Rituximab Antibody Kit ELISA

N° du produit ABIN2862663

Détail du produit

Antigène: Rituximab Antibody

Épitope: Free Chain

Reactivité: Humain, Chemical

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

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

Marque: ImmunoGuide®

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

Analytical Method: Semi-Quantitative

Specificité: Free antibodies against Rituximab (Rituxan®, Mabthera®)B ecause of our special assay design this Antibody to Rituximab ELISA kit provides the advantage of limiting the potential false positive reactions that are related with the presence of RF in serum/plasma samples. A screening test was performed with 64 different native human sera. All samples showed OD450 nm values (ranged from 0.048 to 0.062) less than the mean OD of cut-off controls. This test system measures the concentration of free antibodies directed against Infliximab. It cannot detect these antibodies if the drug already is bound to it.

Sensibilité: 10 ng/mL

Attributs du produit: This test does not measure the antibodies if they already are bound to the drug Rituximab

Ingrédients:

• 1 x 12 x 8 Microtiter Plate Break apart strips pre-coated with the drug Rituximab.
• 1 x 0.5 mL Negative Control Ready to use. Contains human serum and <15 mM NaN3.
• 1 x 0.5 mL Cut-off Control Ready to use. Contains human serum and antibody to Rituximab at 3 AU/mL and <15 mM NaN3.
• 1 x 0.5 mL Positive Control Ready to use. Contains human serum and antibody to Rituximab and <15 mM NaN3.
• 1 x 12 mL Assay Buffer Blue colored. Ready to use. Contains human serum, proteins and <15 mM NaN3.
• 1 x 12 mL Enzyme Conjugate Red colored. Ready to use. Contains horseradish peroxidase(HRP)- conjugated Rituximab, 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:

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

Volume d'échantillon: 10 μL

Durée du test: 2.5 h

Plaque: Pre-coated

Protocole: The Antibody to Rituximab ELISA is a sandwitch type ELISA for the determination of antibodies against Rituximab in serum and plasma samples. During the first incubation period, the drug Rituximab, coated on the wall of the microtiter wells, captures the antibodies to Rituximab in patient serum and plasma samples. After washing away the unbound components from samples, a Peroxidase-labelled Rituximab conjugate is added to each well and then incubated. Antibody to Rituximab, if present in sample, will make a bridge, with its two identical Fab arms, between the Rituximab coated on the well and the other Rituximab 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 an acidic stop solution. The intensity of the reaction color is related with the presence and quantity of antibodies to Rituximab in thesample.

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.

Prélèvement de l'échantillon: Normal serum or plasma collection

Préparation de l'échantillon:

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 10 μL of each Ready-to Use Negative Control, Cut-off Control, Positive Control, and Samples into the respective wells of microtiter plate. Wells A1: Negative Control B1: Negative Control C1: Cut-off Control D1: Cut-off Control E1: Positive Control F1: Positive Control G1 and so on: Samples (Serum/Plasma)
3. Cover the plate with adhesive seal. Shake plate carefully. Incubate 60 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-Rituximab) into each well.
6. Cover plate with adhesive seal. Shake plate carefully. Incubate 60 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 OD620nm is optional) within 15 min after pipetting the Stop Solution.

Calcul des résultats:

For the run to be valid, the OD450 nm of each Positive Control should be ≥ 1.000 and the OD450 nm of each Negative Control should be ≤0.100. 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 Cut-off Controls. The results are expressed in arbitrary units (AU/mL).

Cut-off= 3 AU/mL=OD Cut-off Control

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

Range: &geq, 3 AU/mL (Positive)
Range: < 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 patient's sample = 0.600
Average OD of cut-off controls = 0.150 (3 AU/mL)
Concentration of patient's sample = 0.600/0.150 x 3 AU/mL = 12 AU/mL

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

Restrictions: For Research Use only

Stockage

Buffer: < 15mM NaN3

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: Rituximab Antibody

Classe de substances: Antibody

Sujet: Rituximab is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. The antibody is a glycosylated IgG1 kappa immunoglobulin containing murine light- and heavy-chain variable region sequences (Fab domain) and human constant region sequences (Fc domain). Rituximab has a high binding affinity for the CD20 antigen of 5.2 to 11.0 nM. As with all therapeutic proteins, there is a potential for immunogenicity. It was reported that, using an ELISA assay, human anti-chimeric antibody (HACA) was detected in 4 of 356 (1.1 % ) patients with low-grade or follicular NHL receiving single-agent Rituximab. A total of 273/2578 (11 %) patients with RA tested positive for HACA at any time after receiving Rituximab. A total of 23/99 (23 %) Rituximab-treated patients with WG and MPA tested positive for HACA by 18 months. The clinical relevance of HACA formation in Rituximab-treated patients is unclear. Accoring to the prescribing information, the use of Rituximab might be associated to the development of anti-Rituximab antibodies, even some might be neutralizing, in various percentages of patients during therapy with the drug. The Antibody to Rituximab ELISA Kit can be efficiently used for monitoring Anti- Rituximab antibodies during therapy and offers the scientist a tool for decision on possible preventive measures.

Poids moléculaire: 144 kDa