Which Controls to Use in ELISA Assays?

Which Controls to Use in ELISA Assays?

What is an ELISA?

Enzyme-linked immunosorbent assay is popularly abbreviated as ELISA and has another name, enzyme immunoassay (EIA), defining the same technology. It’s a plate-based assay technique developed to discover and quantify soluble substances such as proteins, antibodies, peptides and hormones. 

In an ELISA, the target macromolecule (the antigen) is palsied on a solid surface and then complexed with an antibody linked to a reporter enzyme. Detection is accomplished by gauging the reporter enzyme’s activity, which must be incubated. Sometimes this incubation is required overnight to get a suitable substrate to produce an assessable product, but many assays have a 3 hour incubation time protocol. 

The most crucial component of an ELISA method is a particular antibody-antigen interaction running the appropriate controls that helps you accurately separate true positive results from potentially false ones. Positive and negative controls will also be helpful if you ever need to adjust your protocol. 

The number and sort of controls for routine ELISA assays are defined, and unless exceptions are stated, these controls should apply to all seed health test methods using an ELISA assay. 

For confidence in ELISA results, it is necessary to have three experimental controls for comparison. A positive control, negative control, and standard control. Both the positive and standard controls are known to contain the protein or peptide of interest. The positive control is to confirm simply if the procedure is performing as intended. A positive control serves three functions. It provides confidence in the results, confirms that negative results are accurate, and assists in any protocol adjustments or optimizations that may be necessary.

The standard control is necessary for the quantification of the experimental results. It contains a known quantity of the target molecule and therefore gives the information necessary to make a standard curve. The standard control can also be useful for optimization, as an unsatisfactory standard curve can be indicative of poor antibody binding or inadequate specificity for the target protein.

The negative control is known to not contain the biomolecule of interest and adds validity to any positive results. A poorly-chosen antibody that does not bind with sufficient specificity will also contribute to a false result in the negative control. If this is the case, further troubleshooting of the experimental design is needed.

There are three major formats of ELISA. 

Several formats are used for ELISAs, but the major ones fall into either direct, indirect, or sandwich capture and detection methods: 

  • Direct ELISA: the antigen is immobilized to the exterior of the multi-well plate indirect ELISA, and it’s detected with an antibody specific for the antigen. The antibody is directly conjugated to HRP (horseradish peroxidase) or other detection molecules. 
  • Indirect ELISA: In indirect ELISA, a technique that uses a two-step process for detection, whereby a primary antibody specific for the antigen affixes to the target. The labelled secondary antibody against the primary antibody’s host species binds to the primary antibody for detection. This method can also detect specific antibodies in a serum sample by substituting the serum for the primary antibody. 
  • Sandwich ELISA: The sandwich ELISA assay is the most widely used ELISA assay format because of its sensitivity and specificity, which indirectly immobilizes and detects the target antigen’s presence. This assay is called ‘sandwich’ because the analyte is measured bound between two primary antibodies, each detecting a different epitope of the antigen–the capture antibody and the detection antibody. 

Overview of Parameters to Consider When Choosing an ELISA Kit

ParameterAcceptance criteria
Sensitivity          Depends on the target protein*
Dynamic rangeDepends on the target protein*
CV (%) for intra-assay precision≤ 10%
CV (%) for inter-assay precision≤ 15%
SpecificityCheck reactivity with highly homologous proteins
% Recovery≥ 80%
Linearity of dilution≤ 15% difference from undiluted samples

Knowing what target protein levels are expected in a sample is essential to check that a given ELISA’s sensitivity and dynamic range are appropriate. Samples with a high target protein concentration can be diluted so that the raw signal falls within the assay’s dynamic range.

  • Sensitivity: Sensitivity is the lower level of protein that the antibody pair used in the ELISA kit can accurately detect.
  • Dynamic range: The dynamic range is defined as the upper and lower concentrations of the target protein that the assay can accurately quantify. Reported sensitivity and dynamic range values can be misleading because they are often determined using standard protein in simple wash buffers. This may not represent the kinetics of detection of an endogenous protein in a biological sample.
  • Coefficient of Variability (CV): The CV (%) or the coefficient of variability indicates how consistent the assay is. CV is generally calculated to assess the inter-assay or plate-to-plate consistency and the intra-assay precision or consistency between duplicates run in the same experiment.
    • Inter-assay % CVs of less than 15 are generally acceptable.
    • Intra-assay % CVs should be less than 10. 
Pre coated elisa kit stored for an hour at room temperature

The antibodies used in the ELISA kit mustn’t cross-react with non-target proteins. These can be proteins of high homology across species. 

  • Per cent recovery: Per cent (%) recovery is determined by spiking a known amount of purified target protein into a biological sample matrix. Typical sample matrices are plasma and serum; samples are cell culture lysates for intracellular proteins. The spiked sample is measured in the ELISA, and the concentration is calculated from the standard curve. This calculated concentration is compared to the known concentration of the protein and is expressed as a percentage. 

For example, 100% recovery means that the observed concentration was the same as the sample’s actual concentration of spiked protein. It suggests that the proteins and other molecules in the sample type did not interfere with the quantification of the protein. If % recovery measures less than 80% for a specific sample type, select different ELISA kits for quantification.

  • Linearity of dilution: The linearity of dilution is an excellent companion to % recovery because it measures native rather than spiked protein in biological samples. ELISA measuring multiple dilutions of known positive samples determines the dilution linearity. The target protein concentration is determined by multiplying the dilution factor by the calculated concentration. 

For best results, the concentration of the unknown samples should be similar for all dilutions. If a difference of more than 20% from the undiluted sample is observed, you should choose a different ELISA kit for accurate quantification.

Validation and Controls

Regardless of which ELISA assay type, sample, or analyte you opt to use, a thorough validation during set-up and vigorous control of every assay run form the foundation of reliable quantitative ELISA results. Helvetica Health Care offers an extensive range of ready-to-use ELISA kits for various research fields. 

Our kits are highly published and well renowned, and we have decades of experience developing and optimizing commercial immunoassays to meet the ever-changing needs of our scientific community. We provide an expansive range of immunoassay kits and HHV-6 IgG antibody and KSHV/HHV8 IgG antibody ELISA kits. 

Please visit the Helvetica Health Care website for further assistance on assay validation. 


Michael Moore is a chartered marketeer and member of the Institute of Marketing with over 35 years experience in the Biotechnology industry, with particular expertise in  International Business Development, distributor management, and international tender business.