6. Antibody Based Experimental Systems

Question 1

How are polyclonal antibodies generated?

Answer 1

• immunize animal with antigen > collect antiserum

Question 2

How are monoclonal antibodies made?

Answer 2

• fuse plasma cell with myeloma tumor cell > hybridization (hybridoma)

Question 3

What is required to generate a B-cell hybridoma?

Answer 3

• myeloma cell/ primary plasma cell
• chemical fusogens
• HGPRT selection

Question 4

What are the 2 main types of immunoprecipitation?

Answer 4

• Solution Immunoprecipitation
  > purify antigen from a heterogeneous mixture of soluble molecules
• Gel Immunoprecipitation
  > assay for the presence of antibodies

Question 5

How does solution immunoprecipitation work?

Answer 5

• multivalent antibodies mixed in solution with polyvalent antigen (more than 1 antibody-binding site/ antigen)
• cross-linking complex is so large it precipitates out of solution
• needs equal concentrations of antigen/ antibody to work

Question 6

How does gel immunoprecipitation work?

Answer 6

• when antigen/ antibody diffuse toward each other in a gel, a visible line of precipitation forms > precipitin line
• diagnostic application to assay for antibody presence

Question 7

In the Ouchterlony gel immunoprecipitation method, antigen/ antibody diffuse toward each other. When does the precipitin line form?

Answer 7

• at a certain concentration > equivalence

Question 8

What kind of antibody does immunoprecipitation use?

Answer 8

• polyclonal antibody > bind to different epitopes
• immunoprecipitation does not require the use of monoclonal antibody

Question 9

What is agglutination?

Answer 9

• results from cross-linking of multivalent antibodies/ antigens
  > visible clumping of complexes formed between cells with antigens/ binding antibodies
• cells with antigen form clumps with antibodies, not soluble antigen

Question 10

What is the difference between immunoprecipitation/ agglutination?

Answer 10

• immunoprecipitation > soluble antigens
• agglutination > antigens bound to cells (not soluble antigen)

Question 11

What is hemagglutination?

Answer 11

• agglutination reaction when antibodies bind antigens on surface of RBCs

Question 12

What are agglutins?

Answer 12

• antibodies that produce agglutination reactions

Question 13

What are hemagglutination inhibition reactions used to detect?

Answer 13

• responses to viruses

Question 14

How do hemagglutination inhibition reactions work?

Answer 14

• some viruses can agglutinate RBCs
• check for antibody against virus in patient serum by adding virus to RBCs
• if agglutination occurs > patient does not have antibodies
• if no agglutination > patient has viral antibodies

Question 15

What are 2 examples of antibody assays based on molecules bound to solid-phase supports?

Answer 15

• ELISA > allows determination of antibody/ antigen
• Western Blotting > identify a specific protein in a complex protein mixture

Question 16

What are the 3 types of ELISA assays?

Answer 16

1. Indirect ELISA > used to measure antibodies
2. Sandwich ELISA > used to measure antigens
3. Competitive ELISA > used to measure antigen levels in pre-incubation solution

Question 17

How does western blotting work?

Answer 17

• technique for detection of a specific protein in a sample
• treat protein mixture with SDS (denaturing detergent)
• separate protein mixture via electrophoresis (by molecular weight)

Question 18

What are some antibody-mediated microscopic visualization methods?

Answer 18

• Immunocytochemistry > on cells
• Immunohistochemistry > on tissue samples
• Immunoelectron microscopy
  > antibodies coupled to electron-dense particles like gold particles
  > electron gold microscopy to detect gold deposits

Question 19

How are immunocytochemistry/ immunohistochemistry different?

Answer 19

• enzyme-conjugated antibodies bind to proteins/ antigens in cells
• Immunocytochemistry > on cells
• Immunohistochemistry > on tissue samples

Question 20

In flow cytometry, what do forward/ side scatter measure?

Answer 20

• FSC > reflects size of cell
• SSC > internal complexity of cell