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Questions and Answers
What is the primary function of a vaccine?
What is the primary function of a vaccine?
Which method is commonly used to produce proteins for vaccines?
Which method is commonly used to produce proteins for vaccines?
What is a significant challenge in developing vaccines for certain diseases?
What is a significant challenge in developing vaccines for certain diseases?
Which of the following is NOT a side effect commonly associated with vaccinations?
Which of the following is NOT a side effect commonly associated with vaccinations?
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Which term best describes small chemical compounds that can elicit an immune response?
Which term best describes small chemical compounds that can elicit an immune response?
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What is the primary purpose of a western blot in molecular biology?
What is the primary purpose of a western blot in molecular biology?
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Why are haptens not considered immunogenic on their own?
Why are haptens not considered immunogenic on their own?
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What is needed for a hapten to function as an immunogen?
What is needed for a hapten to function as an immunogen?
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In immunofluorescence, what is the primary goal?
In immunofluorescence, what is the primary goal?
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What role do carrier proteins play when haptens are involved in immune response?
What role do carrier proteins play when haptens are involved in immune response?
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Study Notes
Flow Cytometry
- Flow cytometry is used to analyze biological samples.
- Samples are labeled with fluorescent markers.
- Cells flow in a single file through a laser beam.
- Fluorescent molecules are activated and emit light.
- Light is filtered and detected by detectors (photomultiplier tube).
- Analog fluorescent signals are converted to digital signals.
Flow Cytometer Components
- Flow system (fluidics): Cells are suspended and passed in a single file.
- Optical system (light sensing): Laser beam scatters light and emits fluorescence to measure size, granularity, and fluorescence intensity.
- Electronic system (signal processing): Emitted light is converted to digital signals for analysis.
Forward Scatter Channel (FSC)
- Light scattered in the forward direction is measured.
- Intensity is related to cell size and refractive index.
Side Scatter Channel (SSC)
- Light scattered at 90 degrees to the laser path is measured.
- Intensity is proportional to the amount of cytosolic structures (granules, inclusions) in the cell.
Study of FSC and SSC
- Allows for differentiation of different cell types.
- Allows identification of lymphocytes, red blood cells, debris, dead cells, granulocytes, and monocytes.
Method Application
- Qualitative and quantitative analysis of cell populations.
- Uses fresh, unfixed tissue or body fluid.
- Measured properties include particle size, granularity, internal complexity, and fluorescence intensity.
- Common materials for analysis: blood, bone marrow aspirate, and lymph node suspensions.
Antibodies in Immuno-Detection Methods
- Immuno blotting (Western blot)
- Immunofluorescence
- Screening of DNA libraries
Western Blot
- A widely accepted technique for detecting specific proteins.
- Used to detect proteins like HIV.
- A set up is used with filter paper, gel, and membrane.
- Protein samples are run through gel electrophoresis.
Immunofluorescence
- Tissue containing the antigen of interest is identified.
- Monoclonal antibodies are added, labeled with a fluorochrome.
- Unbound antibodies are removed.
- Examined under a fluorescence microscope to detect the antigen.
- Used for detecting Toxoplasma and Leishmania.
Screening DNA Libraries
- Radioactively labeled DNA probe is used to identify colonies of bacteria with recombinant plasmids.
- DNA from the colonies is isolated and analyzed.
- The position of colonies with plasmids of interest is determined through autoradiography.
Haptens
- Low molecular weight molecules.
- Small organic molecules that are antigenic but not immunogenic.
- Bind to immune cells but do not induce a humoral or cell-mediated response.
Why Haptens are Not Immunogenic
- By themselves, they cannot function as immunogenic epitopes.
- Multiple hapten molecules coupled to a carrier protein or non-immunogenic homopolymer make them accessible to the immune system, allowing them to function as immunogens.
Hapten-Carrier Conjugates
- Haptens induce an immune response when covalently bound to a larger protein called a carrier protein.
- Carrier proteins are antigenic and immunogenic.
Karl Landsteiner
- 1901: Discovered the A-B-O blood typing system.
- Awarded the Nobel Prize twenty-nine years later.
- Demonstrated that human blood types and properties are not the same.
- Created the key for matching blood types from a donor to a patient.
- Mixing a wrong type could result in problematic and sometimes fatal consequences.
Vaccine
- Biological preparation establishing or improving immunity to a particular disease.
- Edward Jenner, 1796, observed protection against smallpox using cowpox.
Vaccine Design and Production
- Vaccines use either killed or live microbes.
- Vaccination introduces a non-virulent microbe.
- Your body produces antibodies against the microbes, but you avoid the disease.
- Subsequent exposure to that microbe results in a ready antibody supply for defense.
Problems with Vaccines
- Can cause allergic reactions, aches, or pains, and fever.
- Consistent production of certain virus-based vaccines is difficult.
- Vaccination against diseases like HIV/AIDS and malaria is complicated.
Vaccine Production
- Typically, pathogen proteins trigger antibodies.
- Isolate the pathogen gene for antibody production and insert into bacteria like E. coli for large-scale protein production.
- Using biotechnology, scientists developed vaccines against Hepatitis B and Meningitis.
DNA Vaccines
- Injecting naked DNA into muscle or skin cells triggers an immune response.
- Researchers have been trying to produce DNA-based vaccines, but they still have limitations.
Vaccine Delivery Systems
- Current vaccine development often uses live viruses, coat proteins or DNA-based methods.
- Large-scale vaccine production requires costly facilities and procedures.
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Description
This quiz covers the fundamental principles of flow cytometry, including its applications and the main components of a flow cytometer. Participants will learn about the flow system, optical system, and electronic system involved in this analytical technique, as well as the significance of forward and side scatter channels in cell analysis.
