Rituximab (Rituxan®) (mAb-based)

The drug Rituximab (trade name Rituxan® and Mabthera®) is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. The antibody is a glycosylated IgG1 kappa immunoglobulin containing murine lightand heavy-chain variable region sequences (Fab domain) and human constant region sequences (Fc domain). Rituximab is composed of 1,328 amino acids and has an approximate molecular weight of 144 kD. Rituximab has a high binding affinity for the CD20 antigen. The specificity of this testsystem is achieved by using a monoclonal antibody (clon 9D5b) for the coating of the microtiter plate. This antibody is specific for Rituximab only (regardless whether Rituxan® and Mabthera®) and does not cross react with other CD20 catchers. Enzyme immunoassay (ELISA) test kit for the determination of free Rituximab (Mabthera®, Rituxan®) in serum and plasma. The catcher is a highly specific mAb and the detection antibody is a HRP-labeled mAb binding at the Fc part of the drug. This assay design makes sure that there is no cross reactivity with any other CD20 catcher. This kit is for research use only, not for use in diagnostic procedures.
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Properties

Protocol PDF Click for Protocol
Catalog Number TK01645
Species Human
Design Enzyme immunoassay (ELISA) technique.
Standards 5 standards, concentrated.
Controls None provided.
Sample Types Serum and plasma.
Sample Volume 10 µL
Assay Desc. 1 hour incubation (RT) + 30 min. (RT) + 10 min. (RT) = 1 hour, 40 min. total incubation time.
Standard Range 0 / 6.0 - 200.0 ng/mL
Sensitivity 2 ng/mL
Radioactivity
Other Names Mabthera®, Rituxan®.
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 Rituximab (Rituxan®) (mAb-based) 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 Rituximab (Rituxan®) (mAb-based). Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Rituximab (Rituxan®) (mAb-based) 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 Rituximab (Rituxan®) (mAb-based), 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 Rituximab (Rituxan®) (mAb-based). You can calculate the concentration of Rituximab (Rituxan®) (mAb-based) 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 Rituximab (Rituxan®) (mAb-based). And the sample of Rituximab (Rituxan®) (mAb-based) were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of Rituximab (Rituxan®) (mAb-based). And the sample of Rituximab (Rituxan®) (mAb-based) were tested on 3 different plates, 30 replicates in each plate.

Recovery

Matrices listed below were spiked with certain level of Rituximab (Rituxan®) (mAb-based) and the recovery rates were calculated by comparing the measured value to the expected amount of Rituximab (Rituxan®) (mAb-based) in samples.

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 87-101 98
EDTA plasma (n=5) 88-104 95
heparin plasma (n=5) 84-102 95

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rituximab (Rituxan®) (mAb-based) 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 (%) 87-100 90-104 89-107
Average (%) 93 94 93
1:4 Range (%) 91-102 87-107 90-99
Average (%) 93 95 95
1:8 Range (%) 92-101 91-105 89-107
Average (%) 94 94 95
1:16 Range (%) 88-108 93-108 87-108
Average (%) 94 94 93

Stability

The stability of Rituximab (Rituxan®) (mAb-based) is determined by the loss rate of activity. The loss rate of Rituximab (Rituxan®) (mAb-based) 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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