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Osteoprotegerin ELISA

Cat. No.: BI-20403

/ € 540Please Inquire

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Therapeutic Areas
1
Assay Characteristics
  • Category number

    BI-20403

  • Method

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

  • Sample type

    Serum, EDTA plasma, citrate plasma, heparin plasma

  • Sample volume

    20 µl / well

  • Assay time

    4 h / 1 h / 30 min

  • Sensitivity

    0.07 pmol/l

  • Standard range

    0 – 20 pmol/l (= 0 – 400 pg/ml)

  • Conversion factor

    1 pg/ml = 0.05 pmol/l (MW: 19.9 kDa)

  • Specificity

    Recombinant and endogenous human OPG.

  • Precision

    In-between-run (n=12): ≤ 5 % CV

    Within-run (n=5): ≤ 3 % CV

  • Cross-reactivity

    The assay does not cross react with rat or mouse samples.

  • Use

    CE marked – for IVD use in the EU

  • Validation Data

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

Product details

Product Overview

The Osteoprotegerin (OPG) immunoassay is a 4.5 hour, 96-well sandwich ELISA for the quantitative determination of OPG in human serum and plasma. The assay employs human serum-based standards to ensure the measurement of biologically reliable data.

Principle of the Assay

This kit is a sandwich enzyme immunoassay for the determination of OPG in human serum and plasma samples.

The figure below explains the principle of the Osteoprotegerin sandwich ELISA:



In a first step, assay buffer, standard/control/sample and detection antibody (biotinylated monoclonal mouse anti-human OPG) are pipetted into the wells of the microtiter strips, which are pre-coated with polyclonal goat anti-OPG antibody. OPG 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 second 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 OPG. This color change is detectable with a standard microtiter plate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) vs. standard concentration is generated. The concentration of OPG in the sample is determined directly from the dose response curve.

Typical Standard Curve

The figure below shows a typical standard curve for the OPG ELISA. The immunoassay is calibrated against recombinant human OPG:



ELISA Kit Components

Contents

Description

Quantity

PLATE

Polyclonal goat anti-OPG antibody pre-coated microtiter strips in strip holder, packed in aluminum bag with desiccant

12 x 8 tests

WASHBUF

Wash buffer concentrate 20x, natural cap

1 x 50 ml

STD

Standards 1-6, (0; 1.25; 2.5; 5; 10; 20 pmol/l), recombinant human OPG in human serum, white caps, ready to use

6 x 300 µl

CTRL

Control, yellow cap, ready to use, exact concentration see label

1 x 300 µl

ASYBUF

Assay buffer, red cap, ready to use

1 x 25 ml

AB

Monoclonal mouse anti-OPG antibody, biotin labeled, green cap, yellow dye, ready to use

1 x 7 ml

CONJ

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

1 x 22 ml

SUB

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

1 x 22 ml

STOP

Stop solution, white cap, ready to use

1 x 7 ml

Storage instructions: All reagents of the OPG ELISA kit are stable at 4°C (2-8°C) until the expiry date stated on the label of each reagent.

Instructions for use

Sample Collection & Storage

Serum, EDTA plasma, heparin plasma and citrate plasma are 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 & Plasma

Collect venous blood samples in standardized serum separator tubes (SST) or standardized blood collection tubes using EDTA, heparin or citrate as an anticoagulant. For serum samples, allow samples to clot for 30 minutes at room temperature. Perform separation by centrifugation according to the tube manufacturer’s instructions for use. If this is not possible store the samples at 4°C (2-8°C) prior to centrifugation (up to one day). Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or haemolyzed samples may give erroneous results. Samples can undergo at least four 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).

Sample Preparation

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

Samples for which the OD value exceeds the highest point of the standard range can be diluted with STD1 or OPG negative human serum.

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

5.

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

6.

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

7.

Cover the plate tightly and incubate for 4 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 200 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 200 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 (1 pg/ml = 0.05 pmol/l; OPG MW: 19.9 kDa). Respective dilution factors have to be considered when calculating the final concentration of the sample.

The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each kit at production date. Data for OD obtained by customers may differ due to various influences including the normal decrease of signal intensity throughout shelf life. However, this does not affect validity of results as long as an OD of 1.50 or more is obtained for STD6 and the value of the CTRL is within the target range (see label).

Background & Therapeutic Areas

INFORMATION ON THE ANALYTE

OPG Protein

Osteoprotegerin (OPG) or osteoclast inhibitory factor (OCIF) is a glycoprotein of the tumor necrosis factor receptor superfamily encoded by the TNFRSF11B gene. OPG is synthesized as a monomer of 380 amino acids, assembled as a homodimer within the cell and then secreted mainly as a disulfide-linked homodimer into the extracellular compartment. OPG is produced by many different tissues and cell types including osteoblasts, breast tissue, vascular endothelial cells as well as B cells and dendritic cells in the immune system.

Molecular Weight

19.9 kDa

Cellular localization

Extracellular

Post-translational modifications

Glycosylation

Sequence similarities

Member of the tumor necrosis factor receptor superfamily

Alternative Names

TNFRSF11B, OCIF, PDB5, TR1, tumor necrosis factor receptor superfamily member 11b, TNF receptor superfamily member 11b, osteoclastogenesis inhibitory factor

Entrez/NCBI ID

4982 link: https://www.ncbi.nlm.nih.gov/gene/4982

Genecards

GC08M118923 link: https://www.genecards.org/cgi-bin/carddisp.pl?gene=TNFRSF11B

OMIM

602643 link: https://www.omim.org/entry/602643

PDB

3URF link: http://www.rcsb.org/structure/3URF

Protein Atlas

TNFRSF11B link: https://www.proteinatlas.org/ENSG00000164761-TNFRSF11B/tissue

Uniport ID

O00300 link:https://www.uniprot.org/uniprot/O00300

OPG Function

OPG is a negative regulator of bone resorption by acting as decoy receptor for Receptor Activator of NF-κB Ligand (RANKL), thus neutralizing its function in osteoclastogenesis. It is also a decoy receptor for TRAIL and thereby inhibits apoptosis of mature osteoclasts. Hence, OPG is centrally involved in the regulation of bone resorbtion and formation. Since estrogen induces the expression of OPG in osteoblasts, post-menopausal decrease in estrogen levels can lead to the development of osteoporosis due to decreased OPG expression and enhanced RANKL-induced osteoclastogenesis. In contrast to estrogen, glucocorticoids induce the expression of RANKL and decrease the expressioin of OPG, which may lead to glucocorticoid-induced osteoporosis. Proinflammatory cytokines also increase the RANKL/OPG ratio and hence, induce bone loss in diseases like rheumatoid arthritis or periodontitis.

OPG has further been identified to promote tumor growth and survival by inducing tumor vascularization and inhibiting TRAIL-mediated tumor cell death. Dysregulation of the RANKL/OPG system is also common in metastatic bone disease, which is often observed in disseminated cancers of breast, prostate and lung. In multiple myeloma, reduced OPG levels and increased osteoclastogenesis cause the formation of osteolytic bone lesions.

Increased concentrations of OPG, especially in the context of diabetes and chronic kidney disease, are associated with vascular calcification and increased cardiovascular risk.

  • Bone Diseases

    • Osteoporosis

    • Rheumatoid arthritis

    • Osteoarthritis

    • Juvenile idiopathic arthritis

    • Juvenile Paget's disease

    • Paget's disease of the bone

    • Glucocorticoid-induced osteoporosis

    • Ankylosing spondylitis

    • Periodontitis

  • Cardiovascular Diseases

    • Vascular calcification

    • Peripheral artery disease

    • Heart failure

    • Coronary artery disease

  • Oncology

    • Multiple myeloma

    • Hairy cell leukemia

    • Breast cancer

    • Prostate Cancer

    • Lung Cancer

    • Bone metastasis

    • Osteosarcoma

    • Tumor-induced osteomalacia

Literature

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.

Show validation literature

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

  • EMEA/CHMP/EWP/192217/2009 Guideline on Validation of Bioanalytical Methods

  • Bioanalytical Method Validation, Guidance for Industry, FDA, May 2018

Calibration

The OPG immunoassay is calibrated against recombinant human OPG protein (AA 22-194 of Uniprot ID# O00300).

Detection Limit & Sensitivity

To determine the sensitivity of the OPG 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 OPG, with a confidence level of 99%. It is defined as the mean back calculated concentration of standard 1 (0 pmol/l of OPG, 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 OPG, is diluted, measured two times and its concentration is back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.

The following values were determined for the OPG ELISA:

LOD

0.07 pmol/l

LLOQ

0.08 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 (Repeatability or Intra-Assay Precision)

Within-run precision was assessed by measuring two samples of known concentrations five times within one OPG ELISA kit lot by one operater.

ID

n

Mean OPG [pmol/l]

SD [pmol/l]

CV [%]

S1

5

3.2

0.05

2

S2

5

10.1

0.34

3

In-Between Run Precision (Inter-Assay Precision)

In-between-run precision was assessed by measuring two samples twelfe times within two OPG ELISA kit lots by three different operaters.

ID

n

Mean OPG [pmol/l]

SD [pmol/l]

CV [%]

S1

12

3.2

0.10

3

S2

12

9.9

0.5

5

Accuracy

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

The recovery of the OPG ELISA was measured by adding recombinant OPG to human samples containing a known concentration endogenous OPG. 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 OPG ELISA in different sample matrices:

   

% Recovery

Sample Matrix

n

+ 2 pmol/l

+5 pmol/l

+ 10 pmol/l

Mean

Range

Mean

Range

Mean

Range

Serum

3

95

85-106

105

94-111

95

89-99

EDTA plasma

3

100

99-102

93

83-113

108

93-119

Citrate plasma

3

88

82-96

89

76-100

109

99-120

Heparin plasma

3

91

67-118

82

70-95

94

84-110
Show individual measurements

Data showing recovery (R [%]) of recombinant OPG in human serum samples:

   

OPG [pmol/l]

% Recovery

  

Sample Matrix

ID

Reference

+2 pmol/l

+5 pmol/l

+10 pmol/l

+2 pmol/l

+5 pmol/l

+10 pmol/l

  

Serum

s1

3.8

5.5

8.1

13.3

106

94

99

  

Serum

s2

2.6

4.5

8.2

12.3

95

110

97

  

Serum

s3

3.5

5.2

9.0

12.4

85

111

89

  

 

 

 

 

 

 

95

105

95

 Mean

 

 

 

 

 

 

85

94

89

 Min

 

 

 

 

 

 

106

111

99

 Max

Data showing recovery of recombinant OPG in human EDTA plasma samples:

   

OPG [pmol/l]

% Recovery

  

Sample Matrix

ID

Reference

+2 pmol/l

+5 pmol/l

+10 pmol/l

+2 pmol/l

+5 pmol/l

+10 pmol/l

  

EDTA plasma

s1

3.3

5.2

7.4

14.4

99%

83%

112%

  

EDTA plasma

s2

2.5

4.6

6.7

14.4

102%

83%

119%

  

EDTA plasma

s3

3.3

5.3

8.9

12.5

100%

113%

93%

  

 

 

 

 

 

 

100%

93%

108%

 Mean

 

 

 

 

 

 

99

83

93

 Min

 

 

 

 

 

 

102

113

119

 Max

Data showing recovery of recombinant OPG in a human citrate plasma sample:

   

OPG [pmol/l]

% Recovery

  

Sample Matrix

ID

Reference

+2 pmol/l

+5 pmol/l

+10 pmol/l

+2 pmol/l

+5 pmol/l

+10 pmol/l

  

Citrate plasma

s1

2.7

4.4

7.7

12.6

85%

100%

99%

  

Citrate plasma

s2

2.1

4.0

5.9

12.9

96%

76%

108%

  

Citrate plasma

s3

2.8

4.4

7.3

14.8

82%

90%

120%

  

 

 

 

 

 

 

88%

89%

109%

 Mean

 

 

 

 

 

 

82

76

99

 Min

 

 

 

 

 

 

96

100

120

 Max
   

OPG [pmol/l]

% Recovery

  

Sample Matrix

ID

Reference

+2 pmol/l

+5 pmol/l

+10 pmol/l

+2 pmol/l

+5 pmol/l

+10 pmol/l

  

Citrate plasma

s1

3.1

4.4

6.6

11.5

67%

70%

84%

  

Citrate plasma

s2

2.3

4.6

6.4

13.3

118%

82%

110%

  

Citrate plasma

s3

3.1

4.9

7.9

12.0

87%

95%

89%

  

 

 

 

 

 

 

91%

82%

94%

 Mean

 

 

 

 

 

 

67

70

84

 Min

 

 

 

 

 

 

118

95

110

 Max

Dilution Linearity & Paralellism

Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing OPG 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 considered acceptable if the results are within ±20% of the expected concentration.

Dilution linearity was assessed by serially diluting human serum samples spiked with 6 pmol/l recombinant OPG with standard 1.

The table below shows the mean recovery and range of serially diluted recombinant OPG in serum samples:

   

% Recovery of recombinant OPG in diluted samples

Sample

Matrix

n

1+1

Mean

Range

Serum

8

96

84-116
Show individual measurements

Data showing dilution linearity of 6 pmol/l recombinant OPG spiked into human serum samples (reference) containing endogenous OPG:

   

OPG [pmol/l]

% Recovery

  

Sample Matrix

ID

Reference

+6 pmol/l

1+1

1+1

  

Serum

s1

3.7

10.7

5.2

98%

  

Serum

s2

2.3

6.7

3.9

116%

  

Serum

s3

4.4

12.8

5.1

80%

  

Serum

s4

3.4

12.0

5.1

86%

  

Serum

s5

3.3

9.4

5.0

107%

  

Serum

s6

3.6

10.3

5.2

101%

  

Serum

s7

4.4

11.6

4.9

84%

  

Serum

s8

4.7

10.9

5.2

96%

  

 

 

 

 

 

96

 Mean

 

 

 

 

 

80

 Min

 

 

 

 

 

116

 Max

Parallelism was assessed by serially diluting human serum samples containing endogenous OPG with standard 1.

The table below shows the mean recovery and range of serially diluted endogenous OPG in human serum samples:

   

% Recovery of endogenous OPG in diluted samples

Sample

Matrix

n

1+1

1+3

1+7

Mean

Range

Mean

Range

Mean

Range

Serum

3

98

92-102

90

86-94

87

79-86
Show individual measurements

Data showing dilution linearity of endogenous OPG in human serum samples:

   

OPG [pmol/l]

% Recovery

  

Sample Matrix

ID

Reference

1+1

1+3

1+7

1 + 1

1 + 3

1 + 7

  

Serum

s1

8.2

4.1

1.9

0.9

99%

94%

86%

  

Serum

s2

3.6

1.9

0.8

0.4

102%

92%

95%

  

Serum

s3

6.7

3.1

1.4

0.7

92%

86%

79%

  

 

 

 

 

 

 

98

90

87

 Mean

 

 

 

 

 

 

92

86

79

 Min

 

 

 

 

 

 

102

94

95

 Max

Specificity

The OPG ELISA recognizes human endogenous and recombinant OPG. It detects monomeric and dimeric OPG as well as OPG-RANKL complexes. The assay does not cross-react with rat or mouse samples.

Sample Stability

The stability of endogenous OPG was tested by comparing OPG measurements in samples that had undergone four freeze-thaw (F/T) cycles.

For freeze-thaw experiments, samples were collected according to the supplier’s instruction using blood collection devices and stored at -80°C. Reference samples were freeze-thawed once. The mean recovery of sample concentration after four freeze-thaw cycles is 101%.

   

OPG [pmol/l]

% Recovery after 4 (F/T) cycles

  

Sample Matrix

ID

Reference

2x

3x

4x

  

Serum

s1

2.8

2.8

3.7

2.5

86%

  

Serum

s2

3.1

3.8

4.1

3.7

119

  

Serum

s3

5.0

5.4

5.3

5.1

103

  

Serum

s4

2.6

3.0

2.6

2.5

94

  

 

 

 

 

 

 

101

 Mean

Samples can undergo at least up to four freeze-thaw cycles.

Sample Values

OPG Values in Apparently Healthy Individuals

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

A summary of the results is shown below:

   

OPG [pmol/l]

Sample Matrix

n

Mean

Serum

60

2.7

EDTA plasma

6

2.2

Citrate plasma

5

2.3

Heparin plasma

7

2.3

It is recommended to establish the normal range for each laboratory.

Matrix Comparison

To assess whether all tested matrices behave the same way in the OPG ELISA, concentrations of OPG were measured in serum, EDTA, citrate and heparin plasma samples prepared from four apparently healthy donors. Each individual donated blood in all tested sample matrices.

A summary table of OPG levels in various sample matrices is shown below:

 

OPG [pmol/l]

 

Sample ID

Serum

EDTA plasma

Citrate plasma

Heparin plasma

% CV

  

#1

2.8

3.4

2.4

2.9

14

  

#2

2.8

2.8

2.6

2.3

9

  

#3

2.1

3.1

2.3

2.6

17

  

#4

0.9

1.0

0.8

0.8

11

  
         

13

Mean

Citations

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