How to Troubleshoot Inconsistent Results in Your ELISA Kit Protocol?

ELISA stands for Enzyme-Linked Immunosorbent Assay. It is a plate-based technique used to detect and quantify proteins, antibodies, and antigens in a biological sample.

This technique relies on the principle of antigen-antibody interactions. It is categorized in 4 formats – Direct ELISA, Indirect ELISA, Sandwich ELISA, and Competitive ELISA.

Each kit has a standard ELISA kit protocol. However, sometimes, even well-designed kits show unreliable results. While there are several reasons for these results, knowing the potential causes and how to correct them can save you both time and resources. So, here, we will discuss how to troubleshoot variability in the ELISA kit protocol.

Why Inconsistencies Occur During ELISA Test?

ELISA involves multiple steps – coating, blocking, incubation, washing, and detection. Each of these stages requires precision. Even small deviations in pipetting, temperature, or timing can lead to inconsistent readings.

Common signs of inconsistencies include:

• High background noise
• Weak or uneven signal
• Poor reproducibility between wells or plates
• Unexpectedly high or low optical density (OD) values

Inconsistent results don’t always mean that the kit is faulty. However, they usually indicate issues in handling, sample preparation, or environmental control.

How to Troubleshoot Inconsistent Results in ELISA Kit Protocol?

Check Your Sample Preparation

The quality of your sample has a major impact on your ELISA results. Samples, such as serum, plasma, or cell culture supernatant, must be collected, stored, and prepared properly. If the sample is not handled well, the target protein or antibody may break down and give false or inconsistent readings.

• Avoid freeze-thaw cycles: Try not to freeze and thaw the same sample multiple times. Otherwise, this can damage the antibodies and affect the results. Instead, divide the sample into smaller portions before freezing so that you only thaw what you need.
• Ensure correct dilution: A sample with a high or low concentration produces too strong or a weak signal, respectively. This can also lead to background noise. So, make sure you always follow the dilution instructions provided with your ELISA kit.
• Remove particulates: Before using the sample, make sure to centrifuge it to remove any particles or debris that may interfere with the reaction.

Verify Reagent Preparation and Storage

Reagents are a key part of every ELISA test. If they are not prepared or stored correctly, the entire assay can be affected. So, make sure you:

• Check expiration dates: Expired reagents, especially enzyme conjugates and substrates, can lose their activity over time.
• Mix reagents thoroughly: When preparing reagents, mix them well to make sure the solution is uniform. Some reagents, such as standards and controls, can settle or form layers if not mixed properly.

Like preparation, storage conditions are equally important for reagents. Many enzyme-linked reagents must be kept in the refrigerator between 2°C and 8°C. Substrate solutions should be stored away from direct light to prevent damage.

If you get inconsistent results between plates or over time, prepare fresh reagents and compare the outcomes. This will help you identify if reagent quality is the problem.

Ensure Proper Plate Washing Technique

Washing is one of the most crucial steps in the ELISA protocol. If the plate is not washed properly, leftover reagents or unbound antibodies may stay in the wells and cause a high background signal. On the other hand, excessive washing can remove the bound complexes, which lowers the signal.

So, it is important to wash the plate consistently after each step that requires it. To ensure you get consistent results:

• Use an automated washer.
• Ensure complete removal of liquid between washes by tapping plates on absorbent paper.
• Increase the number of washes or the soaking time if the background remains high.

The Bottom Line

While these are the tips you can follow to ensure you get reliable and accurate results, there are many more, such as maintaining consistent incubation conditions, calibrating your equipment regularly, following the kit protocol exactly, standardizing data analysis, etc.

Now that you know how to fix the issues in your ELISA test, what are you waiting for? Find a reliable supplier to buy high-quality ELISA kits and start your research today!

(Disclaimer: Devdiscourse's journalists were not involved in the production of this article. The facts and opinions appearing in the article do not reflect the views of Devdiscourse and Devdiscourse does not claim any responsibility for the same.)