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VET 413: Infectious Disease/Immunology > Serology > Flashcards

Serology Flashcards

1
Q

serology

A

the measurement and characterization of antibodies or antigens in body fluids

mostly done on serum or plasma

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2
Q

is it more useful to detect antigen or antibody

A

antigen because it indicates active or current infection

ex. heart worm, FeLV, parvovirus, some fungi

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3
Q

do the majority of serologic tests detect antigen or antibody

A

antibody - useful for pathogens that may not be in the blood at the time of the test

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4
Q

antibody characteristics

A
  • produced by plasma cells
  • have an Fab and Fc region
    (Fab = variable, binds antigen; Fc = constant)
  • can be monomeric (IgG), dimeric (IgA), or pentameric (IgM)
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5
Q

what is an antigen

A

the entire molecule that is recognized by antibody

usually a protein

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6
Q

what is an epitope

A

the portion of a molecule that is recognized by an antibody

antigens usually have >1 epitope

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7
Q

monoclonal antibodies

A

reagents that are produced to be used in serologic tests

the exogenous antibody required to detect endogenous (patient) antibody

NOT the same as the antibody produced by the patient

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8
Q

how are monoclonal antibodies formed

A
  1. mouse vaccinated with antigen of interest
  2. splenocytes that contain antigen specific plasma cells are harvested and fused with myeloma cells to form a hybridoma
  3. antigen specific hybridomas are selected and propagated
  4. antibodies are harvested from hybridoma supernatant
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9
Q

what are the main serologic tests

A

immunodiffusion
agglutination
ELISA
immunofluorescence

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10
Q

primary binding tests

A

measure immune complexes that form after combining antigen and antibody

requires labeling of a reactant in order to detect the immune complexes

ex: ELISA, immunochromatography

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11
Q

lateral flow assays

A

paper based platforms for the detection and quantification of antigens + antibody

point of care tests done in clinic

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12
Q

immunochromatography

A

lateral flow assay in which antigen solution flows unilaterally through a porous strip

  1. sample placed on sample pad
  2. flows to the conjugate pad containing labeled antibody
    - if antigen present in sample: forms immune complex
  3. flows to the detection zone (test line) where immune complexes are captured by immobilized detection antibody
    - if immune complexes present: color change occurs
  4. flows to the control line where non-antigen related antibodies are captured to induce color change

ex. at home covid tests

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13
Q

ELISA

A

enzyme linked immunosorbent assay

can detect antigen or antibodies

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14
Q

antibody ELISA

A
  1. antigen immobilized
  2. add sample
    - if antibody present: will bind antigen and form immune complex
  3. add conjugate containing detection antibody and enzyme
  4. wash and add substrate
    - if immune complexes formed: color change
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15
Q

antigen ELISA

A
  1. capture antibody immobilized
  2. add sample
    - if antigen present: will bind antibody and form immune complex
  3. add conjugate containing detection antibody and enzyme
  4. wash and add substrate
    - if immune complexes formed: color change
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16
Q

SNAP tests

A

ELISA based test where the flow matrix is connected to reagent reservoirs that are released when “snapped”

solution flows bidirectionally through strip

  1. sample is mixed with labeled detection antibody and placed on sample pad
  2. flows (left to right) to the antigen window containing capture antibodies
    - if antigen present: forms immune complexes on the “dots” in the window
  3. test is snapped to initiate flow of wash and substrate reagent (right to left)
  4. detection antibodies bind immobilized immune complexes to cause color change
17
Q

secondary binding tests

A

measure the downstream effects of immune complex formation after combining antigen and antibody

ex: precipitation or agglutination

18
Q

immunodiffusion

A

precipitation test
detects antibodies in blood

antigen: small and soluble –> forms a lattice and precipitates

antibody amount is constant (based on patient sample)
antigen amount if variable (added to sample to reach optimal proportion)

ex. Coggins test

19
Q

what happens if too much or not enough antigen is added to the patient sample

A

no precipitate will form

20
Q

Coggins test

A

immunodiffusion assay

antigen-antibody complex formation depends on proportions of each (must be in the equivalent zone)

used to diagnose EIA (equine infectious anemia)

produces a QUALITATIVE result (positive vs negative)

21
Q

radial immunodiffusion assay

A

antibody is located in agar and antigen is located in a well –> ring of precipitation indicates a positive result and the area is proportional to the amount of antigen in the well

produces a QUANTITATIVE result using standardized curve

ex. measuring serum ig levels in foals

22
Q

agglutination tests

A

detect antibodies on RBCs in IMHA

antigen: large and particulate –> clumps and agglutinates

ex. Coombs test

23
Q

Coombs test

A

detects autoantibodies on RBCs to test for IMHA

direct: Coombs reagent added to blood and check for agglutination

indirect: Coombs reagent added to donor blood mixed with patient serum and check for agglutination

24
Q

what does a positive vs negative Coombs test indicate

A

positive: confirms there is an immune mediated component to anemia BUT that main mechanism may or may not be immune mediated

negative: does not rule out IMHA

25
Q

functional tests

A

measure the protective effect of antibodies in animal or in vitro

ex. neutralization tests

26
Q

neutralization tests

A

rely on the ability of antibody to neutralize biological activity of antigen

27
Q

qualitative results

A

determine the presence or absence of antibodies

result is positive or negative

28
Q

semiquantitative results

A

determines the levels of antibody or antigen present

results is low vs high levels

29
Q

quantitative results

A

determines the amount of antibody (titer)

achieves an actual measurement by doing serial dilutions

titer: highest dilution that produces a positive result

30
Q

sensitivity

A

ability to correctly identify patients with a disease (true positives)

Sensitivity = TP / (TP + FN)

percentage of true positives out of all disease positives

31
Q

specificity

A

ability to correctly identify patients without a disease (true negatives)

Specificity = TN / (TN + FP)

percentage of true negatives out of all disease negatives

32
Q

why is it a challenge to determine a specific titer cutoff point

A

some disease + animals have low titers and some disease - animals have high titers

there is always going to be overlap, will cause there to always be some false positive and negatives

highly specific tests = insensitive

highly sensitive tests = non specific

33
Q

what are paired titers used for

A

identifying acute infection

acute sample: first sample obtained at presentation

convalescent sample: second titer obtained 3 weeks layer

34
Q

what is the criteria for a positive paired titer

A

convalescent sample has 4x increase in IgG or total Ig compared to acute sample
OR
there is IgM present

VET 413: Infectious Disease/Immunology (38 decks)

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