1

Human Matrix metalloproteinase 9 ELISA
Cat. No.: BA1014
Enzyme Immunoassay for the quantitative determination of Matrix
metalloproteinase 9 (MMP-9) in human plasma and serum.
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.
Increased matrix metalloprotease 9 (MMP9), also called Gelatinase B, after
myocardial infarction (MI) exacerbates ischemia-induced chronic heart
failure (CHF).2 It is also upregulated in the diabetic heart, and ablation of
MMP9 decreases infarct size in the non-diabetic myocardial infarction
heart.3 There is also strong evidence that the increased levels of MMP-9 areassociated with many inflammatory conditions, such as coronary arterydisease, COPD, arthritis, and metabolic syndrome.4 It was also proved thatMMP-9 is associated with progression of atherosclerosis and higher risk ofcardiovascular events. It was also suggested that elevated levels of MMP-9(combined with MMP-2) are associated with highly tumorigenic cancers.
PRINCIPLE OF MMP-9 ELISA
The microtiter plate is coated with the antibody specifically binding theMatrix metalloproteinase 9. 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-9 in the specimen. The
concentration of MMP-9 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 AntibodyStreptavidin-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 vial2x13 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-9 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-9 in Master Standard is20 ng/mL. Use the Master Standard for serial dilution (as below). Mix eachtube thoroughly before the next transfer. The Dilution Buffer serves asBlank. Prepare set of Standard solution as follows:
Volume of Standard

v_02

Std1

Standard 20 ng/mL (lyophilized)

Dilution
Buffer1000 µL

Concentration

Std2

250 µL of Std1

250 µL

10 ng/mL

Std3

250 µL of Std2

250 µL

5 ng/mL

Std4

250 µL of Std3

250 µL

2.5 ng/mL

Std5

250 µL of Std4

250 µL1.25 ng/mL

Std6

250 µL of Std5

250 µL

0.625 ng/mLBlank-200 µL

0 ng/mL

20 ng/mL

2Preparation 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 1x1Prepare 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

1.
2.

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 10 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)
473
348
285

Yield (%)

1250.0
125.0
62.5

102
103102

Human MMP-9 Standard Curve
3.5
32.5
21.5
10.5

Sensitivity

00.625

1.25

2.5

5

1020

Concentration (ng/mL)

PrecisionRESOURCES
1Mean (ng/mL)
527
224

SD
9.7
6.7

CV (%)
23Inter-assay (Run – to – run)
SampleMean (ng/mL)
1389
2255

SD
31
14CV (%)
86v_02

Spike(ng/ml)

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.

The limit of detection, defined as a concentration of human MMP-9 giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 0.16 ng/mL of sample.

Intra-assay
Sample12Sample

Typical standard curve

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

2.

Measuredconcentration(ng/mL)
223
480
357
290

95
95
91
99
104
95

Spiking Recovery

PERFORMANCE CHARACTERISTICS

1.

Yield
(%)

2x4x8xAbsorbance (450/630 nm)

ASSAY PROCEDURE

Expected
concentration(ng/mL)
260
13065
291
145
73

2x4x8xPreparation of samples
Human plasma or serum 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. Pay attention to a possibly elevated serum
level of human MMP-9 due to MMP-9 release by platelets during sampling
process. This may cause variable and irreproducible results. Use a silicabased activator with polymer gel tubes for serum separation (SST/ BD
Vacutainer, Serum gel (coagulation activator) / Sarstedt S-Monovette), or
platelet-poor plasma. Platelet-poor plasma can be prepared in two-stepprocedure: 1/ centrifugation 20 minutes at 1500 x g, and 2/ additional
centrifugation step of the plasma at 10 000 x g for 10 minutes for platelet
removal. Recommended dilution of samples is 1:50, i.e., 5 µL of sample +245 µL of Dilution Buffer, for duplicates and for singlets. Do not store thediluted samples.

Measuredconcentration(ng/mL)
519
247
1235958228915169Łukaszewicz-Zając M, Mroczko B, Słowik A. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in
amyotrophic lateral sclerosis (ALS). J Neural Transm (Vienna). 2014 Nov;121(11):1387-97. doi: 10.1007/s00702-014-12053. Epub 2014 Jul 22. PMID: 25047909; PMCID: PMC4210652.
2(Nandi SS, Katsurada K, Sharma NM, Anderson DR, Mahata SK, Patel KP. MMP9 inhibition increases autophagic flux inchronic heart failure. Am J Physiol Heart Circ Physiol. 2020 Dec 1;319(6):H1414-H1437. doi:
10.1152/ajpheart.00032.2020. Epub 2020 Oct 16. PMID: 33064567; PMCID: PMC7792705.)
3

Yadav SK, Kambis TN, Kar S, Park SY, Mishra PK. MMP9 mediates acute hyperglycemia-induced human cardiac stem cell
death by upregulating apoptosis and pyroptosis in vitro. Cell Death Dis. 2020 Mar 13;11(3):186. doi: 10.1038/s41419-0202367-6. PMID: 32170070; PMCID: PMC7070071.
4

Snitker S. Correlation of Circulating MMP-9 with White Blood Cell Count in Humans: Effect of Smoking. PLoS ONE. 2013;
8(6): e66277