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Human Matrix metalloproteinase 2 ELISA
Cat. No.: BA1023
Enzyme Immunoassay for the quantitative determination of Matrix
metalloproteinase 2 (MMP-2) in human serum and plasma.
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases,
responsible for the integrity of the basement membrane (BM) via
degradation of extracellular matrix and BM components.1 MMPs arepresented in various types of cells, including cardiomyocytes, cerebral
neurons, hepatocytes, and many others, thus influencing various processes.
MMP-2, also called gelatinase A, is reported to play a crucial intracellularrole where it cleaves several sarcomeric proteins inside cardiac myocytes
during oxidative stress-induced injury.2 Elevated levels of MMP-2(combined with MMP-9) are also associated with highly tumorigenic
cancers, such as breast, gastric.
PRINCIPLE OF MMP-2 ELISA
The microtiter plate is coated with the antibody specifically binding theMatrix metalloproteinase 2. The human serum or plasma is incubated in theplate with the capture antibody.
The specimen is washed out and the specifically bound protein is incubatedwith biotin-labelled detection antibody. Following another washing step,
Streptavidin-HRP conjugate is added into the well.
Unbound reagent is then washed out. Horseradish peroxidase (HRP) boundin the complex reacts with the chromogenic substrate (TMB) creating theblue colour. The reaction is stopped by addition of STOP solution (H2SO4).
The absorbance values are measured at 450 nm (optionally 450/630 nm)
and are proportional to the concentration of MMP-2 in the specimen. The
concentration of MMP-2 in unknown samples is determined from thecalibration curve which is created by plotting the absorbance values against
the standard concentration values.

Kit Contents
Item
Antibody Coated Microtiter Plate
Biotin-labelled Antibody
Streptavidin-HRP Conjugate
Master Standard (lyophilized)
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer
Wash Buffer 15x conc.
Substrate Solution
STOP Solution

Qty.
96 wells
13 mL
13 mL
1 vial1 vial1 vial2 x 13 mL
50 mL
13 mL
13 mL

MATERIAL REQUIRED BUT NOT SUPPLIED
1.
2.
3.
4.
5.

Glassware and test tubes
Microtiter plate washer
Precision pipettes (various volumes) with tips
Orbital shaker
Microtiter plate reader capable of measuring absorbanceat 450 nm or 450/630 nm with software for data generation

WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
7.

For research use only
For professional laboratory use
The reagents with different lot numbers should not be mixed
To prevent cross sample contamination, use disposable labware
and pipette tips
To protect laboratory stuff, wear protective gloves andprotective clothing
The substrate solution should remain colourless, keep it
protected from light
The test should be performed at standard laboratory conditions(temperature 25oC ± 2oC).

STORAGE CONDITIONS
1.
2.

The kit must be stored at 2 – 8oC.
The opened components can be stored for one week at 2 – 8oC.

PREPARATION OF REAGENTS
Use new pipette tip for pipetting different reagents and samples
to prevent cross-contamination.
All reagents and samples should be allowed to reach thetemperature 25oC ± 2oC.

Preparation of Standards
Reconstitute lyophilized Human MMP-2 Standard in Dilution Buffer, for thevolume information see the Certificate of Analysis. Let it rehydrate for 15
min prior to use. The concentration of human MMP-2 in Master Standard is
40 ng/mL.
Prepare set of Standard solution as follows:
Use the Master Standard for serial dilution (as below). Mix each tube
thoroughly before the next transfer. The Dilution Buffer serves as Blank.
Volume of Standard

v_02

Std1

Standard 40 ng/mL (lyophilized)

Dilution
Buffer625 µL

Concentration

Std2

250 µL of Std1

250 µL

20 ng/mL

Std3

250 µL of Std2

250 µL

10 ng/mL

Std4

250 µL of Std3

250 µL

5 ng/mL

Std5

250 µL of Std4

250 µL

2.5 ng/mL

Std6

250 µL of Std5250 µL

1.25 ng/mL

Std7

250 µL of Std5

250 µL

0.625 ng/mLBlank-200 µL

0 ng/mL

40 ng/mL2

Preparation of Quality Control A and B

3.

Reconstitute the lyophilized human serum Quality Controls indeionized/distilled water, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:50 in Dilution
Buffer, prior to use, see Preparation of samples.

Accuracy

Dilution linearity
SamplePreparation of Wash Buffer 1x1

Prepare a working solution of Wash Buffer by adding 50 mL of Wash Buffer
15x conc. to 700 mL of deionized/ distilled water (dH2O). Mix well. Store at
4oC for two weeks or at -20oC for long term storage.

2Dilution

SampleSpike(ng/ml)

1250
125
62.5

ASSAY PROCEDURE
1.
2.

Human MMP-2 Standard Curve

Absorbance (450/630 nm)

3Sensitivity

2.5
2
1.5
1
0.5
0Precision

0.625

Intra-assay

1.25

2.5

5

10

20

40

Concentration (ng/mL)

Mean (ng/mL)
175
208

SD
5.1
4.3

CV (%)
32Mean (ng/mL)
252
145

RESOURCES
1 Łukaszewicz-Zając M, Mroczko B, Słowik A. Matrix metalloproteinases (MMPs) andtheir tissue inhibitors (TIMPs) in amyotrophic lateral sclerosis (ALS). J Neural Transm(Vienna). 2014 Nov;121(11):1387-97. doi: 10.1007/s00702-014-1205-3. Epub 2014 Jul
22. PMID: 25047909; PMCID: PMC4210652.

Inter-assay (Run – to – run)

v_02

99
100
100

3.5

The limit of detection, defined as a concentration of human MMP-2 giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 0.25 ng/mL of sample.

Sample12Yield (%)

The standard curve needs to be measured in every test. Most of themicroplate reader can automatically calculate the analyte concentration
using 4-parameter algorithm or alternative functions to fit the standard
points properly. The concentrations need to be multiplied by the dilution
factor, either automatically by reader or manually.

Samples used in the tests were diluted 1:50 as recommended and assayed.
The results are multiplied by the dilution factor.

Sample12Expected
concentration(ng/mL)
458
333
270

Typical standard curve

PERFORMANCE CHARACTERISTICS2.

Measuredconcentration(ng/mL)
208453
334
270

110
114
97107114
99

Spiking Recovery

Do not store the diluted samples.

1.

Yield
(%)

2x4x8xRecommended dilution of samples is 1:50, i.e., 5 µL of sample + 245 µL ofDilution Buffer, for duplicates and for singlets.

Prepare the reagents as described in the previous chapter.
Pipette 100 µL of set of Standards, Quality Controls, diluted
Samples and Dilution Buffer = Blank into each well. Incubate for
1 hour at 25oC ±2oC, shaking at 300 rpm.
3. Wash the wells 3-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
4. Pipette 100 µL of Biotin-labelled Antibody into each well.
Incubate for 1 hour at 25oC ±2oC, shaking at 300 rpm.
5. Wash the wells as described in point 3.
6. Pipette 100 µL of Streptavidin-HRP into each well. Incubate for
30 min at 25oC ±2oC, shaking at 300 rpm.
7. Wash the wells as described in point 3.
8. Pipette 100 µL Substrate solution, incubate for 15 min,at 25oC ±2oC. Avoid exposure to the light during this step.
9. Pipette 100 µL of STOP solution.
10. Read the signal at 450 or 450/630 nm within 15 min.

Expected
concentration(ng/mL)
88
44
2275
37
192x4x8x

Preparation of samples
Human serum or plasma may be used with this assay. For long-term storage
the samples should be frozen at minimum -70oC. Lipemic or haemolytic
samples may cause false results.

Measuredconcentration(ng/mL)
175
96
50
21150
80
43
19SD
21.7
4.3

CV (%)
93

Bassiouni W, Ali MAM, Schulz R. Multifunctional intracellular matrix
metalloproteinases: implications in disease. FEBS J. 2021 Dec;288(24):7162-7182. doi:
10.1111/febs.15701. Epub 2021 Jan 22. PMID: 33405316.
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