Abatacept ELISA Kit (Orencia®) Free Drug

SKU:
HUMB00068
Product type:
Biosimilar ELISA
Application:
ELISA
Reactivity:
Human
€999

Description

Technical Manual

Abatacept (Orencia®) Free Drug ELISA Kit (HUMB00068)

The Assay Genie Abatacept has been developed for the quantitative determination of free Abatacept (Orencia®) in serum and plasma.

Abatacept (Orencia®) Free Drug 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 microtiter plate coated with the reactant for abatacept. After incubation, the wells are washed. Then, horse radish peroxidase (HRP) conjugated probe is added and binds to abatacept captured by the reactant on the surface of the wells. Following incubation wells are washed and the bound enzymatic activity is detected by addition of tetramethylbenzidine (TMB) chromogen substrate. Finally, the reaction is terminated with an acidic stop solution. The colour developed is proportional to the amount of abatacept in the sample or standard. Results of samples can be determined directly using the standard curve.

Abatacept (Orencia®) Free Drug ELISA Kit Product Information

Information Description
Application Free Drug
Required Volume (µl) 10
Total Time (min) 105
Sample Typle Serum, plasma
Number of Assays 96
Detection Limit (ng/mL) 10
Recovery 85-115%
Shelf Life (year) 1

Abatacept (Orencia®) Free Drug ELISA Kit - General Information

Abatacept is licensed for the treatment of rheumatoid arthritis (RA) in combination with methotrexate. The indication includes moderate to severe RA unresponsive to other disease-modifying antirheumatic drugs including at least one tumour necrosis factor (TNF)- a blocker, or where patients have been intolerant of such drugs.Abatacept (CTLA4Ig) is a fusion protein of the extracellular domain of the human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to a modified Fc of human immunoglobulin 1 (IgG1). Abatacept inhibits the activation of T lymphocytes that play an important role in the early stages of pathogenesis of RA.Activation of a T cell requires two signals from the antigen-presenting cell (APC).

The first signal is antigen specific and arises when antigenic peptides are presented to the T cell through the Major Histocompatibility Complex. A second signal, so-called co-stimulation, develops from the interaction between CD80 or CD86 antigen on the APC and CD28 antigen on the T cell. Abatacept binds with the extracellular domain of CTLA-4 to CD80 or CD86 antigen on the APC with a higher affinity than CD28, preventing the essential second signal for T-cell activation. T-cell activation and the production of inflammatory mediators and cytokines (TNF-a, interferon-gamma and interleukin-2) are consequently reduced. Trials in patients with RA have shown that abatacept slows progression of joint damage and improves function.

Abatacept (Orencia®) Free Drug ELISA Kit Contents

Size Kit Contents
1x12x8 Microtiter Plate
Break apart strips. Microtiter plate with 12 rows each of 8 wells coated with reactant.
1 mL (each) Standards A-E (10x)
Standard A: 300 ng/mL
Standard B: 100 ng/mL
Standard C: 30 ng/mL
Standard D: 10 ng/mL
Standard E: 0 ng/mL
Used for the standard curve and control. Contains evolocumab, human serum and stabilizer, <0,1% NaN3.
Standards are prepared concentrated (10x).
1 mL (each) Controls
Control low and high levels (10x)
Contains human serum and stabilizer, <0,1% NaN3.
Controls are prepared concentrated (10x).
1x50 mL Assay buffer
Ready to use. Blue coloured. Contains proteins, <0,1% NaN3.
1x12 mL Horse radish peroxidase conjugated probe.
Ready to use. Red coloured. Contains HRP conjugated probe, stabilizer and preservatives.
1x12 mL TMB substrate solution.
Ready to use. Contains 3,3′,5,5′- Tetramethylbenzidine (TMB).
1x12mL TMB stop solution.
Ready to use. 1N HCl
1x50mL Wash buffer (20x).
Prepared concentrated (20x) and should be diluted with the dilution rate given in the “Pretest setup instructions” before the test. Contains buffer with tween 20.
2x1 Adhesive Foil.
For covering microtiter plate during incubation

Abatacept (Orencia®) Free Drug ELISA Kit Protocol

1 Pipette 100µl of Assay Buffer non-exceptionally into each of the wells to be used.
2 Pipette 100 µL of each diluted Standards, Low level control, High level control and samples into the respective wells of microtiter plate
Wells
A1: Standard A
B1: Standard B
C1: Standard C
D1: Standard D
E1: Standard E
G1: Low level control
H1: High level control
3 Cover the plate with adhesive foil. Briefly mix contents by gently shaking the plate. Incubate 60 minutes at room temperature (18-25°C)
4 Remove adhesive foil. Discard incubation solution. Wash plate three times each with 300 µL Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel
5 Pipette 100 µL Conjugate into each well
6 Cover the plate with adhesive foil. Incubate 30 minutes at room temperature (18-25°C)
7 Remove adhesive foil. Discard incubation solution. Wash plate three times each with 300 µL Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel
8 Pipette 100 µL Substrate into each well
9 Incubate 15 minutes without adhesive foil at room temperature (18-25°C) in the dark
10 Stop the substrate reaction by adding 100 µL 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 OD 450nm with reference wavelength 650 nm (450/650 nm) within 30 minutes after pipetting the Stop Solution.
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