Lab 5: Culturing Hybridoma Cells and Immunodetection

Question 1

How is the secreted antibody titer (monoclonal antibodies raised against human transferrin) determined, and hybridoma cells selected and screened?

Answer 1

Using an indirect Enzyme-Linked ImmunoSorbent Assay (ELISA)

-provide useful concentration of antibodies or antigen

Question 2

How is the antibody specificity tested?

Answer 2

Through a Western Blot

Question 3

How are hybridoma cells selected for using HAT media that supports the growth of only hybridoma cells?

Answer 3

• unfused cells and cells fused to cells other than myelomas will not survive due to the limited growth potential
• unfused myeloma cells and myeloma cells fused to other myeloma cells will be unable to grow

Question 4

What do we want the hybridoma cells we are working with?

Answer 4

IgG monoclonal antibodies to antigen human transferrin

Question 5

What is the difference between sensitivity and specificity?

Answer 5

sensitivity: ability to detect small amounts of antigen or antibody
specificity: ability to discriminate between closely related but antigenically different molecules

Question 6

What is the order for the addition for an Indirect ELISA?

Answer 6

1) coat well with antigen
2) Block
3) incubate with primary antibody
4) wash
5) Incubate with secondary antibody
6) wash
7) add substrate and observe colour change

Question 7

What is the purpose of blocking in indirect ELISA and what is used for it in the lab?

Answer 7

• blocks any unbound sites of the well to prevent non-specific binding of primary or secondary antibodies
• used skim milk dissolved in PBS-Tween

Question 8

What is the purpose of washing in indirect ELISA and what is used for it in the lab?

Answer 8

• removes unbound or excess antibody that is not bound tightly to the solid phase
• PBS-Tween is detergent that disrupts proteins that are not bound tightly

Question 9

How is colour shown using the spectrophotometer? How is colour being developed?

Answer 9

the secondary antibody has an enzyme attached. The secondary antibody detects the presence of the primary antibody and the secondary antibody is raised against the Fc region of the primary antibody. A chromogenic or fluorescent substrate is added

Question 10

What is the substrate used for the Indirect ELISA? What are the conditions the substrate has to be in?

Answer 10

• use PNPP

- substrate dissolved diethanolamine with a pH 9.8 (basic pH optimal for substrate)

Question 11

What is the definition of an antibody titer?

Answer 11

The dilution of antibody that gives half-maximal absorbance (shown between 2 dilution factors)

Question 12

What are the three general steps of Western blotting/immunoblotting?

Answer 12

1) separation of proteins by SDS-PAGE
2) transfer proteins onto membrane for electrophoresis
3) Identification of proteins by specific antibodies

Question 13

What is the purpose of the Western Blot in this lab?

Answer 13

To confirm specificity of monoclonal antibodies produced by hybridoma cell line to antigen Human Transferrin (HT)

Question 14

What is the purpose of the pre-stained molecular weight marker?

Answer 14

1) in SDS to determine the relative molecular weight of the HT antigen on the Coomassie gel
2) to confirm the transfer of separated proteins to the membrane

Question 15

What are some characteristics of the PVDF membrane and what is the purpose of hydrating it with methanol?

Answer 15

• the membrane is hydrophobic and binds proteins that have strong hydrophobic interactions
• methanol coats membrane with polar surface so that aqueous solutions can interact with the membrane

Question 16

How is primary antibody detected in the Western Blot/Immunoblotting?

Answer 16

the bound primary antibody is detected using goat anti-mouse polyclonal secondary antibody covalently linked to alkaline phosphatase

Question 17

what is the substrate used in the Western blot? Where does it produce colour?

Answer 17

The substrates used were BCIP and NBT

Colour where there is a secondary antibody bound to primary antibody, bound to antigen

Question 18

What is the purpose of soaking the PVDF membrane in transfer buffer?

Answer 18

To equilibrate the membrane

Question 19

For the indirect ELISA quantification what is the positive control? Why?

Answer 19

pretested mouse mAb a-HT

Know it can bind to the primary antigen (the human trasnferrin)

Question 20

For the indirect ELISA quantification what are the negative controls? Why?

Answer 20

• mouse mAb a-OTI
• rat mAb a-HT
  Because they are either not for the correct antigen (the OTI) or not the correct antibody (from rat instead of mouse)

Question 21

Why do we perform the Western Blot?

Answer 21

• confirm specificity of monoclonal antibodies to human transferrin
• detect presence of human transferrin antigen in cell lysate

Question 22

What class of primary antibody are we working with?

Answer 22

IgG (monoclonal)— raised against human transferrin

Question 23

What is the media used for sub culturing mouse hybridoma cell line?

Answer 23

Dulbecco’s Modified Eagle Medium (DMEM)

Question 24

What is the secondary antibody for the ELISA?

Answer 24

goat anti-mouse conjugated to alkaline phosphatase

Question 25

What does mouse a-OT1 control for?

Answer 25

controls for binding to primary antigen. Shows that not any mouse IgG antibody can bind human transferrin

Question 26

Why do we see results for rat a-HT?

Answer 26

Shows that the polyclonal antibody can cross-react and recognize the epitope of rat IgG

Question 27

What is the molecular weight of human transferrin?

Answer 27

76-81 kDa