Polyclonal and Monoclonal Ab

1
Polyclonal and Monoclonal Ab

• Antiserum (serum containing Ab) contains a
  mixture of different Ab directed at the numerous
  determinants on an Ag. This mixture of serum Ab
  is called polyclonal Ab and, though they protect
  the host, they do not provide reproducible
  results for use in clinical diagnostic testing.
• Only a few Ab are directed toward each Ag
  determinant. Ab produced by a single B cell (or
  in vitro clones of a single B cell) monoclonal
  Ab. Monoclonal Ab do provide reproducible
  results for clinical testing ex. for
  immunological typing of bacteria, pregnancy
  testing and blood typing.

2
Serology

• Serology the study of Ag-Ab rxns. in vitro.
• Specificity ability of an Ab prep. to recognize
  a single Ag (no cross-rxn, no false pos. rxn).
• Sensitivity the lowest amt. of an Ag that can
  be detected. High sensitivity prevents false
  neg. rxns.
• Precipitation tests are the least sensitive
  serological tests. Enzyme-linked immunosorbent
  assays (ELISA) are among the most sensitive.
• Neutralization interaction of Ab with Ag
  (usually in vivo) to block Ag sufficiently to
  reduce or eliminate its biological activity, ex.
  antitoxin.

3
Precipitation

• Precipitation soluble Ab soluble Ag
  insoluble complex.
• Precipitation rxns are easily observed in vitro.
• Precipitation tests carried out in agar gels
  immunodiffusion tests, used to study specificity
  of Ag-Ab complexes.

4
Agglutination

• Agglutination visible clumping of a particular
  Ag Ab specific for it.
• More sensitive than precipitation tests,
  inexpensive, rapid, widely used in clinical labs
  to ID blood groups, pathogens, and pathogen
  products.
• Direct agglutination soluble Ab Ag that is
  integral part of the surface of a cell or other
  insol. particle.
• Passive agglutination soluble Ab or Ag that
  have been adsorbed or chemically coupled to cells
  or insol. particles such as latex beads or
  charcoal particles, 5X more sensitive than direct
  agglut., ex. latex agglut. test for S. aureus,
  Strep. pyogenes, N. gonorrhoeae, Candida albicans.

5
Immunoelectron Microscopy

• Ab conjugated to heavy metals are used to locate
  Ag (usually proteins such as enzymes) in cells by
  electron microscopy.
• Can also be used to locate pathogens, ex. HIV, in
  cells, but is so expensive that it is reserved
  for only the most specialized clinical research
  settings.

6
Fluorescent Ab

• Fluorescent Ab Ab chemically modified with
  fluorescent dyes.
• Used in clinical applications to diagnose
  suspected pathogens long before primary isolation
  techniques show growth, ex. to diagnose
  Legionella, Bacillus anthracis, viral diseases,
  malignant cells.
• Used in research applications to separate complex
  mixtures of cells, ex. immune cells.
• Disadvantage Ab can show cross reactivity with
  various bacterial species.

7
ELISA

• ELISA Enzyme-Linked Immunosorbent Assay
• Makes use of Ab to which enzymes have been
  covalently bound so that the enzymes catalytic
  properties and the Abs specificity are
  unaltered.
• Enzymes include peroxidase, alkaline
  phosphatase, ?-galactosidase, all of which
  catalyze rxns whose products are colored and can
  be detected in small amts with a
  spectrophotometer.
• ELISA tests have been developed for HIV,
  Salmonella, E. coli toxin, S. aureus enterotoxin,
  Vibrio cholerae, Mycobacterium tuberculosis,
  Mycobacterium leprae, Legionella pneumophila,
  Borrelia burgdorferi, Treponema pattidum, Candida
  what diseases do these organisms cause?

8
Immunoblot

• Immunoblot very sensitive method for detecting
  specific proteins in complex mixtures.
• 1. Protein mixture is separated by
  electrophoresis on a polyacrylamide gel.
• 2. Distinct bands of proteins (each of a certain
  mw) are transferred to a membrane by
  electrophoretic transfer.
• 3. Ab raised against a protein or group of
  proteins from a pathogen are added to the blot.
• 4. Radioactive marker that binds Ag-Ab complexes
  is added.

9
Nucleic Acid Probes

• Genotypic rather than phenotypic characteristics
  are used to ID pathogens.
• Genetic or DNA-based diagnostic procedures based
  on
• 1. NAs can be readily isolated from infected
  tissues.
• 2. NAs can be readily visualized and measured.
• 3. NA sequence of an individual pathogen genome
  is so unique that NA hybridization analysis can
  be used for ID
• 4. NA sequences can be amplified to increase the
  amt of material available for analysis.

10
Nucleic Acid Probes (cont.)

• NA probes usually 20 bases or less,
  single-stranded.
• NA probes bind with single-stranded target DNA to
  form double-stranded molecule.
• Probe is labeled with a reporter molecule, ex.
  radioisotope, enzyme, or fluorescent compound.
• As little as 0.25?g of DNA can be detected.
• PCR can be used to detect and amplify specific
  DNA sequences.
• Advantages stable, can be more specific than Ab
  test, very sensitive.

11
Diagnostic Virology

• Viral pathogens can be diagnosed by the following
  methods
• Virus growth in vitro with cell lines.
• Electron microscopy.
• ELISA
• NA probes and PCR