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ELISA Tips & Tricks

ELISA kits from a vendor are attractive due to their convenience, ease-of-use, and relative economy. The extensive and often complicated optimization process for producing a robust ELISA assay has been done for the user. A well-developed ELISA assay will be robust enough that small variations in assay processes will have minimal effect on results. Regardless, user technique does contribute to successful assay data. Below are some guidelines to consider when using an ELISA kit and will help ensure optimal reproducibility, accuracy, and sensitivity.



Kit Fidelity

Vendors manufacture their kit components and materials uniquely, requiring each kit to be handled carefully.

  • Select the kit which is best suited to your sample and target type. Aside from target, check sample compatibility and assay detection ranges.
  • Read all the kit protocols, sample prep and material prep instructions prior to performing every assay. Individual kit manufacturers employ varying techniques and kit methods may change between lots.
  • Prepare all reagents as directed in the instructions.
  • Ensure all kit components are stored under the recommended conditions. Individual components may have different specific storage requirements prior to, as well as, after being opened or formulated.
  • Utilize only the diluents and buffers contained in the kit for reagent preparations, or those specified by the vendor protocol. Reagents are optimized for individual methods, sample types and even specific targets.
  • Spin reagent tubes briefly prior to opening.


Achieving accurate measurements and good reproducibility within wells or across several different experiments is a primary goal. Inconsistent assay methods will result in less accurate measurements and cause problems when comparing data sets.

  • Perform all wash and reagent addition steps in the same sequence and at the same rate across the well plates for each successive assay step.
  • All wash steps should be performed thoroughly using the exact volume, agitation conditions and cycles as indicated in the protocol. Complete removal of the liquid between washes is essential, but do not allow the microplate wells to become dry at any point in the assay procedure. Cross contamination can be problematic in the liquid removal step, whether by aspiration of flicking. Washing is a highly optimized assay step and often overlooked. Inaccuracy at this juncture will introduce large variability in data results.
  • Conduct all incubation steps using consistent times and conditions across plates and experiments.
  • Ensure all reagents are equilibrated to the same temperature prior to use. Some kits specify performing all assays at slightly elevated temperatures (30°C or 37°C) in order to control for small variations in temporal ambient conditions.
  • Assay in replicate. Outliers are easily identified when multiple data points are obtained. Replicates will improve accuracy by averaging larger data sets.
  • Use only calibrated pipettes.
  • For colorimetric assays, perform color development steps in complete darkness. Aluminum foil will completely block all light.


  • Keep the workbench and work areas clean to prevent contamination and avoid any potential erroneous processes or confusion during processing steps.
  • Never re-use old reagents. If possible, prepare only enough reagent to be used in the immediate experiment. Never return unused reagent back to stock containers.
  • Never re-use wash or reagent troughs or trays.


  • Prepare a fresh standard curve for each assay performed.
  • Dilute the standard within the range indicated by the vendor protocol. Adding points to the top or bottom will not increase sensitivity or dynamic range. However, additional dilution points approaching the top or bottom asymptotes of the standard range may increase accuracy within this range of the curve.
  • Run standards in duplicate.
  • Always run the appropriate negative and blank control wells in addition to the standards.
  • Assays reporting a relative positive/negative cut-off threshold require controls samples. Always run the included positive/negative controls with each experiment in replicate.

Sample Prep

  • Optimize sample dilution level. Analyte measurement accuracy is optimal near the middle of the assay dynamic range. Dilute sample to the appropriate level by evaluating a small sample dilution range prior to performing broad experiments.
  • Plan out your plate loading layouts ahead of time. Utilize a template or map for reference.
  • Avoid samples which contain easily recognizable interference factors such as bilirubin or appear lipemic. Interferences factors will contribute to erroneous results.
  • Always use fresh tips when creating serially diluted standards.


  • Always use a calibrated reading instrument.
  • Calculate you inter- and intra-assay %CV's. This will assist in tracking assay reliability and provide a quick and easy indication if problems exist.
  • Subtract control wells from test wells for more accurate measurements.
  • Become familiar and utilize analysis software providing the most accurate curve fit models. 4- or 5-parameter curve fitting capabilities are recommended.

Technical Support

We know a lot of questions an arise as you are running your ELISA assays. Aviva's scientists are very knowledgeable in the use of immunoassays and are keen to assist with any questions you may have. Please contact use via our online chat, send us an email at, or call us at 858-552-6979.