Bone Sialoprotein ELISA


Bone Sialoprotein is a phosphorylated glycoprotein that comprises 12% of the total ... standards and with the antibody coated on the wells of the microtiter plate.

Offered in the US by ALPCO

www.alpco.com | Phone: (800) 592-5726 | Fax: (603) 898-6854

Arbeitsanleitung/Manual

Bone Sialoprotein ELISA Manual

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Bone Sialoprotein (BSP) ELISA Kit

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For the in vitro determination of underglycosylated bone sialoprotein in serum

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For research use only

+8 °C

Valid from 05.01.2011

K 4222

+2 °C

96

Immundiagnostik AG, Stubenwald-Allee 8a, D 64625 Bensheim Tel.: ++49 6251 70190-0 Fax: ++ 49 6251 849430 e.mail: [email protected] www.Immundiagnostik.com

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Offered in the US by ALPCO

www.alpco.com | Phone: (800) 592-5726 | Fax: (603) 898-6854

Arbeitsanleitung/Manual

Bone Sialoprotein ELISA

1. INTENDED USE The described Assay is intended for the quantitative determination of underglycosylated Bone Sialoprotein in serum. It is for research use only.

2. INTRODUCTION

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Bone Sialoprotein is a phosphorylated glycoprotein that comprises 12% of the total noncollagenous proteins in bone and is thought to be involved in bone mineralization and remodelling. The polypeptide chain of BSP has a molecular weight of 35 kDa. Native BSP has an apparent molecular weight of 70-80 kDa due to complex posttranslational modifications involving glycosylation. BSP is synthesized mainly by skeletal-associated cell types, including fibroblasts, hypertrophic chondrocytes, osteoblasts, osteocytes, osteoclasts, as well as trophoblasts.

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BSP is overexpressed and underglycosylated in human primary breast and prostate cancers that metastasize to the bone, whereby BSP expression correlates with the development of microcalcifications. Underglycosylated BSP is also expressed by other tumours, such as lung cancer, thyroid cancer, multiple myeloma and neuroblastoma, that predominantly metastasise to bones.

3. PRINCIPLE OF THE TEST

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This Enzyme-Linked-Immuno-Sorbent-Assay (ELISA) is designed for the quantitative determination of underglycosylated Bone Sialoprotein in serum. The test principle is based on interaction of the antigen in the samples or standards and with the antibody coated on the wells of the microtiter plate. A peroxidase-conjugated secondary antibody is used for detection and quantification, and tetramethylbenzidine (TMB) as a peroxidase substrate. The enzymatic reaction is terminated by an acidic stop solution. A dose response curve of absorbance unit (optical density, OD at 450 nm) vs. concentration is generated using the values obtained from the standards. Bone Sialoprotein present in the samples is determined directly from this curve.

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Offered in the US by ALPCO

www.alpco.com | Phone: (800) 592-5726 | Fax: (603) 898-6854

Arbeitsanleitung/Manual

Bone Sialoprotein ELISA

4. MATERIAL SUPPLIED Kit Components

Quantity

K 4222MTP

MTP

Microtiter plate, precoated

12 x 8 wells

K 4222WP

WASHBUF

ELISA wash concentrate, 10 x

1 x 100 ml

K 4222PV

SAMPLEBUF

Sample dilution buffer

1 x 15 ml

K 4222K

CONJ

Conjugate (Mouse-anti-BSP, Peroxidaselabeled)

1 x 200 µl

K 4222ST

STD

Standards

2 x 6 vials

K 4222KO1

CTRL

Control, lyophilized

K 4222KO2

CTRL

Control, lyophilized

K 4222TMB

SUB

TMB substrate (Tetramethylbenzidine), ready to use

1 x 15 ml

K 4222AC

STOP

ELISA stop solution, ready to use

1 x 15 ml

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Label

2 x 1 vial 2 x 1 vial

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Cat. No

5. MATERIAL REQUIRED BUT NOT SUPPLIED



Ultra pure water* Precision pipettors calibrated and tips to deliver 5-1000 µl Absorbent paper Foil to cover the microtiter plate Horizontal microtiter plate shaker A multi-channel dispenser or repeating dispenser Centrifuge capable of 3000 x g Vortex-Mixer Standard laboratory glass or plastic vials, cups, etc. Microtiter plate reader at 450 or 405 nm

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• • • • • • • • •

*Immundiagnostik AG recommends the use of Ultra Pure Water (Water Type 1; ISO 3696), which is free of undissolved and colloidal ions and organic molecules (free of particles > 0.2 µm) with an electrical conductivity of 0.055 µS/cm at 25°C (≤18.2 MΩ cm).

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Offered in the US by ALPCO

www.alpco.com | Phone: (800) 592-5726 | Fax: (603) 898-6854

Arbeitsanleitung/Manual

Bone Sialoprotein ELISA

6. PREPARATION AND STORAGE OF REAGENTS Reagents with a volume less than 100 µl should be centrifuged before use to avoid loss of volume.



The WASHBUF (wash buffer concentrate) should be diluted with ultra pure water 1:10 before use (100 ml WASHBUF + 900 ml ultra pure water), mix well. Crystals could occur due to high salt concentration in the stock solutions. The crystals must be redissolved at 37°C in a water bath before dilution of the buffer solutions. The WASHBUF is stable at 2-8°C until the expiry date stated on the label. Diluted buffer solution can be stored in a closed flask at 2-8°C for one month.



The lyophilized STD (standards) and CTRL (controls) are stable at 2-8°C until the expiry date stated on the label. The STD (standards) and CTRL (controls) must be reconstituted with 500 µl of ultra pure water. Allow the vial content to dissolve for 10 minutes at room temperature and mix thoroughly by gentle inversion to insure complete reconstitution. Reconstituted standards and controls can be stored at -20°C for three weeks.



The conjugate (CONJ) must be diluted 1:100 in wash buffer (100 µl CONJ + 10 ml wash buffer). The undiluted conjugate is stable at 2-8 °C until expiry date stated on the label. Diluted conjugate is not stable and cannot be stored.



All other test reagents are ready to use. Test reagents are stable until the expiry date (see label of test package) when stored at 2-8°C.

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7. PRECAUTIONS

Human materials used in kit components were tested and found to be negative for HIV, Hepatitis B and Hepatitis C. However, for safety reasons, all kit components should be treated as potentially infectious. Kit reagents contain sodium azide or thimerosal as bactericides. Sodium azide and thimerosal are toxic. Substrates for the enzymatic color reactions are toxic and carcinogenic. Avoid contact with skin or mucous membranes. Stop solution is composed of sulphuric acid, which is a strong acid. Even diluted, it still must be handled with care. It can cause acid burns and should be handled with gloves, eye protection and appropriate protective clothing. Any spills should be wiped out immediately with copious quantities of water. Reagents should not be used beyond the expiration date shown on the kit label. 12

Offered in the US by ALPCO

www.alpco.com | Phone: (800) 592-5726 | Fax: (603) 898-6854

Arbeitsanleitung/Manual

Bone Sialoprotein ELISA

8. SPECIMEN COLLECTION AND PREPARATION Serum Serum samples should be diluted 1:10 with SAMPLEBUF (sample dilution buffer) before use (e.g. 30 µl sample + 270 µl SAMPLEBUF). Store samples at -20 °C until use.

9. ASSAY PROCEDURE

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Procedural notes Do not interchange different lot numbers of any kit component within the same assay.



Substrate solution should remain colourless until use.



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



Avoid foaming when mixing reagents.



The assay should always be performed according the enclosed manual.

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Offered in the US by ALPCO

www.alpco.com | Phone: (800) 592-5726 | Fax: (603) 898-6854

Arbeitsanleitung/Manual

Bone Sialoprotein ELISA

Test procedure Prior to use in the assay allow all reagents and samples to come to room temperature (18-26 °C) and mix well

2.

Mark the positions of STD (standards), CTRL (controls) and SAMPLE (sample) on a protocol sheet

3.

Take as many microtiter strips (PLATE) as needed from kit. Wash the precoated microtiter plate 5 times by dispensing 250 µl of diluted wash buffer into each well. After the final washing step, the inverted microtiter plate should be firmly tapped on absorbent paper to remove excess solution

4.

For the analysis in duplicate, pipette 100 µl of STD (standards), CTRL (controls) and SAMPLE (sample) into the respective well of the microtiter plate

5.

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

6.

Aspirate the contents of each well. Wash 5 times by dispensing 250 µl of diluted wash buffer into each well. After the final washing step, the inverted microtiter plate should be firmly tapped on absorbent paper to remove excess solution

7.

Add 100 µl of diluted CONJ (conjugate) into each well

8.

Cover the plate tightly and incubate for 1 hour at room temperature (18-26°C) while shaking

9.

Aspirate the contents of each well. Wash 5 times by dispensing 250 µl of diluted wash buffer into each well. After the final washing step, the inverted microtiter plate should be firmly tapped on absorbent paper to remove excess solution

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1.

10. Add 100 µl of SUB (TMB substrate) into each well 11. Incubate for 10-20 min at room temperature in the dark* 14

Offered in the US by ALPCO

www.alpco.com | Phone: (800) 592-5726 | Fax: (603) 898-6854

Arbeitsanleitung/Manual

Bone Sialoprotein ELISA

12. Add 50 µl of STOP (stop solution) into each well, mix thoroughly 13. Determine absorption immediately with an ELISA reader at 450 nm. If the highest extinction of the standards (STD) is above the range of the photometer, absorption must be measured immediately at 405 nm and the obtained results used for evaluation. If possible, the extinctions from each measurement should be compared with extinctions obtained at a reference wavelength, e. g. 595 nm, 620 nm, 630 nm, 650 nm and 690 nm can be used.

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*The intensity of the color change is temperature sensitive. We recommend to observe the color change and to stop the reaction upon good differentiation.

10. EVALUATION OF THE RESULTS

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The following algorithms can be used alternatively to calculate the results. We recommend using the "4-Parameter-algorithm". 4-Parameter-algorithm

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It is recommended to use a linear ordinate for the optical density and a logarithmic abscissa for the concentration. When using a logarithmic abscissa, the zero calibrator must be specified with a value less than 1 (e. g. 0.01). Point-to-point-calculation

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We recommend a linear ordinate for the optical density and a linear abscissa for the concentration. Spline-algorithm

We recommend a linear ordinate for the optical density and a logarithmic abscissa for the concentration. When using a logarithmic abscissa, the zero calibrator must be specified with a value less than 1 (e. g. 0.01). The plausibility of the pairs of values should be examined before the automatic evaluation of the results. If this option is not available with the used program, a control of the paired values should be done manually. Serum samples For calculation of the BSP concentration in serum samples, the result must be multiplied by 10. 15

Offered in the US by ALPCO

www.alpco.com | Phone: (800) 592-5726 | Fax: (603) 898-6854

Arbeitsanleitung/Manual

Bone Sialoprotein ELISA

14. GENERAL NOTES ON THE TEST AND TEST PROCEDURE All reagents in the kit package are for research use only.



Guidelines for medical laboratories should be observed.



Incubation time, incubation temperature and pipetting volumes of the components are defined by the producer. Any variation of the test procedure, which is not coordinated with the producer, may influence the results of the test. Immundiagnostik AG can therefore not be held responsible for any damage resulting from wrong use.



Warranty claims and complaints in respect of deficiencies must be logged within 14 days after receipt of the product. The product shall be send to Immundiagnostik AG along with a written complaint.

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