ELISA- Definition, Principle, Procedure, Types, Steps, Uses

ELISA stands for Enzyme-Linked Immunosorbent Assay. It is a modern molecular technique that can detect the interaction between an antigen and an antibody with the help of an enzyme. An antigen is a foreign substance that can trigger an immune response in the body, and an antibody is a protein that can bind to a specific antigen and neutralize it. The enzyme acts as a marker that can produce a color change when it reacts with a substrate.

ELISA is based on the principle of immunology, which is the study of how the immune system recognizes and responds to foreign invaders. ELISA can measure the amount of antigen or antibody present in a sample, such as blood, serum, saliva, urine, etc. ELISA can also identify the type of antigen or antibody present in a sample by using specific antibodies or antigens that can recognize them.

ELISA has many applications in various fields of science and medicine, such as diagnosis of diseases, detection of allergens, estimation of hormones, monitoring of vaccines, etc. ELISA is a simple, fast, accurate, and cost-effective method that can provide qualitative and quantitative results. ELISA can also be performed in different formats, such as direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA. Each format has its own advantages and disadvantages depending on the purpose and design of the experiment.

In this article, we will discuss the definition, principle, procedure, types, steps, uses, advantages, and limitations of ELISA in detail. We will also provide some examples and illustrations to help you understand this technique better. By the end of this article, you will have a comprehensive overview of ELISA and its applications in various fields. 😊

ELISA is a technique that requires some specific materials and reagents to perform the assay. The following are some of the common ELISA requirements:

• Coated plates (Microtitre plates): These are plastic plates with multiple wells (usually 96) that can hold small volumes of liquid. The wells are coated with antigens or antibodies that are specific to the target molecule of interest. The coating can be done by direct adsorption or by using a linker molecule such as biotin or avidin. The coated plates can be stored at 4°C for future use or used immediately after coating.
• Sample diluents: These are solutions that are used to dilute the sample before applying it to the wells. The sample diluents can vary depending on the type of ELISA and the nature of the sample. For example, some samples may need to be diluted in a buffer that contains a blocking agent such as bovine serum albumin (BSA) or casein to prevent nonspecific binding of other molecules to the coated wells. Some samples may also need to be treated with detergents, enzymes, or heat to reduce interference from other substances or to release the target molecule from the sample matrix.
• Wash buffers: These are solutions that are used to wash away any unbound or excess molecules from the wells after each incubation step. The wash buffers usually contain a mild detergent such as Tween-20 and a buffer such as phosphate-buffered saline (PBS) or Tris-buffered saline (TBS) to maintain the pH and osmolarity of the solution. The wash buffers can also contain preservatives such as sodium azide to prevent microbial growth. The washing step is important to reduce background noise and increase specificity and sensitivity of the assay.
• Enzyme-linked antibodies: These are antibodies that are conjugated to an enzyme that can catalyze a colorimetric or chemiluminescent reaction. The enzyme-linked antibodies can be either primary antibodies that bind directly to the target molecule or secondary antibodies that bind to the primary antibodies. The most common enzymes used in ELISA are horseradish peroxidase (HRP) and alkaline phosphatase (AP), but other enzymes such as beta-galactosidase, glucose oxidase, or luciferase can also be used. The enzyme-linked antibodies can be purchased commercially or prepared in-house by using chemical cross-linkers or recombinant DNA technology.
• Substrates: These are molecules that react with the enzyme-linked antibodies to produce a detectable signal. The substrates can be either chromogenic, which produce a colored product, or chemiluminescent, which produce a light-emitting product. The choice of substrate depends on the type of enzyme used and the detection method. Some examples of chromogenic substrates are o-phenylenediamine (OPD), tetramethylbenzidine (TMB), and p-nitrophenyl phosphate (PNPP) for HRP and AP, respectively. Some examples of chemiluminescent substrates are luminol, acridinium esters, and dioxetanes for HRP and AP, respectively.
• Stop solution: This is a solution that is added to stop the enzyme-substrate reaction and stabilize the signal. The stop solution can be an acid such as sulfuric acid or hydrochloric acid for chromogenic substrates or an alkali such as sodium hydroxide or sodium carbonate for chemiluminescent substrates. The stop solution can also affect the color or intensity of the signal, so it should be chosen carefully.

These are some of the basic ELISA requirements that are needed to perform the assay successfully. However, depending on the type and purpose of ELISA, some additional reagents such as standards, controls, enhancers, blockers, stabilizers, or conjugates may also be required.

ELISA stands for Enzyme-Linked Immunosorbent Assay. It is a modern molecular technique that uses the specific interaction between antigens and antibodies to detect and measure the presence of a target molecule in a sample. The target molecule can be either an antigen or an antibody, depending on the type of ELISA.

The basic principle of ELISA is that antigens and antibodies react specifically to form the Ag-Ab complex. This complex can be detected and quantified by using an enzyme that is linked or attached to one of the components (either the antigen or the antibody). The enzyme acts as a label that can modify a specific substrate to produce a color change within the solution. The color change can be measured by using a colorimeter or a spectrophotometer at a specific wavelength of light. The intensity of the color change is proportional to the amount of the target molecule in the sample.

There are four main types of ELISA: direct, indirect, sandwich and competitive. Each type has a different configuration of the components and a different way of detecting the target molecule. However, they all share the same principle of using an enzyme-linked label and a chromogenic substrate to generate a measurable signal.

ELISA is a simple, sensitive, specific and efficient technique that can be used for various applications in biomedical research, clinical diagnosis and quality control. It can detect and measure different types of molecules, such as proteins, hormones, toxins, pathogens, allergens and drugs. It can also be used for qualitative and quantitative analysis of samples from different sources, such as blood, serum, plasma, urine, saliva and tissue extracts.

ELISA has some advantages over other techniques that use radioactive or fluorescent labels, such as radioimmunoassay (RIA) or fluorescence immunoassay (FIA). ELISA is safer, cheaper and more stable than these techniques. It does not require any special equipment or handling of hazardous materials. It also has a longer shelf life and can be stored at room temperature.

ELISA also has some limitations that need to be considered when performing the assay. ELISA is work-intensive and time-consuming. It requires careful preparation of the reagents and the samples. It also requires multiple washing steps to remove any unbound or nonspecific components that may interfere with the signal. ELISA may also suffer from cross-reactivity or interference from other molecules that have similar structures or properties to the target molecule. Therefore, it is important to optimize the assay conditions and validate the results with appropriate controls and standards.

ELISA stands for Enzyme-Linked Immunosorbent Assay. It is a modern molecular technique for detecting antigen-antibody interaction with the help of an enzyme. There are mainly four types of ELISA based on the different ways of binding the antigen and the antibody. They are:

1. Direct ELISA

2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA

Each type of ELISA has its own procedure, advantages, disadvantages and applications. In this section, we will briefly describe each type and compare them.

Direct ELISA is the simplest and quickest of all other types of ELISA. A single enzyme-linked antibody is used which directly interacts with the antigen present in the sample. The advantage of direct ELISA is that it requires only one antibody and one incubation step, which reduces the time and cost of the assay. The disadvantage of direct ELISA is that it has lower sensitivity and specificity than other types of ELISA, as there is no signal amplification or cross-reactivity reduction. Direct ELISA is mainly used for the identification of biomolecules and for the diagnosis of infections such as Mycoplasma bovis.

Indirect ELISA is the most popular type of ELISA in use. Antibody detection is carried out in indirect ELISA. A secondary antibody linked with the enzyme is used, which binds to the primary antibody that has already bound to the antigen in the sample. The advantage of indirect ELISA is that it has higher sensitivity and specificity than direct ELISA, as the secondary antibody can amplify the signal and reduce cross-reactivity. The disadvantage of indirect ELISA is that it requires two antibodies and two incubation steps, which increases the time and cost of the assay. Indirect ELISA is mainly used for the detection of HIV, Rubella, Dengue viruses, etc. and for the detection of certain drugs in serum.

Sandwich ELISA is a type of ELISA that detects antigens rather than antibodies. Two different antibodies are required: capture antibodies (for attachment sample antigens) and enzyme-linked secondary antibodies. The antigen of interest first binds with the capture antibodies that are coated on the microtitre plate, and then the secondary antibodies bind with another epitope on the antigen, forming a sandwich-like structure with antigen in between antibodies. The advantage of sandwich ELISA is that it has very high sensitivity and specificity, as it can detect multiple epitopes on a single antigen and eliminate nonspecific binding. The disadvantage of sandwich ELISA is that it requires two specific antibodies and two incubation steps, which increases the time and cost of the assay. Sandwich ELISA is mainly used for detection of Rotavirus and enterotoxins of fecal E. coli and for pregnancy test kits.

Competitive ELISA is a type of ELISA that detects antibodies by measuring their inhibition of enzyme-linked antigen binding to immobilized antigen. The basic concept of this type is that there occurs competition of binding between the sample antibodies (if present) and enzyme-linked antigens to the immobilized antigens. If the sample contains specific antibodies of interest, they will bind to the immobilized antigens and prevent enzyme-linked antigens from binding, which results in less color development after adding substrate. Conversely, if the sample does not contain specific antibodies, more enzyme-linked antigens will bind to the immobilized antigens, which results in more color development after adding substrate. The advantage of competitive ELISA is that it has very high specificity, as it can eliminate nonspecific binding and cross-reactivity. The disadvantage of competitive ELISA is that it has lower sensitivity than other types of ELISA, as it relies on inhibition rather than amplification of signal. Competitive ELISA is mainly used for the detection of HIV.

Direct ELISA: Procedure and Applications

Direct ELISA is the simplest and quickest of all other types of ELISA. A single enzyme-linked antibody is used which directly interacts with the antigen present in the sample. This type of ELISA can be used to test specific antibody-to-antigen reactions and to eliminate cross-reactivity between other antibodies.

Direct ELISA Procedure

• Addition of sample (containing antigen) to the well of the microtitre plate

• Antigen gets adsorbed to the surface of the well.
• Washing to remove unbound antigens
• The addition of an enzyme-linked antibody that with the antigens if present.
• Washing to remove unbound antibodies
• Addition of chromogenic substrate
• Visualization of color change and result interpretation

Direct ELISA Applications

• Used in the identification of biomolecules.
• Also for the diagnosis of infections of Mycoplasma bovis.
  
  Indirect ELISA: Procedure and Applications.

Indirect ELISA is a type of ELISA that is used for the detection of antibodies in a sample. It involves two steps of binding: first, the antigen is coated on the plate and incubated with the sample containing the antibody of interest; second, a secondary antibody that is conjugated with an enzyme is added and binds to the primary antibody. The enzyme catalyzes a colorimetric reaction with a substrate, which indicates the presence and amount of the antibody in the sample.

The procedure of indirect ELISA is as follows:

• Coat the microtiter plate wells with the antigen solution and incubate for 2 hours at 37°C or overnight at 4°C.
• Wash the plate with a wash buffer to remove unbound antigens.
• Block the plate with a blocking buffer to prevent nonspecific binding of antibodies.
• Add the sample (serum, plasma, etc.) to the wells and incubate for 1 hour at 37°C.
• Wash the plate again to remove unbound antibodies.
• Add the enzyme-linked secondary antibody that is specific for the primary antibody (e.g., anti-human IgG) and incubate for 1 hour at 37°C.
• Wash the plate again to remove unbound secondary antibodies.
• Add the substrate solution (e.g., TMB for HRP enzyme) and incubate for 15 minutes at room temperature in the dark.
• Stop the reaction by adding a stop solution (e.g., sulfuric acid) and measure the absorbance at 450 nm using a spectrophotometer.

The applications of indirect ELISA are:

• Detection of various viral, bacterial, and fungal infections by measuring the specific antibodies in the sample. For example, indirect ELISA can be used to detect HIV, rubella, dengue, etc.
• Detection of certain drugs or toxins in the sample by measuring the antibodies against them. For example, indirect ELISA can be used to detect cocaine, morphine, tetanus toxin, etc.
• Detection of autoimmune diseases by measuring the autoantibodies in the sample. For example, indirect ELISA can be used to detect rheumatoid arthritis, lupus, etc.

Sandwich ELISA: Procedure and Applications

Sandwich ELISA is a type of ELISA that detects antigens using two different antibodies: a capture antibody and a detection antibody. The capture antibody is coated on the microtitre plate and binds to the antigen of interest in the sample. The detection antibody is linked to an enzyme and binds to a different epitope on the antigen, forming a sandwich-like structure. The enzyme then reacts with a substrate to produce a color change that can be measured by a spectrophotometer.

Sandwich ELISA has some advantages over direct and indirect ELISA, such as:

• It can detect antigens that have multiple epitopes or are multivalent.
• It can increase the specificity and sensitivity of the assay by using two antibodies that recognize different regions of the antigen.
• It can reduce the background noise and nonspecific binding by washing away unbound antigens and antibodies.

Sandwich ELISA has some limitations as well, such as:

• It requires two antibodies that are specific for the same antigen but bind to different epitopes.
• It may not work well for antigens that are small, denatured or have low affinity for the capture antibody.
• It may be affected by cross-reactivity or interference from other molecules in the sample.

Sandwich ELISA has many applications in various fields of research and diagnosis, such as:

• It is widely used for detecting cytokines, growth factors, hormones and other biomarkers in biological fluids.
• It is commonly used for detecting rotavirus and enterotoxins of fecal E. coli in clinical samples.
• It is the basis of many pregnancy test kits that detect human chorionic gonadotropin (hCG) in urine samples.
• It is also used for detecting allergens, toxins, drugs and other substances in food, environmental and forensic samples.

Competitive ELISA: Procedure and Applications

Competitive ELISA is a type of ELISA that detects antibodies by measuring the competition between the sample antibodies and the enzyme-linked antibodies for binding to the antigens. The principle of competitive ELISA is based on the inverse relationship between the amount of sample antibodies and the color intensity of the reaction. The more sample antibodies are present, the less enzyme-linked antibodies will bind to the antigens, and the less color will be produced by the substrate.

Competitive ELISA Procedure

• Coat the microtitre plate wells with a known amount of antigens.
• Wash the plate to remove any unbound antigens.
• Add the serum sample containing the antibodies of interest to the wells.
• Incubate the plate to allow the sample antibodies to bind to the antigens.
• Wash the plate to remove any unbound sample antibodies.
• Add a known amount of enzyme-linked antibodies that are specific for the same antigens.
• Incubate the plate to allow the enzyme-linked antibodies to compete with the sample antibodies for binding to the antigens.
• Wash the plate to remove any unbound enzyme-linked antibodies.
• Add a chromogenic substrate that reacts with the enzyme on the bound antibodies.
• Measure the color intensity of the reaction using a spectrophotometer or an ELISA reader.

Competitive ELISA Applications

Competitive ELISA is mainly used for detecting low concentrations of antibodies or antigens in complex samples. Some of the applications of competitive ELISA are:

• Detection of HIV infection: Competitive ELISA can be used to detect HIV-specific antibodies in serum samples. The antigens used are recombinant HIV proteins or synthetic HIV peptides. A positive result indicates that the sample contains HIV antibodies and thus, the person is infected with HIV.
• Detection of drug residues: Competitive ELISA can be used to detect traces of drugs or their metabolites in urine, blood, or saliva samples. The antigens used are drug conjugates or drug-specific antibodies. A positive result indicates that the sample contains drug residues and thus, the person has consumed or been exposed to drugs.
• Detection of pesticides: Competitive ELISA can be used to detect residues of pesticides or their metabolites in food, water, or soil samples. The antigens used are pesticide conjugates or pesticide-specific antibodies. A positive result indicates that the sample contains pesticide residues and thus, the food, water, or soil is contaminated with pesticides.

ELISA test results can be interpreted in different ways depending on the type and purpose of the test. They can be:

• Quantitative: Data is compared to a standard curve and the concentration of antigens or antibodies in the samples is calculated . A standard curve is a plot of the mean absorbance (y-axis) versus the log concentration (x-axis) of a known, purified antigen . A best-fit curve is drawn through the points on the graph and an equation is derived for calculating the concentration (x) from a given absorbance (y) in the range of the standard curve. The concentration of the target protein in each sample can be determined using the curve or a suitable computer program. A sample of known concentration can be used as a positive control to validate the results.
• Qualitative: Data is expressed as a yes or no answer indicating the presence or absence of a particular antigen or antibody in the sample. A cut-off value is used to determine whether the test result is reactive or non-reactive . Reactive means the value of the test is equal or greater than the cut-off value, while non-reactive means the value of the test is less than the cut-off value . The cut-off value can be calculated as the mean absorbance of negative controls plus two or three standard deviations.
• Semi-quantitative: Data is compared to relative levels of antigens or antibodies in different samples. This method does not provide an exact concentration but rather an estimation based on comparison with other samples or standards.

ELISA test results are usually graphed with optical density versus log concentration to produce a sigmoid curve . The shape and position of the curve can provide information about the sensitivity and specificity of the test, as well as the dynamic range and linearity of the assay .

The coefficient of variation (CV) is a measure of the precision and accuracy of the results. It is calculated as the ratio of the standard deviation to the mean, expressed as a percentage. A low CV indicates high consistency and reliability, while a high CV indicates large variability and error. Some factors that can cause high CV are inaccurate pipetting, splashing of reagents, contamination, cross-reaction, temperature variations, and instability of antibodies or enzymes.

ELISA test results may have some limitations such as false negative, false positive, or indeterminate results. False negative results occur when the test fails to detect an antigen or antibody that is present in the sample. False positive results occur when the test detects an antigen or antibody that is not present in the sample. Indeterminate results occur when the test cannot clearly distinguish between reactive and non-reactive values. Some causes of these errors are low sensitivity or specificity of antibodies, interference from other substances in the sample, improper sample preparation or storage, incorrect assay protocol, or human error .

Therefore, ELISA test results should be interpreted with caution and confirmed by other methods if necessary. ELISA is a powerful technique for detecting antigen-antibody interaction, but it requires careful optimization and validation to ensure accurate and reliable results.

ELISA is a versatile technique that can be used for various purposes in different fields of science and medicine. Some of the common applications of ELISA are:

• Detection of different viral, bacterial and fungal infections: ELISA can identify the presence of specific antigens or antibodies in a sample that indicate an infection by a pathogen. For example, ELISA can detect the antigens of HIV, hepatitis B virus, dengue virus, rotavirus, etc. or the antibodies produced against them in the blood or saliva of the infected person. ELISA can also detect the antigens or antibodies of bacteria such as Mycobacterium tuberculosis, Escherichia coli, Salmonella typhi, etc. or fungi such as Candida albicans, Aspergillus fumigatus, etc. in the body fluids or tissues of the infected person.
• Screening test for HIV infection: ELISA is widely used as a screening test for HIV infection because it is simple, fast and inexpensive. ELISA can detect the presence of HIV antigens or antibodies in the blood or saliva of a person who may have been exposed to the virus. If the test is positive, it means that the person is likely to be infected with HIV and needs further confirmation by other tests such as Western blot or PCR. If the test is negative, it means that the person is unlikely to be infected with HIV and does not need further testing.
• Detection of dengue fever, TB and Hepatitis B infections: ELISA can also be used to diagnose some common infectious diseases such as dengue fever, tuberculosis (TB) and hepatitis B. ELISA can detect the antigens or antibodies of these pathogens in the blood or serum of the infected person. For example, ELISA can detect the NS1 antigen of dengue virus in the acute phase of dengue fever or the IgM and IgG antibodies against dengue virus in the convalescent phase of dengue fever. ELISA can also detect the antigens or antibodies of Mycobacterium tuberculosis in the sputum or serum of a person with TB or the antigens or antibodies of hepatitis B virus in the blood or serum of a person with hepatitis B infection.
• Pregnancy test kits: ELISA is also used to make pregnancy test kits that can detect the presence of human chorionic gonadotropin (hCG) hormone in the urine or blood of a pregnant woman. hCG is a hormone produced by the placenta during pregnancy and its level increases rapidly in the first few weeks of pregnancy. ELISA can detect hCG by using an enzyme-linked antibody that binds to hCG and produces a color change when exposed to a substrate. The color change indicates a positive result for pregnancy and the absence of color change indicates a negative result for pregnancy.
• Qualitative and quantitative estimation of various proteins, hormones and toxins: ELISA can also be used to measure the amount or concentration of various proteins, hormones and toxins in a sample by using specific enzyme-linked antibodies that bind to them and produce a color change when exposed to a substrate. The intensity of the color change is proportional to the amount or concentration of the target molecule in the sample. For example, ELISA can measure the level of insulin, cortisol, thyroid hormones, growth hormones, etc. in the blood or serum of a person by using specific enzyme-linked antibodies that bind to them and produce a color change when exposed to a substrate. ELISA can also measure the level of toxins such as aflatoxin, botulinum toxin, ricin, etc. in food samples by using specific enzyme-linked antibodies that bind to them and produce a color change when exposed to a substrate.
• Detection of different food allergens: ELISA can also be used to detect the presence of different food allergens such as peanuts, eggs, milk, gluten, etc. in food samples by using specific enzyme-linked antibodies that bind to them and produce a color change when exposed to a substrate. The color change indicates a positive result for food allergen and the absence of color change indicates a negative result for food allergen.

ELISA is one of the most widely used techniques in molecular biology and immunology. It has several advantages over other methods of detecting antigen-antibody interactions, such as:

• Simple protocols: ELISA is easy to perform and does not require complex equipment or procedures. The basic steps of ELISA are coating, blocking, incubation, washing and detection. The results can be obtained within a few hours or even minutes.
• Highly specific and sensitive: ELISA relies on the specific binding of antigens and antibodies, which minimizes the chances of false-positive or false-negative results. ELISA can also detect very low levels of antigens or antibodies in the sample, as low as picograms per milliliter.
• Highly efficient: ELISA can process multiple samples simultaneously using microtitre plates that have 96 or more wells. This allows for high-throughput screening and analysis of large numbers of samples in a short time.
• Low-cost reagents: ELISA uses inexpensive reagents such as enzymes, substrates and buffers that are readily available and stable. The cost per test is much lower than other methods such as radioimmunoassay or fluorescence immunoassay.
• No requirement of any unsafe materials: ELISA does not involve the use of any radioactive or toxic substances that may pose health or environmental hazards. ELISA is a safe and eco-friendly technique that can be performed in any laboratory setting.

ELISA is a powerful technique for detecting and quantifying antigens and antibodies, but it also has some limitations that need to be considered. Some of the main limitations are:

• Work-Intensive: ELISA requires multiple steps of washing, incubation, and addition of reagents, which can be time-consuming and laborious. Moreover, each step needs to be performed carefully and accurately to avoid errors and contamination. ELISA also requires specialized equipment such as microtitre plates, pipettes, and ELISA readers, which may not be available or affordable in some settings.
• Preparation of enzyme-linked antibodies: ELISA relies on the availability and quality of enzyme-linked antibodies, which are used to detect the antigen-antibody interaction. However, preparing these antibodies can be difficult and expensive, as they need to be purified, conjugated with the appropriate enzyme, and tested for specificity and activity. Furthermore, different types of ELISA may require different types of enzyme-linked antibodies, which adds to the complexity and cost of the technique.
• Stability of antibodies: Antibodies are sensitive to environmental factors such as temperature, pH, and light, which can affect their stability and function. Therefore, proper refrigeration and storage conditions are required for transport and preservation of antibodies. Otherwise, they may lose their activity or specificity over time, leading to false results or reduced sensitivity. Additionally, some antibodies may cross-react with other antigens or substances in the sample, causing non-specific binding and background noise.

We are Compiling this Section. Thanks for your understanding.