Advanced antibody based immunoassays Flashcards
What does ELISA stand for? And the method
Enzyme linked immunosorbent assay.
- Proteins such as Ag or Ab (w/ Alb) sticks on plastic
- washed
- conjugate Ab is put
- washed
- substrate is added = colour change = detected
Describe the different types of ELISA
- Direct ELISA: 1º Ab conjugate bind to Ag (on plastic) -> substrate added = colour change (bc enzymes)
- Indirect: 2º Ab conjugate binds to 1º Ab (bound to Ag on plastic)= enhance signal
- Sandwich: similar to indirect but 1º Ab is bound to unknown Ag - held by a capture Ab on the plastic
- Competitive: non & labelled Ag compete for binding sites on Ab. Measure the amount of known labelled Ag to determine [ ] of unknown & non-labelled Ag: [unknown Ag] inversely proprt. to intensity of signal
How would you use an ELISA to measure the concentration of a particular serum protein?
(quantitative ELISA give [ ])
- Detect Ag: require capture Ab (on plastic) to bind to unknown Ag & 2º conjugate Ab binds to Ag
- Detect Ab: requires known Ag (on plastic) binds to 1º Ab & 2º conjugate Ab binds to Ab (=enhance signal)
Describe a competitive RIA (radioimmuno assay)
Known amount of radioactive (labelled) Ag competes with unlabelled Ag in a sample for binding sites on 1º Ab = 1º Ab bound to 2nd Ab for detection
- intensity of signal is inversly proportional to [unknown Ag] i.e. low signal = more [unlabled Ag]
What is the difference between Direct and Indirect immunofluorescence?
- Direct: detect Ag or Ag-Ab complexes formed in vivo: distinguish b/w T cells & B cells bc have diff. markers
- Indirect: detect autoantibodies in serum (for biposies): use 2º Ab to inc. signal
Compare and contrast Immunohistochemistry to Immunofluorescence and ELISA methods
- IHC: Ag on tissue; enzyme labelled Ab; apply & wash method like ELISA; view on light microscopy
- IF: view on fluorescent microscopy bc use fluorescent labelled Ab to detect Ag
- ELISA: Ag/Ab on plastic; Ab conjugate can be labelled w/ enzyme
Principles of immunmohistochemistry
- permeate membrane so enzymes get in cell
- add patient serum (to see if they have Ab to Ag on nuclei)
- Wash & add 2º Ab
- Wash & add substrate = colour change
- view under light microscope
What is “antigen retrieval” and in what circumstances is it used?
- process that unmasks epitopes from frozen sections- are embedded in aldehyde fixatives = cross-linking of proteins = keeps epitope intact but also masks epitope . - Must be done before IF & IHC to access epitope
What parameters are measured by Flowcytometry and what information is obtained from each parameter?
Measure:
- FWD scatter = size
- Side scatter = granularity
What cell types have CD3, CD4, CD8 and CD19 expressed on their surface?
- CD3, CD4, CD8 = T cells
- CD19 = B cells
Describe the SDS-PAGE.
Sodium dodecyl sulfate- polyacrylamide gel electrophoresis
- SDS: Anionic detergent - breaks 2º & 3º structure of protein (not break S-S bonds) = linear & have same charge per mass. Then S-S broken
- PAGE: Gel has gradient of pores from big to sml to separate protein sizes (MW) : ease of migration dec. = size inc.
Describe the Western Blot methods.
aka protein blot
- protein electrophoresis in SDS PAGE gel
- protein blot transferred from gel to nitrocellulose (bc current)
- Immuno staining w/ labelled Ab (similar to ELISA)
- Ag-Ab complex detected
What is the advantage of doing a Western Blot to detect a serum protein?
- when you electrophorese the serum you may be able to see the types of protein in the serum from the bands.
- when you add the Ab you’ll be able to see which bands light up (Ag-Ab complex = indicate Ab matches to that protein/s in sample)
difference b/w quantitative & qualitative ELISA
- Quantitative: test how much present
- Qualitative: test presence of something
