1

Human D-dimer ELISA
MATERIAL REQUIRED BUT NOT SUPPLIED

Cat. No.: BA1005
Enzyme Immunoassay for the quantitative determination of D-dimer inhuman serum and plasma.
D-dimer, the final degradation product of cross-linked fibrin, is typicallyelevated in patients with acute venous thromboembolism.1 D-dimer testingis an integral part of validated algorithms for the diagnosis of deep-vein
thrombosis (DVT) and pulmonary embolism (PE)2
PRINCIPLE OF D-DIMER ELISA
The microtiter plate is coated with the antibody specifically binding theD-dimer. The human serum or plasma is incubated in the plate with thecapture antibody.

1.
2.
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WARNINGS AND PRECAUTIONS
1.
2.
3.
4.

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.

5.

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).

7.

The absorbance values are measured at 450 nm (optionally 450/630 nm)
and are proportional to the concentration of D-dimer in the specimen. The
concentration of D-dimer in unknown samples is determined from thecalibration curve which is created by plotting the absorbance values against
the standard concentration values.

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

6.

For research use only
For professional laboratory useThe 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 D-dimer Master Standard in Dilution Buffer,
for the volume information see the Certificate of Analysis. Let it rehydrate
for 15 min The concentration of human D-dimer in Master Standard is
160 ng/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 CONTENTSItem
Antibody Coated Microtiter Plate
Biotin-labelled Antibody
Streptavidin-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 vial
13 mL
50 mL
13 mL
13 mL

Std1

Standard 160 ng/mL (lyophilized)

Dilution
Buffer1000 µL

Concentration

Std2300 µL of Std1

300 µL

80 ng/mL

Std3300 µL of Std2300 µL

40 ng/mL

Std4300 µL of Std3300 µL

20 ng/mL

Std5300 µL of Std4300 µL

10 ng/mL

Std6300 µL of Std5300 µL

5 ng/mLBlank-300 µL

0 ng/mL

160 ng/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 1:3 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 at4oC for two weeks or at -20oC for long term storage.

Accuracy

Dilution linearity
SamplePreparation of samples

Dilution

Measuredconcentration(ng/mL)
75
38
19966
34
168

Expected
concentration(ng/mL)
38
19
933
178

Yield(%)

Measuredconcentration(ng/mL)
52
147
99
68

Expected
concentration(ng/mL)
172
112
82

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.

2x4x8x2

Recommended dilution of samples is 1:3, i.e., for singlets 50 µL of sample +100 µL of Dilution Buffer, for duplicates 100 µL of sample + 200 µL of Dilution
Buffer, respectively.

2x4x8x10199
951029598

Do not store the diluted samples.
Spiking Recovery

ASSAY PROCEDURE
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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 for1 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.

SampleSpike(ng/mL)

1

120
60
30

86
89
84

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 D-dimer Standard Curve
43.5

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:3 as recommended and assayed.
The results are multiplied by the dilution factor.

Absorbance (450/630 nm)

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3Sensitivity

The limit of detection, defined as a concentration of human D-DIMER giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 1.25 ng/mL of sample.
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Precision

Intra-assay
Sample
12

2.5
2
1.5
1
0.5

Mean (ng/mL)
7567SD
5.1
4.6

CV (%)
770510204080160

Concentration (ng/mL)

Inter-assay (Run – to – run)
Sample12Mean (ng/mL)
1895

RESOURCE
SD
0.8
8.3

CV (%)
491

Prisco D, Grifoni E. The role of D-dimer testing in patients with suspectedvenous thromboembolism. Semin Thromb Hemost. 2009 Feb;35(1):50-9. doi:10.1055/s-0029-1214148. Epub 2009 Mar 23. PMID: 19308893.
2WELLS, P.S. (2007), Integrated strategies for the diagnosis of venousthromboembolism. Journal of Thrombosis and Haemostasis, 5: 41-50.
https://doi.org/10.1111/j.1538-7836.2007.02493.x

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