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ELISA KITS - INSTRUCTIONS FOR USE
Procedural Notes - Reagents Preparation - Assay Protocol
PROCEDURAL NOTES
| - | Allow samples and reagents to reach room temperature prior to testing. Do not use water baths to thaw samples or reagents. |
| - | Mix samples and all reagents thoroughly before use. |
| - | Avoid excessive foaming of reagents. Also avoid exposure of reagents to excessive heat or light during storage and incubation. |
| - | Avoid handling the tops of the wells both before and after filling. |
| - | Standards and samples should be assayed in duplicate. |
| - | Run a separate standard curve for each assay. |
| - | Use only coated wells from the same reagent batch for each assay. Also do not mix reagents from different kit lots. |
| - | Perform incubations in a sealed box containing a wet paper towel in order to prevent evaporation. |
REAGENTS PREPARATION
| - | Reconstitute XG00n-Calibrator with 440 µL of deionized water for each calibrator vial. |
| - | Prepare the required amount of XG-DB5 dilution buffer by diluting 5-fold the concentrated solution in deionized water. If crystals appear upon refrigeration, warm the bottle to 37°C with mixing to dissolve. |
| - | Reconstitute 1 tablet of XG-WB2 washing buffer in 500 mL of deionized water. Prepare 1 liter with two tablets of XG-WB2 washing buffer for the entire plate. |
| - | Prepare the required amount of XG-EA enzyme-conjugated secondary antibody solution by dilution with reconstituted XG-DB5 dilution buffer. See product datasheet for exact dilution required. |
| - | Hepa-IC ELISA kit only: Prepare the required amount of chromogen-enzyme substrate solution adding 1 µL of XG-SB enzyme substrate solution per 3 mL of XG-CH4 chromogen solution just before adding it to the assay plate (step 12 of the assay protocol). |
ASSAY PROTOCOL
| 1. | Prepare assay reagents as described above. |
| 2. | Set up the microtiter plate with sufficient wells to enable the running of all required standards and samples. |
| 3. | Remove excess microtiter plate strips from the frame and store in the re-sealable foil bag with the desiccant provided. |
| 4. | Wash the microtiter plate strips three times with XG-WB2 washing buffer (300 µL/well). (Shown in videoclip) |
| 5. | Perform serial dilution of the calibrator in-plate (in duplicate) as shown in videoclip. |
| 6. | Dispense 100 µL/well of a diluted sample (in duplicate). See product datasheet for dilution required. Use XG-DB5 dilution buffer as diluent. |
| 7. | Incubate 1 hour at room temperature. |
| 8. | Wash six times with XG-WB2 washing buffer (300 µL/well). |
| 9. | Add 100 µL/well of diluted XG-EA enzyme-conjugated secondary antibody solution. |
| 10. | Incubate 1 hour at room temperature. |
| 11. | Wash six times with XG-WB2 washing buffer (300 µL/well). |
| 12. | Hepa AFP-IC, Colon-IC and Prostate-IC: apply 100 µL/well of XG-CH3 chromogen solution. Allow color to develop for 10-15 min at room temperature in the dark then apply 100 µL/well of XG-ST3 Stop Solution and measure OD values of each well using an ELISA plate reader equipped with a 450 nm filter. Stopped reaction should be read within 1 hour. Hepa-IC: apply 150 µL/well of prepared chromogen-enzyme substrate solution. Allow color to develop for 20 min. at 37°C in the dark and measure OD values of each well using an ELISA plate reader set to 405 nm. |
| 13. | Elaborate OD values with Xerepro software |
