Immunochemical Techniques Flashcards
(30 cards)
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
What are the most common enzyme labels?
Horse radish peroxidase (used first)
Alkaline phosphatase
ß-galactosidase
Horse radish peroxidase as an enzyme label?
-Composed of TMB, DAB, ABTS
-Photometric
-Cheap, stable, strong signal
-Some chromogenic substrates are mutagenic, cost
Alkaline phosphatase as an enzyme label?
-Composed of p-nitrophenol phosphate
-Dephosphorylates molecules
-Very stable, sensitive, safe
-Source of material disadvantage - calf intestine - quite expensive
ß-galactosidase as an enzyme label?
-Composed of ONPG, CRPG
-No inherent enzyme activity - absent from plasma
-Good detection limits
-Cost - disadvantage
What are the properties of an ideal enzyme label?
-High enzyme activity (Vo) at a low substrate conc (low Km)
-Enzyme stable and active at the assay pH required
-High sensitivity, preferably with a spectrophtometric end point
-Have reactive groups that can covalently link to Ab and Ag with minimum loss of enzyme or immune activities
-Enzyme-labelled conjuagtes are stable
-Not affected by interference issues ( e.g inherent enzyme activity - particulary for homogenous EIA)
What happens in fluoresence?
-Molecules absorb radiant energy and electrons are re-distributed - excited state
-Extra energy emitted as electronds return to ground state - light is emitted (lamba em) at lower energy and a longer wavelength than the original absorbed energy (lambda exc)
What is the Stokes shift and what does this mean?
Stokes shift is the difference between lambda exc (original absorbed light) and lambda em (light emitted)
-larger stokes shift is better discrimmination and more precise detection
-Detection requires instrument capable of measuring fluorescence
What is quantum yield and what is this dependent on?
-The ratio of absorbed to emitted light energy
-dependent on:
–temp
–polarity and pH of solvent
–Conc of fluorophore
-Other quenching effects (molecular O2)
What are fluorochromes?
-Antibodies are conjugated to fluorochromes (fluorescent dyes)
-Polyaromatic hydrocarbons or heterocycles (organic compounds)
-Have different decay times (std is 10-9 to 10-8 s)
-lanthanide chelates (e.g Europium, Terbium) have decay times of 10-5 to 10-2 seconds - facilitated development of time resolved fluorescence (signal lasts longer)
