Immunoassays

Immunoassays

1. Evaluate the role of immunoassays in animal health compared to other options available

   • Competitve assays slower due to incubation period
   • Radio labelled health risks

2. How they work

   • ELISA
     • ENZYME linked immuosorbant assay
       • Horseradish peroxidase (HRP)
         • Conjulated to a labelled molecule producing a flurimetric labelled molecule
         • More stable and less expensive than AP
       • Alkaline phosphotase (AP)
         • ?Removal of phosphate group to be replaced with radioactive labelled phosphate? DNA sampelling?
       • Glucose oxidase
         • ???Somthing to do with glucose???
     • Produce colour change in prescence of reagents, some emit light
     • Indirect ELISA
       • Solution applied to microtiter plate, and charged to join to plastic, followed by a neutral protein to fill in any gaps.
         • Primary antibody added to bind to antigen, plate washed with mild detergent
           • Secndary antibody with conjulated enzyme added to bind to original antibody
             • Enzyme specific substrate added, if reaction occurs between enzyme and substrate a colour change will be observed, stronger clour means greater amount of antigen.
               • Spectrometer can give quantitive value
         • Disadvantages
           • Test analyte will compete with other proteins to stick to microtiter plate
     • Sandwich ELISA
       • A known amount of antibody is applied to plate (capture antibody)
         • Antigen added to plate and binds with capture antibody
           • Second antibody added to sandwich antigen
             • Enzyme bound antibody added to bind to sandwich antibody
               • Substrate added for imunoflorescent detection
     • Competitive ELISA
       • Antibody and sample incubated
         • 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
           • Secondary antibody (specific to primary) coupled to an enzyme is added
             • Substrate added to elicit colour response
   • Lateral flow
     • Detect prescence or abscence of analyte in a substance
       • Measure qualitative, i.e. Yes or No
       • Based on a series of cappillary beds, which transport fluid down a membrane
         • 1) Excess fluid collected on sponge and migrates down bed to conjugate pad where a sample of antibody is stored specific to tested antigen
           • If antigen present it will bind with antibody on conjulated pad,
             • Antigen/antibody complex bound to a capture zone test line producing visible colour line
               • Coloured latex
               • Colloidal gold
     • Easier for home testing
       • Electronic reader required for fluroescence or magnetic
   • Radio Label
     • Radioactive labelled antibody, AP use?

3. Examples of their many uses

   • Fecundity
   • Drug testing
   • Fertility control
     • Hormones in blood and milk (progesterone)
     • Oestrus detection
   • Disease surviellence
     • IBR
     • BVD
     • Johnes
     • Lepto
     • TB?
     • Rotavirus
     • Coronavirus
     • E.Coli K99
   • Nutrition
     • Nutritive value of feedstuff
     • Rumen function

4. Future use

   • Foot and mouth detection
     • Collect pathogens from the air and put into a liquid stream. Liquid is then analysed for pathogen
       • Collected samples are coated in antibodies wich undergo optical change in prescence of antigen
     • Avoid need for animal swabbing

5. Value in animal health compared to other options

6. What are they?

   • A biochemical tests that measures the concentration of a macromolecule in a solution through the use of an antibody or immuno globulin
   • Rely on antibody to recognise a specific antigen and bind a the epitope (specific area of binding
     • Some work in reverse in which an antigen is used to detect the prescence of an antibody
   • Produce a measurable signal to detect prescence of an antigen with some kind of label
     • Emit radiation
     • Change colour
     • Flourescence under light

7. Potential interference

   • TATE and Ward, 2004
     • Tate and Ward 2004
   • Substances which alter the level of measurable anylate within a sample, or the binding capacity of an antiybody may affect specificity
   • Interference may be anylate dependant or independant
     • Independant refers to common interferences such as haemolysis, lipaemia, anticoagulant and storage
     • Dependant interference is reaction between constituents within a sample, often of chemically different but structurally similar mollecules such as other proteins and other antibodies
   • False negatives result in overdosing