Infliximab Total Antibody ELISA 试剂盒

• 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.

• 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.

• 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.

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.

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.

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

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.

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