ELISA Immune complex optimization

The ELISA technique is a widely used and informative immunoassay. However, development and optimization remains the key to a successful ELISA assay. The assembly of a large immune complex can prove challenging with failure or incorrect optimization of any component determining the success of the assay. There are number of factors which can affect ELISA signal generation (see Table 1).

Before beginning your ELISA it is recommended to first generate and optimize a standard curve for your analyte of interest before proceeding with multiple samples of unknown composition. If your standard curve displays the correct range, sensitivity and linearity you may proceed to process the samples.

Factor Variable Characteristic

Blocking buffer

Concentration, cross-reactivity, composition

Capture Antibody

Specificity, affinity, incubation time, temperature

Coupling Buffer

pH, Composition

Detection Antibody

Specificity, affinity, incubation time, temperature, cross-reactivity

Enzyme Conjugate

Concentration, cross-reactivity, enzyme type, conjugate type

Microtiter Plate

Well shape, pre-activation, material

Signal Detection

Exposure time, instrument, filters


Sensitivity, age

Target Antigen

Stability, available epitopes

Table – Factors affecting ELISA Signal generation

Plate type and Adsorption

The absorbance of the colorimetric substances used in ELISA are measured via a laser which shines up the base of each well, therefore it is essential that the plate used is flat bottomed with a clear base. If using fluorescent detection the plate used must be opaque white or black, fluorometric plate readers can measure either above or below the plate. Chemiluminescent detection requires the use of black or white opaque plates with a clear bottom. White plates are preferred for chemiluminescence as they magnify the signal. For fluorescence detection black plates are preferred as they give off lower background.

Plates used for an ELISA may be made from polystrene, polypropylene or polycarbonate. Many of the plates used are gamma irradiated to give a positive charge which aids in coating.

The most commonly used form of protein attachment to plates is passive adsorption. This method relies on hydrophobic interactions in addition to some electrostatic forces. A commonly used coating buffer is carbonate-bicarbonate (0.2M sodium/bicarbonate pH 9.4). The high pH of this solution aids the solubility of many proteins and peptides, and ensures that proteins are unprotonated with an overall negative charge, which helps when binding to a positively charged plate. Other buffer options include Tris-buffered saline (TBS) or phosphate buffered-buffered saline (PBS) at physiological pH. Pre-coated plates are also available depending on the specifics or your assay.


Not all antibodies can be used for ELISA and will require evaluation before commencing with experimental samples. During the adsorption process, the three-dimensional structure of the antigen may be altered and as a result of this may no longer be able to bind its target epitope. Furthermore, if an antibody was raised against a peptide and that peptide sequence represents part of the internal sequence of the antigen of interest the antibody may not bind the whole antigen if immobilized on the plate.

For an antibody to successfully work in an ELISA assay it must bind specifically to the antigen and not cross react with components of the blocking buffer. When two antibodies are required for instance in a sandwich ELISA the selected two antibodies must react with different epitopes on the antigen. If the antigen is immobilized via a capture antibody, the detection antibody must still be able to interact with its own epitope without steric hinderance from the first antibody on the plate. The capture and detection must be raised in different species.

Another important factor to consider when preparing an ELISA assay is the concentration at which the antibodies will be used at. This will require optimization and may may also be determined by the substrate used.

Blocking Buffer

During ELISA a blocking buffer is used to prevent non-specific binding of proteins to the plate. An optimized blocking buffer maximises the signal-to-noise ratio and does react with with the antibodies or target protein. If cross-reactivity is observed a different blocking buffer should be used. Some ELISAs may require the addition of a surfactant such as TWEEN-20 to the blocking buffer. A surfactant may be used to minimise hydrophobic interactions between the blocking buffer proteins, antigen and antibodies. If using TWEEN-20 a final concentration of 0.05% is recommended.

A recommended volume of blocking buffer for a 96 well plates is 300ul per well or enough solution to completely coat the wells.

Target Antigen

The target antigen should be present either in a matrix or a buffer to enable interaction with a pre-coated capture antibody or be coupled directly to the plate. In some exceptional circumstances the 3D structure of the antigen may be altered during adsorption to the plate and will no longer bind the target epitope. If this is the case a plate pre-coated with a binding protein (capture antibody) is recommended.

If the antigen is in the form of a biological sample the effects of the matrix e.g. serum and plasma should be controlled by performing spike in recovery and linearity dilution of experiments.

For quantitative analysis it is essential to include a standard curve of a protein of known concentration. A serial dilution of the known protein is performed and measured, from a standard curve is made plotting the concentration against absorption. From the standard curve the concentration of the samples of unknown concentration are extrapolated.

The concentration of enzyme conjugate used is a very important aspect of ELISA optimization, the amount of enzyme which binds will directly influence the amount of signal generated. Too little enzyme can result in a weak signal with poor signal-to-noise ratio. Too much enzyme may also affect the signal-to-noise ratio, in addition to increasing background noise.

Washing the Plate

Two commonly used washing buffers for ELISA are TBS (Tris-buffered saline) and PBS (Phosphate-buffered saline) with 0.05% TWEEN. To wash the plate the wells should be be repeatedly filled and emptied by aspiration or plate inversion. Following incubation with the coating and detection antibody 3 x 5-minute washes are generally recommended. Following incubation with the enzyme conjugate 6 x 5-minute washes are recommended.

Substrates and signal detection

Substrate choice will depend on the sensitivity of the assay and the equipment available. Chemiluminescent substrates are the most sensitive with antigen detection possible in the sub-picogram per well range. Colorimetric and chemifluorescent substrates are able to detect mid to low-picogram levels of antigen per well.

Colorimetric signal detection is performed using a standard plate reader with the appropriate filters. Chemifluoroescene is measured using a fluorometer whilst, chemiluminescence is measured using a luminometer, some plate readers can however measure Chemiluminescence.