Basis of Immunologic Diagnostics

Question 1

Antigen-Antibody Assay

Answer 1

Precipitation

Flocculation

Neutralization

Complement Activation

Question 2

Precipitation

Answer 2

Mechanism

Ab binds to soluble Ag,
forming a visible precipitate

Examples

Precipitin ring test to
visualize lattice formation in
solution
Immunoelcetrophoresis to
examine distribution of
antigens following
electrophoresis
Outchterlony assay to
compare diverse antigens
Radial immunodiffusion
assay to quantify antigens

Question 3

Flocculation

Answer 3

Mechanism

Ab binds to insoluble
molecules in suspension,
forming visible aggregates

Examples

VDRL test for syphilis

Question 4

Neutralization

Answer 4

Mechanism

Ab binds to virus, blocking
viral entry into target cells
and preventing formation of
plaques

Examples

Plaque reduction assay for
detecting presence of
neutralizing antibodies in
patient sera

Question 5

Complement activation

Answer 5

Mechanism

Ab binds to Ag, inducing
complement activation and
leaving no complement to lyse RBCs

Examples

Complement fixation test for
patient antibodies against
hard-to-culture bacteria such
as Chlamydia

Question 6

Select Antibody- Antigen Assay

Answer 6

Agglutination

Hemagglutination

Question 7

Agglutination

Answer 7

Mechanism

Direct: Ab is used to clump
bacterial cells or other large structures

• Ex: Serotyping bacteria

Indirect: latex beads are
coupled w/ Ag or Ab to look
for Ab or Ag, respectively, in
patient serum

• Ex: Indirect: latex beads are
  coupled w/ Ag or Ab to look
  for Ab or Ag, respectively, in
  patient serum

Question 8

Hemoagglutination

Answer 8

Mechanism

Direct: some bacteria and
viruses cross-link RBCs and
clump them together

• Ex: Diagnosing influenza,
  mumps, and measles

Direct Coombs’ test (DAT):
detects nonagglutating Abs or
complement proteins on RBC in vivo

• Ex: Checking for maternal Abs
  binding to neonatal RBCs

Indirect Coombs’ test (IAT):
screens an individual for Abs
against RBCs Ags (other than
the A and B Ags) that are
unbound in a patient’s serum
in vitro

• Ex: Performing pretransfusion
  blood testing

Viral hemagglutination
inhibition: uses Abs from a
patient to inhibit viral
agglutination

• Ex: Diagnosing various viral
  diseases by the presence of
  patient Abs against the virus

Blood typing and cross-
matching: Detects ABO, Rh,

and minor antigens in the blood

• Ex: Matches donor blood
  recipient immune
  requirements

Question 9

Immunoblots and Enzyme Assays

Answer 9

Immunoblots

Immunostaining

Enzyme-lined immunosorbent (ELISA)

Immunochromatographic

Question 10

Immunoblots

Answer 10

Mechanism

Western blot: detects
the presence of a
particular proteins

• Ex: Detecting the
  presence of HIV
  peptides (or
  peptides from other
  infectious agents)
  in patient sera

Question 11

Immunostaining

Answer 11

Mechanism

Immunohistochemistry:
used to stain specific
cells in a tissue

• Ex: Stain for presence
  of CD8 cells in
  host cells

Question 12

ELISA

Answer 12

Mechanism

Direct ELISA: uses a
single AB to detect the
presence of an antigen

• Ex: Detection of HIV antigen p24 up to one month after being infected

Indirect ELISA:
measures the amount of
Ab produced against an
antigen

• Ex: Detection of HIV Abs in serum

Question 13

Immunochromatographic

Answer 13

Mechanism

Sandwich ELISA:
measures the amount of
Ag bound by the Ab

• Ex: Detection of Abs for various pathogens in patient sera (Rapid strep, malaria dipstick)

Pregnancy test
detecting human
chorionic

Question 14

Flurescent Antibody Techniques

Answer 14

Direct Flurescent Antibody

Indirect Fluorescent Antibody

Flow cytometry

Fluorescence Activated Cell Sorter (FACS)

Question 15

Direct Fluorescent Antibody

Answer 15

Mechanism

Uses fluorogen-Ab
conjugates to label bacteria
from patient samples

Ex: Visualizing Legionella
pneumophila from a throat
swab

Question 16

Indirect Fluorescent Antibody

Answer 16

Mechanism

Detects disease-specific Abs
in patient serum

Ex: Diagnosing syphilis;
detecting antinuclear Abs
(ANA) for lupus and other
autoimmune diseases

Question 17

Flow cytometry

Answer 17

Labels cell membranes with
fluorogen-Ab conjugate
markers excited by a laser;
machine counts the cell and
records the relative
fluorescence

Ex: Counting the number of
fluorescently labeled CD4 or
CD8 cells in a sample

Question 18

Fluorescence Activated Cell Sorter

Answer 18

Form of flow cytometry that
both counts cells and
physically separates them into
pools high and low
fluorescence cells

Ex: Sorting cancer cells