1. No category

How to convert ELISA format assays to xMAP using
MagPlex beads for MAGPIX
Purpose
This publication will provide guidance on how to create a microsphere-based assay utilizing
antibody pairs that you are currently using in an ELISA or a new set of antibodies. With your
xMAP format assay, you will decrease spending on antibodies, use less sample, have the option
of utilizing automation, and see increased sensitivity and dynamic range. The ‘open’ platform of
xMAP allows for rapid addition to an already existing single-plex or multiplex panel (Faucher et al.
2009; Kofoed et al. 2006). This publication specifically addresses how to create a washed
sandwich immunoassay using magnetic beads. If you are interested in building a different type of
assay such as a no-wash, competitive, serology, etc., please see the Luminex FAQ page,
http://www.luminexcorp.com/Support/SupportResources/index.htm. The references at the end of
this document have all utilized xMAP for creation of sandwich immunoassays to replace ELISAs.
A number of publications built assays for human cytokines, and others have developed assays for
porcine (Bjerre et al. 2009, Johannison et al. 2006, Lawson et al. 2010), bovine (Dernfalk et al.
2004, Dernfalk et al. 2007), equine (Wagner et al. 2009 and 2010), canine (Wagner et al. 2011)
and feline (Wood et al. 2011) cytokines as well as plant proteins (Haasnoot et al. 2007). Assays
have been built as just a single-plex due to the advantages previously stated, such as reduced
sample volume used and increased sensitivity (Faucher et al. 2009; Rizzi et al. 2010).
Contents
Purpose ....................................................................................................................................... 1
Contents ...................................................................................................................................... 1
Introduction .................................................................................................................................. 2
Selection of Assay Components ................................................................................................. 2
Bead Coupling ............................................................................................................................. 3
Recommended Protocol for Two-Step Carbodiimide Coupling of Antibodies to MagPlex
Magnetic Carboxylated Microspheres .................................................................................... 5
Confirmation of Coupling ........................................................................................................... 10
Recommended Protocol for Confirmation of Antibody Coupling .......................................... 14
Selecting the best Pairs of Antibodies....................................................................................... 16
Assay Protocol/Performing the Assay ....................................................................................... 16
®
Recommended Protocol for Washed Capture Sandwich Immunoassay Using MagPlex
Microspheres ........................................................................................................................ 18
References ................................................................................................................................ 22
Appendix – Automated Washing Option Using Bio-Tek ELx405 Microplate Washer ............... 25
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Introduction
For the last four decades, enzyme-linked immunosorbent assays (ELISA) developed by Engvall
and Perlmann (1971) have been the primary source for detection of protein biomarkers in
biological samples for both life science research and clinical diagnostics. While widely utilized
(Lequin, 2005), ELISA has limitations. An ELISA is typically performed in a 96-well microplate and
the wells are coated with relatively large quantities of capture antibody. Also, in order to capture
antigen of interest from the sample, large sample volumes are required.
xMAP format assays have the added value of higher throughput, increased flexibility, reduced
sample volume, and lower costs with a similar workflow as ELISA. Luminex’s xMAP Technology
has been utilized in over 8,000 publications (www.luminexcorp.com/bibliography), and can be
applied to both protein and nucleic acid applications. The suspended beads allow for assay
flexibility in a single-plex or multiplex format. Since the beads have the capture antibody
immobilized on their much smaller surface area as compared to a microplate well, smaller sample
volumes are required and non-specific binding is reduced (Carson & Vignali 2000). Recent
enhancements by Luminex include the new MAGPIX system and the introduction of magnetic
microspheres which have reduced the cost of performing a single-plex assay as expensive filter
microplates are no longer required. The magnetic beads also make the workflow easier and are
automation compatible.
Selection of Assay Components
Manufacturers of ELISA kits often sell matched pairs that are easily transferable to beads.
Examples include but are not limited to, DuoSets® from R&D Systems (Wood et al. 2011) and
eBioscience (Rizzi et al. 2010). Many publications list the source, catalog number and clone
number for their antibodies used (Bjerre et al. 2009; Carslon & Vignali 1999; de Jager et al. 2003;
de Jager et al. 2005; de Jager et al. 2009; Dernfalk, et al. 2004; Dernfalk et al. 2007; Lawson et
al. 2010; Ray et al. 2005; Skogstrand et al. 2005). Many of the assays built in these publications
are common and Luminex suggests you use these as a starting point to save time. The Antibody
Resource website (http://www.antibodyresource.com) is a good starting point to search for
antibody suppliers.
When choosing raw materials (antibodies and recombinant proteins), select vendors that have
rigorous quality control procedures and provide as much information as possible about the
antibodies or proteins. Request that the vendor provide purity information from SDS- and nondenaturing-PAGE. Also, request profiles of the antibody from capillary isoelectric focusing to
compare lots from the same vendor. Don’t rely upon the vendor to quality control raw materials
for you. Luminex recommends that you devise your own incoming materials quality control
procedure.
When coupling antibodies to the beads it’s ideal to use a monoclonal antibody as the capture. A
polyclonal or monoclonal can be used as the detection antibody. Monoclonal antibodies used for
capture should be specific for a different epitope than the detection antibody.
Couple every antibody to the microspheres. Try to find antibodies that come in both plain and
biotinylated versions. If you need to biotinylate antibodies there is an example in Lawson et al.
2010. Also, Pierce has a product, EX-Link Sulfo-NHS-LC-Biotin catalog #21335, for this purpose.
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Bead Coupling
Luminex MagPlex superparamagnetic microspheres are covered with approximately 10 million
carboxyl groups for binding proteins or other macromolecules. Proteins are coupled to the beads
by conjugating the free amines of lysine sidechains and the N-terminal amines of proteins to the
carboxyl groups on the beads.
Bead coupling is a two-step procedure. The first step is to activate the microspheres with
EDC/Sulfo-NHS. The excess is removed and then protein is added. After the protein is
conjugated to the beads, the remaining sites are blocked with detergent or BSA.
Proteins conjugate best when maintained in the storage buffer the manufacturer provided but if
interfering substances are present, buffer exchange into an alternative buffer of the same pH is
recommended.
Table 1. Suggested Mag-Plex bead regions to utilize for building an immunoassay
Region
Part #
Region
Part #
Region
Part #
Region
Part #
Region
Part #
12
MC10012
26
MC10026
38
MC10038
52
MC10052
65
MC10065
13
MC10013
27
MC10027
39
MC10039
53
MC10053
66
MC10066
14
MC10014
28
MC10028
42
MC10042
54
MC10054
67
MC10067
15
MC10015
29
MC10029
43
MC10043
55
MC10055
72
MC10072
18
MC10018
30
MC10030
44
MC10030
56
MC10056
73
MC10073
19
MC10019
33
MC10033
45
MC10045
57
MC10057
74
MC10074
20
MC10020
34
MC10034
46
MC10046
61
MC10061
75
MC10075
21
MC10021
35
MC10035
47
MC10047
62
MC10062
76
MC10076
22
MC10022
36
MC10036
48
MC10048
63
MC10063
77
MC10077
25
MC10025
37
MC10037
51
MC10051
64
MC10064
78
MC10078
6
Note: Bead regions in bold are also available in lower concentrations of 2.5 x 10 beads/ml. Part
Number Format: MC100XX-ID (where XX is the region)
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Typically, Luminex suggests coupling approximately 5 µg of antibody per 1 million beads as a
starting point. If optimization is required, try coupling in the 1 to 25 µg per 1 million bead range.
Microsphere coupling reactions can be easily scaled up or down depending upon user needs.
Please refer to Bjerre et al. (2009) for an example of coupling titration results and Table 2 for
bead and reagent information.
Here are some things to keep in mind for coupling reactions:
• Do not couple less than one million microspheres at a time
• Mixing volume is very important for protein coupling and is based on the number of beads
you are coupling and the size of the vessel you are using.
Table 2. Scale-up Information for Bead Coupling
EDC
SulfoNHS
Activation
volume
0.5mg
0.5mg
0.5mg
2.5mg
2.5mg
2.5mg
5mg
5mg
100µl
100µl
100µl
100µl
100µl
500µl
500µl
500µl
Microspheres
1 million
2.5 million
5 million
10 million
12.5 million*
50 million
100 million
200 million
0.5mg
0.5mg
0.5mg
2.5mg
2.5mg
2.5mg
5mg
5mg
Coupling
Volume
S-NHS
(50mg/ml)
EDC
(5mg/ml)
0.5mL
0.5mL
0.5mL
1mL
1mL
2mL
2mL
2mL
10ul
10ul
10ul
10ul
10ul
10ul
100ul
100ul
10ul
10ul
10ul
10ul
10ul
10ul
100ul
100ul
~ # of 96well plates
using
2500
beads/well
4
10
20
40
50
200
400
800
*Standard 1ml vials of microspheres contain 12.5 million microspheres
Coupling reactions of 0.5 to 1ml are performed in 1.5 mL microcentrifuge tubes, and 2ml coupling
reactions are performed in 4mL microcentrifuge tubes or 15ml polypropylene centrifuge tubes.
The stability of your coupled beads will depend upon the antibody coupled to the beads.
Antibody coupled microspheres are stable for approximately one year at 4°C storage.
Important notes about EDC:
.
• EDC and Sulfo-NHS are both water sensitive and once resuspended can only be used
once
• Store EDC and Sulfo-NHS at -20 C in a dessicator
• Aliquot EDC into single-use portions as it is hygroscopic and will degrade if opened
multiple times. Alternatively, convenient single-use 10 mg vials are commercially
available.
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Recommended Protocol for Two-Step Carbodiimide Coupling of
Antibodies to MagPlex
 Magnetic Carboxylated Microspheres
Microspheres should be protected from prolonged exposure to light throughout this
procedure.
Table 3. Required Materials for Bead Coupling
Source
Catalog Number
MagPlex Microspheres
Capture Antibody
Luminex
Various
Vortex Mixer
Sonicator (mini)
Pipettors P10, P20, P100, P1000, 8
ch.
Hemacytometer (Bright Line) OR
automated cell/particle counter
Rotator
1.5 mL co-polymer microcentrifuge
tubes
OR 1.5 mL Eppendorf Protein LoBind
microcentrifuge tubes
2.0 mL screw-cap microcentrifuge
tubes for storage
10 µL pipette tip refills
250 µL pipette tip refills
1000 µL pipette tip refills
Dynal MPC®-S Magnetic Particle
Separator
EDC (1-ethyl-3[3dimethylaminopropyl] carbodiimide
hydrochloride)
Sulfo-NHS (Nhydroxysulfosuccinimide)
VWR
Cole Parmer
Rainin or other
Refer to Table 1
Various (see
references for
examples)
58816-121
08849-00
Various
VWR
15170-172
VWR
USA Scientific
56264-302
1415-2500
Eppendorf
Fisher
Fisher
022431081
13-698-794
05-669-8
Rainin
Rainin
Rainin
Invitrogen
GPS-10G
GPS-250
GPS-1000
120-20D
Pierce
77149 or 22980
Pierce
24510
Product
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Table 4. Recipe for 0.1 M NaH2PO4, pH 6.2 (Activation Buffer)* 250 mL
Reagent
Catalog Number
Sigma S3139
Final
Concentration
0.1 M NaH2PO4
Amount/
250 mL
3g
NaH2PO4 (Sodium phosphate
monobasic, anhydrous)
5 N NaOH
Fisher SS256-500
------
~ 40 drops
Filter Sterilize and store at 4°°C
*
Activation can be performed in 50 mM MES, pH 6.0-6.2 with similar results.
Table 5. Recipe for 0.05 M MES, pH 5.0 (Coupling Buffer)** 250 mL
Reagent
Catalog Number
MES (2[N-Morpholino] ethanesulfonic
acid)
5 N NaOH
Sigma M2933
Fisher SS256500
Final
Concentration
0.05 M MES
Amount/
250 mL
2.44 g
------
~ 5 drops
Filter Sterilize and store at 4°C
** Coupling can be performed in 100 mM MES, pH 6.0 with similar results. For some proteins, better
solubility and better coupling may be achieved at a higher coupling pH.
Table 6. Recipe for PBS, 0.1% BSA, 0.02% TWEEN-20, 0.05% Azide, pH 7.4
(PBS-TBN Blocking/Storage Buffer) 1000 mL
Reagent
PBS, pH 7.4
Catalog
Number
Sigma P3813
BSA
TWEEN® 20
Sigma A7888
Sigma P9416
Sodium Azide
Sigma S8032
Final
Concentration
138 mM NaCl
2.7 mM KCl
0.1% BSA
0.02% TWEEN®
20
0.05% Azide
Amount/
1L
1 packet
1g
0.2 mL
500 mg
Filter Sterilize and store at 4°C
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1. Resuspend the stock uncoupled microsphere suspension according to the
instructions described in the Product Information Sheet provided with your
microspheres.
2. Transfer 5.0 x 106 of the stock microspheres to a USA Scientific microcentrifuge
tube.
3. Place the tube into a magnetic separator and allow separation to occur for 60 to 120
seconds.
4. With the tube still positioned in the magnetic separator, remove the supernatant.
Take care not to disturb the microspheres.
5. Remove the tube from the magnetic separator and resuspend the microspheres in
100 µL dH2O by vortex and sonication for approximately 20 seconds.
6. Place the tube into a magnetic separator and allow separation to occur for 60 to 120
seconds.
7. With the tube still positioned in the magnetic separator, remove the supernatant.
Take care not to disturb the microspheres.
8. Remove the tube from the magnetic separator and resuspend the washed
microspheres in 80 µL 100 mM Monobasic Sodium Phosphate, pH 6.2 by vortex and
sonication for approximately 20 seconds.
9. Add 10 µL of 50 mg/mL Sulfo-NHS (diluted in dH20) to the microspheres and mix
gently by vortex.
10. Add 10 µL of 50 mg/mL EDC (diluted in dH20) to the microspheres and mix gently by
vortex.
11. Incubate for 20 minutes at room temperature with gentle mixing by vortex at 10
minute intervals.
12. Place the tube into a magnetic separator and allow separation to occur for 60 to 120
seconds.
13. With the tube still positioned in the magnetic separator, remove the supernatant.
Take care not to disturb the microspheres.
14. Remove the tube from the magnetic separator and resuspend the microspheres in
250 µL of 50 mM MES, pH 5.0 by vortex and sonication for approximately 20
seconds. See Technical Note 1.
15. Repeat steps 12. and 13. This is a total of two washes with 50 mM MES, pH 5.0.
16. Remove the tube from the magnetic separator and resuspend the activated and
washed microspheres in 100 µL of 50 mM MES, pH 5.0 by vortex and sonication for
approximately 20 seconds.
17. Add 125, 25, 5 or 1 µg protein to the resuspended microspheres. (Note: Luminex
recommends titration in the 1 to 125 µg range to determine the optimal amount of
protein per specific coupling reaction.)
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18. Bring total volume to 500 µL with 50 mM MES, pH 5.0.
19. Mix coupling reaction by vortex.
20. Incubate for 2 hours with mixing (by rotation) at room temperature.
21. Place the tube into a magnetic separator and allow separation to occur for 60 to 120
seconds.
22. With the tube still positioned in the magnetic separator, remove the supernatant.
Take care not to disturb the microspheres.
23. Remove the tube from the magnetic separator and resuspend the coupled
microspheres in 500 µL of PBS-TBN by vortex and sonication for approximately 20
seconds. See Technical Note 2.
24. Incubate for 30 minutes with mixing (by rotation) at room temperature. (Note:
Optional – perform this step when using the microspheres the same day.)
25. Place the tube into a magnetic separator and allow separation to occur for 60 to 120
seconds.
26. With the tube still positioned in the magnetic separator, remove the supernatant.
Take care not to disturb the microspheres.
27. Remove the tube from the magnetic separator and resuspend the microspheres in 1
mL of PBS-TBN by vortex and sonication for approximately 20 seconds. See
Technical Note 3.
28. Repeat steps 25. and 26. This is a total of two washes with 1 mL PBS-TBN.
29. Remove the tube from the magnetic separator and resuspend the coupled and
washed microspheres in 250-1000 µL of PBS-TBN.
30. Count the microsphere suspension by hemacytometer, Beckman Coulter Z2™
Coulter Counter®, Bio-Rad TC10™ Automated Cell Counter (see Figure 1),
Invitrogen Countess® Automated Cell Counter, or Millipore Scepter™ cell counter.
31. Calculation on Hemacytometer: Total microspheres = count (1 corner of 4 x 4
section) x (1 x 104) x (dilution factor) x (resuspension volume in mL)
32. Store coupled microspheres refrigerated at 2-8°C in the dark.
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Figure 1. Example bead count performed on a Bio-Rad TC10 Cell Counter
Technical Note 1: Coupling can be performed in 100 mM MES, pH 6.0 with similar
results. For some proteins, better solubility and better coupling may be achieved at a
higher coupling pH or in a different buffer. If your protein does not couple satisfactorily
under these recommendations, try PBS, pH 7.4 as an alternate coupling buffer.
Table 7. Recipe for PBS, pH 7.4 (Alternate Coupling Buffer)*** 1000 mL
Reagent
PBS, pH 7.4
Catalog
Number
Sigma P3813
Final
Concentration
138 mM NaCl
2.7 mM KCl
Amount/
1L
1 packet
Filter Sterilize and store at 4°C
*** An alternative coupling buffer for proteins that do not couple well in 50-100 mM MES, pH 5.0-6.0.
Technical Note 2: Either PBS-TBN (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide,
pH 7.4) or PBS-BN (PBS, 1% BSA, 0.05% Azide, pH 7.4) may be used as
Blocking/Storage Buffer.
Table 8. Recipe for PBS, 1% BSA, 0.05% Azide, pH 7.4
(PBS-BN Blocking/Storage Buffer) 1000 mL
Reagent
Catalog Number
PBS, 1% BSA, pH 7.4
Sigma P3688
Sodium Azide
Filter Sterilize and store at 4°C
Sigma S8032
Final
Concentration
138 mM NaCl
2.7 mM KCl
1% BSA
0.05% Azide
Amount/
1L
1 packet
500 mg
Technical Note 3: Either PBS-TBN or PBS, 0.05% Tween-20 may be used as Wash
Buffer.
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Confirmation of Coupling
Coupling should be confirmed before proceeding to choose the best detection and capture
antibodies. Confirmation is key to assay troubleshooting and is a good quality control check for
stability over time of your coupled beads. Examples of coupling confirmation can be found in de
Jager et al. (2003), Giavedoni (2005), and Lawson et al. (2010).
The general protocol for coupling confirmation is to combine your coupled beads and an antispecies IgG (e.g. anti-mouse IgG-PE) that is labeled with phycoerythrin. Typically, a two-fold
dilution series starting at 4 µg/ml and ending at 0.0625 µg/ml is sufficient for coupling
confirmation. Table 1 shows a typical assay plate setup for coupling confirmation.
Table 9. Example Plate Layout for Coupling Confirmation
1
2
3
Coupled Bead #1
Coupled Bead #2
Coupled Bead #3
PE Anti-Mouse antibody
(ug/ml)
PE Anti-Mouse antibody
(ug/ml)
PE Anti-Mouse antibody
(ug/ml)
A
4
4
4
B
2
2
2
C
1
1
1
D
0.5
0.5
0.5
E
0.25
0.25
0.25
F
0.125
0.125
0.125
G
0.0625
0.0625
0.0625
H
0
0
0
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What results should be expected from a successful antibody coupling reaction?
Antibody coupling is confirmed by testing the coupled microspheres with dilutions of a
phycoerythrin-labeled anti-species IgG detection antibody. Typical results are shown
below:
Figure 2. Coupling confirmation of well and poorly coupled microspheres
Coupling Confimation
30000
25000
MFI
20000
15000
10000
5000
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
PE-Antimouse Aby (ug/mL)
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Figure 3 shows coupling confirmation of an anti-IL-1 antibody coupled at different concentrations.
Ideally, you want a curve that saturates somewhere between 10,000 and 20,000 MFI. In this
case, you would proceed with either 5 or 25 µg per one million beads.
Figure 3. Coupling confirmation of Four Different Concentrations of IL-1 Antibody
Anti-IL1 Coupling Titration
25000
Median Fluorescence
20000
15000
0.2 ug
1 ug
5 ug
10000
25 ug
5000
0
0
1
2
3
4
5
6
7
8
9
Detection Antibody (ug/mL)
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In the next case (Figure 4), four different concentrations of an anti-IL-2 antibody coupled to four
different bead sets. This is quite different from the previous graph in Figure 3. The three lowest
concentrations (1, 5 and 25 µg) behave almost identically, so you would proceed with using 1µg
of anti-IL-2 per one million beads. There is no reason to use more antibody than necessary.
Using less capture antibody is one of the most significant cost savings features of using beads
over a traditional ELISA. In most situations, 3-10 µg per one million beads is optimal, but you
should titrate each antibody to determine the optimal concentration.
Figure 4. Coupling confirmation of Four Different Concentrations of IL-2 Antibody
Anti-IL2 Coupling Titration
25000
20000
0.2ug
Median Fluorescence
1 ug
5 ug
15000
25 ug
10000
5000
0
0
1
2
3
4
5
6
7
8
9
Detection Antibody (ug/mL)
Things to keep in mind for coupling confirmation:
•
•
•
•
•
•
The biggest loss of microspheres occurs from beads sticking to walls of microcentrifuge
tubes
Use only recommended USA Scientific or Eppendorf Protein low binding tubes
Do not remove too much supernatant during wash steps
The bead pellet is soft and can be disturbed during supernatant removal
Free amines will compete for carboxyl sites on the beads – do not use BSA or other
proteins in your antibody solution
Do not use any buffers containing glycine, TRIS or azide
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Recommended Protocol for Confirmation of Antibody Coupling
Microspheres should be protected from prolonged exposure to light throughout this
procedure.
Table 10. Required Materials for Antibody Coupling Confirmation
Product
Antibody-Coupled MagPlex Microspheres
Lab-Line Microtiter Plate Shaker
Phycoerythrin-Labeled Anti-Species IgG
Detection Antibody
96 well microplate aluminum sealing tape
(to reduce light exposure of samples in a
plate)
96-well round bottom polystyrene solid
plates
Luminex Magnetic Plate Separator
Vortex Mixer
Sonicator (mini)
Pipettors P10, P20, P100, P1000, 8 ch.
MAGPIX
Source
Catalog Number
User
Barnstead International
Various
N/A
4625
Various
Costar
6570
Costar
3789 (white)
3792 (black)
CN-0269-01
58816-121
08849-00
Various
MAGPIX-XPON4.1
Luminex
VWR
Cole Parmer
Rainin or other
Luminex
Table 11. Recipe for PBS, 1% BSA, pH 7.4 (Assay Buffer) 1000 mL
Reagent
Catalog Number
PBS, 1% BSA, pH 7.4
Sigma P3688
Final
Concentration
138 mM NaCl
2.7 mM KCl
1% BSA
Amount/
1L
1 packet
Filter Sterilize and store at 4°C
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1. Select the appropriate antibody-coupled microsphere sets.
2. Resuspend the microspheres by vortex and sonication for approximately 20
seconds.
3. Prepare a Working Microsphere Mixture by diluting the coupled microsphere stocks
to a final concentration of 100 microspheres of each set/µL in PBS-1% BSA. (Note:
50 µL of Working Microsphere Mixture is required for each reaction.) See Technical
Note 1.
4. Prepare two-fold serial dilutions of phycoerythrin-labeled anti-species IgG detection
antibody from 4 to 0.0625 µg/mL in PBS-1% BSA. (Note: 50 µL of diluted detection
antibody is required for each reaction.)
5. Aliquot 50 µL of the Working Microsphere Mixture into the appropriate wells of the
microplate.
6. Add 50 µL of the diluted detection antibody into the appropriate wells of the
microplate.
7. Mix the reactions gently by pipetting up and down several times with a multi-channel
pipettor.
8. Cover the microplate and incubate for 30 minutes at room temperature on a plate
shaker.
9. Place the plate into the magnetic separator and allow separation to occur for 60 to
120 seconds.
10. Use a multi-channel pipette to carefully aspirate the supernatant from each well.
Take care not to disturb the microspheres.
11. Wash each well twice with 100 µL of PBS-1% BSA and aspirate by vacuum
manifold.
12. Resuspend the microspheres in 100 µL of PBS-1% BSA by gently pipetting up and
down five times with a multi-channel pipettor.
13. Analyze 50-75 µL on the Luminex analyzer according to the system manual.
Technical Note 1: Either PBS-1% BSA or PBS-BN (PBS, 1% BSA, 0.05% Azide, pH
7.4) may be used as Assay Buffer.
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Selecting the Best Pairs of Antibodies
Once all antibodies available are coupled to microspheres, the best pairs should be selected by
performing single-plex assays utilizing each combination.
Capture and Detection Antibody Selection
To perform antibody pair selection, prepare a matrix to determine the best pair. Use one high
standard (10,000 pg/ml) as the analyte, as this will allow you to identify the best pairs.
Table 12. Example Matrix and Results for Selection of Antibody Pairs
Capture
Antibodies
Antibody A
Antibody B
Antibody C
Detection Antibodies
Antibody A Antibody B
Antibody C
N/A
3583
12321
14832
7920
N/A
8769
N/A
3281
In this example the combination of Antibody C for Detection and Antibody A for capture produced
the best results.
Assay Protocol/Performing the Assay
Start with the recommended protocol below and work from there. Many of the publications in the
references section have utilized the standard protocol with good results. In some cases you will
need to optimize the protocol. Start with the standard buffers in the protocol.
Outline of Capture Sandwich Protocol:
1. Add 50 ul of microspheres to 50 ul of sample
2. Incubation and wash
3. Add 50 ul of detection antibody to antigen bound microspheres
4. Incubation and Wash
5. Add 50 ul of SAPE to detection antibody labeled microspheres
6. Incubation and wash
If you are designing a multiplex assay, build each assay individually to roughly optimize as you
may need to optimize the multiplex later. Initial rough experiments allow for early assessment of
cross reactivity. Cross-reactivity studies are generally performed by running the assay in a
multiplex format but with only a single antigen added at high concentration (Bjerre et al. 2009;
Joannisson et al. 2006; Kellar et al. 2003).
Multiple parameters of the xMAP sandwich immunoassay can be optimized for the critical
parameters required for your particular assay. Detection antibody concentration can be titrated
for sensitivity (Bjerre et al. 2009). Incubation times can be adjusted to increase sensitivity or
reduce background (Bjerre et al. 2009; Dernfalk et al. 2007). In some cases the best sensitivity
may be obtained by running more than one smaller multiplex panel instead of one large multiplex
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(Bjerre et al. 2009). The amount of beads per well can also affect sensitivity. The range should
be 2000 to 5000 beads per well. A slight increase in sensitivity can be obtained by using fewer
beads, but do not use less than 2000 per well.
Pick a buffer for standards that closely mimics the sample composition. Dilute serum or plasma
1:5 (de Jager et al. 2005). Serum and other biological fluids are available from vendors such as
Bioreclamation (www.bioreclamation.com). Also, samples should be subjected to only a single
freeze-thaw cycle (de Jager et al. 2009), for maximum performance. Aliquot samples if you will
analyze over a period of time. Examples of biological fluids used in xMAP assays include tissue
culture media (de Jager et al. 2003), serum/plasma (Bjerre et al. 2009; de Jager et al. 2005;
Dernfalk et al. 2007; Prabhakar et al. 2002; Ray et al. 2005; Wagner et al. 2009; Wagner et al.
2010), synovial fluid (de Jager et al. 2005), and dried blood spots (Skogstrand et al. 2005).
Validation of immunoassays is described in detail in multiple publications (Dernfalk et al. 2007;
Bjerre et al. 2009; de Jager et al. 2009; Kellar et al. 2005; Ray et al. 2005; Rizzi et al. 2010; Wood
et al. 2011). The parameters selected and optimized will be determined by your particular
research needs. The National Institutes of Health Chemical Genomics Center is a good
resource for general immunoassay validation (http://www.ncgc.nih.gov/guidance/section10.html).
There are also publications that give guidelines for validating assays (Lee et al. 2006; Lee et al.
2009).
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Recommended Protocol for Washed Capture Sandwich Immunoassay
Using Magnetic Microspheres
Microspheres should be protected from prolonged exposure to light throughout this
procedure.
Table 13. Required Materials for a Washed Capture Sandwich Immunoassay
Product
MagPlex Microspheres coupled with
capture antibody
Biotinylated Detection antibody
Recombinant Protein Standards
Streptavidin-R-phycoerythrin (1 mg/ml)
OR PhycoLink® Streptavidin-Rphycoerythrin (≥1 mg)
OR PhycoLink® Streptavidin-Rphycoerythrin (≥1 mg)
Source
User
Catalog Number
NA
Various
Various
Molecular Probes
Prozyme
Various
Various
S-866
PJ31S
Prozyme
OR Streptavidin-Phycoerythrin
Protos Immunoresearch
PJRS20 or PJRS14
(next generation of
PJ31S)
683
96 well microplate aluminum sealing tape
(to reduce light exposure of samples in a
plate)
96-well round bottom polystyrene solid
plates
Luminex Magnetic Plate Separator
Costar
6570
Costar
Luminex
3789 (white)
3792 (black)
CN-0269-01
Lab-Line Microtiter Plate Shaker
Barnstead International
4625
Vortex Mixer
VWR
58816-121
Sonicator (mini)
Cole Parmer
08849-00
Pipettors P10, P20, P100, P1000, 8 ch.
Rainin or other
Various
MAGPIX
Luminex
MAGPIX-XPON4.1
Table 14. Recipe for PBS, 1% BSA, pH 7.4 (Assay Buffer) 1000 mL
Reagent
Catalog Number
PBS, 1% BSA, pH 7.4
Sigma P3688
Final
Concentration
138 mM NaCl
2.7 mM KCl
1% BSA
Amount/
1L
1 packet
Filter Sterilize and store at 4°C
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Table 15. Recipe for PBS, 0.05% TWEEN-20, pH 7.4 (Wash Buffer) 1000 mL
Reagent
Catalog Number
PBS, 0.05% TWEEN® 20, pH 7.4
Sigma P3563
Final
Concentration
138 mM NaCl
2.7 mM KCl
0.05% TWEEN
Amount/
1L
1 packet
Filter Sterilize and store at 4°C
1. Select the appropriate antibody-coupled microsphere sets.
2. Resuspend the microspheres by vortex and sonication for approximately 20 seconds.
3. Prepare a Working Microsphere Mixture by diluting the coupled microsphere stocks to
a final concentration of 100 microspheres of each set/µL in PBS-1% BSA. (Note: 50
µL of Working Microsphere Mixture is required for each reaction.) See Technical
Note 1.
4. Aliquot 50 µL of the Working Microsphere Mixture into the appropriate wells of a
round-bottom plate.
5. Add 50 µL of PBS-1% BSA to each background well.
6. Add 50 µL of standard or sample to the appropriate wells.
7. Mix the reactions gently by pipetting up and down several times with a multi-channel
pipettor.
8. Cover the plate and incubate for 30 minutes at room temperature on a plate shaker
set to approximately 800 rpm. Note: The range for incubation can be from 15
minute to overnight.
9. Place the plate into the magnetic separator and allow separation to occur for 60 to
120 seconds. See Technical Note 2.
10. Use a multi-channel pipette to carefully aspirate the supernatant from each well.
Take care not to disturb the microspheres.
11. Leave the plate in the magnetic separator for the following wash steps:
a. Add 100 µL PBS-1% BSA to each well.
b. Use a multi-channel pipette to carefully aspirate the supernatant from each well.
Take care not to disturb the microspheres.
c. Repeat steps a. and b. above.
12. Remove the plate from the magnetic separator and resuspend the microspheres in
50 µL of PBS-1% BSA by gently pipetting up and down several times using a multichannel pipettor.
13. Dilute the biotinylated detection antibody to 4 µg/mL in PBS-1% BSA. (Note: 50 µL of
diluted detection antibody is required for each reaction.) See Technical Note 3.
14. Add 50 µL of the diluted detection antibody to each well.
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15. Mix the reactions gently by pipetting up and down several times with a multi-channel
pipettor.
16. Cover the plate and incubate for 30 minutes at room temperature on a plate shaker
set to approximately 800 rpm. Note: The range for incubation can be from 15
minutes to several hours).
17. Place the plate into the magnetic separator and allow separation to occur for 60 to
120 seconds.
18. Use a multi-channel pipette to carefully aspirate the supernatant from each well.
Take care not to disturb the microspheres.
19. Leave the plate in the magnetic separator for the following wash steps:
a. Add 100 µL PBS-1% BSA to each well.
b. Use a multi-channel pipette to carefully aspirate the supernatant from each well.
Take care not to disturb the microspheres.
c. Repeat steps a. and b. above.
20. Remove the plate from the magnetic separator and resuspend the microspheres in
50 µL of PBS-1% BSA by gently pipetting up and down several times with a multichannel pipettor.
21. Dilute streptavidin-R-phycoerythrin reporter to 4 µg/mL in PBS-1% BSA. (Note: 50
µL of diluted streptavidin-R-phycoerythrin is required for each reaction.) See
Technical Note 3.
22. Add 50 µL of the diluted streptavidin-R-phycoerythrin to each well.
23. Mix the reactions gently by pipetting up and down several times with a multi-channel
pipettor.
24. Cover the plate and incubate for 30 minutes at room temperature on a plate shaker
set to approximately 800 rpm.
25. Place the plate into the magnetic separator and allow separation to occur for 60 to
120 seconds.
26. Use a multi-channel pipette to carefully aspirate the supernatant from each well.
Take care not to disturb the microspheres.
27. Leave the plate in the magnetic separator for the following wash steps:
a. Add 100 µL PBS-1% BSA to each well.
b. Use a multi-channel pipette to carefully aspirate the supernatant from each well.
Take care not to disturb the microspheres.
c. Repeat steps a. and b. above.
28. Remove the plate from the magnetic separator and resuspend the microspheres in
100 µL of PBS-1% BSA by gently pipetting up and down several times with a multichannel pipettor.
29. Analyze 50-75 µL on the Luminex analyzer according to the system manual.
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Technical Note 1: Either PBS-1% BSA or PBS-BN (PBS, 1% BSA, 0.05% Azide, pH
7.4) may be used as Assay Buffer.
Technical Note 2: For a list of magnetic separator plates, see Recommended Materials
for Magnetic Microspheres. Optimal separation time may vary with the type of separator
used.
Technical Note 3: Concentrations should be optimized for specific reagents, assay
conditions, level of multiplexing, etc. in use. Detection antibody concentrations can
range from 1-4 µg/mL. Streptavidin-PE concentrations can range from 1-4 µg/mL. Start
with the concentrations in the protocol.
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References
References with Customer-Developed xMAP Assays
Bjerre, M., Hansen, T. K., Flyvbjerg, A., and Tonnesen, E. Simultaneous detection of porcine
cytokines by multiplex analysis: Development of magnetic bioplex assay. Vet Immunol
Immunopathol 2009; 130:53-8.
Carson, R. T. and Vignali, D. A. A. Simultaneous quantitation of 15 cytokines using a
multiplexed flow cytometric assay. J Immunol Methods 1999; 227:41-52.
de Jager, W., te Velthuis, H., Prakken, B. J., Kuis, W., and Rijkers, G. T. Simultaneous
detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear
cells. Clin Diagn Lab Immunol 2003; 10:133-9.
de Jager, W., Prakken, B. J., Bijlsma, J. W. J., Kuis, W., and Rijkers, G. T. Improved multiplex
immunoassay performance in human plasma and synovial fluid following removal of interfering
heterophilic antibodies. J Immunol Methods 2005; 300:124-35.
de Jager, W. and Rijkers, G. T. Solid-phase and bead-based cytokine immunoassay: A
comparison. Methods 2006; 38:294-303.
de Jager, W., Bourcier, K., Rijkers, G. T., Prakken, B. J., and Seyfert-Margolis, V. Prerequisites
for cytokine measurements in clinical trials with multiplex immunoassays. BMC Immunology
2009;10:U1-U11.
de Jager W, Prakken B, Rijkers GT. Cytokine Multiplex Immunoassay: Methodology and
(Clinical) Applications. T Cell Protocols. 2008:119-33.
Dernfalk, J., Waller, K. P., and Johannisson, A. Commercially available antibodies to human
tumour necrosis factor-α tested for cross-reactivity with ovine and bovine tumour necrosis
factor-α using flow cytometric assays. Acta Vet Scand 2004; 45:99-107.
Dernfalk, J., Waller, K. P., and Johannisson, A. The xMAP™ technique can be used for
detection of the inflammatory cytokines IL-1β, IL-6 and TNF-α bovine samples. Vet Immunol
Immunopathol 2007; 118:40-9.
Faucher, S., Crawley, A. M., Decker, W., Sherring, A., Bogdanovic, D., Ding, T., Bergeron, M.,
Angel, J. B., and Sandstrom, P. Development of a quantitative bead capture assay for soluble
IL-7 receptor alpha in human plasma. PLoS One 2009; 4:U66-U71.
Giavedoni, L. D. Simultaneous detection of multiple cytokines and chemokines from nonhuman
primates using Luminex technology. J Immunol Methods 2005; 301:89-101.
Haasnoot, W. and du Pre, J. G. Luminex-based triplex immunoassay for the simultaneous
detection of soy, pea, and soluble wheat proteins in milk powder. J Agric Food Chem 2007;
55:3771-7.
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Johannisson, A., Jonasson, R., Dernfalk, J., and Jensen-Waern, M. Simultaneous detection of
porcine proinflammatory cytokines using multiplex flow cytometry by the xMAP™ technology.
Cytometry Part A 2006; 69A:391-5.
Kellar, K. L., Kalwar, R. R., Dubois, K. A., Crouse, D., Chafin, W. D., and Kane, B.-E.
Multiplexed fluorescent bead-based immunoassays for quantitation of human cytokines in
serum and culture supernatants. Cytometry 2001; 45:27-36.
Kellar, K. L. and Douglass, J. P. Multiplexed microsphere-based flow cytometric immunoassays
for human cytokines. J Immunol Methods 2003; 279:277-85.
Kofoed, K., Schneider, U. V., Scheel, T., Andersen, O., and Eugen-Olsen, J. Development and
validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology. Clin
Chem 2006; 52:1284-93.
Lawson, S., Lunney, J., Zuckermann, F., Osorio, F., Nelson, E., Welbon, C., Clement, T., Fang,
Y., Wong, S., Kulas, K., and Christopher-Hennings, J. Development of an 8-plex Luminex
assay to detect swine cytokines for vaccine development: Assessment of immunity after
porcine reproductive and respiratory syndrome virus (PRRSV) vaccination. Vaccine 2010;
28:5356-64.
Prabhakar, U., Eirikis, E. and Davis, H. M. Simultaneous quantification of proinflammatory
cytokines in human plasma using the LabMAP™ assay. J Immunol Methods 2002; 260:207-18.
Ray, C. A., Bowsher, R. R., Smith, W. C., Devanarayan, V., Willey, M. B., Brandt, J. T., and
Dean, R. A. Development, validation, and implementation of a multiplex immunoassay for the
simultaneous determination of five cytokines in human serum. J Pharm Biomed Anal 2005;
36:1037-44.
Rizzi, G., Zhang, Y. J., Latek, R., Weiner, R., and Rhyne, P. W. Characterization and
development of a Luminex®-based assay for the detection of human IL-23. Bioanalysis 2010;
2:1561-72.
Skogstrand, K., Thorsen, P., Norgaard-Pedersen, B., Schendel, D. E., Sorensen, L. C., and
Hougaard, D. M. Simultaneous measurement of 25 inflammatory markers and neurotrophinsin
neonatal dried blood spots by immunoassay with xMAP technology. Clin Chem 2005; 51:185466.
Wagner, B. and Freer, H. Development of a bead-based multiplex assay for simultaneous
quantification of cytokines in horses. Vet Immunol Immunopathol 2009; 127:242-8.
Wagner, B., Freer, H. and Rollins, A. A fluorescent bead-based multiplex assay for the
simultaneous detection of antibodies to B. burgdorferi outer surface proteins in canine serum.
Vet Immunol Immunopathol 2011, doi:10.1016/j.vetimm,2010.12.003
Wood, B., O’Halloran, K. and VandeWoude, S. Development and validation of a multiplex
microsphere-based assay for detection of domestic cat (Felis catus) cytokines. Clin Vaccine
Immunol 2011, doi:10.1128/CVI.00289-10
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Assay Validation References
Lee, J. W., Devanaryan, V., Barrett, Y. C., Weiner, R., Allinson, J., Fountain, S., Keller, S.,
Weinryb, I., Green, M., Duan, L., Rogers, J. A., Millham, R., O’Brien, P. J., Sailstad, J., Khan,
M., Ray, C., and Wagner, J. A. Fit-for-Purpose Method Development and Validation for
Successful Biomarker Measurement. Pharmaceutical Research 2006; 23:312-28.
Lee, J. W. and Hall, M. Method validation of protein biomarkers in support of drug development
or clinical diagnosis/prognosis. J Chromatogr B Analyt Technol Biomed Life Sci 2009;
877:1259-71.
NIH Chemical Genomics Center. Immunoassay Methods.
http://www.ncgc.nih.gov/guidance/section10.html
ELISA References
Engvall, E. The ELISA, Enzyme-Linked Immunosorbent Assay. Clin Chem 2010; 56:319-20.
Lequin, R. M. Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA).
Clin Chem 2005; 51:2415-8.
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Appendix – Automated Washing Option Using Bio-Tek ELx405
Microplate Washer
BioTek ELx405 Microplate Washer – Keypad Programming Instructions
Luminex Mag Bead Assay Wash 96-well Protocol – 2 Cycle Wash with No Final Dispense
Notes:
For use with BioTek-provided magnet P/N 7103016 (Dexter LifeSep 96F technology) and
round-bottom 96-well microplate
Program delays for 60 seconds with 96-well plate on magnet prior to an aspiration
followed by a two cycle wash with 60 second delays after each dispense and ends
with a final aspiration
Instructions created 1/6/2011 by J. Greene, BioTek Instruments with settings determined
by H. Baker, Luminex Corporation
1.) Create SOAK program.
DEFINE → CREATE → MORE → SOAK → name (Ex. SOAK60) note: to get letters,
scroll through with OPTIONS button → ENTER.
SOAK DURATION: 60 SEC → ENTER.
SHAKE BEFORE SOAK?: YES → ENTER.
SHAKE DURATION: 1 SEC → ENTER.
SHAKE INTENSITY: 1 → ENTER.
OK TO SAVE PROGRAM? YES → ENTER.
2.) Create ASPIRATION program.
DEFINE → CREATE → MORE → ASPIR → name (Ex. ASPIR) note: to get letters, scroll
through with OPTIONS button → ENTER.
PLATE TYPE: 96 → ENTER.
ASPIRATE HEIGHT: 58 → ENTER.
HORIZONTAL ASPR POS: -50 → ENTER.
HORIZ Y ASPR POS: 0 → ENTER.
ASPIRATION RATE: 1 → ENTER.
ASPIRATE DELAY: 0 → ENTER.
CROSSWISE ASPIR: NO → ENTER.
OK TO SAVE PROGRAM? YES → ENTER.
3.) Create WASH program.
DEFINE → CREATE → WASH → name (Ex. WASH2X) note: to get letters scroll through
with OPTIONS button → ENTER.
SELECT REAGENT BOTTLE: A → ENTER.
PLATE TYPE: 96 → ENTER.
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METHOD → NUMBER OF CYCLES: 2 → ENTER → SOAK/SHAKE: YES → ENTER →
SOAK DURATION: 60 SEC → ENTER → SHAKE BEFORE SOAK: NO → ENTER →
PRIME AFTER SOAK: NO → ENTER.
DISP →
DISPENSE VOLUME: 100 UL/WELL → ENTER.
DISPENSE FLOW RATE: 9 → ENTER.
DISPENSE HEIGHT: 128 → ENTER.
HORIZONTAL DISP POS: 0 → ENTER.
HORIZ Y DISP POS: 0 → ENTER.
DISABLE ASPIRATE? NO → ENTER.
BOTTOM WASH FIRST?: NO → ENTER.
PRIME BEFORE START?: NO → ENTER.
ASPIR →
ASPIRATE HEIGHT: 58 → ENTER.
HORIZONTAL ASPR POS: -50 → ENTER.
HORIZ Y ASPR POS: 0 → ENTER.
ASPIRATION RATE: 7 → ENTER.
ASPIRATE DELAY: 0 → ENTER.
CROSSWISE ASPIR: NO → ENTER.
FINAL ASPIRATION: YES → ENTER.
FINAL ASPIR DELAY: 0 → ENTER.
MAIN MENU → OK TO SAVE PROGRAM?: YES → ENTER.
4.) Create LINK program.
DEFINE → CREATE → MORE → LINK → name (Ex. LUMINEX) note: to get letters,
scroll through with OPTIONS button → ENTER → MORE → SOAK (scroll through using
OPTIONS button to find SOAK program you made in step # 1) → ENTER → MORE →
ASPIR (scroll through using OPTIONS button to find ASPIR program you made in step #
2) → ENTER → WASH (scroll through using OPTIONS button to find WASH program
you made in step # 3) → ENTER.
MAIN MENU → OK TO SAVE PROGRAM?: YES → ENTER.
5.) Run LINK program.
•
•
After creating LINK program, this is what you will actually run when washing plates.
RUN → MORE → LINK (scroll through with OPTIONS button to find LINK program
you made in step # 4) → ENTER.
6.) Basic maintenance.
•
•
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Before each use:
o RUN → PRIME (using your wash buffer) → scroll through using OPTIONS to
PRIME 200 → ENTER.
End of the day:
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o
o
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Switch to bottle containing rinse liquid (ex. deionized water).
MAINT → scroll through using OPTIONS to OVERNIGHT LOOP → ENTER.
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