An immunological assay, or immunoassay, is a qualitative method of determining biochemical activity using immunological components, typically an antibody or antigen, with high affinity to the target analyte. Immunoassays play a significant role in modern biomedical research and a range of applications in clinical diagnostics, environmental monitoring, pharmaceutical and drug analysis, and even food testing.
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A variety of immunoassays have been developed to enable the detection of analytes through quantitative, semiquantitative, and qualitative analysis, which can be instrumental in a clinical setting, and this article will discuss their function and most critical applications.
What are Immunological Assays?
The term ‘in vitro’ is Latin for ‘in the glass’ and describes experiments that occur outside of a living organism, excluding microorganisms. For example, the response of cells grown in culture to particular chemical or physical stimuli, or the function of proteins, may be investigated in vitro.
Both in vitro and in vivo testing methodologies are used by researchers to advance comprehension of patient diseases, within biomedical research to find novel targets, pharmaceutical research to identify treatments, and within a healthcare setting for diagnosis and treatment guidance.
Enzyme-linked immunosorbent assay (ELISA) is one of the most widely employed immunological assays and is considered the gold standard in bio-molecule recognition and quantification. ELISAs employ enzymes specific to the target analyte, which may be bound to a two-dimensional immobile plate or otherwise to mobile nanoparticles. Once the enzyme encounters the analyte, a strong bond is formed between the two, which may act to quench or cease the ongoing quenching of a nearby fluorophore.
Alternatively, a second antibody complimentary to the enzyme-analyte complex can be utilized as a fluori- or colorimetric indicator. The high sensitivity and adaptability of ELISAs allow them to be utilized in the quantification of a wide variety of biomolecules, including but not limited to antigens, antibodies, proteins, and hormones.
A broad range of non-biological molecules can also be detected, as long as they are capable of interacting with the utilized enzymes, and thus ELISAs are also useful in detecting trace concentrations of drugs, toxins, and other small molecule analytes of interest.
A variety of immunological components may alternatively be employed in other types of assay, including whole immune cells, typically to investigate the probable immune response of humans or animals to a therapeutic prior to in vivo assay. For example, T cell assays utilize whole human T cells and quantify down-stream indicators of T cell activity, while dendritic cell assays utilize whole cells and are intended to examine the probable effects of autoimmune disease treatment.
Where ELISAs are typically employed to both indicate the presence of an analyte and quantify by fluori- or colorimetric intensity, other analysis endpoints may be employed to infer the result of the immunological assay. Commonly, analysis of the gene expression of immune cells following a therapeutic application may be performed using complementary techniques, the health of cells assessed by microscopy, or cell cycle stage determined by imaging and chemical methods.
Flow cytometry is frequently exploited as a high throughput method of cell assessment within immunological studies, able to infer information relating to cell health and, therefore, the outcome of the immunological assay, and further can be used to sort and separate cells for further analysis.
Applications of in vitro Immunological Assays
The World Health Organization (WHO) estimates that there are over 400,000 products currently available for in vitro diagnostic testing, which are used to research the function of and test for a multitude of diseases and treatments. These include laboratory-based tests performed in research or a hospital setting and specialty point-of-care tests that consumers and semi-professionals can execute in the field.
For example, immunological assays are contained within many field-use diagnostic kits, including pregnancy tests and the widely distributed SARS-CoV-2 lateral flow testing devices. These devices contain colorimetric indicators bound with antibodies specific to the target analyte, such as the SARS-CoV-2 spike protein, which then show or change color once bound with the analyte.
Mobile kits such as this are required globally as the first line of defense against the spread of diseases, particularly in poorer regions without ready access to proper laboratory facilities. This can aid in infection control and allow for fast, early, and effective detection. Additionally, diagnostic testing can also aid in monitoring drug resistance and the development of alternative treatments.
Where analytes are in low concentration within a sample, steps may be taken to purify or amplify their presence, improving the sensitivity of subsequent immunological assays. Reverse transcription polymerase chain reaction (RT-PCR) can be used for this purpose when the analyte is a nucleic acid derivative such as RNA or DNA and can be combined with fluorescence spectroscopy to give a real-time quantitative readout of analyte concentration.
Future Outlook
In vitro testing has various advantages over in vivo testing, including greater allowance of examination of the mechanism of action under investigation, the opportunity to generate custom biological systems, and, importantly, reduces the requirement for animal models, potentially refining and replacing them in the future.
Specifically, in vitro immunological assays aim to target and measure analytes for various applications such as diagnostics, as well as in facilitating research into potentially novel treatment options for various diseases and disorders.
Sophisticated immunological assays improve the capacity for early detection of diseases and disorders in patient samples and aid in accurate diagnosis and prognosis. This is significant for the progression of healthcare and medicine as a whole, serving as first line diagnostic tools and investigative platforms for the future treatment of diseases involving immune cells or other immune components.
In vitro immunological assays such as ELISA have become a key stage in the diagnosis of various diseases, including HIV, Lyme disease, hepatitis B, and many more. However, the technique can be time-consuming and skill intensive, particularly where custom assays must be developed, and thus pre-made immunoassay kits offering faster, simpler, and more cost-effective analyte detection are broadly available.
Simple and robust testing kits such as lateral flow assays may be a potential route for developing many novel diagnostic techniques that can be used globally for infection control without access to testing facilities.
Sources:
- Albert-Vega, C. et al. (2018) “Immune functional assays, from custom to standardized tests for precision medicine,” Frontiers in Immunology, 9. Available at: https://doi.org/10.3389/fimmu.2018.02367.
- Alhajj M, Farhana A. Enzyme Linked Immunosorbent Assay. [Updated 2022 Feb 2]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK555922/
- Hu, B. et al. (2020) “Characteristics of SARS-COV-2 and COVID-19,” Nature Reviews Microbiology, 19(3), pp. 141–154. Available at: https://doi.org/10.1038/s41579-020-00459-7.
- In vitro diagnostics - global (no date) World Health Organization. World Health Organization. Available at: https://www.who.int/health-topics/in-vitro-diagnostics#tab=tab_1 (Accessed: February 28, 2023).
- Lankveld, D.P. et al. (2009) “In vitro testing for direct immunotoxicity: State of the art,” Methods in Molecular Biology, pp. 401–423. Available at: https://doi.org/10.1007/978-1-60761-401-2_26.
- Vashist, S.K. and Luong, J.H.T. (2018) “Immunoassays,” Handbook of Immunoassay Technologies, pp. 1–18. Available at: https://doi.org/10.1016/b978-0-12-811762-0.00001-3.
Further Reading