Aflibercept (Eylea®, Zaltrap®)

The drug Aflibercept (trade names Eylea®, Zaltrap®) is a recombinant fusion protein consisting of VEGF-binding portions from the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. Aflibercept has broad affinity for all ligands that bind to these receptors, including isoforms of VEGF-A, VEGF-B, and placental growth factors. Aflibercept demonstrated antitumour effects and antiangiogenic activity as a single agent and enhanced activity in combination with chemotherapy. Identification of biomarkers for (non-)response and risk factors for adverse drug reactions that might be related to serum concentrations and maintaining the effective concentration of Aflibercept in order to potentially avoid some side effects with a reliable method might be beneficial. Enzyme immunoassay (ELISA) for the determination of free Aflibercept in serum and plasma samples. For research use only, not for use in diagnostic procedures.
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Properties

Protocol PDF Click for Protocol
Catalog Number TK01026
Species Human
Design Sandwich enzyme immunoassay (ELISA) technique.
Standards 5 standards, ready to use.
Controls None provided.
Sample Types Serum and plasma.
Sample Volume 10 µL
Assay Desc. 60 min. incubation (RT) + 30 min. (RT) + 15 min. (RT) = 1 hour, 45 min. total incubation time.
Standard Range 0 / 6 - 200 ng/mL
Sensitivity 5 ng/mL
Radioactivity
Other Names Eylea®, Zaltrap®.
Storage 2 - 8°C
Postscript For research use only, not for use in diagnostic procedures.

Intended use

This ELISA kit applies to the in vitro quantitative determination of Aflibercept (Eylea®, Zaltrap®) concentrations in serum, plasma and other biological fluids.

Test principle

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antigen specific to Aflibercept (Eylea®, Zaltrap®). Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Aflibercept (Eylea®, Zaltrap®) and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Aflibercept (Eylea®, Zaltrap®), biotinylated detection antigen and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Aflibercept (Eylea®, Zaltrap®). You can calculate the concentration of Aflibercept (Eylea®, Zaltrap®) in the samples by comparing the OD of the samples to the standard curve.

Typical data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Precision

Intra-assay Precision (Precision within an assay):
3 samples with low, middle,high concentration of Aflibercept (Eylea®, Zaltrap®). And the sample of Aflibercept (Eylea®, Zaltrap®) were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of Aflibercept (Eylea®, Zaltrap®). And the sample of Aflibercept (Eylea®, Zaltrap®) were tested on 3 different plates, 30 replicates in each plate.

Recovery

Matrices listed below were spiked with certain level of Aflibercept (Eylea®, Zaltrap®) and the recovery rates were calculated by comparing the measured value to the expected amount of Aflibercept (Eylea®, Zaltrap®) in samples.

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 92-108 96
EDTA plasma (n=5) 88-103 93
heparin plasma (n=5) 85-99 94

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Aflibercept (Eylea®, Zaltrap®) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

    Serum (n=5) EDTA plasma (n=5) heparin plasma (n=5)
1:2 Range (%) 92-101 88-102 87-102
Average (%) 94 93 93
1:4 Range (%) 91-108 92-100 91-106
Average (%) 95 95 94
1:8 Range (%) 91-100 91-103 93-104
Average (%) 95 93 94
1:16 Range (%) 88-103 94-104 87-100
Average (%) 94 94 93

Stability

The stability of Aflibercept (Eylea®, Zaltrap®) is determined by the loss rate of activity. The loss rate of Aflibercept (Eylea®, Zaltrap®) is less than 5% within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Item Specifications Storage
Micro ELISA Plate(Dismountable) 8 wells ×12 strips -20℃, 6 months
Reference Standard 2 vials
Concentrated Biotinylated Detection Ab (100×) 1 vial, 120 μL
Concentrated HRP Conjugate (100×) 1 vial, 120 μL -20℃(shading light), 6 months
Reference Standard & Sample Diluent 1 vial, 20 mL 4℃, 6 months
Biotinylated Detection Ab Diluent 1 vial, 14 mL
HRP Conjugate Diluent 1 vial, 14 mL
Concentrated Wash Buffer (25×) 1 vial, 30 mL
Substrate Reagent 1 vial, 10 mL 4℃(shading light)
Stop Solution 1 vial, 10 mL 4℃
Plate Sealer 5 pieces
Product Description 1 copy
Certificate of Analysis 1 copy

Other supplies required

Microplate reader with 450 nm wavelength filter
High-precision transfer pipette, EP tubes and disposable pipette tips
Incubator capable of maintaining 37℃
Deionized or distilled water
Absorbent paper
Loading slot for Wash Buffer

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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