Description

Anti-Denosumab (Prolia®) ADA Qualitative ELISA Kit

Enzyme-linked immunosorbent assay for the qualitative determination (screening) of anti-Denosumab (Prolia®) antibodies in serum and plasma.The Assay Genie Antibody to Denosumab (Prolia®) ELISA Kit is intended for the qualitative determination of Denosumab anti-drug antibodies (ADA) in serum and plasma. It is for professional use only.

Anti-Denosumab (Prolia®) ADA Qualitative ELISA Kit test principle

The Assay Genie Antibody to Denosumab (Prolia®) ELISA is a sandwich assay for the determination of anti-Denosumab antibodies in serum and plasma samples. During the first incubation period, antibodies to Denosumab (ATD) in patient serum/ plasma samples are captured by the drug Denosumab (Prolia®) coated on the wall of the microtiter wells. After washing away the unbound components from samples, a peroxidase-labelled specific conjugate is added to each well and then incubated. After a second washing step, the bound enzymatic activity is detected by addition of tetramethylbenzidine (TMB) chromogen-substrate. Finally, the reaction is terminated with an acidic stop solution. The intensity of the reaction colour is directly proportional to the concentration of anti-Denosumab antibodies in sample.

Anti-Denosumab (Prolia®) ADA Qualitative ELISA Product Information

Information	Description
Application	Free drug
Required Volume (uL)	10
Total Time (min)	140
Sample Type	Serum, Plasma
Number of Assays	96
Detection Limit (ng/mL)	plus/minus
Spike Recovery (%)	-
Shelf Life (year)	1
Alternative Names	Anti-RANKL mAb Prolia

Anti-Denosumab (Prolia®) ADA Qualitative ELISA - 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- ligand (RANKL)

Denosumab binds to RANKL, a transmembrane or soluble protein essential for the formation, function, and survival of osteoclasts, the cells responsible for bone resorption. Denosumab (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®) immunogenicity

As with any biologic therapeutic, immunogenicity, in the form of anti-denosumab antibodies can occur. The demonstration of anti-denosumab antibodies during treatment with denosumab (Prolia®) is a major concern. The Assay Genie Anti-Denosumab (Prolia®) ADA Qualitative ELISA Kit can be efficiently used for monitoring anti-denosumab antibodies in biological samples and is for research use only.

Anti-Denosumab (Prolia®) ADA Qualitative 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
1 x 0.25 mL	Reactive Control Ready-to-use. Contains denosumab-reactive reagent, human serum, stabilizers and <0.1% NaN3
1 x 0.5 mL	Negative Control Ready-to-use. Contains human serum, stabilizers and <0.1% NaN3
1 x 12 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 peroxidase (POD) conjugate, stabilizer and preservatives.
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.

Anti-Denosumab (Prolia®) ADA Qualitative ELISA Protocol

Steps	Protocol
1	Pipette 100µl of Assay Buffer non-exceptionally into each of the wells to be used.
2	QUALITATIVE ELISA TEST FORMAT Pipette 10 µL of ready-to use Negative Control, Reactive Control, and Samples into the respective wells of microtiter plate. Wells A1: Negative Control B1: Negative Control C1: Reactive Control D1 and on: Sample (serum/plasma)
3	Cover the plate with adhesive film. Briefly mix contents by gently shaking the plate. Incubate 60 min at room temperature (18-25°C).
4	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.
5	Pipette 100 µL of ready-to use Peroxidase Conjugate into each well.
6	Cover the plate with adhesive foil. Incubate 30 min at room temperature (18- 25°C).
7	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.
8	Pipette 100 µL of TMB Substrate Solution into each well.
9	Incubate 20 min (without adhesive foil) at room temperature (18-25°C) in the dark
10	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.
11	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.