Judith Sunzenauer (last update 9.9.2014)

Human Premixed Multi-Analyte Kit LXSAH

Procedures

Preparation:

• Warm Wash Buffer Concentrate to room temperature and add 20ml Wash Buffer Concentrate to 480ml a.dest
  

• Dilute all plasma and serum samples 1:2 with Calibrator Diluent RD6-52
  

• Reconstitue Standard Cocktails with the correct volume of Calibrator Diluent RD6-52 (see Certificate of analysis) and allow the standard to sit for a minimum of 15min with gentle agitation prior to making dilutions.
  

• Fill 5 Eppendorf Tubes with 200µl Calibrator Diluent RD6-52
  

• Combine 100µl of each Standard Cocktail and 600µl Calibrator Diluent RD6-52 in Tube #1
  

• Make a 3-fold dilution series
  

• Centrifuge the Microparticle Cocktail vial for 30sec at 1000g prior to removing the cap. Gently vortex the vial to resuspend the microparticles, do not invert the vial. Dilute 500µl Microparticle Cocktail with 5ml Diluent RD2-1. PROTECT FROM LIGHT.
  

• Centrifuge the Biotin Antibody Cocktail vial for 30sec at 1000g prior to removing the cap. Gently vortex the vial, do not invert the vial. Dilute 500µl Biotin Antibody Cocktail with 5ml Diluent RD2-1.
  

• Centrifuge the Streptavidin-PE vial for 30sec at 1000g prior to removing the cap. Gently vortex the vial, do not invert the vial. Dilute 100x Streptavidin-PE to a 1x by adding 55µl Strepavidin-PE to 5.5ml Wash Buffer.
  

Instrument Settings:

• Adjust the probe height setting on the Luminex analyzer to avoid puncturing the membrane!
  

1. Assign the bead region for each analyte being measured
   

2. 50 events/bead
   

3. Minimum events:0
   

4. Flow rate: 60µl/min (fast)
   

5. Sample size: 50µl
   

6. Doublet Discriminator gates at approximately 7500 and 15,500
   

7. Collect Median Fluorescence Intensity (MFI)
   

Assay Procedure:

• Prewet the filter-bottomed microplate by filling each well with 100µl of Wash Buffer. Remove the liquid through the filter at the bottom of the plate
  using a vacuum manifold. Blot the bottom of the microplate with a paper towel.
  

• Resuspend the diluted Microparticle Cocktail by inversion and add 50µl to each well.
  

• Add 50µl Sample or Standard per well.
  

• Cover the plate with a foil and incubate for 2h at RT on a horizontal orbital microplate shaker set at 500rpm.
  

• Remove the liquid with a vacuum manifold. Fill each well with 100µl Wash Buffer and remove liquid again. Repeat 2x.
  

• Add 50µl of diluted Biotin Antibody Cocktail to all wells. Cover the plate with a foil and incubate for 1h at RT on a horizontal orbital microplate shaker set at 500rpm.
  

• Remove the liquid with a vacuum manifold. Fill each well with 100µl Wash Buffer and remove liquid again. Repeat 2x.
  

• Add 50µl of diluted Strepavidin –PE to each well. Cover the plate with a foil and incubate for 30min at RT on a horizontal orbital microplate shaker set at 500rpm.
  

• Remove the liquid with a vacuum manifold. Fill each well with 100µl Wash Buffer and remove liquid again. Repeat 2x.
  

• Add 100µl Wash Buffer to each well to resuspend the beads. Cover the plate with a foil and incubate for 2min at RT on a horizontal orbital microplate shaker set at 500rpm.
  

• Blot the bottom of the microplate with a paper towel to prevent wicking.
  

• Read within 90min using the Luminex 100IS or Luminex 200 analyzer.
  

Measuring:

Make sure the laser is warmed up prior to scanning for 30min.

Prime Luminex 200.

Wash Luminex 200 with 70% ethanol.

Wash Luminex 200 with a.dest 2x.

Run Calibrator1 and Calibrator2 if necessary and measure Control1 and Control2 with Luminex 200.

Create New Template for your Luminex Panel.

Create New Lot for your used Standards and calculate concentrations of standard curve.

Create New Batch for each plate. Save and Load.

• After loading your Batch insert plate in the XY plate handling platform.
  

• Fill the reservoir with fresh a.dest.
  

• Click Run Batch to start analysis.
  

Wash Luminex 200 with 70% ethanol.

Wash Luminex 200 with a.dest 3x.

Materials