In medical research, accuracy and precision are crucial. Errors can jeopardize the validity of your findings and lead to wasted time and resources. ELISA is a preferred tool for detecting and quantifying proteins, antibodies, and hormones. But your results may be inaccurate when you miss the procedure, use the wrong reagents, or make other mistakes. So how can you avoid these errors and get the most accurate ELISA results?

This post addresses most ELISA mistakes that can ruin your research, tests, and effectiveness. Understanding the requirements, protocols, and precautions will help eliminate errors and enhance your results. Also, using high-quality ELISA kits from a reputable supplier is critical to the success of your study.

Here are some tips on how to get the most desirable test results on your ELISA analysis.

1. Select Kits Matching Your Needs

The ELISA test you perform depends on the research question. For instance, detecting and quantitating specific analytes requires a competitive or sandwich ELISA. Also, the plate you use matters because it influences the ELISA assay’s working range, sensitivity, and specificity.

Consider other factors like the sample type, detection method, and sample size when selecting ELISA kits. If uncertain, ask the supplier for help according to your needs. Inform them of the expected values, including minimum and maximum concentrations of your analytes.

2. Use Microtubes or Dilution Plates

Various assays require dilution to ensure they are in their detectable ranges. You will use your sample’s stock solution to make a working dilution, which you will use in the assay. But first, prepare a dilution series using microtubes or plates. These instruments will help you make an accurate dilution of your sample.

Use serial tubes or plates and add reagents to prepare a dilution series. Avoid unrecommended containers or items because they can alter the concentrations of your samples. Also, add the same volume of each reagent to every tube or well for consistency.

3. Keep Reagents and Samples at Standard Temperatures

Your ELISA results will be more accurate when you follow the specified storage and incubation temperatures. Most ELISA protocols require storing reagents at -20°C for short-term storage. -80°C is ideal for long-term storage. Use cryogenic conditions (-150 or -196°C) for storing some reagents, such as antibodies. At this stage, your samples and reagents will not degrade. All biological processes will have stopped.

But how do you determine the best temperature for your reagents and samples? Not everything works the same. Some DNA strands may break at -20°C, while others will do so only when frozen at -80°C. Determine the optimal temperature for your samples. Contact your reagent or kit supplier for help with this. Also, avoid frequent freezing and thawing of your analytes as it degrades their quality.

4. Clean Your Plates Thoroughly

Use clean and well-maintained plates to achieve the most accurate ELISA results. After every experiment, soak them in recommended cleaning solutions. For instance, you can use a 0.01 M phosphate-buffered saline (PBS) solution with Tween-20 for cleaning polystyrene plates.

After soaking, rinse the plates with distilled water and let them dry in a laminar. The buffer you use in your assay may also have a cleaning effect. But it is still essential to soak and rinse the plates after each experiment for the best results.

Avoid detergents with cationic groups. These groups will bind to the anionic groups on your plate and compromise the ELISA assay. Also, do not use organic solvents, as they will remove critical coatings.

5. Block Non-Specific Binding Sites

After cleaning your plates, you must block all the non-specific binding sites that can make your ELISA assay less sensitive. Doing this will reduce background noise and interference, giving you accurate results.

To block the sites, incubate your plate with a blocking solution. Bovine serum albumin (BSA) is a universal blocking agent, but you can also use other proteins such as casein and non-fat dry milk. Incubate the plate for about an hour at room temperature or overnight in a fridge. After incubation, discard the blocking solution and rinse it with distilled water or Tris-buffered saline (TBS).

6. Stick to Protocols and Guidelines

Your ELISA results will only be as good as your ability to follow the guidelines and protocols. Every ELISA kit comes with a set of instructions you must follow to the letter. Do not deviate from any steps, no matter how small they may seem. Follow the kit’s instructions even if you have experience with ELISA.

Also, use the recommended reagents and solutions. Do not substitute them with other products, even from the same manufacturer. Even a slight change in reagents can affect your ELISA results. That also covers mixing and interchanging solutions from different ELISA kits. Doing this will give you inaccurate results.

Finally, be careful when handling your samples and reagents. Contaminations and improper storage can lead to significant errors in your ELISA results. Use specific and dedicated pipettes for each reagent. Do not use the same tips or containers for different solutions.


To get the best ELISA results, you must use the correct reagents and follow all the protocols to the letter. Store your samples and reagents properly, and clean your plates thoroughly after each experiment. Also, block all the non-specific binding sites, and be careful when handling your materials. By following these tips, you can avoid common mistakes and get the most accurate ELISA results every time.