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HOWTO ELISA KITS - INSTRUCTIONS FOR USE
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. |
| - | Reconstitute XG00n-C(1-6) 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. |
| - | Prepare the required amount of XG-WB3 washing buffer by diluting the concentrated solution 10-fold in deionized water. |
| 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-WB3 washing buffer (300 µL/well). (Shown in videoclip) |
| 5. | Dispense 100 μL/well of reconstituted calibrators C1, C2, C3, C4, C5 and C6 (in duplicate) in order to obtain a six point calibration curve. For the exact concentration of the reconstituted calibrators please refer to the concentration value (AU/mL) indicated on the labels. |
| 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-WB3 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-WB3 washing buffer (300 µL/well). |
| 12. | 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. |
| 13. | Elaborate OD values with Xerepro software |
| WARNING! The following procedure can only be performed using the same ELISA plate. |
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| INFORMATION ELISA test must be performed as indicated in the instructions for use attached in the kit box. The data processing must be performed with Xerepro version 2.3 or later. |
| Day 1 - preparation of the reference point | |
| 1. | Reconstitute the first calibrator XG00x-C1 with 440 µL of deionized water. |
| 2. | Prepare the reference point as follows: 220µL of XG00x-C1 + 700µL of H2O + 180µL of NOT reconstituted XG-DB5. |
| 3. | Make 5 aliquots of 220µL of the newly prepared reference point. |
| 4. | Freeze the aliquots at -20°C. |
| 5. | Store the reconstituted calibrator (XG00x-C1) and the dilution buffer (XG-DB5) at 2-8°C. |
| Day 2 - to be performed within 48 hours from day 1 | |
| 1. | Dispense 100µL/well in duplicate of reference prepared and freezed the day 1. |
| 2. | Perform the ELISA test following the instructions for use. |
| 3. | Elaborate the OD values using Xerepro software. Leave the cells of reference point empty. |
| Day 3 - to be performed before the expiration date of the kit | |
| 1. | Dispense 100µL/well in duplicate of reference prepared and freezed the day 1. |
| 2. | Perform the ELISA test following the instructions for use. Do not add any calibration point. |
| 3. | Elaborate the OD values using Xerepro software. Use the ODs of the calibrators and reference point (Robs) obtained on day 2, and the ODs of the reference point (Rsim) and samples just obtained. |