Enzyme-linked immunosorbent assay (ELISA) is a labeled immunoassay. To perform an ELISA, researchers immobilize target analytes (such as antigens, recombinant proteins, or peptides) on a microplate. They then incubate the analyte with enzyme-linked antibodies that affinity bind to the antigen, wash the plate to remove unbound materials, and analyze the signal produced by the antigen-antibody interaction. Linking reporter enzymes to antibodies catalyzes certain chemical reactions that convert the substrate to a detectable product and produce measurable signals, enabling researchers to detect the presence and concentration of analytes with a high degree of sensitivity. 

Selecting Enzymes, Detection Methods, and Substrates for ELISA

The exact enzyme that researchers conjugate to the detection antibody significantly impacts the sensitivity and signal-to-noise ratio of the assay. Researchers select an enzyme label that is appropriate for the application as well as the signal detection method that will be employed. Most immunoassays utilize colorimetric, chemiluminescent, or fluorescent substrates that produce signals detectable to the naked eye or with specially designed readers. After selecting the enzyme, researchers chose an appropriate substrate for the intended detection method and the requirements of the assay that will be performed (sensitivity, dynamic range, and reaction speed). Then they optimize the enzyme-substrate reaction with specific incubation times, development conditions, and microplate types for optimal results. 

Colorimetric Detection

Colorimetric detection conjugates Horseradish peroxidase (HRP)- or alkaline phosphatase (AP)-labeled antibodies with a chromogenic substrate solution for AP. This substrate creates a soluble, colored product that absorbs light in the visible wavelength range and accumulates in the well with an optical density (OD) relative to the concentration of the enzyme. Reaction time, temperature, and exposure to light can all impact the measurement of enzymatic activity in ELISA. 

Before taking any readings, researchers mix well contents to evenly disperse the colored reaction product. After the assay produces the desired color intensity, they use a standard absorbance plate reader to measure the level of product absorbance and determine the concentration of the analyte based on a standard curve. There are also many different types of microplate readers available for colorimetric detection. Single-wavelength colorimeters work well for standard ELISA kits with little background noise, while multi-wavelength spectrophotometers are necessary if background noise interferes with the ability to accurately detect the colored reaction product.

Chromogenic ELISA substrates enable direct visualization of the target analyte, offer high reproducibility between plates, and allow researchers to perform kinetic studies on the antigen-antibody interaction. However, this method is less sensitive than chemiluminescent and fluorescent detection. Substrates that generate intensely colored reaction products at very fast rates are ideal for assays that must detect low amounts of the target analyte in the sample. Substrates that produce reaction products over a longer period of time and create a range of color intensities for multiple analyte concentrations are ideal for assays that involve analyte amounts in various concentrations (large dynamic range).

Chemiluminescent Detection

Luminescence refers to the emission of light from a substance as it transitions to a ground state after reaching an electronically excited state. Chemiluminescent detection is a variation of the standard ELISA in which the enzyme causes a chemical reaction that converts a substrate to a reaction product and releases energy in the form of light photons. Although AP equivalents may be used, the most common procedure combines HRP enzymes in a luminol-based enhancer solution with a peroxide buffer. As the enzyme-substrate interacts, the luminol oxidizes and forms an excited state product that generates light as it decays. Researchers measure the light signals with a luminometer plate reader, and the emission of light continues until the substrate is exhausted. 

Chemiluminescent detection is considered more sensitive than colorimetric detection and is ideal for assays that need a large dynamic range, but the signal intensity in ELISAs tends to vary with this substrate more than others. This can create a problem for researchers when reading many plates, as the signal can decay too rapidly before it can be thoroughly measured. Optimizing the assay for the substrate can reduce signal-fade misinterpretations in samples with a low abundance of antigens. Selecting an opaque white microplate made of a specially formulated resin composite is crucial for reducing background noise and unwanted interactions (cross talk) between wells. This type of plate also reflects emitted light into the detector, increasing assay sensitivity.

Fluorescent Detection

Another variation of the colorimetric ELISA, fluorescent immunoassays (ELFIA), convert a non-fluorescent substrate to a reaction product that fluoresces (glows or shines brightly) when exposed to light of a particular wavelength. In most cases, the number of emitted light photons (fluorescence units) is directly proportional to the concentration of the analyte in the sample. Fluorescent detection can use HRP or AP enzymes, but AP enzymes are most popular for this method. Researchers choose the fluorogenic substrate for its ability to emit a quantitative amount of light following excitation, and the enzyme-substrate reaction product must feature distinctly separate wavelengths for excitation and emission. 

Fluorescent assays require black microplates specifically designed for these assays to enable visualization of the cells and absorb stray light or cross-talk during detection. Researchers can increase efficiency and save samples by running dual assays with clear-bottom black plates and fluorometers that can read from the top or bottom of wells. This allows them to read a colorimetric product for one analyte and a fluorescent product for a second in one plate. The best fluorometer will feature a light-sealed and thermally consistent reading compartment, adjustable light detector, numerically adjustable gain, and internal mechanisms for background control.