Bevacizumab ELISA 试剂盒

• 1 x 12 x 8 Microtiter Plate Break apart strips coated with recombinant human vascular endothelial growth factor-A (rhVEGF-A).
• 5 x 0.3 mL Bevacizumab Standards A-E, Concentrate (1000x), 200, 60, 20, 6, and 0 µg/mL Ready to use. Used for construction of the standard curve. Contains Bevacizumab, human serum, proteins, stabilizer and <15mM NaN3..
• 1 x 12 mL Assay Buffer Blue colored. Ready to use. Contains proteins and <15mM NaN3.
• 1 x 60 mL Dilution Buffer, Concentrate (5X), 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.

• 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.

• 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. 6
• Use new disposable plastic pipette tips for each reagent, standard or specimen in order to avoid cross contamination.

Bevacizumab 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.

Dilution of Standards and Samples
The dilutions depicted below are examples of how to obtain final 1:1000 dilution. Standards and samples should be properly diluted as homogenous mixture before starting the assay procedure. It is recommended mixing the standards and samples well to homogenous solution at each dilution step.
1. 10 µL of standard or sample added to 90 µL of 1X dilution buffer, giving the total volume of 100 µL and a dilution of 1:10.
2. 10 µL of 1:10 diluted standard or sample added to 990 µL of 1X dilution buffer, giving the total volume of 1000 µL and a dilution of 1:1000.
3. 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 Dilution Buffer and then retested.

1. Pipette 100 μL of Assay Buffer into each of the wells to be used.
2. Pipette 20 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)
3. Cover the plate with adhesive seal. Shake plate carefully. Incubate 30 min at room temperature (RT) (18-25 °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 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 OD620 nm is optional) within 15 min after pipetting the Stop Solution.

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 we are recommending 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. 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 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.