Immunodiagnostics Flashcards
Describe serum.
-source of antibodies
-portion of blood left after clotting = removes platelets, WBCs, RBCs, clotting factors
Describe serology.
-study of in vitro reactions of antibodies in serum & antigens = microorganisms that cause infectious diseases
Describe requirements for antibody based techniques.
- Antigens = bind to antibody
- Antibodies = generated to detect antigen
- Method of visualizing = antibody-antigen reaction (ex. Dyes, enzymes, etc)
Describe the 2 types of antibodies used in diagnostics & research.
- Polyclonal antibodies = mixture of antibodies specific for epitopes of antigen -> products of diff clones of B cells -> source of polyclonal antibodies is serum (antiserum)
- Monoclonal antibodies = antibodies specific for single antigenic epitope -> products of single B cell clone
Describe polyclonal antibody production.
-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
Describe monoclonal antibody production.
Describe detecting antibody antigen reactions.
• Enzyme-linked immunosorbent assays (ELISA)
• Immunofiltration technique
• Immunohistochemistry
• Immunofluorescent microscopy
• Precipitation assays
• Immunoblotting
• Agglutination
• Hemagglutination inhibition assays
• Complement fixation test
• Virus neutralization test
Describe enzyme linked immunosorbent assay (ELISA).
- Create solid phase - coat plate w known antigen
- Add serum containing antibodies that you’re looking for (source of primary antibodies)
- Add secondary antibodies against species from which the serum originated = labeled w enzyme
- Add substrate for the enzyme used to label secondary antibodies
- Look at color change / read optical density in spectrophotometer
Describe sandwich ELISA.
- Create solids phase - coat plates w known antibodies
- Add serum containing antigen you’re looking for
- Add antibodies against antigen you’re looking for - these antibodies are NOT labeled w enzyme (secondary antibodies)
- Add detecting antibody against secondary antibodies - these antibodies ARE labeled w enzyme
- Add substrate for enzyme used to label detecting antibodies
- Observe color change or read optical density in spectrophotometer
Describe SNAP tests.
modification of ELISA
Describe immunohistochemistry.
-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
Describe immunofluorescence.
-same principle as immunohistochemistry
-fluorescent labeled antibodies used instead of enzyme labeled antibodies
-samples: tissue or cells on slides
Describe immunoprecipitation assays.
-antigens & antibodies can form complexes
-small & soluble
>in vivo = type III hypersensitivity
>in vitro = form visible precipitation
Describe the formation of precipitates.
-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
Describe the different types of immunoprecipitation assays.
- Solution based immunoprecipitation
-quantitative or qualitative - 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)
Describe radial immunodiffusion.
-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
Describe immunoblotting.
‘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
Describe virus neutralization.
-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)
Describe agglutination.
-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
Describe hemagglutination inhibition assay.
-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
Describe dog blood antigens.
-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
Describe cat blood antigens.
-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
Describe horse blood antigens.
-Aa, Qa, Ca
Describe typing feline blood.
-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
Describe blood typing card.
Describe typing gels.
-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
Describe membrane dipstick.
-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
Describe cross matching of feline A, B, AB blood types.
