ELISA tests are widely used in medical diagnostics, research, and Drug Development and various fields of biology. However, since they are often run manually, there are chances of human error that may cause high background and therefore incorrect results and quantification of analyte. Krishgen’s scientific team has put together a list of causes of that may cause high background in ELISA results, and some quick tips on how to prevent it.

1. Sample Quality:

One of the main sources of high background noise in ELISA is the quality of the samples. Contaminants, such as endotoxins, detergents, or proteins, can interfere with the binding of the antibodies or the enzyme substrate and generate false-positive signals.

Prevention: To prevent this, use clean and sterile equipment and reagents, store and handle your samples properly, and avoid repeated freeze-thaw cycles. Also dilute your samples appropriately to avoid saturation or interference effects.

2. Cross-reactivity:

Cross-reactivity of ELISA occurs when an antibody raised against one specific antigen recognizes two antigens that have similar structural regions. This caused nonspecific binding and background noise.   

Prevention: To prevent this use highly specific antibodies and optimizing assay conditions. Also, Try an antibody that recognizes a different epitope.

3. Poor Dilutions:

Using a high concentration of primary or secondary antibodies without appropriate dilution can increase background noise.

Prevention: To prevent this balance the sensitivity and specificity by optimizing the antibody dilution. 

4. Contaminated Reagents:

Contaminated reagents, plates, or buffer solutions can introduce additional proteins or particles that can bind nonspecifically to the plate surface, causing a high background.

Prevention: Make sure water is not contaminated. Also, Prepare blocking and wash buffers fresh.

5. Incubation Conditions:

Short incubations can interfere with formation of antigen-antibody complexes and inhibit color development.

Prevention: To prevent this, follow the recommended incubation conditions for each step of the ELISA and Optimize substrate incubation time.

6. Sample Matrix Effects:

Complex matrices such as serum, plasma may contain substances that interfere with the assay or contribute to nonspecific binding.

Prevention: To prevent this/ to reduce matrix effects it is necessary to do pre-treatment or dilution.

7. Inadequate Blocking:

Another common cause of high background noise in ELISAs is the use of an inadequate or unoptimized concentration of blocking buffer.

Prevention: it is necessary to optimize your blocking buffer solution by increasing its concentration. You can also prevent it by adding a small amount of a non-ionic detergent (i.e. Tween 20) as a cost-effective and stable option.

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