Tips For Better ELISA Results

The ELISA assay is a rapid method used to detect the amount of a protein of interest in experimental samples. While this process has become increasing automated and less labour intensive, it is still important to carry out these assays with care to maintain accuracy and maximise the usability of your results. The following are a few tips to keep in mind:

  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.
  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. Many ELISA kits come with a loading template. Write out exactly what samples and what dilutions you will be analysing. You can tick off each sample as you have loaded 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 absorbance values for your experimental samples of interest.
  5. Monitor data quality with replicates. Carry out your standard curve and samples in duplicate or triplicate. You should be aiming for an r2 value as close to 1 as possible.
  6. Minimize variability. Take care with your pipetting and make sure your pipettes are properly calibrated.
  7. Coat your plates all at once. To minimize inter-plate variability it can be possible to coat all the plates your kit provides reagents for and store at -20°C until use. Double check the kit insert to make sure this is possible.
  8. Set up an analysis template. Save yourself time and have an excel template prepared to automatically calculate your standard curve and sample protein levels.
  9. Back up your data! Keep at least one electronic copy of both your raw and analysed data. Label each file clearly with the date and analyte. 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.

What are your tips for successful ELISAs?


  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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