Denosumab (Prolia®) ELISA Kit

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SKU:
HUMB00036
€999

Description

Denosumab (Prolia®) ELISA Kit

Enzyme-linked immunosorbent assay for the quantitative determination of Denosumab (Prolia®) in serum and plasma. ELISA Genie Denosumab ELISA has been especially developed for the quantitative analysis of Denosumab in serum and plasma samples between the Cmin and Cmax range of concentrations indicated in the pharmacokinetics section of the technical manual.

Denosumab (Prolia®) ELISA Kit test principle

Solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. Standards and samples (serum or plasma) are incubated in the microtitre plate coated with the reactant specific for Denosumab (Prolia®). Following incubation wells are washed and then horse radish peroxidase (HRP) is added and binds to Denosumab. After incubation, the wells are washed, and the bound enzymatic activity is detected by addition of chromogen-substrate. The colour developed is proportional to the amount of Denosumab (Prolia®) in the sample or standard. Results of samples can be determined directly using the standard curve.

Denosumab (Prolia®) Product Information

Information Description
Application
Free drug
Required Volume (μl)
10
Total Time (min)
70
Sample Type
Serum, Plasma
Number of Assays
96
Detection Limit (ng/mL)
3 (ng/mL)
Spike Recovery (%)
85-115%
Shelf Life (year)
1

Alternative Names

Anti-RANKL mAb

Prolia

Denosumab (Prolia®) - Key Information

Denosumab (Prolia®) mode of action

Denosumab (Prolia®) has a mechanism of action unlike other osteoporosis treatments. Administered every six months as a subcutaneous injection, Denosumab (Prolia®) is the only therapy that targets RANK Ligand, an essential regulator of osteoclasts (the cells that break down bone). Through highly specific inhibition of RANK ligand, Denosumab (Prolia®) decreases bone resorption, and improves bone density at all measured skeletal sites. Denosumab (Prolia®) is reimbursed for women and men aged 70 or over with a Bone Mineral Density (BMD) T-score of -2.5 or less.

Denosumab (Prolia®) uses

Denosumab (Prolia®) is used for the treatment of osteoporosis in postmenopausal women to reduce the risk of vertebral, non-vertebral and hip fractures and to increase bone mass in men with osteoporosis at increased risk of fracture.

Denosumab (Prolia®) treatment

Denosumab (Prolia®) is a twice-yearly injection under the skin, usually administered by a general practitioner or practice nurse, and works by targeting the cells that break down bone (osteoclasts) thereby making bone less susceptible to osteoporosis-related fractures.

Denosumab (Prolia®) and the receptor activator of nuclear factor kappa-Β ligand (RANKL)

Denosumab (Prolia®) binds to RANKL, a transmembrane or soluble protein essential for the formation, function, and survival of osteoclasts, the cells responsible for bone resorption. Prolia prevents RANKL from activating its receptor, RANK, on the surface of osteoclasts and their precursors. Prevention of the RANKL/RANK interaction inhibits osteoclast formation, function, and survival, thereby decreasing bone resorption and increasing bone mass and strength in both cortical and trabecular bone.

Denosumab (Prolia®) clinical studies

In clinical studies, treatment with 60 mg of Prolia resulted in reduction in the bone resorption marker serum type 1 C-telopeptide (CTX) by approximately 85% by 3 days, with maximal reductions occurring by 1 month. CTX levels were below the limit of assay quantitation (0.049 ng/mL) in 39-68% of subjects 1-3 months after dosing of Prolia. At the end of each dosing interval, CTX reductions were partially attenuated from a maximal reduction of > 87% to <45% (range: 45% to 80%), as serum denosumab levels diminished, reflecting the reversibility of the effects of Prolia on bone remodelling. These effects were sustained with continued treatment. Upon reinitiation, the degree of inhibition of CTX by Prolia was similar to that observed in patients initiating Prolia treatment. Consistent with the physiological coupling of bone formation and resorption in skeletal remodelling, subsequent reductions in bone formation markers (i.e., osteocalcin and procollagen type 1 N-terminal peptide [PlNP]) were observed starting 1 month after the first dose of Prolia.

After discontinuation of Prolia therapy, markers of bone resorption increased to levels 40-60% above pre-treatment values but returned to baseline levels within 12 months. In a study conducted in healthy male and female volunteers (n = 73, age range: 18 to 64 years) following a single subcutaneously administered Prolia dose of 60 mg after fasting (at least for 12 hours), the mean maximum denosumab concentration (Cmax) was 6.75 mcg/mL (standard deviation [SD] = 1.89 mcg/mL). The median time to maximum denosumab concentration (Tmax) was 10 days (range: 3 to 21 days).

After Cmax, serum denosumab concentrations declined over a period of 4 to 5 months with a mean half-life of 25.4 days (SD = 8.5 days; n=46). No accumulation or change in denosumab pharmacokinetics with time was observed upon multiple dosing of 60 mg subcutaneously administered once every 6 months. Prolia pharmacokinetics were not affected by the formation of binding antibodies. A population pharmacokinetic analysis was performed to evaluate the effects of demographic characteristics.

Denosumab (Prolia®) ELISA Kit Contents

Size Kit Contents

1 x 12 x 8

Microtiter Plate

Break apart strips. Microtiter plate with 12 rows each of 8 wells coated with reactant

8 x 0.3 mL

Denosumab Standards A-F, High Level Control, Low Level Control


300, 100, 30, 10, 3 and 0 ng/mL. Ready to use. Used for construction of the
standard curve. Contains denosumab (Prolia®), human serum, stabilizer
and <0.1% NaN3

1 x 50 mL

Assay Buffer
Blue coloured. Ready to use. Contains proteins and <0.1% NaN3.

1 x 12 mL

Peroxidase Conjugate
Red coloured. Ready to use. Contains horse radish peroxidase (HRP) and
stabilizers.

1 x 12 mL

TMB Substrate Solution
Ready to use. Contains TMB

1 x 12 mL

TMB Stop Solution
Ready to use. 1N HCl

1 x 50 mL

Wash Buffer concentrate (20x)
Contains Buffer with Tween 20.

2 x 1

Adhesive Foil
For covering of Microtiter Plate during incubation.


Denosumab (Prolia®) ELISA Protocol

Steps Protocol

1

Pipette 100 µL of each ready-to use Standards, High Level Control, Low Level Control or diluted Serum Samples into the respective wells of microtiter plate.


Wells
A1: Standard A
B1: Standard B
C1: Standard C
D1: Standard D
E1: Standard E
F1: Standard F
G1: High Level Control
H1: Low Level Control
A2 and on: Sample (serum/plasma)

2

Cover the plate with adhesive foil. Incubate 30 min at room temperature (18- 25°C).

3

Remove adhesive foil. Discard incubation solution. Wash plate 3 times each with 300µL of diluted. Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.

4

Pipette 100 µL of ready-to use Peroxidase Conjugate into each well.

5

Cover the plate with adhesive foil. Incubate 30 min at room temperature (18- 25°C).

6

Remove adhesive foil. Discard incubation solution. Wash plate 3 times each with 300 µL of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.

7

Pipette 100 µL of TMB Substrate Solution into each well.

8

Incubate 10 min (without adhesive foil) at room temperature (18-25°C) in the dark

9

Stop the substrate reaction by adding 100 µL of Stop Solution into each well. Briefly mix contents by gently shaking the plate. Colour changes from blue to yellow.

10

Measure optical density with a photometer at 450/650 nm within 30 min after pipetting of the Stop Solution.

Trademarks

Prolia® is a trademark of Amgen Inc.

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