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MEDL 210 > Module 13 > Flashcards

Module 13 Flashcards

1
Q

What is a label?

A

Reagent component that produces a measurable signal.

Usually detected by photometry.

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2
Q

What is a capture antibody?

A

Reagent antibody designed to capture the analyte of interest.

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3
Q

What is a conjugate?

A

Reagent antibody with attached label.

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4
Q

What is a tracer?

A

Reagent antigen with attached label.

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5
Q

What are the different types of signals in immunoassays?

A

Radioactive isotopes- iodine or cobalt

Fluorescent- fluorescein or phycoerythrin

Enzymes and substrates- enzyme catalyzes substrate into detectable product, horseradish peroxidase and alkaline phosphatase

Chemiluminescent- substances luminesce due to chem reaction, acrindinium esters and isoluminol

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6
Q

What are the different types of antigen-antibody reaction classifications?

A

Competitive/limited- uses one antibody, tracer competes with analyte for capture antibody binding sites, indirect relationship between amount of nature and signal

Noncompetitive/excess reagent/two site/sandwich- two antibodies, sample is added and binds to capture antibody, conjugate is added after washing, binds to analyte, signal is directly proportional to the amount of analyte present

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7
Q

What are the different signal property classifications?

A

Heterogeneous- labels produce the same signal when bound and unbound, requires separation step, can wash away potential interferences

Homogeneous- labels produce different signals whether bound or unbound

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8
Q

What are the different types of timing regarding immunoassays?

A

Simultaneous- one step, add everything together

Sequential- sample is added with capture antibody first and then reagent antibody is added, two step

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9
Q

What are radioimmunoassays (RIA)?

A

Use radioactive isotopes as labels.

Good sensitivity for thyroid hormones.

Not used due to safety concerns.

Competitive and heterogeneous.

Variation- measure the radioactivity of the wash solution

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10
Q

What are immunoradiometric assays?

A

Same as RIA but a labelled antigen is used instead of an antibody.

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11
Q

What are immunoassays and their benefits and used?

A

Antigen-antibody reactions are used to ID and quantify analytes.

High levels of specificity and sensitivity, good for analytes with low concentrations.

Hormones, therapeutic drugs, vitamins, serological markers, metabolites, cancer markers, cardiac markers.

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12
Q

How are enzyme immunoassays (EIA) used?

A

Enzyme conjugated to a reagent component produces a signal by acting on a substrate.

Signal can be measured spectrophotometrically.

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13
Q

How is ELISA (one step) performed?

A

Capture AB is coated on well.

Sample is added, analyte binds.

Tracer binds remaining sites.

Wash removes excess tracer.

Substrate is added and converted by enzyme.

Signal measured in indirectly proportional.

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14
Q

How is ELISA (two step) performed?

A

Capture AB is coated on well.

Sample added, analyte binds.

Conjugate is added, binds to analyte.

Wash.

Substrate added, enzyme produces product.

Signal produced is measured and directly proportional.

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15
Q

How is EMIT performed?

A

Capture AB and sample is incubated, analyte binds.

Tracer is added, binds remaining sites, inactivates enzyme activity.

Substrate is added.

Unbound tracer is free to convert substrate (homogeneous).

Signal produced is measured and is directly proportional.

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16
Q

What type of enzyme does CEDIA use?

A

Recombinant enzyme produced in two parts (donor and acceptor).

Must be combined to form an active enzyme.

One part is attached to a tracer which will compete with the analyte.

When bound, it can’t combine and become active.

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17
Q

How is CEDIA performed?

A

Capture AB, sample and tracer are incubated together.

Other part of the enzyme is added with the substrate.

Free antigen can bind with other enzyme structure that can convert substrate.

Signal measured is directly proportional.

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18
Q

How is MEIA performed?

A

Micro particles coated with capture AB are mixed with sample, analyte binds.

Mixture is applied to glass fiver matrix, bound microparticles are captured.

Conjugate is added, binds analyte.

Substrate is added, phosphatase enzyme converts MUR to MU, which is a fluorescent product.

Signal is measured, directly proportional to concentration.

19
Q

How do enzyme immunoassays work?

A

An enzyme labelling system is used to produce a signal.

Enzyme is conjugated onto a reagent and a substrate is provided.

Enzyme catalyzes a conversion, the product is measured.

20
Q

How is one site ELISA performed?

A

Capture AB is coated on well.

Sample is added analyte binds.

Optional wash.

Tracer binds remaining sites.

Wash step.

Substrate is added, product is produced.

Signal is measured, indirectly proportional to concentration.

21
Q

What type of assay is one site ELISA?

A

Heterogeneous and competitive

22
Q

How is two site ELISA performed?

A

Capture AB coated on well.

Sample added, analyte binds.

Optional wash.

Conjugate is added, binds analyte.

Wash step.

Substrate is added, product produced.

Signal is measured, directly proportional.

23
Q

What type of assay is two step ELISA?

A

Noncompetitive heterogeneous

24
Q

How is EMIT performed?

A

Capture AB and sample incubated, analyte binds.

Tracer is added, binds to remaining sites and the enzyme is inactivated.

Substrate is added.

Unbound tracer is free to convert substrate.

Signal is produced, directionally proportional.

25
Q

What type of assay is EMIT?

A

Competitive and homogeneous

26
Q

What type of enzymes does CEDIA use?

A

Recombinant enzyme produced in two parts (donor and acceptor).

The two must be combined to form a complete and active enzyme.

27
Q

How is CEDIA performed?

A

Capture AB, sample and tracer (with part of the enzyme) are incubated together.

The second part of the enzyme is added with the substrate.

Free antigen can bind with the second part forming a complete enzyme that can act on the substrate.

Signal is measured, directly proportional.

28
Q

What kind of assay is CEDIA?

A

Competitive and homogeneous

29
Q

How is MEIA performed?

A

Microparticles coated with capture AB are mixed with sample, analyte binds.

Applied to glass fibre matrix, microparticles bound to analyte are captured.

Conjugate added, binds analyte.

Substrate is added, phosphatase enzyme cleaves MUP to MU (fluorescent product).

Fluorescent signal is measured, directly proportional.

30
Q

What type of assay is MEIA?

A

Noncompetitive and heterogeneous

31
Q

How does FPIA work?

A

Uses a fluorescent tracer that rotates when free.

When bound it remains fixed.

When polarized excitation light is used, emitted light will only be polarized in the same plane if it’s bound.

32
Q

How is FPIA performed?

A

Analyte and fluorescent tracer are incubated with capture AB.

Mixture is exposed to polarized excitation light.

Polarization of the emitted light is measured, indirect relationship.

33
Q

What type of assay is FPIA?

A

Competitive and homogeneous

34
Q

What are the advantages of CLIA?

A

Uses labels that produce light due to a chemical reaction giving the best sensitivity.

Can detect low levels of analyte.

35
Q

What does CLIA require?

A

Trigger activation to start the chemical reaction.

36
Q

What are the types of CLIA?

A

Competitive/onesite- indirect, heterogeneous

Noncompetitive/twosite- direct, heterogeneous

37
Q

What is the high dose effect and how can it be corrected?

A

High levels of analytes give falsely low results because there isn’t enough capture AB and some is washed away.

High initial sample dilution.

38
Q

What are heterophile ABs?

A

ABs produced against the ABs of other species.

39
Q

Why are heterophile ABs a problem?

A

Potential interferences if developed to species used as reagent ABs.

Cross binding of conjugate and capture AB without analyte.

Binding to conjugate.

40
Q

What is a common heterophile AB?

A

Human anti-mouse AB (HAMA).

41
Q

What are other potential interferences experienced with immunoassays?

A

Unexpected/abnormal proteins

Analyte fragments

Drugs and metabolites

42
Q

What techniques can be used to identify or account for interferences?

A

Repeat testing with different methodology.

Repeat testing with different sample types.

Running serial dilutions of samples.

Use of blocking agents to address interferences.

43
Q

What is serology and how is it performed?

A

Measures the ABs in patient samples, can use any immunoassay.

Surface antigen binds ABs, labelled anti human globulin (AHG) is added.

44
Q

Why does serology require a wash?

A

AHG is nonspecific

MEDL 210 (12 decks)

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