Trastuzumab specific Kit ELISA

N° du produit ABIN3172722

Le kit ELISA Trastuzumab specific (ABIN3172722) Chemical et Humain est un kit ELISA Colorimetric conçu pour quantifier Chemical et Humain Trastuzumab specific.

Aperçu rapide pour Trastuzumab specific Kit ELISA (ABIN3172722)

Antigène: Trastuzumab specific

Reactivité: Chemical, Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 0-300 ng/mL

Application: ELISA

Type d'échantillon: Serum, Plasma (EDTA - heparin)

Détail du produit

Seuil minimal de détection: 0 ng/mL

Fonction: Enzyme immunoassay for the specific and quantitative determination of free Trastuzumab in serum and plasma.

Analytical Method: Quantitative

Specificité: There is no cross reaction with any other proteins present in native human serum. A screening test was performed with 48 different native human sera. All produced OD450/620 nm values (ranged from 0.028 to 0.136) less than the mean OD (0.214) of standard D (10 ng/mL). In addition, binding of Trastuzumab is inhibited by recombinant human HER2 in a concentration dependent manner. Therefore, the Trastuzumab ELISA (mAb-Based) measures the biologically active free form of Trastuzumab, i.e. not pre-occuppied by HER2. No cross reaction was observed with sera spiked with the other therapeutic antibodies including Infliximab, Adalimumab, Etanercept, Rituximab, Tocillizumab, Omalizumab and Bevacizumab at concentrations up to 2 mg/mL. All produced mean OD450/620 nm values ranged from 0.013 to 0. 019.

Sensibilité: 10 ng/mL

Attributs du produit: Trastuzumab ELISA (mAb-based) Enzyme immunoassay for the specific quantitative determination of free Trastuzumab in serum and plasma.

Ingrédients:

• 1 x 12 x 8 Microtiter Plate Break apart strips coated with anti-Trastuzumab monoclonal antibody.
• 5 x 0.5 mL Trastuzumab Standards A-E 300, 100, 30, 10, and 0 ng/mL Ready to use. Used for construction of the standard curve. Contains Trastuzumab, serum, proteins, stabilizer and <15mM NaN3.
• 2 x 50 mL Assay Buffer Blue colored. Ready to use. Contains proteins and <15mM NaN3.
• 1 x 12 mL Enzyme Conjugate Red colored. Ready to use. Contains horseradish peroxidase(HRP)-conjugated anti-human IgG mouse monoclonal antibody, Proclin® and stabilizers.
• 1 x 12 mL TMB Substrate Solution Ready to use. Contains 3,3',5,5'-Tetramethylbenzidine (TMB).
• 1 x 12 mL Stop Solution Ready to use. 1 N Hydrochloric acid (HCl).
• 1 x 50 mL Wash Buffer, Concentrate (20x) Contains buffer, Tween® 20 and KathonTM.
• 2 x 1 Adhesive Seal For sealing microtiter plate during incubation.

Matériel non inclus:

• Micropipettes (< 3 % CV) and tips to deliver 5-1000 μL.
• Bidistilled or deionised water and calibrated glasswares (e.g. flasks or cylinders).
• Wash bottle, automated or semi-automated microtiter plate washing system.
• Microtiter plate reader capable of reading absorbance at 450 nm (reference wavelength at 600-650 nm is optional).
• Absorbent paper towels, standard laboratory glass or plastic vials, and a timer.

Information d'application

Indications d'application:

• Before performing the assay, samples and assay kit should be brought to room temperature (about 30 minutes beforehand) and ensure the homogeneity of the solution.
• All Standards should be run with each series of unknown samples.
• Standards should be subject to the same manipulations and incubation times as the samples being tested.
• All steps of the test should be completed without interruption.
• Use new disposable plastic pipette tips for each reagent, standard or specimen in order to avoid cross contamination.

Commentaires:

ELISA Kits are suitable also for using by an automated ELISA processor.

Durée du test: 1.5 h

Plaque: Pre-coated

Protocole: This ELISA is based on Trastuzumab-specific mouse monoclonal antibody (catcher Ab, clone VME-5A6b). Standards and diluted samples are incubated in the microtiter plate coated with IG-VME-5A6b mAb. After incubation, the wells are washed. A horseradish peroxidase (HRP)-conjugated anti-human IgG monoclonal antibody is added and binds to the Fc part of Trastuzumab. Following incubation, wells are washed and the bound enzymatic activity is detected by addition of chromogen-substrate. The colour developed is proportional to the amount of Trastuzumab in the sample or standard. Results of samples can be determined by using the standard curve. Binding of Trastuzumab to the solid phase, pre-coated with VME-5A6b, is inhibited by human HER2 in a concentration dependent manner. Therefore, the Trastuzumab ELISA (mAb-Based) measures the free form of Trastuzumab.

Préparation des réactifs:

Wash Buffer: Dilute 10 mL Wash Buffer (up to 200 mL) at the ratio of 1:20 with distilled water.
Warm up at 37 °C to dissolve crystals. Mix vigorously.
Store at 2-8 °C for up to 4 weeks.
Prepare Wash Buffer before starting the assay procedure.

Préparation de l'échantillon:

Serum/ Plasma: Initially dilute the Serum/ Plasma (Sample) at the ratio of 1:50 with Assay Buffer.
Sample : Assay Buffer Relation can be 1:50-1:250.
For dilution at 1:50, 10 μL Sample + 490 μL Assay Buffer
For dilution at 1:250, 20 μL of 1:50 diluted Sample + 80 μL Assay Buffer
Samples with a drug concentration above the measuring range should be rated as ">highest standard". The result should not be extrapolated. The sample in question should be further diluted with Assay Buffer and then retested

Serum, Plasma (EDTA, Heparin): The usual precautions for venipuncture should be observed. It is important to preserve the chemical integrity of a blood specimen from the moment it is collected until it is assayed. Do not use grossly hemolytic, icteric or grossly lipemic specimens. Samples appearing turbid should be centrifuged before testing to remove any particulate material.

Storage: 2-8 °C &leq,-20 °C (Aliquots)
Keep away from heat or direct sun light.
Avoid repeated freeze-thaw cycles.
Stability: 3 days at 2-8 °C, 6 months at -20 °C

Procédure de l'essai:

1. Pipette 100 μL of Assay Buffer into each of the wells to be used.
2. Pipette 20 μL of each Ready-to Use Standard, and Diluted Samples into the respective wells of the microtiter plate. Wells A1: Standard A B1: Standard B C1: Standard C D1: Standard D E1: Standard E F1 and so on: Diluted samples (Serum/Plasma)
3. Cover the plate with adhesive seal. Shake plate carefully. Incubate 30 min at room temperature (RT, 20-25 °C).
4. Remove adhesive seal. Aspirate or decant the incubation solution. Wash the plate 3 X 300 μL of Diluted Wash Buffer per well. Remove excess solution by tapping the inverted plate on a paper towel.
5. Pipette 100 μL of Enzyme Conjugate (HRP-anti human IgG mAb) into each well.
6. Cover plate with adhesive seal. Shake plate carefully. Incubate 30 min at RT.
7. Remove adhesive seal. Aspirate or decant the incubation solution. Wash the plate 3 X 300 μL of Diluted Wash Buffer per well. Remove excess solution by tapping the inverted plate on a paper towel.
8. Pipette 100 μL of Ready-to-Use TMB Substrate Solution into each well.
9. Incubate 15 min at RT. Avoid exposure to direct sunlight.
10. Stop the substrate reaction by adding 100 μL of Stop Solution into each well. Briefly mix contents by gently shaking the plate. Color changes from blue to yellow.
11. Measure optical density (OD) with a photometer at 450 nm (Reference at OD620 nm is optional) within 15 min after pipetting the Stop Solution.

Calcul des résultats:

A standard curve should be calculated using the standard concentration (X-axis) versus the OD450 (or OD450/620) values (Y-axis). This can be done manually using graph paper or with a computer program. Concerning the data regression by computer, it is recommended to primarily use the "4 Parameter Logistic (4PL)" or alternatively the "point-to-point calculation". In case of manual plot there are 2 options: Semilog graph or linear graph . Semilog graph paper is available at http://www.papersnake.com/logarithmic/semilogarithmic/. The concentration of the samples can be read from this standard curve as follows. Using the absorbance value for each sample, determine the corresponding concentration of the drug from the standard curve. This value always has to be multiplied by the individual dilution factor. If any diluted sample is reading greater than the highest standard, it should be further diluted appropriately with Assay Buffer and retested. Also this second dilution has to be used for calculation the final result

Précision du teste: Intra-assay CV: <10%.
Inter-assay CV: <10%.

Recovery rate was found to be >95% with native human serum and plasma samples when spiked with exogenous Trastuzumab at 10 μg/mL.

Restrictions: For Research Use only

Stockage

Agent conservateur: Sodium azide

Précaution d'utilisation: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Stock: 4 °C

Stockage commentaire: The kit is shipped at ambient temperature and should be stored at 2-8°C.
Keep away from heat or direct sun light.
The storage and stability of specimen and prepared reagents is stated in the corresponding chapters.
The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2-8°C.

Date de péremption: 24 months

Détail du antigène

Antigène: Trastuzumab specific

Sujet: Trastuzumab 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. 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.