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Tips for ELISA Failure


Although ELISA is a relatively mature protein quantification method, there are still many factors that lead to the failure of an ELISA assay due to its micro-volume reaction system. Here are some suggestions with the ELISA assay for reference:

Reagent Preparation Before Assay

1. Check the expiration date of the kit. The kit is not supposed to be used within 1 month, store the items -20℃ or 4℃ separately once the kit is received.

2. Check for signs of instability or deterioration in reagent solutions, such as precipitation or discoloration and so on.

3. Use clean plastic disposable pipettes, tips, and containers for reagents preparation and storage. Avoid cross-contamination of reagents by changing pipette tips between addition of each standard, sample and reagent.

4. All reagents or liquids should be prepared for use as needed and balanced to room temperature to use. Prior to assay, the frozen samples should be slowly thawed and centrifuged to remove precipitates.

5. All working soulution must be prepared just before use.

6. Centrifuge the standard at 10,000×g for 1 minute before use.

7. Avoid bubbles in diluenting. Bubbles generated during vortex or inverting could be removed by centrifuging at a relatively low speed.

8. Ensure accurate pipetting during standard working solution preparation. After the fully mixture of gradient standard working solution, added them into wells in duplicate from the lowest concentration to the highest concentration with the same pipette, which can effectively reduce the error of the pipette tips.

9. Avoid mixing other components or contamination in all reagents preparation and dilute them thoroughly.

Assay Procedure

10. ELISA experimental procedures and precautions of different suppliers may be different, and many unsatisfactory experimental results can be avoided by carefully reviewing and understanding the contents of the instructions. Please strictly follow the instructions and do not perform other operation steps based on previous operating experience.

11. It is recommended that samples and standards should be run in duplicate to improve assay accuracy.

12. Cover the sealer tightly to reduce the influence of edge effect.

13. Ensure that specified incubation time and temperature have been guaranteed. The incubation temperature should be controlled accurately, 37±1℃ would be better.

14. Refuse improper incubation conditions, such as water bath or incubator with unstable temperature or shaking.

15. Centrifuge the Concentrated Ab and HRP Conjugate at 800×g for 1 minute prior to dilution.

16. Make sure the whole sample adding time controlled within 10 minutes.

17. Use fresh wash buffer. Pat dry the plate with absorbent paper in each washing step and avoid reusing the absorbent paper. Add samples in time after washing, and don’t let the plate dry.

18. Auto washer is available in ELISA assay. However, multi-channel pipettes are more recommended as auto washers may introduce buffer mixing/contamination or improper parameter settings, which may lead to unexpected results. However, whether you choose auto washer or multi-channel pipette, it is necessary to ensure accuracy operation.

19. The soaking time in washing step should be 1-2 min. Not be too long or too short.

Color Development and Reading

20. Control the color reaction time properly (4 high-concentration standards show a clear blue gradient).

21. The microplate reader need to be preheated for 10-15 min before measurement and set shaking step before reading.

22. Use the microplate reader to read the OD value immediately after the plate is terminated.

Hoping that customers would obtain satisfactory test results with Elabscience® ELISA kits.

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