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Immunology > Immunological Diagnostics > Flashcards

Immunological Diagnostics Flashcards

1
Q

Antigens

A
  • indicates active infection

- not always circulating in the blood

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2
Q

Antibodies

A
  • active infection
  • exposure and subsequent immunity
  • vaccination
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3
Q

DNA

A
  • active infection

- presence does not always equal causation

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4
Q

What are the 4 things we can test for?

A
  • antigens
  • antibodies
  • DNA
  • cell surface markers
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5
Q

Sensitivity

A

High sensitivity picks up any remote chance that an animal is infected/affected
- ex: pancreatic snap tests

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6
Q

Specificity

A

Something can have high sensitivity, but low specificity

- chance for cross reactivity

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7
Q

Primary binding tests

A

Directly detects antigen binding to antibody (immune complexes)
- one of reactants (either ag or ab) must be chemically labeled for detection

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8
Q

What are the chemical labels used for primary binding tests?

A
  • radioisotopes
  • fluorescent dyes
  • enzymes
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9
Q

What do primary binding tests detect?

A

Either antigen or antibody

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10
Q

Immunofluorescence assays

A

Direct or indirect fluorescent antibody tests

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11
Q

Immunoenzymes assays

A

Enzyme-linked immunosorbent assays

  • ELISA
  • western blot
  • immunohistochemistry
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12
Q

Radioimmnoassays

A

Uses radioisotopes

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13
Q

Chemiluminescent immunoassays

A

Better than RIA

- commonly used in labs

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14
Q

Epitope

A

Something on the antigen that the antibody recognizes and binds to

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15
Q

Antigen-antibody recognition

A

Specific recognition between antigen and a monoclonal antibody specific for a certain target
- target can be the patient antibody or the patient antigen

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16
Q

Patient antigen reacts with test antibody

A
  • patient antigen in blood

- patient antigen in tissue

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17
Q

Patient antibody reacts with test antigen

A

Patient serum applied to test antigen

18
Q

IFA

A

Immunofluorescense assays

  • direct fluorescent antibody: detects specific Ag in patient sample
  • indirect fluorescent antibody: detects patient antibody specific to Ag
19
Q

Direct fluorescent antibody

A

Patient tissue or fluid with suspected antigen is fixed to a slide and incubated with commercial antibody
- ex: rabies detection, FeLV test

20
Q

Indirect fluorescent antibody

A

Commercial antigen is pre-placed on slide and incubated with patient serum

  • bound Ab is detected with FITC-labeled antiglobulin (antiglobulins bind antibodies)
  • fluorescence indicates patient has Ab to specific antigen
21
Q

ELISA

A

Enzyme-linked because an enzyme is conjugated to an antibody

  • enzyme changes color when mixed with a substrate
  • can detect patient antibodies or antigens
22
Q

Immunosorbent

A

Relating to or denoting techniques making use of the absorption of antibodies by insoluble preparations of antigens

23
Q

ELISA method

A
  • add patient serum to plate of commercial Ag
  • bound Ab is identified by adding solution with antiglobulin that is linked to enzyme
  • when a solution with enzyme substrate is added, a color change is produced
24
Q

Color change is proportional to amount of ______

A

Antibody in patient’s serum

25
Q

Disposable immunoassays

A

Nylon membrane coated with Ab

  • patient sample with suspected Ag is applied
  • if Ag binding occurs, a positive result is detected by a dot or + sign
26
Q

Immunochromotography

A

Lateral flow test

  • antigen solution (patient sample) flows thru a porous strip and meets labeled commercial Ab to form an immune complex
  • positive indicated by a blue or pink color
27
Q

Western Blot

A

Identifies a specific protein in a complex mixture

- use clinically as a confirmation of other test results (rule out false positives)

28
Q

Western Blot method

A
  • electrophoresis of a protein mixture on gels –> resolves proteins to a single band
  • blotting of bands to a nitrocellulose membrane via electric current
  • enzyme or immunoassay used to visualize transferred protein
29
Q

IHC

A

Enzymes are used as the label and are conjugated to Ab or antiglobulin to locate specific antigen in tissue

  • diagnosis of disease and determination of tissue type in tumors (ex: actin Ab used to determine if skeletal muscle in origin)
  • bound Ab detected by brown color
30
Q

What is the difference between direct and indirect IHC methods?

A

Direct: primary Ab labelled
Indirect: secondary Ab (antiglobulin) labelled

31
Q

RIA

A

Highly sensitive

  • expensive
  • detection of trace drugs
32
Q

Antibody titration

A

Testing for amount of circulating antibodies present in the serum to a given pathogen
- performed for infectious diseases (lepto, tick-borne diseases)

33
Q

Using titers

A

Antibody levels checked at initial presentation

  • disease usually likely if initial titers are extremely high
  • recheck 2-3 weeks later (can be falsely decreased from antibiotics)
  • 4-fold rise in titers confirms infection
34
Q

Serum neutralization

A

Incubate different concentrations of serum with virus

35
Q

Microscopic agglutination tests

A

Serum dilutions incubated with the organism

- titer is the highest dilution that causes agglutination of 50% of the organisms

36
Q

Polymerase chain reaction

A

Amplifies DNA specific to the organism you are looking for

  • positive PCR: target DNA found in patient sample
  • risks: contamination, sensitivity and specificity issues
37
Q

PCR DNA extraction

A
  • blood: mycoplasma, hepatozooan
  • urine: leptospirosis
  • bone marrow
  • fluids: feline infectious peritonitis
38
Q

RT-PCR

A

Reverse transcriptase

  • used when starting sample is RNA
  • must reverse transcribe RNA to DNA before amplification
39
Q

Quantitative

A

Amplify and quantitate the amount of target DNA

- can get absolute copy number or perform relative quantification

40
Q

PCR only works when ___

A

Actual organism is present in patient blood or sample

41
Q

Flow cytometry

A

Identifies cell surface antigen (NOT immune complex!)

- most commonly used for immunophenotyping (lymphoma, leukemia)

42
Q

Immunophenotyping

A

Useful for assessing leukocyte populations

  • lymphoma: all B or T cells
  • leukemia: look for markers of mature leukocytes
  • use for transplantations

Immunology (28 decks)

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