Immunoassays: Types, Principles, and Applications in Biology and Medicine

What are immunoassays?

Testing methods used to detect or measure antigens (AG) or antibodies (AB) in a sample.

What does AG stand for in immunoassays?

Antigen, which can be a hormone, vitamin, or drug found in biological samples.

What does AB stand for in immunoassays?

Antibody, which are proteins created in response to antigenic stimulators.

What significant development in immunoassays occurred in 1960?

Yalow and Berson released the radioimmunoassay (RIA) for insulin.

What is the role of a label in immunoassays?

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

What is the structure of an antibody?

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

What are the two types of chains in an antibody?

Heavy chains and light chains.

What are the variable regions of an antibody responsible for?

They form the antigen-binding sites.

How are antibodies classified into isotypes?

Based on the number of Y units and the type of heavy chain.

What is the function of IgA antibodies?

They are found in mucosal areas and prevent colonization by pathogens.

What is the primary class of antibodies used as immunochemical reagents?

Immunoglobulin G (IgG).

What is the difference between polyclonal and monoclonal antibodies?

Polyclonal antibodies are derived from different plasma cell lines, while monoclonal antibodies come from a single plasma cell line.

What is a hybridoma?

A cell line created by fusing mouse spleen cells with myeloma cells to produce monoclonal antibodies.

What is an immunogen?

A chemical substance capable of causing an antibody-mediated immune response.

What is a hapten?

A small chemical determinant that can stimulate an immune response when conjugated to a larger carrier protein.

What factors can affect the binding strength between an antibody and an antigen?

Cation salts, anionic salts, and the presence of linear polymers.

What is the difference between affinity and avidity in antibody-antigen interactions?

Affinity is the strength of a single binding site, while avidity refers to the overall strength of binding considering all binding sites.

What types of labels have replaced radioactive isotopes in immunoassays?

Enzymes and fluorescent labels, which were later replaced by chemiluminescent labels.

What is the significance of chemiluminescent labels in immunoassays?

They can produce light or luminescence that can be detected with a photodetector.

What is the structure of IgM antibodies?

They are pentameric and serve as the first response antibodies.

What is the role of IgE antibodies?

They bind to allergens and trigger histamine release, also protecting against parasitic worms.

What is the function of IgD antibodies?

Their function is unclear but they work with IgM in B cell development.

What is the primary function of IgG antibodies?

They provide the majority of antibody-based immunity against invading pathogens.

How can monoclonal antibodies be prepared?

Through immunization with a non-purified antigen.

What are the advantages of using monoclonal antibodies in immunoassays?

They provide specific reagents, yield unlimited quantities, and have consistent affinity and specificity.

What are some disadvantages of using monoclonal antibodies?

They may exhibit insufficient reactivity in precipitation/agglutination and difficulty in characterizing heterogeneous antigens.

What is the significance of the C-terminal constant region of the heavy chain in antibodies?

It interacts with other molecules in the immune system and determines the overall class of the antibody.

What is the purpose of Comboed AGs in RIA procedures?

They are prepped by the manufacturer for use in immunoassays.

How is conjugation accomplished in immunoassays?

Through chemical reactions that convert complex AGs to labels or other materials.

What instrument is required for detecting fluorescent labels?

A fluorimeter.

What is the role of the photodetector in a fluorimeter?

It produces an electrical signal when light strikes the cathode.

What is the purpose of a calibration material in fluorimetry?

To relate detector response to the concentration of the analyte.

What is the primary method used in Western Blotting?

Separation of proteins via gel electrophoresis followed by immunoassay investigation.

What does an Eastern Blot detect?

Post-translational modifications such as sugars, lipids, and phosphates.

What is the first step in the Western Blot process?

Denaturing proteins by boiling the sample.

What is the purpose of SDS in SDS-PAGE?

To give proteins a negative charge for separation by weight.

What type of membrane is typically used in Western Blotting?

PVDF or nitrocellulose membrane.

What is the function of a blocking agent in Western Blotting?

To fill empty protein binding locations on the membrane to prevent non-specific binding of antibodies.

What is the role of the primary antibody in Western Blotting?

To bind specifically to the protein of interest.

What does a secondary antibody do in Western Blotting?

It binds to the primary antibody and contains a label for detection.

What is a competitive immunoassay?

An assay where an unlabelled analyte competes with a labelled analyte to bind to an antibody.

What is the difference between homogenous and heterogenous competitive immunoassays?

Homogenous measures unbound labelled analyte, while heterogenous measures bound labelled analyte after washing away unbound ones.

What is a non-competitive immunoassay?

An assay where the unknown analyte binds to labelled antibodies, and the signal is directly proportional to the analyte concentration.

What is the principle of the two-site non-competitive immunoassay?

An unknown analyte is bound to an unlabelled antibody, and then labelled antibodies are added to bind to the analyte.

What is the purpose of an Enzyme-Linked Immunosorbent Assay (ELISA)?

To detect and quantify soluble substances like peptides, proteins, antibodies, and hormones.

What is the first step in an ELISA?

Coating the plate with the antigen.

What is the function of the blocking step in an ELISA?

To cover unsaturated surface-binding sites of the microplate wells.

What are the three formats of ELISA?

Direct, Indirect, and Sandwich ELISA.

What distinguishes a Sandwich ELISA?

The analyte is bound between two antibodies, enhancing sensitivity and specificity.

What is the principle behind the Enzyme Multiplied Immunoassay Technique (EMIT)?

It is a competitive homogenous immunoassay based on enzyme inhibition proportional to the analyte amount.

What is the role of the reporter enzyme in ELISA?

To produce a measurable product upon incubation with a substrate.

What is the role of glucose-6-phosphate dehydrogenase (G6PDH) in the described immunoassay?

G6PDH is conjugated to a drug and its activity is measured colorimetrically to determine the concentration of the sample analyte.

What type of immunoassay is CEDIA?

CEDIA is a competitive homogenous immunoassay.

How does CEDIA work?

It allows enzyme fragments to assemble into a functional enzyme in the presence of an analyte, with formation inhibited by an antibody.

What is the principle behind Fluorescence Polarization Immunoassay (FPIA)?

FPIA is based on the principle of fluorescence polarization, where the rotational velocity of a fluorescent molecule affects the detection of emitted light.

What happens to the rotational velocity of a fluorescent molecule when an antibody binds to it?

The binding slows down the rotational velocity, allowing for increased measurement of fluorescence.

What is the hook effect in immunoassays?

The hook effect occurs when high concentrations of analyte overwhelm the binding capacity of the assay, leading to falsely low readings.

How can the hook effect be validated?

By diluting the sample and re-running the immunoassay to see if the results increase, indicating the presence of the hook effect.

What are heterophile antibodies?

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

How do human anti-animal antibodies affect immunoassays?

They bind to animal antibodies used in immunoassays, potentially causing false positive or negative results.

What is the significance of using blocking agents in immunoassays?

Blocking agents are used to prevent interference from heterophile antibodies and improve assay accuracy.

What is the consequence of high concentrations of antibodies in immunoassays?

Excess antibodies can lead to impaired immunocomplex formation and inaccurate results.

What is the relationship between enzyme activity and analyte concentration in CEDIA?

Enzyme activity is directly proportional to the amount of analyte in the patient sample.

What is the role of fluorescein in FPIA?

Fluorescein is a fluorescent molecule used in the assay, excited at 490nm and reemitting fluorescence at 520nm.

What happens to the dose response curve when analyte concentration exceeds antibody levels?

The curve flattens and may become negatively sloped, indicating the hook effect.

What is the impact of heterophile antibodies on assay results?

They can bind to multiple antigens and reagents, leading to false results in immunoassays.

What is the typical occurrence rate of human anti-animal antibodies in patient samples?

They are reported to occur in 30-40% of patient samples.

What is the primary challenge posed by heterophile antibodies in immunoassays?

They can lead to inaccurate results by binding to capture or detection antibodies.

What is the effect of dilution on immunoassay results when the hook effect is present?

Dilution may lead to higher readings, indicating interference from the hook effect.

What is the function of the antibody in the CEDIA assay?

The antibody binds to the analyte-enzyme complex, inhibiting the formation of the functional enzyme.

What is the expected outcome when a patient sample contains the analyte of interest in CEDIA?

The analyte will bind to the antibody, releasing the analyte-enzyme complex to form a functional enzyme.

What is the significance of measuring fluorescence in FPIA?

The amount of polarized fluorescence emitted is inversely proportional to the analyte concentration in the serum sample.

What is the role of NAD+ in the G6PDH assay?

NAD+ is a substrate that, along with glucose-6-phosphate, is used to produce a measurable product in the assay.

How does the presence of analyte in a patient sample affect the CEDIA assay?

It competes with the enzyme conjugate for the antibody, allowing for the formation of functional enzyme.

What is the expected result of an immunoassay if the analyte concentration is within the optimal range?

The assay will provide accurate and quantitative results.

What is the primary mechanism of interference caused by heterophile antibodies?

They bind non-competitively to various components of the detection system.