Experimental Tools and Techniques Flashcards
(26 cards)
What occurs when a plasma cell and a myeloma cell undergo hybridization?
A hybridoma is formed
What occurs when two plasma cells undergo hybridization?
The cell dies within a few days
What does HGPRT do?
It catalyzes a step in the DNA synthesis salvage pathway
What does HAT selection media do?
aminopterin inhibits de novo DNA synthesis so cells must use a salvage pathway (ex. HGPRT mutants can’t grow); supplemented with hypoxanthine and thymidine to support nucleotide synthesis
What hybridomas survive?
Only hybridomas that gain WT HGPRT will survive
What type of experimental technique is used for ABO blood typing?
Agglutination
Hemagglutination from influenza causes agglutination of ___
RBCs
What is used to type influenza viral infection?
hemagglutination inhibition
The disappearance of the button in hemagglutination inhibition assays indicates: ___
Decreased levels of anti-viral antibodies
What does ELISA do?
It detects proteins and can be quantifiable (protein concentration)
What are the three different types of ELISA?
-Indirect
-Sandwich
-Competitive
What are the steps involved in indirect ELISA?
-Antigen coated well
-Wash
-Add specific antibody to be measured
-Wash
-Add enzyme-conjugated secondary antibody
-Wash
-Add substrate and measure colour
What are the steps involved in sandwich ELISA?
-Antibody coated well
-Wash
-Add antigen to be measured
-Wash
-Add enzyme-conjugated secondary antibody
-Wash
-Add substrate and measure colour
What is colour used to measure in indirect ELISA?
Antibodies
What is colour used to measure in sandwich ELISA?
Antigens
What are the steps of competitive ELISA?
-Pre-incubate antibody with antigen to be measured
-Add Ag-Ab mixture to antigen-coated well
-Wash
-Add enzyme-conjugated secondary antibody
-Wash
-Add substrate to measure colour
What is colour used to measure in competitive ELISA?
Used to measure antigen levels in pre-incubation solution
What does the ELISPOT do?
Detects proteins secreted by cells
Can be quantifiable (# of secreting cells)
What mAb-based method is used for detecting proteins in a COVID test and pregnancy tests?
Lateral flow assay architecture
What does Western blotting (immunoblotting) do?
-Detects specific proteins at specific sizes
-Semi-quantitative
During Western Blotting what does SDS do?
SDS coats proteins with a negative charge so proteins migrate according to size not innate charge
What is equilibrium analysis used for?
To determine antibody affinity
If an antibody had a lower affinity, would there be more or less free ligands compared to an antibody with a higher affinity?
More
What is an essential aspect of confocal fluorescence microscopy?
It removes light outside of the focal plane
