Aug 02, 2017
- Ligia Pinto1,
- Troy Kemp1
- 1Human Papilloma Virus Immunology Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research
External link: https://doi.org/10.1371/journal.pone.0182359
Protocol Citation: Ligia Pinto, Troy Kemp 2017. Luminex Milliplex Cytokine/Chemokine 9-plex MAG. protocols.io https://dx.doi.org/10.17504/protocols.io.hvhb636
Manuscript citation:
Dyke ALV, Kuhs KAL, Shiels MS, Koshiol J, Trabert B, Loftfield E, Purdue MP, Wentzensen N, Pfeiffer RM, Katki HA, Hildesheim A, Kemp TJ, Pinto LA, Chaturvedi AK, Safaeian M (2017) Associations between self-reported diabetes and 78 circulating markers of inflammation, immunity, and metabolism among adults in the United States. PLoS ONE 12(7): e0182359. doi: 10.1371/journal.pone.0182359
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: May 09, 2017
Last Modified: February 04, 2018
Protocol Integer ID: 5769
Abstract
Luminex Milliplex Cytokine/Chemokine 9-plex MAG manufacturer's protocol
PREPARATION OF SAMPLES/REAGENTS FOR IMMUNOASSAY
Preparation of Serum/Plasma Thaw Time: Thaw the samples completely on ice, mix well by shaking on plate shaker for 1 min. at RT (20-25°C) and centrifuge (1,700 xg, 10 minutes, 4°C) prior to use in the assay to remove particulates.
Preparation of Antibody-Immobilized Beads Sonicate each antibody-bead vial for 30 seconds; vortex for 1 minute. Add 150 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 portions may be stored at 2-8°C for up to one month.Example: When using 9 antibody-immobilized beads, add 150 μL from each of the 9 bead sets to the Mixing Bottle. Then add 1.65 mL Bead Diluent.
Preparation of Quality Controls Reconstitution Time: 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 and then transfer the controls to appropriately labeled polypropylene microfuge tubes. Unused portion may be stored at £ -20°C for up to one month.
Preparation of Wash Buffer Bring the 10X Wash Buffer to room temperature and mix to bring all salts into solution. Dilute 30 mL of 10X Wash Buffer with 270 mL deionized water. Store unused portion at 2-8°C for up to one month.
Preparation of Serum Matrix Reconstitution Time: Add 1.0 mL Assay Buffer 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.
Preparation of Human Metabolic Hormone Panel Standard Reconstitution Time: 1.) Prior to use, reconstitute the Human Metabolic Hormone Panel Standard with 250 μL deionized water to give STD7. Invert the vial several times to mix. Vortex the vial for 10 seconds. Allow the vial to sit for 5-10 minutes and then transfer the standard to an appropriately labeled polypropylene microfuge tube. This standard will be termed STD7; the unused portion may be stored at £ -20°C for up to one month.
) Preparation of Working Standards. Label six polypropylene microfuge tubes STD6, STD5, STD4, STD3, STD2, and STD1.Add 200 μL of Assay Buffer to each of the six tubes.-Prepare serial dilutions by adding 100 μL of STD7 reconstituted standard to the STD6 tube, mix well and transfer 100 μL of the STD6 standard to the STD5 tube, mix well and transfer 100 μL of the STD5 standard to the STD4 tube, mix well and transfer 100 μL of the STD4 standard to STD3 tube, mix well and transfer 100 μL of the STD3 standard to the STD2 tube and mix well, transfer 100 μL of the STD2 standard to the STD1 tube and mix well. The 0 pg/mL standard (Background) will be Assay Buffer.StandardVolume of Deionized Water to Add (mL)Volume of Standard to AddOriginal (STD7)2500Standard Concentration (pg/ml)Volume of Assay Buffer to Add (mL)Volume of Standard to AddSTD6200100 mL of STD7STD5200100 mL of STD6STD4200100 mL of STD5STD3200100 mL of STD4STD2200100 mL of STD3STD1200100 mL of STD2After dilution, each tube has the following concentrations for each analyte:Standard Tube #GIP (pg/ml)Ghrelin, GLP-1, Glucagon, PP, PYY (pg/ml)Amylin (pg/ml)C-Peptide (pg/ml)Insulin, Leptin (pg/ml)1
7
7
4
6
22
2
2
3205.8
53
7
5
9
31,2354
1370.4740.71,8523,7045
21,1112,2225,55611,1116
73,3336,66716,66733,33372,00010,00020,00050,000100,000IMMUNOASSAY PROCEDURE Allow all reagents to warm to room temperature (20-25°C) before use in the assay. Run the standards, controls, and samples in duplicate.
Prewet plate by pipetting 200 μL of Assay Buffer into each well of the MAG Plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25°C).
Decant Assay Buffer and remove residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times.10) Add 25 μL of each Standard or Control into the appropriate wells. Add 25 μL Assay Buffer to the 0 pg/mL standard (Background).
Add 25 μL of Assay Buffer to the sample wells.
Add 25 μL of the Serum Matrix solution to the background, appropriate standards, and control wells.
Add 25 μL of Sample into the appropriate wells.
Vortex Mixing Bottle and add 25 μL of the mixed Beads to each well. (Note: During addition of Beads, shake bead bottle intermittently to avoid settling. Due to the composition of magnetic beads, you may notice a slight color in the bead solution. This does not affect the performance of the beads or the kit).
Seal the plate with a plate sealer, cover it with the lid. Wrap a rubber band around the plate holder, plate and lid and incubate with agitation on a plate shaker overnight (16-18 hours) at 4°C.
Gently remove fluid by aspiration.
Wash plate 3 times with 200 μL/well of Wash Buffer, removing Wash Buffer by aspiration between each wash.
Add 50 μL of Detection Antibodies into each well. (Note: Allow the Detection Antibodies to warm to room temperature prior to addition.)
Seal, cover with lid, and incubate with agitation on a plate shaker for 30 minutes at room temperature. DO NOT WASH AFTER INCUBATION.20) Add 50 μL Streptavidin-Phycoerythrin to each well containing the 50 μL of Detection Antibodies.
Seal, cover with lid and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25°C).
Gently remove all contents by aspiration.
Wash plate 3 times with 200 μL/well Wash Buffer, removing Wash Buffer by aspiration between each wash.
Add 100 μL of Sheath Fluid to all wells. Resuspend the beads on a plate shaker for 5 minutes.
Run plate on Luminex 100™ IS.
Save and analyze the data using Bio-Plex Manager software.EQUIPMENT SETTINGSEvents: 50, per bead region Sample Size: 50 μLGate Settings 5000 to 25,000Time Out 60 secondsQUALITY CONTROLSThe ranges for each analyte in Quality Control 1 and 2 are provided on the card insert or can be located at the MILLIPORE website www.millipore.com/techlibrary/index.do using the catalog number as the keyword.