Immunoassays

Question 1

What are immunoassays?

Answer 1

Testing methods used to detect and measure either antigens or antibodies in a sample.

Question 2

What is an antigen?

Answer 2

A hormone, vitamin, or drug found in biological samples such as blood.

Question 3

What are antibodies?

Answer 3

Proteins created in response to antigenic stimulators.

Question 4

Who developed the first radioimmunoassay for insulin?

Answer 4

Yalow and Berson in 1960.

Question 5

What is a label in the context of immunoassays?

Answer 5

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

Question 6

What is the structure of antibodies?

Answer 6

Composed of 4 polypeptide chains arranged in a Y-shape.

Question 7

What are the two types of chains in antibodies?

Answer 7

Heavy chain and light chain.

Question 8

What determines the class of each antibody?

Answer 8

The C-terminal constant region of the heavy chain.

Question 9

How many isotypes of antibodies are there in mammals?

Answer 9

Five: IgG, IgM, IgA, IgD, and IgE.

Question 10

What is the function of IgA?

Answer 10

Found in mucosal areas, prevents colonization by pathogens.

Question 11

What is the primary role of IgG?

Answer 11

Major antibody in serum, provides immunity against invading pathogens.

Question 12

What are monoclonal antibodies?

Answer 12

Antibodies derived from a single plasma cell line or clone.

Question 13

What are the advantages of using monoclonal antibodies?

Answer 13

Well defined reagents, unlimited quantity, consistent affinity and specificity.

Question 14

What is an immunogen?

Answer 14

A chemical substance capable of inducing an immune response.

Question 15

What is a hapten?

Answer 15

Chemical determinants that stimulate antibody synthesis when bound to a carrier.

Question 16

What is the difference between affinity and avidity?

Answer 16

Affinity refers to the interaction energy of a single binding site; avidity is the overall strength of binding.

Question 17

What type of labels were first used in immunoassays?

Answer 17

Radioactive isotopes.

Question 18

What is the purpose of a fluorimeter?

Answer 18

To detect fluorescent labels in immunoassays.

Question 19

What is a Western blot?

Answer 19

A method for separating proteins via gel electrophoresis followed by immunoassay.

Question 20

What is the purpose of blocking agents in Western blots?

Answer 20

To fill empty protein binding locations on the membrane.

Question 21

What is a competitive immunoassay?

Answer 21

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

Question 22

What are the two types of competitive immunoassays?

Answer 22

• Homogenous
• Heterogenous

Question 23

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

Answer 23

A plate-based assay technique designed for detecting and quantifying soluble substances.

Question 24

What are the general steps in an ELISA?

Answer 24

• Coating/capture
• Plate blocking
• Probing/detection
• Signal Measurement

Question 25

What is the role of the secondary antibody in an indirect ELISA?

Answer 25

To detect the primary antibody that is bound to the antigen.

Question 26

What is the purpose of signal measurement in immunoassays?

Answer 26

Detection of the signal generated via the direct or secondary tag on the specific antibody.

Question 27

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

Answer 27

A technique used to detect and quantify proteins or antigens.

Question 28

List the formats of ELISA.

Answer 28

* Direct * Indirect * Sandwich

Question 29

What characterizes a Sandwich ELISA?

Answer 29

The analyte is bound between two primary antibodies, each detecting a different epitope.

Question 30

Why is the Sandwich ELISA format highly used?

Answer 30

It offers high sensitivity (signal amplification) and specificity.

Question 31

What does EMIT stand for?

Answer 31

Enzyme Multiplied Immunoassay Technique.

Question 32

What principle does EMIT operate on?

Answer 32

Enzyme inhibition being proportional to the amount of analyte in the patient sample.

Question 33

What enzyme is commonly used in EMIT?

Answer 33

Glucose-6-phosphate dehydrogenase.

Question 34

How does a competitive homogenous immunoassay work?

Answer 34

It measures the concentration of analyte by competition with a conjugated enzyme.

Question 35

What is the function of the Cloned Enzyme Donor Immunoassay (CEDIA)?

Answer 35

It allows enzyme fragments to assemble into a fully functional enzyme based on the presence of an analyte.

Question 36

What is a specific enzyme example used in CEDIA?

Answer 36

β-galactosidase.

Question 37

What is the role of anti-analyte antibodies in CEDIA?

Answer 37

They bind to the analyte-enzyme complex, inhibiting the formation of the functional enzyme.

Question 38

What does Fluorescence Polarization Immunoassay (FPIA) measure?

Answer 38

The polarization of fluorescence emitted by a fluorescent molecule.

Question 39

What is the hook effect?

Answer 39

A phenomenon where high concentrations of analyte impair immunocomplex formation, affecting assay results.

Question 40

What must be validated to avoid the hook effect?

Answer 40

Questionable sample results using dilution protocol.

Question 41

What are heterophile antibodies?

Answer 41

Antibodies produced against poorly defined antigens, generally weak with multi-specific activities.

Question 42

What are human anti-animal antibodies?

Answer 42

Antibodies developed as a result of treatment with animal immunoglobulins.

Question 43

How do heterophile antibodies interfere with immunoassays?

Answer 43

They bind to the conjugate, enzyme, or other parts of the detection system.

Question 44

What is a common type of human anti-animal antibody?

Answer 44

Human anti-mouse antibodies (HAMA).

Question 45

What percentage of patient samples may contain human anti-animal antibodies?

Answer 45

30–40%.

Question 46

Fill in the blank: The _______ effect is also known as the prozone effect.

Answer 46

hook

Question 47

True or False: The amount of polarized fluorescence emitted in FPIA is directly proportional to the analyte concentration.

Answer 47

False