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A novel assay for MMP-9 activity—correlation with ELISA
and application to biological fluid measurements
S. Capper, V. Evans, R. Hanemaaijer*, M. Sully, H.Visser*, and J.Verheijen*
Amersham Biosciences UK Ltd., Amersham Place, Little Chalfont, Buckinghamshire, UK
*Gaubius Laboratory, TNO-PG, Leiden, The Netherlands
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range of 0.5–8 ng/ml with a sensitivity of 0.5 ng/ml
can be achieved using a 6 h incubation.
A novel and specific immunocapture activity assay for the
measurement of either total or active MMP-9 in biological
samples has been developed. The assay is quantitative and
convenient, yielding results which correlate well with those
obtained by more conventional procedures such as ELISA.
Either total (latent and active) levels of MMP-9 or
endogenous levels of active MMP-9 can be measured
by using buffer with or without p-aminophenylmercuric
acetate (APMA), respectively. Total MMP-9 can be quantified by treating immunocaptured MMP-9 with APMA
and then performing the assay. ProMMP-9, activated in
parallel for both types of samples, is used as the standard.
Introduction
Over-expression and activation of matrix metalloproteinases (MMPs), or an imbalance between active
MMPs and tissue inhibitors of metalloproteinases
(TIMPs), have been suggested as key factors in the
pathogenesis of diseases characterized by the breakdown
of extracellular matrix. The ability to determine not only
the total amount of enzyme expressed, but also the proportion subsequently activated or possessing the latent
potential to become active, is instrumental in research
involving these diseases.
The anti-MMP-9 antibody used in the assay recognizes
both the pro and active forms of MMP-9 and does not
cross-react with other MMPs or TIMPs. Both pro- and
active MMP-9/TIMP-1 complexes, as well as active
MMP-9/TIMP-2 complexes, have shown some degree
of reactivity in the assay.
In addition to human samples, the assay is applicable
to the detection of MMP-9 activity in rabbit and
mouse samples.
We have developed and validated a novel and specific
immunocapture assay for MMP-9 activity in biological
samples including cell culture supernatants, serum,
plasma, and saliva. The assay is specific for MMP-9,
quantitative, and convenient. It offers distinct advantages
over more cumbersome methods such as zymography.
Validation studies
Cell culture: Cell culture samples were assayed in
parallel using the MMP-9 Activity Assay and the
MMP-9, Human, ELISA (RPN2614). The ELISA is
specific for proMMP-9 and has a sensitivity range of
1–32 ng/ml (protocol for cell culture measurements).
Levels of proMMP-9 detected by ELISA and total
MMP-9 detected by the activity assay showed a good
degree of correlation (Fig 1). Absolute levels of MMP-9
were two-fold higher in the ELISA than in the activity
assay, possibly reflecting the contribution of MMP-9/
TIMP-1 complex to the measured levels. Because TIMP-1
inhibits urokinase cleavage by MMPs, this complex is
detected with reduced (39%) cross-reactivity in the
activity assay. The demonstration of significant levels
of TIMP-1 in these samples, using a TIMP-1 specific
ELISA assay (RPN2611), supports this hypothesis
(data not shown).
MMP-9 Activity Assay
The MMP-9 Activity Assay uses an anti-MMP-9
antibody coated onto the wells of a 96-well plate to
capture MMP-9 from standards, biological fluids, or
cell culture media. The detection reaction uses a
modified pro-urokinase in which the activation sequence,
normally recognized by plasmin (Pro-Arg-Phe-Lys ⇓
Ile-Ile-Gly-Gly), is replaced by a sequence specifically
recognized by MMPs (Arg-Pro-Leu-Gly ⇓ Ile-Ile-Gly-Gly).
The modified pro-urokinase is cleaved by the captured
MMP to form active urokinase, which is then detected
using a chromogenic peptide substrate, S-2444™. The
resulting absorbance is read at 405 nm. Through the
use of standard curves, the absorbance at 405 nm can
be related back to the quantity of MMP-9 in the sample.
Serum and plasma: Figure 2 shows the latent (total)
MMP-9 levels in serum and in various plasma preparations as determined by the MMP-9 Activity Assay.
Although normal levels are detectable in most of the
The detection reaction is performed at 37 °C for 2–6 h.
The normal assay range is 2–32 ng/ml for a 2 h incubation,
with a sensitivity of 1 ng/ml. A more sensitive assay
Life Science News 2, 1999 Amersham Biosciences
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total MMP-9 by
Activity Assay (ng/ml)
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Fig 1. Correlation between MMP-9 levels in U937 cell cultures
assayed by ELISA and the MMP-9 Activity Assay. Cell culture samples
were assayed in parallel using the MMP-9 Activity Assay and the
MMP-9, Human, ELISA (RPN2614). Latent (total) activity was determined using the MMP-9 Activity Assay. The ELISA is specific for
proMMP-9. U937 cell cultures were assayed after stimulation with the
following reagents: phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), 1α,25-(OH)2D3 (TRK810), interferon-γ (IFN-γ) and
interleukin-4 (IL-4). Samples were assayed as a population of samples
of varying concentration with no attempt to segregate them by treatment.
assay
MMP-9 Activity
Assay
125I-collagen
degradation (1)
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6.7 ± 5.4
(n = 5)
MMP-9 form
active
(p < 0.05)
latent
(p < 0.0001)
active
latent
(p = 0.11)
MMP-9 activity
controls
Sjögren’s patients
0.8 ± 0.3
2.5 ± 1.3
9.4 ± 1.2
26.2 ± 3.2
0
36.3 ±6.5
0
44.5 ± 4.6
The MMP-9 Activity Assay can be used to measure levels
of MMP-9 in a variety of biological samples, including
cell culture fluids, serum, plasma, and saliva. Some of the
key advantages of the new assay are demonstrated in the
study of Sjögren’s patients. Because of the specificity and
ability of the MMP-9 Activity Assay to discriminate
endogenous active levels of MMP-9 from total levels, the
assay can clearly detect specific increases in MMP-9
levels in these patients.
80
18.6 ± 7.6
(n = 5)
R
Conclusions
Serum levels of MMP-9 measured using the activity assay
were similar to those reported in the literature using
ELISA. It should be noted that levels in serum are higher
than those observed in plasma, presumably reflecting the
additional contribution from monocytes, neutrophils, and
other cellular components which lyse during clotting.
17.3 ± 8.0
(n = 5)
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Table 1. MMP-9 activity in saliva from patients with Sjögren’s syndrome
samples, the results suggest that some EDTA-treated
plasma samples may be difficult to assay reliably because
of low levels of MMP-9. This may be due to some
deleterious effect of EDTA on MMP folding and activity.
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Saliva samples from control and Sjögren’s patients showed
no significant differences when assayed for total MMP
using the radiolabelled substrate assay (Table 1). Levels
of active MMP were undetectable by this assay. However,
when samples were assayed using the MMP-9 Activity
Assay, both active and total levels of MMP-9 were
significantly different between controls and patients.
proMMP-9 by ELISA (ng/ml)
70
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In a study performed at Gaubius Laboratory (Leiden,
The Netherlands), levels of MMP-9 activity in saliva
samples from patients with Sjögren’s syndrome were
determined using the new assay. MMP-9 Activity Assay
results were compared with results obtained using an
assay based on radiolabelled gelatinated collagen (1).
15
0
N
MMP-9 in Sjögren’s patients
r = 0.7784
y = 0.432x + 0.685
0
MMP-9 conc. (ng/ml)
I
38.4 ± 21.3
(n = 5)
While MMPs have been implicated in this disease for
some time, it has, until now, proved difficult to demonstrate specific activation of an individual MMP. In
addition, the sensitivity of the new assay, when compared
with conventional substrate degradation assays, has
allowed the determination of endogenous active levels
of MMP-9.
50
40
30
20
10
0
plasma
citrate
plasma
heparin
plasma
EDTA
serum
Reference
sample
1. Hanemaaijer, R. et al., Matrix Biology 17, 657–665 (1998).
Fig 2. Measurement of total MMP-9 levels in serum and plasma
samples using the MMP-9 Activity Assay. Serum and plasma were
separated from whole blood samples collected from healthy volunteers.
Plasma samples were collected in heparin, EDTA, or citrate. All samples
were stored at -70 °C until assayed. Serum and plasma samples were
diluted 1:32 in assay buffer and assayed by the MMP-9 Activity Assay
using the 6 h detection incubation protocol. Means for each sample
are represented by horizontal lines. Numbers at the top of each
sample column represent mean ± standard deviation.
ORDERING INFORMATION
MMP-9 Activity Assay
Life Science News 2, 1999 Amersham Biosciences
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96 wells
RPN2630
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