What are your tips?

Enzyme-linked immunosorbent assays (ELISA or EIA) are tests that detect and measure antibodies in your blood. It can determine the presence of antibodies, or proteins that are produced in response to harmful substances called antigens, that are related to certain infectious conditions. ELISA has been used to diagnose HIV, Lyme disease, pernicious anemia, rotavirus, squamous cell carcinoma, syphilis, toxoplasmosis, and varicella-zoster virus. It is often used as a screening tool before more in-depth tests are ordered. That is why is it important to ensure accurate results are found. Below are are a few tips to increase your efficiency while improving overall accuracy in conducting ELISAs.

Improve Efficiency

  • Tell Yourself What to Do - Writing out the instructions in your own words and mentally running through the procedure before executing it will help avoid any surprises.
  • Use a contrast background - Use a dark sheet of paper or cloth underneath your well plate to create a contrasting background for your samples to stand out against. It will help detect whether or not a reagent has already been added to the well.
  • Create a template of where to add samples on a plate - Save your time by not having to label every new plate, just use a template noting where to add what.
  • Arrange samples in the order they will be used - Position samples differently after placing a sample in a well. (If it was to the left of the plate before using it, place it on the right when you are finished). This may help lessen chances of error.

Keep Consistency - Watch out for these mistakes!

Small differences in pipetting can lead to large variations in data, making your results less usable. For instance, when adding the same volume of solution to multiple wells or making serial dilutions, it can be hard to keep track of whether or not the last drop in the pipette tip made it to the well. It will do well in avoiding contamination and increasing accuracy, too.

  • Avoid using the same tip for each well . It can lead to an accumulation of bubbles. Tiny differences in volume input, due to different levels of wetting inside the tips with each successive use, can also affect the standard curve and data accuracy.
  • Ergo: Change tips every well! Using the same tip can thus result in faulty pipetting. In this case, it is advisable to change the tips between wells to ensure consistency. Making this change in consistency can make a big difference in the accuracy and repeatability of your results.
Ensure Experiments can be Reproduced 

Reproducibility may be affected by mishandling inconsistent preparation of the kit reagents or even samples. To avoid mishaps through this issue:

  • Ensure all reagents are at room temperature for approximately 30 minutes before every assay. Many reagents contain temperature-dependent components that may come out of solution when cold. Room temperature reagents allows consistent binding kinetics and consistent color development in all following assays you do. Temperature changes can also affect assay behavior.
  • Samples should be managed and handled by researchers in a consistent manner.Do this by:
    - introducing consistent sample collections protocols
    - minimizing freeze-thaw cycles
    - optimizing dilution for your sample group in the assay ahead of time
    This way your samples should be falling in the most linear portion of the standard curve.
 Last but not least, Follow Recommendations!
They are there for a reason.

  • Use the provided/recommended diluents and buffers

Use the recommended diluents only for your standard curve and sample preparation for the best results. In assay development, manufacturers find optimal conditions for not only curve performance, but samples detection as well. Detergents, ionic strength, pH etc. may play a crucial part in finding the most conducive environment for a happy assay.

  • Follow recommended incubation times, temperatures and conditions

Achieving complete color development is as simple as adhering to the incubation guidelines in the assay manual.

Temperature, time, and shaking can greatly affect the status of the assay if they are not followed. Accuracy and consistency of plate incubation handling can really help enhance assay performance.

  • Include background and control well suggested in the protocolInclude all of the suggested background wells in your assay to allow for needed troubleshooting, if issues arise. Some assays call for multiple control wells or background wells, but they serve a slightly different purpose. If you are unclear about their uses, just reach out to technical support for clarification.

 

 

 

Related

References

5 Ways to Improve Your ELISAs - Bitesize Bio. (2012). Retrieved June 06, 2016, from http://bitesizebio.com/8041/5-ways-to-improve-your-elisas/

ELISA. (n.d.). Retrieved June 06, 2016, from http://www.healthline.com/health/elisa

TechNote: 10 Tips for Successful ELISA Assay. (n.d.). Retrieved June 06, 2016, from http://www.enzolifesciences.com/science-center/technotes/2015/may/10-tips-for-successful-elisa-assay/

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