ab205810
ADHP HRP substrate
Instructions for Use
In the presence of H2O2, HRP converts ADHP to the fluorogenic
product, resorufin.
This product is for research use only and is not intended for diagnostic
use.
Version 1 Last Updated 10 July 2015
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
3
GENERAL INFORMATION
3.
4.
5.
6.
7.
8.
PRECAUTIONS
STORAGE AND STABILITY
MATERIALS SUPPLIED
MATERIALS REQUIRED, NOT SUPPLIED
LIMITATIONS
TECHNICAL HINTS
4
4
4
5
5
6
ASSAY PREPARATION
9.
REAGENT PREPARATION
7
ASSAY PROCEDURE
10.
ASSAY PROCEDURE
8
DATA ANALYSIS
11.
TYPICAL DATA
9
RESOURCES
12.
13.
TROUBLESHOOTING
NOTES
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11
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1
INTRODUCTION
1. BACKGROUND
The HRP substrate is a combination of 10-Acetyl-3,7dihydroxyphenoxazine (ADHP), a sensitive substrate for HRP, and
ADHP Dilution Buffer, a stabilized H2O2 solution. In the presence of
H2O2, HRP converts ADHP to the fluorogenic product, resorufin.
Resorufin is a highly fluorescent molecule, and should be used with
excitation filters in the range of 530 - 540nm, with bandwidths ≤ 30nm.
The emission filter should be in the range of 590 - 600nm, with
bandwidths ≤ 30nm.
The signal in the wells should be
developed
for
around
10
minutes. Best results will be
obtained if the microplates are
developed in the dark, e.g. by
covering the microplate with foil.
The Stop Solution provided can
be used for stopping HRPmediated conversion of ADHP to
resorufin. Once added to the
wells, signal in the wells is stable
for 60 minutes. However, ADHP
remaining in the wells is still lightsensitive, and should be handled
under low-light conditions until
the microplate is read.
2. ASSAY SUMMARY
Remove appropriate number of
antibody
coated
well
strips.
Equilibrate all reagents to room
temperature.
Prepare
all
the
reagents, standards and samples as
instructed. Plates are pre-coated with
PTX3.
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2
INTRODUCTION
Add standard or sample to each well used. Incubate at room temperature.
Aspirate and wash each well. Add prepared HRP labeled secondary detector
antibody. Incubate at room temperature.
Aspirate and wash each well. Add TMB Substrate to each well and incubate. Add Stop
Solution at a defined endpoint and record color development.
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3
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
All reagents are stable as supplied at +2-8°C.
Do not freeze reagents. Refer to list of materials supplied for storage
conditions of individual components. Note the storage conditions for
individual prepared components in the Reagent Preparation section.
5. MATERIALS SUPPLIED
Item
ADHP Dilution Buffer
ADHP (100X)
Stop Solution
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Quantity
1 x 15 mL
1 x 120 µL
1 x 2 mL
Storage
Condition
(Before
Preparation)
RT
RT
RT
4
GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:
7. LIMITATIONS

Exact conditions may vary from assay to assay, a standard curve
should be generated for every assay performed.

Bacterial or fungal contamination of either samples or reagents or
cross-contamination between reagents may cause erroneous
results.

Disposable pipette tips, flasks or glassware are preferred, reusable
glassware must be washed and thoroughly rinsed of all detergents
before use.

Improper or insufficient washing at any stage of the procedure will
result in either false positive or false negative results. Completely
empty wells before dispensing fresh 1X Wash Buffer. Do not allow
wells to sit uncovered or dry for extended periods.

Avoid exposure of reagents to excessive heat or light during
storage and incubation.

Optical density values obtained for duplicates should be within
10% of the mean. Duplicate values that differ from the mean by
greater than 10% should be considered unreliable and should be
repeated.
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GENERAL INFORMATION
8. TECHNICAL HINTS

Avoid preparation or use under bright lights or direct sunlight –
ADHP is sensitive to bright light.

For best results we recommend preparing the Substrate Mix
immediately prior to use. It is not stable for long periods once
mixed.

Kit components should be stored as indicated. All the reagents
should be equilibrated to room temperature before use. Samples
should be thawed at room temperature. Do not use water baths to
thaw samples.

Use a clean disposable plastic pipette tip for each reagent,
standard, or specimen addition in order to avoid crosscontamination.

Thoroughly mix the reagents and samples before use by agitation
or swirling.

All residual washing liquid must be drained from the wells by
efficient aspiration or by decantation followed by tapping the plate
forcefully on absorbent paper. Never insert absorbent paper
directly into the wells.

When pipetting reagents, maintain a consistent order of addition
from well-to-well. This will ensure equal incubation times for all
wells.

Many individual components contain preservative. Appropriate
protective clothing, glasses and gloves should be worn at all times
when handling kit reagents to avoid direct skin contact
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ASSAY PREPARATION
9. REAGENT PREPARATION
Before testing, allow ADHP to thaw prior to use – it is solid at 4°C.
9.1
Substrate Mix
Dilute ADHP 100-fold with ADHP Dilution Buffer A (e.g. add
20 μL ADHP to 2 mL of the ADHP Dilution Buffer)
immediately prior to use. The Substrate Mix is then ready to
use. Prepare enough to use 100 µL/well.
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ASSAY PROCEDURE
10. ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to room
temperature prior to use.

Pipetting Hints: Use different tips to pipette the buffer,
standards, samples, and conjugate. Before pipetting each
reagent, equilibrate the pipette tip in that reagent (i.e., slowly
fill the tip and gently expel the contents, repeat several times).
Do not expose the pipette tip to the reagent(s) already in the
well.
10.1 Following washing, add 100 µL/well of Substrate Mix. Cover
microplate with foil, and incubate for 10 minutes at room
temp on a microplate shaker (~300 rpm).
10.2 Add 10 µL/well Stop Solution, and mix for 30 seconds on a
microplate shaker.
10.3 Read fluorescence signal with a compatible filter set (e.g.
540/590 nm).
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DATA ANALYSIS
11. TYPICAL DATA
RFU (540/590)
10 8
10 7
10 6
10 5
10 4
10 3
0.01
0.1
1
10
100
1000 10000
Test Analyte (pM)
Figure A
Absorbance (450 nm)
1000
100
10
1
0.1
0.01
0.01
0.1
1
10
100
1000 10000
Test Analyte (pM)
Figure B
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DATA ANALYSIS
Figure 1.
Comparison of ADHP fluorescence (A) and TMB
colorimetric (B) assay readouts. Using an ELISA based format,
standard curves were generated for a test analyte with concentrations
ranging from 40 fM up to 10 nM. After washing away unbound
reactants, bound HRP was reacted with ADHP or TMB substrate for 10
mins before stopping each reaction with its respective stop solution.
Fluorescence was measured at 540/590 using a FlexStation II384
(Molecular Devices) and absorbance was measured at 450 nm using
an EnVision 2102 Multilabel Reader (Perkin Elmer).
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RESOURCES
12. TROUBLESHOOTING
Problem
Possible Causes
Standard degraded
Poor Standard Curve
Curve doesn’t fit scale
Pipetting Error
Plate washings too
vigorous
No Signal
Wells dried out
High Background
Low sensitivity
Solutions
Store and handle
standard as
recommended
Try plotting using
different scales
Use calibrated pipettes
and proper pipetting
technique.
Check and ensure
correct pressure in
automatic wash system.
Pipette wash buffer
gently if washes are
done manually.
Do not allow wells to dry
out. Cover the plate for
incubations.
Wells are insufficiently
washed
Contaminated wash
buffer
Waiting too long to read
the plate after adding
stop solution
Standard is degraded
Wash wells as per
protocol
Prepare fresh wash
buffer
Read plate within 30
minutes
Mixing or substituting
reagents from other kits
Avoid mixing
components
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Replace standard
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RESOURCES
13. NOTES
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Japan
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RESOURCES
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