Rat-MID Osteocalcin ELISA

For the quantification of osteocalcin in rat serum and plasma

The Rat-MID™ Osteocalcin ELISA should be used for the in vitro determination of osteocalcin in rat serum and plasma only. Nordic Bioscience Diagnostics a/s is not responsible for any other use of the kit

or consequence hereof than the one specified above. Neither for misuse e.g. uses deviating from the

procedure described in this manual.

Furthermore, Nordic Bioscience Diagnostics a/s is not to be made responsible for any diagnoses or conclusions made by the user or third party based on the results obtained with the Rat-MID™ Osteocalcin ELISA nor for any consequences such interpretations may cause.

Nordic Bioscience Diagnostics a/s

Herlev Hovedgade 207, 2730 Herlev, Denmark

www.nordicbioscience.com

REF: 7OSC4000

0207A

INTRODUCTION

Intended use

The Rat-MID™ Osteocalcin ELISA is an enzyme-linked immunosorbent assay for the quantitative

determination of osteocalcin in rat serum and plasma. The assay is for research-use-only.

Summary and explanation of the test

Osteocalcin or bone Gla protein (BGP), is a major non-collagenous protein of the bone matrix. It has

a molecular weight of approximately 6000 Dalton and consists in most species of 49 amino acids; however, rat osteocalcin consist of 50 amino acids (1-4). Osteocalcin has a unique property for binding to calcium facilitated by the presence of 2-3 gamma-carboxyglutamic acids at position 17, 21 and 24 (5). The mid-molecular part of osteocalcin, especially the amino acids between 20 and 30, exhibits a high degree of inter-species conservatism (4).

The bone forming cells, osteoblasts, synthesizes osteocalcin. After production it is partly incorporated

into the bone matrix and partly delivered to the circulatory system. Circulating osteocalcin is associated with changes in the rate of bone turnover and regarded as a specific marker for bone formation (4).

The clinical utility of rat osteocalcin as a marker for bone turnover has been evaluated in several preclinical settings. It has been reported that the serum level increases after ovariectomy and this estrogen-deficiency-induced state can be prevented by treatment with either estrogen, selective estrogen receptor modulators (SERMs) or bisphosphonates (6-10).

Principle of the procedure

The Rat-MID™ Osteocalcin ELISA is based upon the competitive binding of a monoclonal antibody to soluble osteocalcin or to immobilized osteocalcin. Briefly, the monoclonal antibody is raised against human osteocalcin and recognizes the mid-molecular part (amino acids 21-29) of the molecule (11). Synthetic human osteocalcin is used for standardization. Parallelism is observed with purified rat

osteocalcin and rat serum.

During the Pre-incubation step, biotinylated osteocalcin is immobilized by binding to the streptavidin

coated microtitre wells. The wells are emptied and washed, and standards, control, or unknown serum samples are pipetted into appropriate wells, followed by a solution of monoclonal antibody. Following the Primary-incubation step the wells are emptied and washed. In the Secondary-incubation step a solution of peroxidase conjugated anti-mouse immunoglobulins is added and binds to the monoclonal antibody. After the third washing step a chromogenic substrate (TMB) is added and the colour reaction is stopped

with sulfuric acid. Finally, the absorbence at 450 nm is measured. The absorbence level is inversely

related to concentration of osteocalcin in the sample.

PRECAUTIONS

Storage

Store the package upon receipt at 2-8°C. Under these conditions the reagents are stable up to the expiry date stated on each vial.

Following reconstitution the Standards and the Control should be stored below -18°C, and should only be frozen and thawed twice. When Primary antibody and the Primary Incubation Buffer have been mixed, the remaining solution should be stored at 2-8°C for maximum 30 days or below -18°C. The remaining reagents and immuno strips should be stored at 2-8°C.

Warnings

The Rat-MID™ Osteocalcin ELISA is for research-use-only.

The following precautions should be observed in the laboratory:

Do not eat, drink or smoke where immuno diagnostic materials are being handled.
Do not pipette by mouth.
Wear gloves when handling immuno diagnostic materials.
Do not use reagents beyond their expiration date and do not mix reagents from different lots of kits.

All reagents and laboratory equipment should be handled and disposed of as if they were infectious.

MATERIAL

Specimen collection

When collecting blood take care to avoid haemolysis. Separate the serum from the cells within 3 hours after collection of blood. It is recommended to freeze (<-18°C) samples immediately after use.

Materials supplied

Before opening the kit, please read the section on Precautions. The kit contains reagents sufficient for 96

determinations.

Streptavidin coated microtitre plate (MTP)

Microwell strips (12 pcs. of 1x8 wells) pre-coated with streptavidin. Supplied in a plastic frame.

Osteocalcin Standard (Vial A)

One vial (lyophilized) containing a PBS-buffered solution with protein stabilizer and preservative. Reconstitute with 5.0 mL of distilled water. The standard must be stored at or below -18°C after use.

Osteocalcin Standards (Vial B-F)

Five vials (lyophilized) containing synthetic human osteocalcin in a PBS-buffered solution with protein stabilizer and preservative. Reconstitute each of the vials with 0.5 mL of distilled water. The standard must be stored below -18°C after use, and must only be frozen and thawed twice. Please refer to the

enclosed Technical Data Sheet for the exact concentrations.

Control (Vial CO)

One vial (lyophilized) containing synthetic human osteocalcin in a PBS-buffered solution with protein stabilizer and preservative. Reconstitute with 0.5 mL of distilled water. The control must be stored below -18°C after use, and must only be frozen and thawed twice. Please refer to the enclosed Technical Data

Sheet for control range.

Biotinylated Osteocalcin (Vial no. 1)

One vial (min. 12.0 mL) of a ready for use solution containing biotinylated synthetic human osteocalcin in a PBS-buffered solution with protein stabilizer and preservative.

Primary Antibody (Vial no. 2)

One vial (min. 0.5 mL) of a concentrated solution of a monoclonal antibody specific the mid-molecular part (amino acids 21-29) of osteocalcin, in a buffered solution with protein stabilizer and preservative. Mix vials 2 and 3: 1+100 before use.

Primary Incubation Buffer (Vial no. 3)

One vial (min. 20.0 mL) of a ready-for-use PBS-buffered solution with protein stabilizer, detergent and preservative for dilution of Primary Antibody. Mix vials 2 and 3: 1+100 before use.

Secondary Antibody (Vial no. 4)

One vial (min. 12.0 mL) of a ready-for-use solution of a peroxidase conjugated antibody specific for mouse IgG in a buffered solution with protein stabilizer and preservative.

Substrate Solution (Vial TMB)

One vial (min. 12.0 mL) of a ready-for-use tetramethylbenzidine (TMB) substrate in an acidic solution.

Stopping Solution (Vial ST)

One vial (min. 12.0 mL) of ready-for-use 0.18 M sulfuric acid.

Washing Solution. (Vial W)

One vial (min. 20.0 mL) of a concentrated washing solution containing detergent and preservative. Dilute 1+50 in distilled water before use.

Sealing tape

Adhesive film for covering wells during incubation.

Materials required – not supplied

Containers for preparing the dilutions of the Primary Antibody and the Washing Solution
Precision micropipettes to deliver 20 μL
Distilled water
Precision 8 or 12-channel multipipette to deliver 100 μL and 150 μL
Microtitre plate mixing apparatus (300 rpm)
ELISA plate reader with both 450 nm and 650 nm filters

ASSAY PROCEDURE

Prior to use, equilibrate all solutions to room temperature. Perform the assay at room temperature (18-22°C). Determine the number of strips needed for the entire experiment. It is recommended to test all samples in duplicate. In addition, for each ELISA plate 14 wells are recommended for standards and 2 wells are recommended for the Control.

1. Pre-incubation

Add 100 μL of Biotinylated Osteocalcin (vial no. 1) to each well, cover with sealing tape, and incubate for 30±5 minutes at room temperature (18-22°C) on a microtitre plate mixing apparatus (300 rpm).

2. Washing

Wash the immuno strips 5 times manually with Washing Solution (vial W). Make sure that the wells are completely emptied after each washing cycle. Using an automated plate washer, follow the instructions of the manufacturer or the guidelines of the laboratory. Usually 5 washing cycles are

adequate.

3. Primary incubation

A: Prior to use mix the solutions in vial no. 2 (Primary Antibody) and vial no. 3 (Primary Incubation Buffer) in the volumetric ratio 1+100 in an empty container. Mix carefully and avoid formation of foam.

B: 20 μL of either Standards (vial A-F), Control (vial CO) or unknown samples are pipetted into appropriate wells followed by 150 μL of the mixture of Primary Antibody in Primary Incubation Buffer. Cover the immuno strips with sealing tape and incubate for 60±5 minutes at room temperature (18-22°C) on a microtitre plate mixing apparatus (300 rpm).

4. Washing

See step 2

5. Secondary incubation

Add 100 μL of the Secondary Antibody (Vial No. 4) to each well, cover with sealing tape, and incubate for 60±5 minutes at room temperature (18-22°C) on a microtitre plate mixing apparatus (300 rpm).

6. Washing

See step 2

7. Incubation with chromogenic substrate solution

Pipette 100 μL of the Substrate Solution (vial TMB) into each well and incubate for 15±2 minutes at room temperature (18-22°C) in the darkness on the microtitre plate mixing apparatus (300 rpm). Use

sealing tape.

8. Stopping of colour reaction

Pipette 100 μL of the Stopping Solution (vial ST) into each well.

9. Measurement of absorbance

The absorbance is measured within two hour at 450 nm. It is recommended to use the reading at

650 nm as reference.

Limitations of the procedure

If the absorbance of a sample is lower than Standard F, it is recommended to dilute the sample to be diluted in rat serum with a low osteocalcin concentration.

QUALITY CONTROL

Good Laboratory Practice requires the use of quality control specimens in each series of assays in order

to check the performance of the assay. Controls should be treated as unknown samples, and the results

analyzed with appropriate statistical methods.

RESULTS

Calculation of results

Calculate the mean of the duplicate absorbance determinations. Construct a standard curve on log-

linear graph paper by plotting the mean absorbencies of the six standards A-F (ordinate) against the corresponding osteocalcin concentrations (abscissa). Determine the osteocalcin concentration of the controls and each patient sample by interpolation.

Alternatively, a four-parametric logistic curve fit can be used.

Example: