Infliximab Total Antibody ELISA Kit

Produktnummer ABIN2862668

Produktdetails

Target: Infliximab Total Antibody

Bindungsspezifität: Free & Bound

Reaktivität: Chemical, Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Verwendungszweck: Enzyme immunoassay for the semi-quantitative determination of total (free and drug-bound) antibodies to Infliximab (ATI) in human serum and plasma.

Marke: ImmunoGuide®

Proben: Serum, Plasma (EDTA - heparin)

Analytische Methode: Semi-Quantitative

Spezifität: Free and bound antibodies against Infliximab (Remicade®, Remsima®)S creening test was performed with 59 different native human sera. All produced OD450nm values (ranged from 0.081 to 0.146) less than the mean OD of cut-off controls.

Sensitivität: 10 tAU/mL

Produktmerkmale: This test measures the total (free and bound) antibody concentration directed against the drug Infliximab. It can detect the drug-bound antibody even in a 20 fold excess of the drug.

Bestandteile:

• 1 x 12 x 8 Microtiter Plate Break apart strips pre-coated with the drug Infliximab.
• 1 x 0.5 mL Positive Control Ready to use. Contains human serum with antibody to Infliximab.
• 1 x 0.5 mL Negative Control Ready to use. Contains human serum.
• 1 x 0.5 mL Cut-off Control Ready to use. Contains human serum with antibody to Infliximab and Infliximab.
• 1 x 12 mL Dissociation Buffer Ready to use. Contains buffer.
• 1 x 12 mL Assay Buffer Ready to use. Contains buffer.
• 1 x 6 mL Enzyme Conjugate Red colored. Ready to use. Contains horseradish peroxidase(HRP)-conjugated Infliximab, 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.
• 1 x 1 Polypropylene Microtiter Plate For the usage of sample and control dilution.
• 2 x 1 Adhesive Seal For sealing microtiter plate during incubation.

Benötigtes Material:

• Micropipettes (< 3 % CV) and tips to deliver 5-1000 μL.
• Ultrapure 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:

Semiquantitative measurement with reference to a cut-off control
10 tAU/ml (sensitivity in the well)T
otal Antibody to Infliximab ELISA is suitable also for using by an automated ELISA processor.

Probenmenge: 5 μL

Testdauer: 1.5 h

Plattentyp: Pre-coated

Protokoll: The Antibody to Infliximab ELISA has been desighed for the determination of total antibodies (free and Infliximab-bound) against Infliximab in serum and plasma samples. During sample preparation, the Infliximab-bound anti-Infliximab antibodies are separated from the complex in order to acquire free anti-Infliximab antibodies. During the incubation period, antibodies to Infliximab (ATI) are replaced and bridged in the well with its Fab arms, between the Infliximab coated on the well and the conjugated Infliximab labelled with peroxidase. After washing away the components the bound enzymatic activity is detected by addition of tetramethylbenzidine (TMB) chromogen-substrate. Finally, the reaction is terminated with stop solution. The positive reaction is related with the presence of ATI in the sample. The semi-quantitative interpretation is made by using the cut-off control.

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.

Probennahme: Normal serum or plasma collection

Aufbereitung der Proben:

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 50 μL of Assay Buffer into the wells to be used.
2. Pipette 50 μL of Enzyme Conjugate (HRP-Infliximab) into each well.
3. Pipette 20 μL of Diluted Controls and Samples (as described in section 10.2.) into the respective wells of microtiter plate. Wells A1: Diluted Positive Control B1: Diluted Negative Control C1: Diluted Negative Control D1: Diluted Cutoff Control E1: Diluted Cutoff Control F1 and so on: Diluted Samples (Serum/Plasma)
4. Cover the plate with adhesive seal. Shake plate carefully. Incubate 60 min at room temperature (RT) (20-25 °C).
5. 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.
6. Pipette 100 μL of Ready-to-Use TMB Substrate Solution into each well.
7. Incubate 20 min at RT. Avoid exposure to direct sunlight.
8. 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.
9. Measure optical density (OD) with a photometer at 450 nm (Reference at OD620nm is optional) within 15 min after pipetting the Stop Solution.

Ergebnisberechnung:

For the run to be valid, the OD450 nm of the Positive Control should be ≥ 0.500 and the OD450 nm of each Negative Control should be ≤0.200. If not, improper technique or reagent deterioration may be suspected and the run should be repeated. The results are evaluated by dividing all individual results by the mean OD450nm of the Cutoff Controls. The results are expressed in total arbitrary units (tAU/mL).

Cutoff =10 tAU/mL= OD Cutoff Control

Samples which have an equal and higher optical density (OD) than the mean OD of cutoff controls are positive.

Range: &geq, 10 tAU/mL (Positive)
Range: < 10 tAU/mL (Negative)

The results themselves should not be the only reason for any therapeutical consequences. They have to be correlated to other clinical observations.

Sample calculation for a positive sample:
OD of patient's sample = 0.900
Average OD of cutoff controls = 0.300 (10 tAU/mL)
Concentration of patient's sample = 0.900/0.300 x 10 tAU/mL = 30 tAU/mL 8

Testpräzision: Intra-assay CV: <10%.
Inter-assay CV: <10%

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Buffer: < 15mM NaN3

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: Infliximab Total Antibody

Substanzklasse: Antibody

Hintergrund: Infliximab is a chimeric monoclonal antibody and used to treat auto-immune disorders. Infliximab reduces the amount of active human tumour necrosis factor alpha (hTNFα) in the patient by binding to it and preventing it from signaling the receptors for TNFα on the surface of various cell types. TNFα is one of the key cytokines that triggers and sustains the inflammatory reactions. Infliximab is used for the treatment of psoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis. This drug is approved by FDA. One of the major concern is the potential development of anti-Infliximab antibodies (ATI) which in turn may interfere with Infliximab efficacy as mainly judged by observing the relapse of signs and symptoms of disease and necessitate dose-escalation or potentially ending up the treatment. In this context, demonstration of anti-Infliximab antibodies during treatment with Infliximab has a major concern and monitoring for the presence of specific antibodies during clinical trials is an important issue for follow up of the treatment regimens. The Total Antibody to Infliximab ELISA Kit can be efficiently used for measuring free and Infliximab-bound antibodies against Infliximab. This test kit can measure any antibodies against Inflixima even in the presence of high excess of Infliximab. But the most convenient time for sampling serum or plasma is the time just before the next administration of the therapeutic antibody. In combination with the drug level determination, the Total Antibody to Infliximab ELISA Kit offers the clinician a tool for monitoring and optimizing the therapy early on. And might be a possible decision on preventive measures to reduce anti-Infliximab antibodies during therapy.

Molekulargewicht: 144 kDa