Trastuzumab (Herceptin®, Herclon®) ELISA Kit

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SKU:
HUMB00030
€899

Description

Trastuzumab (Herceptin®, Herclon®) ELISA Kit

Enzyme-linked immunosorbent assay for the quantitative determination of free Trastuzumab (Herceptin®, Herclon®) in serum and plasma and is for research use only. ELISA Genie Trastuzumab ELISA has been developed for the quantitative analysis of free trastuzumab in serum and plasma samples at high specificity.

Trastuzumab (Herceptin®, Herclon®) ELISA Kit test principle

Solid phase enzyme-linked immunosorbent assay (ELISA). Standards and samples (serum or plasma) are incubated in the microtitre plate coated with ligand antigen (recombinant human HER2/ErbB2/CD340) for trastuzumab (Herclon®, Herceptin®). After incubation, the wells are washed. In the next step, a horse radish peroxidase (HRP) conjugated is added. Following incubation, wells are washed, and the bound enzymatic activity is detected by addition of TMB chromogen-substrate. The colour developed is proportional to the amount of trastuzumab in the sample or standard. Results of samples are determined using the standard curve constructed.

Trastuzumab (Herceptin®, Herclon®) ELISA Product Information

Information Description
Application
Free drug
Required Volume (μl)
10
Total Time (min)
70
Sample Type
Serum, Plasma
Number of Assays
96
Detection Limit (ng/mL)
2 (ng/mL)
Spike Recovery (%)
85-115%
Shelf Life (year)
1

Alternative Names

Human Epidermal Growth Factor Receptor 2 (HER2)

Herceptin

Herclon

Trastuzumab ELISA - Key Information

Trastuzumab (Herceptin®, Herclon®) mode of action

Trastuzumab (Herclon®, Herceptin®) 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 is composed of 1,328 amino acids and has a molecular weight of 148 kDa. Trastuzumab has anti-tumor activity against HER2-positive human breast tumor cells in laboratory models and is active for the treatment of women with HER2-overexpressing breast cancers. Trastuzumab was approved in 1998 for clinical use for HER2-overexpressing metastatic breast cancer.

Trastuzumab exerts its anti-tumor effects by several mechanisms that are not yet completely understood. In HER2 overexpressing cells, trastuzumab markedly down-regulates HER2 expression by accelerating receptor endocytosis and degradation and inhibits cell cycle progression by inducing the formation of p27Kip1/Cdk2 complexes. Trastuzumab also induces antibody-dependent cell-mediated cytotoxicity against the HER2 expressing tumor cells in animal models. This process is regulated by antibody receptors FcγRIII and FcγRIIB on myeloid cells. Other additional mechanisms that have been proposed include suppression by trastuzumab of angiogenesis and metastasis.

Trastuzumab (Herceptin®, Herclon®) uses

Trastuzumab is used to treat metastatic breast cancer and stomach cancer. It is effective against tumors that posesss HER2/neu protein overexpression. Traztuzumab can be used alone or in combination with other chemotherapeutic medication. Traztuzumab is used in cancers that are HER2 receptor positive.

Trastuzumab (Herceptin®, Herclon®) and the HER2/neu protein

The HER (or ErbB) family of transmembrane tyrosine kinase receptors is composed of four members, HER1 to HER4 . HER2, a ligand-less Mr (molecular mass of the repeat unit) 185,000 receptor encoded by the neu proto-oncogene, is overexpressed in 25-30% of human breast cancer and has been associated with enhanced tumor aggressiveness and a high risk of relapse and death.

Recent evidence indicates that HER2 amplifies the signal provided by other receptors of the ErbB family by heterodimerizing with them. The important biological role of HER2 in the signaling network that drives epithelial cell proliferation and transformation, together with its extracellular accessibility and its overexpression in some human tumors led to considering HER2 as an appropriate target for tumor-specific therapies.

The neu gene encodes a 185-kDa transmembrane glycoprotein, referred to as p185neu, HER2, or erbB-2, possessing intrinsic protein tyrosine kinase activity. The receptor consists of an extracellular domain, with four subdomains including two cysteine rich domains, a transmembrane domain, and an intracellular domain, consisting of a juxtamembrane region, a tyrosine kinase domain, and a carboxyl tail harboring autophosphorylation sites HER2 is homologous to, but distinct from, other members of the erbB family, which includes the epidermal growth factor receptor (EGFR or erbB-1), erbB-3, and erbB-4.

The binding of cognate growth factors to these receptors regulates cell growth, proliferation, and diferentiation through the activation of receptor tyrosine kinases, triggering an incompletely defined signal transduction cascade. Signal transduction by these receptors is believed to involve dimerization and oligomerization, both in the form of homo-oligomers and hetero-oligomers in various erbB receptor combinations.

Trastuzumab (Herceptin®, Herclon®) pharmacokinetics

The pharmacokinetics of trastuzumab according to the prescribing data; The pharmacokinetics of trastuzumab were studied in women with metastatic breast cancer. Short duration intravenous infusions of 10 to 500 mg trastuzumab once weekly demonstrated dose-pendent pharmacokinetics. Mean half-life increased and clearance decreased with increasing dose level.

The half-life averaged 2 and 12 days at the 10 and 500 mg dose levels, respectively. The volume of distribution of trastuzumab was approximately that of serum volume (44 mL/kg). At the highest weekly dose studied (500 mg), mean peak serum concentrations were 377 mcg/mL. In studies using an initial dose of 4 mg/kg followed by a weekly dose of 2 mg/kg, a mean half-life of 6 days (range 4 1-32 days) was observed.

Between weeks 16 and 32, trastuzumab serum concentrations reached a steady state with mean trough and peak concentrations of approximately 9 mcg/mL and 123 mcg/mL, respectively. In a study of women receiving adjuvant therapy for breast cancer, a mean half-life of trastuzumab of 16 days (range: 11-23 days) was observed after an initial dose of 8 mg/kg followed by a dose of 6 mg/kg every three weeks.

Between weeks 6 and 37, trastuzumab serum concentrations reached a steady state with mean trough and peak concentrations of 63 mcg/mL and 216 mcg/mL, respectively. In this context, identification of biomarkers for (non-)response and risk factors for adverse drug reactions relating to serum concentrations and therapeutic drug monitoring of trastzumab might be very helpful.

Trastuzumab (Herceptin®, Herclon®) ELISA Kit Contents

Size Kit Contents

1 x 12 x 8

Microtiter Plate

Break apart strips. Microtiter plate with 12 rows each of 8 wells coated with reactant

7 x 0.3 mL

Trastuzumab Standards A-E, Concentrate (10X),High Level Control, Low Level Control


3000; 1000; 333; 111; 0 nanogram/mL.
Ready to use. Contains trastuzumab, human serum, stabilizers and <0.1% NaN3.

1 x 30 mL

Assay Buffer
Blue coloured. Ready to use. Contains proteins and <0.1% NaN3.

1 x 12 mL

Horse radish peroxidase-Conjugated Probe.

Red coloured. Ready to use. Contains HRP-probe, stabilizer and preservatives.

1 x 12 mL

TMB Substrate Solution
Ready to use. Contains TMB

1 x 12 mL

TMB Stop Solution
Ready to use. 1N HCl

1 x 50 mL

Wash Buffer concentrate (20x)
Contains Buffer with Tween 20.

2 x 1

Adhesive Foil
For covering of Microtiter Plate during incubation.

2 x 1

Semi-Log Graph Paper
For constructing standard curve and calculation of results.


Trastuzumab (Herceptin®, Herclon®) ELISA Protocol

Steps Protocol

1

Dilute each of the standards and samples (serum/plasma) using Diluted Assay Buffer as described in “Dilution of Standards and Samples (serum/plasma)” section of the technical manual.

2

Pipette 10 µL of each ready-to use Standards, High Level Control, Low Level Control and Diluted Samples into the respective wells of microtiter plate.


Wells
A1: Standard A
B1: Standard B
C1: Standard C
D1: Standard D
E1: Standard E
F1: High Level Control
G1: Low Level Control
H1 and on: Sample (Serum / Plasma) A

3

Cover the plate with adhesive foil. Incubate 30 min at room temperature (18- 25°C).

4

Remove adhesive foil. Discard incubation solution. Wash plate 3 times each with 300µL of diluted. Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.

5

Pipette 100 µL of ready-to use HRP-Conjugated Probe into each well.

6

Cover the plate with adhesive foil. Incubate 30 min at room temperature (18- 25°C).

7

Remove adhesive foil. Discard incubation solution. Wash plate 3 times each with 300 µL of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.

8

Pipette 100 µL of TMB Substrate Solution into each well.

9

Incubate 10 min (without adhesive foil) at room temperature (18-25°C) in the dark

10

Stop the substrate reaction by adding 100 µL of 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 450/650 nm within 15 min after pipetting of the Stop Solution.

Trademarks

Herceptin® is a registered trademark of Genentech, Inc.

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