Overcome Elisa Common Test Issues

ELISA (Enzyme-Linked Immunosorbent Assay) is a technique used to detect the presence of peptides, proteins and other antibodies in the blood. The ELISA test is widely used to quantify biological molecules. It is a common test used to diagnose diseases such as HIV, Lyme, pernicious anemia, rotavirus, syphilis, etc. The experiment is simple and easy to perform, but it is exceptionally sensitive.

The test identifies the presence of specific molecules and determines their concentration. Detection is accomplished by the interaction of antibody and antigen. It is also detected by monitoring enzyme activity by incubation with a substrate.

Common problems with ELISA experiments

The results of the experiment are based on the color of the substrate. The reliability and accuracy of ELISA experiments depend on the use of proper technique and attention to detail. As with any bioassay, there may also be difficulties in interpreting ELISA results. Highly concentrated samples or diluted samples will have a huge impact on test results. Preparing different concentrations and blocking solutions, using standard diluent solutions, and choosing a sample substrate based on the amount of antigen can optimize the experiment. However, sometimes the experiment does not yield optimal results and the ELISA controls will indicate a problem.

Problems with ELISA are categorized as low signal, no signal, high background signal, inconsistent result, and samples out of range. There is a high probability of making mistakes that will result in false negatives or false positives. Error in plate handling, plate washing instrumentation, buffers, reagent usage, and assay procedures are likely to cause ELISA experiment problems.

Here are the possible reasons as well as some solutions for performing an ELISA experiment under uniform conditions.

1. Misuse of reagent

If the result is blank or has minimal signal, it may be due to reagent misuse or a procedural error. Improperly mixed reagent or adding too much detection reagent results in poor standard curve or high background signal.


Avoid using reagents from different lots as each reagent kit is optimized for the specific ELISA kit. Their reuse can affect the ability to detect, resulting in inconsistent values.

Mixing or substituting reagents from different kits may affect test quality.

Ensure reagents are fresh by avoiding the use of contaminated glassware during reagent preparation.

 Reagents must be diluted correctly to ensure accurate results.

To maintain consistency between all test wells, reagents and samples must be mixed well before pipetting onto plates.

Reagents should be stored at room temperature to avoid edge effects.