Omalizumab specific ELISA Kit

Produktnummer ABIN3172725

Produktdetails

Target: Omalizumab specific

Reaktivität: Chemical, Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Verwendungszweck: Enzyme immunoassay for the specific and quantitative determination of free Omalizumab in serum and plasma.

Marke: ImmunoGuide®

Proben: Serum, Plasma (EDTA - heparin)

Analytische Methode: Quantitative

Spezifität: There is no cross reaction with any other proteins present in native human serum. A screening test was performed with different native human sera. All produced OD450/620 nm values (ranging from 0.038 to 0.113) less than the mean OD (0.267) of standard D (10 ng/mL). In addition, human IgE antibody was also separately tested at concentrations up to 100 μg/mL and cross reaction was NOT observed. No cross reaction was observed with sera spiked with the other therapeutic antibodies including Infliximab, Adalimumab, Etanercept, Rituximab, Trastuzumab, Tocillizumab and Bevacizumab at concentrations up to 2 mg/mL. All produced mean OD450/620 nm values ranging from 0.019 to 0.101.

Sensitivität: 10 ng/mL

Produktmerkmale: Omalizumab ELISA (mAb-based) Enzyme immunoassay for the specific quantitative determination of free Omalizumab in human serum and plasma.

Bestandteile:

• 1 x 12 x 8 Microtiter Plate Break apart strips coated with anti-Omalizumab monoclonal antibody.
• 5 x 0.5 mL Omalizumab Standards A-E 300, 100, 30, 10, and 0 ng/mL Ready to use. Used for construction of the standard curve. Contains Omalizumab, 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.

Benötigtes Material:

• 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.

Anwendungsinformationen

Applikationshinweise:

• 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.

Kommentare:

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

Testdauer: 1.5 h

Plattentyp: Pre-coated

Protokoll: This ELISA is based on Omalizumab-specific monoclonal antibody (catcher Ab, clone Ulkr4H1). Standards and diluted samples are incubated in the microtiter plate coated with IG-Ulkr4H1 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 Omalizumab. Following incubation, wells are washed and the bound enzymatic activity is detected by addition of a chromogen-substrate. The colour developed is proportional to the amount of Omalizumab in the sample or standard. Results for the unknown samples can be determined by using the standard curve.

Aufbereitung der Reagenzien:

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.

Aufbereitung der Proben:

Serum/ Plasma: Initially dilute the Serum/ Plasma (Sample) at the ratio of 1:100 with Assay Buffer.
Sample : Assay Buffer Relation can be 1:100-1:500.
For dilution at 1:100, 5 μL Sample + 495 μL Assay Buffer
For dilution at 1:500, 50 μL of 1:100-Diluted Sample + 200 μL Assay Buffer
Diluted 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

Testdurchführung:

1. Pipette 10 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)
2. Pipette 100 μL of Assay Buffer into each of the wells used.
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 10 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 of the Stop Solution.

Ergebnisberechnung:

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

Testpräzision: 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 Omalizumab at 30,10 or 3 μg/mL.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Konservierungsmittel: Sodium azide

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

Lagerung: 4 °C

Informationen zur Lagerung: 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.

Haltbarkeit: 24 months

Antigendetails

Target: Omalizumab specific

Hintergrund: Omalizumab (trade name Xolair®) is a recombinant DNA-derived humanized IgG1κ monoclonal antibody and it binds to human immunoglobulin E (IgE). The molecular weight of omalizumab is 149 kilodlatons and is produced by Chinese hamster ovary cell suspension culture. Omalizumab inhibits the binding of IgE to IgE receptor (FcεRI) on the surface of mast cells and basophils. Therefore, the Omalizumab is expected to limit the degree of release of mediators of the allergic response from the FcεRI bearing cells. The Omalizumab ELISA (mAb-based) is developed for the specific measurement of Omalizumab in serum, plasma and other biological fluids by the advantage of using a site-directed IG-Ulkr4H1 mouse monoclonal antibody (mAb) specific for Omalizumab only. Binding of Omalizumab to the solid phase, pre- coated with IG-Ulkr4H1, is inhibited by human IgE in a concentration dependent manner. Therefore, the Omalizumab ELISA (mAb-based) measures the free form of Omalizumab only. The choice of specifically measuring the free form allows investigators to analyze the concentration-effect relationship. The Omalizumab ELISA (mAb-based) kit can be efficiently used for monitoring free Omalizumab levels in serum and plasma.

Molekulargewicht: 149 kDa