Introduction

Recognizing a specific chemical entity amidst a complex matrix is crucial in scientific research and it has applications that span from clinical, to environmental to quality control studies. It is no surprise thus, that for this purpose, several assays have been developed through the decades. Among the most successful methods immunoassays stand out, particularly the ELISA test (Enzyme-Linked-Immunosorbent-Assay), firstly described in 1971 by Engvall & Perlmann. Their assay took advantage of the accuracy and selectivity of the interaction between an immunoglobulin and its specific antigen, while the activity of an enzyme previously bound to the antibody was fundamental to highlight the results. The technique quickly emerged as a gold standard in the field and caught the interest of many manufacturing companies. By the 1980s, fully automated ELISAs were being commercialized, furtherly contributing to the accessibility and spreading of this technique (Lequin 2005). Today, ELISA is a common laboratory practice all over the world, and it is constantly developed, in an effort to reach higher accuracy, throughput, multiplexed data and miniaturization (Mendoza et al. 1999). The interest of companies has diversified and gotten more and more specific over time, developing extremely specific kits for various markers. An explicative example is the one of Mercodia, an Uppsala-based business, world-leading in the development of bioassays for diabetes biomarkers.

Fundamental concept of ELISA

Although several variants of the ELISA method have been developed, the basic principle remains the same. Recognition between an antigen and its specific antibody (either mono- or polyclonal) is exploited by labeling one of the two with an enzyme. The subsequent addition of the enzyme substrate activates a reaction that colors the solution, and the intensity is assessed using a microtiter plate reader, that reports semi-quantitative data. Common enzymes are alkaline phosphatase, β-galactosidase or horseradish peroxidase, that interact with fluorogenic or chromogenic compounds (Sakamoto et al. 2018). 

The variations on the theme of ELISA (Figure 1) can be categorized into “direct” and “indirect” tests, depending on the observed entity, either the antigen (direct) or its antibody (indirect). Direct ELISA closely follows Engvall & Perlmann’s assay (1971). The antigen is absorbed on a polymeric surface. After a washing step mediated by a blocking solution to avoid non-specific absorption, a dispersion with labeled antibodies is added, allowed to react, and washed away. The enzyme substrate will then allow target detection. Optimization of this technique was achieved with “competitive assays” (Engvall et al. 1971), where the antibody is physically absorbed to the surface, while a known concentration of the antigen is purified and labeled with the enzyme. The labeled antigen is added to the sample and the reaction develops. In this variant, the higher the antigen concentration, the lower will be the detected signal, as interaction of the tagged antigen with the antibody will be inhibited. An alternative is “sandwich ELISA”, which exploits two distinct recognition antibodies. A “capture” immunoglobulin is absorbed, and, after sample addition, a ”primary” enzyme-labeled one is mixed in solution to display the results (Belanger et al. 1973).

Indirect ELISA exploits higher selectivity and standardization, allowed by the employment of secondary antibodies. The protocol for this assay also starts with manipulation and absorption of the antigen, followed by blocking and incubation of the primary antibodies. The concentration of these components will be detected by secondary labeled antibodies directed against their fragment crystallizable (Fc) region (Lindström & Wager 1978). Sandwich and competitive indirect ELISAs are also available. The former uses 3 different immunoglobulins, capture, primary and secondary. The latter focuses instead on the competitive binding of a known concentration of antigen bound to the surface, and the one in the sample itself that is injected in solution with the primary antibody. Much like direct assays, here the detected signal lowers the higher is the amount of target in the sample (Sakamoto et al. 2018). 

Chart, scatter chart

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Figure 1. Variants of the ELISA test. a) direct; b) competitive; c) sandwich; d) indirect; e) indirect and competitive f) indirect and sandwich. In the picture the test is shown as single step, but every reagent is added separately, divided by a washing step.

References

Belanger L, Sylvestre C, Dufour D. 1973. Enzyme-linked immunoassay for alpha-fetoprotein by competitive and sandwich procedures. Clinica Chimica Acta 48: 15–18.

Engvall E, Jonsson K, Perlmann P. 1971. Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin g, by means of enzyme-labelled antigen and antibody-coated tubes. Biochimica et Biophysica Acta (BBA) – Protein Structure 251: 427–434.

Engvall E, Perlmann P. 1971. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8: 871–874.

Holst JJ, Wewer Albrechtsen NJ. 2019. Methods and Guidelines for Measurement of Glucagon in Plasma. International Journal of Molecular Sciences 20: E5416.

Lequin RM. 2005. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical Chemistry 51: 2415–2418.

Lindström P, Wager O. 1978. IgG autoantibody to human serum albumin studied by the ELISA-technique. Scandinavian Journal of Immunology 7: 419–425.

Liu D, Li X, Zhou J, Liu S, Tian T, Song Y, Zhu Z, Zhou L, Ji T, Yang C. 2017. A fully integrated distance readout ELISA-Chip for point-of-care testing with sample-in-answer-out capability. Biosensors & Bioelectronics 96: 332–338.

Ma L, Abugalyon Y, Li X. 2021. Multicolorimetric ELISA biosensors on a paper/polymer hybrid analytical device for visual point-of-care detection of infection diseases. Analytical and Bioanalytical Chemistry 413: 4655–4663.

Mendoza LG, McQuary P, Mongan A, Gangadharan R, Brignac S, Eggers M. 1999. High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA). BioTechniques 27: 778–780, 782–786, 788.

Sakamoto S, Putalun W, Vimolmangkang S, Phoolcharoen W, Shoyama Y, Tanaka H, Morimoto S. 2018. Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. Journal of Natural Medicines 72: 32–42.

Wewer Albrechtsen NJ, Kuhre RE, Windeløv JA, Ørgaard A, Deacon CF, Kissow H, Hartmann B, Holst JJ. 2016. Dynamics of glucagon secretion in mice and rats revealed using a validated sandwich ELISA for small sample volumes. American Journal of Physiology Endocrinology and Metabolism 311: E302-309.

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