TITLE: MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A - Immunology Multiplex Assay
AUTHORS: Jen Frye

[DESCRIPTION]
This protocol describes lab processing as well as data processing for the MILLIPLEX‱ Human Cytokine/Chemokine/Growth Factor Panel A - Immunology Multiplex Assay

[STEPS]
SECTION: Sample preparation
1. Thaw plasma samples completely on ice.15 min 

SECTION: Sample preparation
3. Centrifuge thawed samples at 5000 x g @ 4°C for 10 minutes to remove particulates.5000 x g, 10 min, 4 °C 

SECTION: Sample preparation
2. Mix well by vortexing.
SECTION: Reagent preparation
6. Preparation of Quality Controls
SECTION: Reagent preparation
6.1. Before use, reconstitute Quality Control 1 and Quality Control 2 with 250 µL deionized water. Invert the vial several times to mix and vortex. Allow the vial to sit for  5-10 minutes. Transfer the reconstituted Quality Control 1 and Quality Control 2 into two polypropylene microfuge tubes. Unused portion may be stored at -20 °C for up to one month.10 min 

SECTION: Reagent preparation
5. Preparation of Antibody-Immobilized Beads
SECTION: Reagent preparation
5.1. For individual vials of beads, sonicate each antibody-bead vial for 30 seconds in an ultrasonic waterbath; vortex for 1 minute. Add 60 µL from each antibody-bead vial to the Mixing Bottle and bring final volume to 3.0 mL with Bead Diluent. Vortex the mixed beads well. Unused portion may be stored at 2-8 °C for up to one month.
SECTION: Reagent preparation
5.2. Example 1: When using 24 antibody-immobilized beads, add 60 µL from each of the 24 bead vials to the Mixing Bottle. Then add 1.56 mL Bead Diluent.
SECTION: Reagent preparation
7. Preparation of Wash Buffer
SECTION: Reagent preparation
7.1. Bring the 10X Wash Buffer to room temperature and mix to bring all salts into solution. Dilute 60 mL of 10X Wash Buffer with 540 mL deionized water. Store the unused portion at 2-8 °C for up to one month.
SECTION: Reagent preparation
8. Preparation of Serum Matrix
SECTION: Reagent preparation
8.1. Add 1.0 mL deionized water to the bottle containing lyophilized Serum Matrix. Mix well. Allow at least 10 minutes for complete reconstitution. Leftover reconstituted Serum Matrix should be stored at -20 °C for up to one month.10 min 

SECTION: Reagent preparation
9. Preparation of Human Cytokine/Chemokine/Growth Factor Panel A Standard
SECTION: Reagent preparation
9.1. Prior to use, reconstitute the Human Cytokine/Chemokine/Growth Factor Panel A Standard with 250 µL deionized water. Refer to table below for analyte concentrations. Invert the vial several times to mix. Vortex the vial for  10 seconds. Allow the vial to sit for 5-10 minutes. Transfer the reconstituted Positive Control into a polypropylene microfuge tube. This will be used as Standard 7; the unused portion may be stored at -20 °C for up to one month.10 min 

SECTION: Reagent preparation
9.2. Preparation of Working Standards 
Label 6 polypropylene microfuge tubes Standard 1 through Standard 6. Add  200 µL of Assay Buffer to each of the 6 tubes.  Prepare serial dilutions by adding 50 µL of the reconstituted standard to the Standard 6 tube, mix well and transfer 50 µL of Standard 6 to the Standard 5 tube, mix well and transfer 50 µL of Standard 5 to the Standard 4 tube, mix well and transfer 50 µL of Standard 4 to the Standard 3 tube, mix well and transfer 50 µL of Standard 3 to the Standard 2 tube, mix well and transfer 50 µL of Standard 2 to the Standard 1 tube and mix well. The 0 pg/mL standard (Background) will be Assay Buffer.
 
 
 
 
 
 
  

SECTION: Immunoassay Procedure
10. Diagram the placement of Standards 0 (Background), Standard 1 through  7, Controls 1 and 2, and Samples on Well Map Worksheet in a vertical configuration.  (Note: Most instruments will only read the 96-well plate vertically by default.)  It is recommended to run the assay in duplicate.
SECTION: Reagent preparation
4. Allow all reagents to warm to room temperature (20-25 °C) before use in the assay.24 Room temperature 

SECTION: Immunoassay Procedure
11. Add 200 µL of Wash Buffer into each well of the plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25 °C).  

SECTION: Immunoassay Procedure
12. Decant Wash Buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times.
SECTION: Immunoassay Procedure
13. 

SECTION: Immunoassay Procedure
14. Add 25 µL of Assay Buffer to the sample wells.
SECTION: Immunoassay Procedure
15. Add 25 µL of appropriate matrix solution to the background, standards, and control wells. When assaying serum or plasma, use the Serum Matrix provided in the kit.
SECTION: Immunoassay Procedure
16. Add 25 µL of Sample (neat) into the  appropriate wells.
SECTION: Immunoassay Procedure
17. Vortex Mixing Bottle and add 25 µL of the Mixed or Premixed Beads to each well using a multichannel pipet.
SECTION: Immunoassay Procedure
18. Seal the plate with a plate sealer. Wrap the plate with foil and incubate with agitation on a plate shaker   overnight (18 hours)  at 2-8 °C. Alternatively, incubate for 2 hours  at room temperature (20-25 °C).  

SECTION: Immunoassay Procedure
19. Gently remove well contents and wash plate 3 times following instructions below:

 Magnetic plate washer (Cat. No. 40-094, 40-095, 40-096 and 40-097)
Rest plate on magnet for 60 seconds to allow complete settling of magnetic beads. 1 min 
Remove well contents by aspiration.  Wash plate with 200 µL of Wash Buffer by adding Wash Buffer, shaking for 30 seconds30 s , letting beads settle for 60 seconds 1 min and removing well contents as previously described after each wash.  Please note that after the final aspiration, there will be approximately 25 µL of residual wash buffer in each well. This is expected when using the BioTek‱ plate washer and this volume does not need to be aspirated
from the plate.

SECTION: Immunoassay Procedure
20. Add 25 µL of Detection Antibodies into each well using a multichannel pipet.
 

SECTION: Immunoassay Procedure
21. Seal, cover with foil and incubate with agitation on a plate shaker for 1 hour at room temperature (20-25 °C).   
 


SECTION: Immunoassay Procedure
22. Add 25 µL Streptavidin-Phycoerythrin to each well containing the 25 µL of Detection Antibodies.
SECTION: Immunoassay Procedure
23. Seal, cover with foil and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25 °C).  

SECTION: Immunoassay Procedure
24. Gently remove well contents and wash plate 3 times following instructions below:

 Magnetic plate washer (Cat. No. 40-094, 40-095, 40-096 and 40-097)
Rest plate on magnet for 60 seconds to allow complete settling of magnetic beads. 1 min 
Remove well contents by aspiration.  Wash plate with 200 µL of Wash Buffer by adding Wash Buffer, shaking for 30 seconds30 s , letting beads settle for 60 seconds 1 min and removing well contents as previously described after each wash.  Please note that after the final aspiration, there will be approximately 25 µL of residual wash buffer in each well. This is expected when using the BioTek‱ plate washer and this volume does not need to be aspirated
from the plate.

SECTION: Immunoassay Procedure
25. Add 150 µL of Wash Buffer to all wells. Resuspend the beads on a plate shaker for 5 minutes.  

SECTION: Immunoassay Procedure
26. Run plate on Luminex‱ 200‱ instrument with xPONENT® software.
SECTION: Immunoassay Procedure
27. Save and analyze the Median Fluorescent Intensity (MFI) data using a 4-parameter logistic curve-fitting method for calculating analyte concentrations in samples (Belysa 1.2.1 Software - Millipore).
 

SECTION: Data Transfer
28. Raw data
 Analyte concentrations from each well are measured twice (since the assay is ran in duplicate). The raw data Excell file has the following tabs.

Summaries tab: This tab contains the mean analyte concentrations from the two measurements.
Analyte-Specific Tabs: Each of these tabs is dedicated to a single analyte and displays the individual concentration results for the two measurements from each well.

SECTION: Data Transfer
29. Processed Data
The raw data is first processed by core G, followed by an additional processing round by the data science core. 
SECTION: Data Transfer
29.1. Core G Processing
The analyte concentration summaries from the "Summaries" tab of the raw data are imputed with

"ND": If the analyte concentration falls below the limit of detection (LOD).
"%MFIe": The analytes CV% of the MFI is above 30%. 
"QCe": The quality controls (QC1 and QC2) fall outside of the range specified by Millipore for the specific analyte.  

Then, the summaries with the imputed values are split into the Sample Data tab, QC Data Runs tab, and the Internal Cntrl data tab. Two additional tabs are included to explain the imputed values (Data key & explanation)  and the minimum detectable concentrations for each metabolite (Imputed ND values).
SECTION: Data Transfer
29.2. Data Science Core Processing
Analyte concentrations for each sample are taken from the Sample data tab of the core G processed data and matched with the HML_ID for the participant. Then the following transformations were made on the data. 

Analytes labeled "ND" were replaced with 1/2 of the LOD. Millipore provided the LOD for each analyte. 
Analytes labeled "%MFIe" were replaced with the batch average concentration for that analyte. 
Analytes that were imputed with "QCe" are left missing (NA).  

SECTION: Data Transfer
30.