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Dickkopf-1 (DKK-1) ELISA

Cat. No.: BI-20413

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Therapeutic Areas
dkk-1 standard curve
Assay Characteristics
  • Category number

    BI-20413

  • Method

    Sandwich ELISA, HRP/TMB, 12×8-well detachable strips

  • Sample type

    Serum

  • Sample volume

    20 µl / well

  • Assay time

    2 h / 1 h / 30 min

  • Sensitivity

    1.7 pmol/l (= 43.0 pg/ml)

  • Standard range

    0 – 160 pmol/l (= 0 – 4102 pg/ml)

  • Conversion factor

    1 pg/ml = 0.039 pmol/l (MW: 25.8 kDa)

  • Precision

    In-between-run (n=9): ≤3 % CV

    Within-run (n=5): ≤ 3 % CV

  • Cross-reactivity

    Human DKK-1 only, no cross-reactivity or interference with recombinant human DKK-2, DKK-4, Kremen-1, Kremen-2 or LRP-6 is observed.

  • Validation Data

    See validation data tab for: precision, accuracy, diltuion linearity, values for healthy donors, etc.

  • Regulatory status

    CE marked – for IVD use in the EU

Product details

Product overview

The DKK-1 immunoassay is a 3.5 hour, 96-well sandwich ELISA for the quantitative determination of DKK-1 in human serum. The assay employs human serum-based standards to ensure the measurement of biologically reliable data.

Principle of the Assay

The DKK-1 ELISA kit is a sandwich enzyme immunoassay for the quantitative determination of DKK-1 in human serum samples.



In a first step, assay buffer, standard/control/sample and detection antibody (biotinylated polyclonal rabbit anti-human DKK-1) are pipetted into the wells of the microtiter strips, which are pre-coated with monoclonal mouse anti-human DKK-1 antibody. DKK-1 present in the standard/control/sample binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody. In the washing step all non-specific unbound material  is removed. In a next step, the conjugate (streptavidin-HRP) is pipetted into the wells and reacts with the detection antibody. After another washing step, the substrate (TMB, tetramethylbenzidine) is pipetted into the wells. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of DKK-1. This color change is detectable with a standard microtiter plate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) versus standard concentration is generated using the values obtained from the standard.  The concentration of DKK-1 in the sample is determined directly from the dose response curve.

Typical Standard Curve

The figure below shows a typical standard curve for the DKK-1 ELISA. The immunoassay is calibrated against recombinant human DKK-1.



Kit Components

Contents

Description

 Quantity

PLATE

Monoclonal mouse anti-human DKK-1 antibody pre-coated microtiter strips in strip holder packed in an aluminum bag with desiccant

12 x 8 tests

WASHBUF

Wash buffer concentrate 20x, natural cap

1 x 50 ml

STD

Standards 1-6, (0; 10; 20; 40; 80; 160 pmol/l), recombinant human DKK-1 in human serum, white caps, lyophilized

6 vials

CTRL

Control, yellow cap, lyophilized, exact concentration after reconstitution see label

1 vial

ASYBUF

Assay buffer, red cap, ready to use

1 x 10 ml

AB

Polyclonal rabbit anti-human DKK-1 – biotin labeled, green cap, ready to use

1 x 7 ml

CONJ

Conjugate (streptavidin-HRP), amber cap, ready to use

1 x 13 ml

SUB

Substrate (TMB solution), amber bottle, blue cap, ready to use

1 x 13 ml
STOP STOP solution, white cap, ready to use 1 x 7 ml

Storage instructions: All reagents of the DKK-1 ELISA kit are stable at 2-8°C until the expiry date stated on the label of each reagent.

Instructions for use

Sample Collection & Storage

Serum is suitable for use in this assay. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards and controls. The sample collection and storage conditions listed are intended as general guidelines.
 

Serum

 
Collect venous blood samples in standardized serum separator tubes (SST). Allow samples to clot for 30 minutes at room temperature. Perform separation by centrifugation according to the tube manufacturer’s instructions for use. Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or hemolyzed samples may give erroneous results. Samples can undergo up to three freeze-thaw cycles. 

Reagent Preparation

Wash Buffer

 

1.

Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature.

2.

Dilute the WASHBUF concentrate 1:20, e.g. 50 ml WASHBUF + 950 ml distilled or deionized water. Only use diluted WASHBUF when performing the assay.

The diluted WASHBUF is stable up to one month at 4°C (2-8°C).
 

Standards & Controls for Serum Measurements

 

1.

Pipette 200 µl of distilled or deionized water into each standard (STD) and control (CTRL) vial. The exact concentration is printed on the label of each vial.

2.

Leave at room temperature (18-24°C) for 15 min. Swirl gently.

Reconstituted STDs and CTRLs are stable at -25°C or lower until expiry date stated on the label. Avoid more than one freeze-thaw cycle.

Sample Preparation

Bring samples to room temperature and mix gently to ensure the samples are homogenous. We recommend duplicate measurements for all samples.

Samples for which the OD value exceeds he highest point of the standard range can be diluted with STD1 (standard 1).

Assay Protocol

Read the entire protocol before beginning the assay.

1.

Bring samples and reagents to room temperature (18-24°C).

2.

Mark positions for STD/CTRL/SAMPLE (standard/control/sample) on the protocol sheet.

3.

Take microtiter strips out of the aluminum bag. Store unused strips with desiccant at 4°C in the aluminum bag. Strips are stable until expiry date stated on the label.

4.

Pipette 50 µl ASYBUF (assay buffer, red cap) into each well.

5.

Add 20 µl STD/CTRL/SAMPLE (standard/control/sample) in duplicates into the respective wells, swirl gently.

6.

Add 50 µl AB (biotinylated anti-DKK-1 antibody, green cap) into each well, swirl gently.

7.

Cover the plate tightly and incubate for 2 hours at room temperature (18-24°C).

8.

Aspirate and wash wells 5x with 300 µl diluted WASHBUF (wash buffer). After the final wash, remove remaining WASHBUF by strongly tapping the plate against a paper towel.

9.

Add 100 µl CONJ (conjugate, amber cap) into each well.

10.

Cover tightly and incubate for 1 hour at room temperature (18-24°C).

11.

Aspirate and wash wells 5x with 300 µl diluted WASHBUF (wash buffer). After the final wash, remove remaining WASHBUF by strongly tapping the plate against a paper towel.

12.

Add 100 µl SUB (substrate, blue cap) into each well.

13.

Incubate for 30 min at room temperature (18-24°C) in the dark.

14.

Add 50 µl STOP (stop solution, white cap) into each well, swirl gently.

15.

Measure absorbance immediately at 450 nm with reference 630 nm, if available.

Calculation of Results

Read the optical density (OD) of all wells on a plate reader using 450 nm wavelength (reference wavelength 630 nm). Construct a standard curve from the absorbance read-outs of the standards using commercially available software capable of generating a four-parameter logistic (4-PL) fit. Alternatively, plot the standards’ concentration on the x-axis against the mean absorbance for each standard on the y-axis and draw a best fit curve through the points on the graph. Curve fitting algorithms other than 4-PL have not been validated and will need to be evaluated by the user.

Obtain sample concentrations from the standard curve. If required, pmol/l can be converted into pg/ml by applying a conversion factor (DKK-1: 1 pg/ml = 0.039 pmol/l; MW: 25.8 kDa). Respective dilution factors have to be considered when calculating the final concentration of the sample.

The quality control protocol supplied with the kit shows the results of the final release QC for each kit. ODs obtained by customers may differ due to various influences including a normal decrease of signal intensity throughout shelf life. However, this does not affect validity of results as long as an OD of 1.5 or higher is obtained for STD 6 and the value of the CTRL is within the target range (see label).

Background & Therapeutic Areas

Information on the Analyte

DKK-1 protein

DKK-1 is a 25.8 kDa secreted protein functioning as antagonist of the canonical Wnt signaling pathway. It is a member of the dickkopf-related protein family characterized by two cysteine-rich domains separated by a linker region. Binding of DKK-1 to the LRP5/6 co-receptor inhibits the interaction with Wnt and by forming a ternary complex with transmembrane Kremen-1 or -2, promotes the internalization of LRP5/6.

Molecular Weight

25.8 kDa

Cellular localization

Extracellular

Post-translational modifications

Glycosylation

Sequence similarities

Member of the dickkopf-related protein family

Alternative Names

Dickkopf-1, DKK1, dickkopf WNT signaling pathway inhibitor, dickkopf (Xenopus laevis) homolog 1, dickkopf homolog 1 (Xenopus laevis), dickkopf related protein-1, dickkopf-related protein 1, SKdickkopf-1 like

Entrez/NCBI ID

22943 link: https://www.ncbi.nlm.nih.gov/gene/22943

Genecards

GC10P052314 link: https://www.genecards.org/cgi-bin/carddisp.pl?gene=DKK1

OMIM

605189 link: http://omim.org/entry/605189

Protein Atlas

DKK1 link: https://www.proteinatlas.org/ENSG00000107984-DKK1/tissue

Uniport ID

O94907 (DKK1_HUMAN) link: https://www.uniprot.org/uniprot/O94907

DKK-1 Function

 

The Wnt signaling pathway is involved in embryonic development, tissue differentiation and homeostasis as well as carcinogenesis. As soluble inhibitor of Wnt signaling, DKK-1 influences cell fate, proliferation, migration, polarity and gene expression.

Wnt signaling is particularly important in bone homeostasis as it induces the differentiation of osteoblasts while inhibiting osteoclastogenesis. DKK-1 is expressed in mature osteoblast and osteocytes and regulates the differentiation of osteoblasts. Thus, DKK-1 is centrally involved in the regulation of bone remodeling and its dysregulation is associated with bone pathologies.

Moreover, DKK-1 has emerged as a biomarker of cancer progression and prognosis as well as potential therapeutic target in various types of malignancies. In a range of different types of cancers, DKK-1 is further associated with formation of bone metastasis and osteolytic bone lesions.

  • Bone Disease

    Osteoporosis

    Rheumatoid arthritis

    Ankylosing spondylitis

    Axial spondyloarthritis

    Osteoarthritis

    Cancer-induced bone disease

    Psoriatic arthritis

    Primary hyperparathyroidism

    Osteonecrosis

    Osteopathy in type 1 diabetes

    Osteogenesis imperfecta

  • Cancer

    Breast cancer

    Prostate cancer

    Multiple myeloma

    Cervical cancer

    Hepatocellular carcinoma

    Non-small cell lung cancer

    Papillary thyroid cancer

    Lung cancer

    Bone metastasis

    Cutaneous malignant melanoma

    Monoclonal gammopathy of undetermined significance (MGUS)

    Pancreatic ductal adenocarcinoma

    Osteosarcoma

    Chondrosarcoma

    Esophageal squamous cell carcinoma

    Papillary thyroid cancer

    Bladder cancer

    Gastric cancer

    Colorectal cancer

    Chronic lymphocytic leukemia

    Pancreatic cancer

  • Cardiovascular disease

    Abdominal aortic calcification

  • Other

    Systemic mastocytosis

    Nonalcoholic fatty liver disease

    Type 2 Diabetes Mellitus

    Anorexia nervosa

    Acute infections

    HIV-associated neurocognitive impairment

     

Literature

Molecular bases of the regulation of bone remodeling by the canonical Wnt signaling pathway.
Glass, D.A., Karsteny, G., 2006. Curr Top Dev Bio 73, 43-84.

PMID: 16782455

Wnt/β-catenin signaling: components, mechanisms, and diseases.
MacDonald, B.T., Tamai, K., He, X., 2009. Dev Cell 17, 9-26.
PMID: 19619488

The emerging role of Dickkopf-1 in bone biology: is it the main switch controlling bone and joint remodeling?
Daoussis, D., Andonopoulos, A.P., 2011. Semin Arthritis Rheum 2, 170-177.
PMID: 21435697

Wnt signaling in cancer.
Zahn, T., Rindtorff, N., Boutros, M., 2017. Oncogene 36, 1461-1473.
PMID: 27617575

Dickkopf-1 as a mediator and novel target in malignant bone disease.
Rachner, T.D., Göbel, A., Benad-Mehner, P., Hofbauer, L.C., Rauner, M., 2014. Cancer Lett 346, 172-177.
PMID: 24462802

Rationale for targeting the Wnt signalling modulator Dickkopf-1 for oncology.
Kagey, M.H., He, X., 2017. Br J Pharmacol 174, 4637-4650.
PMID: 28574171

Severe abdominal aortic calcification in older men is negatively associated with DKK1 serum levels: the STRAMBO study.
Szulc, P., Schoppet, M., Rachner, T.D., Chapurlat, R., Hofbauer, L.C., 2014. J Clin Endocrinol Metab 99, 617-624.
PMID: 24276456

Validation Data

All Biomedica ELISAs are validated according to international FDA/ICH/EMEA guidelines. For more information about our validation guidelines, please refer to our quality page and published validation guidelines and literature.

1.          ICH Q2(R1)  Validation of Analytical Procedures: Text and Methodology

2.          EMEA/CHMP/EWP/192217/2009 Guideline on bioanalytical method validation

3.          Bioanalytical Method Validation, Guidance for Industry, FDA, May 2018

Calibration

The DKK-1 immunoassay is calibrated against recombinant DKK-1 protein (Uniprot ID O94907 (link: https://www.uniprot.org/uniprot/O94907)).

Detection Limit & Sensitivity

To determine the sensitivity of the DKK-1 ELISA, experiments measuring the lower limit of detection (LOD) and the lower limit of quantification (LLOQ) were conducted.

The LOD, also called the detection limit, is the lowest point at which a signal can be distinguished above the background signal, i.e. the signal that is measured in the absence of DKK-1, with a confidence level of 99%. It is defined as the mean back calculated concentration of standard 1 (0 pmol/l of DKK-1, five independent measurements) plus three times the standard deviation of the measurements.

The LLOQ, or sensitivity of an assay, is the lowest concentration at which an analyte can be accurately quantified. The criteria for accurate quantification at the LLOQ are an analyte recovery between 75 and 125% and a coefficient of variation (CV) of less than 25%. To determine the LLOQ, standard 2, i.e. the lowest standards containing DKK-1, is diluted, measured and its concentration back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.  

The following values were determined for the DKK-1 ELISA:

LOD

1.7 pmol/l

LLOQ

1.25 pmol/l

Precision

The precision of an ELISA is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators at different locations using different ELISA lots (in-between-run precision or reproducibility).
 

Within-Run Precision

 
Within-run (intra-assay precision) was assessed by measuring two samples of known concentration five times within one DKK-1 ELISA lot by one operator. 

ID

n

Mean DKK-1 [pmol/l]

SD [pmol/l]

CV [%]

S1

5

19.9

0.5

3

S2

5

80.1

2.7

3

In-Between-Run Precision

 
In-between-run (inter-assay precision) was assessed by measuring two samples nine times within two DKK-1 ELISA kit lots by two different operators.

ID

n

Mean DKK-1 [pmol/l]

SD [pmol/l]

CV [%]

S1

9

19.7

0.6

3

S2

9

80.4

2.1

3

Accuracy

The accuracy of an ELISA is defined as the precision with which it can recover samples of known concentrations.

The recovery of the DKK-1 ELISA was measured by adding recombinant DKK-1 to human samples containing a known concentration endogenous DKK-1. The % recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value. All our ELISAs are expected to have % recovery rates within 15% of the nominal value of the sample.

This table shows the summary of the recovery experiments in the DKK-1 ELISA in serum matrix:

 

 

% Recovery

Sample Matrix

n

+40 pmol/l , DKK-1

+80 pmol/l, DKK-1

Mean

Range

Mean

Range

Serum

7

93%

86-103%

96%

87-106%

 

+ Individual measurements
 

Recovery of spiked samples was tested by adding two concentrations of human recombinant DKK-1 (40 pmol/l + 80 pmol/l) to human serum.

Data showing spike/recovery of human serum samples:

 

DKK-1 [pmol/l]

% Recovery

Sample ID

Reference

+40 pmol/l

+80 pmol/l

+40 pmol/l

+80 pmol/l

#s1

6

43

88

96%

103%

#s2

10

38

79

86%

87%

#s3

12

43

82

92%

94%

#s4

29

50

90

93%

92%

#s5

41

57

98

93%

92%

#s6

50

61

104

92%

95%

#s7

61

70

122

103%

106%
     

Mean [%]

93

96

Dilution Linearity & Parallelism

Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing DKK-1 behave in a dose dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted clinical samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in clinical samples and provides evidence that the endogenous analyte behaves in same way as the recombinant one. Dilution linearity and parallelism are assessed for each sample type and are acceptable if the results are within ±20% of the expected concentration.

Dilution linearity was assessed by serially diluting samples spiked with recombinant DKK-1 with STD1 (standard 1).

The table below shows the mean recovery and range of serially diluted recombinant DKK-1:

 

 

% Recovery of recombinant DKK-1 in diluted samples

Sample

Matrix

n

1+1

1+3

1+7

Mean 

Range

Mean 

Range

Mean 

Range

Serum

3

106

91-116

102

93-109

102

100-105

 

+ Individual measurements
Data showing dilution linearity of recombinant DKK-1 in human serum samples:
   

DKK-1 [pmol/l]

% Recovery

Sample matrix

ID

Reference

1+1

1+3

1+7

1+1

1+3

1+7

Serum

s1

80

36.2

16.8

8.6

91

93

102

Serum

s2

94.2

54.5

29.8

15.0

116

109

100

Serum

s3

135

75

37.1

19.5

111

99

105
       

 

Mean

106

102

102
       

 

Min

91

93

100
       

 

Max

116

109

105

Parallelism was assessed by serially diluting samples containing endogenous DKK-1 with with STD1 (standard 1).

The table below show the mean recovery and range of serially diluted endogenous DKK-1:

 

 

 

% Recovery of endogenous DKK-1 in diluted samples

Sample

Matrix

n

1+1

1+3

1+7

Mean 

Range

Mean 

Range

Mean 

Range

Serum

4

109

108-112

104

99-107

100

90-114

 

+ Individual measurements
Data showing dilution linearity of endogenous DKK-1 in human serum samples:
   

DKK-1 [pmol/l]

% Recovery

Sample matrix

ID

Reference

1+1

1+3

1+7

1+1

1+3

1+7

Serum

s1

 51.8 28.5 14.8 8.4 110 103 114

Serum

s2

 30.2 16.3 8.7 3.9 108 107 90

Serum

s3

 47.6 26.5 14.0 6.6 112 106 95

Serum

s4

 42.8 23.1 11.4 5.7 108 99 100
          Mean 109 104 100
          Min  108 99 90
          Max 110 107 114

Specificity

The specificity of an ELISA is defined as its ability to exclusively recognize the analyte of interest.

The specificity of the ELISA was established through competition experiments, which measure the ability of the antibodies to exclusively bind DKK-1.
 

Competition of Signal

 
Competition experiments were carried out by pre-incubating human samples with an excess of coating antibody. The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample but without the pre-incubation step. The mean competition was 98%, demonstrating that this assay is specific for DKK-1.

   

 

DKK-1 [pmol/l]

% Competition

Sample matrix

ID

Reference

Reference + capture AB

Serum

s1

40

0

100

Serum

s2

42

5

89

Serum

s3

85

0

100

Serum

s4

255

22

92

Serum

s5

16

0

100

Serum

s6

44

0

100

Serum

s7

142

3

98

Serum

s8

86

2

97

Serum

s9

106

3

97

Serum

s10

17

0

100

Serum

s11

46

0

100

Serum

s12

26

0

100

Serum

s13

56

1

98

Serum

s14

51

3

95
      Mean

98

Cross-Reactivity

The cross-reactivity of the DKK-1 ELISA was tested with recombinant DKK-2, which is 42% identical to DKK-1. The DKK-1 ELISA does not cross-react with DKK-2. No cross-reactivity or interference with recombinant human DKK-4, Kremen-1, Kremen-2 or LRP-6 is observed.

Sample Stability

The stability of endogenous DKK-1 was tested by comparing measurements in samples that had undergone six freeze-thaw cycles.

For freeze-thaw experiments, samples were collected according to the supplier’s instruction using blood collection devices and stored at -80°C. The mean recovery of sample concentration after an additional three freeze-thaw (F/T) cycles is 93%.

   

DKK-1 [pmol/l]

% Recovery

Sample matrix

ID

Reference

3x F/T cycles

6x F/T cycles

3x F/T cycles

6x F/T cycles

Serum

s1

43

40

40

93

92

Serum

s2

70

63

62

90

89

Serum

s3

46

46

43

94

93

Serum

s4

21

21

19

96

88

Serum

s5

30

30

31

100

103

Serum

s6

41

39

38

95

91

Serum

s7

29

26

27

89

92

Serum

s8

46

43

40

92

87

Serum

s9

152

139

131

91

86

Serum

s10

106

99

93

94

88
       

Mean

93

91

All samples should undergo a maximum of three freeze-thaw cycles.

Sample Values

DKK-1 Values in Apparently Healthy Individuals

 
To provide expected values for circulating DKK-1, a panel of samples from apparently healthy donors was tested.

A summary of the results is shown below:

 

  

DKK-1 [pmol/l]

Sample Matrix

n

Mean

Median

Minimum

Maximum

Serum

51

35

34

5

70

We recommended establishing the normal range for each laboratory.
 

DKK-1 Values in an Osteoporosis Panel

 
In addition to samples from apparently healthy donors, a panel of samples from osteoporosis patients was tested.

A summary of the results is shown below:

 

  

DKK-1 [pmol/l]

Sample Matrix

n

Mean

Median

Minimum

Maximum

Serum

48

69

60

25

150

A comparison of apparently healthy individuals and patients with osteoporosis shows that DKK-1 is increased in osteoporosis patients:



Automation

The assay was programmed on the ETI-Max3000. Standards and samples were tested and show a CV of <7% in the low to high calibration range. The coefficient of determination is >0.998 for a logit-log transformation.

An assay protocol for the ETI-max3000 is available on request.

Citations

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