Analytical techniques for TDM Flashcards
What are the 2 main analytical techniques used in TDM
- seperation science techniques- HPLC, GC
2.Immunoassays
what is an immunoassay
based on antibody antigen interactions
a type of bioassay where we are using the antibody as a recognition site for a molecule or analyte of interest
what does ELISA stand for
Competitive enzyme linked immunosorbent assay
describe the process of ELISA
- Add sample of drug molecules that we want to measure
- add antibodies
- will bind to the drug and at the bottom of the plate - washing- only left with the antibodies that a=managed to have the ability to bind to the bottom of the plate
- add substrate- will convert in the presence of the enzyme to another product
- we can then measure this using spectroscopy
How do you analyse results from ELISA
- if we have a low concentration of analyte, we get a high signal
- if we have a high concentration of analyte, we get a low signal
- inverse linear relationship between concentration and absorbance in this assay
what is fluorescence polarisation immunoassay
- looking at the admitted polarised flourescence intensity of a particular species
describe the process of fluorescence polarisation immunoassays
- we have an antibody that is specific to the analyte we are interested in and these antibodies are initially bound to an analyte that has a fluorescent tag
- the flourescent tag will emit fluorescence radiation when a polarised light is placed on it
- when there is no analyte that we want to measure, we get a very big fluorescent signal - when we add our biological sample, it will compete on the antibody site and displace the fluorescent tagged analyte analogues
- lack of polarised fluorescent intensity
what is enzyme multiplied immunoassay technique
similar in principle to flourescence polarised immunoassay, but the enzyme is the key driver of this mechanism
describe the process of EMIT
- Antibody with an analyte and an enzyme bound to it
- reagent cannot be converted into a product and no signal can be detected because the active site of the enzyme is not available for the substrate to be converted into a product
- once we add our analyte, it will displace the analyte that is enzymatically bound
- enzyme now becomes active so can convert that substrate into a product
- the conversion generates a product that has colour
- measure using calorimetry
describe the correlation in EMIT
As you increase the amount of analyte, you increase the absorbance that can be measured
what is luminescent oxygen channel immunoassay
a reaction mechanism that is associated with measuring the behaviour of singular oxygen
describe the process of LOCI
- in the reagent there are 2 parts: an antibody bound to a microbe that has a singlet oxygen based on it, which has an excitation associated with it; and another micro particle that has an antibody placed within it bound to luminescent bead
- when we add the analyte, the 2 ends of the antibody bind together
- there is a transfer in the reaction of single oxygen that creates a hopping mechanism which generates emission of signal on the secondary placed bead
What is kinetic interaction of micro particle in solution
based on a principle around nano chemistry where particles have an absorbance when they aggregate
describe the process of KIMS
- when Microparticles bind together, they have a formation of conjugates which generates a signal
- micro particle is placed in a solution with antibodies and these antibodies will bind to the drugs and form a agglomerate solution
- as the molecular size of these particles has changed, we see an absorbance of light
what is cloned enzyme donor immunoassay
an assay that utilises an enzymatic donor fragment that is placed within the analyte
