Unlocking the secrets: A proven guide to mastering ELISA assay troubleshooting

Dec 01, 2023

ELISAs are widely used to quantify or detect antigens, hormones and antibodies. ELISA assays rely on an antibody that’s specific for the target of interest, and detection methods may be direct, two step, or rely on two types of antibodies (capture and detection). When running ELISAs it’s not unusual to encounter various types of assay issues.

However, these issues result in reduced accuracy, increased variability, wasted resources, and delayed results, compromising the reliability of data interpretation and the reproducibility of experimental findings. Below are some of the common issues experienced when running ELISA assays and how to effectively troubleshoot and resolve them.

High Background

Could be caused by Try this
Too few washes/low wash buffer volumeIncrease number of washes/ensure wash buffer completely fills plate well.
Blocking buffer issue Use fresh batch of blocking buffer.
Non-specific antibody binding Titrate antibodies/try different blocking agent/change antibody.
Assay/matrix component causing interference Switch out assay component(s). Test matrix only to determine background levels.
Antibody concentration too high Titrate primary and secondary antibody and use negative and positive (known protein) controls.
If using, Strepavidin-HRP conjugate concentration is too high Check dilution. Titrate to find optimal concentration.
Non-specific interactionsAdd additional protein to assay buffers and wash buffer.
Contaminated buffersRemake all buffers.

No signal/Low signal

Could be caused by Try this
Incorrect antibody used Check assay components used. Include a positive control (known protein).
Assay component not added Check protocol and steps.
Antibody too dilute Titrate primary and secondary antibody – use controls.
Primary and secondary antibody mismatchEnsure secondary antibody is against the host species the primary was raised in.
If using HRP – can be inhibited by azide Ensure that SA-HRP dilution buffer does not contain azide.
Not enough target for detection Increase sample concentration.
Non-binding (eg: tissue culture) plate used for coating Use a high-binding plate from a reliable source.
Issue with coating buffer or wash bufferChange coating buffer and try different (low salt) wash buffer.

OriGene resources for ELISAs

OriGene offers a comprehensive catalog of mouse and rabbit monoclonal antibodies validated for ELISA assays. In addition, our ELISA kits allow specific, quantitative measurements of disease-related proteins, including cytokines, chemokines and signaling targets. OriGene’s MVPro recombinant proteins and over-expression lysates are ideal for use as ELISA controls.


Contact technical support - techsupport@origene.com


  1. https://www.origene.com/products/assay-kits/elisa-kits