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 enzyme’s catalytic properties and the Ab’s 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