One of the most widely-used forms of off-the-shelf kit, enzyme-linked immunosorbent assays (ELISAs) are microplate-based immunoassays which rely on adsorption of a molecule to a solid surface. This solid surface could be the microplate itself, or alternatively a specialised bead may be used as the solid phase. Easy to perform, quantitative, and sensitive, ELISA has utility across every field of research. It also benefits from applicability to automation, meaning it is ideally-suited to high throughput screening campaigns.
Popular ELISA formats include sandwich, direct (antigen-down), and competitive ELISA. In a sandwich ELISA, the solid phase is coated with an antigen-specific antibody, which captures the antigen from a test sample. Antigen binding is detected directly, using an enzyme-conjugated antigen-specific antibody, or indirectly with an antigen-specific antibody and an enzyme-conjugated anti-species antibody. The main advantages of a sandwich ELISA are its specificity and sensitivity, making it an excellent choice to analyse complex samples.
During a direct ELISA, the solid phase is coated with the antigen, which is then used to capture antigen-specific antibodies from a test sample. An enzyme-conjugated anti-species antibody is usually employed for detection. This ELISA format is faster than sandwich ELISA as it involves fewer incubation steps, however assay sensitivity is reduced since no secondary antibody is used. Direct ELISA is often used for allergy testing.
A competitive ELISA is used when only one antibody is available for the target analyte, or to detect small analytes which cannot bind more than one antibody simultaneously. Both a sandwich ELISA and a direct ELISA can be adapted to a competitive format. During a competitive ELISA, any antigen present in the test sample competes with a known amount of labelled reference antigen to become immobilised on the solid phase. Those samples which contain a high concentration of antigen give rise to a low signal by preventing the labelled reference antigen from binding.
Commonly-used enzymes for all three ELISA formats include horseradish peroxidase (HRP) and alkaline phosphatase (AP). These are typically paired with chromogenic substrates to produce a quantifiable colour change upon catalysis of the reaction. PNPP is a popular chromogenic substrate for AP, while TMB, ABTS and OPD are widely-used for colorimetric detection with HRP. Chemiluminescent and fluorescent ELISA substrates can also be paired with these enzymes.
Chemiluminescent immunoassay (CLIA) is a similar technique to ELISA, reliant on chemiluminescent detection. Chemiluminescence delivers increased sensitivity and a greater dynamic range than chromogenic detection, making CLIA ideal to detect low-abundance analytes.
The process of chemiluminescence involves conversion of a specialised substrate to product with the associated release of light energy which can be quantified. During CLIA, this reaction is typically catalysed by enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), which are provided as antibody conjugates. CLIA detection requires a dedicated reader, capable of measuring a chemiluminescent readout.
Enzyme-linked immunospot (ELISpot) assays are used to detect substances secreted by cells which have been cultured in vitro. To achieve this, the cells are grown on a surface which has been coated with a specific capture antibody. As the cells secrete biomolecules such as cytokines, these become bound to the immobilised antibody. Following removal of the cells, the secreted biomolecules can be visualised through direct or indirect detection in a method analogous to ELISA.
Horseradish peroxidase (HRP) or alkaline phosphatase (AP) are widely-used for ELISpot detection. By pairing these with a precipitating substrate, rather than the soluble substrates which are commonly employed for traditional ELISA, discrete spots are produced. Each spot corresponds to an individual cell that has secreted the biomolecule of interest and can be analysed using an ELISpot reader.
ELISpot is one of the most sensitive assays available for single-cell analysis, making it perfect to study low abundance cell populations. It is straightforward to perform and suitable to evaluate large sample numbers. Since the plated cells can be treated in situ with a wide range of stimuli, ELISpot is useful to better understand complex cellular signalling pathways.
Like an ELISpot assay, FluoroSpot is used to detect and analyse the secreted products of plated cells. However, instead of relying on a colorimetric readout, FluoroSpot uses fluorescently-labelled antibodies for detection. By measuring the intensity of the fluorescent readouts, the amount of each secreted product can be quantified.
Highly-sensitive and easy to perform, FluoroSpot can be used to analyse the secretion of multiple analytes from the same cell. It can also distinguish between different cell types in the same assay well and can inform the relative robustness of similar cell types within an assay by comparing spot sizes and fluorescent intensities. A further utility of FluoroSpot is to investigate the downstream effects of biomolecular co-secretion.
Analysis of FluoroSpot data requires a specialised reader, capable of measuring the volume of each spot and of detecting different wavelengths. Careful selection of fluorophores allows simultaneous analysis of multiple secreted molecules which may exhibit different kinetics, while fixed camera settings can eliminate user bias.