Immuno 14 - Immunodiagnostic Techniques

Question 1

sensitivity, specificity, false positive, false negative

Answer 1

• Sensitivity: the probability that a test
  result will be positive when a disease is present (want to avoid false negatives)
• Specificity: the probability that a test will be negative when the disease is absent (want to avoid false positives)

Test errors:
* False‐positive result: a positive result on a sample that is actually negative
* False‐negative result: a negative result on a sample that is actually positive

Question 2

flow cytometry

Answer 2

• The fluidics system of a flow cytometer is designed to arrange cells within a sample into single file so they can be interrogated one at a time by a laser
• A narrowing central chamber that contains the sample to be analyzed is
  surrounded by a sheath containing faster moving fluid (i.e., the “sheath fluid”)
• This creates a physical process known hydrodynamic focusing that causes cells to exit the central chamber (and pass by a laser beam) one at a time

Question 3

what flow cytometry can tell us about a cell

Answer 3

• Its relative size based on forward‐scattered light (FSC) – think of this as measuring the size of the shadow made by the cell
• Its relative granularity or internal complexity based on side‐ scattered light (SSC; 90o angle to laser beam)
• Its relative fluorescence intensity (FL1, FL2, FL3, etc.)
• Measured by detectors

Question 4

forward vs side scatter

Answer 4

• Plotting forward scatter vs. side scatter data allows cell subsets to be differentiated based on their physical characteristics (e.g., size and complexity/granularity)

Question 5

flow cytometry; surface vs intracellular staining

Answer 5

• Antibodies added to cells bind to antigens expressed on the surface
• If cells are fixed and then permeabilized, antibodies can bind to intracellular antigens
• An additional permeabilization step can allow antibodies to access the nucleus

Question 6

ELISA

Answer 6

• Antigen is bound to the wells in a styrene plate
• Bound antibodies from a serum sample are detected using an
  enzyme‐labeled anti‐
  immunoglobulin
• Addition of the enzyme substrate leads to a color change proportional to the amount of bound antibody
• The color change can be read in an ELISA reader (a special spectrophotometer)

Question 7

sandwich ELISA

Answer 7

• Antigen (e.g., from a serum sample) is bound to the plate by means of an antibody
• Bound antigen is detected by sequential addition of a second antigen‐specific antibody and an
  enzyme‐labeled antiglobulin
• Addition of the enzyme substrate
  leads to a color change proportional to the amount of bound antigen

Question 8

immunofiltration; snap tests

Answer 8

• An immunofiltration technique
• Capture antibody is immobilized on
  a membrane
• It binds to antigens in the test sample (e.g., serum)
• Reagent samples are allowed to
  flow through sequentially
• The final labeled product is seen as
  a colored bar or dot
• In practice this method is used in
  the form of a plastic‐mounted kit
• Can also be used to detect antibodies (i.e., the antigen is bound to the membrane and binds Abs in the test sample)

Question 9

immunochromatography

Answer 9

• A sample containing an antigen flows through a porous strip
• Positive reactions are shown by the appearance of a coloured band

Question 10

virus neutralization assay (plaque reduction)

Answer 10

• The test virus is plated on a “permissive cell line” (i.e., one that supports the replication of the virus)
• Virus replication can be observed via the development of “plaques” in a confluent monolayer of cells (i.e., clear circles develop where the virus is spreading and killing cells)
• The virus is incubated with various dilutions of serum from the animal being tested
• If virus‐specific antibodies are present, they will neutralize the virus (i.e., prevent it from infecting cells) and no plaques will form in the permissive cell monolayer
• The more dilute the serum can be and still prevent plaque formation, the higher the antibody titer

Question 11

virus hemoagglutination inhibition assay

Answer 11

• Some viruses will attach to red blood cells and cause them to agglutinate (i.e., hemagglutination)
• Influenza viruses are one example; they express hemagglutinins
• If virus‐specific antibodies are present in serum, they will neutralize the virus and prevent/inhibit hemagglutination
• The more dilute the serum can be and still inhibit hemagglutination, the
  higher the antibody titer

Question 12

PCRs

Answer 12

• By performing a cycle of reactions repeatedly, it is possible to produce large amounts of DNA coding for a
  gene of interest
• Once produced in sufficient amounts, this DNA can be detected by gel electrophoresis

reverse transcription PCR (RT-PCR)
* To detect RNA, it must first be transcribed into cDNA using reverse
transcriptase
* Then PCR can amplify the cDNA
* This can be used to detect RNA viruses