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Immunology > Immunology Exam 3 (Serology, Precip/Agglut, Immunoassays) > Flashcards

Immunology Exam 3 (Serology, Precip/Agglut, Immunoassays) Flashcards

1
Q

Antigen-Antibody interactions are affected by 3 forces:

A
  • Antibodys’ various affinity and avidity towards antigens
  • Law of mass action
  • Precipitation curve/rate
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2
Q

What are the 3 main types of UNLABELED antibody-antigen assays?

A

Precipitation
Agglutination
Flocculation

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3
Q

What is precipitation rxn?

A

Soluble antigen and antibody form insoluble complexes that settle at the bottom of the container

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4
Q

What is agglutination rxn?

A

Soluble antibodies bind to antigens on cell surfaces which causes the cells to aggregate/clump and form insoluble complexes

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5
Q

What is flocculation rxn?

A

Soluble antibodes combine with antigen to form insoluble complexes that have a lighter density than solution so they float on the surface of the solution

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6
Q

What is affinity? What antibody has the highest affinity?

A

INITIAL force of attraction between FAB region and epitope of antigen
IgG has highest affinity

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7
Q

What is avidity? What antibody has the highest avidity?

A

OVERALL STRENGTH of antibody-antigen interactions and is the sum of all affinity
IgM has the highest avidity

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8
Q

The number of Fab binding sites per Ig is referred to as its ________.

A

Valency

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9
Q

What is the valency of IgM? IgG? IgA? IgD? IgE?

A

IgM = 10
IgG = 2
IgA = 4
IgD = 2
IgE = 2

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10
Q

What is “K” in the law of mass action? What does increased/decreased K result in?

A

K is a constant for each antibody and is based upon affinity and avidity.

Inc K = Inc Ab:Ag complexes = more product = higher sensitivity bc easier to detect

Dec K = Dec Ab:Ag complexes = less product = more reactants and substrate needed

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11
Q

Prozone

A

Zone of antibody excess
Produces a false negative reaction, dilute source of antibody

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12
Q

Postzone

A

Zone of antigen excess
Produces a false neg or falsely low pos, dilute source of antigen (aka patient sample)
ASSOCIATED W/ THE HOOK EFFECT

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13
Q

What is the optimal area for visible lattice formation for precipitation/agglutination?

A

Zone of equivalence

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14
Q

What is the Hook Effect?

A

Excess antigen present in sample that is creating a falsely low positive or false negative. Specimen must be diluted to visualize the true result.

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15
Q

Turbidimetry

A
  • Measures turbidity of sample
  • Detects how much light is able to pass through sample and into detector
  • Light reduction inversely proportional to sample concentration, AKA less light that is able to go through = more turbidity/higher concentration
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16
Q

Nephelometry

A
  • Measures scattered light as the light passes through the solution
  • Opposite of turbidimetry
  • Light scattering is directly proportional to sample concentration, AKA less light scatter = lower concentration
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17
Q

Passive diffusion rate is affected by:

A
  • Size of particles (larger migrate slower)
  • Temperature (warmer is faster)
  • Gel viscosity (more viscous = less migration)
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18
Q

What is the passive immunodiffusion technique?

A

Uses medium such as agarose gel to measure precipitation reactions between antibodies and antigens with NO electrical current applied

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19
Q

What is radial immunodiffusion?

A

Single diffusion technique where only the antigen is mobile. The reagent antibody is distributed in the gel.

Antigen concentration can be determined by plotting concentration of antigen (x) vs the diameter of the precipitation ring (y).

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20
Q

What is Mancini End-Point Method?

A

Radio immunodiffusion technique with prolonged incubation
The ring diameter squared is directly proportional to the concentration of the antigen.

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21
Q

What is Ouchterlong Double Diffusion?

A

Multiple wells with Ab + Ag independently diffusing across the medium with both Ab and Ag being mobile

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22
Q

What is the purpose of Ouchterlong Double Diffusion?

A

To see if there are shared epitopes between different antigens against the same antibody

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23
Q

There are 3 possible precipitation patterns for the Ouchterlong Double Diffusion test. Describe “identity” pattern.

A

An arc is formed due to fusion of Ab/Ag, indicating serological identity of shared epitope to Fab site

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24
Q

There are 3 possible precipitation patterns for the Ouchterlong Double Diffusion test. Describe “non-identity” pattern.

A

X pattern forms due to lack of fusion between Ab/Ag, indicating no shared epitopes between Ag/Ab.

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25
Q

There are 3 possible precipitation patterns for the Ouchterlong Double Diffusion test. Describe “partial identity” pattern.

A

T pattern forms due to partial fusion of Ab/Ag. This indicates that the antigens share a common epitope, but one antigen has higher expression of the epitope of interest so the open end (aka “alligator mouth”) will face the antigen with higher expression of the epitope.

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26
Q

Cathode

A

Negatively charged, attracts cations (which are positively charged)

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27
Q

Anode

A

Positive charged, attracts anions (which are negatively charged)

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28
Q

Electrophoresis

A

Molecules are separated out based on their electrical charge through a gel

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29
Q

What does electrophoresis look like for someone with hypogammaglobulinemia?

A

No gamma region, lacking Igs

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30
Q

What does electrophoresis look like for someone with multiple myeloma?

A

LARGE gamma region, indicating monoclonal gammopathy

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31
Q

**Know the normal/abnormal patterns of electrophoresis.

A
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32
Q

**Know how to interpret IFEs

A
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33
Q

What is immunofixation electrophoresis?

A

Qualitative technique to see increases or decreases of Igs in patient serum.

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34
Q

Which assays would be best for monoclonal gammopathy diagnosis?

A

Electrophoresis (screen)
IFE (confirmatory)

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35
Q

Immunoglobulins that combine with exposed/surface antigens to form insoluble agglutination are known as __________.

A

Agglutinins

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36
Q

Direct agglutination

A

Agglutination that results from antigens naturally found on a particle or cell

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37
Q

Hemagglutination

A

Agglutination that involves antigens on natural cells, erythrocytes (blood bank)

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38
Q

Passive agglutination

A

Indirect agglutination where artificial particles are coated with antigens and you are trying to detect ANTIBODY

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39
Q

Reverse passive agglutination

A

Indirect agglutination where artificial antibodies are used to detect ANTIGEN

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40
Q

Passive vs reverse passive agglutination

A

Passive: detecting antibody
Reverse passive: detecting antigen

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41
Q

Describe agglutination inhibition reaction.

A

If patient sample has hapten of interest, there will be no agglutination = positive result

If patient does not have hapten of interest, agglutination occurs = negative result

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42
Q

What is the PRINCIPLE (from chart) of the Nephelometry technique?

A

Light that is scattered at an angle is measured, indicating the amount of antigen/antibody present.

43
Q

What is the PRINCIPLE (from chart) of the Radial immunodiffusion technique?

A

Antigen diffuses out into a gel that is infused with antibody, measurement of the precipitin ring diameter indicates the concentration of the antigen

44
Q

What is the PRINCIPLE (from chart) of the Ouchterlony double diffusion technique?

A

Both antigen and antibody diffuse out from wells in a gel. The patterns of precipitate lines formed indicate the relationship of the antibodies.

45
Q

What is the PRINCIPLE (from chart) of the Immunofixation electrophoresis technique?

A

Electrophoresis of serum followed by direct application of reagent antisera to the gel.

46
Q

What type of reaction is this?:
Patient serum is reacted with antigen that is naturally found on a particle. Agglutination indicates the presence of patient antibody to a natural antigen.

A

Direct agglutination

47
Q

What type of reaction is this?:
Patient sample is reacted with particles coated with antigens not normally found on their surfaces. Agglutination indicates the presence of patient antibody to an artificially attached antigen.

A

Indirect (passive) agglutination

48
Q

What type of reaction is this?:
Patient sample is reacted with particles that are coated with reagent antibody. Agglutination indicates the presence of specific antigen in the patient sample.

A

Reverse passive agglutination

49
Q

What type of reaction is this?:
Haptens are attached to carrier particles. Particles compete with patient antigens for a limited number of antibody sites. Lack of agglutination is a positive test, indicating the presence of antigen in the patient sample.

A

Agglutination inhibition

50
Q

What type of reaction is this?:
RBCs spontaneously agglutinate if viral particles are present. Lack of agglutination is a positive test, indicating the presence of patient antibody.

A

Hemagglutination inhibition

51
Q

Serology

A

The study of liquid/fluid components in the blood, serum, that contains proteins for testing (especially antibodies)

52
Q

Serum vs plasma

A

Serum has no clotting factors
Plasma has clotting factors

53
Q

Serum is collected in which type of tube?

A

Non-anticoagulated (Red top, SST, or tiger top)

54
Q

How is serum for serology testing stored if testing is delayed?

A

Between 2-8C for up to 70 hours or frozen at -20C or below

55
Q

Complement may interfere with certain test results. How do you inactivate complement?

A

Heat sample to 56C for 30 minutes

56
Q

Volumetric vs Graduated pipettes

A

Volumetric: deliver only one volume, labeled TD (to deliver) allow for exact measurements

Graduated: labeled TC (to contain) and the last drop must be forced out to accurately deliver liquid, used for serology

57
Q

What is the formula for dilutions?

A

1 Amount of solute (serum)
________ = _____________________________
Dilution Total volume (Solute + diluent)

Cross multiply

58
Q

Formula for compound dilutions

A

C1 (starting concentration) x V1 (starting volume) = C2 (ending concentration) x V2 (ending volume)

59
Q

A ____-fold increase is associated with a clinical significant finding.

A

4-fold

60
Q

What is the titer?

A

The last tube in which a positive reaction is visible in a serial dilution of serum

61
Q

Sensitivity

A

The likelihood of correctly identifying a disease even in low concentrations

62
Q

Specificity

A

The likelihood of correctly identifying a disease or its analyte when there are other diseases/analytes that are similar to it

63
Q

PPV

A

The likelihood that a person who truly has a disease will also test positive for the disease

64
Q

NPV

A

The likelihood that a person who truly does not have a disease will also test negative for the disease

65
Q

What is the formula for sensitivity?

A

(TP/TP + FN) x 100

66
Q

What is the formula for specificity?

A

(TN/TN + FP) x 100

67
Q

What is the formula for PPV?

A

(TP/TP + FP) x 100

68
Q

What is the formula for NPV?

A

(TN/TN + FN) x 100

69
Q

Which is more sensitive/specific: Unlabeled immunoassays or labeled immunoassays?

A

Labeled immunoassays

70
Q

Name a few examples of something that can be a biomarker/analyte

A

Proteins (most of the time)
Hormones
Tumor markers
Drugs
Antibodies
Antigens

71
Q

What type of immunoassay has the highest sensitivity?

A

Chemiluminescent assays

72
Q

What type of immunoassay has the lowest sensitivity?

A

Colorimetric

73
Q

Immunoassays that capture patient antibody are referred to as _________ assays.

A

Indirect assays

74
Q

Immunoassays that capture patient antigen are referred to as __________ assays.

A

Direct/capture

75
Q

What are “labels”?

A

Detection molecules that produce a signal in the form of light, color, or fluorescence

76
Q

Heterogenous assays

A

Physical separation of bound/free analytes are required prior to detection

77
Q

Homogenous assays

A

Do not need physical separation of bound and free analytes prior to detection, do not require washing

78
Q

Competitive assays

A

Unknown concentration of analyte in patient competes with excess reagent analyte for reagent antibody Fab binding sites. Excess labeled analyte will ensure that all Fab binding sites are saturated if the patient sample is truly negative.

If patient analyte present = low signal = pos result

If patient analyte NOT present = high signal = neg result

INDIRECTLY PROPORTIONAL

79
Q

What is a major type/another name for a non-competitive assay?

A

Sandwich assay

80
Q

Non-competitive assays

A

Excess capture/reagent antibodies are added to a stationary/immobilized medium, patient sample added and if present will be captured by reagent antibody. Labeled antibody is added that binds to a different epitope of the same antigen forms a sandwich.

If patient analyte present = high signal = pos result

If patient analyte NOT present = low signal = neg result

DIRECTLY PROPORTIONAL

81
Q

Which type of immunoassay is a health hazard to work with and hard to safely dispose of reagents?

A

Radioimmunoassays

82
Q

T/F: EIAs can be homogenous or heterogenous.

A

True

83
Q

What are common enzyme conjugates used in EIAs?

A

Horseradish peroxidase
Alkaline phosphatase
B-D galactosidase
G6PD

84
Q

Non-competitive assays are ________ proportional to the signal produced while competitive assays are __________ proportional to the signal produced.

A

Non-competitive = DIRECT
Competitive = INDIRECT

85
Q

What is a Biotin-Avidin Labeled Immunoassay?

A

Biotin used as a label with a capture assay with streptavidin bound to a solid-phase material, but dietary biotin can interfere with tests where biotin is used

86
Q

Highest interfering substances occur with ___________/__________ assays.

A

Heterogenous/sandwich (non-competitive) assays

87
Q

What is an example of antigen interference in immunoassays?

A

High Dose Hook Effect / Postzone
Excess antigen causing false neg/low pos

88
Q

WHY does the hook effect occur? (elaborate)

A

There are limited capture antibodies. So, when excess antigen is added it can only bind so much. During the wash step, the excess antigen is removed since it is unbound, causing a falsely low result.

89
Q

How do you correct the hook effect?

A

Perform dilution
Re-run sample

90
Q

What are heterophile antibodies?

A

Human antibodies made against animal proteins due to previous exposure, can interfere with non-competitive assays that use AHG to capture human IgG

91
Q

Heterophile antibodies can lead to false ________ reactions.

A

It can lead to both false positive and false negative reactions depending on where it binds. False increases are more common.

92
Q

What is an advantage/disadvantage of homogenous assays?

A

Adv: No wash steps required, faster
Disadv: Can lead to sensitivity/specificity errors

93
Q

What are homogenous EIAs usually done to detect?

A

Small molecular weight molecules like haptens/hormones/drugs

94
Q

Describe a homogenous immunoassay

A

Directly proportional
Low patient antigen = low signal = neg
High patient antigen = high signal = pos

Reagent antibody is in solution as patient antigen and enzyme-labeled antigen are added together

95
Q

Describe the reactions of EMIT

A

Higher patient analyte = less antibodies bound to enzyme = more enzymes with open active site = subtrate allows for detectable signal = positive

Lower patient analyte = more antibodies bound to enzyme = antibody binding blocks active site of enzyme = substrate will not produce a signal = negative

DIRECTLY PROPORTIONAL

96
Q

Describe CMIA (Chemiluminescent Microparticle Assay)

A

More patient analyte present = less reagent antigen w/ acridium ester binds to microparticles = low signal = positive result

Less patient analyte present = more reagent antigen w/ acridium ester binds to microparticles = high signal = negative result

INDIRECTLY PROPORTIONAL
Often used for phenytoin and TDM

97
Q

Fluorescent assays

A

Reagent antibodies labeled with fluorophores or chlurochromes absorb energy

Used for flow cytometry, direct/indirect IFAs

98
Q

DFA vs IFA

A

DFA - looking for antigen, uses monoclonal reagent antibodies (ex. acridine orange or legionella pneumophila staining kits)

IFA - looking for antibody (sandwich assay), two step process, uses AHG with fluorescent tag which binds to PT antibody if present (ex. ANA testing, ANCAs)

99
Q

What can occur if you forget the wash step in immunoassays?

A

False negative
Any antibody will bind to AHG, so if wash step is skipped, unwanted antibodies will still be present and can bind to AHG, and unwanted antibodies will not produce a signal. Leading to a False negative.

100
Q

Describe fluorescence polarization immunoassays

A

Larger molecules will spin slowly and polarize light slowly while small molecules spin quickly and polarize light cannot be captured well.

More analyte = slower spinning = less polarized light = positive result

Less analyte = faster spinning = more polarized light = negative result

101
Q

Two step method non-competitive test example

A

C. diff quik chek
Line = positive

102
Q

One step method non-competitive test example

A

Uses thin layer chromatography
Pregnancy test
Line = positive

103
Q

One step method competitive test example

A

Drug tests
No line = positive

Immunology (6 decks)

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