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Flashcards in Immunotechniques Deck (49):

Describe the affinity constant and give examples of weak and high affinity constants of antibodies:

Ka= Ag:Ab/AgAb (potency)
Biotin:Advidin - 10^15
Weak: Ag:Ab - 10^6
Strong: Ag:Ab - 10^12


Describe the properties of monoclonal antibodies (vs. polyclonal):

Highly specific for a single epitope on a multivalent antigen
Produced by hybridoma
Potentially indefinite supply of same antibody

Potentially not as forgiving as polyclonals


Describe the properties of polyclonal antibodies (vs. monoclonal):

More tolerant of small changes in the nature o the antigen e.g. polymorphism, heterogeneity in glycosylation, slight denaturation
More robust detection because they target multiple epitopes

Requires whole animal to be sacrificed to isolate antiserum
Limited supply


Describe the steps in monoclonal antibody generation:

Inject antigen into animal
Isolate antibody producing plasma cells
Fuse to cancerous plasma cells to make hybrid
Hybridoma cells grow in culture
Clone individual hybridoma colonies
Test clones for desired antibody
Hybridoma tumours are kept alive in mouse and desired clones are cultures and frozen
Monoclonal antibodies are purified


Describe basic polyclonal antibody generation:

Inoculate animal with antigen
Collect serum (antiserum)
Determine tire


Describe the advantages of using chicken IgY polyclonal:

Chickens are not mammals so they are able to make more high-avidity antibodies to mammalian antigens
Most ethical way of producing polyclonal antibodies (just collect eggs)
A single chicken can produce and enormous amount of antibodies (up to 3g of IgY per month, 10-20x the amount of a rabbit)
By having the IgY packaged conveniently in eggs, you can collect and store eggs over a long period of time and retroactively purify the IgY from the eggs of desired tire/avidity
HOWEVER majority of rabbit/mouse are better characterised


Describe immunoassays:

Use antibody:antigen complexes to generate a signal that can be measured
The analyte may be either an antibody or antigen
They are quantitative, where a standard curve of concentration is known


Describe bioassay measurement of secreted biological molecules:

Response of an animal or animal organ/cells
Measures activity not structure
Lacks sensitivity, precision, accuracy, reproducibility
Expensive, time consuming, ethical issues
Large sample requirement, not automatable


Describe immunoassay measurements of secreted biological molecules:

Small volumes, precise, sensitive, reproducible
Can detect molecular determinants
Requires antibody and antigen for calibration
Can require expensive equipment, modest expertise
Fast, automatable


Describe assay sensitivity requirements for:
Steroid hormones (total)
Steroid hormones (free)
Plasma proteins
Protein hormones

10^-6 - 10^-4
10^-9 - 10^-7
10^12 - 10^-10
10^-9 - 10^-7
10^-12 - 10^-10


Describe immunoassay labels:

A label is a molecule that will react as part of the assay, so a change in signal can be measured.
Can be a:
Radioactive compound
Enzyme which can cause a change of colour
Substance that produces light
The label can be attached to the antibody or antigen


List the components of immunoassays:

Microtitre 96-well plate
Standard solution (known amount of antigen of interest)
Sample (e.g. serum)
Diluent solution
Detection antibody
Wash solution - buffer that removes unbound antibodies
Substrate - typically a substrate for an enzyme which produces a measurable signal
Stop solution - stops further production of signal


Describe the basic differences between competitive and non-competitive immunoassays:

Competitive: Unlabelled analyte (usually antigen) in the test sample is measured by its ability to compete with labelled antigen in the immunoassay
Non-competitive - analyte is bound between two highly specific antibody reagents


Describe the competitive immunoassay graph:

Inverse correlation with signal detected
The unlabelled antigen blocks the ability of the labelled antigen to bind because that binding site on the antibody is already occupie
Less label measured = more of the unlabelled (test sample) antigen is present


Describe the non-competitive immunoassay graph:

Measurement of labelled analyte (usually antibody) is directly proportional to the amount of antigen present in the sample
The more antigen that is present, the more labelled antibody that will bind


Describe the two steps in competitive immunoassays:

1. Both the labelled antigen reagent an the unlabelled specimen compete for a limited amount of antibody
2. The antibody concentration of the reaction solution is present in excess in comparrison to the concentration of antigen - antibody reagent is first incubated with specimen containing antigens of interest, then in the second step, labelled antigen is added


Describe the differences between homogenous and heterogenous immunoassays:

Do not require separation of bound Ab-Ag complex
Generally applied to the measurement of small analytes such as abused and therapeutic drugs
Much easier and faster to perform

Those that do require separation of bound Ab-Ag complex


List the immunoassay detection techniques:

Radioimmunoassays (RIA)
Enzyme immunoassay (EIA)
Fluorescence polarisation immunoassay (MEIA)
Chemiluminescent magnestic immunoassay (CMIA)


Describe radioimmunoassays and their advantages and disadvantages:

Utilise radioactive isotopes as a label and the amount of radioactivity measured is indicative of the amount of labelled analyte present
Labelling relatively easy
Specificity of detection
Labelling retains molecular structure
Handling and disposal problems
Limited shelf life
Long counting times
Expensive counting equipment needed


Describe enzyme immunoassays:

Utilise enzyme labels that convert substrate to colour, fluorescence, light
ELISA an example of a non-competitive assay combining enzyme-antibody label reagent with a solid phase bound antibody (bound to microwell plates or beads)


Describe fluorescence polarisation immunoassays:

An homogenous competitive assay
Antigen from specimen and antigen-fluorescein labelled reagent compete for binding sites on the antibody
Reaction carried out in a single reaction solution, and bound Ab-AgF compelx does not require a wash step

Used to provide accurate and sensitive measurement of small toxicology analytes such as therapeutic drugs, drugs of abuse, hormones etc.


List the FPIA reagents:

S - Antibody reagent - antiserum to analyte
T - Tracer - fluorescein-labelled analyte
P - Pretreatment detergent - facilitate release of drug from serum proteins


Describe the principles of FPIA:

Larger molecules (Ab-AgF) rotate more slowly in solution than smaller molecules (AgF) do - distinguish by polarised light
Bigger complex = slower = light through polarised filter (not in little)


Describe microparticle enzyme immunoassays:

Utilises the isolation of antibody/antigen complexes on a solid phase of small beads (microparticles)
The compartments (all suspended in a specific buffer optimised for the assay) include:
Microparticle-antibody solid phase
Antibody-enzyme conjugate
Enzyme substrate


Describe chemiluminescent magnetic immunoassays (CMIA):

Produces light when combined with a trigger reagent
Amount of signal measured is directly proportional to the amount of analyte present in the sample
e.g. sulfupropyl group (improves water stability, leads to more efficient reaction) and sulfonamide group (improves stability)


Describe some factors impacting immunolabelling:

Want to standardise everything, there is so much variability
Interfering substances present in blood
Problems inherent in the measuring technology and/or lab technique


Describe optimisation of immunolabelling:

Antibody tritration and sample dilution
Control material
Eliminate interference


Describe these terms:

Hits the right target
Hits the same target
Ability to exclude false negatives
Ability to exclude false positives


Describe the standard/calibration curve:

Correlates certain values of signal on an instrument to known analyte concentrations
Important to use the same solvent between known analyte preps and patient samples to ensure that the signal response of the calibration curve mimics the signal from patient samples


Describe controls and their importance:

Samples that contain known concentrations of analyte
Used to monitor the accuracy and precision performance of an assay and analyser
Controls should be tracked over time
Need negative controls (don't add antibody) and positive controls (known concentrations/sample definitely having antigen)


Describe antibody titration and sample dilution (issues):

Potential for a high dose hook effect with non-competitive immunoassay designs - very high concentrations of antigen in the patient sample bind to all available sites and saturate them on the antibody solid phase and antibody labelled conjugate, preventing the sandwich formation
Measured level of analyte may be significantly lower than the actual level in the sample


Describe immunohistochemistry (IHC):

Conducted on this sections of tissue or smears/layers of cells (immunocytochemistry)
Gives visual information on localisation of specific proteins
Not strictly quantitative (but commonly used as such)
There are different antigens to localise to different cellular compartments - need to know the antigen looking for


Describe tissue preparation in IHC:

Goal is to maintain tissue/cellular architecture
For tissue - snap-freezing, fixatives, whole animal perfusion with BNF or tissue dissection and immersion in fixative
For cells - remove tissue culture media, add paraformaldehyde, wash and proceed with ICC, need constant fixing method


Describe the different types of tissue sections for immuno:

Fresh frozen tissue
Fixed tissue
Cyrostat sections (free floating 40um or slide moutned 10-20um)
Paraffin embedding (thin, mounted 3-10um)
Vibratome sections (thicker, free floating 50-70um)
Observe via confocal microscopy


Describe the primary antibody for immuno:

Monoclonal or polyclonal against antigen of interest at appropriate dilution in an immunobuffer
Non-specific binding and specificity:
Inclusion of appropriate control (omit primary antibody)
Antigen preadsorption
Western blot validation
Isotype control
Positive tissue control


Describe the secondary antibody in IHC (signal generation and detection):

Amplification methods need to consider abundance of protein of interest
Enzyme substrate colour vs. fluorescence
Enzyme colour - good first approach, permanent record
Fluorescence - fluorophones, visual clarity, good for dual labelling applications, confocal microscopy
Counterstain for a second marker and colour is more permanent than fluorescence


Describe IHC double-labelling:

Visualisation fo more than one protein of interest
Colocalisation/subcellular compartmentalisation
Ideally, primary antibodies used are generated in different species
Optimise conditions for each primary antibody, then the combination
Ideally no significant overlap of emission spectrums or use substrate colours that can easily be distinguished
Inclusion of controls important


Name three things you might troubleshoot for in IHC:

No staining
High background
Non-specific staining


Describe western blotting:

Used for detecting and characterising proteins
Can be used to validate IHC results
Gives information on abundance, size and structure/isoforms
Uses an antibody specific for the protein of interest to locate it after separation by SDS-PAGE (gel electrophoresis)


Describe the basic western blotting antibody procedure:

Sample preparation
Protein transfer to a membrane
Incubate membrane with antibody
Detect antibody
Reveals protein of interest on gel


Describe lysis buffer selection for western blotting:

Use protease and phosphatase inhibitor cocktails and work on ice during cell/tissue homogenisatioN


Describe sample preparation for western blotting:

Protein concentration of sample determined
Sample heated in SDS-PAGE buffer to release subunits and denature proteins
Load equal amounts of proteins per sample per lane
Standards: Defined molecular weights, typically biotinylated or pre-stained
Have positive control with protein of interest


Describe SDS-PAGE in western blotting:

Polyacrylamide gel formed by polymerisation of acrylamide and bis-acrylamide monomers
Electrophosesis on poly-acrylamide gel (7.5-15%) separates on basis of size (5-500,000 kDa)
Proceed to protein transfer to a membrane for western blots or stain with coomassie blue for protein visualisation (good for high abundance histone molecules)


Describe immunodetection in western blotting:

Block excess binding sites on membrane the membrane with bulk protein
Dilute chosen antibody in suitable buffer (monoclonal or polyclonal to protein of interest/housekeeping genes)
Wash extensively
Incubate with anti-species-specific IgG antibody coupled to enzyme or biotin
Complete incubations if necessary with streptavidin-enzyme-enzyme conjugate


Describe some labels for immunodetection:

Horseradish peroxidase signal captured by light sensitive film or CCD camera imaging
ALP may be use to label streptavidin or protein-A or -G
Visualised by chemiluminescence


Describe some problems when western blotting:

Do not use for quantification (it is qualitative)
Failure to transfer properly to membrane
Re-optimise transfer times - check with Ponceau stain
Failure of antibody to recognise protein after electrophoresis and transfer
Use methods to enrich protein of interest (differential centrifugation, immunoprecipitation)
Lack of specificity of antibody
Post-translational modification
High background
Non-linearity of detection
Loading equivalence


List some factors that you might troubleshoot for in western blotting:

No signal
High background
Multiple bands


Describe immunoblotting:

Technique for detecting, analysing and identifying proteins, similar to western blotting but protein samples are not separated electrophoretically (spotted through circular templates directly onto the membrane or paper substrate)
Concentration of proteins in crude preparations can be estimated semi-quantitatively if there is purified protein and specific antibody against it


Describe high content screening and digital pathology applications of immuno techniques:

Look at population heterogeneity to bypass average effect as it looks at every cell
Used in quantification
Automated microscopy and analysis to save time, remove biase
Screening and hypothesis-driven research