Immunochemical Techniques Flashcards
What are immunochemical techniques?
Biochemical techniques which employ antibodies or antibody-related reagents for the selective determination of an analyte in a sample - based on antigen(Ag):Antibody(Ab) rxns
What 2 classes are immunochemical techniques divided into?
-Assays using non-labelled reagents e.g immunoelectrophoresis
-Assays using labelled reagents e.g enzyme immunoassays
What are advantages of immunochemical techniques?
-Highly specific - binding between Ag and Ab
-Sensitive
-Easily automated
-Good reproductability
-Can be both qualitative and quantitative
-Wide scope - all clinical disciplines, research, food, environmental, pharmaceutical
What does immunoassays using labelled reagents branch into?
-Homogenous - do not require separation of bound Ab-Ag* from the free components
-Heterogenous - require separation of bound Ab-Ag* complex from free compounds - can be competitive or non-competitive
What are immunoassay requirements?
-Antibodies, signal-generating label, separation matrices
Characteristics of these immunoassay requirements?
Anibodies - success of immunoassay depends on use of right antibody - polyclonal or monoclonal
Signal generating labels - radioisotopes, enzymes, fluorescent tags/probes, chemi-luminescent probes
Separation matrices - depends on the formulations and labelling systems used
How is heterogenous non-competitive (sandwich IA) immunoassay formed?
- Excess of primary antibody immobolised to solid support.
- Prime/wash to remove unbound or loosely bound Ab
- Add sample/control/standard. Analyte binds to the primary Ab. Fractional occupancy is directly proportional to the analyte
- Wash - removes unbound material
- Add excess of secondary antibody, labelled with a reporter, this antibody is directed against a different epitope on the analyte
6.Wash - to remove unbound secondary antibody - Measure the signal - signal directly proprtional to conc of analyte
Heterogenous Competitive immunoassay formulation?
- Limited fixed amount of antibody immobilised to a solid support
- Sample/Standard/Control is co-incubated with a limited fixed amount of reporter labelled analyte
- Competition between sample/standard/ control analytes and labelled analyte for fixed limited number of antigen binding sites on the antibody
- Law of mass action - the entity present in highest conc has the higher occupancy
- Wash - to remove unbound material
- Measure the signal
- Measured signal is indirectly propertional to the conc of test analyte
Homogenous Competitive immunoassay formulation
- Limited fixed amount of antibody is immobilised to a solid support
- Sample/Standard/Control is co-incubated with a limited fixed amount of reporter labelled analyte
- On incubation the sample/standard/control analyte competes with the labelled analyte for the fixed limited number of antigen binding sites on the antibody
- Law of mass action - entity present in highest conc has higher occupancy
- In homogenous assay formulations the binding event between the Ab and labelled antigen results in modulation of the signal (usually by 100%) e.g inactive label when binds or activate signal when binds
- Measure signal - directly proportional to test analyte
What kinds of reporter labels can be used for immunoassays?
-Radioactive labels
-Enzyme labels
-Fluorescent labels
-Luminescent labels
-Miscellaneous e.g DNA probes
Why are enzyme labels used in someimmunoassays? (EIA)
-Enzymes are specific in their action
-Enzyme-substrate reactions can produce easily, observable, measureable colour
-Can couple an enzyme molecule to one of the immuno-analytical reagents (analyte or antibody)
Suitable formulations for EIA - Heterogenous non-competitive, heterogenous competitive, homogenous competitive
What are the 4 type of ELISAs - Enzyme-linked immunosorbent assays
-Direct
-Indirect
-Sandwich
-Competitive
Look at pictures
Method to Sandwich ELISA
- Plate coated w capture antibody
- Sample added, if antigen present it binds to capture antibody
- Detecting antibody added, binds to antibody
- Enzyme-linked secondary antibody added and binds to detecting antibody
- Substrate added and converted by enzyme into a detectable form
(target antigen is sandwiched between antibodies-capture antibody and specific antibody)
What are the requirements of ELISA?
-Test sample is immobilised on a solid support- either non-specifically via adsorption to the surface or specifically using e.g a capture enzyme
-A detection Ab is added which can i) be covalently linked to the enzyme, or ii) a secondary Ab is added that is linked to the enzyme
-Wash with detergent to remove unbound material
-Add substrate to react which produces a visible signal/colour
