Experimental Methods

Question 1

What are polyclonal antibodies?

Answer 1

a mixture of different antibodies directed toward a variety of epitopes on immunizing antigen

Question 2

How are polyclonal antibodies generated?

Answer 2

• inject host animal with antigen
• the antigen results in a B cell response and plasma cells will secrete antibody
• collect serum from host and isolate the antibodies

Question 3

What are monoclonal antibodies?

Answer 3

antibodies that are generated from a single B cell clone

Question 4

How are monoclonal antibodies (mAb) generated?

Answer 4

• isolate a single B cell and fuse the B cell with myeloma cell, creating a hybridoma
• collect antibodies produced from hybridomas

Question 5

How are monoclonal antibodies modified?

Answer 5

conjugating them to other compounds, but leave antigen binding sites available to bind antigen

Question 6

What can monoclonal antibodies be conjugated to?

Answer 6

• biotin
• HRP
• fluorophore
• magnetic bead
• agarose

Question 7

What are the uses for polyclonal antibodies?

Answer 7

• immunoprecipitation
• hemagglutination

Question 8

What is immunoprecipitation?

Answer 8

antibody-antigen complexes are precipitated from solution

Question 9

What is immunoprecipitation used for?

Answer 9

isolation of proteins

Question 10

What is hemagglutination?

Answer 10

clumping of antibody-RBC complexes

Question 11

What is hemagglutination used for?

Answer 11

blood typing

Question 12

What are the uses for monoclonal antibodies?

Answer 12

• enzyme-linked immunosorbent assays (ELISAs)
• western blotting
• immunohistochemistry

Question 13

What do ELISAs utilize?

Answer 13

monoclonal antibodies conjugated to enzymes that produce color changes

Question 14

What are the types of ELISAs?

Answer 14

• indirect
• sandwich

Question 15

What are indirect ELISAs used for?

Answer 15

to detect presence of antibodies in a sample

Question 16

How are indirect ELISAs performed?

Answer 16

• coat plate with antigen
• add sample that contains antibodies (typically serum)
• add antibody-enzyme complex
• add enzyme substrate
• measure color change

Question 17

What are sandwich ELISAs used for?

Answer 17

to detect presence of an antigen

Question 18

How are sandwich ELISAs performed?

Answer 18

• coat plate with mAbs specific to antigen
• add mAb conjugated to enzyme that is specific for a different epitope on antigen
• add enzyme substrate
• detect color change

Question 19

What is western blotting used for?

Answer 19

used to quantify specific proteins in a sample

Question 20

How is western blotting performed?

Answer 20

• separate proteins in a mixture by molecular weight using gel electrophoresis
• transfer bands to a membrane using electrophoresis
• incubate membrane with enzyme conjugated to mAb
• add enzyme substrate
• use imaging software to view bands

Question 21

What is immunohistochemistry used for?

Answer 21

to detect presence of antigen within tissues

Question 22

How is immunohistochemistry performed?

Answer 22

• add enzymes conjugated mAbs to tissue slices on microscope slide
• add enzyme substrate
• view presence of antigen with microscope