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Tips For Better ELISA Results

Tips For Better ELISA Results

The ELISA (enzyme-linked immunosorbent assay) is a rapid method used to detect the amount of a protein of interest in clinical and experimental samples. There are a number of ELISA formats to choose from, depending on your research needs. These include direct ELISA, indirect ELISA, competitive ELISA and sandwich ELISA. We have previously covered the basic steps common to all ELISA formats, and you can read more about the different ELISA formats here. While this process has become increasing automated and less labor-intensive, it is still important to carry out these assays with care to maintain accuracy and maximize the usability of your results. Keep the following top tips in mind for good ELISA results every time:

  1. Read the instructions. Sit down the day before you plan to carry out the ELISA and read through the instruction booklet and/or protocol. Make sure you have all your reagents to hand. Write our your own protocol if it will make it easier to perform the ELISA on the day.
  2. Familiarize yourself with the equipment. If it’s your first time using the lab plate washer and spectrophotometer, ask a colleague to show you how they are used.
  3. Plan your plate layout. Many ELISA kits come with a loading template. Write out exactly what samples and what dilutions you will be analyzing, and record what will go into each well. If you don’t have a template, make your own 96-well plate layout on your computer and print it out before you start. You can tick off each sample as you load them.
  4. Don’t waste precious samples. Carry out a trial run of a standard curve and a few of your samples at varying dilutions first. This will give you an idea of the expected absorbance values for your unknown samples of interest, as well as helping you figure out how much you might need to dilute your samples.
  5. Monitor data quality with replicates. Prepare your standard curve and unknown samples in at least duplicate or triplicate if possible. You should be aiming for an r2 value as close to 1 as possible.
  6. Minimize variability. Take care when pipetting and make sure that your pipettes are properly calibrated.
  7. Coat your plates all at once. To minimize inter-plate variability aim to coat all the plates your kit provides reagents for, and store the coated plates at -20°C until use. Double check the kit insert before you start to make sure that your plates can be stored after coating.
  8. Set up an analysis template. Save yourself time and have an excel template prepared to automatically generate your standard curve and calculate sample protein concentrations.
  9. Back up your data! Keep at least one electronic copy of both your raw and analyzed data. Label each file clearly with the date and analyte under investigation. You never know when you may need to revisit the data.
  10. Take your time. Don’t be overly ambitious and perform more than one or two ELISA plates at a time. This will only lead to mistakes, potentially skewing your results and wasting precious samples and expensive reagents.
  11. Document everything. This is critical so that if things go wrong you can easily troubleshoot your problematic ELISAs.

What are your tips for successful ELISA results?

Further Reading

Shah K, Maghsoudlou P. 2016. Enzyme-linked immunosorbent assay (ELISA): the basics. Br J Hosp Med (Lond). 77(7):C98-101.

Originally published in 2013. Updated and republished in May 2017.

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Image Credit: woody1778a


  1. Poonam Pawar on February 1, 2013 at 10:59 am

    Dear astarothcy;

    If you are using an in house protocol then this seems to be a problem at coating or blocking step as well..
    The pipetting plays a major role here again

    • astarothcy on February 1, 2013 at 2:33 pm

      Thanks! Could you please be more specific? What aspect of the coating or blocking step could lead to the end-of-run effect? How do I do it in a way that avoids the issue?

  2. astarothcy on January 23, 2013 at 3:41 pm

    I have been having some trouble with a home-made sandwich ELISA. What I observe is a “gradient” of ODs across the plate, meaning that if I just load the same sample in every well of the plate, the readings get progressively lower from left to right. This is apparently also called the “end of run” effect. There can be as much as a two-fold difference from the leftmost to the rightmost column. This is obviously quite bad and I cannot figure out how to fix it. I’m just wondering if you have heard of this before and know what causes it/how to prevent it. I made a post on a relevant forum where I describe most of what I’ve tried:

    At the end of the thread I say that the effect is gone, however that seems to have been a fluke as I still get the effect and its extent varies widely from day to day, but it’s always there.

    • Ellen Moran on January 24, 2013 at 1:17 am

      This does sound very frustrating! The fact that the gradient is in the same direction as your loading does suggest a handling/pipetting issue. Have you tried merely doing half a plate at a time?

    • astarothcy on January 25, 2013 at 6:31 am

      No, but that would substantially increase my reagent use and workload since I would need to load two standard curves per run, plus blanks, so would have very few wells per run for actual samples. I’ve actually recently discovered that we have been making up our PBS stocks incorrectly (using a recipe for anhydrous Sodium Phosphate while we actually had the heptahydrate) so I will try again with correctly made PBS. I doubt it will make a big difference, but you never know. I’ve always seen this as a drying issue, I believe the plate is drying during handling despite me working up to pipetting very quickly and drying minimally after washes. There is an extremely elaborate way to figure out exactly where the issue lies, by methodically controlling every step of the assay, but I am too busy with other experiments for that.

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