20.4 EIAs and ELISAs
3 min read•june 18, 2024
immunoassays are powerful tools for detecting specific molecules in biological samples. These techniques use enzyme-labeled antibodies or antigens to produce measurable signals, enabling the detection of various substances like viruses, bacteria, and antibodies.
ELISA, a type of enzyme immunoassay, is widely used in medical diagnostics and research. It can be performed in different formats, such as direct, indirect, and , each with unique advantages for detecting antigens or antibodies in samples like blood or urine.
Enzyme Immunoassays and ELISA Techniques
EIA vs FEIA vs ELISA techniques
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- Enzyme Immunoassay () uses an or to detect the presence of a specific or (, hepatitis)
- Enzyme catalyzes a reaction producing a detectable signal such as color change (), (), or
- Includes ELISA and FEIA as specific types of EIA
- Fluorescent Enzyme Immunoassay (FEIA) is a type of EIA that uses an enzyme-labeled antibody or antigen
- Enzyme catalyzes a reaction producing a fluorescent signal (, )
- More sensitive than colorimetric EIA due to the use of fluorescence detection enables detection of lower concentrations of analytes
- Enzyme-Linked Immunosorbent Assay (ELISA) is a type of EIA that uses an enzyme-labeled antibody or antigen
- Enzyme catalyzes a reaction producing a colorimetric signal (, )
- Performed on a solid surface coated with an antigen or antibody (, beads)
- Widely used for the quantitative detection of antigens or antibodies in a sample (serum, plasma, urine)
Immunohistochemistry vs immunocytochemistry applications
- detects antigens in tissue sections preserving tissue architecture
- Involves fixing and sectioning tissue samples before applying labeled antibodies (paraffin-embedded, frozen)
- Allows for the localization of antigens within specific cell types or structures (tumor cells, lymphocytes)
- Applications in immune response analysis include:
- Identifying immune cell infiltrates in tissues during an immune response (CD4+ T cells, macrophages)
- Assessing the expression of cytokines, chemokines, or other immune-related molecules in tissue samples (IL-6, TNF-α)
- detects antigens in individual cells or cell smears without preserving tissue architecture
- Involves fixing cells on a slide before applying labeled antibodies (cytospin, smear)
- Allows for the detection of antigens in specific cell types (lymphocytes, monocytes)
- Applications in immune response analysis include:
- Detecting the presence of specific immune cell populations in blood or other fluid samples (B cells, NK cells)
- Assessing the expression of cell surface markers or intracellular molecules in isolated immune cells (CD3, CD19)
Direct and indirect ELISA methods
- detects the presence and quantity of a specific antigen in a sample
- Principles:
- Antigen is immobilized on a solid surface (microtiter plate)
- Enzyme-labeled specific to the antigen is added
- is added, and the enzyme catalyzes a reaction producing a detectable signal
- Applications include:
- Detecting the presence of a specific antigen in a sample (viral proteins, bacterial antigens)
- Quantifying the amount of antigen present in a sample (pg/mL, ng/mL)
- Principles:
- detects the presence and quantity of specific antibodies in a sample
- Principles:
- Antigen is immobilized on a solid surface (microtiter plate)
- Unlabeled specific to the antigen is added
- Enzyme-labeled specific to the primary antibody is added
- Substrate is added, and the enzyme catalyzes a reaction producing a detectable signal
- Applications include:
- Detecting the presence of specific antibodies in a sample (serum antibodies against a pathogen)
- Quantifying the amount of antibody present in a sample (, concentration)
- Allows for increased sensitivity and specificity compared to due to the use of a amplifies signal
- Principles:
Additional ELISA types and steps
- : A highly specific and sensitive method where the antigen is "sandwiched" between two antibodies
- : Used when the antigen is small and has only one epitope, involving competition between sample antigen and added labeled antigen
- Common steps in ELISA protocols:
- : Prevents non-specific binding of antibodies to the plate surface
- : Removes unbound reagents between each step of the assay
- : Enhances the sensitivity of the assay through various methods
- : The final step where the enzyme converts the substrate into a detectable signal
Key Terms to Review (57)
Antibody: An antibody is a large, Y-shaped protein produced by the immune system to identify and neutralize foreign substances, such as bacteria and viruses. Antibodies play a crucial role in the context of EIAs and ELISAs, which are immunoassay techniques used to detect and measure the presence of specific antibodies or antigens in a sample.
Antigen: An antigen is any substance that induces an immune response in the body, specifically by triggering the production of antibodies. They are typically foreign proteins or polysaccharides found on the surface of pathogens like bacteria and viruses.
Antigen: An antigen is a substance, typically a protein or polysaccharide, that is capable of stimulating an immune response and triggering the production of antibodies. Antigens are central to the topics of polyclonal and monoclonal antibody production, as well as enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs).
Blocking Step: The blocking step is a critical component in enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs), where it serves to prevent non-specific binding of antibodies or other proteins to the solid support, ensuring accurate and reliable results.
Chemiluminescence: Chemiluminescence is the emission of light as a result of a chemical reaction, where the energy released from the reaction is converted into light energy. This process is commonly used in various analytical techniques, including enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs), to detect and quantify specific analytes in a sample.
Chromogen: A chromogen is a substance that can be converted into a colored compound, often used in immunoassays to indicate the presence of specific antigens or antibodies. In laboratory analysis, chromogens are key components for visualizing results in assays like EIAs and ELISAs.
Colorimetric: Colorimetric refers to a quantitative analytical technique that measures the intensity of color in a solution to determine the concentration of a specific substance. It is a widely used method in various fields, including biochemistry, analytical chemistry, and clinical diagnostics.
Competitive ELISA: Competitive ELISA, or competitive enzyme-linked immunosorbent assay, is an analytical technique used to detect and quantify specific antigens in a sample by measuring the ability of the sample to compete with a known amount of labeled antigen for a limited number of antibody binding sites.
Constant region: The constant region is the part of an antibody's structure that remains the same among different antibodies of the same class. It is responsible for mediating interactions with immune cells and effector functions.
Direct ELISA: Direct ELISA is a laboratory technique used to detect antigens in a sample using an enzyme-linked antibody that directly binds to the target antigen. It provides quick and specific results with fewer steps compared to other ELISA methods.
Direct ELISA: Direct ELISA (Enzyme-Linked Immunosorbent Assay) is an immunoassay technique used to detect and quantify specific antigens in a sample. It involves the direct attachment of an enzyme-labeled antibody to the target antigen, allowing for the measurement of the antigen's presence and concentration.
Direct immunofluorescence assay (DFA): Direct immunofluorescence assay (DFA) is a laboratory technique used to detect specific antigens in tissue samples by employing fluorescently-labeled antibodies. It allows for the visualization of antigen-antibody complexes under a fluorescence microscope.
EIA: EIA, or Enzyme Immunoassay, is a widely used analytical technique that employs enzymes to detect and quantify specific target molecules, such as proteins, hormones, or other biomolecules, in a sample. EIAs are commonly utilized in various fields, including clinical diagnostics, environmental monitoring, and research applications.
Enzyme: Enzymes are biological catalysts that accelerate the rate of chemical reactions in living organisms without being consumed or altered themselves. They play a crucial role in facilitating and regulating the vast array of biochemical processes that sustain life, including those involved in EIAs and ELISAs.
Enzyme immunoassays (EIAs): Enzyme immunoassays (EIAs) are biochemical tests that use antibodies and color change to identify the presence of a substance, typically an antigen or antibody. They are widely used for detecting and quantifying specific proteins in samples.
Enzyme-Labeled Antibody: An enzyme-labeled antibody is an immunological detection technique where an antibody is conjugated with an enzyme, allowing for the indirect detection and quantification of a target antigen through a colorimetric or fluorescent signal generated by the enzymatic reaction.
Enzyme-Labeled Antigen: An enzyme-labeled antigen is a molecule that consists of an antigen (a substance that can induce an immune response) chemically coupled to an enzyme. This complex is used in various immunoassay techniques, such as Enzyme-Linked Immunosorbent Assays (ELISAs) and Enzyme Immunoassays (EIAs), to detect and quantify the presence of specific antibodies or antigens in a sample.
Enzyme-linked immunosorbent assays (ELISAs): Enzyme-linked immunosorbent assays (ELISAs) are laboratory techniques used to detect and quantify substances such as proteins, antibodies, and hormones. They rely on enzyme-mediated color changes to indicate the presence of a target molecule.
Enzyme-Substrate Reaction: An enzyme-substrate reaction is a fundamental process in biochemistry where an enzyme, a biological catalyst, binds to and facilitates the conversion of a substrate, a reactant molecule, into one or more products. This reaction is central to various biological processes, including metabolism, cell signaling, and enzymatic assays such as EIAs and ELISAs.
FEIA: FEIA, or Fluorescent Enzyme Immunoassay, is a type of immunoassay technique that utilizes fluorescent labels to detect and quantify target analytes in a sample. It combines the specificity of antibody-antigen interactions with the sensitivity of fluorescence detection, making it a powerful tool for various applications in the field of diagnostics and research.
Fluorescein: Fluorescein is a fluorescent dye that emits green light when exposed to blue or ultraviolet light. It is commonly used in various biomedical and diagnostic applications, including Enzyme-Linked Immunosorbent Assays (ELISAs) and Enzyme Immunoassays (EIAs).
Fluorescence: Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. This phenomenon is widely used in various analytical techniques, including Enzyme-Linked Immunosorbent Assays (ELISAs) and Fluorescent Antibody Techniques, to detect and quantify specific molecules or cells.
Fluorescence microscope: A fluorescence microscope is a type of optical microscope that uses fluorescence to generate an image. It relies on the emission of light by fluorophores to visualize structures within biological specimens.
Fluorescent enzyme immunoassays (FEIAs): Fluorescent enzyme immunoassays (FEIAs) are laboratory techniques used to detect the presence of antibodies or antigens in a sample by using fluorescent-labeled enzymes. The fluorescence emitted is measured to determine the concentration of the target molecule.
Fluorogen: A fluorogen is a molecule that becomes fluorescent upon binding with another specific molecule or undergoing a chemical reaction. Fluorogens are commonly used in biological assays to detect the presence of target substances.
HIV: HIV (Human Immunodeficiency Virus) is a virus that targets the immune system, specifically CD4 cells (T cells), leading to a progressive failure of the immune system. If left untreated, HIV can lead to AIDS (Acquired Immunodeficiency Syndrome).
Immunoblot: An immunoblot, also known as a Western blot, is a laboratory method used to detect specific proteins in a sample using antibody binding. It is commonly used to analyze the immune response and protein expression.
Immunochromatographic assays: Immunochromatographic assays are rapid diagnostic tests that detect the presence of specific antigens or antibodies in a sample using a combination of immunochemical reactions and chromatography. These assays are commonly used in point-of-care testing due to their simplicity and quick results.
Immunocytochemistry: Immunocytochemistry is a technique that uses antibodies to detect and visualize specific proteins or other molecules within cells. It combines the specificity of antibody-antigen interactions with microscopy to provide information about the localization and distribution of target molecules in cellular structures.
Immunocytochemistry (ICC): Immunocytochemistry (ICC) is a laboratory technique used to visualize the presence and localization of specific proteins or antigens in cells by using labeled antibodies as specific probes. It combines immunological and biochemical methods to identify cellular components.
Immunofiltration: Immunofiltration is a laboratory technique that combines filtration and immunoassay principles to detect specific antigens or antibodies in a sample. It involves passing a liquid sample through a membrane, which captures the target molecule using an antigen-antibody reaction.
Immunohistochemistry: Immunohistochemistry (IHC) is a powerful technique that combines immunology and histology to detect and visualize specific proteins or antigens within cells and tissues. It is widely used in both research and clinical settings to study the distribution and localization of target molecules in biological samples.
Immunohistochemistry (IHC): Immunohistochemistry (IHC) is a laboratory technique used to visualize the presence and localization of specific antigens in tissue sections by using labeled antibodies. It combines histological, immunological, and biochemical techniques to identify cells or tissue components.
Immunostaining: Immunostaining is a laboratory technique used to detect specific proteins or antigens in cells or tissue sections using antibodies. It is widely utilized in microbiology to study the immune response.
Indirect ELISA: Indirect ELISA is a laboratory technique used to detect antibodies in a sample by using an antigen-coated plate and an enzyme-linked secondary antibody. It is commonly employed in diagnostics to measure immune responses.
Indirect ELISA: Indirect ELISA (Enzyme-Linked Immunosorbent Assay) is a type of immunoassay used to detect and quantify the presence of specific antigens or antibodies in a sample. It involves a two-step process where the target antigen is first captured on a solid surface, and then a labeled secondary antibody is used to detect the bound antigen.
Lateral flow tests: Lateral flow tests are a type of rapid immunoassay used to detect the presence or absence of a target analyte, such as an antigen or antibody, within a liquid sample. These tests provide results within minutes and are commonly used for point-of-care testing.
Microtiter Plate: A microtiter plate, also known as a microplate or microwell plate, is a flat plate with multiple small wells used as miniature test tubes. These plates are commonly employed in enzyme-linked immunosorbent assays (EIASs) and enzyme-linked immunosorbent assays (ELISAs), allowing for the simultaneous processing and analysis of multiple samples in a standardized and high-throughput manner.
O-phenylenediamine: o-Phenylenediamine is a colorless, crystalline organic compound with the chemical formula C6H8N2. It is a diamine derivative of benzene, with two amino groups attached to adjacent carbon atoms on the benzene ring. This compound is widely used in various applications, particularly in the context of enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs).
Polyclonal antibody: Polyclonal antibodies are a mixture of antibodies produced by different B cell clones in the body, each recognizing a different epitope on the same antigen. They are commonly used in research and diagnostic applications due to their ability to recognize multiple epitopes.
Pregnancy tests: Pregnancy tests are diagnostic tools that detect the presence of human chorionic gonadotropin (hCG) in urine or blood to determine pregnancy. They often utilize enzyme-linked immunosorbent assays (ELISAs) for high sensitivity and specificity.
Primary antibody: A primary antibody is an immunoglobulin that binds specifically to a target antigen, enabling its detection and study. It is the first antibody used in immunoassays like ELISA to detect the presence of an antigen.
Primary Antibody: A primary antibody is a specific antibody that binds directly to a target antigen in various immunoassays, such as Enzyme-Linked Immunosorbent Assays (ELISAs) and Enzyme Immunoassays (EIAs). It is a crucial component in these techniques used to detect and quantify analytes of interest.
Reverse transcriptase PCR (RT-PCR): Reverse transcriptase PCR (RT-PCR) is a laboratory technique used to amplify and detect RNA sequences by converting them into complementary DNA (cDNA) using reverse transcriptase. This method is essential for studying gene expression and detecting RNA viruses.
Rhodamine: Rhodamine is a fluorescent dye that is commonly used in various biomedical and scientific applications, particularly in the context of enzyme-linked immunosorbent assays (ELISAs) and fluorescent antibody techniques. It is known for its bright, reddish-pink color and its ability to fluoresce when exposed to light of a specific wavelength.
Sandwich ELISA: Sandwich ELISA is an immunoassay technique used to detect and quantify specific antigens in a sample. It involves an antigen being 'sandwiched' between two layers of antibodies.
Sandwich ELISA: Sandwich ELISA is a type of enzyme-linked immunosorbent assay (ELISA) used to detect and quantify specific target analytes, such as proteins, in a sample. It is called a 'sandwich' ELISA because the target analyte is 'sandwiched' between two antibodies - a capture antibody and a detection antibody.
Secondary antibody: A secondary antibody is an antibody that binds to a primary antibody to assist in detection, sorting, and purification processes. It is often conjugated with a marker, such as an enzyme or fluorescent dye, for visualization.
Secondary Antibody: A secondary antibody is an antibody that binds to a primary antibody, which has already bound to its target antigen. Secondary antibodies are used in various immunological techniques, such as Enzyme-Linked Immunosorbent Assays (ELISAs) and Enzyme Immunoassays (EIAs), to amplify the signal and facilitate the detection of the target analyte.
Signal Amplification: Signal amplification refers to the process of increasing the strength or intensity of a signal, such as a biological or electrical signal, to make it more detectable or easier to measure. This concept is particularly important in the context of enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs), where it helps enhance the sensitivity and accuracy of these analytical techniques.
Spectrophotometer: A spectrophotometer is an analytical instrument used to measure the amount of light that a sample absorbs. It quantifies concentrations of compounds in solutions by measuring absorbance at specific wavelengths.
Substrate: In the context of biochemistry, a substrate is a molecule that is acted upon by an enzyme to undergo a chemical reaction. Substrates are the starting materials in enzymatic reactions, which are then converted into different products by the catalytic activity of the enzyme.
Tetramethylbenzidine: Tetramethylbenzidine (TMB) is a colorless, organic compound that is commonly used as a chromogenic substrate in enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs). It is a sensitive and versatile reagent that undergoes a color change when oxidized, allowing for the detection and quantification of target analytes in these immunoassay techniques.
Titer: Titer refers to the quantitative measurement of the amount or concentration of a specific substance, such as an antibody or antigen, in a sample. It is a crucial concept in the context of Enzyme-Linked Immunosorbent Assays (ELISAs) and Enzyme Immunoassays (EIAs), which are widely used analytical techniques in microbiology and immunology.
TORCH test: The TORCH test is a blood test used to screen for infections that can cause birth defects in a fetus. It includes tests for Toxoplasma, Other agents, Rubella, Cytomegalovirus (CMV), and Herpes Simplex Virus (HSV).
Washing Step: The washing step is a crucial component in various analytical techniques, such as Enzyme-Linked Immunosorbent Assays (ELISAs) and Enzyme Immunoassays (EIAs), where it serves to remove unbound or excess reagents from the reaction surface, ensuring the accuracy and specificity of the final results.
Western blot: Western blot is a lab technique used to detect specific proteins in a sample through gel electrophoresis and antibodies. It is widely utilized for confirming the presence of viral proteins and immune responses.