Immunoassay

• ALSO KNOWN AS: Bioassay, biochemical assay, immunochemical assay, immunodiagnosis, immunologic test

Definition

An immunoassay is a laboratory technique or method that quantifies the presence or concentration of a substance by immunochemical means.

Diagnosis of Infectious Agents

Unmanageable infectious disease can be a major burden to global health, even causing societal and civil disruption. Therefore, a key strategy in lessening this burden is rapid, accurate diagnosis of infectious agents, which allows for appropriate intervention by treatment with drugs that slow the spread of infection to others. Today’s infectious disease diagnostics are still rooted in classical methods from the 20th century. Microscopic- and culture-based methodologies are considered the gold standard for diagnosing infectious agents, ahead of antigen detection and immunoserology. However, limitations for these classical methodologies include microscopy’s poor sensitivity, time-consuming culturing techniques, the biohazard risk of uncultured or fastidious organisms, and skilled worker and manual labor requirements.

Disease-related biomarkers are increasingly used as an alternative diagnostic tool to identify infected persons and populations. A biomarker refers to a specific substance related to the disease in question. The biomarker can be used to examine health status, organ function, and the condition and progression of a disease. Protein and polysaccharide biomarkers are often measured with immunoassays (defined in the following section). Immunoassay detection can accelerate diagnosis or supplement classical microbiology assays by detecting pathogen-associated proteins or host-produced antibodies to the pathogen in an infected person’s samples.

While many diagnostic immunoassays are used for infectious disease, some challenges with false-positive and false-negative results remain. These results may come from assay reagents cross-reacting with similar structures in the sample. Also, false results may be caused by nonspecific biomarker expression, or lack thereof. Therefore, these indirect methods tend to be undervalued compared with traditional diagnostic tools for infectious agents.

Immunoassay Technologies and Technical Considerations

Diagnostic immunoassays for infectious disease are based on antibody recognition of proteins or peptides specific for the pathogen of interest. The antibodies employed work as a unique analytical reagent equipped with distinct specificity and remarkable binding affinity to detect specific antigens or other antibodies. Immunoassays share four common elements: immobilized capture reagents (usually antibodies), binding of a protein or peptide analyte/antigen of interest, detection with an antibody or antiserum, and an indirect signal readout. Most immunoassays also contain separation steps to remove unbound reagents. The indirect signal readout is directly or inversely proportional to the analyte concentration in the sample. Thus, the signal relates to either the detection of a pathogen-specific antibody or the detection of a pathogenic antigen (or antigens) in the sample. Because of the high specificity and affinity of antibodies used, unpurified blood or other fluid samples can be applied in immunoassays without prior purification. Antibodies can specifically pick out the analyte of interest.

Signal measurement requires a detectable label to quantify the amount of captured antigen or antibody. The label is a molecule that reacts during the assay producing a quantifiable signal. A variety of labels are available, including enzymes, gold, silver, and selenium colloidal particles, radioactive elements, coenzymes, electroactive groups, fluorescent, and phosphorescent and chemiluminescent dyes. Signal interpretation calls for reference to a calibrator that resembles the attributes of the analyte in the sample. Calibrators with known analyte concentrations allow for the correlation of signal strength in the assay with the analyte concentration in the sample.

Independently of their technology, what determines the efficacy of any immunoassay is the capacity to form antigen-antibody complexes and the ability to detect such immunocomplexes. Highly specific antibody reagents with preferably high affinity are at the heart of successful immunoassays. High specificity allows for minimal cross-reactivity with unrelated proteins while high affinity can translate into high analytical sensitivity.

The first immunoassays were introduced in the 1960s as radioimmunoassays (RIAs) to measure insulin and thyroxine. RIAs typically use radioactive iodine; labels and the amount of radioactivity measured indicate the amount of analyte accounted for. Despite their advantage to detect very low quantities of analytes, the use of RIAs has decreased in clinical laboratories because of issues concerning isotope handling and the disposal of radioactive materials; RIAs were replaced by the enzyme immunoassay (EIA). In EIAs, enzyme labels such as horseradish peroxidase, a-galactosidase, and alkaline phosphatase are utilized instead of radioactive isotopes. During a substrate reaction, the enzyme mediates light emission, color change, or other measurable signals.

Now, enzyme-linked immunosorbent assay (ELISA) is the most common format, in which a solid-phase, bound, capture reagent is combined with an enzyme-labeled detection antibody. The immobilized capture reagent can be an antibody specific for the analyte or a target (such as a receptor), binding the analyte out of the sample. The bound antigen subsequently is recognized by the detection antibody.

In addition to ELISA, the main formats employed in the specific detection of infectious disease agents include lateral flow systems, flow cytometry, and fluorescent polarization. Lateral flow assays, also known as hand-held assays (HHA), are simple immunochromatographic assays, or strip tests. Best known as home pregnancy tests, lateral flow assays are often deployed as point-of-care testing for nose and throat swabs and for urine, fecal, or blood samples because of their simplicity and quick results (within minutes; traditional plate-based assays or immunoanalyzers take much longer).

Many immunoassays are now fully automated on immunoanalyzers. The method of choice (for example, lateral flow, RIA, immunoanalyzer, and flow cytometry) depends on the priorities of the application, such as simplicity versus speed. Molecular diagnostic methods (for example, polymerase chain reaction and microarray technology) have started to complement or even replace immunoassays for the detection of infectious diseases. One example of a molecular diagnostic method is testing for the human immunodeficiency virus (HIV).

Verification of Assay Performance

Assay sensitivity and specificity are essential to accurately determine the presence and concentration of the analyte. Clinical sensitivity and specificity refer to the assay’s false-negative and false-positive rates when applied to patient samples. Analytical sensitivity and specificity refer to the assay’s ability to recognize the analyte at low concentrations with minimal cross-reactivity.

While clinical assay performance is determined during initial validation, analytical performance is assessed both during validation and during daily quality controls. Quality assessment ensures the collected data are true and accurate. The main issues facing immunoassays include cross-reactivity (that is, nonspecificity) of the reagents used, interferences from sample components, and assay variability within each assay or across repeat measurements. Researchers are working on developing immunoassays with better clinical performance through the development of highly specific reagents, through increased speed with automation, and through novel technologies and greater analytical sensitivity.

Impact

The main benefit of any immunoassay is its speed, flexibility, cost efficiency, and relative simplicity, allowing it to be deployed in central laboratories and in the field. Field use is particularly critical to obtaining quick diagnosis of infectious disease and to administering appropriate treatment.

Immunoassay specificity plays a critical role in characterizing and distinguishing methicillin-resistant Staphylococcus aureus (MRSA) infections from themethicillin-sensitive (MSSA) strain. MRSA is a multi-drug-resistant aggressive bacterial infection that affects people in hospitals, prisons, and nursing homes. Another advantage of immunoassays is that they are safe to perform and can be adapted to several formats, which range from strip tests to test-tube assays, 96 well microplates, and high throughput analyzers, combined with complex microarray or biosensor systems. Automation has helped streamline workflow and increase productivity in central laboratory settings, while manual versions can be used in universities, colleges, and other low-volume settings.

Although there are several test systems to detect antigens and antibodies, detection methods to diagnose disease differ from those to verify immunity. Therefore, it is critical that the right criteria for the assay selected is clear and appropriate for each application. Thus, detection of antibodies can be utilized to verify and measure a person’s immunity to bacteria, viruses, and fungi.

Detection of immunity to rubella virus and varicella zoster virus, for example, are common screening tests performed in the health care industry because both viral infections can spread and be fatal in immunocompromised persons, infants, and unvaccinated adults. During prenatal screening, immunity is characterized in addition to past medical history by the TORCH panel, which uses ELISA methodology to test for antibodies against four TORCH organisms: Toxoplasma gondii (toxoplasmosis), rubella (German measles), cytomegalovirus (CMV), and herpes simplex virus (HSV). Pretransplant immunity screening for recipients and donors also makes use of immunoassays to determine CMV seroreactivity because transplant recipients are prone to develop CMV infections.

During the Covid-19 pandemic, scientists raced to develop immunoassay technologies to help detect the virus and slow down its spread. Technology quickly advanced to easy at-home antigen lateral flow immunoassay devices that detect the nucleocapsid protein antigen from the SARS-CoV-2 virus in as little as ten minutes. This technology has assisted with slowing the infection rate of the disease.

The impact of immunoassay technologies is not restricted to medical diagnosis; it is applied also in pharmaceutical development, veterinary medicine, forensic toxicology, military-based medicine, environmental monitoring, and food sciences. Pregnancy and ovulation home-tests have brought the technology to the general public. Point-of-care diagnostics devices are being improved and expanded in scope, potentially allowing immunoassays to play an even greater role in personalized medicine and infectious disease testing. Tabletop-sized automation and quantitative, sensitive strip tests will improve accurate and early diagnosis and turnaround time, will decrease costs, and will reduce the burdens of infectious diseases and related outbreaks.

Bibliography

Cavanaugh, Bonita Morrow. Nurse’s Manual of Laboratory and Diagnostic Tests. 4th ed. Philadelphia: F. A. Davis, 2003.

“Immuview COVID-19 Antigen Home Test.” Food and Drug Administration, 2022, www.fda.gov/media/164139/download. Accessed 14 Nov. 2024.

Pagana, Kathleen Deska, and Timothy J. Pagana. Mosby’s Diagnostic and Laboratory Test Reference. 9th ed. St. Louis, Mo.: Mosby/Elsevier, 2009.

Wild, David, ed. The Immunoassay Handbook. 3d ed. Boston: Elsevier, 2005.