Chem Path Flashcards
Define avidity
Function of strength and number of binding sites
Define affinity
Strength of association
Define immunoassay
Any analytical method which uses Abs as reagents, the results from which assist in diagnostic interpretation
What is an homogenous assay
Signal changes when Ab and Ag bind. Therefore no separation step is required
What is an heterogenous assay
Requires separation of bound and unreacted molecules to determine/assess result
What are the three types of assay and an eg of each
Immunometric assay - ELISA, IFMA, IRMA
Equivalence precipitation - gel diffusion
Immunoassay (competitive) - RIA, FIA
How does one produce polyclonal antisera?
Inject animal with Ag (possibly needs to be bound to haptogen to increase immunogenicity)
Gives rise to mixed/polyclonal population = greater affinity than monoclonal Abs
How does one produce monoclonal Abs?
Inject animal with Ag.
Isolate one plasma cell and fuse that cell to a tumour cell.
Screen hybridoma and allow clonal expansion
Advantage of monoclonal Ab
Highly specific
Disadvantage of monoclonal Ab
Lower affinity
Not truly representative of analytes with multiple molecular forms
Why characterize Abs?
Determine diagnostic performance of test
What factors are assessed when characterizing Abs
Affinity
No. of binding sites
Specificity
How do we characterize Abs practically
Titre (affinity and concentration using dilution curve) Scatchard analysis (higher the affinity constant [K] better the sensitivity, also looks at no of binding sites) Cross reaction studies (use structurally related molecules)
Methods of Ab purification
Ig precipitation Ion-exchange chromatography Affinity chromatography Ab fragments Protein A/G chromatography
Types of label
Enzyme linked Flourophores Chemiluminescense Radioisotopes Particle precipitatates Etc etc
Eg of a homogenous assay
EMIT
Enzyme-multiplied immunoassay technique
What is turbidimetry
Measure of loss of intensity of transmitted light due to the scattering effect of particles in a solution
What is Nephelometry
The measure of scattered light at a given angle to the transmitted light
Eggs of substances that can cause assay interference
Human anti mouse Ab
Complement
Rheumatoid factor
What is the hook effect
Production of artefactually low results from samples that have very high level of Ag.
Most commonly found in immunometric assays
What is only prerequisite for analyte in capillary GC
Analyte must be volatile
Pros of GC-MS
Reliable and reproducible
Wide range of analytes
Technically straight forward
Cons of GC-MS
Some analytes require time consuming extraction etc
Cost (equipment, training, libraries, isotopes)
What is Trinder’s reagent
Contains mercuric chloride and HCl to precipitate proteins
