ProcartaPlex™ Human Antibody Isotyping
Panel 7-Plex
USER GUIDE
Catalog Number EPX070-10818-901
Publication Number MAN0024721
Revision A.0 (30)
For Research Use Only. Not for use in diagnostic procedures.
Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, Austria
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
End User License Agreement for Luminex Assay Products: By opening the packaging containing this Assay Product (which contains
fluorescently labeled microsphere beads authorized by Luminex Corporation) or using this Assay Product in any manner, you are
consenting and agreeing to be bound by the End User Terms and Conditions and the End User License Agreement available at
https://www.luminexcorp.com/end-user-terms-and-conditions/#sec3. If you do not agree to all of the terms and conditions, you must
promptly return this Assay Product for a full refund prior to using it in any manner.
Revision history: Pub. No. MAN0024721
Revision Date Description
A.0 (30) 5 February 2021 new manual
TRADEMARKS: xMAP, Luminex 100/200, MAGPIX, INTELLIFLEX and FLEXMAP 3D are trademarks of Luminex Corp. and Bio-Plex is a
trademark of Bio-Rad Laboratories Inc. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise
specified.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex .......... 4
Introduction .................................................................... 4
Contents and storage ............................................................ 5
Required materials not supplied ................................................... 5
Precautions and technical hints ................................................... 6
Workflow ....................................................................... 7
■
CHAPTER 2 Methods ............................................................... 8
Sample preparation ............................................................. 8
Plasma sample preparation .................................................. 8
Serum sample preparation ................................................... 8
Cell culture supernatant preparation ........................................... 8
Dilution of serum, plasma, and cell culture supernatant samples .................. 9
Preparation of reagents .......................................................... 9
Prepare 1X Wash Buer ..................................................... 9
Prepare 1X Universal Assay Buer (UAB) ...................................... 9
Prepare 1X Detection Antibody Mix .......................................... 10
Prepare Standard Mix ...................................................... 10
Prepare 3-fold serial dilution ................................................ 10
Assay protocol ................................................................. 12
Instrument settings ............................................................. 14
Analyze results ................................................................ 14
■
APPENDIX A Recommended plate layout ...................................... 16
ProcartaPlex
™
Human Antibody Isotyping Panel 7-Plex User Guide
3
1
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are available from
thermofisher.com/support.
Introduction
The ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex has been optimized for detection of multiple
analytes from serum, plasma, and cell culture supernatants.
The panel is provided with individual vials of 1X capture and 50X detection reagents and is not
combinable with simplexes or other panels.
ProcartaPlex™ Human Antibody
Isotyping Panel 7-Plex
ProcartaPlex™ preconfigured panels are extensively tested for analyte combinability, interference and
cross-reactivity to provide the highest level of validation and precision. All ProcartaPlex™ panels are
supplied with the necessary reagents to perform the assay.
Analytes
IgG1 IgG2 IgG3
IgG4 IgA IgE
IgM
For detailed product information, visit thermofisher.com/procartaplex
4
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
Contents and storage
Upon receipt, store the kit at 2°C to 8°C. When stored as indicated, all reagents are stable until the
expiration date.
Contents Amount
Isotyping Standard Mix (lyophilized) 2 each
Isotyping Detection Antibody Mix (50X) 1 x 70 μL
Capture Bead Mix (1X) 1 x 5 mL
Streptavidin-PE (SA-PE) (1X) 1 x 5 mL
Wash Buer (10X) 1 x 25 mL
Reading Buer (1X) 1 x 40 mL
Universal Assay Buer (10X) 1 x 35 mL
Detection Antibody Diluent (1X) 1 x 3 mL
Chapter 1
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex
Contents and storage
1
8-Tube Strip 2 each
Flat Bottom 96-well Plate, black 1 each
Microplate Lid 1 each
Plate Seals 8 each
Retain the lot-specific Certificate of Analysis that contains the product expiration date. The Certificate
of Analysis also contains important information such as bead number, analyte names and highest
standard concentration required for the assay setup on the xMAP instrument.
CAUTION! This kit contains materials with small quantities of sodium azide. Sodium azide reacts
with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a
large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin and
mucous membranes. In case of contact, rinse aected area with plenty of water. Observe all federal,
state and local regulations for disposal.
Required materials not supplied
• xMAP™ instrument
• Hand-Held Magnetic Plate Washer (Cat. No. EPX-55555-000)
• Deionized water
• Fresh cell culture medium for running cell culture supernatant samples
• Vortex mixer (e.g., Cat. No. 88882010)
• Microcentrifuge
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
5
Chapter 1 ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex
1
Precautions and technical hints
• Adjustable single and multichannel pipettes with disposable tips and low volume reservoirs (e.g.,
Cat. No. 95128093)
• Beakers, flasks, and cylinders necessary for preparation of reagents
• Orbital microplate shaker with at least 1.5 mm or 0.059 inch orbit diameter capable of maintaining a
speed of 600 ± 50 rpm (e.g., Cat. No. 88882006)
Note: The use of rockers or large orbit shakers may cause adverse results.
Precautions and technical hints
1. Thoroughly read this User Guide and Certificate of Analysis prior to using the kit.
2. All chemicals should be considered potentially hazardous.
3. To avoid cross-contamination, do not invert the assay plate during the assay or allow contents
from one well to mix with another well.
4. Use a multichannel pipette and reagent reservoirs whenever possible to achieve optimal assay
precision.
5. Ensure that the xMAP™ instrument has been properly calibrated and set up prior to preparing and
running the assay.
6
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
Workflow
1. Vortex capture beads for 30 sec. Add 50 µL of the capture beads to each well.
2. Remove liquid.
Note: Wash the plate after adding the beads.
1. Add the following according to sample type
-For serum and plasma samples: Add 25 µL of Universal Assay Buer, then add 25 µL of standards
or prediluted samples. For background wells, add 50 µL of 1X UAB.
-For cell culture supernatant samples: Add 50 µL of standards or prediluted samples. For
background wells, add 50 µL of cell culture medium.
2. Seal the plage and incubate with shaking at room temp for 2 hr.
3. Wash plate twice.
Chapter 1 ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex
Workflow
Assay protocol
Prepare antigen standard
Add capture beads
Add samples and standards
1
Prepare and add detection antibody
1. Add 25 µL of Detection Antibody Mix (1X).
2. Seal the plate and incubate with shaking at room temp for 30 min.
3. Wash plate twice.
Add Streptavidin-PE
1. Add 50 µL of Streptavidin-PE.
2. Seal the plate and incubate with shaking at room temp for 30 min.
3. Wash plate twice.
Resuspend beads
1. Add 120 µL of Reading Buer.
2. Seal the plate and shake at room temp for 5 min.
Acquire data on xMAP™system
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
7
2
Sample preparation
Thaw frozen serum and plasma samples on ice and mix well by vortexing. Centrifuge at 10,000 × g for
5–10 minutes to pellet out particulates. Avoid multiple freeze/thaw cycles. If samples are high in lipid
content, centrifuge at 10,000 × g for 10 minutes and transfer contents to a new tube.
Plasma sample preparation
1.
Collect samples in sodium citrate or EDTA tubes. If using heparin as an anticoagulant, no more
than 10 IU of heparin per mL of blood collected should be used to prevent assay interference that
can result in a false positive signal.
2.
Centrifuge samples at 1,000 × g at 4°C for 10 minutes within 30 minutes of collection.
3.
Collect the plasma fraction. Use immediately or store aliquots at –80°C.
Methods
Serum sample preparation
1.
Allow blood to clot for 20–30 minutes at 20–25°C.
2.
Centrifuge at 1,000 × g for 10 minutes at 20–25°C.
3.
Collect the serum fraction. Alternatively, a serum separator tube can be used following the
manufacturer’s instructions.
4.
Use immediately or store aliquots at –80°C. Avoid multiple freeze/thaw cycles.
Cell culture supernatant preparation
1.
Centrifuge samples at 1,400 rpm for 10 minutes at 4°C to remove particulates.
2.
Aliquot the clarified medium into clean polypropylene microcentrifuge tubes.
3.
Use immediately or store aliquots at –80°C. Avoid multiple freeze/thaw cycles.
8
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
Chapter 2 Methods
Preparation of reagents
Dilution of serum, plasma, and cell culture supernatant samples
The analytes included in the panel typically have high serum, plasma, and cell culture supernatant
concentrations. We recommend that you dilute serum and plasma samples 1:20,000 in 1X Universal
Assay Buer (UAB) according to the following scheme to ensure that they fall within range of the assay.
To prepare 1X UAB, see Prepare 1X Universal Assay Buer on page 9.
Dilute cell culture supernatant samples 1:200 in cell culture medium (CCM) according to the following
scheme.
1:20,000 Dilution Volume of Sample Volume of UAB
Dilution 1 (1:200) 10 μL 1,990 μL
Dilution 2 (1:100) 10 μL of Dilution 1 990 μL
1:200 Dilution Volume of Sample Volume of CCM
Dilution 1 (1:200) 10 μL 1,990 μL
2
Preparation of reagents
Before starting with the assay protocol, define the plate map. Mark the standard, sample and
background wells using the plate map found in Appendix A, “Recommended plate layout” to determine
the number of wells used.
Prepare 1X Wash Buer
Bring the Wash Buer Concentrate (10X) to room temperature and vortex for 15 seconds. Mix 20 mL of
the Wash Buer Concentrate (10X) with 180 mL ddH2O. Mix gently to avoid foaming. Wash Buer (1X)
can be stored at 2–8°C for up to 6 months.
Note: Additional Wash Buer Concentrate (200 mL, Cat. No. EPX-66666-001) can be purchased
separately for automated plate washers.
Prepare 1X Universal Assay Buer (UAB)
Note: 1X UAB is required for the preparation of standards and dilution of serum and plasma samples
only. If working with cell culture supernatant samples, use the cell culture medium as a diluent.
Mix 10 mL of 10X Universal Assay Buer (UAB) with 90 mL ddH2O. Mix gently to avoid foaming. 1X
UAB can be stored at 2° to 8 °C for up to 30 days.
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
9
Chapter 2
2
Preparation of reagents
Methods
Prepare 1X Detection Antibody Mix
Detection antibody is provided at a 50X concentration and requires dilution prior to use. The steps
below provide diluted Detection Antibody Mix for a 96-well plate.
1.
Add 60 μL of Detection Antibody Mix concentrate to a mixing bottle.
2.
Add Detection Antibody Diluent (1X) to a final volume of 3 mL if using the entire 96-well plate.
Prepare Standard Mix
This kit is supplied with one lyophilized Standard Mix for generation of standard curves. Two vials of
each Standard Mix are provided to permit the user to run the assay twice if running a partial plate.
For experiments measuring serum or plasma samples, use 1X UAB as the diluent to reconstitute and
dilute the standard. For experiments measuring cell culture supernatant samples, use fresh cell culture
medium as the diluent.
Note: Change pipette tips after each dilution step and avoid air bubbles.
1.
Centrifuge the standard mix stock vial at 2,000 x g for 10 seconds.
2.
Add 250 μL of diluent to the stock vial.
3.
Vortex the vial at high speed for 30 seconds and centrifuge at 2,000 x g for 10 seconds to collect
contents at the bottom of the vial.
4.
Incubate on ice for 10 minutes to ensure complete reconstitution.
Prepare 3-fold serial dilution
1.
Label the tubes in the 8-Tube Strip: Std1, Std2, Std3, Std4, Std5, Std6 and Std7.
2.
Add 200 μL of the reconstituted standard mix into Std1 tube.
3.
Add 150 μL of diluent into Std2–Std7 tubes.
4.
Transfer 75 μL from Std1 tube into Std2 tube.
5.
Mix by pipetting up and down 10 times.
6.
Transfer 75 μL of the mixed standards from Std2 tube into Std3 tube using new pipette tip.
7.
Mix by pipetting up and down 10 times.
8.
Repeat steps 4–7 for tubes Std4–Std7, changing pipette tips between dilution steps.
10
9.
Add 150 μL of diluent to the last tube of the 8-Tube Strip to serve as a background.
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
Transfer 200 μL
75 μL 75 μL 75 μL 75 μL
75 μL
75 μL
std 1 std 2 std 3 std 4 std 5 std 6 std 7
Antigen
Standard
Vial
Use Assay Buffer
or Cell Culture
Medium
for Background
Chapter 2 Methods
Preparation of reagents
10.
Keep tubes on ice until ready to use.
Note: Use the reconstituted standard immediately. The reconstituted standard cannot be stored.
Discard unopened standard vials if the entire plate was used in a single experiment.
2
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
11
2
Chapter 2
Assay protocol
Methods
Assay protocol
1.
Add Capture Bead Mix to the plate.
a.
Vortex the 1X Capture Bead Mix vial for 30 seconds at high speed.
b.
Using a multichannel pipette, add 50 μL of the Capture Bead Mix to each well of the plate.
2.
Wash beads using a Hand-Held Magnetic Plate Washer.
Note: To avoid loss of beads, secure the plate using the clamps on both sides of the Hand-Held
Magnetic Plate Washer during this procedure.
Note: This protocol was developed using the Hand-Held Magnetic Plate Washer (Cat. No.
EPX-55555-000). Other washers should be validated by the end user.
a.
Place the plate on the Hand-Held Magnetic Plate Washer and wait 2 minutes to allow the
beads to settle on the bottom of each well.
b.
Remove the liquid by quickly inverting the washer/plate assembly over a sink or waste
container.
c.
Gently blot the inverted washer/plate assembly onto several layers of paper towels or
absorbent surface to remove any residual liquid.
d.
Add 150 μL of 1X Wash Buer into each well and wait 30 seconds.
e.
Remove the liquid by quickly inverting the washer/plate assembly over a sink or waste
container.
f.
Gently blot the inverted washer/plate assembly onto several layers of paper towels or
absorbent surface to remove any residual liquid.
g.
Remove the plate from the magnet and proceed to step 3.
3.
Add samples and standards to the plate.
a.
Serum and plasma: Add 25 μL of 1X UAB to each well followed by 25 μL of prepared
standards or prediluted samples as defined on the plate layout. Add an additional 25 μL
of 1X UAB to the wells designated as backgrounds. Cell culture supernatants: Add 50 μL
prepared standards or prediluted samples as defined on the plate layout. Add 50 μL of cell
culture medium to the wells designated as backgrounds.
b.
Seal the plate using one of the provided Plate Seals and cover with the provided Microplate
Lid. Shake at 600 rpm for 2 hours at room temperature.
12
Note: For those wishing to perform the assay over two days, the 96-well plate can be incubated
overnight. Shake the 96-well plate for 30 minutes at room temperature at 600 rpm, then transfer
the plate to 4°C and store on a level surface. After overnight incubation, shake the plate for an
additional 30 minutes at room temperature at 600 rpm.
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
4.
Remove and discard the Plate Seal. Wash the plate following the steps below.
a.
Place the plate on the Hand-Held Magnetic Plate Washer and wait 2 minutes to allow particles
to settle on the bottom of each well.
b.
Remove the liquid by quickly inverting the washer/plate assembly over a sink or waste
container.
c.
Gently blot the inverted washer/plate assembly onto several layers of paper towels or
absorbent surface to remove any residual liquid.
d.
Add 150 μL of 1X Wash Buer into each well and wait 30 seconds.
e.
Remove the liquid by quickly inverting the washer/plate assembly over a sink or waste
container.
f.
Gently blot the inverted washer/plate assembly onto several layers of paper towels or
absorbent surface to remove any residual liquid.
g.
Repeat steps 4d-4f once more for a total of two washes.
Chapter 2
Assay protocol
Methods
2
h.
Remove the plate from the magnet and proceed to the next step.
5.
Add Isotyping Detection Antibody Mix to the plate.
a.
Using a multichannel pipette, add 25 μL of the detection antibody solution (1X) to each well of
the plate. Gently tap the plate to evenly distribute the solution in the wells.
Note: A narrow trough reservoir for multichannel pipetting is recommended to be used to
prevent volume loss.
b.
Seal the plate using a new Plate Seal and cover with the provided Microplate Lid. Shake at
600 rpm for 30 minutes at room temperature.
6.
Wash the plate following step 4.
7.
Add Streptavidin-PE (SA-PE) to the plate.
a.
Add 50 μL of SA-PE solution to each well.
b.
Seal the plate using new Plate Seal and cover with the provided Microplate Lid. Shake at 600
rpm for 30 minutes at room temperature.
8.
Wash the plate following step 4.
9.
Prepare the plate for analysis on a xMAP™ instrument.
a.
Add 120 μL of reading buer into each well.
b.
Seal the plate using new Plate Seal and cover with the provided Microplate Lid. Shake at 600
rpm for 5 minutes at room temperature.
10.
Remove the Plate Seal and run the plate on a xMAP™ instrument.
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
13
Chapter 2
2
Instrument settings
Methods
Instrument settings
Follow the recommended guidelines and procedures for calibration and verification of the instrument.
Laser-based systems require 30 minutes to warm up prior to use.
™
™
Acquisition
volume
[1]
50 μL
30 μL 40 sec MagPlex
50 μL 60 sec MagPlex
50 μL 60 sec MagPlex
Instrument
MAGPIX
INTELLIFLEX
FLEXMAP 3D
Luminex™ 100/200
Bio-Rad™ Bio-Plex
[1]
™
™
™
MAGPIX volume can be changed during the run to optimize bead count.
Timeout
(optional)
N/A N/A N/A Standard PMT 50
Note: To assure a good bead count, the probe height must be adjusted to the plate provided in the kit.
We recommend using two 5.08 mm spacer disks to adjust the sample probe height for Mylar-bottom
plates.
Analyze results
The concentration of the samples can be calculated by plotting the expected concentration of the
standards against the NET MFI generated by each standard. For Bio-Plex™ Manager, plot standard
concentrations against FI-Bkgd. A 4PL or 5PL algorithm is recommended for the best curve fit. Analyze
the assayed samples according to the operation manual for the Luminex™ or Bio-Plex™ instrument.
Bead type DD gate Reporter gain
™
4,000–13,000 Standard PMT 50
™
7,500–25,000 Standard PMT 50
™
5,000–25,000 Standard PMT 50
Min. bead
count
14
We oer a free and robust analysis software package for data analysis. To analyze the data, follow the
instructions below or contact our technical support.
1.
Export the run data in .csv format and navigate to the ProcartaPlex™ Analysis App on Thermo
Fisher Connect: https://apps.thermofisher.com/apps/procartaplex
Note: Before exporting .csv raw data from Bio-Plex™ Manager, please make sure to set 'Analytes
Labels' under 'Document Export Properties' to 'Name (Region)'. The .csv raw data exported as
Report Type ‘xPONENT’ from INTELLIFLEXTM instruments are supported.
2.
Upload the .csv files to the ProcartaPlex™ Analysis App to analyze the run data. The intuitive
software features 4PL/5PL curve fit optimization, group-wise statistical and heat map analysis.
Users can export detailed reports including images for presentations and publications.
Note: The serum and plasma samples have been diluted 1:20,000 and cell culture supernatant
samples 1:200, which must be accounted for in the software analysis.
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
Chapter 2 Methods
Analyze results
2
IMPORTANT! For ProcartaPlex
tools, and common troubleshooting questions visit thermofisher.com/procartaplexsupport
For more complete troubleshooting questions and answers, visit our FAQ database at
thermofisher.com/procartaplexfaqs
™
getting started guides, technical literature, protocol support
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
15
A
Recommended plate layout
Standards
1 1 1 1 9 9 17 17 25 25 33 33
2 2 2 2 10 10 18 18 26 26 34 34
3 3 3 3 11 11 19 19 27 27 35 35
4 4 4 4 12 12 20 20 28 28 36 36
5 5 5 5 13 13 21 21 29 29 37 37
6 6 6 6 14 14 22 22 30 30 38 38
7 7 7 7 15 15 23 23 31 31 39 39
[1]
Bkgd
[1]
Background
A
B
C
Bkgd 8 8 16 16 24 24 32 32 40 40
1 2 3 4 5 6 7 8 9 10 11 12
Samples
16
D
E
F
G
H
ProcartaPlex™ Human Antibody Isotyping Panel 7-Plex User Guide
ProcartaPlex Human Antibody Isotyping Panel 7-Plex_UG_MAN0024721-v1-GUID-6813B3D8-8598-4351A12B-348FB7BA7E02-2021/01/14 15:39:00 en
21:25:30.099Z
thermofisher.com/support | thermofisher.com/askaquestion
thermofisher.com
5 February 2021
Loading...