Introduction

Enzyme immunoassays (EIAs) use the catalytic properties of enzymes to detect and quantify immunologic reactions. Enzyme-linked immunosorbent assay (ELISA) is a heterogeneous EIA technique commonly used in clinical analyses.[1] In ELISA, one of the reaction components is either nonspecifically adsorbed or covalently bound to the surface of a solid phase, such as a microtiter well, magnetic particle, or plastic bead. This attachment facilitates efficient separation of bound and free-labeled reactants.[2]

In the most common ELISA approach, an aliquot of the sample or calibrator containing the antigen (Ag) to be quantified is added to a solid-phase antibody (Ab) and allowed to bind. After washing, an enzyme-labeled antibody is introduced, forming a “sandwich complex” of solid-phase Ab–Ag–Ab–enzyme. Unbound antibody is removed by washing, and the enzyme substrate is added. The amount of product generated is proportional to the antigen concentration in the sample.[1]

Unlike traditional ELISA, the competitive ELISA method is used to measure low-molecular-weight antigens or haptens. In this format, the antigen in the sample competes with a labeled antigen for binding to a limited amount of solid-phase antibody. The signal produced is inversely proportional to the concentration of antigen in the sample.[3]

Specific antibodies in a sample can also be quantified using an ELISA procedure in which the antigen, rather than the antibody, is bound to a solid phase. An enzyme-labeled secondary antibody specific for the analyte antibody is then added.[4] This format, known as an indirect ELISA, is commonly used to detect antiviral antibodies. ELISA assays have been widely applied to detect antibodies to viruses and autoantigens in serum or whole blood.[5][6]

Recent advancements have led to the development of ultrasensitive ELISA techniques capable of detecting biomarkers at femtomolar concentrations via enzymatic amplification. In addition, enzyme conjugates coupled to substrates that generate visible reaction products have been used to develop ELISA-type assays that can be visually interpreted. These assays are very useful for screening, point-of-care testing, and home testing.[7][8]

Etiology and Epidemiology

The direct ELISA technique was developed simultaneously by 2 independent research teams: Engvall and Perlman, and Van Weemen and Schuurs. ELISA evolved from the radioimmunoassay by replacing radioactive iodine-125 labels with enzyme-conjugated antigens or antibodies. The method was initially used to quantify immunoglobulin G levels in rabbit serum. Within the same year, scientists quantified human chorionic gonadotropin (hCG) in urine using horseradish peroxidase. Since then, the ELISA method has been used across a wide range of applications and has become a routine laboratory research and diagnostic method worldwide.[1]

Early ELISA methodologies used chromogenic reporter molecules and substrates to generate an observable color change indicating the presence of the antigen. Further advancement in the ELISA technique led to the development of fluorogenic, quantitative PCR-based, and electrochemiluminescent reporters to generate signals.[9] However, some of these techniques do not rely on enzyme-linked substrates but instead use nonenzymatic reporters that still utilize the underlying principles of ELISA.[10]

A more recent development, reported in 2012, is an ultrasensitive enzyme-based ELISA that manipulates nanoparticles as chromogenic reporters. This technique can generate a color signal visible to the naked eye, with blue indicating a positive result and red indicating a negative result. However, this method is qualitative and can only determine whether an analyte is present or absent, not its concentration.[11]

Specimen Requirements and Procedure

ELISAs are performed in polystyrene plates, typically 96-well plates, that are coated to bind proteins strongly.[2] Depending on the ELISA type, testing may require a primary and/or secondary detection antibody, an analyte or antigen, a coating antibody or antigen, buffer, wash solutions, and a substrate or chromogen.[4] The primary detection antibody specifically binds the protein of interest, whereas the secondary detection antibody is enzyme-conjugated and binds to a primary antibody that is not enzyme-conjugated.[7]

An ELISA immunoassay consists of 4 main steps:

• Coat the plate with an antigen or an antibody
• Block nonspecific binding sites with bovine serum albumin (BSA)
• Detect bound antigen or antibody using an enzyme-linked system
• Measure the signal after substrate addition

Detection is performed by adding a substrate that generates a color. Although a variety of substrates are available for ELISA detection, the most commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase.[4] HRP uses hydrogen peroxide as a substrate, producing a blue color change, whereas alkaline phosphatase typically uses p-nitrophenyl phosphate as its substrate and yields a yellow nitrophenol product after incubation at room temperature for approximately 15 to 30 minutes.[2]

Between each of the 4 primary steps, the plate undergoes a wash step using a buffer solution, such as phosphate-buffered saline (PBS) with a nonionic detergent, to remove unbound material. The wells are typically washed 2 or more times during each wash cycle, depending on the specific protocol.[7]

In a usual ELISA protocol, serial dilutions of known concentrations are added to the wells of the plate. After the results are measured, a standard curve is generated from the serial dilutions data, plotting concentration on a logarithmic x-axis and absorbance on a linear y-axis.[12]

The 4 major types of ELISA are mentioned below.

• Direct ELISA: This method uses an antigen-coated plate to detect antibodies.
• Indirect ELISA: This method uses an antigen-coated plate to detect antigens or antibodies.
• Sandwich ELISA: This method uses an antibody-coated plate to detect antigens.
• Competitive ELISA: This method is used to detect antibodies.

Direct ELISA

Both direct and indirect ELISAs begin by coating antigens onto the ELISA plates.[13][14] The initial binding step involves adding antigens to the plates, which are incubated for 1 hour at 37 °C or overnight at 4 °C. Once the incubation step is complete, the plates are washed to remove any unbound antibodies, and the remaining binding sites on the ELISA plate are blocked using agents such as bovine serum albumin, ovalbumin, aprotinin, or other animal proteins.[2] This second step is crucial because it prevents the binding of any nonspecific antibodies to the plate and minimizes false-positive results. After adding the buffer, the plates are washed again, and a selected enzyme-conjugated primary detection antibody is added. The plates are then incubated for an additional 1 hour.[15]

In a direct ELISA, the primary detection antibody binds directly to the protein of interest.[16] The plate is then washed to remove any unbound antibodies. An enzyme, such as alkaline phosphatase or HRP, is added to the plate, which results in a color change. This color change in the sample occurs either through the hydrolysis of phosphate groups on the substrate by alkaline phosphatase or through the oxidation of substrates by HRP.[15] 

The advantages of using direct ELISA include the elimination of secondary antibody cross-reactivity and a faster assay workflow due to fewer steps compared with indirect ELISA. However, disadvantages include lower sensitivity than other ELISA formats and higher reagent costs.[4] Recent developments have reported novel enzyme conjugates, such as engineered glucose oxidase variants, that enhance signal stability and enable multiplexed direct ELISA workflows, thereby improving throughput.[17]

Indirect ELISA

The steps of indirect ELISA are similar to those of direct ELISA, with the addition of an extra wash step and differences in the antibodies added after the buffer is removed.[18] Indirect ELISA requires 2 antibodies: a primary detection antibody that binds to the protein of interest and a secondary enzyme-linked antibody that is complementary to the primary antibody.[12] The primary antibody is added first, followed by a wash step. The enzyme-conjugated secondary antibody is then added and incubated. Afterward, the steps are the same as the direct ELISA, which includes a wash step, the addition of substrate, and the detection of a color change.[19]

The indirect ELISA has a higher sensitivity than the direct ELISA.[19] Indirect ELISA is also less expensive and more flexible because a wide range of primary antibodies can be used. The only major disadvantage of this ELISA format is the risk of cross-reactivity involving the secondary detection antibodies.[4] Recent studies have highlighted the use of recombinant hypervalent secondary antibodies that bind multiple enzyme reporters per antigen-antibody complex, significantly amplifying the signal and extending the sensitivity of indirect ELISA into the low attomolar range for certain viral targets. [6]

Sandwich ELISA

Unlike direct and indirect ELISA, the sandwich ELISA technique begins with a capture antibody coated onto the wells of the plate.[12] This method is called a “sandwich” because the antigens are sandwiched between 2 layers of antibodies—the capture antibody and the detection antibody.[2] After the capture antibody is added to the plates, the plates are then covered and incubated overnight at 4 °C. Once the coating step is complete, the plates are washed with PBS and then buffered or blocked with BSA. The blocking step is carried out at room temperature for at least 1 to 2 hours. Finally, the plates are washed with PBS again before the antigen is added.[20]

The antigen of interest is added to the plates to bind to the capture antibody and incubated for 90 minutes at 37 °C. The plates are then washed, and the primary detection antibody is added, and the plates are incubated for another 1 to 2 hours at room temperature, followed by a buffer wash. Next, the secondary enzyme-conjugated antibody is added and incubated for an additional 1 to 2 hours. The plate is rewashed, and the substrate is added to produce a color change.[2]

The sandwich ELISA method has the highest sensitivity among ELISA formats.[20] However, its major disadvantages include the time and expense required, as well as the need for matched antibody pairs (divalent or multivalent antigens) and secondary antibodies.[4] To address these limitations, a universal platform using high-affinity DNA oligonucleotide-tagged antibodies has been developed. This approach allows compatible detection and capture antibodies to be used interchangeably, thereby drastically reducing development time and cost for novel assays.[21]

Competitive ELISA

The competitive ELISA technique is used to detect the presence of antibodies specific to antigens in a test serum.[22] This method utilizes 2 specific antibodies: an enzyme-conjugated antibody and the antibody present in the test serum (if the serum is positive). When both antibodies are added to the wells, they compete for binding to the antigen. A color change indicates a negative test result, as the enzyme-conjugated antibody binds the antigen rather than the antibodies in the test serum.[4] In contrast, the absence of color indicates a positive test result and the presence of antibodies in the test serum.

Competitive ELISA has relatively low specificity and is not suitable for dilute samples. However, its benefits include minimal sample purification requirements, the ability to measure a wide range of antigens within a given sample, applicability to small antigens, and low assay variability.[15]

Direct Cellular ELISA

This technique is used to detect cell surface antigens and receptors. Cells are exposed to enzyme-linked antibodies that specifically bind to surface molecules. After washing off any unbound conjugate, a substrate is added, and the resulting enzyme reaction indicates the amount of the target antigen present on the cells.[3]

Indirect Cellular ELISA

This method is used to quantify antibodies directed against cell-surface antigens. Cells are first incubated with antibody-containing samples, followed by the removal of any unbound antibodies. Enzyme-labeled secondary antibodies, specific to the primary antibodies, are then added. After washing off excess secondary conjugate, a substrate is introduced to produce a measurable signal.[3]

Cellular ELISA techniques are being integrated with high-content imaging systems. High-throughput, multiplexed indirect cellular ELISA platforms have been developed that can simultaneously quantify antibody binding to multiple live-cell surface receptors within a single well, enabling rapid screening for autoimmune and anti-receptor antibodies in patient sera.[23][24]

Diagnostic Tests

ELISAs are widely used in diagnostic testing.[1][4] ELISA can be performed on a broad range of biological fluids, including blood, saliva, urine, milk, cerebrospinal fluid, amniotic fluid, gastric juice, semen, pleural fluid, peritoneal fluid, synovial fluid, bronchoalveolar lavage fluid, as well as cyst fluids (such as ovarian or hydatid cysts) and fluids from various fistulas. These applications are used in both research and diagnostic settings.[3][25] Some uses of ELISA are listed below.

Detection and Measurement of Antibodies in Blood

• Autoantibodies (eg, anti–double-stranded DNA, anti-desmoglein 1, and antinuclear antibodies)
• Antibodies against infectious diseases (antibacterial, antiviral, and antifungal), including hepatitis A, B, and C, and HIV

Detection and Quantification of Tumor Markers

• Prostate-specific antigen
• Carcinoembryonic antigen

Detection and Quantification of Hormone Levels

• Luteinizing hormone
• Follicular-stimulating hormone
• Prolactin
• Testosterone
• hCG

Tracking Disease Outbreaks

• Cholera
• HIV
• Influenza

Detection of Past Exposures

• HIV
• Lyme disease
• Hepatitis
• COVID-19 [26]

Screening Donated Blood for Possible Viral Contaminants

• Anti-HIV-1/2
• Anti-hepatitis C virus
• Hepatitis B surface antigen

Detection of Drug Abuse

• Amphetamine
• Methamphetamine
• 3,4-Methylenedioxymethamphetamine
• Cocaine
• Benzoylecgonine

Interfering Factors

Factors that interfere with appropriate ELISA testing can arise at any phase of the testing process, beginning with specimen collection. The quality and integrity of the assay plate, coating buffer, capture antibody, blocking buffer, target antigen, detection antibody, enzyme conjugate, wash steps, substrate, and signal detection can all interfere with proper ELISA testing.[1] Some of the factors that can interfere with ELISA testing are listed below.

• Assay plate: Well shape and quality, plate material, potential preactivation, and uniformity of coating.[2]
• Buffers: Contamination and pH.[19]
• Capture and detection antibodies: Incubation time, temperature, specificity, titer, and affinity.[15]
• Blocking buffer: Concentration, cross-reactivity, and contamination.[20]
• Target antigen: Conformation, stability, and epitopes.[4]
• Enzyme conjugate: Type, concentration, functional activity, and cross-reactivity.[27]
• Washes: Contamination, frequency, volume, duration, and composition.[1]
• Substrate: Quality and manufacturer variability.[2]
• Detection: Instrument-dependent factors.[27]
• Reader or operator error.[4]

The increasing demand for point-of-care ELISA testing in resource-limited settings has highlighted new categories of environmental interferents, particularly during ambient-temperature incubations. Studies reported in 2025 emphasize that ambient humidity, atmospheric particulate matter, and diurnal temperature fluctuations greater than 5 °C can significantly alter antigen-antibody binding kinetics and enzyme-substrate reaction rates, leading to batch-to-batch variability in field-deployed tests. 

Advancements in machine learning for quality control now enable the real-time detection of assay interference by analyzing raw kinetic readout data from plate readers. These algorithms can identify anomalies caused by subtle factors, such as incomplete washing or low-level substrate contamination, that were previously undetectable by standard endpoint absorbance thresholds.[28][29]

Research on next-generation blocking agents has identified new sources of interference. Although traditional protein blockers such as BSA remain common, 2025 investigations revealed that certain commercial recombinant protein blockers contain trace contaminants that bind to Fc regions with low affinity. This interaction can lead to nonspecific signal elevation in sandwich ELISA formats that use monoclonal detection antibodies.[6]

Finally, the increasing use of multiplexed ELISA panels has introduced a novel interference mechanism, known as assay crosstalk. Recent studies have documented that enzymatic products from a single assay well, particularly when using high-sensitivity chemiluminescent substrates, can diffuse as aerosols during plate handling and generate false-positive signals in adjacent wells. this effect necessitates the use of strict physical barriers or staggered development times in multiplex protocols.[30][31][32]

Results, Reporting, and Critical Findings

Data collected from ELISA tests can be quantitative, qualitative, or semiquantitative.[1] Quantitative results are plotted and compared with a standard curve to determine analyte concentration. Qualitative results indicate the presence or absence of a specific antigen or antibody in a sample.[2] Semiquantitative results compare the intensities of signals to estimate relative antigen levels in a sample.[27]

After color development is measured, results are plotted using either manual methods (on a graph paper) or software.[1] Typically, optical density is plotted against the logarithm of concentration, producing a sigmoidal curve. Known analyte concentrations are used to generate the standard curve, and concentrations of unknown samples are determined by comparing their values with the linear portion of that curve.[22]

Clinical Significance

ELISAs are used across a wide range of clinical and laboratory settings, including rapid antibody screening for HIV, detection of other viruses (such as COVID-19; IDSA Guidelines on the Diagnosis of COVID-19: Serologic Testing), identification of bacterial and fungal pathogens, diagnosis of autoimmune diseases, detection of food allergens, blood typing, presence of the pregnancy hormone hCG, laboratory and clinical research, applications in forensic toxicology, and many other diagnostic settings.[33][34][35][36]

In HIV testing, a blood or saliva specimen is collected, and screening is typically performed using indirect ELISA-based tests.[7] ELISA serves as a screening tool for HIV detection, but it is not diagnostic. Diagnosis requires confirmatory Western blot testing due to the risk of false-positive results. Another virus, Molluscum contagiosum virus (MCV), which commonly infects the skin of children and young adults, can also be detected by ELISA testing.[37] ELISA testing in this setting is currently being evaluated to assess global MCV seroprevalence.[38]

ELISA has also been used to detect autoantibodies to desmogleins 1 and 3, as well as bullous pemphigoid antigen 180, which are implicated in pemphigus and bullous pemphigoid, respectively.[39][40] In food allergy, the evolution of the ELISA has played an important role in allergy research and diagnosis. Ultrasensitive ELISA variants have been developed to detect allergens at picogram concentrations, which is critical given the potentially life-threatening public health impact of food allergies.[41][42]

Quality Control and Lab Safety

As with quantitative procedures, results from qualitative and semiquantitative examinations must be verified for accuracy before being reported to the requesting health care provider.[43] Laboratories must establish a quality control program for all qualitative and semiquantitative tests.[44][45][46] When establishing this program, policies should be defined, staff should be trained, responsibilities assigned, and adequate resources ensured. All quality control data must be completely documented and reviewed by the quality manager and the laboratory director.[47]

Positive and negative controls are recommended for many qualitative and semiquantitative tests, including some procedures that use special stains or reagents and assays with endpoints such as agglutination or color change.[48] These controls should generally be used with each test run.[49] Their use of controls also helps validate new lot numbers for test kits or reagents, monitor temperatures in storage and testing areas, and assess assay performance when testing is performed by new personnel.[50]

When traditional controls are used for qualitative or semiquantitative tests, laboratories should test control materials in the same manner as patient samples; include both positive and negative controls, preferably once each day of testing or at least as frequently as recommended by the manufacturer; select positive controls with values close to the assay cutoff to ensure detection of weak positive reactions; and, for agglutination procedures, include a weak positive control in addition to a negative control and a stronger positive control.[44]

Basic safety rules for laboratory conduct should be observed at all times when working in a laboratory.[51] All specimens, control materials, and calibrators should be considered potentially infectious. Standard precautions should be followed when handling laboratory reagents.[52] Disposal of all waste materials should comply with local regulations. Personnel should wear gloves, a laboratory coat, and safety glasses when handling human blood specimens. All plastic tips, sample cups, and gloves that come into contact with blood should be disposed of in biohazard waste containers, and all disposable glassware should be discarded in approved sharps containers.[53]

Work surfaces should be protected with disposable absorbent benchtop paper, which should be discarded into biohazard waste containers weekly or whenever blood contamination occurs. Work surfaces should be wiped down at least once a week. All equipment should be routinely inspected for wear or deterioration and maintained in accordance with the manufacturer’s requirements. Records of certification, maintenance, and repairs should be retained for the life of the equipment.[54] 

Computers and instrumentation should be clearly labeled to indicate whether gloves are required. Inconsistent glove use when handling keyboards or keypads can be a source of contamination. Jewelry should be avoided in the laboratory because it can pose multiple safety hazards. Safety requirements specific to laboratory operations should be outlined in the appropriate laboratory standard operating procedures.[55]

The implementation of digital quality control (QC) platforms, now mandated in many accredited laboratories, has transformed the traditional QC review process. These systems use AI-driven statistical process control to analyze multiparameter QC data in real time, automatically flagging shifts or trends before they exceed acceptable limits and correlating them with variables such as reagent lot changes or environmental conditions. [56][57]

Laboratory safety protocols were updated to address risks posed by novel signal amplification reagents used in next-generation rapid tests. Certain high-potency enzymatic substrates now require handling in designated containment areas due to the potential for aerosolization, which may cause false-positive results in adjacent tests and possible respiratory sensitization.

Furthermore, the widespread adoption of automated ELISA plate handlers and multiplex analyzers has introduced new ergonomic and safety considerations. Reassessment of risk highlighted the need for specific lock-out and tag-out procedures during maintenance to prevent unexpected mechanical movement, as well as regular decontamination cycles for robotic grippers, which can accumulate biohazardous material not visible during routine inspection.

Enhancing Healthcare Team Outcomes

ELISA testing plays a critical role in medical care and scientific research. The use of this technique has expanded beyond core clinical pathology laboratories into point-of-care settings, global health surveillance networks, and advanced discovery research, reinforcing its status as an indispensable analytical platform. Effective implementation depends on a highly collaborative workflow that requires coordinated efforts among phlebotomists to ensure specimen integrity, laboratory scientists to execute assays accurately, bioinformaticians to analyze high-dimensional data, and clinicians to interpret results, establish diagnoses, and communicate findings in a patient-centered manner.

Recent advancements have primarily focused on augmentation rather than replacement, enhancing traditional ELISA with new capabilities. Key innovations include the integration of ultrasensitive digital and single-molecule ELISA, such as Simoa and dELISA, which enable the detection of neurological biomarkers, such as tau and alpha-synuclein, at femtomolar levels, thereby revolutionizing early neurodegenerative disease research. Furthermore, the growing use of multiplexed bead-based and on-chip array platforms now allows simultaneous quantification of dozens of cytokines, chemokines, or autoantibodies from a single microsample, accelerating progress in systems immunology and personalized medicine. Efforts to decentralize testing have also led to the successful deployment of smartphone-based paper microfluidic ELISA readers in resource-limited settings, enabling quantitative analysis for tropical disease surveillance without the need for traditional laboratory infrastructure.

The clinical impact of these evolving technologies is profound. Beyond its historical success in HIV diagnosis and pregnancy testing, ELISA has evolved to underpin critical advances in cancer liquid biopsies, including the detection of exosomal proteins; therapeutic drug monitoring of biological agents; and rapid serological profiling during pandemic responses. The integration of artificial intelligence for automated plate image analysis and predictive quality control further standardizes outputs and reduces turnaround times.

As a mature yet dynamically evolving technology, ELISA continues to adapt to emerging needs. The future of ELISA lies in hybrid systems that combine immunoassay specificity with the sensitivity of nucleic acid amplification (eg, immuno-PCR) and in fully automated, walk-away platforms for high-throughput biomarker validation. The continued refinement of ELISA ensures that it will remain vital for improving early diagnosis, enabling precision medicine, and responding to emerging public health challenges globally.

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Disclosure: Muhammad Zubair declares no relevant financial relationships with ineligible companies.

Disclosure: Charanjeet Singh declares no relevant financial relationships with ineligible companies.

Disclosure: Aisha Farhana declares no relevant financial relationships with ineligible companies.