Instruction Manual Cat. No. PK-EL-KB20492

Human Sclerostin ELISA Kit

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Contents Intended Use 3 Introduction 3 Material Supplied 4 Material Required but not Supplied 4 Reagents and Sample Preparation 5 Principle of the Assay 6 Assay Protocol 6 Calculation of Results 7 Assay Characteristics 8 Precision 8 Technical Hints 9 Precautions 9 Literature 10 Ordering Information 12

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Intended Use

This PromoCell ELISA is intended for the quantitative determination of Sclerostin in human serum, EDTA-plasma or heparin-plasma. It is for in vitro research use only.

Introduction

Canonical Wnt-signalling plays an important role in the regulation of bone homeostasis by promoting the development of osteoblasts. Negative regulators of the Wnt pathway are important new therapeutic targets for the treatment of diseases with enhanced bone resorption. One of these molecules is Sclerostin, a 22.5 kDa secreted glycoprotein, which acts by binding to the Wnt-coreceptor LRP5 thus preventing the binding of Wnt molecules. Sclerostin is nearly exclusively produced in osteocytes. Therefore, it is considered a clinical marker which provides highest bone specificity. Possible Indications

Osteoporosis

Cancer Induced Bone Diseases

Rheumatoid arthritis

Chronic inflammation

Kidney diseases

Therapy monitoring of anabolic treatment

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Material Supplied

Content Kit Components Quantity

PLATE polyclonal goat anti human Sclerostin antibody, pre-coated microtiter strips in a strip holder, in aluminium bag with desiccant

12 x 8 tests

WASHBUF Wash buffer, natural cap, concentrate 20x

1 x 50 ml

ASYBUF Assay buffer, red cap, ready to use 1 x 20 ml

AB monoclonal mouse anti human Sclerostin antibody – biotin labelled, green dye, green cap, ready to use

1 x 7 ml

STD Standards 1-6, (0; 15; 30; 60; 120; 240 pmol/l), white caps, lyophilised

6 vials lyophilised

CTRL Control, yellow cap, lyophilised (exact concentration on the label)

1 vial lyophilised

CONJ Conjugate, (streptavidin-HRPO), 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

2 self-adhesive plastic films Instruction manual for use, QC protocol

Material Required but not Supplied

Precision pipettes calibrated to deliver 20 μl, 50 μl, 150 μl, 200 μl, 400 μl, and disposable tips

Distilled or deionised water

Plate washer is recommended for washing, alternative multichannel pipette or manifold dispenser

ELISA reader capable of measuring absorbance at 450 nm (with correction wavelength at 630 nm)

Software for calculation of results

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Reagents and Sample Preparation

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

Collect venous blood samples by using standardized blood collection tubes for serum or plasma. We recommend performing plasma or serum 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 plasma or serum samples should be measured as soon as possible. For longer storage aliquot samples and store at -20°C, for long time storage at -70°C. All samples should undergo only 4 freeze-thaw cycles. Lipemic or haemolysed samples may give erroneous results. Samples should be mixed well before assaying. We recommend duplicates for all values.

Dilution of samples with assay buffer or reducing the sample volume gave linear results over a 4-fold range. This documents that it is not necessary to dilute samples with Sclerostin concentrations higher than 240 pmol/l alternatively, 10μl of sample can be used.

Reconstitute as follows:

WASHBUF (Wash buffer): Dilute the concentrate 1:20: e.g. 50 ml WASHBUF + 950 ml distilled water. Crystals in the buffer concentrate will dissolve at room temperature. The diluted buffer is stable at 2-8°C until expiry date stated on the label. Use only diluted WASHBUF (Wash buffer) during the assay procedure.

STD (Standards) and CTRL (Control): Dissolve each in 400 μl deionised or distilled water at room temperature for 15 min. Mix well (Vortex mixer). Reconstituted STD and CTRL are stable at -20°C until expiry date stated on the label. Avoid repeated freeze thaw cycles.

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Principle of the Assay

Assay Protocol

All reagents and samples must be at room temperature (18-26°C) before use in the assay.

Mark position for BLANK/STD/SAMPLE/CTRL (Blank/Standard/Sample/Control) on the protocol sheet.

Take microtiter strips out of the aluminium bag, take a minimum of one well as Blank. Store unused strips with desiccant at 4°C (2-8°C) in the aluminium bag. Strips are stable until expiry date stated on the label.

1. Add 150 μl ASYBUF (red cap) into each well.

2. Add 20 μl STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective well, except blank.

3. Add 50 μl AB (biotinylated anti Sclerostin antibody, green cap, green dye) into each well, except blank, swirl gently.

4. Cover tightly and incubate over night (18-24h) at room temperature (18-24°C) in the dark. Attention: Incubation higher than room temperature reduces the Top-OD.

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

6. Add 200 μl CONJ (Conjugate, amber cap) into each well.

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

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

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

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

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

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

Calculation of Results

Subtract the blank OD from all wells. Construct the standard curve from the OD values of the STD. Use commercially available software. Obtain sample concentration from this standard curve. The assay was evaluated with 4PL algorithm. Different curve fitting methods need to be evaluated by the user.

If 10μl samples are used the concentration has to be multiplied by 2.

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The quality control 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 and/or due to the normal decrease of signal intensity during shelf life. However, this does not affect validity of results as long as an OD of 1.00 or higher is obtained for the standard with the highest concentration and the control value is in range (target range see label).

Assay Characteristics Values from apparently healthy individuals (n=9):

Human sera Mean: 19.3 pmol/l Min: 10.9 pmol/l Max: 28.7 pmol/l

Human EDTA-plasma Mean: 26.2 pmol/l Min: 17 pmol/l Max: 37.7 pmol/l

Human Heparin-plasma Mean: 21.4 pmol/l Min: 13.7 pmol/l Max: 31.6 pmol/l

It is recommended to establish the normal range for

each laboratory. Standard range: 0 to 240 pmol/l Conversion factor pg/ml to pmol/l:

1 pg/ml = 0.044 pmol/l (MW: 22.5 kD)

Cross reactivity:

88% homology to mouse sclerostin, 90% homology to rat sclerostin The ELISA does not cross react with neither of these species.

Sample volume:

20 μl human EDTA plasma, Heparin plasma, serum, cell culture supernatant

Detection Limit: (0 pmol/l + 3 SD): 2.6 pmol/l Incubation time: 18-24 h / 1 h / 30 min

Precision

Intra-Assay: 2 samples of known concentrations were tested 16 times Inter-Assay: 3 samples of known concentrations were tested in 3 assays from 3 different operators

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Inter-Assay (n=3)

Sample 1 Sample 2 Sample 3

Mean (pmol/l) 44 127 150

SD (pmol/l) 2.6 3.8 4.5

CV% 6 3 3

Intra-Assay (n=16)

Sample 1 Sample 2

Mean (pmol/l) 54 154

SD 2.6 7.3

CV% 5 5

Technical Hints

Do not mix or substitute reagents with those from other lots or sources.

Do not mix stoppers and caps from different reagents or use reagents between lots.

Do not use reagents beyond expiration date.

Protect reagents from direct sunlight.

Substrate solution should remain colourless until added to the plate.

To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

Avoid foaming when mixing reagents.

Precautions

All test components of human source were tested with 3rd generation tests against HIV-Ab and HBsAg and were found negative.

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Nevertheless, they should be handled and disposed of as if they were infectious. Liquid reagents contain ≤0.1% Proclin 300 as preservative. Avoid contact with skin and mucous membrane. Proclin 300 is not toxic in concentrations used in this kit. It may cause allergic skin reactions – avoid contact with skin or eyes. Do not pipette by mouth. Do not eat, drink, smoke or apply cosmetics where reagents are

used. Avoid all contact with reagents by using gloves. Sulfuric acid is irritating to eyes and skin. Avoid contact with skin

and mucous membrane. Irritations are possible – Flush with water if contact occurs!!

Literature

1) Gaudio A et al.: Increased Sclerostin Serum Levels Associated with Bone Formation and Resorption Markers in Patients with Immobilization-Induced Bone Loss. J Clin Endocrinol Metab, (May 2010); 95: 2248 – 2253. 2) Mirza F et al.: Serum Sclerostin Levels Negatively Correlate with Parathyroid Hormone Levels and Free Estrogen Index in Postmenopausal Women. J Clin Endocrinol Metab (2010), Apr 2010; 95: 1991 – 1997 3) Kneissel M: The Promise of Sclerostin Inhibition for the Treatment of Osteoporosis. IBMS BoneKEy (2009), 6: 259- 264. 4) Terpos E et al.: High Serum Sclerostin Correlates with Advanced Stage, Increased Bone Resorption, Reduced Osteoblast Function, and Poor Survival in Newly-Diagnosed Patients with Multiple Myeloma. Blood (2009), 114: 425. 5) Yuen H et al.: The Prognostic Significance of BMP-6 Signalling in Prostate Cancer. Mod Path (2008), 21(12):1436-1443.

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6) Appel H et al.: Altered Skeletal Expression of Sclerostin and its Link to Radiographic Progression in Ankylosing Spondylitis. Arthritis Rheum (2009), 60(11): 3257-3262. 7) Wehmeyer C et al.: The Role of the WNT inhibitor sclerostin in rheumatoid arthritis. Ann Rheum Dis, (Mar 2010); 69: A21 doi:10.1136/ard.2010.129593n. 8) Choi Y et al.: Promising Bone-Related Therapeutic Targets for Rheumatoid Arthritis. Nat Rev Rheumatol (2009), 5(10): 543-548. 9) Eddleston et al.: A Short Treatment with an Antibody to Sclerostin can Inhibit Bone Loss in an Ongoing Model of Colitis. J Bone Miner Res (2009), 24(10): 1662-1671. 10) M Lee and NC Partridge: Parathyroid Hormone Signalling in Bone and Kidney. Curr Opin Nephrol Hypertens (2009), 18(4): 298-302. 11) Lin C et al.: Sclerostin mediates bone response to mechanical unloading through antagonizing Wnt/beta-catenin signaling. J Bone Miner Res, (Oct 2009); 24(10): 1651-61.

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www.promocell.com

Ordering Information

Product Name Product Description Size Catalog Number

Sclerostin ELISA Kit, human

Human Sclerostin ELISA 96 Tests PK-EL-KB20492 For in vitro research use only. Not for diagnostic or therapeutic procedures.

06/2013

PromoCell GmbH Sickingenstr. 63/65 69126 Heidelberg Germany USA / Canada Phone: 1 – 866 – 251 – 2860 (toll free) Fax: 1 – 866 – 827 – 9219 (toll free) Deutschland Telefon: 0800 – 776 66 23 (gebührenfrei) Fax: 0800 – 100 83 06 (gebührenfrei) France Téléphone: 0800 90 93 32 (ligne verte) Téléfax: 0800 90 27 36 (ligne verte) United Kingdom Phone: 0800 – 96 03 33 (toll free) Fax: 0800 – 169 85 54 (toll free) Other Countries Phone: +49 6221 – 649 34 0 Fax: +49 6221 – 649 34 40 Email: info@promocell.com