Immunoassays Flashcards
What are immunoassays?
Testing methods used to detect and measure either antigens or antibodies in a sample.
What is an antigen?
A hormone, vitamin, or drug found in biological samples such as blood.
What are antibodies?
Proteins created in response to antigenic stimulators.
Who developed the first radioimmunoassay for insulin?
Yalow and Berson in 1960.
What is a label in the context of immunoassays?
A compound attached to an antibody or antigen that helps observe the interaction.
What is the structure of antibodies?
Composed of 4 polypeptide chains arranged in a Y-shape.
What are the two types of chains in antibodies?
Heavy chain and light chain.
What determines the class of each antibody?
The C-terminal constant region of the heavy chain.
How many isotypes of antibodies are there in mammals?
Five: IgG, IgM, IgA, IgD, and IgE.
What is the function of IgA?
Found in mucosal areas, prevents colonization by pathogens.
What is the primary role of IgG?
Major antibody in serum, provides immunity against invading pathogens.
What are monoclonal antibodies?
Antibodies derived from a single plasma cell line or clone.
What are the advantages of using monoclonal antibodies?
Well defined reagents, unlimited quantity, consistent affinity and specificity.
What is an immunogen?
A chemical substance capable of inducing an immune response.
What is a hapten?
Chemical determinants that stimulate antibody synthesis when bound to a carrier.
What is the difference between affinity and avidity?
Affinity refers to the interaction energy of a single binding site; avidity is the overall strength of binding.
What type of labels were first used in immunoassays?
Radioactive isotopes.
What is the purpose of a fluorimeter?
To detect fluorescent labels in immunoassays.
What is a Western blot?
A method for separating proteins via gel electrophoresis followed by immunoassay.
What is the purpose of blocking agents in Western blots?
To fill empty protein binding locations on the membrane.
What is a competitive immunoassay?
An assay where unlabelled analyte competes with labelled analyte to bind to an antibody.
What are the two types of competitive immunoassays?
- Homogenous
- Heterogenous
What is an Enzyme-Linked Immunosorbent Assay (ELISA)?
A plate-based assay technique designed for detecting and quantifying soluble substances.
What are the general steps in an ELISA?
- Coating/capture
- Plate blocking
- Probing/detection
- Signal Measurement
What is the role of the secondary antibody in an indirect ELISA?
To detect the primary antibody that is bound to the antigen.
What is the purpose of signal measurement in immunoassays?
Detection of the signal generated via the direct or secondary tag on the specific antibody.
What is the Enzyme-Linked Immunosorbent Assay (ELISA)?
A technique used to detect and quantify proteins or antigens.
List the formats of ELISA.
- Direct
- Indirect
- Sandwich
What characterizes a Sandwich ELISA?
The analyte is bound between two primary antibodies, each detecting a different epitope.
Why is the Sandwich ELISA format highly used?
It offers high sensitivity (signal amplification) and specificity.
What does EMIT stand for?
Enzyme Multiplied Immunoassay Technique.
What principle does EMIT operate on?
Enzyme inhibition being proportional to the amount of analyte in the patient sample.
What enzyme is commonly used in EMIT?
Glucose-6-phosphate dehydrogenase.
How does a competitive homogenous immunoassay work?
It measures the concentration of analyte by competition with a conjugated enzyme.
What is the function of the Cloned Enzyme Donor Immunoassay (CEDIA)?
It allows enzyme fragments to assemble into a fully functional enzyme based on the presence of an analyte.
What is a specific enzyme example used in CEDIA?
β-galactosidase.
What is the role of anti-analyte antibodies in CEDIA?
They bind to the analyte-enzyme complex, inhibiting the formation of the functional enzyme.
What does Fluorescence Polarization Immunoassay (FPIA) measure?
The polarization of fluorescence emitted by a fluorescent molecule.
What is the hook effect?
A phenomenon where high concentrations of analyte impair immunocomplex formation, affecting assay results.
What must be validated to avoid the hook effect?
Questionable sample results using dilution protocol.
What are heterophile antibodies?
Antibodies produced against poorly defined antigens, generally weak with multi-specific activities.
What are human anti-animal antibodies?
Antibodies developed as a result of treatment with animal immunoglobulins.
How do heterophile antibodies interfere with immunoassays?
They bind to the conjugate, enzyme, or other parts of the detection system.
What is a common type of human anti-animal antibody?
Human anti-mouse antibodies (HAMA).
What percentage of patient samples may contain human anti-animal antibodies?
30–40%.
Fill in the blank: The _______ effect is also known as the prozone effect.
hook
True or False: The amount of polarized fluorescence emitted in FPIA is directly proportional to the analyte concentration.
False
