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Immunoassay
Immunoassay
Immunoassay
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Immunoassay
Illustration of the basic components of an immunoassay, which includes an analyte (green), an antibody (black), and a detectable label (yellow)
MeSHD007118

An immunoassay (IA) is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoassay is often referred to as an "analyte" and is in many cases a protein, although it may be other kinds of molecules, of different sizes and types, as long as the proper antibodies that have the required properties for the assay are developed. Analytes in biological liquids such as serum or urine are frequently measured using immunoassays for medical and research purposes.[1]

Immunoassays come in many different formats and variations. Immunoassays may be run in multiple steps with reagents being added and washed away or separated at different points in the assay. Multi-step assays are often called separation immunoassays or heterogeneous immunoassays. Some immunoassays can be carried out simply by mixing the reagents and samples and making a physical measurement. Such assays are called homogeneous immunoassays, or less frequently non-separation immunoassays.

The use of a calibrator is often employed in immunoassays. Calibrators are solutions that are known to contain the analyte in question, and the concentration of that analyte is generally known. Comparison of an assay's response to a real sample against the assay's response produced by the calibrators makes it possible to interpret the signal strength in terms of the presence or concentration of analyte in the sample.

Principle

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Immunoassays rely on the ability of an antibody to recognize and bind a specific macromolecule in what might be a complex mixture of macromolecules. In immunology the particular macromolecule bound by an antibody is referred to as an antigen and the area on an antigen to which the antibody binds is called an epitope.

In some cases, an immunoassay may use an antigen to detect for the presence of antibodies, which recognize that antigen, in a solution. In other words, in some immunoassays, the analyte may be an antibody rather than an antigen.

In addition to the binding of an antibody to its antigen, the other key feature of all immunoassays is a means to produce a measurable signal in response to the binding. Most, though not all, immunoassays involve chemically linking antibodies or antigens with some kind of detectable label. A large number of labels exist in modern immunoassays, and they allow for detection through different means. Many labels are detectable because they either emit radiation, produce a color change in a solution, fluoresce under light, or can be induced to emit light.

History

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Rosalyn Sussman Yalow and Solomon Berson are credited with the development of the first immunoassays in the 1950s. Yalow accepted the Nobel Prize for her work in immunoassays in 1977, becoming the second American woman to have won the award.[2]

Immunoassays became considerably simpler to perform and more popular when techniques for chemically linked enzymes to antibodies were demonstrated in the late 1960s.[3]

In 1983, Professor Anthony Campbell[4] at Cardiff University replaced radioactive iodine used in immunoassay with an acridinium ester that makes its own light: chemiluminescence. This type of immunoassay is now used in around 100 million clinical tests every year worldwide, enabling clinicians to measure a wide range of proteins, pathogens and other molecules in blood samples.[5]

By 2012, the commercial immunoassay industry earned US$17,000,000,000 and was thought to have prospects of slow annual growth in the 2 to 3 percent range.[6]

Labels

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Immunoassays employ a variety of different labels to allow for detection of antibodies and antigens. Labels are typically chemically linked or conjugated to the desired antibody or antigen.

A sandwich ELISA run on a microtitre plate

Enzymes

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Possibly one of the most popular labels to use in immunoassays is enzymes. Immunoassays which employ enzymes are referred to as enzyme immunoassays (EIAs), of which enzyme-linked immunosorbent assays (ELISAs) and enzyme multiplied immunoassay technique (EMIT) are the most common types.

ELISA plate showing various cortisol levels

Enzymes used in ELISAs include horseradish peroxidase (HRP), alkaline phosphatase (AP) or glucose oxidase. These enzymes allow for detection often because they produce an observable color change in the presence of certain reagents. In some cases these enzymes are exposed to reagents which cause them to produce light or chemiluminescence. There are several types of ELISA: direct, indirect, sandwich, competitive.[7]

Radioactive isotopes

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Radioactive isotopes can be incorporated into immunoassay reagents to produce a radioimmunoassay (RIA). Radioactivity emitted by bound antibody-antigen complexes can be easily detected using conventional methods.

RIAs were some of the earliest immunoassays developed, but have fallen out of favor largely due to the difficulty and potential dangers presented by working with radioactivity.[8][9]

DNA reporters

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A newer approach to immunoassays involves combining real-time quantitative polymerase chain reaction (RT qPCR) and traditional immunoassay techniques. Called real-time immunoquantitative PCR (iqPCR) the label used in these assays is a DNA probe.[10][11]

Fluorogenic reporters

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Fluorogenic reporters like phycoerythrin are used in a number of modern immunoassays.[12] Protein microarrays are a type of immunoassay that often employ fluorogenic reporters.[13]

Electrochemiluminescent tags

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Some labels work via electrochemiluminescence (ECL), in which the label emits detectable light in response to electric current.[14][15]

Label-free immunoassays

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While some kind of label is generally employed in immunoassays, there are certain kinds of assays which do not rely on labels, but instead employ detection methods that do not require the modification or labeling the components of the assay. Surface plasmon resonance is an example of technique that can detect binding between an unlabeled antibody and antigens.[16] Another demonstrated labeless immunoassay involves measuring the change in resistance on an electrode as antigens bind to it.[17]

Classifications and formats

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In a competitive, homogeneous immunoassay unlabeled analyte displaces bound labelled analyte, which is then detected or measured.

Immunoassays can be run in a number of different formats. Generally, an immunoassay will fall into one of several categories depending on how it is run.[18]

Competitive, homogeneous immunoassays

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In a competitive, homogeneous immunoassay, unlabelled analyte in a sample competes with labeled analyte to bind an antibody. The amount of labelled, unbound analyte is then measured. In theory, the more analyte in the sample, the more labelled analyte gets displaced and then measured; hence, the amount of labelled, unbound analyte is proportional to the amount of analyte in the sample.

Homogeneous competitive assays: FPIA, EMIT, LOCI, KIMS and CEDIA.[19] See section text for details.
  • The fluorescence polarization immunoassay (FPIA) measures the fluorescence polarization signal after incubation, without separating bound and free labels. Free labeled analyte analog molecules are added to the sample, and their Brownian motion differs when bound to a large antibody (Ab) versus free in solution. The analyte competes for binding to the Ab, and if the labeled analyte binds to the Ab, a signal is produced. The signal intensity is inversely proportional to the analyte concentration.[19]
  • In the enzyme multiplied immunoassay technique (EMIT), free analyte analog molecules labeled with an enzyme (e.g., glucose-6-phosphate dehydrogenase enzyme) compete with the analyte being tested. The active enzyme reduces NAD (no signal) to NADH (which absorbs at 340 nm), so absorbance is monitored at 340 nm. When the labeled analyte binds to the Ab, the enzyme becomes inactive, and a signal is generated by the free label. The signal intensity is directly proportional to the analyte concentration.[19]
  • The luminescent oxygen channeling immunoassay (LOCI) generates singlet oxygen species in microbeads coupled to the analyte, and when the analyte binds to the respective Ab molecule, coupled to another kind of bead, the analyte reacts with singlet oxygen, generating chemiluminescence signals proportional to the concentration of the analyte-Ab complex.[19]
  • In the kinetic interaction of microparticle in solution (KIMS) and particle enhanced turbidimetric inhibition immunoassay (PETINIA), free antibodies bind to drug microparticle conjugates to form aggregates that absorb in the visible range in the absence of the analyte. In the presence of the analyte, the Ab binds to the free analyte, preventing microparticle aggregation and causing a reduction in absorbance. The signal is inversely proportional to the analyte concentration.[19]
  • The cloned enzyme donor immunoassay (CEDIA) involves genetically engineering an enzyme (e.g., beta-galactosidase) into two inactive fragments: a small enzyme donor (ED) conjugated with the drug analog, and a larger enzyme acceptor (EA). When the two fragments associate, the full enzyme converts a substrate into a cleaved colored product. If drug analyte molecules are present, they compete with the ED-labeled drug in solution for the limited Ab sites. Free ED-labeled drug analog will bind to EA, generating a colorimetric signal directly proportional to the amount of analyte.[19]
Two-site, noncompetitive immunoassays usually consist of an analyte "sandwiched" between two antibodies. ELISAs are often run in this format.

Competitive, heterogeneous immunoassays

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As in a competitive, homogeneous immunoassay, unlabelled analyte in a sample competes with labelled analyte to bind an antibody. In the heterogeneous assays, the labelled, unbound analyte is separated or washed away, and the remaining labelled, bound analyte is measured.

One-site, noncompetitive immunoassays

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Mixing a sample with labelled antibodies, the targeted analyte is bound by labelled antibodies. The unbound, labelled antibodies are washed away, and the bound, labelled antibodies are measured. The intensity of the signal is directly proportional to the amount of analyte in the sample.

Two-site, noncompetitive immunoassays

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The analyte in the unknown sample is bound to the antibody site, then the labelled antibody is bound to the analyte. The amount of labelled antibody on the site is then measured. It will be directly proportional to the concentration of the analyte because the labelled antibody will not bind if the analyte is not present in the unknown sample. This type of immunoassay is also known as a sandwich assay as the analyte is "sandwiched" between two antibodies.

Examples

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Clinical tests

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A wide range of medical tests are immunoassays, called immunodiagnostics in this context. Many home pregnancy tests are immunoassays, which detect the pregnancy marker human chorionic gonadotropin.[20] More specifically, they are qualitative tests that detect whether hCG is present, using a lateral flow setup.[21] The COVID-19 rapid antigen test is also a qualitative, lateral-flow test.[22]

Other clinical immunoassays are quantitative; they measure amounts. Immunoassays can measure levels of CK-MB to assess heart disease, insulin to assess hypoglycemia, prostate-specific antigen to detect prostate cancer, and some are also used for the detection and/or quantitative measurement of some pharmaceutical compounds (see Enzyme multiplied immunoassay technique for more details).[23]

Drug testing also starts with a quick qualitative immunoassay.[24]

Sports anti-doping analysis

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Immunoassays are used in sports anti-doping laboratories to test athletes' blood samples for prohibited recombinant human growth hormone (rhGH, rGH, hGH, GH).[25]

Research

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Photoacoustic Immunoassay

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The photoacoustic immunoassay measures low-frequency acoustic signals generated by metal nanoparticle tags. Illuminated by a modulated light at a plasmon resonance wavelength, the nanoparticles generate strong acoustic signal, which can be measured using a microphone.[26] The photoacoustic immunoassay can be applied to lateral flow tests, which use colloidal nanoparticles.[27]

See also

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Immunoassay

View on Grokipedia
from Grokipedia
An immunoassay is a biochemical technique that exploits the highly specific interaction between an and its corresponding to detect and quantify target molecules, known as , in complex biological or environmental samples. This method relies on the principle of - binding, where the antibody serves as a selective to capture the analyte, enabling sensitive measurement even at low concentrations. Immunoassays are widely valued for their high specificity, sensitivity, and versatility across diverse fields, including clinical diagnostics, pharmaceutical analysis, and . The foundational principle of immunoassays involves either competitive or non-competitive formats to generate a measurable signal proportional to the concentration. In competitive assays, the in the sample competes with a labeled (such as an antigen analog) for limited binding sites, resulting in an inverse relationship between signal intensity and amount. Non-competitive formats, like sandwich assays, use two : one to capture the and another labeled detection to form a "sandwich" complex, producing a signal directly proportional to the level. Detection methods vary, including radioactive isotopes in radioimmunoassays (), enzymes in enzyme-linked immunosorbent assays (), , , or electrochemical signals, each chosen based on the required sensitivity and safety profile. Common types of immunoassays include , developed in the late 1950s by Rosalyn Yalow and Solomon Berson for measuring insulin levels, which earned Yalow the 1977 Nobel Prize in Physiology or Medicine. ELISA, introduced in the 1970s as a safer alternative to RIA, encompasses subtypes such as direct, indirect, sandwich, and competitive formats, widely used for detecting proteins, hormones, and pathogens. Other variants include immunoassays (FIA) for rapid and multiplex assays that simultaneously measure multiple analytes. Immunoassays find extensive applications in healthcare for diagnosing diseases through biomarker detection, such as cardiac troponins for or HIV antibodies for screening. In pharmaceuticals, they support by quantifying therapeutic levels, assessing , and evaluating . Environmental uses involve detecting pollutants like pesticides or in water and soil samples. Despite advantages like high throughput and minimal , limitations such as potential and matrix effects necessitate validation and controls for accuracy.

Fundamentals

Definition and Principle

An immunoassay is a biochemical technique that utilizes the specific binding affinity of antibodies to detect and quantify analytes, such as antigens or small-molecule haptens, in complex samples including , , or environmental media. Antibodies, also known as immunoglobulins, are Y-shaped glycoproteins produced by plasma cells as part of the adaptive ; they consist of two heavy chains and two light chains linked by disulfide bonds, with the variable regions at the tips of the Y-arms forming antigen-binding sites (paratopes) that recognize unique structural features (epitopes) on target molecules. This high-affinity interaction, often with dissociation constants in the nanomolar to picomolar range, enables precise identification of analytes even at trace levels. The fundamental principle of an immunoassay exploits the reversible, non-covalent binding between an (Ab) and its corresponding (Ag), forming a stable Ab-Ag complex that can be detected through associated signals or physical changes. In a typical setup, the sample containing the is incubated with antibodies immobilized on a solid surface or in solution, allowing the Ab-Ag complex to form; unbound components are then separated, and the bound complex is measured to infer analyte concentration. This process mimics natural immune recognition but is engineered for analytical purposes, providing a foundation for sensitive quantification without requiring prior purification of the sample. The technique was first demonstrated in the for insulin detection, highlighting its potential for measuring endogenous substances. Key performance characteristics of immunoassays include sensitivity, which refers to the assay's ability to detect low concentrations (often down to picograms per milliliter), specificity, which measures the selective recognition of the target over similar molecules, the (LOD), defined as the lowest concentration reliably distinguishable from (typically calculated as three times the standard deviation of the blank), and , which quantifies unintended binding to non-target substances that can compromise accuracy. These attributes stem from the antibody's structural versatility and immune system's evolutionary optimization for discrimination, allowing immunoassays to achieve trace-level in diverse matrices while minimizing false positives.

Historical Development

The development of immunoassay techniques began in the mid-20th century with the pioneering work of Rosalyn Yalow and Solomon Berson, who introduced in 1959 as a method to measure endogenous plasma insulin levels in humans. This innovation leveraged the specific binding between antigens and antibodies, combined with radioactive labeling, to achieve unprecedented sensitivity for quantifying peptide hormones at picomolar concentrations. Yalow's contributions were recognized with the in Physiology or Medicine in 1977, highlighting RIA's transformative role in and clinical diagnostics. A major milestone came in 1971 with the introduction of the by Eva Engvall and Peter Perlmann, independently developed by Anton Schuurs and Bauke van Weemen, providing a safer alternative to RIA by replacing radioactive isotopes with labels that produce a colorimetric signal upon substrate reaction. This technique enabled broader adoption due to its simplicity and lack of hazards. The first commercial ELISA kits emerged in 1976, facilitating routine laboratory use for detecting analytes like human choriogonadotropin. By the , concerns over and safety prompted a widespread shift to non-isotopic labels, including enzymes, fluorophores, and chemiluminescent markers, which improved accessibility and reduced regulatory burdens in clinical settings. A pivotal event was the 1975 invention of by Georges Köhler and , enabling the production of monoclonal antibodies with uniform specificity and affinity, which revolutionized immunoassay reproducibility and specificity for diverse antigens. This earned them the 1984 and facilitated the standardization of antibody reagents in assays. In the , the rise of automated immunoassay systems marked a shift toward high-throughput clinical applications, with fully automated platforms from multiple manufacturers becoming available by 1994, allowing random-access testing for hormones, tumor markers, and infectious agents. Organizations like the (WHO) began establishing international standards for key analytes, such as peptide hormones and antibodies, to ensure traceability and harmonization across global laboratories by the late . These efforts culminated in standardized reference materials that supported consistent clinical interpretations and inter-laboratory comparability.

Detection Methods

Labeled Detection Systems

Labeled detection systems in immunoassays rely on attaching detectable tags to antibodies or antigens to generate measurable signals proportional to the concentration. These labels convert the specific binding event into a quantifiable output, such as light, color, or , enabling high sensitivity through signal amplification or direct emission. Common strategies include enzymatic, radioactive, fluorescent, luminescent, and other specialized labels, each offering distinct mechanisms for detection while addressing challenges like and assay throughput. Enzymatic labels, such as (HRP) and (AP), function by catalyzing the conversion of substrates into detectable products, providing substantial signal amplification due to the catalytic turnover where a single can produce thousands of product molecules per minute. For instance, HRP oxidizes substrates like tetramethylbenzidine (TMB) to yield a colorimetric signal at 450 nm, while AP hydrolyzes p-nitrophenyl phosphate (pNPP) for at 405 nm; these reactions can also be adapted for chemiluminescent or fluorescent outputs using specialized substrates. This amplification enhances sensitivity, achieving detection limits in the pg/mL range for analytes like (0.2 ng/mL with HRP), making enzymatic labels a cornerstone of (ELISA) since their development in the . Radioactive labels, historically pivotal in radioimmunoassays (), utilize isotopes like (^125I) or (^3H) that emit beta or gamma detectable by scintillation counters. ^125I, with a of approximately 60 days, is incorporated via iodination of residues on proteins, emitting low-energy gamma rays for precise quantification, while ^3H, with a longer of 12.3 years, relies on beta emission but requires more complex handling. These labels enabled early RIA sensitivities down to 24 pg/mL for analytes like , but their use has declined since the due to hazards, waste disposal issues, and regulatory constraints, favoring non-isotopic alternatives. Fluorescent labels, including fluorophores like fluorescein isothiocyanate (FITC) and semiconductor quantum dots, produce light emission upon excitation, allowing direct visualization or quantification via fluorescence spectroscopy. FITC, excited at 494 nm and emitting at 518 nm, conjugates easily to proteins via its isothiocyanate group, though it suffers from photobleaching; quantum dots, nanoscale semiconductor particles, offer superior photostability, broad excitation spectra, and narrow emission peaks for multiplexing, achieving detection limits around 1-10 ng/mL in fluorescence polarization immunoassays. Luminescent labels extend this capability: chemiluminescent acridinium esters generate light through oxidation without external excitation, yielding signals up to 100 times stronger than luminol-based systems with limits near 5 fmol/L, while electrochemiluminescent ruthenium complexes, such as [Ru(bpy)_3]^{2+}, produce light via electrochemical stimulation for high-sensitivity assays down to pg/mL levels with low background noise. These non-radioactive options emerged prominently in the 1980s and 1990s, improving safety and enabling time-resolved fluorescence to minimize autofluorescence. Other labels include biotin-streptavidin systems for indirect signal enhancement and DNA reporters for nucleic acid amplification. Biotin, a small vitamin, binds streptavidin with extremely high affinity (K_d ≈ 10^{-15} M), allowing multiple enzyme or fluorophore conjugates to attach to a single biotinylated antibody, amplifying signals by several orders of magnitude to reach fg/mL sensitivities in enhanced ELISA formats. DNA reporters, such as oligonucleotide-tagged antibodies, enable post-binding PCR amplification of the signal, providing exponential gains for ultra-low analyte detection. These approaches, popularized in the 1990s, add versatility without direct labeling of the immunoreagent. Overall, labeled detection systems offer high sensitivity, often detecting analytes at pg/mL concentrations, with enzymatic and luminescent methods providing superior signal-to-noise ratios (up to 10^3-10^6) compared to direct fluorescent labels due to amplification. Their advantages include adaptability to various formats and established protocols, though selection depends on factors like speed and equipment availability.

Label-Free Detection Systems

Label-free detection systems in immunoassays enable the direct monitoring of antibody-antigen (Ab-Ag) binding events by exploiting changes in physical or optical properties at the sensor surface, without the need for exogenous labels such as fluorescent or enzymatic tags. These methods provide real-time insights into biomolecular interactions, contrasting with labeled approaches that rely on signal amplification from reporters, which can sometimes introduce artifacts or require additional washing steps. Surface plasmon resonance (SPR) is a prominent optical technique for label-free immunoassays, where polarized light excites surface plasmons on a thin film, generating an evanescent wave that penetrates approximately 100-200 nm into the adjacent medium. Upon Ab-Ag binding near the surface, the increases, shifting the resonance angle and producing a measurable signal in real time. The first demonstration of SPR for biosensing, including an immunoassay concept, occurred in 1983, marking the inception of label-free SPR-based detection. Commercial instruments like Biacore, introduced in the early 1990s, have since facilitated widespread use by integrating for continuous flow and sensorgram analysis.01721-3) Quartz crystal microbalance (QCM) operates on piezoelectric principles, utilizing a quartz crystal oscillator whose resonant frequency decreases proportionally to the mass adsorbed on its surface during Ab-Ag interactions. This frequency shift, governed by the Sauerbrey relation for thin films, allows detection of mass changes as low as 1-10 ng/cm², enabling label-free quantification of binding events. QCM is particularly suited for immunoassays in viscous media or with larger analytes, as it measures total mass loading including hydration effects. Other label-free methods include , such as (BLI), which detects optical thickness changes from biomolecular layers using white light interference patterns for real-time kinetic analysis. Impedance spectroscopy monitors alterations at surfaces due to binding-induced changes in charge transfer resistance, offering portability for point-of-care applications. Optical waveguide techniques, like waveguide evanescent field sensors, confine light within a to probe variations near the surface, achieving high sensitivity for integrated immunosensors. These systems excel in providing real-time kinetic parameters, such as the association rate constant (k_on) and dissociation rate constant (k_off), which yield the equilibrium dissociation constant (K_D = k_off / k_on) for affinity measurements, often in the nanomolar to picomolar range for high-affinity interactions. Advantages include shortened times—typically minutes versus hours for labeled methods—and the avoidance of label-induced perturbations, facilitating studies of native binding dynamics. However, limitations arise from potentially lower sensitivity compared to labeled fluorescent assays, with detection limits around 1-100 ng/mL for SPR and QCM versus sub-picomolar for fluorescence-based systems, necessitating optimized surface chemistries to mitigate nonspecific binding.

Assay Classifications

Competitive Assays

Competitive immunoassays operate on a principle where the in the sample competes with a labeled analog for a limited number of binding sites on specific . In this format, the amount of labeled bound to the antibody decreases as the concentration of unlabeled increases, resulting in a signal that is inversely proportional to the concentration. This competition-based mechanism is particularly suited for detecting small molecules, or haptens, which cannot be captured by two antibodies simultaneously due to their size. Homogeneous competitive assays eliminate the need for a separation step, allowing direct measurement of the reaction mixture. A prominent example is fluorescence polarization immunoassay (FPIA), in which a fluorescently labeled (tracer) competes with the sample for binding. When the tracer binds to the , its slows due to the larger complex size, increasing the polarization of emitted upon excitation with plane-polarized light. Conversely, higher concentrations leave more free tracer, which rotates rapidly and exhibits lower polarization. Another homogeneous variant is the enzyme multiplied immunoassay technique (EMIT), where the competes with an enzyme-labeled for binding; unbound enzyme-labeled remains active, producing a measurable colorimetric signal proportional to presence. Heterogeneous competitive assays incorporate a separation step, such as washing, to distinguish bound from free components. The classical (), developed by Yalow and Berson in 1960, exemplifies this approach: a radiolabeled competes with the sample for sites, after which bound and free fractions are separated (e.g., via ), and in the bound fraction is quantified, inversely reflecting analyte levels. The binding dynamics follow the equilibrium association constant KaK_a, defined as Ka=[Ag−Ab][Ag][Ab],K_a = \frac{[Ag-Ab]}{[Ag][Ab]}, where [Ag−Ab][Ag-Ab] is the concentration of the antigen-antibody complex, and [Ag][Ag] and [Ab][Ab] are the free concentrations of antigen and antibody, respectively; this equation underpins the affinity governing competition. Quantification relies on calibration curves, often fitted using logit-log transformations for linearization or four-parameter logistic models to capture the sigmoidal response of signal versus log analyte concentration. These assays excel in detecting haptens such as steroids (e.g., ) and drugs (e.g., or THC in urine screens), achieving sensitivities typically in the range of 10−910^{-9} to 10−1210^{-12} M due to the high affinity of antibodies for small-molecule targets. For instance, RIA enables insulin quantification at picomolar levels, while FPIA detects therapeutic drugs like with nanomolar precision, making competitive formats indispensable for pharmacokinetic monitoring and .

Noncompetitive Assays

Noncompetitive immunoassays, also referred to as immunometric assays, enable direct quantification of analytes through binding without a step, resulting in a signal that increases proportionally with concentration. In the core mechanism, a capture is immobilized on a solid support to bind the target , followed by the addition of a detection that recognizes a distinct, non-overlapping on the and is typically conjugated to a label such as an , , or radioisotope. The resulting "sandwich" complex generates a measurable signal—via enzymatic reaction, , or —that correlates linearly or sigmoidally with the captured amount, offering improved sensitivity over competitive formats for suitable targets. One-site variants of noncompetitive assays utilize a single antibody species, where the sample is first mixed with an excess of labeled to form an immune complex, which is then captured on an immobilized anti-immunoglobulin or secondary binder to separate bound from free label; this approach is particularly applicable to soluble antigens and avoids the need for dual epitope recognition. In contrast, two-site or sandwich variants employ two distinct antibodies with high specificity: the capture antibody secures the analyte, and the labeled detection binds the second , enhancing overall assay precision and reducing . These two-site formats are exemplified in enzyme-linked immunosorbent assays (ELISA) for detecting macromolecules like cytokines, where the dual-antibody strategy achieves detection limits in the picomolar range for targets such as interleukin-6. Key advantages of noncompetitive assays include the absence of competition, which yields higher specificity for macromolecules exceeding 5 that possess multiple distinct epitopes, enabling reliable detection without the inverse signal-concentration relationship seen in competitive methods. In multiplex configurations, where multiple analytes are assayed simultaneously, signal amplification strategies—such as tyramide deposition or labels—can further boost sensitivity by orders of magnitude, facilitating high-throughput analysis of complex samples like serum. The dose-response curve in noncompetitive immunoassays is commonly fitted using the four-parameter logistic (4PL) model to interpolate analyte concentrations from observed signals: y=bottom+top−bottom1+10(log⁡EC50−x)⋅HillSlopey = \text{bottom} + \frac{\text{top} - \text{bottom}}{1 + 10^{(\log\text{EC}_{50} - x) \cdot \text{HillSlope}}} Here, yy represents the measured response, xx is the logarithm of the analyte concentration, "bottom" and "top" denote the lower and upper asymptotes of the curve, EC50\text{EC}_{50} is the concentration producing half-maximal response, and HillSlope indicates the curve's steepness; this model ensures accurate quantification across a wide dynamic range without deriving underlying kinetics.

Applications

Clinical Diagnostics

Immunoassays play a pivotal role in clinical diagnostics by enabling the sensitive and specific detection of biomolecules such as hormones, proteins, and antibodies in samples, facilitating diagnosis, monitoring, and therapeutic decision-making. These assays are integral to routine healthcare, supporting applications from prenatal screening to management. In healthcare settings, immunoassays underpin a significant portion of testing, with results influencing over 70% of clinical decisions globally. Common clinical tests include hormone assays like (TSH) for thyroid function evaluation and (hCG) for pregnancy confirmation, both utilizing (ELISA) or similar formats. such as or T are detected via high-sensitivity immunoassays to diagnose , providing results within minutes to hours. For infectious diseases, assays target antigens like HIV p24 for early detection or antibodies against for serology, often employing chemiluminescent or fluorescent detection. Point-of-care (POC) immunoassays, such as lateral flow assays, deliver rapid results using minimal sample volumes (e.g., a throat swab for group A in rapid strep tests), enabling bedside without specialized equipment. In high-throughput laboratories, automated chemiluminescent platforms process hundreds of samples per hour for tests like or hormone panels, enhancing efficiency in hospital settings. These systems integrate with electronic records (EHRs), supporting by linking results to patient histories for timely interventions. FDA-approved multiplex panels, such as the OVA1 test for risk assessment, simultaneously measure biomarkers including CA-125 alongside apolipoprotein A1, , , and to aid surgical decision-making. (PSA) immunoassays, while often uniplexed, contribute to multiplex strategies in protocols. Despite these advances, challenges persist, including interference from heterophilic antibodies that can cause false positives in sandwich formats by bridging capture and detection antibodies. across kits remains an issue due to lot-to-lot variability, potentially affecting result comparability and requiring rigorous validation.

Non-Clinical Uses

Immunoassays find extensive application in non-clinical settings, where their high specificity and sensitivity enable rapid detection of target analytes in diverse matrices such as pharmaceuticals, environmental samples, and forensic specimens. These techniques support , , and safety monitoring across industries, often serving as screening tools before confirmatory analyses. In pharmaceutical analysis, immunoassays are employed for drug purity testing and pharmacokinetic studies, particularly in assessing levels in biologics. For instance, enzyme-linked immunosorbent assays (ELISAs) detect anti-drug antibodies to evaluate during non-clinical safety evaluations, ensuring product quality and stability. These assays facilitate the quantification of therapeutic concentrations in formulations, aiding in the validation of processes as per FDA guidelines. Environmental and food safety monitoring relies on immunoassays to detect pesticides, biotoxins, and pathogens. ELISAs, for example, provide a sensitive screening method for this in water samples, with detection limits as low as 0.1 ppb, allowing for cost-effective compliance with environmental regulations. In , immunoassays target mycotoxins such as aflatoxins and ochratoxins in grains and nuts; lateral flow assays enable on-site detection of these biotoxins at levels below regulatory thresholds, preventing contamination in the . Additionally, these methods identify pathogens like E. coli in water, supporting rapid in agricultural runoff. Forensic and anti-doping applications utilize immunoassays for screening steroids, (EPO), and other prohibited substances in urine. (WADA)-compliant assays detect recombinant EPO through followed by immunoblotting, a form of immunoassay that identifies doping manipulations with high specificity. Toxicology screening employs competitive ELISAs for THC metabolites and opioids, offering initial qualitative results with cutoffs such as 50 ng/mL for THC-COOH, though confirmatory is required for positives. These tools are integral to forensic investigations and athlete compliance testing. Other non-clinical uses include veterinary diagnostics and agricultural residue monitoring. In , ELISAs measure rabies virus-neutralizing antibodies in animal serum to confirm vaccination efficacy, with titers above 0.5 IU/mL indicating protection as per standards. Immunoassays also monitor pesticide residues like in crops, ensuring adherence to maximum residue limits set by regulatory bodies. Portable immunoassay kits, such as strip tests for aflatoxins, facilitate field use in , delivering results in 5-10 minutes without equipment and aligning with EPA-approved methods in the 4000 series for pollutant screening.

Emerging Developments

Advanced Detection Techniques

Advanced detection techniques in immunoassays have evolved to surpass the limitations of conventional labeled systems, such as or , by leveraging novel physical and chemical principles for enhanced sensitivity, specificity, and capabilities. These methods enable detection at ultralow concentrations, often in complex biological matrices, and facilitate applications in challenging environments like deep tissues. Photoacoustic immunoassay employs light-absorbing labels, typically plasmonic nanoparticles, that convert absorbed laser energy into waves, generating detectable acoustic signals for quantification. This approach was first demonstrated in the mid-2010s, building on earlier photoacoustic principles from the , and offers advantages over traditional optical methods by minimizing light scattering in turbid media. The technique's strength lies in its ability to provide high-contrast imaging in deep tissues, up to several centimeters, making it particularly suitable for detection of cancer s such as or tumor-associated antigens. Digital immunoassays, exemplified by the Simoa technology developed by Quanterix, achieve single-molecule counting through bead-based partitioning, where paramagnetic beads coated with capture antibodies isolate target analytes into femtoliter-volume wells for enzymatic amplification and digital readout. This method delivers femtogram per milliliter (fg/mL) sensitivity, orders of magnitude lower than conventional ELISAs, enabling early detection of low-abundance proteins in blood or . The Simoa platform was commercially launched in 2014, with Breakthrough Device designations, such as for light chain assays in (2022) and phospho-tau assays in (2021), supporting its use in monitoring neurodegeneration. Surface-enhanced Raman scattering (SERS) immunoassay utilizes tags functionalized with Raman reporter molecules, providing unique spectral fingerprints for detection with minimal background interference. These tags, often or silver nanostructures, amplify Raman signals by factors exceeding 10^6, allowing label-free-like specificity while enabling multiplexed analysis of over 10 analytes simultaneously through distinct spectral peaks. This capability has been demonstrated in lateral flow assays for simultaneous quantification of multiple mycotoxins or cytokines, enhancing throughput in clinical and . Mass spectrometric immunoassay (MSIA) combines affinity capture with , where antibodies immobilized on surfaces selectively bind targets from complex samples, followed by direct and MS analysis for precise molecular weight determination and quantification. Unlike labeled methods, MSIA eliminates the need for tags, reducing variability and enabling detection of proteoforms with high accuracy. It effectively handles isobaric interferences—overlapping masses from different species—through high-resolution MS fragmentation patterns, making it ideal for distinguishing post-translational modifications in biomarkers like apolipoproteins.

Integration with Other Technologies

Immunoassays have been increasingly integrated with and technologies to enable miniaturized, automated processes that enhance portability and efficiency in point-of-care settings. These systems utilize microscale channels, typically on the order of micrometers, to automate fluid handling, mixing, and washing steps, reducing sample volumes to nanoliters and assay times to minutes compared to traditional formats. For instance, paper-based microfluidic analytical devices (μPADs) incorporate hydrophilic channels on hydrophobic paper substrates to facilitate capillary-driven flow, enabling low-cost, disposable immunoassays for detecting biomarkers like infectious disease antigens at the point of care without requiring external pumps. Recent advancements, such as 3D-printed μPADs, further support automated immunoassays by integrating stacking layers for sequential reactions, achieving detection limits in the picomolar range for proteins. Nanotechnology has augmented immunoassay sensitivity and specificity through nanomaterials that facilitate separation and signal amplification. Magnetic beads, often coated with antibodies, enable rapid immunocapture and magnetic separation of target analytes from complex matrices, minimizing non-specific binding and simplifying downstream processing in heterogeneous assays. Quantum dots provide multicolor fluorescence for multiplexed detection due to their size-tunable emission spectra, while gold nanoparticles enhance signals via localized surface plasmon resonance, amplifying colorimetric or fluorescent readouts by factors of up to 100-fold in lateral flow formats. Between 2023 and 2025, surface-enhanced Raman scattering (SERS)-active nanosensors, incorporating plasmonic nanostructures like gold-silver core-shell particles, have advanced immunoassay performance by enabling single-molecule detection of biomarkers through vibrational fingerprinting, with applications in extracellular vesicle analysis. The incorporation of artificial intelligence (AI) and automation has optimized immunoassay workflows, particularly for data-intensive applications. algorithms excel in for multiplexed assays, employing to deconvolute overlapping spectra or classify profiles from imaging data, improving accuracy in heterogeneous samples by reducing false positives by up to 20%. In pharmaceutical screening, high-throughput robotic systems automate liquid handling and plate-based immunoassays, enabling the evaluation of thousands of compounds daily for drug-target interactions, with integrated AI for real-time in kinetic binding data. Multiplexing capabilities have expanded through hybrid platforms that simultaneously quantify dozens to hundreds of analytes. The Luminex xMAP technology employs polystyrene beads internally dyed with distinct ratios of red and infrared fluorophores, allowing suspension-based immunoassays to detect over 100 analytes per sample via , offering a 10- to 100-fold throughput advantage over single-plex . CRISPR-based reporters further enable ultra-sensitive multiplexing by coupling Cas12a or Cas13 nucleases to immunoassay outputs; upon target recognition, collateral cleavage of reporter molecules generates amplified fluorescent or colorimetric signals, achieving attomolar detection limits for antibodies or antigens in a single reaction. These integrations have driven market expansion, with the global immunoassay sector projected to reach $35.5 billion in 2025, fueled by and microfluidic advancements that reduce operational costs and enhance .

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