Evolocumab ELISA Kit (Repatha®) Qualitative

SKU:
HUMB00065
€899

Description

Technical Manual

Evolocumab (Repatha® ) Antibody screening - Qualitative ELISA Kit (HUMB00065)

The Assay Genie Evolocumab (Repatha® ) Antibody screening - Qualitative ELISA Kit has been developed for the determination of Evolocumabin serum and plasma samples.

Evolocumab (Repatha® ) 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 evolocumab. After incubation, the wells are washed. Then, horse radish peroxidase (HRP) conjugated probe is added and binds to evolocumab antibodies captured by the drug evolocumab 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 evolocumab antibodies in the sample or controls. The results can be evaluated with using cut-off value.

Evolocumab (Repatha® ) Antibody screening - Qualitative ELISA Kit Product Information

Information Description
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

Evolocumab (Repatha® ) Antibody screening - Qualitative ELISA Kit - General Information

Proprotein convertase subtilisin/kexin type 9 (PCSK9) was discovered in 2003 and subsequently emerged as a novel target for LDL-C-lowering therapy. By inactivating PCSK9, evolocumab upregulates LDL receptors causing increased catabolism of LDL-C and the consequent reduction of LDL-C levels in blood. Overall, evolocumab has had notable efficacy, with LDL-C reduction ranging from 53% to 75% in monotherapy and combination therapies, and is associated with minor adverse effects.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases plasma low-density lipoprotein cholesterol (LDL-C) by decreasing expression of the LDL receptor on hepatic cells. Evolocumab is a human monoclonal immunoglobulin G2 that binds specifically to human PCSK9 to reduce LDL-C. Evolocumab exhibits nonlinear kinetics as a result of binding to PCSK9. Elimination is predominantly through saturable binding to PCSK9 at lower concentrations and a nonsaturable proteolytic pathway at higher concentrations.

Evolocumab (Repatha® ) Antibody screening - Qualitative 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 (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

Evolocumab (Repatha® ) 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
Wells
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.
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Additional Information

Product type:
Biosimilar ELISA
Application:
ELISA
Reactivity:
Human
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