7. Immunoassays and Antibody Structure

What are immunoassays used for?

Immunoassays are testing methods used to detect and measure either antigens or antibodies in a sample.

What is an antigen?

An antigen may be a hormone, vitamin, or drug found in biological samples such as blood.

What are antibodies?

Antibodies are proteins created in response to antigenic stimulators.

What do immunoassays provide?

Immunoassays provide a very sensitive, specific, and reliable method.

Who developed the radioimmunoassay for insulin?

Yalow and Berson developed it in 1960.

What is a label in the context of immunoassays?

A label refers to a compound attached to an antibody or antigen that helps observe their interaction.

What is the structure of antibodies?

Antibodies are composed of 4 polypeptide chains forming a Y-shaped unit.

What distinguishes light chains in antibodies?

The light chains can be classified as either kappa (κ) or lambda (λ) type.

What determines the antibody class?

The C-terminal constant region of the heavy chain interacts with other molecules in the immune system.

List the five isotypes of antibodies in mammals.

IgG, IgM, IgA, IgD, and IgE.

What is the function of IgA antibodies?

IgA protects mucosal areas, preventing colonization by pathogens and is resistant to digestion.

What is the main function of IgG antibodies?

IgG provides the majority of antibody-based immunity against invading pathogens.

What type of antibodies are derived from a single plasma cell line?

Monoclonal antibodies are derived from a single plasma cell line.

What is an immunogen?

An immunogen is a chemical substance capable of inducing an immune response.

Define a hapten in immunology.

A hapten is a small chemical that stimulates an immune response only when bound to a larger carrier protein.

What does the affinity of an antibody refer to?

Affinity refers to the thermodynamic energy of interaction of a single antibody binding site and its corresponding epitope.

What does avidity describe in the context of antibodies?

Avidity describes the overall strength of binding of an antibody to an antigen, considering all binding sites.

What is radioimmunoassay (RIA)?

Radioimmunoassay is a technique using radioactive isotopes as labels.

What are some modern replacements for radioactive isotopes in immunoassays?

Enzymes and fluorescent labels are modern replacements.

What is the purpose of a blocking agent in Western Blot?

It fills empty protein binding sites on the membrane to prevent non-specific antibody sticking.

What is a two-site non-competitive immunoassay?

It’s an assay where an unknown analyte binds to an unlabelled antibody, and a labeled antibody is added to measure the signal.

What does ELISA stand for?

Enzyme-Linked Immunosorbent Assay.

What is the principle behind EMIT?

EMIT works on the principle of enzyme inhibition being proportional to the amount of analyte in the sample.

How does the CEDIA assay function?

CEDIA uses enzyme fragments that assemble into a functional enzyme depending on the presence of an analyte.

What is fluorescence polarization immunoassay (FPIA)?

FPIA is a competitive homogeneous immunoassay that measures the rotational movement of fluorescent molecules.

What is the hook effect in immunoassays?

The hook effect occurs when antibody or analyte concentrations are too high, impairing immunocomplex formation.

What are heterophile antibodies?

Heterophile antibodies are produced against poorly defined antigens and can interfere with immunoassays.

What is a characteristic of human anti-animal antibodies?

They can bind to animal antibodies used in immunoassays, competing with test antigens.

Why are monoclonal antibodies preferred over polyclonal antibodies?

Monoclonal antibodies provide a well-defined reagent with consistent affinity and specificity.

What method is used in Western blotting to separate proteins?

Proteins are separated by gel electrophoresis.

What is the purpose of washing the membrane in a Western blot?

Washing removes excess unbound antibodies.

How are antigens detected in ELISA?

Antigens are detected by measuring the activity of a reporter enzyme that produces a measurable product.

What is the advantage of using a sandwich ELISA?

It's highly sensitive due to signal amplification and specificity, binding between two antibodies.

What are the general types of immunoassay?

Competitive and non-competitive immunoassays.

What is the role of the substrate in Enzyme-Linked Immunosorbent Assay?

The substrate reacts with the enzyme to produce a measurable product.

What does SDS-PAGE stand for and its role?

SDS-PAGE is a technique that separates proteins based on their molecular weight.

How are patient antibodies used in Western blotting?

Patient serum is used as the primary antibody to detect specific proteins from pathogens.

What does each antibody isotype differ in?

Biological properties, functional locations, and ability to deal with different antigens.

How does a competitive immunoassay function?

Unlabelled analytes compete with labelled analytes to bind to an antibody.

What influences the strength of the reaction between an antibody and antigen?

Factors such as cation salts and polymers present in the solution.

What technique visualizes the signals from fluorescently labelled antibodies?

A fluorescent microscope is used.

Describe a major disadvantage of using monoclonal antibodies.

Some monoclonal antibodies can exhibit cross-reactivity due to similar epitopes.

What happens in a sandwich ELISA?

An antigen is captured between two antibodies, facilitating measurement of the analyte.

Why are blocking agents important in assays?

They prevent non-specific binding of antibodies to the test surface.