ELISA

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.

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.

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Contents
What is ELISA?
Four operational methods of ELISA
Causes of false positive, high background, low signal of ELISA
ELISA procedure
Differences between ELISA and PCR?

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.

ROCKER BioWasher 100 ELISA Washer, specially designed height of tubes, prevents cross-contamination.
BioWasher 100 ELISA Microplate Washer
BioWasher 100-T ELISA Microplate Washer

BioWasher 100-T Microplate ELISA Washer

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
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:

Applications