ProcartaPlex™

ProcartaPlex™
®
ProcartaPlex
Multiplex Immunoassay
Using Magnetic Beads
For Serum, Plasma, and Cell Culture Supernatant Samples
Instructions for
Rat Isotyping Assays
North America
Technical Support:
Research Products:
888-810-6168
858-642-2058
[email protected]
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877-726-8559
858-642-2058
[email protected]
Customer Service:
888-999-1371
858-642-2058
[email protected]
Fax: 858-642-2046
Europe/International*
Technical Support:
+43 1796 40 40 120
[email protected]
Customer Service:
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[email protected]
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Bender MedSystems GmbH
Campus Vienna Biocenter 2
1030 Vienna, Austria
www.eBioscience.com
* Customers in countries where direct sales are not avaliable may
contact their eBioscience distributor listed at www.eBioscinece.
com/distributors *
19.10.2015 Rev.03
For research use only.
Not for use in diagnostic procedures.
Limited License
Subject to the eBioscience, an Affymetrix Company, terms and conditions that govern your use of eBioscience products, eBioscience grants
you a non-exclusive, non-transferable, non-sub licensable license to use this eBioscience product only in accordance with the manual and
written instructions provided by eBioscience. You understand and agree that, except as expressly set forth in the eBioscience terms and
conditions, no right or license to any patent or other intellectual property owned or licensable by eBioscience is conveyed or implied by this
eBioscience product. In particular, no right or license is conveyed or implied to use this eBioscience product in combination with a product
not provided, licensed, or specifically recommended by eBioscience for such use.
Citing ProcartaPlex® Immunoassay in Publications
When describing a procedure for publication using this product, please refer to it as the ProcartaPlex® Multiplex Immunoassay from
eBioscience.
Disclaimer
eBioscience, Inc. reserves the right to change its products and services at any time to incorporate technological developments. This manual is
subject to change without notice.
Although this manual has been prepared with every precaution to ensure accuracy, eBioscience, Inc. assumes no liability for any errors or
omissions, not for any damages resulting from the application or use of this information.
Trademarks
©2014 Affymetrix Inc. All rights reserved. Affymetrix®, eBioscience® and ProcartaPlex® are trademarks or registered trademarks of
Affymetrix, Inc. Luminex®, xMAP®, MAGPIX®, Luminex® 100/200™, and FLEXMAP 3D® are registered trademarks of the Luminex Corporation.
All other trademarks are the property of their respective owners.
2
Contents
Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
How it Works . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Materials Provided and Storage Conditions . . . . . . . . . . . . . . . . . . . . 4
Precautions and Technical Hints . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Required Equipment and Materials Not Supplied . . . . . . . . . . . . . . . 5
Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Assay Protocol Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Preparation of Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Setup of the Luminex Instruments . . . . . . . . . . . . . . . . . . . . . . . . . 11
Analyzing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Recommended and Blank Plate Layout . . . . . . . . . . . . . . . . . . . . . . 13
3
Intended Use
This user manual is for a ProcartaPlex® Rat Antibody Isotyping Panel from the eBioscience division
of Affymetrix to perform quantitative, multiplexed protein measurements from serum, plasma, and
cell culture supernatant samples using magnetic beads technology from Luminex®. Other biological
samples might be suitable for use in the assay.
NOTE: For the most current version of user documentation, go to our website at www.ebioscience.com
NOTE: ProcartaPlex Rat Antibody Isotyping Panel cannot be combined with other ProcartaPlex pre-mixed Panels
and Simplex Kits.
How it Works
ProcartaPlex Immunoassays incorporate magnetic microsphere technology licensed from the
Luminex Corporation to enable the simultaneous detection and quantitation of multiple protein
targets in diverse matrices. The platform allows the simultaneous detection from a single sample
of up to 100 protein targets on the Luminex 200/100 and FLEXMAP 3D® platforms and 50 protein
targets on the MAGPIX® platform.
Materials Provided and Storage Conditions
ProcartaPlex Immunoassay Kits contain the components listed below. Refer to the Certificate of
Analysis for quantities and details of components supplied. Expiration date is stated on the kit when
stored between 2-8°C. Do not use past kit expiration date.
Components Supplied
Antigen Standards, premixed
Detection Antibody, premixed (50X) 1
Antibody Magnetic Beads, premixed1
Wash Buffer Concentrate (10X)1
Detection Antibody Diluent1
Universal Assay Buffer Concentrate (10X)1
Reading Buffer1
PCR 8-Tube Strip
96-Well Flat Bottom Plate
Black Microplate Lid
Plate Seals
1
Contains sodium azide. See WARNING.
WARNING: All chemicals should be considered potentially hazardous. We recommend that this product and its components
be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice.
This kit contains small quantities of sodium azide. Sodium azide is highly toxic and reactive in the pure form. At this product’s
concentration, though not classified as hazardous, build up of sodium azide may react with lead and copper plumbing to
form highly reactive explosive metal azide. Dispose of the product in accordance with all State and local regulations.
4
Precautions and Technical Hints







Thoroughly read this user manual and Certificate of Analysis that is included with the assay
kit. The product insert may contain specific instructions for proper use of your kit.
For Luminex 100/200 and FLEXMAP 3D instruments initiate the startup protocol to warm up
the lasers for at least 30 minutes. Ensure that the Luminex machine is calibrated according
to the manufacturer’s instructions. MAGPIX instrument doesn't require additional warm up.
When working with samples and standards, change the pipette tips after every transfer and
avoid creating bubbles when pipetting.
During the incubation steps, cover the 96-Well Flat Bottom Plate with the Black Microplate
Lid provided in the kit to minimize exposure of the beads to light.
Be careful not to invert the 96-Well Flat Bottom Plate during the assay or allow contents
from one well to mix with another well.
Use a multi-channel pipette and reagent reservoirs whenever possible to achieve optimal
assay precision.
Store the reconstituted standards (including standard diluent sets) on ice before adding to
the 96-Well Flat Bottom Plate.
Required Equipment and Materials Not Supplied






MAGPIX, Luminex® 100/200, FLEXMAP 3D, or Luminex-based Instrument.
Glass-distilled or deionized water.
Adjustable single and multi channel pipettes with disposable tips.
Multichannel pipette reservoir.
Beakers, flasks, cylinders necessary for preparation of reagents.
Hand-Held Magnetic Plate Washer, Vortex mixer and Microtiter plate shaker.
Sample Preparation
For frozen samples, thaw samples on ice and mix well by vortexing followed by
centrifugation to remove particulates. Avoid multiple freeze/thaw cycles.
 If there is a high lipid content in the sample, centrifuge at 10,000 x g for 10 min at 2-8 °C
and transfer contents to a new tube.
For preparation instruction of tissue homogenates and lysates from cultured cells refer to
www.ebioscience.com/resources/best-protocols/multiplexing.htm

Plasma Sample Preparation
Action
A.
Collect samples in sodium citrate or EDTA tubes. When using heparin as an anticoagulant, no
more than 10 IU of heparin per mL of blood collected should be used since an excess of heparin
may give falsely high values of some of the analytes.
B.
Centrifuge samples at 1,000 x g at 4 °C for 10 min within 30 min of blood collection.
C.
Collect the plasma fraction.
D.
Use immediately or aliquot and store at -80 °C.
5
Serum Sample Preparation
We recommend to spin down serum samples at 1000 x g for 10 min at 20-25 °C before running
the assay.
Action
A.
Allow blood to clot for 20-30 min at 20-25 °C.
B.
Centrifuge at 1,000 x g for 10 min at 20-25 °C.
C.
Collect the serum fraction. (Alternatively, use any standard serum separator tube following the
manufacturer's instructions.)
D.
Use immediately or aliquot and store at -80 °C.
Diluting Samples with High Concentration of Target Analytes
The analytes included in the kit have high serum or plasma concentrations.
1:40.000-fold dilution in Universal Assay Buffer (1X) is appropriate for most serum and plasma
samples. For samples that yield results outside the range of the standard curve, a lower or higher
dilution might be required. or cell culture supernatant, a 1:200 dilution in cell culture medium is
suggested.
Each serum or plasma sample has to be diluted 1:40000 according to following scheme:
1:200 Dilution
Sample Volume
UAB Volume
Dilution 1 (1:200)
10 µL
1990 µL
Dilution 2 (1:200)
10 µL
1990 µL
If the samples are taken from cell culture supernatant prepare a 1:200 dilution:
1:200 Dilution
Sample Volume
CCM Volume
Dilution (1:200)
10 µL
1990 µL
6
Assay Protocol Overview
Prepare Antigen Standard
Add Antibody Magnetic Beads
Vortex magnetic capture beads for 30 seconds
Add 50 μL of the Antibody Magnetic Beads to each well. Remove liquid. If required, add
next type of Antibody Magnetic Beads. Repeat, until all Beads needed are in the Plate.
Wash the plate
Cell Culture Supernatant Samples
Serum and Plasma Samples
Add to the beads:
Add to the beads:
• 25 μL of Universal Assay Buffer
• 25 μL of standards, controls, or samples
• 50 μL of standards, controls, or samples
Seal Plate. Incubate with shaking for
60 min at room temperature
Wash beads (3X)
Detection Antibody
Preparation
Add Detection Antibody
Add 25 μL of Detection Antibody Mix (1X)
Seal Plate. Incubate with shaking for
30 min at room temperature
Wash beads (3X)
Resuspend Beads
Add 120 μL of Reading Buffer
Seal plate and shake for 5 minutes
at room temperature
Acquire Data on Luminex® 100/200,
Magpix®, or FlexMAP 3D® system
7
Preparation of Reagents
Antigen Standard
Carefully review the Certificate of Analysis for kit specific Antigen Standard preparation
instructions. Each kit is shipped with two identical vials of each premixed antigen standard set from
the same lot to permit the user to run the assay twice if running a partial plate.
Step
Step 1.
Reconstitution
and pooling of
Standards
Step 2. Prepare
3-Fold Serial
Dilution
Action
A.
Centrifuge the antigen standard set vial at 2000 x g for 10 sec.
B.
Add 250 μL of sample type specific buffer into the standard set vial. For
serum or plasma samples, use Universal Assay Buffer and for cell culture
supernatant samples use the cell culture media that was used to culture
the cells.
C.
Gently vortex the vial for 30 seconds and centrifuge at 2000 x g for 10
seconds to collect contents at the bottom of the vial.
D.
Incubate on ice for 10 min to ensure complete reconstitution.
A.
Refer to Certificate of Analysis for the value of each premixed standard
with assigning S1 values for each analyte for the current lot.
B.
Prepare a 3-fold serial dilution of the reconstituted standard(s) using the
PCR 8-tube strip provided. Label tubes Std1, Std2, Std3, Std4, Std5, Std6
and Std7.
C.
Add 200 μL of the reconstituted antigen standards into the first tube of
the strip tube and label as Standard 1 (Std1).
D.
Add 150 μL of sample type specific standard buffer into Std tubes 2-7. Use
Universal Assay Buffer for serum or plasma samples and cell culture media for
culture supernatant samples.
E.
Transfer 75 μL of the reconstituted antigen standards from Tube 1 into
Tube 2.
F.
Mix by pipetting up and down for a total of 10 times.
G.
Change the pipette tip and transfer 75 μL of the mixed standards from
Tube 2 into Tube 3.
H.
Mix by pipette up and down 10 times.
I.
Repeat steps E-H for Std tubes 4-7.
J.
Add 200 μL of Universal Assay Buffer or cell culture media into tube 8 which
serves as a blank. Keep on ice until ready to use.
Transfer 200μL
75 μL
75 μL
75 μL
75 μL
75 μL
75 μL
Antigen
Standard Vial
Std 1
Std 2
8
Std 3
Std 4
Std 5
Std 6
Std 7
Use Assay Buffer or
Cell Culture Media
for Blanks
Assay Protocol
Step
Action
Bring the Wash Buffer Concentrate (10X) to room temperature and vortex
for 15 seconds. Mix 20 mL of the Wash Buffer Concentrate (10X) with 180
mL ddH2O. Wash Buffer (1X) can be stored at 2-8 °C for up to 6 months.
Mark the standard, sample and blank wells using the plate map at the end
of this manual.
Step 1. Prepare 1X
Wash Buffer
Step 2. Define the
plate map
Step 3a. Add the
Antibody Magnetic
Beads
A.
Vortex the Antibody Magnetic Beads for 30 sec.
B.
Add 50 μL of the Antibody Magnetic Beads to each well. Use a multichannel pipette for this step as well as the steps below.
Step 4. Wash
Antibody Magnetic
Beads
A.
Securely insert the 96-Well Flat Bottom Plate into the Hand-Held
Magnetic Plate Washer and ensure that the plate is held in place by
the tabs and wait 2 min to allow the Antibody Magnetic Beads to
accumulate on the bottom of each well.
B.
Remove the liquid in the wells by quickly inverting the Hand-Held
Magnetic Plate Washer and 96-Well Flat Bottom Plate assembly over a
sink or waste container. Do not remove the 96-Well Flat Bottom Plate
from the Hand-Held Magnetic Plate Washer. Blot the inverted assembly
onto several layers of paper towels or absorbent surface to remove any
residual solution.
C.
Add 150 μL of Wash Buffer (1X) into each well and wait 30 seconds
to allow the beads to accumulate on the bottom of each well.
D.
Remove the Wash Buffer in the wells by quickly inverting the plate and
then blotting onto an absorbent towel to remove any residual
solution.
Remove the 96-Well Flat Bottom Plate from the Hand Held Magnetic
Plate Washer and proceed to the next step.
A. This kit is supplied with 10X Universal Assay Buffer. Prepare a 1X
working concentration of Universal Assay Buffer by mixing 10 mL of
the 10X Universal Assay Buffer with 90 mL ddH2O.
E.
Step 5. Add sample
type-specific buffer,
samples, standards
and blanks and
incubate
Step 6. Wash the
96-Well Plate
B.
Add 25 μL of Universal Assay Buffer (1X) to each well for serum and
plasma followed by 25 μL of standards or samples into dedicated
wells. For cell culture supernatant samples, add 50 μL standards or
samples into dedicated wells.
C.
For wells designated as blanks, add an additional 25 μL of Universal
Assay Buffer for serum or plasma samples. For cell culture supernatant
samples, add 50 μL of cell culture medium.
D.
Seal the plate with the provided Plate Seal. Cover the plate with the
Black Microplate Lid and shake at 500 rpm for 60 min at room
temperature (RT).
Wash plate for a total of three washes using "Step 4. Wash Antibody
Magnetic Beads".
9
Step
Step 7. Prepare 1X
Detection Antibody
Mixture
Action
A.
Detection antibody is provided at 50X concentration. Add 60 μL of
each detection antibody concentrate to the mixing bottle and bring
volume of the mixing bottle to a total of 3 mL using detection
antibody diluent.Table below is an example for 48 and 96-wells.
48-wells
96-wells
Detection Antibody
30 µL
60 µL
Detection Antibody
Diluent
1470 µL
2940 µL
Total
1500 µL
3000 µL
Step 8. Add Detection A. Add 25 μL of Detection Antibody Mixture (1X) to each well.
Antibody Mixture and B. Seal the plate with a new Plate Seal, cover plate with Black Microplate
Lid and incubate 30 min on a plate shaker at RT at 500 rpm.
incubate
Step 9. Wash the
96-Well Plate
Wash plate for a total of three washes using "Step 4. Wash Antibody
Magnetic Beads".
Step 10. Prepare the A. Add 120 μL of Reading Buffer into each well.
96-Well Plate for
B. Seal the plate with a new Plate Seal, cover plate with Black Microplate
Lid and incubate 5 min on a plate shaker at RT at 500 rpm.
Analysis on a Luminex
Instrument
C. Remove Plate Seal and run the plate on a Luminex Instrument.
10
Setup of the Luminex Instruments
Instrument
Sample Size
DD Gate
Timeout
Bead Event/Bead
Region
Luminex 100/200
FLEXMAP 3D
50 µL
5,000 - 25,000
60 sec
50-100
MAGPIX
50 µL
N/A
N/A
50-100
Prior to running the assay, ensure that the probe height has been calibrated with 96-Well Flat
Bottom Plate supplied with the kit. Failure to adjust the probe height can cause damage to the
instrument or low bead count. The Luminex system allows for calibration of Low and High RP1
target values. We recommend RP1 Low target value settings for ProcartaPlex Immunoassays. Please
refer to the Certificate of Analysis provided with the kit for bead region and analyte associations
when entering the information into the Luminex Acquisition Software.
NOTE: If there is a malfunction of the Luminex Instrument or software during the run, the 96-Well Flat Bottom
Plate can be re-read. Remove the 96-Well Flat Bottom Plate from the instrument, insert the 96-Well Flat Bottom
Plate into the Hand-Held Magnetic Plate Washer, wait 2 min, then remove the buffer in the wells by quickly
inverting the 96-Well Flat Bottom Plate over a sink or waste container. Blot the assembly onto several layers of
paper towels to remove any residual solution. Resuspend the beads in 120 µL of Reading Buffer, remove from
the Hand-Held Magnetic Plate Washer, seal the 96-Well Flat Bottom Plate with a new Plate Seal and Lid and
shake at 500 rpm for 5 min at room temperature. The assayed samples may take longer to read since there will
be less beads in the well.
Analyzing Results
The concentration of the samples can be calculated by plotting the expected concentration of the
standards against the MFI generated by each standard. A 4PL or 5PL algorithm is recommended for
the best curve fit. Analyze the assayed samples according to the operation manual for the Luminex
Instrument (e.g MAGPIX®, Luminex® 100/200™, FLEXMAP 3D®). We offer a free and robust
analysis software package for data analysis. ProcartaPlex Analyst 1.0 can be download at:
www.ebioscience.com/resources/procartaplex-analyst-1.0-software.htm
11
Troubleshooting
Observation
Probable Cause
Recommend Solution
Low Flow Rate
Samples/beads are stuck in flow cell
Remove the 96-Well Plate and perform a wash and rinse
cycle.
High CVs
Samples and antigen standards not stored
on ice
Prepare the samples and standards on ice before setting
up the assay.
Contamination from
re-using the Plate Seal
Use a new Plate Seal for each incubation step.
Incomplete washing
After adding the standards and samples, it is very
important that any excess standards are removed during
the wash step.
Contamination from contents from
adjacent wells
Avoid splashing the Wash Buffer during wash steps into
adjacent wells.
Poor pipetting techniques
Use a multichannel pipettor and careful pipette
techniques. Avoid touching pipette tips to sides of the
wells when adding Wash Buffer.
Limited dynamic
range for BioPlex
software users
Instrument calibrated at high PMT settings
Calibrate the instrument using the CAL2 Low RP1 target
value.
Low bead count
Volume of bead solution is too low
Add 120 μL Reading Buffer into each well and shake at
500 rpm for 5 min at room temperature to resuspend
beads prior to reading on the Luminex Instrument.
High bead aggregation
Vortex the bead suspension well before using in the assay
and ensure that the beads are properly mixed during the
incubation steps.
Dyes contained in the beads are photobleached from overexposure to light
Store bead solution and the 96-well plate in the dark.
Samples causing the instrument to clog
Remove the 96 Well Flat Bottom Plate and perform a
wash and rinse to the instrument. Rerun the assay with
further dilution of samples
Probe height is incorrect
Refer to the Luminex Manual for proper adjustment of
the needle height.
Instrument needle is partially clogged
Replace or clean needle according to the manufacturer's
recommendations.
Beads stuck to the bottom of the plate
Confirm that the plate shaker is set to 500 rpm and
shaking for at least 5 min before reading.
Air bubble in the sample loop
Refer to the Luminex manual for proper removal of the air
bubble.
Low signal or
sensitivity
Standards not reconstituted and diluted
correctly
Prepare fresh antigen standards following the
instructions in the Preparing Antigen Standards section.
Poor recovery
Did not use appropriate cell culture media
to prepare the standards
Use the same cell culture media that is used to culture the
cells.
Samples and antigen standards were not
stored on ice
Prepare the samples and standards on ice before setting
up the assay.
12
Recommended and Blank Plate Layout
Standards
Samples
Standard 1
Standard 1
1
1
9
9
17
17
25
25
33
33
Standard 2
Standard 2
2
2
10
10
18
18
26
26
34
34
Standard 3
Standard 3
3
3
11
11
19
19
27
27
35
35
Standard 4
Standard 4
4
4
12
12
20
20
28
28
36
36
Standard 5
Standard 5
5
5
13
13
21
21
29
29
37
37
Standard 6
Standard 6
6
6
14
14
22
22
30
30
38
38
Standard 7
Standard 7
7
7
15
15
23
23
31
31
39
39
Blank
Blank
8
8
16
16
24
24
32
32
40
40
3
4
1
2
5
6
7
A
B
C
D
E
F
G
H
13
8
9
10
11
12
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