Homogeneous immunoassays
Homogeneous immunoassays are a type of immunological test used primarily for detecting substances, such as illicit drugs, in bodily fluids like urine, plasma, or blood serum. They are particularly favored by forensic scientists due to their low cost, minimal sample size requirements, and high sensitivity. Unlike heterogeneous immunoassays, homogeneous methods do not require the separation of bound and unbound components, allowing for quicker and easier testing processes. These assays often utilize enzyme-linked antibodies, which produce a measurable color change upon reaction with specific substrates, signaling the presence of the target analyte.
Homogeneous immunoassays can detect a range of substances, including hormones, markers of cardiovascular damage, infectious agents, and various drugs of abuse such as opiates and amphetamines. While they serve as effective screening tools, they may produce false positives and lack specificity compared to confirmatory methods, such as gas chromatography-mass spectrometry (GC-MS). Despite these limitations, continuous advancements in technology are enhancing the sensitivity and accuracy of immunoassays, enabling their application in new areas, including viral testing for influenza. Overall, homogeneous immunoassays play a critical role in the rapid detection and analysis of various substances in clinical and forensic settings.
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Homogeneous immunoassays
DEFINITION: Immunological test used to detect substances such as illicit drugs in bodily fluids.
SIGNIFICANCE: Forensic scientists often use the homogeneous enzyme immunoassay process to screen samples of urine, plasma, or blood serum for the presence of illicit substances. This method has some distinct advantages over other assay methods: It is low in cost, requires only a small sample size, and is highly sensitive. In addition, automated instrumentation provides speed and precision.
Immunoassays combine biochemistry with the specific binding tendency of antibody to antigen to produce a measurable signal. Immunoassays may be heterogeneous or homogeneous. Heterogeneous immunoassay requires the additional step of separating bound and unbound components, whereas homogeneous immunoassay returns a measurement without separating the antigen and antibody. Enzyme assays use an enzyme-bound antibody to detect the antigen. The enzyme then signals with a color reaction when exposed to the substrate. Because the homogeneous assay is quicker and easier, it is often the test preferred by forensic scientists in cases involving drugs of abuse.
In response to an antigen, which is any substance the body considers as foreign, specific antibodies bind in lock-and-key fashion, forming a complex that awaits destruction by other components of the immune system. Immunoassays take advantage of this binding specificity. The analyte (that is, the substance being analyzed) may be either antibody or antigen. Analytes may be hormones (such as testosterone or cortisol), markers of cardiovascular damage (such as creatinine or myoglobin), hepatitis strains (such as hepatitis B), tumor markers (such as certain proteins), congenital infectious agents (such as rubella), or metabolics (such as folate or vitamin B12). Examples of drugs of abuse that are amenable to homogeneous immunoassay are opiates, amphetamines, barbiturates, benzodiazepines, and metabolites. In addition to testing for drugs of abuse, immunoassays are useful in testing for therapeutic drug levels in the blood.
Typically, a toxicologist must use a screening process to determine the presence and identity of unknown drugs in a sample. A number of immunoassay screening techniques are suitable for this purpose. These include radioimmunoassay (RIA), enzyme-multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), cloned enzyme donor immunoassay (CEDIA), and fluorescence polarization immunoassay (FPIA). Automated ELISA screening has largely replaced older immunoassay techniques such as RIA and EMIT.
Although immunoassays are cost-effective and quick screening tools, they may fall short in revealing the potency, amount, and time of use of any drug found to be present. Further, these tests may give false positives and in some cases lack specificity. In addition, cross-reactivity can occur when the chemical properties of legitimate pharmaceuticals resemble the chemical properties of illicit drugs. Immunoassays are most useful for discriminating between the presence and absence of suspected drugs, although improvements in detection equipment and increasingly sensitive labels are advancing the sensitivity of immunoassay systems, allowing for the accurate measurement of an ever-increasing range of analytes. Confirmation testing, however, may need to rely on gas chromatography-mass spectrometry (GC-MS) tests, which are both more accurate and more expensive than immunoassay tests. Continued advancements in the development of homogeneous immunoassays allowed for the development of new types of viral testing, with researchers utilizing them to test for the presence of influenza.
Bibliography
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Kim, Hong-Rae, et al. "Homogeneous One-Step Immunoassay Based on Switching Peptides for Detection of the Influenza Virus." Analytical Chemistry, 28 June 2022, doi.org/10.1021/acs.analchem.2c00716. Accessed 15 Aug. 2024.
Mieczkowski, Tom, ed. Drug Testing Technology: Assessment of Field Applications. Boca Raton, Fla.: CRC Press, 1999.
Smith, Frederick P., ed. Handbook of Forensic Drug Analysis. Burlington, Mass.: Elsevier Academic Press, 2005.
Zhang, Jian-Hua, et al. "Homogeneous Electrochemical immunoassay Using an Aggregation-Collision Strategy for Alpha-Fetoprotein Detection." Analytical Chemistry, 24 Jan. 2023, pubs.acs.org/doi/10.1021/acs.analchem.2c05193. Accessed 15 Aug. 2024.