

Wash the plate 3 times then add detection antibody solution to each well. Seal the plate and incubate at RT with shaking for 1 hours.

STEP 3: Wash and Read

Wash the plate 3 times then add 150μL of MSD GOLD Read Buffer to each well.

STEP 4: Analyze the plate on an MSD instrument.