Enzyme-Linked Immunosorbent Assay (ELISA) is a powerful and widely used laboratory technique in the field of immunology. It plays a pivotal role in detecting and quantifying specific proteins, antibodies, or antigens in biological samples. ELISA offers high sensitivity, specificity, and versatility, making it an indispensable tool in various areas such as clinical diagnostics, biomedical research, and pharmaceutical development.

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By discussing ELISA, we gain insights into its significance in disease diagnosis, vaccine development, drug discovery, and understanding immune responses. Understanding the principles, methodologies, and advancements in ELISA enables researchers and healthcare professionals to enhance their analytical capabilities, improve patient care, and advance scientific knowledge in immunological studies. Therefore, exploring the topic of ELISA is crucial for anyone involved in the fields of immunology, diagnostics, and biomedical research.

 

 

 

What is ELISA?

 

Enzyme-Linked Immunosorbent Assay (ELISA), also known as enzyme immunoassay (EIA) or enzyme-linked assay, is a sensitive and specific molecular biology analysis technology widely used in biology, biochemistry, and medical research. It mainly utilizes the specific bond between an antigen and antibody on a solid surface, such as a microwell plate, and employs enzyme-labeled antibodies or antigens to detect target molecules in the sample. The enzymes used for labeling are commonly known as reporter enzymes, with horseradish peroxidase (HRP) and alkaline phosphatase (AP) being common examples.

ELISA offers numerous advantages over other immunoassay techniques. Firstly, it demonstrates higher sensitivity and specificity compared to alternative immunological detection methods. ELISA can detect very low concentrations of biomolecules, including proteins, antibodies, and cytokines, and can distinguish between similar molecules. This enhances the reliability and precision of ELISA in diagnosing diseases, monitoring conditions, and detecting pathogens.

In terms of operation, ELISA is simple, fast, and easily automated, reducing manual errors and enhancing experiment stability and repeatability. Moreover, ELISA requires a small amount of sample, preserving precious samples. Additionally, ELISA can be applied for high-throughput detection, enabling simultaneous testing of multiple samples, thus improving detection efficiency and saving time and costs. Furthermore, ELISA is a non-radioactive detection method, eliminating radiation hazards and complying with environmental and health regulations. Consequently, ELISA finds wide applications in clinical diagnosis, life science research, and other fields.

 

ELISA can be classified into three categories based on different analytical purposes: qualitative, quantitative, and semi-quantitative.

• Qualitative ELISA is employed to determine the presence or absence of specific biomolecules, such as pathogen antigens or antibodies, in a sample. Typically, a single antibody is used to detect the sample, and if the target molecule is present, a signal will be generated; otherwise, no signal will be observed.
• Quantitative ELISA is used to measure the concentration of a target molecule in a sample. This method usually involves constructing a standard curve for quantification. During analysis, the absorbance value of the sample corresponds to a known sample concentration on the standard curve, allowing for the calculation of the target molecule&#;s concentration in the sample.
• Semi-quantitative ELISA is an analytical method that lies between qualitative and quantitative approaches. It is commonly used to estimate the relative concentration of target molecules in a sample. This method involves comparing the sample with a standard substance of known concentration to determine the relative concentration of the target molecule in the sample.

 

 

Four operational methods of ELISA

 

ELISA can be classified into four methods: Direct ELISA, Indirect ELISA, Sandwich ELISA, and Competitive ELISA.

 

Direct ELISA

Direct ELISA is commonly used for antigen detection. In this process, only one primary antibody is required, and the analyte can be detected without complicated steps, making it the simplest ELISA method. The operational procedure involves immobilizing the target antigen onto a solid surface, typically a 96-well plate, and then utilizing an enzyme-labeled primary antibody to detect the antigen under examination.

Due to the simplicity of the reaction process, the direct method allows for rapid analysis of results and avoids non-specific interaction reactions caused by secondary antibodies. However, since only one antibody is used, the direct method lacks signal amplification capability, resulting in reduced detection sensitivity. Additionally, labeling the primary antibodies with enzymes is time-consuming and costly.

 

Indirect ELISA

 

Indirect ELISA is a detection method that uses a primary antibody combined with an enzyme-labeled secondary antibody, and is usually used to detect an unknown primary antibody. Among them, the most common application example is to detect AIDS virus (human immunodeficiency virus, HIV).
The steps of this method are to first immobilize the known antigen on the solid surface, add the sample, and then add the enzyme-labeled secondary antibody to detect the antibody to be tested. Since two kinds of antibodies are used, the indirect method has higher sensitivity and can use a variety of primary antibodies, which is more flexible in application.
However, in indirect ELISA, there may be interactions between the secondary antibodies, resulting in non-specific signals and affecting the accuracy of the experimental results. To solve this problem, a blocking solution can be added during the experiment to reduce non-specific interactions. In this way, the indirect method can detect the antibody to be tested more accurately and obtain reliable results.

 

Sandwich ELISA

 

Indirect ELISA is a detection method that utilizes a primary antibody in combination with an enzyme-labeled secondary antibody. It is commonly employed to detect unknown primary antibodies, with one of the most frequent applications being the detection of the AIDS virus (human immunodeficiency virus, HIV).

The steps of this method involve immobilizing the known antigen on a solid surface, adding the sample, and subsequently introducing the enzyme-labeled secondary antibody to detect the target antibody. By employing two types of antibodies, the indirect method offers higher sensitivity and allows for the use of various primary antibodies, making it more versatile in application.

However, in indirect ELISA, there is a possibility of interactions occurring between the secondary antibodies, resulting in non-specific signals that can affect the accuracy of experimental results. To address this issue, a blocking solution can be added during the experiment to reduce non-specific interactions. This enables the indirect method to achieve more accurate detection of the target antibody and obtain reliable results.

 

Competitive ELISA

 

Competition ELISA, also referred to as competition inhibition ELISA, involves the competition between the antigen to be tested and the enzyme-labeled biomolecule for binding to the antigen or antibody immobilized on a solid surface. When the concentration of the antigen to be tested is higher in the specimen, it will bind to more antibodies, leading to reduced binding of the enzyme-labeled antibody and a lower signal value in the color reaction. Thus, the concentration of the antigen to be tested is inversely proportional to the color reaction. This method is commonly employed for the detection of haptens or small molecule antigens.

 

  Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA How it work Immobilize the target antigen on a solid surface, and use the enzyme-labeled primary antibody to detect the antigen to be tested Immobilize the known antigen on the solid surface, add the primary antibody, and then add the enzyme-labeled secondary antibody to detect the antibody to be tested The two antibodies specifically identify the antigen in the specimen twice Use the antigen to be tested and the enzyme-labeled biomolecule to compete for binding to the antigen or antibody immobilized on the solid surface Advantage(s) &#; Faster
&#; No secondary antibody interaction &#; Higher sensitivity
&#; Able to use a variety of primary antibodies, high flexibility
&#; Lower cost &#; Not requiring purified sample
&#; Higher sensitivity
&#; High specificity &#; Not requiring purified sample
&#; Able to detect most antigens in a single sample
&#; Lower variability Disadvantage(s) &#; Lower sensitivity
&#; False positives or interference may occur
&#; Specific primary antibody is required
&#; Enzyme-labeled primary antibody is required, which is time-consuming and expensive &#; Secondary Antibody Interactions &#; Specific primary and secondary antibodies are required
&#; The antigen to be tested must be a multivalent antigen
&#; Time-consuming and costly analysis &#; Lower specificity, not suitable for dilute samples Suitable target(s) Antigen Antibody Macromolecular antigen Haptens, small molecule antigens

 

 

Causes of false positive, high background, low signal of ELISA

 

ELISA often encounters unstable results, including false positives, high background values, or no/low signal. False positives or high background values can be attributed, in part, to incomplete removal of unbound antibodies, antigens, or impurities between steps, leading to background signals or nonspecific interactions.

 

 

ELISA procedure

 

To minimize the occurrence of false positives and high background values, it is essential to perform &#;wash&#; steps between each stage of ELISA. This step helps eliminate the interference caused by non-specific or low-affinity reactants. The wash buffer typically used is phosphate-buffered saline (PBS) or TBS buffer, and it is advisable to include an appropriate concentration of surfactant, such as 0.01% to 0.1% Tween-20 or Triton X-100, to reduce non-specific binding in samples.

The washing process holds great importance. Generally, each wash step necessitates 2-5 washes with buffer. If manual washing is employed, it is crucial to shake off any residual reaction solution and promptly add the subsequent reaction solution to avoid excessive drying of the microwell plate.

 

Manual washing

Microplate washer

Suction

Micropipette with manual spin dry

Dispenser

Dispensing

Micropipette and wash bottle

Dispenser

Microplate placement

Lay flat

Lay flat or horizontal

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Liquid residue

Tilt manually

Suggested to use tilt-able equipment

Autoclavable

Depending on the dispenser

Usually autoclavable

Cost

Lower

Total cost of the whole system (including vacuum pump, etc.) is to be considered

 

 

Differences between ELISA and PCR?

 

With the advancement of analytical technology, it is now possible to analyze a wide range of substances and molecules using various technologies. ELISA and PCR (Polymerase Chain Reaction) are often compared to each other. The following is a comparison table between these two methods.

  ELISA PCR How it work Uses the specific combination between antigen and antibody, and use enzyme to catalyze the color reaction Amplify DNA fragments to detect pathogens or detect genes of interest Detection target Protein DNA Sensitivity Higher Lower Specificity Lower Higher Processing time Longer Shorter Cost Lower Higher Comparison for detecting coronavirus Sample Serum Saliva Analytical target Antibody DNA after RNA reverse transcription Purpose Confirming whether the human body has been infected by the virus Detecting whether the virus is present in the human body.

 

Therefore, in the field of epidemiology, combining ELISA with PCR for virus or organism detection, such as Monkeypox virus (MPV), Hantavirus, Norovirus, etc., can yield a more comprehensive detection effect.

Another technique worth mentioning is the combination of ELISA with PCR, known as PCR-ELISA. This method enables direct quantification of DNA immobilized on a solid surface through PCR. However, since this article primarily focuses on the analysis method of ELISA, it will not discuss PCR-ELISA.

 

ELISA Applications&#;

 

• Clinical medicine: diagnosis of cancer, autoimmune disease, infectious disease, etc.
• Virus testing: coronavirus (SARS-CoV-2), human immunodeficiency virus (HIV), West Nile Virus, etc.
• Point-of-care testing: home pregnancy tests
• Academic research: DNA/RNA quantification, cell signaling, metabolic pathway, gene expression and regulation, etc.
• Food Safety: Food Allergens

 

 

References:

Advantages of ELISA for Accurate and Accelerated Drug Discovery [Podcast]

Since its discovery nearly 50 years ago, the Enzyme-Linked Immunosorbent Assay (ELISA) has become the gold standard in diagnostics and drug evaluation studies. Using an ELISA, one can quantify a molecule of interest (e.g., protein, peptide, or hormone) in a liquid sample by immobilizing it on a microplate, using specific antibodies to bind that molecule, and detecting the particular binding with high sensitivity. The technique has remained popular, despite the emergence of other methods like flow cytometry, due to its specificity and versatility.

Uncover the detailed discussion of how our experts use ELISA assays in their research, the specific benefits of this kind of immunoassay, and what research directions have been opened up through the use of ELISA, and much more in our recent podcast:

Three accomplished scientists discuss what makes the ELISA stand out and how it can be adapted to the rapidly evolving drug discovery scene. Key learning points:

Dr. Ana Miletic is a senior research scientist at Kyowa Kirin. She focused on immune cell signaling and development during her Ph.D. in immunology at Washington University and did a postdoc at Sanford Burnham Prebys Medical Discovery Institute. She worked on monoclonal antibodies at eBioscience and Becton, Dickinson, and Company (BD) before joining Kyowa Kirin six years ago as a project leader on early-stage drug discovery programs.

Anna Dey, MSc in biotechnology at Johns Hopkins University, has worked in biotechnology for 17 years, mainly at cancer research laboratories. She is the senior research associate at Kyowa Kirin, working on antibody-based therapeutics for autoimmune diseases.

Dr. Cathy Olsen works at Molecular Devices as a senior application scientist and is involved in the development of a broad range of applications for microplate-based detection systems &#; including cell-based, biochemical and imaging assays. She has a PhD in Cell and Developmental Biology from the University of California, Davis.

ELISA applications in drug discovery and development

Our three experts discuss how they use ELISA assays to support their research in drug discovery and development.

Efficacy of therapeutic antibodies against cytokines

For Kyowa Kirin, the ELISA is a critical checkpoint to assess the efficacy of therapeutic antibodies against inflammatory cytokines. By measuring the levels of cytokines, such as interferon-gamma, IL-8, and TNF-alpha, Dey and her colleagues assess the ability of their antibody clones to block the production of these cytokines. This helps them select the most effective antibody clones.

Assess the pharmacokinetic properties of therapeutic antibodies

ELISAs have also helped Dr. Miletic and Ms. Dey assess the pharmacokinetic properties of therapeutic antibodies. In these studies, the team measures the presence of the antibody in a patient's blood at various time points. This process helps them determine the half-life of the therapeutic, information that guides their selection of the most stable antibody clones.

Assess the reduced levels of p53 in knockout cells

Molecular Devices has demonstrated the value of a variety of ELISAs for a wide range of research approaches, including CRISPR knock-out experiments. One example is the knock-out of the p53 gene, which encodes a tumor suppressor. Using an ELISA to assess the reduced levels of p53 in knockout cells demonstrates the usefulness of the method for gaining insights into gene function, which could ultimately drive the development of novel targeted therapies.

Advantages of ELISA assays compared to other immunoassays

The three examples above demonstrate the versatility of ELISAs, but additional factors distinguish them from other immunoassays.

Cathy Olsen emphasizes the flexibility and sensitivity of ELISAs: &#;ELISAs can be very highly sensitive and specific. They are typically available with different readouts, depending on what is needed. Colorimetric ELISAs that use absorbance detection as a readout has been around for 30 years. Still, there are also fluorescent and luminescent readouts that can give you better sensitivity and better dynamic range. Thanks to high sensitivity, an ELISA can detect low-abundance molecules in the sample.&#;

&#;&#;fluorescent and luminescent readouts can give you better sensitivity and better dynamic range&#; and, detect low-abundance molecules in the sample.&#;

For Ana Miletic, specificity is a critical attribute of ELISAs, which allows them to distinguish between closely related proteins. That said, the choice of antibody is the key. &#;An ELISA is only as good as the antibodies used in it. The backbone of any ELISA assay, which makes it a great assay, is the use of particular, high-affinity antibodies to your target of interest.&#; In other words, the correct antibody choice helps ensure the target analyte is detected and minimizes the risks of cross-reactivity with similar molecules.

&#;The backbone of any ELISA assay, which makes it a great assay, is the use of particular, high-affinity antibodies to your target of interest.&#;

Another advantage of the ELISA is its simplicity, with only plates, pipettes to add samples, and plate readers needed (a microplate washer can be added to ease the workload). Despite their undemanding design, ELISAs can provide a wide range of readouts. More importantly, since most of the equipment is automation-friendly, one can make ELISAs even more accessible and streamlined.

ELISA instrumentation and automation workflow solutions

An automated ELISA workflow involves several upgrades to the manual version, but the extent of automation ultimately depends on throughput needs.

Automated liquid handlers are very useful for sample preparation and reagent addition, while robotic arms can handle the transfer of plates between different sections of the system. Combining these two elements means one does not have to manually pipette reagents or carry the plates from the bench to the liquid handling system, back, and to the plate washer and reader. Since the liquid handler and robotic arm handle these steps, the researcher earns more time to run more focus-intensive experiments.

Two additional components make the workflow even more streamlined. One of them is the automation scheduling software that coordinates the workflow timeline and sequence to get more timely, accurate results. Cell-based assays require a stable environment for cells (e.g., 37oC, 5% CO2), and an automated incubator is used to maintain these conditions consistently.

Nevertheless, Cathy Olsen considers the plate reader a key to success in automated ELISA. &#;The quality of results depends very heavily on accurately detecting the signal in the ELISA plate. When you analyze your data, you want to get accurate results and quantitation of that target protein, which falls on the plate reader.&#; A plate reader&#;s software may offer automated analysis using protocols configured for a particular assay.

The automated ELISA workflow has two positive outcomes. First, as mentioned before, the walkaway time significantly increases, allowing researchers to multitask. More importantly, it can reduce human error with uniform sample preparation and a well-monitored liquid handling procedure.

What the Future Holds for ELISA

Many scientists continue to harness the qualitative and quantitative powers of ELISAs, and a current market analysis report predicts that the global research ELISA market will rise from US$519.4 million in to US$754.38 million by .

ELISAs continue to serve as the benchmark for diagnosis by detecting specific antigens in bodily fluids, and healthcare professionals use them in various fields, including HIV testing, pregnancy tests, and the detection of infectious diseases.

In the recent COVID-19 pandemic, ELISAs have played a significant role in expanding our understanding of the disease mechanisms involved by enabling the detection of antibodies against the spike protein, nucleocapsid antigen, and other viral proteins. By measuring antibodies produced in response to vaccination, researchers could also assess the potential efficacy of a vaccine and optimize its formulation.

ELISAs can also help to elucidate the working mechanism of a drug. Even in the presence of a target protein, one needs to investigate how targeting a specific protein works. By introducing a drug molecule to different cell types and quantifying the levels of target proteins in these cells, researchers can deduce the impact of the drug on various cellular processes.

Future work must focus on addressing some of the limitations of ELISA to expand its potential. One key area is increasing sensitivity to accurately detect low levels of antigens in a sample, which can be achieved through signal amplification techniques. Additionally, optimization of blocking agents can reduce background noise and prevent non-specific antibody binding or cross-reactivity with similar antigens. Lastly, the addition of multiplexing is an upgrade enabling the quantification of multiple proteins, peptides, and small molecules in the sample simultaneously.

Discuss your automated ELISA solution

Automation does not have to be overwhelming &#; there are a lot of flexibility and options available. Our five ready-made workcells for ELISA workflows offer automation solutions ranging from simple plate-loading capabilities to more advanced, completely automated workcells and the best part, it can be customized and built over time. Automating labor-intensive plate-based assays increases walkaway time, throughput, and reproducibility by reducing a researcher&#;s need to engage in common, repetitive, hands-on tasks.

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