Immunoassays

jamesryeomans
Mind Map by , created over 6 years ago

Degree FAHAW Mind Map on Immunoassays, created by jamesryeomans on 05/18/2013.

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jamesryeomans
Created by jamesryeomans over 6 years ago
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Immunoassays
1 Evaluate the role of immunoassays in animal health compared to other options available
1.1 Competitve assays slower due to incubation period
1.2 Radio labelled health risks
2 How they work
2.1 ELISA
2.1.1 ENZYME linked immuosorbant assay
2.1.1.1 Horseradish peroxidase (HRP)
2.1.1.1.1 Conjulated to a labelled molecule producing a flurimetric labelled molecule
2.1.1.1.2 More stable and less expensive than AP
2.1.1.2 Alkaline phosphotase (AP)
2.1.1.2.1 ?Removal of phosphate group to be replaced with radioactive labelled phosphate? DNA sampelling?
2.1.1.3 Glucose oxidase
2.1.1.3.1 ???Somthing to do with glucose???
2.1.2 Produce colour change in prescence of reagents, some emit light
2.1.3 Indirect ELISA
2.1.3.1 Solution applied to microtiter plate, and charged to join to plastic, followed by a neutral protein to fill in any gaps.
2.1.3.1.1 Primary antibody added to bind to antigen, plate washed with mild detergent
2.1.3.1.1.1 Secndary antibody with conjulated enzyme added to bind to original antibody
2.1.3.1.1.1.1 Enzyme specific substrate added, if reaction occurs between enzyme and substrate a colour change will be observed, stronger clour means greater amount of antigen.
2.1.3.1.1.1.1.1 Spectrometer can give quantitive value
2.1.3.1.2 Disadvantages
2.1.3.1.2.1 Test analyte will compete with other proteins to stick to microtiter plate
2.1.4 Sandwich ELISA
2.1.4.1 A known amount of antibody is applied to plate (capture antibody)
2.1.4.1.1 Antigen added to plate and binds with capture antibody
2.1.4.1.1.1 Second antibody added to sandwich antigen
2.1.4.1.1.1.1 Enzyme bound antibody added to bind to sandwich antibody
2.1.4.1.1.1.1.1 Substrate added for imunoflorescent detection
2.1.5 Competitive ELISA
2.1.5.1 Antibody and sample incubated
2.1.5.1.1 Antibody/antigen complexes added to a antigen coated well, (the more antigen present the less antibody available to bind to well wall antigen). Well then washed and unbound antibody removed
2.1.5.1.1.1 Secondary antibody (specific to primary) coupled to an enzyme is added
2.1.5.1.1.1.1 Substrate added to elicit colour response
2.2 Lateral flow
2.2.1 Detect prescence or abscence of analyte in a substance
2.2.1.1 Measure qualitative, i.e. Yes or No
2.2.1.2 Based on a series of cappillary beds, which transport fluid down a membrane
2.2.1.2.1 1) Excess fluid collected on sponge and migrates down bed to conjugate pad where a sample of antibody is stored specific to tested antigen
2.2.1.2.1.1 If antigen present it will bind with antibody on conjulated pad,
2.2.1.2.1.1.1 Antigen/antibody complex bound to a capture zone test line producing visible colour line
2.2.1.2.1.1.1.1 Coloured latex
2.2.1.2.1.1.1.2 Colloidal gold
2.2.2 Easier for home testing
2.2.2.1 Electronic reader required for fluroescence or magnetic
2.3 Radio Label
2.3.1 Radioactive labelled antibody, AP use?
3 Examples of their many uses
3.1 Fecundity
3.2 Drug testing
3.3 Fertility control
3.3.1 Hormones in blood and milk (progesterone)
3.3.2 Oestrus detection
3.4 Disease surviellence
3.4.1 IBR
3.4.2 BVD
3.4.3 Johnes
3.4.4 Lepto
3.4.5 TB?
3.4.6 Rotavirus
3.4.7 Coronavirus
3.4.8 E.Coli K99
3.5 Nutrition
3.5.1 Nutritive value of feedstuff
3.5.2 Rumen function
4 Future use
4.1 Foot and mouth detection
4.1.1 Collect pathogens from the air and put into a liquid stream. Liquid is then analysed for pathogen
4.1.1.1 Collected samples are coated in antibodies wich undergo optical change in prescence of antigen
4.1.2 Avoid need for animal swabbing
5 Value in animal health compared to other options
6 What are they?
6.1 A biochemical tests that measures the concentration of a macromolecule in a solution through the use of an antibody or immuno globulin
6.2 Rely on antibody to recognise a specific antigen and bind a the epitope (specific area of binding
6.2.1 Some work in reverse in which an antigen is used to detect the prescence of an antibody
6.3 Produce a measurable signal to detect prescence of an antigen with some kind of label
6.3.1 Emit radiation
6.3.2 Change colour
6.3.3 Flourescence under light
7 Potential interference
7.1 TATE and Ward, 2004

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7.2 Substances which alter the level of measurable anylate within a sample, or the binding capacity of an antiybody may affect specificity
7.3 Interference may be anylate dependant or independant
7.3.1 Independant refers to common interferences such as haemolysis, lipaemia, anticoagulant and storage
7.3.2 Dependant interference is reaction between constituents within a sample, often of chemically different but structurally similar mollecules such as other proteins and other antibodies
7.4 False negatives result in overdosing

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