20.5 Fluorescent Antibody Techniques
3 min read•june 18, 2024
techniques revolutionize microbial detection and analysis. These methods use glowing markers attached to antibodies, making it easier to spot specific microbes or molecules. They're faster and more sensitive than older methods, allowing scientists to see things that were once invisible.
These techniques come in two flavors: direct and indirect. Direct methods are quick and simple, while indirect methods offer more sensitivity. Both are crucial in microbiology, helping researchers identify pathogens, study cell populations, and even sort specific cell types for further study.
Fluorescent Antibody Techniques
Advantages of immunofluorescent antibody assays
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- Increased sensitivity
- Fluorescent labels amplify signal enabling improved detection of low abundance targets (single molecules or cells)
- Improved specificity
- Antibodies bind with high affinity to specific reducing non-specific binding
- Multiple fluorescent labels allow simultaneous detection of several targets (, proteins, or cells)
- Rapid and simple protocols
- Shorter incubation times compared to enzyme-based assays (ELISA or Western blot)
- Fewer steps and reagents required streamlining workflow
- Compatibility with live cells
- Fluorescent labeling does not require cell or preserving native cellular state
- Allows real-time monitoring of cellular processes and interactions (protein trafficking, cell signaling)
Direct vs indirect fluorescent antibody techniques
- Direct fluorescent antibody technique
- Primary antibody directly conjugated to fluorescent label (fluorophore)
- Faster protocol with fewer steps reducing time and resources
- Reduced risk of cross-reactivity and non-specific binding improving signal-to-noise ratio
- Suitable for detecting abundant (cell surface markers, viral proteins)
- Applications: Microorganism identification (bacteria, fungi), cell surface marker detection (CD antigens, receptors)
- Indirect fluorescent antibody technique
- Unlabeled primary antibody binds to target antigen
- Fluorescently labeled binds to primary antibody
- Signal amplification due to multiple secondary antibodies binding each primary antibody increasing sensitivity
- Higher sensitivity for detecting low abundance antigens (intracellular proteins, rare )
- Applications: Intracellular antigen detection (cytokines, transcription factors), histopathology (tissue sections), immunohistochemistry (cancer markers)
Principles of fluorescence in antibody techniques
- : Light emission by a substance after absorbing light or other electromagnetic radiation
- : absorb light at a specific wavelength (excitation) and emit light at a longer wavelength (emission)
- : Specific interaction between antibody and antigen, allowing targeted fluorescent labeling
- : Reduction in fluorescence intensity due to molecular interactions or environmental factors
Flow cytometry for cell population quantification
- Cells in suspension labeled with fluorescent antibodies specific to target antigens (surface or intracellular markers)
- Labeled cells passed through a flow cell in a single file allowing individual cell analysis
- Lasers excite the fluorescent labels as cells pass through the interrogation point
- Scattered light and emitted fluorescence collected by detectors
- (FSC) correlates with cell size distinguishing cell types
- (SSC) correlates with cell granularity or complexity indicating cell state
- Signals converted to digital data for each individual cell enabling high-throughput analysis
- Data analysis software generates plots and statistics
- Populations identified based on their fluorescence intensity and light scattering properties
- Quantification of cell subsets as a percentage of total cells or absolute counts (cells/μL)
- Applications: (lymphocyte subsets), cell cycle analysis (DNA content), apoptosis detection (annexin V)
Process and applications of FACS
- Process:
- Cells labeled with fluorescent antibodies and analyzed by
- Desired cell populations identified based on their fluorescent and light scattering properties
- Sorting parameters set to define the target population (gates and thresholds)
- Cells passed through a vibrating nozzle, forming droplets containing single cells
- Droplets containing target cells electrically charged
- Charged droplets deflected by an electric field into collection tubes
- Sorted cells collected for further analysis or culture
- Applications:
- Isolation of rare cell populations (stem cells, circulating tumor cells) for research and therapeutics
- Purification of specific cell subsets for functional studies (T cell subsets, B cell stages)
- Single-cell cloning for monoclonal antibody production (hybridoma technology)
- Enrichment of genetically modified cells after transfection or transduction (reporter gene expression)
- Detection and isolation of microbial cells from environmental samples (water quality testing, food safety)
Key Terms to Review (51)
Antibody-Antigen Binding: Antibody-antigen binding is the specific interaction between an antibody and its complementary antigen. This fundamental process is central to the adaptive immune response, allowing the body to recognize and neutralize foreign pathogens and substances.
Antigen-Antibody Complex: An antigen-antibody complex is the stable, noncovalent interaction that forms when an antibody binds to its specific antigen. This complex is a fundamental concept in immunology and is crucial for understanding the mechanisms behind fluorescent antibody techniques.
Antigens: Antigens are molecules or molecular structures that can be recognized by the immune system, specifically by antibodies, B cells, or T cells. They trigger an immune response when detected in the body.
Antigens: Antigens are any substances that can induce an immune response and bind to specific antibodies or receptors on immune cells. They are a crucial component of the adaptive immune system, playing a central role in vaccine development and fluorescent antibody techniques.
Antinuclear antibodies: Antinuclear antibodies (ANAs) are autoantibodies that target substances found in the nucleus of cells. They are commonly used as markers in the diagnosis of autoimmune diseases.
Automated, cell-counting: Automated cell-counting is a technique that uses automation and technology to count cells, often using image analysis or flow cytometry. It ensures accuracy and efficiency in quantifying cell populations.
Confocal Microscopy: Confocal microscopy is an advanced imaging technique that uses a focused laser beam to scan a specimen point-by-point, producing high-resolution, three-dimensional images. This method is particularly useful in the context of fluorescent antibody techniques, as it allows for the visualization of specific cellular structures and biomolecules with exceptional clarity and detail.
Direct fluorescent antibody (DFA) tests: Direct fluorescent antibody (DFA) tests use fluorescent-labeled antibodies to directly detect the presence of specific antigens in a sample. They are commonly used in microbiology to identify pathogens quickly and accurately.
Direct Immunofluorescence: Direct immunofluorescence is a technique used in fluorescent antibody techniques to directly label an antigen with a fluorescent dye. It involves the use of a primary antibody that is directly conjugated to a fluorescent molecule, allowing for the visualization and localization of a specific target within a sample.
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.
Epitopes: Epitopes are specific regions on an antigen that are recognized and bound by antibodies or T-cell receptors. They play a crucial role in the specificity of the immune response.
Epitopes: Epitopes, also known as antigenic determinants, are the specific regions on the surface of an antigen that are recognized and bound by antibodies or T cell receptors. These unique molecular structures on the surface of pathogens or foreign substances are crucial for the activation of the adaptive immune response.
Excitation and Emission: Excitation and emission are fundamental concepts in the field of fluorescence, which is a key technique used in fluorescent antibody methods. Excitation refers to the process of absorbing energy by a fluorescent molecule, causing it to enter an excited state. Emission is the subsequent release of that absorbed energy in the form of light, allowing the molecule to return to its ground state.
FACS: FACS, or Fluorescence-Activated Cell Sorting, is a specialized type of flow cytometry that uses fluorescent dyes or antibodies to identify and separate specific cell populations from a heterogeneous sample. It is a powerful technique used in various fields, including immunology, cell biology, and microbiology, to analyze and sort cells based on their unique physical and chemical properties.
Fixation: Fixation is the process of stabilizing and preserving biological specimens, such as cells or tissues, to maintain their structural and chemical integrity for microscopic examination or further analysis. This technique is crucial in both staining microscopic specimens and fluorescent antibody techniques.
Flow cytometry: Flow cytometry is a laboratory technique used to detect and measure physical and chemical characteristics of a population of cells or particles. It uses fluorescently labeled antibodies to analyze cell populations in suspension.
Flow Cytometry: Flow cytometry is a powerful analytical technique that allows for the rapid measurement and analysis of multiple physical and chemical characteristics of individual cells or particles as they flow in a fluid stream through a beam of light. This technology has become an essential tool in the fields of immunology, cell biology, and microbiology, particularly in the context of T Lymphocytes and Cellular Immunity, as well as Fluorescent Antibody Techniques.
Fluorescein Isothiocyanate: Fluorescein isothiocyanate (FITC) is a fluorescent dye commonly used in fluorescent antibody techniques to label and visualize specific biomolecules or cellular structures. It is a derivative of the fluorescent compound fluorescein, which emits a bright green fluorescence when excited by blue or ultraviolet light.
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.
Fluorescence Microscopy: Fluorescence microscopy is a powerful imaging technique that utilizes the phenomenon of fluorescence to visualize and study biological samples. It involves the use of fluorescent probes or markers that emit light when excited by a specific wavelength of light, allowing for the selective labeling and detection of specific molecules or structures within cells and tissues.
Fluorescence Quenching: Fluorescence quenching is a process in which the emission of fluorescent light from a fluorophore is reduced or suppressed due to various interactions or energy transfers. This phenomenon is widely utilized in fluorescent antibody techniques, providing valuable insights into molecular interactions and environmental changes.
Fluorescence-activated cell sorter (FACS): A fluorescence-activated cell sorter (FACS) is a specialized type of flow cytometry that sorts a heterogeneous mixture of cells into distinct populations based on fluorescent labeling. It enables researchers to isolate specific cell types for further analysis or experimentation.
Fluorescent Antibody: A fluorescent antibody is an antibody molecule that has been conjugated with a fluorescent dye or fluorophore. This technique allows for the visualization and identification of specific target molecules or cells through the emission of fluorescent light when the antibody binds to its target.
Fluorescent antibody (FA) techniques: Fluorescent antibody (FA) techniques are laboratory methods used to detect specific antigens or antibodies by tagging them with a fluorescent dye. These techniques are widely used in immunology to visualize and identify cellular components under a fluorescence microscope.
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.
Fluorophores: Fluorophores are molecules that can absorb light energy and then re-emit that energy as light of a longer wavelength. This property of fluorescence is a fundamental principle underlying the use of fluorescent antibody techniques in various areas of microbiology and immunology.
Forward Scatter: Forward scatter, in the context of fluorescent antibody techniques, refers to the light scattering that occurs when a beam of light hits a particle or cell at a shallow angle, typically less than 20 degrees from the incident light. This scattered light provides information about the size and granularity of the particle or cell being analyzed.
Group A strep: Group A strep (GAS) is a bacterium known as Streptococcus pyogenes, responsible for illnesses ranging from mild infections to severe diseases. It is often detected using immunological techniques including fluorescent antibody methods.
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).
Humanized monoclonal antibodies: Humanized monoclonal antibodies are engineered antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibodies produced naturally in humans. This reduces the immune response against them when used therapeutically.
Immunofluorescence: Immunofluorescence is a technique that uses fluorescent dyes or labels to visualize the presence and location of specific proteins, such as antibodies or antigens, within cells or tissues. It is a powerful tool for detecting and analyzing antigen-antibody complexes and has various applications in the field of microbiology.
Immunoglobulins: Immunoglobulins are specialized glycoproteins produced by B cells that function as antibodies in the immune response. They play a critical role in identifying and neutralizing pathogens such as bacteria and viruses.
Immunoglobulins: Immunoglobulins, also known as antibodies, are glycoprotein molecules produced by plasma B cells that recognize and bind to specific antigens, initiating an immune response. They play a crucial role in the humoral immune system and are involved in various immunological processes described in the topics of 18.2 Major Histocompatibility Complexes and Antigen-Presenting Cells, 18.4 B Lymphocytes and Humoral Immunity, 19.4 Immunodeficiency, and 20.5 Fluorescent Antibody Techniques.
Immunophenotyping: Immunophenotyping is the process of identifying and characterizing cells based on their expression of specific cell surface markers or antigens. It is a powerful tool used in the diagnosis and monitoring of various immunological disorders and diseases.
Indirect fluorescent antibody (IFA) tests: Indirect fluorescent antibody (IFA) tests are a diagnostic tool used to detect the presence of specific antibodies in a patient’s serum. The technique involves binding antibodies to antigens on a slide and using a secondary, fluorescently-labeled antibody to visualize the interaction under a microscope.
Indirect Immunofluorescence: Indirect immunofluorescence is a technique used in fluorescent antibody techniques to detect the presence of specific antigens or target molecules in a sample. It involves the use of a primary antibody that binds to the target, followed by a fluorescently labeled secondary antibody that recognizes the primary antibody, amplifying the signal and enabling visualization under a fluorescence microscope.
Legionella pneumophila: Legionella pneumophila is a Gram-negative bacterium that is the primary cause of Legionnaires' disease, a severe form of pneumonia. It thrives in aquatic environments and can infect humans through inhalation of contaminated water droplets.
Monoclonal Antibodies: Monoclonal antibodies are identical antibodies produced by a single clone of B cells, each recognizing a specific epitope on an antigen. They are a crucial tool in immunology, diagnostics, and therapeutic applications, with their ability to target specific molecules with high affinity and specificity.
Permeabilization: Permeabilization is the process of temporarily increasing the permeability of cell membranes to allow the passage of molecules or substances that would normally be excluded. This is an important technique used in various biological and biomedical applications, such as fluorescent antibody techniques.
Polyclonal antibodies: 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 laboratories to detect antigens due to their high sensitivity.
Polyclonal Antibodies: Polyclonal antibodies are a diverse mixture of antibodies produced by multiple B-cell lineages in response to an antigen. They recognize multiple epitopes on the same antigen and are commonly used in various immunological techniques, such as those covered in Chapters 20.1 and 20.5.
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.
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.
Side Scatter: Side scatter (SSC) is a parameter in flow cytometry that measures the amount of light scattered at an angle (typically 90 degrees) to the path of the incident laser beam. This scattered light provides information about the internal complexity or granularity of a cell, such as the size and shape of the cell's nucleus, the amount and type of cytoplasmic granules, and the roughness of the cell surface.
Strep throat: Strep throat is a bacterial infection caused by Streptococcus pyogenes, characterized by inflammation and pain in the throat. It is highly contagious and spreads through respiratory droplets.
Streptococcus pyogenes: Streptococcus pyogenes is a Gram-positive bacterium responsible for various infections in humans. It is known for causing diseases such as strep throat, scarlet fever, and rheumatic fever.
Syphilis: Syphilis is a sexually transmitted infection caused by the bacterium Treponema pallidum. It progresses through distinct stages and can lead to serious health complications if left untreated.
Systemic lupus erythematosus: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the immune system attacking its own tissues, leading to inflammation and tissue damage. It often affects the skin, joints, kidneys, brain, and other organs.
Titer: Titer refers to the concentration of a substance in a solution, often used to measure the level of antibodies or antigens in a serum. It is determined by serially diluting the sample and identifying the highest dilution at which activity is still detectable.
VDRL test: The VDRL test, or Venereal Disease Research Laboratory test, is a blood test used to detect antibodies produced in response to the bacterium Treponema pallidum, which causes syphilis. It is a non-treponemal test that identifies reagin antibodies.