W7.1

Question 1

Specimen Types

Answer 1

Serum, urine, cerebrospinal fluid, tissue

Question 2

Serum (as a specimen)

Answer 2

Includes all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances
(e.g., drugs and microorganisms)

Question 3

Potential Techniques/Assays that could be performed

Answer 3

• Agglutination/Precipitation
• ELISAs (looking for biomarkers in theplasma/serum/urine/CSF)
• PCR? Western Blots? Southern Blots?
• Flow Cytometry (a way to determine more directly if your immune cells are
  just hanging out or all kinds of fired up)

Question 4

Antibodies

Answer 4

Stimulation of B lymphocytes by antigens causes the cell to undergo proliferation and differentiation = end product is an antibody (or immunoglobulin)

Question 5

Monoclonal antibodies

Answer 5

• Purified antibodies cloned from a single cell

- Bind to a specific antigen (a specific epitope on that antigen)

Question 6

Polyclonal antibodies

Answer 6

a bunch of antibodies from several cells that bind the same antigen
(probably multiple different epitopes)

Question 7

Discovery of technique to produce monoclonal antibodies:

Answer 7

• 1975: Niels Jerne, Georges Kohler and Cesar Milstein
• 1984: Nobel Prize for Physiology and Medicine “for theories concerning the specificity in development and control of the immune system and discovery of the principle for production of monoclonal antibodies”

Question 8

Production of Monoclonal Antibodies

Answer 8

• Immunization of mouse with specific antigen
• Harvesting of spleen cells
• Spleen cells are combined with myeloma cells in the presence of polyethylene glycol (PEG)

Question 9

PEG

Answer 9

polyethylene glycol

- (surfactant) brings about fusion of plasma cells with myeloma cells, producing a hybridoma

Question 10

hat

Answer 10

hypoxanthine, aminopterin, and thymidine (selective medium)

• Medium is used to separate the hybridoma cells by allowing them to grow selectively
• Note: myeloma cells usually grow indefinitely yet cannot due to the components of the medium
• Selective properties of the medium include:
• Pathway that builds DNA from degradation of old nucleic acids is blocked as the myeloma cell line is deficient in the required enzymes HGPRT (hypoxanthine-guanine phosphoribosyltransferase) and thymidine kinase
• Pathway that builds DNA from new nucleotides is blocked by the presence of aminopterin

Question 11

hybridoma

Answer 11

???

Question 12

Clones

Answer 12

Production of Monoclonal Antibodies

• Hybridoma cells are diluted and screened for the presence of the desired antibody (looking for the correct clone)
• Screened using enzyme immunoassay
• Clones are grown in mass cultures; produce a permanent and uniform supply of monoclonal antibodies

Question 13

Diagnostic Applications for Monoclonal Antibody

Answer 13

• Identifying and quantifying hormones
• Typing tissue and blood
• Identifying infectious agents
• Identifying tumor antigens and autoantibodies

Question 14

Therapeutic Agents (monoclonal antibody)

Answer 14

(allows higher concentration of drugs to reach desired site)

• As direct therapeutic agents
• Opsonization to help destroy disease-causing microbes
• Treatment of cancer (bind and destroy)
• Immunosuppression of organ transplantation
• Targeting agents in therapy

Question 15

Immunological techniques

Answer 15

• Electrophoresis
• Precipitation and Agglutination reactions
• Labeled immunoassays
  Other: Molecular assays, Flow cytometry