ELISA Flashcards
Describe how a direct ELISA works
Ag is coated to a multi-well plate and it is detected by an Ab that has been directly conjugated to an enzyme
What are the advantages and disadvantages of a direct ELISA?
advantages:
- quick: only one Ab and fewer steps (also less places to fuck up)
- cross-reactivity of a secondary Ab is eliminated
disadvantages:
- immunoreactivity of the primary Ab may be adversely affected by labeling with enzymes or tags
- labeling of the primary Ab for each specific ELISA system is time consuming and expensive
- no flexibility in choice of the primary Ab label from one experiment to another
- minimal signal amplification
How does an indirect ELISA work?
Ag is coated to a multi-well plate –> add a primary Ab specific for the Ag –> add a enzyme-labeled secondary Ab that is anti-species
What is an indirect ELISA used for? Give examples of tests that use this.
used for testing the amount of Ab to an Ag in serum
tests = HIV test and COVID-19 serological test
What are the advantages and disadvantages of an indirect ELISA?
advantages:
- many labeled secondary Abs are commercially available
- lots of flexibility: the same secondary Ab can be used for detection of any Ab made in a species
- maximum immunoreactivity of the primary Ab is retained because it is not labeled
- very sensitive/good amplification: because each primary Ab contains several epitopes that can be bound by the labeled secondary Ab
disadvantages:
- cross-reactivity might occur with the secondary Ab –> non-specific signal/higher background
- extra incubation step is requires –> more time and more places to fuck up
How does a sandwhich ELISA work?
Capture Ab coated well –> add Ag to be measured –> add enzyme-conjugated second Ab (detection Ab) –> add substrate and measure colour
What are the advantages and disadvantages of a sandwhich ELISA?
advantages:
- highly specific: the Ag is specifically captured and detected
- suitable for crude/impure samples: the Ag doesn’t require purification prior to measurement
- flexibility and sensitive: both direct or indirect detection methods can be used
disadvantages:
- requires matched Ab. pairs, where each Ab is specific for a different epitope of the Ag
Describe how the rapid COVID-19 test uses the same principles as a sanwhich ELISA
Before use: Ab against Ag linked to a detector molecule in the area where the solution will be added; Ab against Ag (diff epitope) further up; control Ab (against the detector Ab)
Positive result: detector Ab binds to spike where it will be captured by another Ab against spike and a control Ab (Postive result and positive control)
Negative result: detector Ab goes past the capture Abs but is captured by the control Ab (negative result and positive control)
What are four factors that affect the sensitivity of a sandwhich ELISA?
- the number of molecules of the first Ab that are bound to the solid phase
- the avidity of the first Ab to the Ag
- the avidity of the second Ab to the Ag
- the specific activity of the enzyme attatched to the detection Ab (direct) or secondary Ab (indirect)
How does a competitive ELISA work?
Incubate primary/detection Ab with Ag –> add these complexes to wells that have been coated with the Ag –> after incubation, any unbound Ab is washed off –> there will be a smaller smount of primary Ab available to bind to the Ag coated on the well –> smaller signal
What is an advantage of a competitive ELISA?
highly sensitive to compositional differences in complex Ag mixtures
What is the Ab titre in an ELISA?
the smallest concentration of Ab that gives a positive result
What are the three types of substrates for detection in an ELISA? What signal do they give? What instrument is required to read them?
- chromogenic –> colour –> spectrophotometer
- fluorogenic –> fluorescence –> fluorometer
- chemiluminscent –> light –> luminometer
What substrate is used for alkaline phosphate and horseradish perioxidase?
AP = pNPP
HP = lumino
What is the purpose of an ELISPOT assay?
measures molecules secreted from cells –> spot size is proportional to secretion
