ELISA dilution ratio and why it is important to get this right

What is an ELISA dilution ratio

The ELISA dilution ratio can be defined as a unit volume of the sample that needs to be combined with an appropriate diluent in to obtain the desired final total volume. For example, a dilution ratio of 1:8, indicates that there needs to be 1 part of sample to 8 parts of diluent to give a final volume of 9 parts.

There are many reasons why it is important to perform this initial sample dilution step. The sample needs to be in a concentration range that can be detected by the specific ELISA assay to obtain accurate and meaningful results. If the sample concentration is outside the detection range, this may lead to the assay not working, samples not being detected or providing false results.

There are many factors that need to be considered when calculating the optimal dilution ratio. By carefully considering all these factors it is possible to work-out a ratio that will maximise the specificity and sensitivity of the ELISA assay that is being performed. This will ultimately lead to accurate and trustworthy results being obtained.

How to decide the ELISA dilution ratio

Some of the factors that need to be considered when calculating what dilution ratio to use for a sample include the sensitivity and detection range of the assay, the amount of target antigen that is present within the sample and what the affinity the of the antibody that is being used in the assay has to the antigen that is going to be detected.

Listed below are some steps that can be taken to help reach an optimal dilution ratio.

• It is important to initially start with a high dilution of the sample (for example 1:100 or 1:1000). This will mean that if the target antigen present within the sample is at low concentration, then they will be picked up by the sensitivity of the assay.
• Next a series of serial dilution of the sample need to be investigated (for example, 1:10, 1:100, 1:500, 1:1000). For each dilution that is being tested, it is critical that exact manufacturer’s instructions are followed step by step when performing the ELISA assay.
• The results for each dilution can be plotted and the shape of the curve is observed.
• If the curve is relatively flat and does not increase significantly with higher dilutions used. This will indicate that the assay has sufficient sensitivity and that the highest dilution can be used.
• If the curve continues to increase significantly with higher dilutions. Then this indicates that the assay could benefit from a higher dilution ratio to improve sensitivity. In this situation, it is advised to the repeat the assay using a set of higher dilution ratio (for example 1:2000, 1:4000, 1:5000). Then repeat the ELISA assay and observe the shape of the curve on the graph.

By following the above steps and observing the shape of the curve on the graph. It is possible to determine the optimal dilution ratio that will provide the best sensitivity and results for the specific ELISA procedure that is being undertaken.

Factors affecting ELISA dilution ratio

There are many factors that are important and will need to be considered when calculating the optimal dilution ratio to use when performing an ELISA (enzyme-linked immunosorbent assay).

Some of the more common factors will be discussed in more detail below.

1. Assay sensitivity: This is the minimum levels of the antigen that can be detected using an ELISA kit.
2. Antigen concentration: For low amounts of antigen, it is recommended to have a high dilution ratio. Whereas, for high antigen concentrations then it is advised to have a low dilution ratio. This will ensure that sensitivity and the results generated are not compromised.
3. Antibody to antigen affinity: Generally, it is widely accepted that the higher the affinity between the two molecules, the lower the dilution ratio.

By obtaining the right balance between the factors listed above. It is possible to have the optimal dilution ratio.

Advantages of using a correct ELISA dilution ratio

There are many advantages to using the best dilution ratio.

• It will lead to improving the sensitivity of the assay and even help to measure very low concentrations of antigens present within a given sample. This can be extremely important when investigating the early stages of many cancers because the target antigen is only present at very low levels. For this to be detected all the conditions for the assay need to be optimised.
• It can make the specificity for the assay better. This will eliminate any false positives or false negative results being obtained. This can be very critical when life threatening medical conditions are being investigated.
• It will lead to more accurate results that consistent, reproducible and trustworthy.
• It will help to reduce the cost by decreasing the amount of reagents and samples that are going to be used. Also, it may also be possible to reduce the amount of waste that will need to dispose.

Final thoughts

It is important to work out the dilution ratio that will be suitable for measuring the concentration of the unknown antigen that is present within a given sample. Furthermore, this also will improve the sensitivity and the specificity of the test procedure.

Related Pages

1. ELISA Principle
2. ELISA Protocol
3. ELISA Sample Preparation
4. Analysing ELISA Data
5. General ELISA FAQ
6. ELISA Trouble Shooting FAQ
7. Comparison Between ELISA And EIA
8. Different ELISA Detection Strategies