Description

Datasheet

Anti-Etanercept (Enbrel®) ADA Qualitative ELISA Kit

Enzyme-linked immunosorbent assay for the qualitative determination of antibodies to etanercept in serum and plasma.The Assay Genie Anti-Etanercept (Enbrel®) ADA Qualitative ELISA Kit can be efficiently used for monitoring anti-Etanercept antibodies and is for research use only. Qualitative detection of Etanercept anti-drug antibodies (ADA) can be acheived with this Assay Genie ELISA kit.

Anti-Etanercept (Enbrel®) ADA Qualitative ELISA Kit test principle

The Assay Genie Antibody to Etanercept (Enbrel®) ELISA is a sandwich assay for the determination of antibodies against etanercept in serum and plasma samples. During the first incubation period, the drug etanercept coated on the wall of the microtiter wells captures the antibodies to etanercept in patient serum and plasma samples. After washing away the unbound components from samples, a Peroxidase labelled 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 antibodies to etanercept in sample.

Anti-Etanercept (Enbrel®) 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	Tumour Necrosis Factor Receptor (TNFR) Enbrel

Anti-Etanercept (Enbrel®) ADA Qualitative - Key Information

Etanercept (Enbrel®) mode of action

Etanercept (Enbrel®) is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumour necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. The Fc component of etanercept contains the CH2 domain, the CH3 domain and hinge region, but not the CH1 domain of IgG1. Etanercept consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons.

Etanercept binds specifically to tumour necrosis factor (TNF) and blocks its interaction with cell surface TNF receptors. Elevated levels of TNF are found in involved tissues and fluids of patients with rheumatoid arthritis (RA), psoriatic arthritis, ankylosing spondylitis (AS), and plaque psoriasis. Two distinct receptors for TNF (TNFRs), a 55 kilodalton protein (p55) and a 75 kilodalton protein (p75), exist naturally as monomeric molecules on cell surfaces and in soluble forms. Biological activity of TNF is dependent upon binding to either cell surface TNFR. Etanercept is a dimeric soluble form of the p75 TNF receptor that can bind to two TNF molecules. Etanercept inhibits binding of both TNF alpha and TNF beta (lymphotoxin alpha [LT alpha]) to cell surface TNFRs, rendering TNF biologically inactive.

Etanercept (Enbrel®) uses

Etanercept is a tumor necrosis factor (TNF) inhibitor used to treat various autoimmune conditions including certain types of arthritis; rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis. Etanercept is also used to treat particular skin conditions such as plaque psoriasis.

Etanercept (Enbrel®) immunogenicity

The use of etanercept was associated to the development of anti-etanercept antibodies in various percentages of patients during therapy with the drug Enbrel®. This might lead to severe complications. The Assay Genie Anti-Etanercept (Enbrel®) ELISA Kit can be efficiently used for monitoring Etanercept anti-drug antibodies (ADA) antibodies and is for research use only.

Anti-Etanercept (Enbrel®) 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 etanercept.
1 x 0.25 mL	Reactive Control Ready-to-use. Contains etanercept-reactive reagent, human serum, stabilizers and <0.1% NaN3.
1 x 0.5 mL	Negative Control Ready-to-use. Contains human serum 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-Etanercept (Enbrel®) ADA Qualitative ELISA Protocol

Steps	Protocol
1	Pipette 100µl of Assay Buffer non-exceptionally into each of the wells to be used.
2	Pipette 10 µL of ready-to use Negative Control, Positive Control, and Samples into the respective wells of microtiter plate. Wells A1: Negative Control B1: Negative Control C1: Positive Control D1 and on: Sample (Serum/Plasma)
3	Cover the plate with adhesive foil. Incubate 30 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.