6 Tips For A Successful ELISA Assay Validation

By | November 15, 2021

In research, an ELISA assay validation is a scientific technique that uses living organisms to measure the concentration of an analyte in the environment. Ecologists or environmental scientists often use this technique to determine toxic substances in water or soil that could be hazardous to nearby species.

What is an ELISA assay?

An ELISA assay is an immunoassay where you can test for specific antigens (such as proteins) on the surface of particular cells or tissues (e.g., blood cells, tissue culture cells). It is usually the preferred method for detecting analytes in biological fluids due to its high sensitivity and specificity. This is because it does not rely on the “willingness” of the body to eliminate toxins, which can vary between individuals (and even within individual animals).

6 Successful Tips For Elisa Assay Validation

For a successful and reliable ELISA assay, you should:

Be Thorough Throughout the Samples Present

Be thorough when preparing the samples. The accuracy and reliability of your results will depend on how carefully you train your models. You should also keep your reagents refrigerated, if possible – this will ensure that they remain sterile.

Avoid Using Frozen Samples

Do not use frozen samples. While some detection systems can measure frozen blood samples, using fresh samples for an ELISA assay is preferable. Avoid cross-reacting antibodies. For a successful ELISA, the specific antibodies used in the assay should not react with any other types of target analytes. It is because different antibodies may interfere with the binding of your target analyte and reduce its specificity. 

Don’t Let Your Samples Sit 

Do not let your samples sit while waiting for them to be tested. If you have a lot of samples to test, try to push each sample immediately after receiving it from the field. Also, avoid using pipette tips made from PVC. Polyvinyl chloride is an ordinary plastic used for manufacturing pipette tips, and it can cause a problem when using some types of immunoassay kits. 

Ensure Proper Solutions

Ensure that your pipette tips are clean when you draw up solutions. If possible, try to use new pipette tips when drawing up solutions. This measure will prevent contamination of your samples. In addition, you can use small filters to filter the solutions in-situ, if needed, and it  will also reduce the possibility of contamination.

Ensure Clean Equipment

Ensure that your equipment is as clean as possible. One way to do this is to wash your equipment with detergent and warm water immediately after use rather than wait for the equipment to dry. Do not use undiluted reagents. If undiluted reagents touch your skin, they may be absorbed into your bloodstream and cause a reaction or poisoning. This can be fatal if you have ingested undiluted reagents. Finally, always wear gloves in ELISA lab when handling reagents.

Use Only Sterile Samples

Be sure that your samples are sterile (if they need to be clean, of course). If you are transporting samples from the field in plastic bags, make sure they are free of cracks or holes. If necessary, you can also place them in sterile containers for transport.


An ELISA assay generally works by placing a sample on a platform and allowing the system to measure how much of a target analyte binds to specific antibodies. The target analyte typically works  as a marker. This enables you to determine if a sample has bound enough of the target analyte for your assay system to react positively.

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