Abatacept (Orencia®) Antibody screening - Qualitative ELISA Kit (HUMB00069)
The Assay Genie Abatacept ELISA has been developed for the qualitative determination of antibodies to abatacept in serum and plasma.
Abatacept (Orencia®) Antibody screening - Qualitative ELISA Kit Test Principle
Solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. Controls and samples (serum or plasma) are incubated in the microtiter plate coated with the drug abatacept. After incubation, the wells are washed. Then, horse radish peroxidase (HRP) conjugated probe is added and binds to abatacept antibodies captured by the drug abatacept 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 antibodies in the sample or controls. The results can be evaluated with using cut-off value.
Abatacept (Orencia®) Antibody screening - Qualitative ELISA Kit Product Information
|Application||Antibody screening - Qualitative|
|Required Volume (µl)||10|
|Total Time (min)||140|
|Sample Typle||Serum, plasma|
|Number of Assays||96|
|Detection Limit (ng/mL)||+/-|
|Spike recovery (%)||-|
|Shelf Life (year)||1|
Abatacept (Orencia®) Antibody screening - Qualitative 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®) Antibody screening - Qualitative ELISA Kit Contents
|1x12x8||Microtiter Plate |
Break apart strips. Microtiter plate with 12 rows each of 8 wells coated with reactant.
|1 mL (negative) |
0,3 mL (positive)
|Control Negative & Positive |
Ready to use. Contains human serum and stabilizer, <0,1% NaN3.
|1x12 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®) Antibody screening - Qualitative ELISA Kit Protocol
|1||Pipette 100µl of Assay Buffer non-exceptionally into each of the wells to be used.|
|2||Pipette 10 µL of each Negative control, Positive control and samples into the respective wells of microtiter plate |
A1: Negative control
B1: Negative control
C1: Positive control
D1 and on: Samples
|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 60 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 20 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.|