ELISA Lab

What are the two traditional types of antibodies produced?

Polyclonal antibodies and monoclonal antibodies.

What is the main advantage of polyclonal antibodies?

They are simple and inexpensive to produce.

What is a major disadvantage of polyclonal antibodies?

No two batches will be exactly the same.

How are monoclonal antibodies produced?

By fusing B cells from immunized mice with tumor cells to create hybridomas.

What is the purpose of hybridoma cells?

They can be cultured indefinitely to produce monoclonal antibodies.

What is recombinant DNA technology used for in antibody production?

To produce antibodies that resemble human antibodies for therapeutic use.

What is the significance of 'humanized' antibodies like Herceptin?

They can be used as therapeutic agents without eliciting an immune response.

What is the process of hybridoma immortalization?

Combining mouse monoclonal antibody genes with human antibody genes to produce humanized antibodies.

What is phage display?

A technique to generate novel antibodies by inserting antibody genes into bacteriophages.

How are antibodies used in diagnosis and research?

As labeling tools that are linked to detectable signals.

What is ELISA?

Enzyme-Linked Immunosorbent Assay, a method to detect antigens using enzyme-linked antibodies.

What happens in the first step of an ELISA?

Antigens attach to the bottom surface of a test tube.

What is the role of the HRP enzyme in ELISA?

It catalyzes a reaction with a substrate to produce a detectable color change.

What is the main difference between polyclonal and monoclonal antibodies?

Polyclonal antibodies are a mix from multiple B cell clones, while monoclonal antibodies are from a single B cell clone.

What is the function of the B cell population in phage display?

To create a library of antibodies that can be screened for specific antigen binding.

What is the advantage of using bacteria for antibody production?

It requires less time and expense compared to culturing mouse hybridoma cells.

Why are antibodies considered 'magic bullets' in medical therapies?

They can specifically target and deliver drugs to tumor cells.

What is the primary use of labeled antibodies?

To visualize the presence of specific antigens in various assays.

What is the outcome of the antibody binding step in ELISA?

Antibodies linked to HRP enzyme bind specifically to the antigens.

What is a hybridoma?

A hybrid cell line created by fusing B cells with tumor cells.

What is the significance of the timeline in antibody production?

It illustrates the evolution of techniques from polyclonal to monoclonal to phage display.

What are the two methods used to engineer antibodies?

Hybridoma immortalization and phage display.

What type of cells are typically used to produce polyclonal antibodies?

Animals such as rabbits, goats, or sheep.

What is the main challenge with traditional antibodies made in animals?

They are recognized as foreign by the human immune system.

What is the purpose of amplifying antibody DNA in phage display?

To create a diverse library of antibodies for screening against specific antigens.

What is the final step in the ELISA process?

Adding a substrate that reacts with the enzyme to produce a visible signal.

What is the role of the antigen in antibody production?

It stimulates the immune response to produce specific antibodies.

What is the main advantage of using indirect detection of antibodies?

It reduces time and cost by using one type of enzyme-linked secondary antibody for multiple primary antibodies.

How does the indirect detection method amplify the signal?

The primary antibody has many different epitopes, allowing multiple secondary antibodies to bind, increasing the signal around the antigen.

What are the four steps in an indirect ELISA?

1. Antigen binding, 2. Primary antibody binding, 3. Secondary antibody binding, 4. Detection.

What is the role of secondary antibodies in immunoassays?

They recognize and bind to primary antibodies, allowing for visualization of the antigen.

How are secondary antibodies produced?

By injecting antibodies from one species into a different species to generate antibodies against the first species' antibodies.

What is immunostaining used for?

To localize antigens in cells or tissues and distinguish between different cell types.

How can immunostaining assist in cancer diagnosis?

It reveals variations in cell surface proteins that help identify cancer cells, which may look identical to normal cells.

What is fluorescence-activated cell sorting (FACS)?

A technique that uses fluorescently labeled antibodies to separate labeled and unlabeled cells using lasers.

What does immunoblotting (western blotting) determine?

It provides information about a protein's size and relative abundance in a sample.

What is the purpose of SDS-PAGE in western blotting?

To separate proteins by size before transferring them to a membrane for analysis.

What is dot blotting?

A rapid screening technique where a sample is spotted onto a membrane for quick determination of protein presence.

What are dipstick tests used for?

To provide quick positive or negative results for various substances, including pregnancy and drug use.

How do sandwich immunoassays work?

They use multiple antibodies to capture and detect the target antigen, often producing a visible signal.

What is the role of colloidal gold in dipstick tests?

It is used as a colored label that produces a visible band in positive assays.

What happens if no pink stripe appears in the control zone of a dipstick test?

It indicates that the test did not function properly.

What is the significance of the primary antibody in the indirect detection method?

It binds specifically to the antigen and serves as an antigen for the secondary antibody.

What is the main purpose of using enzyme-linked secondary antibodies?

To enable the visualization of the primary antibody-antigen complex through a color change or light emission.

What does the term 'epitope' refer to in the context of antibodies?

The specific part of an antigen that is recognized and bound by an antibody.

What is the function of the HRP enzyme in the detection process?

It catalyzes a reaction with a substrate to produce a color change, indicating a positive result.

How can immunostaining help in understanding protein function?

By showing the localization and abundance of a protein in different cell types or stages of development.

What is the advantage of using polyclonal secondary antibodies?

They can bind to multiple epitopes on the primary antibody, enhancing signal amplification.

What type of samples can be analyzed using western blotting?

Lysed cells or tissue samples that have been separated by size.

What is the purpose of the control zone in dipstick tests?

To ensure the test is functioning correctly by providing a reference for comparison.

What does the term 'immunoassay' refer to?

A biochemical test that measures the presence or concentration of a substance through antigen-antibody interactions.

What is the outcome of the reaction between HRP enzyme and the substrate TMB?

A color change in the solution, indicating the presence of the target antigen.

What is the purpose of vaccination?

To give resistance to infectious diseases before exposure.

What do booster shots do?

They invoke the secondary immune response to maintain antibody levels.

What are live attenuated vaccines?

Vaccines made from weakened microbes that are nonpathogenic.

Give an example of a live attenuated vaccine.

Vaccines against polio (Sabin type), measles, mumps, or smallpox.

What are killed or inactivated vaccines?

Vaccines made from microbes killed by heat or chemicals.

What is a disadvantage of killed vaccines?

They usually do not provoke as strong an immune response as live vaccines.

Provide an example of a killed vaccine.

Vaccines against rabies, cholera, polio (Salk type), or influenza.

What are subunit vaccines?

Vaccines made from pieces of microbes, consisting of one or more antigens.

Name an example of a subunit vaccine.

Vaccines against hepatitis B, anthrax, or tetanus.

What are DNA vaccines?

Vaccines that use cloned DNA coding for microbial antigens injected into the patient.

Are DNA vaccines currently available?

No, but some are in clinical trials.

What are mRNA vaccines?

Vaccines consisting of mRNA molecules encoding the antigen.

What is a significant advantage of mRNA vaccines?

They have increased stability and immunogenicity due to chemical modifications.

What is an example of an antibody vaccine?

Human monoclonal antibodies prepared against specific antigens.

What are postexposure vaccines?

Vaccines used to treat a disease after exposure.

What is the effectiveness of postexposure rabies vaccination?

100% effective if begun promptly after exposure.

What is the timeframe for administering smallpox vaccination postexposure?

Effective if administered 2-3 days postexposure; may prevent fatality if given up to 5 days later.

What is the role of immune serum globulin in immunotherapy?

Administered after exposure to hepatitis to provide immediate immunity.

What is equine antivenin used for?

To treat snakebite exposure.

What are the primary methods used to diagnose infectious diseases?

Observing symptoms and performing laboratory tests.

What can diagnostic tests look for in infectious diseases?

Microorganisms, microbial products, or the body's immune response to the disease agent.

What types of body fluids can laboratory tests examine for signs of disease?

Blood, urine, stool samples, cerebrospinal fluid, and saliva.

Which US agency regulates laboratory tests for infectious diseases?

The Food and Drug Administration (FDA).

What was one of the first methods used to detect microorganisms in clinical samples?

Using antisera directed against specific microbes with fluorescently labeled antibodies.

What are culture methods in diagnostic testing?

Growing microorganisms from clinical samples on culture media to observe their growth and appearance.

What is an immunoassay, and what is one example?

A test that identifies microbe-specific antibodies in serum; an example is ELISA.

What is an agar diffusion assay?

A test where antisera and antigens are placed in agar, and visible precipitation bands form upon binding.

What is the purpose of immunofluorescence assays (IFA)?

To detect specific microorganisms using fluorescently labeled antibodies.

What occurs during agglutination tests?

Visible precipitates appear when antibodies and specific antigens come into contact.

What are immunochromatography tests?

Card or dipstick-based immunoassays used for rapid detection of specific pathogens.

What do microplate tests like ELISA or RIA detect?

Microbial antigens, microbial products, and antibodies against microorganisms.

What is the role of molecular methods in diagnostics?

Detection of microbial RNA or DNA and microbial drug resistance.

What does AST stand for in the context of infectious disease testing?

Antimicrobial susceptibility testing.

What is microscopy used for in diagnosing infectious diseases?

Visual identification of microorganisms based on staining or physical characteristics.

What is an example of a microorganism detected by electron microscopy?

Ebolavirus.

What types of parasites can be identified using light microscopy?

Protozoa and helminthes.

What is the significance of testing for microbial RNA and DNA?

It has become common for diagnosing infectious diseases in recent years.

What does PCR stand for in molecular methods?

Polymerase Chain Reaction.

What are the three basic categories of problems with the immune system?

Hypersensitivity, immunodeficiency, and autoimmune diseases.

What occurs during a hypersensitive reaction?

The immune system overreacts to an antigen, which can result in illness or death.

What is an allergen?

An antigen that causes a hypersensitive reaction, such as food or pollen.

What are the four types of hypersensitive reactions?

1) Anaphylactic reactions (allergies), 2) Cytotoxic reactions, 3) Immune complex reactions, 4) Delayed-type hypersensitivity.

What is an example of an anaphylactic reaction?

Food allergies or pollen allergies.

What is a cytotoxic reaction?

A hypersensitive reaction such as transfusion reactions and Rh incompatibility reactions.

What is an immune complex reaction?

A hypersensitive reaction like farmer's lung caused by inhaling mold spores.

What is delayed-type hypersensitivity?

A hypersensitive reaction such as contact dermatitis from poison ivy.

What does immunodeficiency mean?

An inability to mount an effective immune response, leading to increased vulnerability to infections.

What are the two types of immunodeficiency?

1) Primary immunodeficiency (genetic basis), 2) Secondary immunodeficiency (external causes).