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Questions and Answers
Direct Immunofluorescence (DIF) detects ______ in a sample using a primary antibody directly conjugated to a fluorescent dye.
Direct Immunofluorescence (DIF) detects ______ in a sample using a primary antibody directly conjugated to a fluorescent dye.
antigens
Indirect Immunofluorescence Assay (IDIFA) detects ______ in a patient’s serum by binding them to antigens and using a secondary fluorescent antibody.
Indirect Immunofluorescence Assay (IDIFA) detects ______ in a patient’s serum by binding them to antigens and using a secondary fluorescent antibody.
antibodies
In DIF, the ______ antibody is fluorescently labeled.
In DIF, the ______ antibody is fluorescently labeled.
primary
In IDIFA, both the ______ and secondary antibody are fluorescently labeled.
In IDIFA, both the ______ and secondary antibody are fluorescently labeled.
DIF involves a single step: antigen + ______ antibody.
DIF involves a single step: antigen + ______ antibody.
IDIFA involves two steps: antigen + primary antibody, then ______ antibody.
IDIFA involves two steps: antigen + primary antibody, then ______ antibody.
DIF identifies ______ directly in tissue or cells.
DIF identifies ______ directly in tissue or cells.
IDIFA detects ______ present in a serum sample.
IDIFA detects ______ present in a serum sample.
DIF is highly specific but limited to the ______ antibody.
DIF is highly specific but limited to the ______ antibody.
IDIFA is more versatile, as different primary antibodies can use the same ______ antibody.
IDIFA is more versatile, as different primary antibodies can use the same ______ antibody.
Immunofluorescence (IF) is a technique that visualizes a specific ______ by binding a specific antibody conjugated with a fluorescence dye.
Immunofluorescence (IF) is a technique that visualizes a specific ______ by binding a specific antibody conjugated with a fluorescence dye.
Fluorescent dyes absorb ______ wavelength UV radiation and emit light of a longer wavelength, often in the visible spectrum.
Fluorescent dyes absorb ______ wavelength UV radiation and emit light of a longer wavelength, often in the visible spectrum.
Fluorescein isothiocyanate (FITC) is a common fluorescent dye that emits ______ light.
Fluorescein isothiocyanate (FITC) is a common fluorescent dye that emits ______ light.
Tetramethylrhodamine, another fluorescent dye, emits ______ light.
Tetramethylrhodamine, another fluorescent dye, emits ______ light.
Immunofluorescence can be used to make ______ more readily visible to the naked eye.
Immunofluorescence can be used to make ______ more readily visible to the naked eye.
Direct Immunofluorescence (DIF) detects specific ______ in tissue sections or cells using a single, fluorescently labeled primary antibody.
Direct Immunofluorescence (DIF) detects specific ______ in tissue sections or cells using a single, fluorescently labeled primary antibody.
In DIF, a fluorescently labeled antibody is applied to the sample and allowed to ______ to the antigen if present.
In DIF, a fluorescently labeled antibody is applied to the sample and allowed to ______ to the antigen if present.
Indirect Immunofluorescence Assay (IDIFA) detects the presence of specific ______ in a biological sample, typically serum.
Indirect Immunofluorescence Assay (IDIFA) detects the presence of specific ______ in a biological sample, typically serum.
A slide is coated with a specific ______ in IDIFA, and the sample containing antibodies is applied to the slide.
A slide is coated with a specific ______ in IDIFA, and the sample containing antibodies is applied to the slide.
In IDIFA, a fluorescently labeled ______ antibody binds to the patient's antibodies, leading to fluorescence.
In IDIFA, a fluorescently labeled ______ antibody binds to the patient's antibodies, leading to fluorescence.
Flashcards
Direct Immunofluorescence (DIF)
Direct Immunofluorescence (DIF)
Assay that detects antigens using a directly conjugated fluorescent primary antibody.
Indirect Immunofluorescence Assay (IDIFA)
Indirect Immunofluorescence Assay (IDIFA)
Assay that detects antibodies in serum by binding to antigens and using a secondary fluorescent antibody.
Fluorescence Label
Fluorescence Label
A fluorescent dye attached to antibodies for visualization under a microscope.
Specificity
Specificity
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Sensitivity
Sensitivity
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Applications of DIF
Applications of DIF
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Applications of IDIFA
Applications of IDIFA
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Time and Cost of DIF
Time and Cost of DIF
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Time and Cost of IDIFA
Time and Cost of IDIFA
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Steps in DIF
Steps in DIF
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Immunofluorescence (IF)
Immunofluorescence (IF)
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Fluorescence Dye
Fluorescence Dye
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Fluorescein Isothiocyanate (FITC)
Fluorescein Isothiocyanate (FITC)
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Tetramethylrhodamine
Tetramethylrhodamine
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Sample Preparation in DIF
Sample Preparation in DIF
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Patient Serum Incubation in IDIFA
Patient Serum Incubation in IDIFA
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Fluorescence Detection
Fluorescence Detection
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Antigen Coating
Antigen Coating
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Study Notes
Immunofluorescence
- Immunofluorescence is a technique used to visualize specific antigens.
- It involves binding a specific antibody chemically conjugated with a fluorescent dye to the antigen.
- Fluorochromes are dyes that absorb short wavelength UV radiation and emit longer wavelength fluorescence (visible light).
- Fluorescein isothiocyanate (FITC) emits green light. Tetramethylrhodamine emits red light.
- This technique can make viral plaques more visible to the human eye.
Principle of Immunofluorescence (IF)
- IF is based on the specific antigen-antibody interaction.
- A labeled antibody binds to its corresponding antigen.
- Exposure to ultraviolet (UV) or specific wavelengths of light causes the fluorescent dye to emit visible light.
- This visualization allows observation of the antigen-antibody complexes.
Requirements of Immunofluorescence
- A slide
- A tagged antibody
- A fluorescent microscope
- Specimens
Methods of Immunofluorescence Assay
- There are two types: direct and indirect.
Direct Immunofluorescence Assay (DIFA)
- A single fluorescently labeled primary antibody directly binds to the target antigen.
- The sample preparation includes fixing tissue sections or cells on a slide.
- The fluorescently labeled antibody is applied to the sample.
- Incubation allows the antibody to bind to the antigen if it is present.
- Using a fluorescence microscope, the presence of fluorescence confirms the target antigen.
Indirect Immunofluorescence Assay (IDIFA)
- Detects antibodies in serum.
- A slide or substrate is coated with a specific antigen.
- The patient's serum sample (containing antibodies) is applied to the slide.
- If target antibodies are present, they bind to the antigen.
- A fluorescently labeled secondary antibody that binds to the patient's antibodies is added.
- Using a fluorescence microscope, the presence of fluorescence confirms the specific antibodies.
Result
- Confocal images can detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus.
Comparison of Direct and Indirect IF
| Aspect | Direct IF | Indirect IF |
|---|---|---|
| Definition | Detects antigens using a primary antibody directly conjugated to a fluorescent dye. | Detects antibodies using a secondary antibody binding to primary antibodies labeled with target antigen. |
| Fluorescence Label | Primary antibody is labeled. | Secondary antibody is labeled. |
| Steps | Single step: Antigen + labeled antibody. | Two steps: Antigen + primary antibody + secondary antibody. |
| Purpose | Identifies antigens directly. | Detects antibodies in a sample. |
| Specificity | Highly specific to the labeled antibody. | More versatile, as different primary antibodies can use the same labeled secondary antibody. |
| Sensitivity | Lower sensitivity due to lack of signal amplification. | Higher sensitivity due to amplification from secondary antibody binding. |
| Applications | Pathology, detecting tissue-bound antigens | Serological tests, autoimmune, infectious disease studies |
| Time and Cost | Faster and generally cheaper | More time-consuming and potentially more expensive |
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