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Troubleshooting a Faulty ELISA

Posted in: Protein Expression and Analysis
Troubleshooting a Faulty ELISA

Is ELISA giving you the blues? The frustrating kind, not the lovely kind you get while watching the enzyme substrate reaction!

This age old assay has the perks of being quick and fairly simple to perform, but when conditions are not perfect, ELISAs can deliver less than optimal results, and fail to be consistent and reproducible. When this happens, it’s helpful to know how to tweak the protocol in order to save the day (read: money and time).

Here are a few common problems that you can run into with ELISAs, and tips on how to solve them.

Problem: Low Signal Intensity

If you get multiple reads below the lower limit of absorbance (<000), and the concentration of your target analyte falls outside the range of your standard curve, your analyte concentration may be too low detect. Alternatively, optimal conditions for detection may not have been met. In this case, the signal can be amplified.


  • Increase the incubation times for your antibodies. If the protocol instructs you to incubate your antibodies for 2 hours at room temperature and you get lackluster results, try incubating overnight at 4 °C. This extra time will allow maximal antibody binding, and should lead to an increase in the signal.
  • Increase the concentration of the secondary antibody-enzyme conjugate. The E in ELISA stands for enzyme (typically horseradish peroxidase/HRP) – which reacts with the substrate (typically TMB) to yield a vibrant blue color. Increasing the HRP concentration by 50%, or doubling it, will produce a stronger color.
  • Protect the TMB substrate from light to maximize its performance. Keeping your ELISA plate in the dark while the color develops produces a stronger signal, as TMB is very sensitive to light.

Problem: High Background

The blank wells on the ELISA plate serve as a negative control. Any values that are markedly above 0 indicate an issue with background. Alternatively, you might be detecting something other than the analyte you want to assay.


  • Are you washing thoroughly in accordance with the protocol? We had a multichannel pipette in the lab that could pipette up to 200 µL, and my protocol asked for 300 µL of wash buffer. I thought, “Surely I can just use 200 and wash it a few more times and be fine, right?” Wrong! The volume required to cleanse the well completely is important. Due to capillary action, solutions on the surface of the well are slightly elevated, so wash buffer needs to reach the very top of the well in order to wash it completely. Be careful though, because washing more than is necessary can weaken your signal.

Problem: Unexpected ELISA Data

You expect to have higher values in one sample than another, and instead your values are all over the place. Concentrations are far from what you expect, and you can pin the differences down to variation.


  • Is your standard curve correct? Did the protocol specify linear, 4 or 5-parametric curves? Double check that you’re using them.
  • Do your standards have logical values? If your standards are not decreasing/increasing linearly they will throw off the calculations of your sample concentrations.

Problem: Interference

Interfering substances like detergents or NADH (present in serum) can influence your absorbance readings and skew your results.


  • Read the protocol. Protocols for assays that are sensitive to interfering substances will typically tell you what they are and how to avoid them. For example, if using a serum assay that hemoglobin interferes with, it is advised not to use serum samples that are hemolyzed (have a red color from lysed red blood cells)
  • You may have no choice but to use samples with interfering substances. If the concentration of your antigen of interest is high, use a very low but still easily detectible dilution of your sample, and you might be able to dilute out the interfering substance.

Keep these tips in mind the next time your ELISA isn’t going to plan. For more reading on everything concerning ELISAs feel free to check out our archives here.

What are your biggest ELISA challenges? Let us know by writing in the comments section!

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Image Credit: mike krzeszak


  1. Venkanna Bayya on August 13, 2018 at 10:49 am

    some online protocols saying that, after addition of capturing anti-body the plate was incubated at 37C for 4 hr and then followed by 2-8 C over night.
    my concerns are

    1. How the 37C temperature impacts the capturing anti-body.
    2. What was the reason for incubating the plate at 37C.

    • Omonse Talton on September 24, 2018 at 10:09 pm

      Hi Venkanna, can you link a protocol? Typically, sandwich ELISAs done using a kit already have the capture antibody immobilized to the plate.
      The addition of other antibodies usually comes with the option of incubating for a short time (~1-4hrs) at room temperature OR overnight at 4C, not usually both. 37c is normal body temperature, but can be used instead of room temperature sometimes. The temperature used is optimal for antibody binding and the incubation is to properly coat the plate (maximize antibody binding). Hope this helps!

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