Immunodiagnostics Flashcards

1
Q

Describe serum.

A

-source of antibodies
-portion of blood left after clotting = removes platelets, WBCs, RBCs, clotting factors

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2
Q

Describe serology.

A

-study of in vitro reactions of antibodies in serum & antigens = microorganisms that cause infectious diseases

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3
Q

Describe requirements for antibody based techniques.

A
  1. Antigens = bind to antibody
  2. Antibodies = generated to detect antigen
  3. Method of visualizing = antibody-antigen reaction (ex. Dyes, enzymes, etc)
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4
Q

Describe the 2 types of antibodies used in diagnostics & research.

A
  1. Polyclonal antibodies = mixture of antibodies specific for epitopes of antigen -> products of diff clones of B cells -> source of polyclonal antibodies is serum (antiserum)
  2. Monoclonal antibodies = antibodies specific for single antigenic epitope -> products of single B cell clone
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5
Q

Describe polyclonal antibody production.

A

-inoculate animal w antigen X
-wait 3-4wk & inoculate w antigen again (booster)
-2 wk later collect blood & separate serum
-purify antibodies on affinity columns or ammonium sulfate precipitation

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6
Q

Describe monoclonal antibody production.

A
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7
Q

Describe detecting antibody antigen reactions.

A

• Enzyme-linked immunosorbent assays (ELISA)
• Immunofiltration technique
• Immunohistochemistry
• Immunofluorescent microscopy
• Precipitation assays
• Immunoblotting
• Agglutination
• Hemagglutination inhibition assays
• Complement fixation test
• Virus neutralization test

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8
Q

Describe enzyme linked immunosorbent assay (ELISA).

A
  1. Create solid phase - coat plate w known antigen
  2. Add serum containing antibodies that you’re looking for (source of primary antibodies)
  3. Add secondary antibodies against species from which the serum originated = labeled w enzyme
  4. Add substrate for the enzyme used to label secondary antibodies
  5. Look at color change / read optical density in spectrophotometer
Indirect ELISA
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9
Q

Describe sandwich ELISA.

A
  1. Create solids phase - coat plates w known antibodies
  2. Add serum containing antigen you’re looking for
  3. Add antibodies against antigen you’re looking for - these antibodies are NOT labeled w enzyme (secondary antibodies)
  4. Add detecting antibody against secondary antibodies - these antibodies ARE labeled w enzyme
  5. Add substrate for enzyme used to label detecting antibodies
  6. Observe color change or read optical density in spectrophotometer
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10
Q

Describe SNAP tests.

A

modification of ELISA

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11
Q

Describe immunohistochemistry.

A

-frozen, formalin fixed tissue at necropsy/biopsy
-thin sections of formalin fixed, paraffin embedded tissues, thin sections of frozen tissues or cells attached to slides
-in situ = detect tissue proteins or pathogen antigens in tissue
*ex. Anti von willebrand factor antibodies or canine distemper virus

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12
Q

Describe immunofluorescence.

A

-same principle as immunohistochemistry
-fluorescent labeled antibodies used instead of enzyme labeled antibodies
-samples: tissue or cells on slides

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13
Q

Describe immunoprecipitation assays.

A

-antigens & antibodies can form complexes
-small & soluble
>in vivo = type III hypersensitivity
>in vitro = form visible precipitation

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14
Q

Describe the formation of precipitates.

A

-precipitation of antigen & antibody complexes in a precipitation assays exploits the conc of antibody & antigen (zone of equivalence) to measure their quantities (titer)
-antibody + antigen in equal amounts = precipitation
-too much antigen or antibody = no precipitation

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15
Q

Describe the different types of immunoprecipitation assays.

A
  1. Solution based immunoprecipitation
    -quantitative or qualitative
  2. Gel based immunoprecipitation
    -qualitative
    -‘agar gel immunodiffusion’
    -antigen & antibody deposited in sep wells in agar gel
    -2 substances diffuse in gel towards each other
    -@ zone of equivalence = antigen-antibody complex form visible precipitate (arc or straight line)
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16
Q

Describe coggins test.

A

EX OF AGAR GEL IMMUNODIFFUSION ASSAY
-detect antibodies against equine infectious anemia virus

17
Q

Describe radial immunodiffusion.

A

-modification of agar gel immunodiffusion assay to measure titers
-quantitative
-antigen or antibody in gel
-one component diffuses into gel from precipitate
-precipitate forms in a circle around the well as sample diffuses
-area of circle proportional to conc of antigen or antibody within sample

18
Q

Describe immunoblotting.

A

‘Western blotting’
-not routine diagnostics
-proteins sep based on size using polyacrylamide gel electrophoresis
-sep proteins transferred from the gel onto a nitrocellulose membrane
-membrane incubated w primary antibody against protein of interest
-secondary antibody conjugated to enzyme applied followed by substrate
-substrate may make color or chemiluminescence

19
Q

Describe virus neutralization.

A

-used to detect virus specific antibodies in serum
-serum mixed w virus & added to cell mono layer
-incubation (2-3d) cell monolayer inspected for cell damage
-serum contains antibodies to virus, they bind the virus = neutralizing it -> no damage should occur to cell monolayer (POSITIVE)
-serum doesnt have antibodies against virus = virus infect monolayer -> cause cell damage (NEGATIVE)

20
Q

Describe agglutination.

A

-reaction between particular antigen & antibody or when soluble antigen is coated onto latex beads
-antibody will cross link particulate antigen = agglutination of antigen
*determine blood group in dog & cats

21
Q

Describe hemagglutination inhibition assay.

A

-viruses can agglutinate RBCs = hemagglutination
-ability of antibodies to inhibit hemagglutination of RBCs by viruses = HAI
-if antibodies against virus are present = inhibit hemagglutination of RBCs
-used in diagnostic virology

C = POSITIVE *if hemagglutination is NOT inhibited = NEGATIVE
22
Q

Describe transfusion reactions.

A

Loss of blood from:
-direct hemorrhage
-indirectly immune mediated process

23
Q

Describe barriers to blood transfusion.

A

-MHC molecules differ between individuals
-RBCs dont express either MHC I or II
-they do express potentially antigenic molecules called RBCs

24
Q

Describe alloantibodies.

A

-adverse reactions happen in blood transfusions bc animals have naturally occurring antibodies to diff RBC anitgens called alloantibodies
*antibodies of one individuals that react against alloantigen of another individual of the same species

25
Q

Describe dog blood antigens.

A

-most imp antigen in blood group incompatibility = dog erythrocyte antigen 1 (DEA1)
-untransfused dogs dont have sig amount of naturally occurring alloantibodies to DEA1
-if exposed to DEA 1.1 pos blood cells = dogs lacking DEA 1.1 will make lg amounts of antiDEA1.1 antibodies

26
Q

Describe cat blood antigens.

A

-types A, B, AB, Mik
-some untransfused cats have naturally occurring anti blood group antibodies
-cats that have type B blood make high titer anti A antibodies
-cats w type B blood = strong transfusion reaction to type A on first transfusion

27
Q

Describe horse blood antigens.

A

-Aa, Qa, Ca

28
Q

Describe diagnosis of blood type incompatibilities.

A

-to prevent or anticipate transfusion reactions due to diff blood types = know recipients & donors blood types & compatibility
>blood typing = patient & donor blood
>cross matching = patient & donor blood

29
Q

Describe blood typing.

A

-monoclonal/polyclonal antibodies that recog the common erythrocyte antigens of a species
-antibodies bind to the blood type antigens on the surface of erythrocytes = cross link these cells & cause them to agglutinate
*typing cards, typing gels, membrane dipsticks

30
Q

Describe typing feline blood.

A

-drop of blood added to card/gel containing anti A antibodies
-anti A antibodies bind to any erythrocyte A antigens present & cause the blood agglutinate
-confirms blood to be type A
-type B blood (doesnt possess an A antigen) is added = anti A antibodies will not bind & thus no agglutination
-blood added to a card w anti B antibodies cause agglutination of type B blood but not of type A

31
Q

Describe blood typing card.

A
32
Q

Describe typing gels.

A

-use monoclonal antibodies against RBC antigens suspended in gel matrices in test tubes
-blood applied to tube filters thru gel
-if RBCs possess the antigen to the antibody in gel = erythrocyte will agglutinate & become suspended in the gel
-if RBCs dont possess antigen to antibody in gel = pass thru gel & settle at bottom of tube

33
Q

Describe membrane dipstick.

A

-uses anti RBC monoclonal antibodies bound to lines on dipstick
-dipstick dipped into diluted blood & erythrocyte move up dipstick via capillary action
-antigen pos RBCs bind to monoclonal antibody at line

34
Q

Describe cross matching.

A

-saline agglutination of RBCs
-major & minor cross matches
-sera & RBCs obtained from recipient & donor
*major = recipient serum mixed w donor erythrocyte to test if recipient has alloantibodies that would destroy donor RBCs
*minor = donor serum mixed w recipient erythrocyte to test if donor has alloantibodies that would destroy recipients RBCs

35
Q

Describe cross matching of feline A, B, AB blood types.

A
White box = no cross reaction occurs = safe