I
N
N
O
V
A
T
I
O
N
S
F
O
R
U
Methods to increase the sensitivity of the MMP-9
Activity Assay
R. Hanemaaijer, N. van Lent, H.Visser, and J.Verheijen
Gaubius Laboratory, TNO-PG, Leiden, The Netherlands
C
E
L
L
B
I
O
L
O
G
Y
A
S
S
A
Y
S
The sensitivity of the MMP-9 Activity Assay is easily
increased to 60 pg/ml by simple modifications to
the protocols. These modifications include longer
incubation times for the detection step and the
incorporation of lower dilutions of the MMP-9
standard for the reference line.
Introduction
Matrix metalloproteinases (MMPs) play an important
role in many (patho)physiological processes like
angiogenesis, tumour growth, and metastasis, and in
diseases with a degradation component such as
rheumatoid arthritis and periodontitis (1, 2). Unlike
serine proteases (e.g. urokinase and plasmin), MMP
enzyme activities are difficult to assay because MMPs
recognize the amino acids on both sides of the cleavage
site. Thus, chromogenic and fluorogenic substrates, in
which a non-peptide bond is cleaved, cannot easily be
used to assay for MMPs. Also, highly specific peptide
substrates are difficult to design because the proteolytic recognition sequences of the various MMPs
overlap considerably (3, 4).
Fig 1. Schematic representation of the MMP-9 Activity Assay. Active
MMP-9 is captured by a specific antibody and can be measured
directly after addition of modified urokinase (UKcol) and peptide substrate. Latent MMP-9 is also captured, but first must be activated by
APMA, before activity can be measured.
Dilutions of the MMP-9 standard, ranging from
0.06–32 ng/ml, were used. After addition of detection
enzyme and substrate, the A405 was immediately
measured. The plate was then incubated at 37 ˚C, and
the A405 was measured at 20–30 min intervals for 9.5 h
plus a 20 h point.
We developed a general MMP-specific substrateproenzyme by modifying the plasmin-specific activation site of pro-urokinase into a site only recognized
by MMPs (4). Upon activation by MMPs this substrate-proenzyme can be assayed using a chromogenic
substrate. We adapted this general MMP activity assay
into an MMP-9-specific activity assay by introducing
antibodies that capture MMP-9 from biological fluids
(5, 6; Fig 1). The method measures active or latent
(total) MMP-9 in a sample and is available as a kit
from Amersham Biosciences (6).
The data were analysed in two different ways. In the
endpoint method, the ∆A405 at 2, 4, 6, 9.5, and 20 h
was plotted versus the MMP-9 concentration. In the
kinetic method, the ∆A405 for each sample was plotted
against the square of the incubation time. The slope
of these lines (∆A405/h2) is a measure of the enzyme
activity. The endpoint method is easily plotted,
whereas the kinetic method requires a computerized
data analysis system.
As developed, the MMP-9 Activity Assay is useful for
assay ranges of 2–32 ng/ml and 0.5–8 ng/ml. Many
biological samples contain small quantities of MMP-9,
so a highly sensitive assay would be desirable. In this
article we present methods to increase the assay
sensitivity to 60 pg/ml.
Increasing assay time lowers detection to 60 pg/ml
The kinetic analysis data in Figure 2A show that the
∆A405 is linearly related to the square of the incubation
time for a wide range of MMP-9 concentrations. Such
plots can be used to determine unknown MMP-9
concentrations (Fig 2B), with the correlation coefficient providing information on the reliability of the
obtained value.
Ultra-sensitive MMP-9 Activity Assay and analyses
The MMP-9 Activity Assay was used according to
supplied protocols with the following exceptions.
Life Science News 3, 1999 Amersham Biosciences
1
M
N
O
2A
A
T
I
O
0.5
0.4
∆A405
0.6
N
S
3A
1 ng/ml
2 ng/ml
4 ng/ml
12 ng/ml
24 ng/ml
28 ng/ml
32 ng/ml
0.7
∆A
V
F
O
0.20
0.60
0.16
0.45
0.12
0.08
0.04
0.00
5
0.3
10 15 20 25 30
conc. MMP-9 (ng/ml)
3C
R2 = 0.997
0
5
3D
t = 6 hr
M
0.30
0.00
35
U
t = 4 hr
0.15
R2 = 0.995
0
R
3B
t = 2 hr
∆A405
N
10 15 20 25
conc. MMP-9 (ng/ml)
30
t = 9.5 hr
0
0
2
4
6
8
10
12
14
16
18
∆A405
0.1
20
t2 (h)
2B
35
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
∆A405
0.2
R2 = 0.994
1.00
0.80
0.60
0.40
0.20
0.00
3E
R2 = 0.945
0
0 2.5 5 7.5 10 12.5 15 17.5 20
conc. MMP-9 (ng/ml)
t = 9.5 hr
1
2 3 4 5 6
conc. MMP-9 (ng/ml)
7
8
t = 20 hr
3F
30
20
15
0.30
0.25
0.20
0.15
0.10
0.05
0.00
∆A405
25
∆A405
activity (1 000 × ∆A405/h2)
I
R2 = 0.930
0
10
5
R2 = 0.993
5
10
15
20
25
30
2
R2 = 0.860
0
0.1 0.2 0.3 0.4 0.5
conc. MMP-9 (ng/ml)
0.6
Fig 3. Analysis of the useful assay range of MMP-9 Activity Assay. The
activity of various concentrations MMP-9 was measured using the
MMP-9 Activity Assay format. The obtained ∆A405 were plotted versus
MMP-9 concentration at various time intervals. The ∆A405 was measured after 2 (A), 4 (B), 6 (C), 9 .5 (D, E) and 20 h (F). The figures
show the assay range for endpoint measurements at the indicated
time intervals.
0
0
0.4
0.8
1.2
1.6
conc. MMP-9 (ng/ml)
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
35
conc. MMP-9 (ng/ml)
Fig 2. Time and concentration dependency of MMP-9 Activity Assay.
A. Each line represents a single concentration of activated MMP-9 in
which the A405 is plotted against t2 (as indicated). B. Analysis of the
activity of various concentrations of MMP-9. Activities, expressed as
1 000 × ∆A405/h2, were plotted against the MMP-9 concentration.
Activity was measured for a reaction time period between 1 and 20 h.
Adaptation of the MMP-9 Activity Assay into a higher
sensitivity assay is easily performed. One simply
makes additional dilutions of the MMP-9 standard
(0.5, 0.25, 0.125, and 0.063 ng/ml) for the reference
measurement. Then one takes additional measurements at longer time intervals (e.g. 2, 4, 6, 9 h, and
overnight). The assay is simple, and samples containing very high or very low amounts of MMP-9 can be
analysed in the same assay.
The simpler endpoint analysis plots the ∆A405 versus
MMP-9 concentration at various times. The 2 and 6 h
incubation times produced assay ranges of 4–32 ng/ml
and 1–16 ng/ml, respectively (Fig 3A, 3C). Increasing
the incubation time to 9.5 or 20 h produced assay
ranges of 0.25–7 ng/ml and 0.06–0.5 ng/ml, respectively (Fig 3D, 3F). Obviously, longer incubation times
extend the assay range, allowing measurements of very
low MMP-9 levels or very small sample volumes.
References
1. Birkedal-Hansen, H. et al., Crit. Rev. Oral Biol. Med. 4,
197–250 (1993).
2. Cawston, T. Pharmacology & Therapeutics 70,
163–182 (1996).
3. Nagase, H. and Fields, G., Biopolymers 40, 399–416
(1996).
4. Verheijen, J. et al., Biochem. J. 323, 603–609 (1997).
5. Hanemaaijer, R. et al., Matrix Biol. 17, 657–665
(1998).
6. Capper, S. et al., Life Science News 2, 8–9 (1999).
The MMP-9 Activity Assay Kit contains all reagents
for this modified protocol. For the full assay range, we
recommend using a reference line of 0.06–32 ng/ml.
Conclusions
The modified MMP-9 Activity Assay offers several
advantages over zymography, ELISA, and Western
blotting for MMP-9 detection. The MMP-9 Activity
Assay allows for a continuous analysis instead of a
single measurement, considerably increasing the assay
range. With measurements at various times to 20 h,
the assay range of 2–32 ng/ml (after 2 h) can be
increased to 0.06–32 ng/ml. Also these MMP-9 concentrations can be measured on the same plate with
this assay. There is no need to repeat the experiment at
higher dilutions as with zymography, ELISA, or
Western blotting.
ORDERING INFORMATION
MMP-9 Activity Assay
Life Science News 3, 1999 Amersham Biosciences
2
96 wells
RPN2630