Developed & Manufactured by Biomedica

FREE soluble RANKL HS ELISA

Cat. No.: BI-20462

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Therapeutic Areas
1
2
Assay Characteristics
  • Method

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

  • Sample type

    Serum, heparin plasma

  • Sample volume

    150 µl / well

  • Assay time

    2 h / overnight / 1 h / 30 min

  • Sensitivity

    0.01 pmol/l (= 0.2 pg/ml)

  • Standard range

    0 – 2 pmol/l (= 0 – 40 pg/ml)

  • Conversion factor

    1 pg/ml = 0.05 pmol/l (MW: 20 kDa; monomer) or 1 pmol= 20 pg/ml

  • Specificity

    Endogenous and recombinant free soluble RANKL

  • Precision

    In-between-run (n=12): ≤ 3 % CV

    Within-run (n=5): ≤ 5 % CV

  • Cross-reactivity

    The sequence homology to various primates is >95%. It is likely that the assay can be used for these species.

  • 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 FREE soluble RANKL HS immunoassay is an overnight, 96-well sandwich ELISA for the quantitative determination of free soluble RANKL in serum and heparin plasma.

Principle of the Assay

This kit is a sandwich enzyme immunoassay for the quantitative determination of free soluble RANKL in human serum and heparin plasma samples.

The figure below explains the principle of the FREE soluble RANKL HS ELISA:

In a first step, wells which are pre-coated with recombinant Osteoprotegerin (OPG), RANKL’s interaction partner, are prewashed to ensure optimal sensitivity. Thereafter, assay buffer is pipetted into the wells of the microtiter strips followed by the addition of standard/control/sample. Any soluble RANKL present in the standard/control/sample binds to the pre-coated OPG in the well. After incubation, a washing step is applied where all non-specific unbound material is removed. In a next step, biotinylated detection antibody (polyclonal goat anti-sRANKL) is pipetted into the wells and reacts with the sRANKL present in the sample, forming a sandwich. Next, all unbound antibody is removed during another washing cycle. In the following step, the conjugate (streptavidin-polyHRP) is added and reacts with the detection antibody. After a final 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 soluble RANKL present in the sample. 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 standards. The concentration of soluble RANKL in the sample is determined directly from the dose response curve.

Typical Standard Curve

The figure below shows a typical standard curve for the free soluble RANKL ELISA. The immunoassay is calibrated against recombinant RANKL peptide:

ELISA Kit Components   

Contents

Description

Quantity

PLATE

Human recombinant OPG 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 (0; 0.0625; 0.125; 0.25; 0.5; 1; 2 pmol/l), lyophilized, white caps

7 vials

CTRL

Control A+B, recombinant human RANKL in human serum, yellow caps, lyophilized, exact concentration see label

2 vials

ASYBUF

Assay buffer, red cap, ready to use

1 x 7 ml

AB

Goat polyclonal biotinylated anti-sRANKL antibody, green cap, ready to use

1 x 22 ml

CONJ

Conjugate (streptavidin-polyHRP), 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 free soluble RANKL ELISA kit are stable at 4°C until the expiry date stated on the label of each reagent.

Instructions for use

Sample Collection & Storage

Serum and heparin 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. Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or haemolyzed samples may give erroneous results. Samples should undergo three freeze-thaw cycles only.

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

 

1.

Pipette 700 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 (18-26°C) for 15 min. Vortex gently.

Reconstituted STDs and CTRLs are stable at -25°C or lower until expiry date stated on the label. STDs and CTRLs are stable for three freeze-thaw cycles.

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 a serum sample with a low OD value.

Assay Protocol

Read the entire protocol before beginning the assay.

1.

Bring reagents and samples to room temperature (18-26°C).

2.

Mark position 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 (2-8°C) in the aluminum bag. Strips are stable until expiry date stated on the label.

4.

Prewash wells with 300 µl diluted WASHBUF (wash buffer) five times.

Remove remaining WASHBUF by strongly tapping plate against paper towel after the last wash.

5.

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

6.

Add 150 µl STD/CTRL/SAMPLE in duplicates into the respective wells.

7.

Cover the plate tightly and incubate for 2 hours at room temperature (18-26°C).

8.

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

9.

Add 200 µl AB (biotinylated anti-sRANKL antibody, green cap) into each well. Swirl gently.

10.

Cover tightly and incubate at 4°C (2-8°C) overnight (18-24 hours).

11.

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

12.

Add 200 µl CONJ (conjugate, amber cap) into each well, swirl gently. 

13.

Cover tightly and incubate for 1 hour at room temperature in the dark.

14.

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

15.

Add 200 µl SUB (substrate, blue cap) into each well. 

16.

Incubate for 30 min at room temperature in the dark.

17.

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

18.

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 to pg/ml by applying a conversion factor (sRANKL: 1 pg/ml = 0.05 pmol/l (MW: 20 kDa; monomer) or 1 pmol= 20 pg/ml). 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 lot. Data for optical density 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 the standard with the highest concentration and the values of the CTRLs are within the target range (see labels).

Background & Therapeutic Areas

INFORMATION ON THE ANALYTE

RANKL Protein

RANKL, the receptor activator of nuclear factor kappa B ligand, a member of the tumor necrosis factor (TNF) family (http://www.uniprot.org/uniprot/O14788), is the main stimulatory factor for the formation of mature osteoclasts and is essential for their survival. RANKL activates its specific receptor RANK, located on osteoclasts and dendritic cells. The effects are counteracted by Osteoprotegerin (OPG) which acts as an endogenous soluble receptor antagonist (see: OPG ELISA, cat.no. BI-20403).

The major source of RANKL are osteocytes, former osteoblasts that become embedded within the mineralized bone matrix. RANKL is a ~35 kD type II transmembrane-type protein and is cleaved to release a soluble biologically active product that forms a homotrimer.

Molecular Weight

20 kDa (soluble form)

Cellular localisation

Intracellular, Membrane, Secreted

Post-translational modifications

Cleavage, glycosylation

Sequence similarities

Tumor necrosis factor (TNF) cytokine family

Alternative Names

TNF Superfamily Member, Tumor Necrosis Factor (Ligand) Superfamily, Member TNF-Related Activation-Induced Cytokine, Osteoclast Differentiation Factor, Osteoprotegerin Ligand, TRANCE, RANKL, OPGL, ODF, Receptor Activator Of Nuclear Factor Kappa B Ligand, Tumor Necrosis Factor Ligand Superfamily Member, Tumor Necrosis Factor Superfamily Member, Tumor Necrosis Factor Ligand 6B, CD254 Antigen, HRANKL, TNLG6B, CD254, OPTB2, SOdf

Entrez/NCBI ID

8600
https://www.ncbi.nlm.nih.gov/gene/8600

Genecards

TNFSF11
https://www.genecards.org/cgi-bin/carddisp.pl?gene=TNFSF11&keywords=tnfsf11

OMIM

602642: https://www.omim.org/entry/602642

PDB

3URF http://www.rcsb.org/structure/3URF;
1S55: http://www.rcsb.org/structure/1S55 ;
1JTZ: http://www.rcsb.org/structure/1JTZ ;
3ME2: http://www.rcsb.org/structure/3ME2 ;
4E4D: http://www.rcsb.org/structure/3URF ;
4GIQ: http://www.rcsb.org/structure/4GIQ ;
3QBQ: http://www.rcsb.org/structure/3QBQ ;
5BNQ: http://www.rcsb.org/structure/5BNQ ;
1IQA: http://www.rcsb.org/structure/1IQA;  

Pfam

PF00229: http://pfam.xfam.org/family/PF00229

Protein Atlas

TNFSF11 https://www.proteinatlas.org/ENSG00000120659-TNFSF11/tissue

Uniport ID

O14788: https://www.uniprot.org/uniprot/O14788

RANKL Function

RANKL and its specific receptor RANK are not only key regulators of bone remodeling but also play an essential role in immunobiology, e.g. lymph node formation, establishment of the thymic microenvironment, mammary gland development during pregnancy, bone metastasis in cancer and sex-hormone, progestin-driven breast cancer, thermoregulation, and finally in the development of type 2 diabetes mellitus (Danks and Takayanagi, 2013; Gonzalez-Suarez et al., 2010; Hanada et al., 2010, 2009; Kiechl et al., 2016; Nakashima et al., 2011; Nelson et al., 2012; Schramek et al., 2010).

  • Bone Disease

    Postmenopausal and senile osteoporosis (Anagnostis et al., 2018; Hofbauer and Schoppet, 2004; Stuss et al., 2016; Tella and Gallagher, 2014)

    Glucocorticoid induced osteoporosis (Compston, 2018; Komori, 2016)

    Disease with locally increased resorption activity

    Rheumatoid Arthritis (Chiu and Ritchlin, 2017; Kurz et al., 2015; Remuzgo-Martínez et al., 2016; Tanaka et al., 2014; Tanaka and Ohira, 2018)

    HIV associated bone loss (Negredo et al., 2015; Pietschmann et al., 2016; Titanji, 2017)

    Ankylosing spondylitis (Caparbo et al., 2018; Perpétuo et al., 2017)

  • Cancer

    Malignant solid tumors (de Groot et al., 2018)

    Breast cancer (Kiechl et al., 2016, 2016; Lüftner et al., 2018; Rachner et al., 2018; Rao et al., 2018; Sarink et al., 2017; Sigl et al., 2016)

    Multiple myeloma (Raje et al., 2019; Terpos et al., 2017)

  • Endocrine disorders

    Test

  • Inflammation

    Test

  • Inflammatory diseases

    Test

  • Lung

    Test

  • Metabolic Disease

    Type 1 diabetes mellitus (Chrysis et al., 2017; Grzelak et al., 2018; Starup-Linde et al., 2016)

    Type 2 diabetes mellitus (Grigoropoulou et al., 2011; Harper et al., 2016; Jansen et al., 2018; Mantovani et al., 2018)

     

     

  • Pulmonology

    Test

  • Respiratory diseases

    Test

Literature

Role of the RANK/RANKL Pathway in Multiple Myeloma.
Raje, N.S., Bhatta, S., Terpos, E., 2019. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 25, 12–20.
PMID: 30093448

The role of sclerostin/dickkopf-1 and receptor activator of nuclear factor kB ligand/osteoprotegerin signalling pathways in the development of osteoporosis in patients with haemophilia A and B: A cross-sectional study.
Anagnostis, P., Vakalopoulou, S., Christoulas, D., Paschou, S.A., Papatheodorou, A., Garipidou, V., Kokkoris, P., Terpos, E., 2018. Haemophilia 24, 316–322.

Monocytes from male patients with ankylosing spondylitis display decreased osteoclastogenesis and decreased RANKL/OPG ratio.
Caparbo, V.F., Saad, C.G.S., Moraes, J.C., de Brum-Fernandes, A.J., Pereira, R.M.R., 2018. Osteoporos. Int. J. Establ. Result Coop. Eur. Found. Osteoporos. Natl. Osteoporos. Found. USA.
PMID: 30006885

Glucocorticoid-induced osteoporosis: an update.
Compston, J., 2018. Endocrine 61, 7–16.
PMID: 29691807; PMCID: PMC5997116

The anti-tumor effect of RANKL inhibition in malignant solid tumors - A systematic review.
de Groot, A.F., Appelman-Dijkstra, N.M., van der Burg, S.H., Kroep, J.R., 2018. Cancer Treat. Rev. 62, 18–28.
PMID: 29154022

Neuropeptide B and neuropeptide W as new serum predictors of nutritional status and of clinical outcomes in pediatric patients with type 1 diabetes mellitus treated with the use of pens or insulin pumps.
Grzelak, T., Wedrychowicz, A., Grupinska, J., Pelczynska, M., Sperling, M., Mikulska, A.A., Naughton, V., Czyzewska, K., 2018. Arch. Med. Sci. 14.

Bone mineral density and markers of bone turnover and inflammation in diabetes patients with or without a Charcot foot: An 8.5-year prospective case-control study.
Jansen, R.B., Christensen, T.M., Bülow, J., Rørdam, L., Holstein, P.E., Jørgensen, N.R., Svendsen, O.L., 2018. J. Diabetes Complications 32, 164–170.
PMID: 29196119

Therapeutic approaches for protecting bone health in patients with breast cancer.
Lüftner, D., Niepel, D., Steger, G.G., 2018. Breast Edinb. Scotl. 37, 28–35.
PMID: 29073497

Association between non-alcoholic fatty liver disease and bone turnover biomarkers in post-menopausal women with type 2 diabetes.
Mantovani, A., Sani, E., Fassio, A., Colecchia, A., Viapiana, O., Gatti, D., Idolazzi, L., Rossini, M., Salvagno, G., Lippi, G., Zoppini, G., Byrne, C.D., Bonora, E., Targher, G., 2018. Diabetes Metab.
PMID: 30315891

Prognostic value of RANKL/OPG serum levels and disseminated tumor cells in non-metastatic breast cancer.
Rachner, T.D., Kasimir-Bauer, S., Göbel, A., Erdmann, K., Hoffmann, O., Browne, A.J., Wimberger, P., Rauner, M., Hofbauer, L.C., Kimmig, R., Bittner, A.-K., 2018. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res.
PMID: 30425091

RANKL and RANK: From Mammalian Physiology to Cancer Treatment.
Rao, S., Cronin, S.J.F., Sigl, V., Penninger, J.M., 2018. Trends Cell Biol. 28, 213–223.
PMID: 29241686

Mechanisms and therapeutic targets for bone damage in rheumatoid arthritis, in particular the RANK-RANKL system.
Tanaka, Y., Ohira, T., 2018. Curr. Opin. Pharmacol. 40, 110–119.
PMID: 29702364

Denosumab: targeting the RANKL pathway to treat rheumatoid arthritis.
Chiu, Y.G., Ritchlin, C.T., 2017. Expert Opin. Biol. Ther. 17, 119–128.
PMID: 27871200; PMCID: PMC5794005

Osteoprotegerin, RANKL, ADMA, and Fetuin-A serum levels in children with type I diabetes mellitus.
Chrysis, D., Efthymiadou, A., Mermigka, A., Kritikou, D., Spiliotis, B.E., 2017. Pediatr. Diabetes 18, 277–282.
PMID: 27028343

Ankylosing Spondylitis Patients Have Impaired Osteoclast Gene Expression in Circulating Osteoclast Precursors.
Perpétuo, I.P., Caetano-Lopes, J., Vieira-Sousa, E., Campanilho-Marques, R., Ponte, C., Canhão, H., Ainola, M., Fonseca, J.E., 2017. Front. Med. 4.

Circulating RANKL and RANKL/OPG and Breast Cancer Risk by ER and PR Subtype: Results from the EPIC Cohort.
Sarink, D., Schock, H., Johnson, T., Overvad, K., Holm, M., Tjønneland, A., Boutron-Ruault, M.-C., His, M., Kvaskoff, M., Boeing, H., Lagiou, P., Papatesta, E.-M., Trichopoulou, A., Palli, D., Pala, V., Mattiello, A., Tumino, R., Sacerdote, C., Bueno-de-Mesquita, H.B.A., van Gils, C.H., Peeters, P.H., Weiderpass, E., Agudo, A., Sánchez, M.-J., Chirlaque, M.-D., Ardanaz, E., Amiano, P., Khaw, K.T., Travis, R., Dossus, L., Gunter, M., Rinaldi, S., Merritt, M., Riboli, E., Kaaks, R., Fortner, R.T., 2017. Cancer Prev. Res. Phila. Pa 10, 525–534.
PMID: 28701332; PMCID: PMC5603271

Mechanisms of bone destruction in multiple myeloma.
Terpos, E., Christoulas, D., Gavriatopoulou, M., Dimopoulos, M.A., 2017. Eur. J. Cancer Care (Engl.) 26.
PMID: 28940410

Beyond Antibodies: B Cells and the OPG/RANK-RANKL Pathway in Health, Non-HIV Disease and HIV-Induced Bone Loss.
Titanji, K., 2017. Front. Immunol. 8, 1851.
PMID: 29312334; PMCID: PMC5743755

Vascular calcification in type-2 diabetes and cardiovascular disease: Integrative roles for OPG, RANKL and TRAIL.
Harper, E., Forde, H., Davenport, C., Rochfort, K.D., Smith, D., Cummins, P.M., 2016. Vascul. Pharmacol. 82, 30–40.
PMID: 26924459

Aberrant regulation of RANKL/OPG in women at high risk of developing breast cancer.
Kiechl, S., Schramek, D., Widschwendter, M., Fourkala, E.-O., Zaikin, A., Jones, A., Jaeger, B., Rack, B., Janni, W., Scholz, C., Willeit, J., Weger, S., Mayr, A., Teschendorff, A., Rosenthal, A., Fraser, L., Philpott, S., Dubeau, L., Keshtgar, M., Roylance, R., Jacobs, I.J., Menon, U., Schett, G., Penninger, J.M., 2016. Oncotarget 8, 3811–3825.
PMID: 28002811; PMCID: PMC5354797

Glucocorticoid Signaling and Bone Biology.
Komori, T., 2016. Horm. Metab. Res. Horm. Stoffwechselforschung Horm. Metab. 48, 755–763.
PMID: 27871116

Immunology of Osteoporosis: A Mini-Review.
Pietschmann, P., Mechtcheriakova, D., Meshcheryakova, A., Föger-Samwald, U., Ellinger, I., 2016. Gerontology 62, 128–137.
PMID: 26088283; PMCID: PMC4821368

Expression of osteoprotegerin and its ligands, RANKL and TRAIL, in rheumatoid arthritis.
Remuzgo-Martínez, S., Genre, F., López-Mejías, R., Ubilla, B., Mijares, V., Pina, T., Corrales, A., Blanco, R., Martín, J., Llorca, J., González-Gay, M.A., 2016. Sci. Rep. 6, 29713.
PMID: 27403809; PMCID: PMC4940734

RANKL/RANK: from bone loss to the prevention of breast cancer.
Sigl, V., Jones, L.P., Penninger, J.M., 2016. Open Biol. 6.
PMID: 27881737; PMCID: PMC5133443

Differences in biochemical bone markers by diabetes type and the impact of glucose.
Starup-Linde, J., Lykkeboe, S., Gregersen, S., Hauge, E.-M., Langdahl, B.L., Handberg, A., Vestergaard, P., 2016. Bone 83, 149–155.
PMID: 26555635

Assessment of OPG, RANKL, bone turnover markers serum levels and BMD after treatment with strontium ranelate and ibandronate in patients with postmenopausal osteoporosis.
Stuss, M., Sewerynek, E., Król, I., Stępień-Kłos, W., Jędrzejczyk, S., 2016. Endokrynol. Pol. 67, 174–184.
PMID: 26884284

Effects of Antitumor Necrosis Factor Therapy on Osteoprotegerin, Neopterin, and sRANKL Concentrations in Patients with Rheumatoid Arthritis.
Kurz, K., Herold, M., Russe, E., Klotz, W., Weiss, G., Fuchs, D., 2015. Dis. Markers, 276969.
PMID: 26576067; PMCID: PMC4631883

Switching from tenofovir to abacavir in HIV-1-infected patients with low bone mineral density: changes in bone turnover markers and circulating sclerostin levels.
Negredo, E., Diez-Pérez, A., Bonjoch, A., Domingo, P., Pérez-Álvarez, N., Gutierrez, M., Mateo, G., Puig, J., Echeverría, P., Escrig, R., Clotet, B., 2015. J. Antimicrob. Chemother. 70, 2104–2107.
PMID: 25769303

IL-6 in Inflammation, Immunity, and Disease.
Tanaka, T., Narazaki, M., Kishimoto, T., 2014. Cold Spring Harb. Perspect. Biol. 6, a016295–a016295

Prevention and treatment of postmenopausal osteoporosis.
Tella, S.H., Gallagher, J.C., 2014. J. Steroid Biochem. Mol. Biol. 142, 155–170.
PMID: 24176761; PMCID: PMC4187361

Immunology and bone.
Danks, L., Takayanagi, H., 2013. J. Biochem. (Tokyo) 154, 29–39.
PMID: 23750028

RANKL employs distinct binding modes to engage RANK and the osteoprotegerin decoy receptor.
Nelson, C.A., Warren, J.T., Wang, M.W.-H., Teitelbaum, S.L., Fremont, D.H., 2012. Struct. Lond. Engl. 1993 20, 1971–1982.
PMID: 23039992; PMCID: PMC3607351

The role of the osteoprotegerin/RANKL/RANK system in diabetic vascular disease.
Grigoropoulou, P., Eleftheriadou, I., Zoupas, C., Tentolouris, N., 2011. Curr. Med. Chem. 18, 4813–4819
PMID: 21919846

Evidence for osteocyte regulation of bone homeostasis through RANKL expression.
Nakashima, T., Hayashi, M., Fukunaga, T., Kurata, K., Oh-Hora, M., Feng, J.Q., Bonewald, L.F., Kodama, T., Wutz, A., Wagner, E.F., Penninger, J.M., Takayanagi, H., 2011. Nat. Med. 17, 1231–1234.
PMID: 21909105

RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis.
Gonzalez-Suarez, E., Jacob, A.P., Jones, J., Miller, R., Roudier-Meyer, M.P., Erwert, R., Pinkas, J., Branstetter, D., Dougall, W.C., 2010. Nature 468, 103–107.
PMID: 20881963

Physiology and pathophysiology of the RANKL/RANK system.
Hanada, R., Hanada, T., Penninger, J.M., 2010. Biol. Chem. 391, 1365–1370.
PMID: 21087090

Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer.
Schramek, D., Leibbrandt, A., Sigl, V., Kenner, L., Pospisilik, J.A., Lee, H.J., Hanada, R., Joshi, P.A., Aliprantis, A., Glimcher, L., Pasparakis, M., Khokha, R., Ormandy, C.J., Widschwendter, M., Schett, G., Penninger, J.M., 2010. Nature 468, 98–102.
PMID: 20881962; PMCID: PMC3084017

Central control of fever and female body temperature by RANKL/RANK.
Hanada, R., Leibbrandt, A., Hanada, T., Kitaoka, S., Furuyashiki, T., Fujihara, H., Trichereau, J., Paolino, M., Qadri, F., Plehm, R., Klaere, S., Komnenovic, V., Mimata, H., Yoshimatsu, H., Takahashi, N., von Haeseler, A., Bader, M., Kilic, S.S., Ueta, Y., Pifl, C., Narumiya, S., Penninger, J.M., 2009. Nature 462, 505–509.
PMID: 19940926

Clinical implications of the osteoprotegerin/RANKL/RANK system for bone and vascular diseases.
Hofbauer, L.C., Schoppet, M., 2004. JAMA 292, 490–495.
PMID: 15280347

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.

- CPMP/ICH/381/95: ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology

- EMEA/CHMP/EWP/192217/2009 Guideline on bioanalytical method validation

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

Calibration

The free soluble RANKL immunoassay is calibrated against human recombinant RANKL protein.

Detection Limit & Sensitivity

To determine the sensitivity of the free soluble RANKL 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 RANKL, with a confidence level of 99%.

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%.

The following values were determined for the free soluble RANKL ELISA:

LOD

0.01 pmol/l

LLOQ

0.008 pmol/l

Independently, an external clinical research organization (CRO) determined the LOD as 0.009 pmol/l and the LLOQ at 0.0038 pmol/l.

+ Show Individual measurements

The lower limit of detection (LOD) of free sRANKL is 0.009 pmol/l and determined by adding 3 SD to the mean of a standard zero study.

Measurement

Soluble RANKL [pmol/l]

1

0.003

2

0.008

3

0.001

4

0.002

5

0.003

6

0.002

7

0.001

8

0.001

9

0.002

10

0.002

11

0.003

12

0.003

13

0.003

14

0.002

15

0.002

16

0.003

17

0.002

Mean

0.003

SD

0.002

% CV

62.7%

LOD

0.009

The lower limit of quantitation was determined by the Sample B and verified by QC1, Sample A and QC 1-2.

Measurement

QC1 [pmol/l]

Sample A [pmol/l]

Sample B [pmol/l]

QC 1-2 [pmol/l]

1

0.046

0.044

0.035

0.020

2

0.045

0.054

0.038

0.027

3

0.046

0.050

0.038

0.014

4

0.045

0.048

0.034

0.016

5

0.048

0.050

0.034

0.017

6

0.046

0.047

0.043

0.021

7

0.039

0.047

0.033

0.026

8

0.046

0.046

0.035

0.021

9

0.046

0.048

0.040

0.015

10

0.045

0.052

0.033

0.015

11

0.046

0.047

0.040

0.017

12

0.045

0.050

0.042

0.020

13

0.046

0.047

0.042

0.028

14

0.045

0.046

0.040

0.028

15

0.046

-

0.040

-

Mean [pmol/l]:

0.045

0.048

0.038

0.020

SD [pmol/l]:

0.002

0.003

0.004

0.005

CV [%]:

4.2%

5.5%

9.3%

24.9%

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 using different ELISA lots (in-between-run precision or reproducibility).

 

Within-Run Precision (repeatability or intra-assay precision)

 

Within-run / intra-assay precision was assessed by measuring 2 samples of known concentrations 5 times within one free soluble RANKL ELISA kit lot by one operator.

Intra-assay (n=5)

Sample 1

Sample 2

Mean [pmol/l]

0.12

0.98

SD [pmol/l]

0.006

0.009

CV (%)

5

1

Independently, within-run / intra-assay precision was assessed by an independent CRO by measuring 5 samples 15 times.

Intra-assay (n=15)

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Mean [pmol/l]

0.045

0.102

0.142

0.242

0.628

SD [pmol/l]

0.002

0.007

0.006

0.006

0.008

CV (%)

4.2

6.6

4.4

2.5

1.3

 

Show individual measurements

 

Measurement

QC1 [pmol/l]

QC2 [pmol/l]

QC3 [pmol/l]

QC4 [pmol/l]

QC5 [pmol/l]

1

0.046

0.098

0.149

0.245

0.620

2

0.045

0.098

0.146

0.242

0.609

3

0.046

0.103

0.137

0.240

0.621

4

0.045

0.092

0.142

0.246

0.632

5

0.048

0.102

0.146

0.233

0.636

6

0.046

0.103

0.135

0.242

0.629

7

0.039

0.099

0.135

0.244

0.642

8

0.046

0.105

0.141

0.254

0.625

9

0.046

0.104

0.138

0.233

0.627

10

0.045

0.095

0.141

0.241

0.627

11

0.046

0.102

0.150

0.242

0.638

12

0.045

0.119

0.132

0.233

0.624

13

0.046

-

0.141

0.237

0.630

14

0.045

-

0.144

0.250

0.627

15

0.046

-

0.155

0.246

0.634

Mean [pmol/l]

0.045

0.102

0.142

0.242

0.628

SD [pmol/l]

0.002

0.007

0.006

0.006

0.008

CV %

4.2

6.6

4.4

2.5

1.3

 

In-Between-Run Precision (inter-assay precision)

 

In-between-run /intra-assay precision was assessed by measuring 2 samples 12 times within free soluble RANKL ELISA kit lots by three different operators.

Inter-assay (n=2)

Sample 1

Sample 2

Mean [pmol/l]

0.12

1.00

SD [pmol/l]

0.004

0.02

CV (%)

3

2

Independently, in-between-run /intra-assay precision was assessed by an independent CRO by measuring 4 samples of known concentrations 7 times.

Intra-assay (n=7)

Sample 1

Sample 2

Sample 3

Sample 4

Mean [pmol/l]

0.114

0.161

0.241

0.550

SD [pmol/l]

0.02

0.01

0.00

0.0

CV (%)

16.2

6.3

1.8

2.1

 

Show Individual measurements

Measurement

QC1 [pmol/l]

QC2 [pmol/l]

QC3 [pmol/l]

QC4 [pmol/l]

1

0.093

0.145

0.239

0.545

2

-

0.151

0.242

0.544

3

-

0.160

0.249

0.573

4

0.101

0.164

0.239

0.545

5

0.113

0.165

0.245

0.538

6

0.139

0.176

0.237

0.557

7

0.126

0.163

0.238

0.550

Mean [pmol/l]

0.114

0.161

0.241

0.550

SD [pmol/l]

0.02

0.01

0.00

0.0

CV %

16.2

6.3

1.8

2.1

Accuracy

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

The recovery of the human free soluble RANKL ELISA was measured by adding human recombinant soluble RANKL to human samples containing a known concentration endogenous RANKL. The % recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value.

This table shows the summary of the recovery experiments in the free soluble RANKL ELISA in different sample matrices:

 

  

% Recovery

 

 

+ 0.25 pmol/l

+ 1 pmol/l

Sample Matrix

n

Mean

Range

Mean

Range

Serum

8

91

70 – 104

84

67 – 100

Heparin plasma

8

91

67 – 119

83

67 - 106

Please note: Recovery of recombinant RANKL is a complicated issue in serum/plasma samples as the samples contain a binding factor that influences the recovery. This observation is different from a sample spiked with endogenous soluble RANKL, as the sample has already reached its equilibrium with OPG (and other potential binding factors).

Show Individual measurements

Data showing % recovery of recombinant RANKL in human serum samples:

   

RANKL c[pmol/l]

% Recovery

Sample Matrix

ID

Reference

 +0.25 pmol/l

 +1 pmol/l

 +0.25 pmol/l

 +1 pmol/l

Serum

s1

1.01

1.19

1.70

70

70

Serum

s2

0.47

0.73

1.47

104

104

Serum

s3

0.27

0.53

1.20

103

103

Serum

s4

0.23

0.43

1.04

77

77

Serum

s5

0.05

0.29

0.83

98

98

Serum

s6

0.07

0.25

0.74

70

70

Serum

s7

0.52

0.76

1.39

98

98

Serum

s8

0.27

0.48

1.23

85

85
       

Mean

91

84
       

Min

70

67
       

Max

104

100

Data showing % recovery of recombinant RANKL in human heparin plasma samples:

   

RANKL c[pmol/l]

% Recovery

Sample Matrix

ID

Reference

 +0.25 pmol/l

 +1 pmol/l

 +0.25 pmol/l

 +1 pmol/l

Heparin plasma

h1

0.31

0.55

1.29

98

98

Heparin plasma

h2

0.18

0.44

0.99

102

81

Heparin plasma

h3

0.39

0.64

1.42

98

103

Heparin plasma

h4

0.49

0.79

1.55

119

106

Heparin plasma

h5

0.06

0.23

0.73

67

67

Heparin plasma

h6

0.04

0.24

0.89

79

85

Heparin plasma

h7

0.19

0.37

0.96

74

77

Heparin plasma

h8

0.10

0.31

0.85

84

76
       

Mean

91

86
       

Min

67

67
       

Max

119

106

Accuracy was also assessed by an external CRO in serum and recovery was determined to be between 80 and 113%.

Show Individual measurements

Experiment: Recovery was determined by mixing samples with different standards in a 75:25 ratio.

 

Sample value

[pmol/l]

Added values

[pmol/l]

Expected values

[pmol/l]

Assay   values

[pmol/l]

Recovery [%]

Sample C

0.113

0.125

0.116

0.131

113

0.250

0.148

0.148

100

0.500

0.210

0.215

102

1.000

0.335

0.298

89

2.000

0.585

0.480

82

Sample D

0.118

0.125

0.120

0.115

96

0.250

0.152

0.138

91

0.500

0.214

0.184

86

1.000

0.339

0.274

81

2.000

0.589

0.472

80

 

 

 

 

Mean R [%]:

92

Parallelism

Tests of parallelism ensure that samples containing endogenous soluble RANKL behave in a dose dependent manner and are not affected by matrix effects. Parallelism refers to dilution linearity in clinical samples.

Parallelism was assessed by serially diluting samples containing endogenous soluble RANKL with serum-based standard matrix.

 

 

% Recovery of endogenous soluble RANKL in diluted samples

 

 

1+1

1+3

Sample Matrix

n

Mean

Mean

Serum

8

112

135

Heparin plasma

8

121

 -

Serum

8

112

81-113

 

Show Individual measurements
   

Soluble RANKL [pmol/l]

% Recovery

Sample matrix

ID

Reference

1+1

1+3

1+1

1+3

Serum

s1

647

308

121

95

93

Serum

s2

1280

725

302

113

118

Serum

s3

372

186

53

100

72

Serum

s4

953

488

199

102

105

Serum

s5

1440

713

316

99

110

Serum

s6

1466

754

307

103

105

Serum

s7

603

284

83

94

69

Serum

s8

837

375

154

90

92

Serum

s9

1608

846

511

105

127

Serum

s10

1612

860

540

107

134

Serum

s11

1155

618

337

107

117

Serum

s12

1359

691

395

102

116
       

Mean

101

105
       

Min

90

69
       

Max

113

134

Parallelism was independently validated in serum by an external CRO:

 

 

% Recovery of endogenous soluble RANKL in diluted samples

   

1+1

Sample

Matrix

n

Mean

SD

Serum

8

96

±12

 

Show Individual measurements

Diluted in a very low pool serum, results not corrected by dilution factor (2).

Sample

Dilution

free sRANKL [pmol/l]

Recovery [%] from neat

Pool A

neat

0.229

-

 1/2

0.093

81

Pool B

neat

0.243

-

 1/2

0.110

91

Pool C

neat

0.258

-

 1/2

0.113

88

Pool D

neat

0.199

-

 1/2

0.098

98

Sample 1

neat

0.300

-

 1/2

0.169

113

Sample 2

neat

0.229

-

 1/2

0.120

105

 

 

 

 

Mean R [%]

96

±

12

Specificity

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

The specificity of the free soluble RANKL ELISA was established through competition experiments, which measure the ability of the antibodies to exclusively bind soluble RANKL.
 

Competition of Signal

 
Competition experiments were carried out by pre-incubating human samples with an excess of OPG. 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. Mean competition was 100 % in both serum and heparin plasma.

Serum:

   

Soluble RANKL [pmol/l]

 

Sample matrix

ID

Reference

Reference + 1µl OPG

% Competition

Serum

s1

1.01

0.00

100

Serum

s2

0.47

0.00

100

Serum

s3

0.27

0.00

100

Serum

s4

0.23

0.00

100

Serum

s5

0.05

0.00

90

Serum

s6

0.07

0.00

100

Serum

s7

0.52

0.03

94

Serum

s8

0.27

0.00

100

Serum

s9

0.37

0.00

99

Serum

s10

0.47

0.00

100

Serum

s11

0.40

0.01

99

Serum

s12

0.10

0.00

100

Serum

s13

0.45

0.00

99
     

Mean

100

Heparin plasma:

   

Soluble RANKL [pmol/l]

 

Sample matrix

ID

Reference

Reference + 1µl OPG

% Competition

Heparin plasma

h1

0.31

0.00

100

Heparin plasma

h2

0.18

0.00

100

Heparin plasma

h3

0.39

0.00

100

Heparin plasma

h4

0.49

0.00

100

Heparin plasma

h5

0.06

0.00

100

Heparin plasma

h6

0.04

0.00

100

Heparin plasma

h7

0.19

0.01

95

Heparin plasma

h8

0.10

0.00

100
     

Mean

100

Cross-Reactivity

Primates: The sequence homology of human sRANKL (soluble form, aa 140-317) to various primate species is >95%. It is likely that the assay can be used for these species. Internal validations have not been carried out.

Sample Stability

We recommend performing serum or plasma separation by centrifugation as soon as possible, e.g. 20 min at 2000 x g, preferably at 4°C (2-8°C). If this is not possible store the samples at 4°C (2-8°C) prior to centrifugation (up to one day).

The acquired serum or plasma samples should be measured as soon as possible. For longer storage aliquot samples and store at -25°C, for long time storage at -80°C. The stability of endogenous soluble RANKL was tested by comparing RANKL measurements in samples that had undergone 3 freeze-thaw cycles.
 

Freeze-Thaw Stability

 
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 in serum after 3 freeze-thaw cycles is 92%.

 

Soluble RANKL c[pmol/l]

ID

Reference

1x

3x

CV (%)

% Recovery after 3 freeze/thaw cycles

s1

0.13

0.10

0.10

1

76

s2

0.13

0.12

0.11

7

87

s3

0.27

0.28

0.23

9

87

s4

0.67

0.62

0.58

7

86

s5

0.18

0.16

0.18

6

95

s6

0.78

0.75

0.74

3

94

s7

0.24

0.22

0.25

6

104

s8

0.33

0.31

0.34

4

103

s9

0.23

0.22

0.21

4

92
     

Mean

7

92

Samples can undergo at least up to 3 freeze-thaw cycles.

Independently, an external CRO determined mean recovery after 4 freeze-thaw cycles to be 102% in serum samples.

 

Soluble RANKL c[pmol/l]

 

 

ID

Reference (1x)

2x

3x

4x

CV (%)

% Recovery after 4 freeze/thaw cycles

s1

0.131

0.131

0.138

0.140

7

107

s2

0.132

0.133

0.128

0.131

1

99

s3

0.121

0.119

0.122

0.119

2

98

s4

0.113

0.116

0.124

0.118

5

105

s5

0.183

0.190

0.195

0.187

2

102

s6

0.189

0.189

0.187

0.184

3

97
     

 

Mean

7

102

 

Whole Blood Stability

 
Human serum sample concentrations which were stored for 20h in whole blood at room temperature show a CV of 3%.

Human Heparin plasma sample concentrations which were stored for 20h in whole blood at room temperature show a CV of 6%.

Free sRANKL is stable in whole blood for 20h at room temperature (18-26°C).

Experiment:

Stability of free sRANKL in whole blood was tested in serum and Heparin plasma samples, directly after collection and after 2h, 4h and 20h.

 

Duration between blood draw and sample prep [h]

 

 

 

 

0

2

4

20

 

 

Sample Matrix

ID

Soluble RANKL [pmol/l]

Mean 

% CV

Serum

s1

0.33

0.32

0.36

0.31

0.33

6

Serum

s2

0.33

0.33

0.33

0.33

0.33

0

Serum

s3

0.40

0.39

0.37

0.36

0.38

5

Serum

s4

0.22

0.22

0.21

0.22

0.22

2

 

 

 

 

          

Mean

3

 

 

Duration between blood draw and sample prep [h]

 

 

 

 

0

2

4

20

 

 

Sample Matrix

ID

Soluble RANKL [pmol/l]

Mean 

% CV

Heparin plasma

h1

0.35

0.34

0.34

0.30

0.33

7

Heparin plasma

h2

0.36

0.34

0.37

0.33

0.35

5

Heparin plasma

h3

0.39

0.39

0.40

0.38

0.39

3

Heparin plasma

h4

0.27

0.24

0.23

0.21

0.24

10

 

 

 

 

          

Mean

6

 

Sample Stability at Room Temperature

 
Serum sample stability at room temperature was assessed by an independent external CRO:

Sample ID

T-0H pmol/l

T-3H pmol/l

%

variation with T-0H

T-6H pmol/l

%

variation with T-0H

T-24H pmol/l

%

variation with T-0H

C1

0.130

0.134

3.1

0.133

2.3

0.125

-3.8

C2

0.134

0.133

-0.7

0.134

0.0

0.127

-5.2

D1

0.110

0.117

6.4

0.120

9.1

0.110

0.0

D2

0.124

0.119

-4.0

0.122

-1.6

0.111

-10.5

E1

0.176

0.185

5.1

0.190

8.0

0.183

4.0

E2

0.195

0.193

-1.0

0.193

-1.0

0.180

-7.7

At 24 hours 6/6 results have variation <11 %. free sRANKL is stable up to 24 hours at Room Temperature.

 

Sample Stability at +4°C

 

Serum sample stability at +4°C was assessed by an independent external CRO:

Sample ID

T-0H pmol/l

T-3H pmol/l

%

variation with T-0H

T-6H pmol/l

%

variation with T-0H

T-24H pmol/l

%

variation with T-0H

C1

0.133

0.121

-9.0

0.118

-11.3

0.116

-12.8

C2

0.117

0.120

2.6

0.119

1.7

0.119

1.7

D1

0.110

0.111

0.9

0.109

-0.9

0.098

-10.9

D2

0.107

0.106

-0.9

0.108

0.9

0.117

9.3

E1

0.184

0.189

2.7

0.197

7.1

0.190

3.3

E2

0.192

0.193

0.5

0.194

1.0

0.193

0.5

At 24 hours 6/6 results have variation <13 %. free sRANKL is stable up to 24 hours at +4°C.

Sample Values

Free Soluble RANKL Values in Apparently Healthy Individuals

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

A summary of the results is shown below:

 

  

Soluble RANKL [pmol/l]

Sample Matrix

n

Median

Minimum

Maximum

Serum

32

0.15

0.03

0.48

Heparin plasma

8

0.15

0.02

0.40

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

An apparently healthy cohort (n=88) was also measured by an external CRO:

Serum

Free soluble RANKL [pmol/l]

Mean value

0.132

SD

0.092

Mean -SD

0.040

Mean +SD

0.224

n=

88

Percentile 5%

0.025

Percentile 95%

0.301

Percentile 2.5%

0.021

Percentile 97.5%

0.356

Screening of 30 sera from apparently healthy men yielded the following results: 

Serum

Free soluble RANKL [pmol/l]

Mean value

0.153

SD

0.103

Mean -SD

0.050

Mean +SD

0.256

n

30

Percentile 5%

0.026

Percentile 95%

0.342

Percentile 2.5%

0.022

Percentile 97.5%

0.381

Screening of 28 sera from apparently healthy pre-menopausal women yielded the following results:  

Serum

Free soluble RANKL [pmol/l]

Mean value

0.121

SD

0.089

Mean - SD

0.032

Mean + SD

0.210

n

28

Percentile 5%

0.022

Percentile 95%

0.282

Percentile 2.5%

0.020

Percentile 97.5%

0.329

Screening of 30 sera from apparently healthy postmenopausal women yielded the following results:

Serum 

Free soluble RANKL [pmol/l]

Mean value

0.123

SD

0.081

Mean -SD

0.042

Mean +SD

0.204

n

30

Percentile 5%

0.030

Percentile 95%

0.266

Percentile 2.5%

0.029

Percentile 97.5%

0.274

Matrix Comparison

To assess whether the two tested matrices behave the same way in the free soluble RANKL ELISA, concentrations of RANKL were measured in serum and heparin plasma samples prepared from 18 apparently healthy donor. Each individual donated blood in both tested sample matrices. 

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

 

Soluble RANKL [pmol/l]

 

Sample ID

Serum

Heparin plasma

% CV

#1

0.16

0.15

0

#2

0.20

0.23

12

#3

0.15

0.17

12

#4

0.09

0.13

25

#5

0.07

0.09

16

#6

0.35

0.40

9

#7

0.17

0.16

6

#8

0.17

0.16

7

#9

0.02

0.02

14

#10

0.15

0.15

0

#11

0.08

0.10

14

#12

0.26

0.28

5

#13

0.06

0.05

9

#14

0.20

0.18

5

#15

0.27

0.29

5

#16

0.07

0.10

26

#17

0.15

0.19

16

#18

0.06

0.05

5
 

 

Mean

10
Citations

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