Optimizing your ELISA Assays

No life science or diagnostic lab can live without it: the ELISA assay. The article explains the principle of ELISA assays and presents variables that determine the sensitivity and precision of your measurements.

Dr Andrea Krumm Dr Andrea Krumm (10)

What is an ELISA assay?

Enzyme-Linked Immunosorbent Assays, typically known as ELISAs, are “immunosorbent” because they utilize immunoglobulins (antibodies) adsorbed on an immobile surface to remove a specific antigen from solution, or vice versa. The antibody-antigen complex is “enzyme-linked” or bound to an enzyme that catalyzes a detectable reaction. Usually the signal is a color, but chemiluminescent or fluorescent signals are also used. The intensity of the detectable signal is proportional to the amount of antigen that initially bound the antibody and can therefore be used to measure the antigen concentration in the analyte solution.


Fig. 1: Assay principle of ELISAs. A capture antibody (green) that specifically binds the analyte is immobilized on a microplate. It serves to bind and capture the analyte (blue) on the plate. A second antibody (red) that likewise binds the analyte carries an enzyme (yellow). In a last step, a substrate is added (grey) that is converted by the enzyme to a chromophore or fluorophore. The more analyte is found in the sample, the more enzyme is bound, and the more substrate is converted. Therefore, the signal intensity correlates with the concentration of analyte.

This popular assay is a simple and fairly quick laboratory method to measure the presence or quantity of biomolecules with a high level of accuracy. Usually performed in a 96-well microplate, ELISAs can process a large number of samples within a few hours. An ELISA can typically detect between 0.01 ng and 0.1 ng of analyte per mL, making it highly sensitive and an established benchmark in antigen quantitation.

Nonetheless, there are many pitfalls that can decrease your throughput, accuracy and the overall performance of ELISAs. The purpose of this note is to provide researchers like you with tips to improve the quality and throughput of your ELISAs.

Ensure sample integrity 

Your ELISA is only as good as the quality of the materials you start off with. The first step to a successful ELISA technique is making sure that samples have been collected properly and are stored under the right conditions. For example, samples may contain interfering substances such as antigen-binding proteins, or anticoagulants that may need to be removed by centrifugation, or diluted out before you begin the assay. Another common pitfall is using samples that have gone through multiple freeze-thaw cycles. Avoid this by aliquoting samples prior to freezer storage.

Stick to optimized incubation parameters

Much of the work of an ELISA takes place during the incubation steps, when antibodies bind to antigens and substrates react with enzymes. There are optimum conditions for these crucial events to take place, such as the right temperature, light exposure or shaking intensity. Being too lax in adhering to the optimized incubation time and temperature can cause evaporation of the well contents or a build-up of condensation on the plate sealer. These discrepancies alter the concentrations of  key reactants in the wells and affect the accuracy of the ELISA read-out. Ensuring that your ELISA is incubated under the right conditions is, therefore, very important. One way this can be achieved is with the CLARIOstar Plus plate reader, which has integrated incubation and shaking capabilities.

Do not skimp on your washing 

Background noise can interfere with your ELISA output and diminish the power of your assay. Much of the background noise in ELISAs comes from inadequately blocking and washing the plates. The wash steps are a major culprit, as it is important to wash wells thoroughly and remove the entire wash buffer after each wash.

ELISAs typically require multiple wash steps in the workflow, and it is almost impossible to ensure that you are washing the plate the same way during every wash. Advancements in immuno-assays, such as ELISAONE™ or SPARCL technology, overcome this problem by limiting the number of wash steps. This overcomes the drawback of making sure repeated washes are identical. Other ELISAs, such as this competitive assay from PAIA Biotech to quantify IgG, have revolutionized bead-based assays by eliminating wash steps altogether, reducing user error.

Block out the background noise

Blocking steps are optimized to coat untargeted binding sites to prevent detection antibodies from non-specifically binding to the plate’s surface. A wide range of sophisticated commercial blocking buffers are available that contain unrelated large and small proteins or protein fragments, as well as preservatives to stabilize the working environment. When you need maximal blocking strength, a viable option is to use a synthetic blocking formula to thwart any non-specific interference.

Use the right plates to avoid the edge and hook on your ELISAs

It is important to use the right kind of plates for your ELISAs. Polystyrene or polyvinyl chloride 96-well flat-bottomed plates are most commonly used for ELISAs. Using the right kind of ELISA plate helps maintain consistency, minimizes both edge and hook effects and maintains conditions required for downstream optical data collection steps.

An edge effect develops due to discrepancies in the manufacturing of ELISA plates where the outer wells behave differently, giving unexpected read-outs inconsistent with the internal wells. This is best controlled by using triplicates for all samples and being mindful of large variations in readings from wells at the edges. 

A hook effect, on the other hand, is seen when antigen levels are very high in the analyte. In such cases there is not enough antibody in the well to bind to the antigen specifically and the readout is lower than expected. The best way to prevent a hook effect is to test a large number of dilutions of each sample in pilot assays before embarking on a large assay.

Optimize signal intensity to detect the maximum number of samples

Whether you use samples with a small concentration of your antigen of interest or have had to dilute out interfering substances, you may run into problems with signal detection.

Fig. 2: Quantification range of ELISAs. ELISA kits provide standards with a known concentration of the analyte to prepare a standard curve (line 3 and 12). The analyte concentration of the sample needs to be in the range covered by the standard curve. Otherwise assay optimization is required. Here are some steps you can take to increase the intensity of your signal:

  • In the ELISA principle, secondary antibodies are typically bound to an enzyme that reacts with a substrate to give off the signal that is measured by the microplate reader. There are two alterations to this reaction that can improve signal intensity: (i) increasing the incubation time of the enzyme-substrate reaction and (ii) increasing the amount of enzyme bound to the antibodies. 
  • Similarly, increasing the quantity of your antibodies to ensure that all the available substrate is bound, and increasing antibody binding times (within the defined limits of the protocol) can increase the signal intensity.

If the signal is higher than the detection range of the ELISA reader, there are also some steps you can take to reduce signal intensity:

  • The easiest method is sample dilution. Adding assay buffer to your samples will decrease the concentration of analyte, thus decreasing sample intensity to a readable level.
  • High signal intensity may also be due to overexposure of the enzyme substrate (typically 3,3′,5,5′-Tetramethylbenzidine, also known as TMB) to light and oxygen. Quick addition of enzyme substrate to the wells, and running the enzyme-substrate reaction in a dark incubator, can resolve this issue.

In some cases, there is a huge variability among sample signal intensities due to differences in treatment group, or response. To capture this variation, consider using a fluorescent or luminescent substrate and a microplate reader with a high dynamic range of detection. A microplate reader with a high dynamic range is capable of measuring a wide range of sample dilutions by detecting their corresponding signal intensities in a single detection cycle.

Don’t allow your plate to dry out

The reactions of an ELISA must take place in solution and drying out your wells in between the addition of reagents can negatively affect your output. It is important to prepare your workspace with thoughtful ergonomic efficiency in order to have each reagent ready in step-wise order, so that reagents can quickly be added to wells after wash steps to prevent plates drying out. 

Other things to consider:

Making the most of your materials 

  • Multiuse Plates: Each ELISA kit comes with a list of included materials. Most of these are standard; buffers, enzyme conjugates and wash solutions, but the plates can vary. Get the microplates that best fit your needs. While many ELISA kits provide plates conjugated to a specific antibody, coating plates with the antibodies of interest individually allows assaying many different types of antigens. In this case, you won’t have to discard plates due to antibody expiration and you have the flexibility to use multiple types of antibodies on one plate, both making your ELISA more cost-effective..
  • Multifunctional Plate Readers: ELISA kits also come with a list of materials needed, but not included. Combination equipment can be very handy here in reducing your lab footprint. For example, a microplate reader that also doubles in function as a shaker and an incubator saves you from buying three separate sets of equipment.  
  • Multi-mode Plate Readers: The enzyme substrate is the step that reveals the analyte concentration. Multiple enzyme-substrate pairs exist that have different reading wavelengths. The range of colorimetric ELISA readouts is mainly between 400 and 500 nm. Additionally, alternative substrates exist leading to fluorescence or luminescence signals to detect analyte concentration. These two methods not only exhibit a higher dynamic range, but are much more sensitive than colorimetric ELISAs, which allows for miniaturization of the assay to reduce sample costs. Considering these alternative methods of detection, and the multiple enzyme-substrate wavelengths, it is cost-effective and pragmatic to use a multi-mode plate reader. The requirements that the reader must meet for certain enzyme-substrate combinations are listed in the table below.

Table 1: Common enzyme-substrate combinations of ELISA assays and corresponding microplate reader requirements.


Enzyme Substrate Detection Mode Reading Wavelength (nm)
Horseradish peroxidase (HRP) OPD (o-Phenylenediamine) Absorbance 450 (unstopped)
492 (stopped)
TMB (3,3′,5,5′-Tetramethylbenzidine)  Absorbance 630 (unstopped)
450 (stopped)
ABTS ((2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid])  Absorbance 414
Luminol Luminescence Detect all light
Luciferin Luminescence Detect all light
HPPA (3-p-hydroxy phenyl proprionic acid) Fluorescence Detect all light
Alkaline Phosphatase (Alk-phos) pNPP (p-Nitrophenyl Phosphate)  Absorbance 405
AMPPD (3-(2'-spiroadamantane)-4-methyl-4-(3'-phosphoryloxyphenyl-1, 2-dioxetane)) Luminescence Detect all light
4-MUP (4-methyl umbelliferyl phosphate) Fluorescence Excitation 360
Emission 440
ONPG (o-Nitrophenyl-ß-D-galactopyranosidase)  Absorbance 420
AMPGD (3-(2'-spiro ada mantane)-4-methoxy-4-(3'-ßD-galactopyranosyloxy phenyl-1,2-dioxetan) Luminescence Detect all light
MUG ((4-methylumbelliferyl galactoside) Fluorescence Excitation 360
Emission 440
Urease Urea bromocresol  Absorbance 590


Increased throughput = increased productivity

Technological upgrades like the ELISAONE™ assay increase throughput by reducing assay time to as little as 1 hour from start to finish. Coupled with a rapid multi-mode microplate reader such as BMG LABTECH´s Omega series of readers, VANTAstar, CLARIOstar Plus or PHERAstar FSX, time-saving, and space-saving ELISAs are at your fingertips, maximizing the productivity of your research team.



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