Immunology Techniques

Question 1

What is immunoassay?

Answer 1

• a biochemical test that measures the presence or conc of a specific molecule through the use of an antibody or antigen.
• the molecule detected by the immunoassay is the ‘analyte’ and is in many cases a protein

Question 2

What is polyclonal antibody generation?

Answer 2

• antibodies produced by several clones of B cell recognising different parts of the same antigen

Question 3

What is monoclonal antibody generation?

Answer 3

• single clone- recognises the same epitope within the same antigen

Question 4

What can be added to antibodies in vaccines in order to elicit a stronger response?

Answer 4

• adjuvants (alum, CFA, poly I:C)
• mix to stimulate the innate IC

Question 5

What are the adv and disadv of bleeding subject and generating polyclonal antibodies?

Answer 5

• adv:
  
  • a mixture of ab directed toward a variety of epitopes are formed, good for agglutination and immunoprecipitation
• disadv:
  
  • may produce cross-reactivites against other ag
  • different bleed-points may yield different degress of affinity

Question 6

How do you produce mass quantities of single-specificity Ab?

Answer 6

• fuse the B cells producing this antibody with myeloma (tumour) cells
• the myeloma fuse and can then proliferate continuously
• all the B cells will produce the same antibody from the same clone

Question 7

How can monoclonal antibodies be modified for use in lab etc?

Answer 7

• binding sites of mAb retained, Fc region replaced with other molecule
• conjugation of Fc region with other molecules
  
  • biotin/enzymes used in ELISA assays
• abzymes- mAb that mimic enzymes by binding and stabilizing transition states of chemical reactions

Question 8

What are the ways in which immunoprecipitation can be performed?

Answer 8

• in solution
• in gel matrices

Question 9

How is immunoprecipitation performed in solution?

Answer 9

• formation of such large immune comlexes that they fall out of solution (precipitate)
  
  • only occurs when Ag/Ab concs are roughly equal
• used to purify antigen molecules/ remove antigens from solution

Question 10

How is immunoprecipitation of soluble antigens performed in gel matrices?

Answer 10

• Ouchterlony method
  
  • Ag placed in center well, serum samples placed in outer wells
  • visible ‘precipitin line’ forms where large immune complexes form

Question 11

What are Hemagglutination reactions used for?

Answer 11

• used to detct any Ag conjugated to the surface of rbcs
• used for blood typing
• easy, sentitive, cheap

Question 12

What are hemagglutination inhibition reactions used for?

Answer 12

• used to detect the presence of viruses and of antiviral Ab

Question 13

What is bacterial agglutination used for?

Answer 13

• used to detect antibodies to bacteria
• can provide quantitative info about serum antibacterial Ab concs
• can be diagnostic

Question 14

What are Radioimmunoassays used to measure?

Answer 14

• concs of biologically relevant proteins/hormones in body fluids

Question 15

Describe the process of Indirect ELISAs

Answer 15

• detects presence and concs of Ab in a sample
• method of choice to detect presence of serum Ab against a wide array of pathogens

Question 16

Describe Sandwich ELISA

Answer 16

• measures Ab presence and levels
• useful for measurement of soluble cytokine concs

Question 17

Describe the process of Competetive ELISA

Answer 17

• measures amount of Ag in a sample
• more Ag in original sample, lower the final signal
• can also be used to measure specific Ab
  
  • competition between serum antibodies and an enzyme-conjugated Ag-specific mAb

Question 18

What are the available enzyme systems for ELISA assays?

Answer 18

• Alkaline phosphatase
• Horseradish peroxidase

Question 19

What are the various substrates used in an ELISA?

Answer 19

• chromogenic = coloured product, quick yes/no response
• fluorogenic = gives off fluorescent light that can be detected and measured
• chemiluminogenic = chrmical reaction that gives off light that can be detected and measured

Question 20

How are ELISAs modified using biotin-streptavidin?

Answer 20

• bind biotin to antibody Fc region
• bind strepavidin to enzyme molecule
• allows yse of a variety of primary biotin-tagged Ab in different reactions with just 1 enzyme-conjugated stock of strepavidin
  
  • saves money and provides flexibility in experimental design

Question 21

What do ELISPOT assays measure?

Answer 21

• molecules secreted by individual cells

Question 22

How can Western blotting be used to identify a specific protein?

Answer 22

• IDs and quantifies (roughly) specific protein in a complex protein mixture
  
  • sample prepared in a loading buffer
  • proteins separated on polyacrtlamide gel by electrophoresis
  • proteins transferred from gel to membrane
  • identification by enzyme-tagged specific Ab binding to Ag, followed by substrate exposure
  • expensive and high level of skill

Question 23

What is the different between immunocytochemistry and immunohistochemistry?

Answer 23

• immunocytochemistry = performed on isolated cells
• immunohistochemistry = performed on tissue sections