Golimumab Antibody ELISA Kit

Produktnummer ABIN2862664

Produktdetails

Target: Golimumab Antibody

Bindungsspezifität: Free Chain

Reaktivität: Chemical, Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Verwendungszweck: Enzyme immunoassay for the semi-quantitative determination of free antibodies to Golimumab in human serum and plasma samples.

Marke: ImmunoGuide®

Proben: Serum, Plasma (EDTA - heparin)

Analytische Methode: Semi-Quantitative

Spezifität: Free antibodies against Golimumab (Simponi®)S creening test was performed with 90 different native and RF-negative human sera and additionally 2 different ATI positive sera. All produced OD450nm values (ranged from 0.044 to 0.136, OD450nm of ATI sera were 0.060 and 0.059 respectively) less than the cut-off value (3x0.060).

Sensitivität: 10 ng/mL

Produktmerkmale: This test does not measure the antibodies if they already are bound to the drug Golimumab

Bestandteile:

• 1 x 12 x 8 Microtiter Plate Break apart strips pre-coated with the drug Golimumab.
• 1 x 2 mL Positive Control Ready to use. Contains reactive antibody, proteins, stabilizer and <15 mM NaN3.
• 1 x 2 mL Negative Control Ready to use. Contains human serum, stabilizer and <15 mM NaN3.
• 1 x 60 mL Assay Buffer Blue colored. Ready to use. Contains proteins and <15 mM NaN3.
• 1 x 12 mL Enzyme Conjugate Red colored. Ready to use. Contains horseradish peroxidase(HRP)-conjugated Golimumab, 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:

Antibody to Golimumab ELISA is suitable also for using by an automated ELISA processor.

Probenmenge: 5 μL

Testdauer: 2.5 h

Plattentyp: Pre-coated

Protokoll: The Antibody to Golimumab ELISA is a sandwich type ELISA for the determination of free antibodies against Golimumab in serum and plasma samples. During the first incubation period, antibodies to Golimumab in patient serum/plasma samples are captured by the drug Golimumab coated on the wall of the microtiter wells. After washing away the unbound components from samples, a peroxidase-labelled Golimumab conjugate is added and then incubated. Antibody to Golimumab, if present in sample, will make a bridge, with its two identical Fab arms, between the Golimumab coated on the well and the other Golimumab labeled with peroxidase. After a second washing step, 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 to the presence of antibodies to Golimumab in the sample.

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: Dilute Serum/ Plasma (Sample) at the ratio of 1:101 with Assay Buffer.
For dilution at 1:101, 5 μL Sample + 500 μL Assay Buffer
Negative and Positive Controls are ready-to-use and should NOT be diluted with the assay buffer.

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 100 μL of each Ready-to Use Negative Control, Positive Control, and 1:101 Diluted Samples (as described in section 10.2) into the respective wells of microtiter plate. Wells A1: Negative Control B1: Negative Control C1: Positive Control D1 and so on: Diluted samples (Serum/Plasma)
2. Cover the plate with adhesive seal. Shake plate carefully. Incubate 60 min at room temperature (RT) (18-25 °C).
3. 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.
4. Pipette 100 μL of Enzyme Conjugate (HRP-Golimumab) into each well.
5. Cover plate with adhesive seal. Shake plate carefully. Incubate 60 min at RT.
6. 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.
7. Pipette 100 μL of Ready-to-Use TMB Substrate Solution into each well.
8. Incubate 20 min at RT. Avoid exposure to direct sunlight..
9. 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.
10. 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 ≥ 1.000 and the OD450 nm of each Negative Control should be ≤0.150. 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 Negative Controls. The results are expressed in arbitrary units (AU/mL).

Range: > 3 AU/mL (Positive)
Range: &leq, 3 AU/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 sample = 0.740 Cut-off = 3 x
Average OD of Negative Controls = 3 x 0.100 = 0.300 = 3 AU/mL
Concentration of sample = 0.740/0.100 = 7.4 AU/mL (positive)

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: Golimumab Antibody

Substanzklasse: Antibody

Hintergrund: Golimumab is a human monoclonal antibody that binds to both the soluble and transmembrane bioactive forms of human TNFα. This interaction prevents the binding of TNFα to its receptors, thereby inhibiting the biological activity of TNF. Golimumab has been proven effective in the treatment of Rheumatoid Arthritis (RA), Ankylosing Spondylitis (AS), Psoriatic Arthritis (PsA) or Ulcerative Colitis (UC). Antibodies to Golimumab were detected in 57 (4 % ) of Golimumab -treated patients across the Phase 3 RA, PsA, and AS trials through Week 24. The data from the literature demonstrated that Anti-Drug Antibody positivity was significantly associated with low Golimumab levels and poor therapeutic response. The positive correlation between serum drug trough levels and therapeutic response indicates that drug monitoring could be useful for optimising the dosing of biologics in a personalised therapy strategy. The Antibody to Golimumab ELISA Kit can be efficiently used for monitoring Anti- Golimumab antibodies during therapy and offers the clinician a tool for decision on possible preventive measures to reduce Anti-Golimumab antibodies.

Molekulargewicht: 144 kDa