ab138914 –
Polyethylene Glycol
(PEG) ELISA Kit
Instructions for Use
For the competitive quantitative measurement of PEGylated molecules
in plasma, serum and cell culture media.
This product is for research use only and is not intended for diagnostic
use.
Version 2 Last Updated 4 December 2014
Table of Contents
INTRODUCTION
1.
BACKGROUND
2
2.
4
ASSAY SUMMARY
GENERAL INFORMATION
3.
PRECAUTIONS
5
4.
STORAGE AND STABILITY
5
5.
MATERIALS SUPPLIED
5
6.
MATERIALS REQUIRED, NOT SUPPLIED
6
7.
LIMITATIONS
6
8.
TECHNICAL HINTS
7
ASSAY PREPARATION
9.
REAGENT PREPARATION
8
10.
STANDARD PREPARATIONS
9
11.
PLATE PREPARATION
13
ASSAY PROCEDURE
12. ASSAY PROCEDURE
14
DATA ANALYSIS
13. CALCULATIONS
15
14.
TYPICAL DATA
16
15.
ASSAY SENSITIVITY
20
16.
ASSAY SPECIFICITY
20
RESOURCES
17. TROUBLESHOOTING
21
18.
23
NOTES
Discover more at www.abcam.com
1
INTRODUCTION
1. BACKGROUND
Abcam’s Polyethylene Glycol (PEG) RabMab® in vitro ELISA (EnzymeLinked Immunosorbent Assay) kit is designed for accurate competitive
quantitative measurement of PEGylated molecules in plasma, serum
and cell culture media.
Abcam’s Polyethylene Glycol (PEG) RabMab® ELISA Kit operates on
the basis of competition between enzyme HRP conjugated PEG and
PEG labeled molecules for a limited number of binding sites on the
surface of 96-wells coated with anti-PEG RabMab® antibody. The
extent of color development resulting from interaction between HRP
and the substrate TMB is inversely proportional to the amount of
PEGylated molecules in the sample. For example, the absence of
PEGylated molecules in the sample will result in a bright blue color,
whereas the presence of PEGylated molecules will result in decreased
or no color development.
Polyethylene glycol (PEG) is an O-CH2-CH2 polymer, which is watersoluble, nontoxic, nonantigenic, and biocompatible. Covalent
conjugation of PEG to therapeutic proteins increases the in vivo
stability by protecting the protein from degradation, masking its
immunogenic sites and reducing clearance. Typically, PEGylation uses
nonspecific reactions with nucleophilic residues and produces mixtures
of PEGylated positional isomers. Qualitative and quantitative analysis
of PEGylated molecules is important for both drug development and
clinical application. This kit is developed to determine levels of
PEGylated molecules in samples such as serum, plasma or cell culture
medium via ELISA.
Users must PEGylate their interested molecules. For pharmaco kinetic
experiments, the PEGylated molecules will be used to construct
standard curves.
Discover more at www.abcam.com
2
INTRODUCTION
The use of this kit requires the end user to have at least 250 ng of
PEGylated compound of interest to use as reference standard. The
included mPEG-BSA is a reference sample only.
2. ASSAY SUMMARY
Remove appropriate number of
RabMab® antibody coated well
strips. Equilibrate all reagents to
room temperature. Prepare all
the reagents, samples, and
standards as instructed.
Mix 1X PEG-HRP with the
standard series, test samples or
controls. Add to appropriate
wells.
Incubate
at
room
temperature.
Aspirate and wash each well.
Add substrate solutions. When
color develops add the Stop
Solution. Immediately begin
recording
the
color
development. The presence of
PEGylated molecules in the
Discover more at www.abcam.com
3
INTRODUCTION
sample will result in decreased or no color development.
Discover more at www.abcam.com
4
GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.
All kit components have been formulated and quality control tested to
function successfully as a kit. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
Store kit at +2-8ºC immediately upon receipt.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in the Reagent Preparation section.
5. MATERIALS SUPPLIED
Amount
Storage
Condition
(Before
Preparation)
1 x 96 well plate
+2-8°C
1 x 200 ng
+ 2-8°C
PEG conjugated HRP (60X)
1 x 150 µL
+2-8°C
Antigen/Antibody Diluent Buffer (1X)
1 x 20 mL
+ 2-8°C
ELISA Washing Buffer (10X)
1 x 12 mL
+2-8°C
TMB A Substrate Solution (1X)
1 x 7 mL
+ 2-8°C
TMB B Substrate Solution (1X)
1 x 7 mL
+2-8°C
Stop Solution (1X)
1 x 11 mL
+ 2-8°C
Item
Dry Plate w/ stripwells (12 x 8 well
RabMab® antibody coated strips)
Reference Standard (PEG-BSA)
Discover more at www.abcam.com
5
GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

Deionized water

PEGylated sample of interest (at least 250 ng to use as a
standard)

Pipettors and pipette tips of various sizes

Rotating shaker

Microtiter plate reader
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures.

Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.
Discover more at www.abcam.com
6
GENERAL INFORMATION
8. TECHNICAL HINTS

Samples generating values higher than the highest standard
should be further diluted in the appropriate sample dilution buffers.

Avoid foaming
components.

Avoid cross contamination of samples or reagents by changing tips
between sample, standard and reagent additions.

Ensure plates are properly sealed or covered during incubation
steps.

Complete removal of all solutions and buffers during wash steps.

This kit is sold based on number of tests. A ‘test’ simply
refers to a single assay well. The number of wells that contain
sample, control or standard will vary by product. Review the
protocol completely to confirm this kit meets your
requirements. Please contact our Technical Support staff with
any questions.
or
bubbles
Discover more at www.abcam.com
when
mixing
or
reconstituting
7
ASSAY PREPARATION
9. REAGENT PREPARATION

Equilibrate all reagents and samples to room temperature (1825°C) prior to use.

Store all buffers and reagents at 4˚C when not in use.
9.1
1X PEG conjugated HRP
Equilibrate PEG conjugated HRP (60X) to room temperature
before diluting to 1X with Antigen/Antibody Diluent Buffer to
1X.
9.2
1X ELISA Washing Buffer
Equilibrate ELISA washing buffer (10X) to room temperature
before diluting to 1X with deionized water.
Discover more at www.abcam.com
8
ASSAY PREPARATION
10. STANDARD PREPARATIONS

Prepare serially diluted standards immediately prior to use.
Always prepare a fresh set of standards for every use.
10.1 PEG - BSA Reference Standard Dilution
10.1.1 Label seven tubes with BSA reference Standards
#1- 7.
10.1.2 Reconstitute lyophilized BSA reference with 1 mL of
distilled water. Leave the reconstituted standard at
room temperature for at least 20 minutes and mix
gently. For PEG-BSA, this reconstitution produces a
stock solution of 200 ng/mL. Transfer 350 μL of this
stock solution to tube #1 to create PEG-BSA
Standard #1.
10.1.3 Prepare PEG-BSA Standard #2 by adding 75 μL
distilled water into tube #2 then transferring 225 μL
from PEG-BSA Standard #1 to tube #2.
Mix
thoroughly and gently.
10.1.4 Prepare PEG-BSA Standard #3 by adding 67 μL
distilled water into tube #3 then transferring 133 μL
from PEG-BSA Standard #2 to tube #3. Mix
thoroughly and gently.
10.1.5 Using the table below as a guide to create PEG-BSA
Standards #4 through #6.
10.1.6 PEG-BSA Standard #7 contains no protein and is
the Blank control.
Discover more at www.abcam.com
9
ASSAY PREPARATION
PEGBSA
Standard
#
1
2
3
4
5
6
7 (Blank)
Sample to
Dilute
Standard #1
Standard #2
Standard #3
Standard #4
Standard #5
N/A
Volume
of
Diluent
(µL)
See Step 10.1.2
225
75
133
67
100
100
40
160
40
160
NA
200
Volume to
Dilute
(µL)
Discover more at www.abcam.com
Starting
Conc.
(ng/mL)
Final
Conc.
(ng/mL)
200
150
100
50
10
0
150
100
50
10
2
0
10
ASSAY PREPARATION

For the PEGylated sample of interest, make a series dilution
using either a 2, 3 or 4-fold dilution depending on number of
data points desired. Start with between 2,000 and 3,000 ng/mL
of PEGylated sample and dilute down to about 2 ng/mL.

Example below with PEG-Mouse-IgG as the PEGylated sample
of interest, using a 4-fold dilution from a stock solution at
2,500 ng/mL.
10.2 PEGylated Sample Standard Dilution
10.2.1 Label seven tubes with PEG-Mouse-IgG Standards
#1 - 7.
10.2.2 Reconstitute / dilute PEG-Mouse-IgG with distilled
water to produce a stock solution of 2,500 ng/mL.
Transfer 200 μL of this stock solution to tube #1 to
create PEG-Mouse-IgG Standard #1.
10.2.3 Add 150 μL distilled water into tube #2 - 6 and
150 μL distilled water into tube #7.
10.2.4 Prepare PEG-Mouse-IgG Standard #2 by
transferring
50
μL
from
PEG-Mouse-IgG
Standard #1 to tube #2. Mix thoroughly and gently.
10.2.5 Prepare PEG-Mouse-IgG Standard #3 by
transferring 50 μL from PEG-Mouse-IgG Standard
#2 to tube #3. Mix thoroughly and gently.
10.2.6 Using the table below as a guide, repeat for tubes
#4 through #6.
10.2.7 Sample Standard #7 contains no protein and is the
Blank control.
Discover more at www.abcam.com
11
ASSAY PREPARATION
PEGMouseIgG
Standard
#
1
2
3
4
5
6
7 (Blank)
Sample to
Dilute
Standard #1
Standard #2
Standard #3
Standard #4
Standard #5
N/A
Volume to
Dilute
(µL)
Volume
of
Diluent
(µL)
See Step 10.2.2
50
150
50
150
50
150
50
150
50
150
N/A
150
Discover more at www.abcam.com
Starting
Conc.
(ng/mL)
Final
Conc.
(ng/mL)
2,500
625
157
39
9.8
0
625
157
39
9.8
2.4
0
12
ASSAY PREPARATION
11. PLATE PREPARATION

The 96 well plate strips included with this kit are supplied ready to
use. It is not necessary to rinse the plate prior to adding reagents.

For each assay performed, a minimum of 2 wells must be used as
blanks, omitting primary antibody from well additions.

For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).

Well effects have not been observed with this assay.

Bring stripped microtiter plate to room temperature. Keep
appropriate numbers of strips for an experiment and remove extra
strips from microtiter plate by evenly pushing the bottoms of the
microwell strips.

Replace unrequired strips immediately into the bag, seal and store
at 4°C.
Discover more at www.abcam.com
13
ASSAY PROCEDURE
12. ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to room
temperature prior to use.

Prepare all reagents, working standards and samples as
directed.

It is recommended to assay all standards, controls and
samples in duplicate.
12.1 Mix 25 µL of PEG-HRP (diluted with 1X Antigen/Antibody
Diluent Buffer to 1X) with 25 µL of the standard series (i.e.
PEG-BSA, PEG-IgG), test samples or control.
12.2 Add the 50 µL mixture to each well. Incubate for 45 min at
room temp on a shaker.
12.3 Aspirate each well and wash with 250 µL 1X Wash Buffer.
Repeat 3 times.
12.4 Combine TMB A and TMB B (1:1) Add 100 µL of combined
substrate solution to each well.
Note: Volume of each TMB substrate needed = 50 μL x (# of
wells +1)
12.5 Cover to protect from light and incubate at room temperature
for 15 minutes.
12.6 Add 100 µL of stop solution to each well and tap plate to
ensure thorough mixing.
12.7 Determine the optical density at 450 nm using a microplate
reader within 30 minutes.
Discover more at www.abcam.com
14
DATA ANALYSIS
13. CALCULATIONS
Calculate the average absorbance values (A450) for each set of
standards (or references) and samples. Construct a standard curve by
setting concentration of each point in the standard serial dilution in
ng/mL along the X axis and the mean absorbance obtained for each
standard point as Y axis. Create a curve for the plotted standard
dilution series using a power trend line. The curve will generate the
equation: y=Ax-B and also generate a R2 value.
Using the mean absorbance value for each sample, determine the
corresponding concentration of sample in ng/mL (x) from the equation:
x=10^((log A – log y) / B).
14. TYPICAL DATA
TYPICAL STANDARD CURVE – Data provided for demonstration
purposes only. A new standard curve must be generated for each
assay performed.
Discover more at www.abcam.com
15
DATA ANALYSIS
PEG-MouseIgG ng/mL
2,500
O.D 450 nm
0.076
625
0.097
157
39
9.8
2.4
0.250
0.455
0.571
0.631
Figure 1. Pharmacokinetic data with PEG ELISA Kit (ab138914). Using the
fitted curve (y = 1.11x-0.34), the IgG concentration (x) for each available OD
(y) can be calculated as: 10^((log(1.11)-log(y))/0.34)
Discover more at www.abcam.com
16
DATA ANALYSIS
Figure 2. Pharmacokinetic data with PEG ELISA Kit (ab138914).
Plot of PEGylated IgG [ng/mL] concentration over time (h). Using four different
rat models, also tested negative controls.
Figure 3. Pharmacokinetic data with PEG ELISA Kit (ab138914). Plot of
PEGylated IgG (ng/mL) concentration over time (hours) in a rat model.
Compared against negative control.
Discover more at www.abcam.com
17
DATA ANALYSIS
Figure 4. Sample data with PEG ELISA Kit (ab138914). Plot of mIgG-PEG
concentration [ng/mL] detection over time (h).
Figure
5.
Sample
data
with
PEG
ELISA
Kit
Competition between PEGylated molecules and HRP-mPEG
Discover more at www.abcam.com
(ab138914).
18
DATA ANALYSIS
RECOVERY – (The recovery of BSA-PEG spiked to Human serum,
plasma, and cell culture medium was evaluated).
Serum (n=6)
Average %
Recovery
113
Plasma (n=6)
100
85-114
Cell Culture Media (n=6)
106
85-117
Sample Type
Range (%)
90-122
PRECISION–(Intra-Assay) Cell Culture Media
n=
Mean (ng/mL)
SD
%CV
1
20
40.42
6.31
15.62
2
20
20.96
3.44
16.42
3
20
60.29
5.12
8.49
PRECISION–(Intra-Assay) Serum
n=
Mean (ng/mL)
SD
%CV
1
20
51.83
10.2
19.7
2
20
24.02
3.4
14.3
3
20
68.81
3.33
4.85
PRECISION–(Inter-Assay) Cell Culture Media
n=
Mean (ng/mL)
SD
%CV
1
20
41.46
3.99
9.67
2
20
24.84
2.55
10.28
3
20
57.96
8.31
14.34
PRECISION–(Inter-Assay) Serum
n=
Mean (ng/mL)
SD
%CV
1
20
46.67
4.14
8.87
Discover more at www.abcam.com
2
20
31.36
5.34
17.01
3
20
61.24
11.14
18.19
19
DATA ANALYSIS
15. ASSAY SENSITIVITY
For mouse IgG, 25 ng/mL of mPEG-mouse-IgG competes against
mPEG-HRP 50% efficiently.
16. ASSAY SPECIFICITY
Monomethoxy PEG (mPEG), with the molecular weight about 5 kDa, is
used to immunize rabbits to generate anti-PEG antibody and modify
BSA and mouse-IgG in our experiments. Rabbit monoclonal RabMab®
Anti-PEG specifically recognizes the methoxy group of mPEG.
Discover more at www.abcam.com
20
RESOURCES
17. TROUBLESHOOTING
Problem
Cause
Solution
Poor standard
curve
Improper standard
dilution
Confirm dilutions made
correctly
Standard improperly
reconstituted (if
applicable)
Briefly spin vial before
opening; thoroughly
resuspend powder (if
applicable)
Standard degraded
Store sample as
recommended
Curve doesn't fit scale
Try plotting using different
scale
Incubation time too short
Try overnight incubation at
4 °C
Target present below
detection limits of assay
Decrease dilution factor;
concentrate samples
Precipitate can form in
wells upon substrate
addition when
concentration of target is
too high
Increase dilution factor of
sample
Using incompatible
sample type (e.g. serum
vs. cell extract)
Detection may be reduced
or absent in untested
sample types
Sample prepared
incorrectly
Ensure proper sample
preparation/dilution
Wells are insufficiently
washed
Wash wells as per protocol
recommendations
Contaminated wash
buffer
Make fresh wash buffer
Low signal
High
background
Discover more at www.abcam.com
21
RESOURCES
Problem
Large CV
Low sensitivity
Cause
Solution
Waiting too long to read
plate after adding STOP
solution
Read plate immediately
after adding STOP solution
Bubbles in wells
Ensure no bubbles present
prior to reading plate
All wells not washed
equally/thoroughly
Check that all ports of plate
washer are
unobstructed/wash wells
as recommended
Incomplete reagent
mixing
Ensure all reagents/master
mixes are mixed
thoroughly
Inconsistent pipetting
Use calibrated pipettes and
ensure accurate pipetting
Inconsistent sample
preparation or storage
Ensure consistent sample
preparation and optimal
sample storage conditions
(e.g. minimize
freeze/thaws cycles)
Improper storage of
ELISA kit
Store all reagents as
recommended. Please
note all reagents may not
have identical storage
requirements.
Using incompatible
sample type (e.g. Serum
vs. cell extract)
Detection may be reduced
or absent in untested
sample types
Discover more at www.abcam.com
22
RESOURCES
18. NOTES
Discover more at www.abcam.com
23
RESOURCES
Discover more at www.abcam.com
24
RESOURCES
Discover more at www.abcam.com
25
RESOURCES
Discover more at www.abcam.com
26
UK, EU and ROW
Email: technical@abcam.com | Tel: +44-(0)1223-696000
Austria
Email: wissenschaftlicherdienst@abcam.com | Tel: 019-288-259
France
Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96
Germany
Email: wissenschaftlicherdienst@abcam.com | Tel: 030-896-779-154
Spain
Email: soportecientifico@abcam.com | Tel: 911-146-554
Switzerland
Email: technical@abcam.com
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
US and Latin America
Email: us.technical@abcam.com | Tel: 888-77-ABCAM (22226)
Canada
Email: ca.technical@abcam.com | Tel: 877-749-8807
China and Asia Pacific
Email: hk.technical@abcam.com | Tel: 108008523689 (中國聯通)
Japan
Email: technical@abcam.co.jp | Tel: +81-(0)3-6231-0940
www.abcam.com | www.abcam.cn | www.abcam.co.jp
Copyright © 2014 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.
RESOURCES
27