1

Human Osteopontin ELISA
Cat. No.: BA1028
Enzyme Immunoassay for the quantitative determination of Osteopontin inhuman serum and plasma.
Osteopontin (OPN), a secreted multifunctional phosphoprotein, is amember of the small integrin-binding ligand N-linked glycoprotein (SIBLING)
family of cell matrix proteins and participates in many biological activities.
OPN has been demonstrated to be closely related to the occurrence anddevelopment of many bone-related diseases, such as osteoporosis,
rheumatoid arthritis, and osteosarcoma.1 Besides that, it functions as aproinflammatory cytokine and promotes cell-mediated immune responses,
and also has protective functions such as biomineralization [20,22,23] andwound healing, and is also a strong predictor of adverse outcomes inpatients with CVDs [30–32]. Thus, OPN is not only a risk factor but also a
potential therapeutic target for CVDs.2

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.

PRINCIPLE OF KL-6 ELISA

5.

The microtiter plate is coated with the antibody specifically binding theOsteopontin. The human serum or plasma is incubated in the plate with thecapture 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 Osteopontin in the specimen.
The concentration of Osteopontin in unknown samples is determined fromthe calibration curve which is created by plotting the absorbance values
against the standard concentration values.

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 KL-6 Master Standard in Dilution Buffer, forthe volume information see the Certificate of Analysis. Let it rehydrate for
15 min The concentration of human KL-6 in Master Standard is4000 pg/mL
Prepare set of Standard solution as follows:
Use the Master Standard to produce a dilution series (as below). Mix eachtube thoroughly before the next transfer. The Dilution Buffer serves asBlank.
Volume of Standard

KIT CONTENTS
ItemAntibody Coated Microtiter Plate
Biotin-labelled AntibodyStreptavidin-HRP Conjugate
Master Standard
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer
Wash Buffer 15x conc.
Substrate Solution
STOP Solution
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Qty.
96 wells
13 mL
13 mL
1 vial1 vial1 vial2x13 mL
50 mL
13 mL
13 mL

Std1

Standard 10 pg/mL (lyophilized)

Dilution
BufferSee CofA

Concentration

Std2300 µL of Std1

300 µL

5 pg/mL

Std3300 µL of Std2300 µL

2.5 pg/mL

Std4300 µL of Std3300 µL

1.25 pg/mL

Std5300 µL of Std4300 µL0.625 pg/mL

Std6300 µL of Std5300 µL

0.156 pg/mLBlank-300 µL0 pg/mL

10 pg/mL

Preparation of Quality Control A and B
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 1:25 prior to use,
see Preparation of samples.

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Preparation of Wash Buffer 1x3.

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.

Accuracy

Dilution linearity
SamplePreparation of samples

Dilution

Measuredconcentration(ng/mL)
54.4
28.4
15.0
7.8
39.8
19.8
10.1
4.7

Expected
concentration(ng/mL)
27.2
13.6
6.8
19.9
9.9
5.0

Yield(%)

Measuredconcentration(ng/mL)
8.8
28.2
13.4
10.7

Expected
concentration(ng/mL)
33.8
15.0
11.9

Yield(%)

1

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.

2x4x8x2Recommended dilution of samples is 1:25, i.e., for singlets 6 µL of sample +144 µL of Dilution Buffer, for duplicates 10 µL of samples + 240 µL of Dilution
Buffer, respectively.

2x4x8x104111
115
99101
95

Do not store the diluted samples.
Spiking Recovery
ASSAY PROCEDURE
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 for2 hours 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 2 hours 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
20 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 20 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.

SampleSpike(Pg/mL)

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25.0
6.3
3.1

1.
2.

83
89
90

Typical standard curve
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.

Human OPN Standard Curve
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3.5

PERFORMANCE CHARACTERISTICS

31.

Absorbance (450/630 nm)

Samples used in the tests were diluted 1:25 as recommended and assayed.
The results are multiplied by the dilution factor.
Sensitivity

The limit of detection, defined as a concentration of human Osteopontin
giving absorbance higher than absorbance of blank + 3 standard deviations,nis better than 1.56 pg/mL of sample.
2.

Precision

Mean (ng/mL)
8.8
54.4

SD
0.4
2.1

1.5
1Mean (ng/mL)
7.8
52.3

0CV (%)
4.3
3.9

62.5

125250

500

1000

2000

4000

Concentration (pg/mL)

RESOURCEInter-assay (Run – to – run)
Sample122

0.5Intra-assay
Sample
12

2.5

SD
0.2
2.1

CV (%)
2.8
3.9

1

Si J, Wang C, Zhang D, Wang B, Zhou Y. Osteopontin in Bone Metabolism andBone Diseases. Med Sci Monit. 2020 Jan 30;26:e919159. doi:
10.12659/MSM.919159. PMID: 31996665; PMCID: PMC7003659.
2Shirakawa K, Sano M. Osteopontin in Cardiovascular Diseases. Biomolecules.
2021 Jul 16;11(7):1047. doi: 10.3390/biom11071047. PMID: 34356671;
PMCID: PMC8301767

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