Trastuzumab (Herceptin®) (mAb-based)

The drug Trastuzumab (trade names Herceptin®, Herclone®) is a recombinant DNA-derived humanized monoclonal antibody that selectively targets the extracellular domain of the human epidermal growth factor receptor 2 protein (HER2). The antibody is an IgG1 kappa that contains human framework regions with the complementarity-determining regions of a murine anti p185 HER2 antibody that binds to HER2. Trastuzumab blood concentrations throughout the dosing interval expected to be remaining above those considered necessary for anticancer activity. Furthermore, in a separate analysis, patients with the lowest Trastuzumab serum trough concentrations had the highest rate of disease progression and shortest overall survival. According to the prescribing information, the steady-state mean trough (Cmin) and maximum serum concentrations of Trastuzumab were reported to be 47.4 (5-115) μg/mL and 179 (107-309) μg/mL for the patients with primary tumor type of either breast cancer or metastatic breast cancer dosing 8mg/kg +6mg/kg q3w, 66.1 (14.9-142) μg/mL and 109 (51.0 209) μg/mL for the patients with breast cancer dosing 4mg/kg +2mg/kg qw, In this context, 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 Trastuzumab in order to potentially avoid some side effects with a reliable method might be beneficial. Enzyme immunoassay (ELISA) for the determination of free Trastuzumab (Herceptin®, Herclon®) in serum and plasma. For research use only, not for use in diagnostic procedures.
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Properties

Protocol PDF Click for Protocol
Catalog Number TK01728
Species Human
Design 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) + 10 min. (RT) = 1 hour, 40 min. total incubation time.
Standard Range 0 / 6 - 200 ng/mL
Sensitivity 2 ng/mL
Radioactivity
Other Names Herceptin®, Herclon®.
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 Trastuzumab (Herceptin®) (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 Trastuzumab (Herceptin®) (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 Trastuzumab (Herceptin®) (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 Trastuzumab (Herceptin®) (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 Trastuzumab (Herceptin®) (mAb-based). You can calculate the concentration of Trastuzumab (Herceptin®) (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 Trastuzumab (Herceptin®) (mAb-based). And the sample of Trastuzumab (Herceptin®) (mAb-based) were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of Trastuzumab (Herceptin®) (mAb-based). And the sample of Trastuzumab (Herceptin®) (mAb-based) were tested on 3 different plates, 30 replicates in each plate.

Recovery

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

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 87-106 97
EDTA plasma (n=5) 88-101 95
heparin plasma (n=5) 89-98 94

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Trastuzumab (Herceptin®) (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 (%) 89-100 90-106 93-99
Average (%) 94 93 94
1:4 Range (%) 93-102 90-105 90-104
Average (%) 94 94 95
1:8 Range (%) 93-101 92-99 89-102
Average (%) 94 95 95
1:16 Range (%) 90-103 89-99 93-103
Average (%) 94 94 93

Stability

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