Lab 5: Culturing Hybridoma Cells and Immunodetection Flashcards
How is the secreted antibody titer (monoclonal antibodies raised against human transferrin) determined, and hybridoma cells selected and screened?
Using an indirect Enzyme-Linked ImmunoSorbent Assay (ELISA)
-provide useful concentration of antibodies or antigen
How is the antibody specificity tested?
Through a Western Blot
How are hybridoma cells selected for using HAT media that supports the growth of only hybridoma cells?
- 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
What do we want the hybridoma cells we are working with?
IgG monoclonal antibodies to antigen human transferrin
What is the difference between sensitivity and specificity?
sensitivity: ability to detect small amounts of antigen or antibody
specificity: ability to discriminate between closely related but antigenically different molecules
What is the order for the addition for an Indirect ELISA?
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
What is the purpose of blocking in indirect ELISA and what is used for it in the lab?
- blocks any unbound sites of the well to prevent non-specific binding of primary or secondary antibodies
- used skim milk dissolved in PBS-Tween
What is the purpose of washing in indirect ELISA and what is used for it in the lab?
- 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
How is colour shown using the spectrophotometer? How is colour being developed?
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
What is the substrate used for the Indirect ELISA? What are the conditions the substrate has to be in?
- use PNPP
- substrate dissolved diethanolamine with a pH 9.8 (basic pH optimal for substrate)
What is the definition of an antibody titer?
The dilution of antibody that gives half-maximal absorbance (shown between 2 dilution factors)
What are the three general steps of Western blotting/immunoblotting?
1) separation of proteins by SDS-PAGE
2) transfer proteins onto membrane for electrophoresis
3) Identification of proteins by specific antibodies
What is the purpose of the Western Blot in this lab?
To confirm specificity of monoclonal antibodies produced by hybridoma cell line to antigen Human Transferrin (HT)
What is the purpose of the pre-stained molecular weight marker?
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
What are some characteristics of the PVDF membrane and what is the purpose of hydrating it with methanol?
- 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
How is primary antibody detected in the Western Blot/Immunoblotting?
the bound primary antibody is detected using goat anti-mouse polyclonal secondary antibody covalently linked to alkaline phosphatase
what is the substrate used in the Western blot? Where does it produce colour?
The substrates used were BCIP and NBT
Colour where there is a secondary antibody bound to primary antibody, bound to antigen
What is the purpose of soaking the PVDF membrane in transfer buffer?
To equilibrate the membrane
For the indirect ELISA quantification what is the positive control? Why?
pretested mouse mAb a-HT
Know it can bind to the primary antigen (the human trasnferrin)
For the indirect ELISA quantification what are the negative controls? Why?
- 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)
Why do we perform the Western Blot?
- confirm specificity of monoclonal antibodies to human transferrin
- detect presence of human transferrin antigen in cell lysate
What class of primary antibody are we working with?
IgG (monoclonal)— raised against human transferrin
What is the media used for sub culturing mouse hybridoma cell line?
Dulbecco’s Modified Eagle Medium (DMEM)
What is the secondary antibody for the ELISA?
goat anti-mouse conjugated to alkaline phosphatase
What does mouse a-OT1 control for?
controls for binding to primary antigen. Shows that not any mouse IgG antibody can bind human transferrin
Why do we see results for rat a-HT?
Shows that the polyclonal antibody can cross-react and recognize the epitope of rat IgG
What is the molecular weight of human transferrin?
76-81 kDa
