Immunochemical Techniques Flashcards
Immunochemical techniques definition
Biochemical techniques that employ antibodies or antibody related reagents for the selective determination of an analyte in a sample
Based on antigen-antibody rxns
2 classifications of immunochemical techniques
- assays using non labelled reagents - immunoelectrophoresis
- assays using labelled reagents - enzyme immunoassays
Advantages of immunochemical techniques
- highly specific as Ag & Ab binding
- sensitive
- easily automated
- good reproducibility
- can be qualitative & quantitative
- wide scope - all clinical disciplines, research, pharma etc
Immunoassays using labelled reagents can be one of two things…
- Homogenous
- Heterogenous
Homogenous immunoassay
Does not require separation of bound Ab-Ag from the free components
Heterogenous immunoassay
Does require the separation of bound Ab-Ag complex from free components
Heterogenous immunoassays can be either….
Non competitive or competitive
Immunoassay requirements
- Antibodies - polyclonal/monoclonal
- Signal generating labels
- Separation matrices
What does the success of the immunoassay depend on
The use of the right antibody
Examples of signal generating labels
Radioisotopes, enzymes, fluorescent tags/probes, chemi luminescent probes
Steps in assay formulation of heterogenous non competitive (sandwich IA)
- Excess of primary antibody immobilised to solid support
- 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 to remove unbound material
- Add excess of secondary antibody, labelled w a reporter. This antibody directed against a different epitope on the analyte
- Wash to remove unbound secondary antibody
- Measure the signal. Signal directly proportional to concentration of analyte
Steps in assay formulation of heterogenous competitive
- limited fixed amount of antibody immobilised to a solid support
- sample/standard/control is co-incubated w a limited fixed amount of reporter labelled analyte
- competition between sample/standard/control analytes & labelled analyte for fixed limited number of antigen binding sites on the antibody
- law of mass action - the entity present in highest conc has higher occupancy
- wash to remove unbound material
- measure the signal
- measured signal is indirectly proportional to the conc of test analyte
Steps in assay formulation of homogeneous competitive
- limited fixed amount of Ab immobilised to a solid support
- sample/standard/control is co-incubated w a limited fixed amount of reporter labelled analyte
- on incubation the sample/standard/control analyte competes w the labelled analyte for the fixed limited number of antigen binding sites on the Ab
- law of mass action - entity present in highest conc has higher occupancy
- in homogenous assay formulations the binding event between the Ab & labelled antigen results in modulation of the signal - e.g: inactive label when binds or activate signal when binds
- measure signal - directly proportional to test analyte
5 reporter labels for IA
- radioactive labels
- enzyme labels
- fluorescent labels
- luminescent labels
- miscellaneous (e.g; DNA probes)
Advantages of using enzyme labels in immunoassays
- Enzymes are specific in their action
- enzyme substrate rxns produce easily observable, measurable colour
what are the most commercially available enzyme immuno assays
enzyme linked immunosorbent assays (ELISAs)
Sandwich ELISA method steps
- Plate coated w capture antibody
- Sample added, if antigen present it binds to capture antibody
- Detecting antibody added, bonds to antibody
- Enzyme linked secondary antibody added & binds to detecting antibody
- Substrate added & converted by enzyme into a detectable form
Requirements of ELISA
- Immobilised test sample on a solid support - either non specifically (via adsorption to the surface) or specifically (use of capture Ab)
- Detection Ab added - can be either covalently linked to enzyme or is a secondary Ab added that is linked to the enzyme
- Wash w detergent to remove unbound material
- Add substrate to react which produces a visible signal/colour
Label - Substrates - Advantages - Disadvantages: Horse radish peroxidase (HRP)
Label: HRP
Substrates: TMB, DAB, ABTS-photometric
Advantages: Cheap, stable, strong signal
Disadvantages: Some chromogenic substrates are mutagenic
Label - Substrates - Advantages - Disadvantages: Alkaline phosphatase
Label: Alkaline phosphatase
Substrates: p-nitrophenol phosphate-dephosphorylates molecules
Advantages: v stable, sensitive, safe
Disadvantages: Source material (calf intestine) expensive
Label - Substrates - Advantages - Disadvantages: B-galactosidase
Label: B-galactosidase
Substrates; ONPG, CRPG
Advantages: no inherent enzyme activity, good detection limits
Disadvantages: Cost
IAs with Fluorescent labels (IFA) - what is fluorescence
Molecules absorb radiant energy & electrons are re distributed
IAs with Fluorescent labels (IFA) - Principle of Fluorescence
Light emitted (lambda em) at lower energy and a longer wavelength than the original absorbed energy (lambda exc)
IAs with Fluorescent labels (IFA) - What is the difference betweeen lambda exc and lambda em known as
The stokes shift
IAs with Fluorescent labels (IFA) - What does a larger Stokes shift mean
Better discrimination & more precise detection
IAs with Fluorescent labels (IFA) - what is the quantum yield
The ratio of absorbed to emitted light energy
IAs with Fluorescent labels (IFA) - what does the quantum yield depend on
- Temperature
- Polarity & pH of solvent
- Concentration of fluorophore
- Other quenching effects - molecular O2
IAs with Fluorescent labels (IFA) - what are the antibodies conjugated to
Fluorochromes (fluorescent dyes)
Luminescent labels - chemiluminescence
Light emission that occurs during the course of a chemical rxn
Luminescent labels - bioluminescence
Type of chemiluminescence found in biological systems, due to presence of specific catalytic proteins
What are the two major types of application of luminescent labels
a) the use of a component of the chemi/bio luminescence reactions as a label
b) the use of the rxn itself to detect a conventional enzyme label
