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Luke Droney
IMMUNOPRECIPITATION IN GELS Explain the specific characteristics of gels commonly used in immunochemistry laboratories Be able to prepare gels for use in the laboratory for specific purposes including the use of templates and punching wells/troughs Explain the nature of antigen-‐antibody reactions in gels, particularly with regard to
-‐ Zones of antibody excess, equivalence and antigen excess (prozone effect) -‐ Principles of Ouchterlony testing -‐ Radial immunodiffusion assays including end-‐point and fixed-‐time assays
Interpret assays based on gel diffusion methods
-‐ Fungal and avian precipitins -‐ Testing for anti-‐ENA antibodies, including identity, non-‐identity and partial
identity Immunoprecipitation: A protein purification method that involves the formation of an antibody-‐protein complex to separate the protein of interest. Explain the specific characteristics of gels commonly used in immunochemistry laboratories Agarose beads/resin:
• Major solid phase support used for immunochemistry historically • Highly porous • Cheap and no specialised equipment required for setup • High potential binding capacity (can actually be problematic due to
insufficient protein binding) • Easy recovery of sample and storage • Don’t have a uniform pore size • Good for electrophoresis of large proteins (>200kDa)
Polyacrylamide:
• Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Pore size is controlled by modulating the concentrations of acrylamide and bis-‐acrylamide powder used in creating a gel.
• Acrylamide is a potent neurotoxin in its liquid and powdered forms. • Mostly used for protein analysis, often used to separate different proteins or
isoforms of the same protein into separate bands. These can be transferred onto a nitrocellulose or PVDF membrane to be probed with antibodies and corresponding markers, such as in a western blot.
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Starch: • Partially hydrolysed potato starch makes for another non-‐toxic medium for
protein electrophoresis. The gels are slightly more opaque than acrylamide or agarose. Non-‐denatured proteins can be separated according to charge and size. They are visualised using Napthal Black or Amido Black staining. Typical starch gel concentrations are 5% to 10%
Explain the nature of antigen-‐antibody reactions in gels, particularly with regard to
-‐ Zones of antibody excess, equivalence and antigen excess (prozone effect) -‐ Principles of Ouchterlony testing -‐ Radial immunodiffusion assays including end-‐point and fixed-‐time assays
Immunoprecipitation curve:
• Requires bivalent antibody and antigen with at least two antigenic determinants
• Three main areas • Antibody excess – all available antigen bound by antibody, only simple
complexes form. Little to no cross-‐linking of antigen and minimal precipitation/lattice formation.
• Equivalence – optimal concentration reached at which every antibody attached to two antigens and maximum lattice formation occurs.
• Antigen excess – available antibody used, precipitation does not occur due to lack of free antibody.
Prozone effect:
• The presence of excess antigen, in relationship to antibody concentration, resulting in increased solubility of immune complexes, decreased apparent reactivity and underestimation of antigen quantity.
Post-‐zone effect:
• The converse of the prozone effect; the presence of a marked excess of antibody in relationship to antigen concentration, resulting in increased solubility of immune complexes and decreased test reactivity
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Immunoprecipitation kinetics:
• Immune complexes begin to form immediately after addition of antigen to antibody
• Peak formation occurs as early as 20 seconds • Reaction continues until equilibrium is reached (peak light scatter). • After equilibrium is reached there is a decrease in scatter as some complexes
precipitate out of solution. • End-‐point nephelometers utilize the equilibrium phase of the reaction • Rate nephelometers utilize the peak rate of Ag-‐Ab reaction
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4. Radial immunodiffusion (RID) - A simple method for measurement of any protein for which antiserum exists - Antiserum is incorporated into an agar gel which is poured into a plate and
allowed to set - Holes are then cut into the gel and the serum containing the protein of
interest is placed into the holes o Serum diffuses o Forms an immunoprecipitate that appears as a white halo around the
well o The antigen concentration is proportional to the square of the
diameter of the halo (see diagram below) o IgA-‐deficient individuals often have antibodies to ruminant proteins
(incl. Igs) and as the antisera incorporated into the gel are typically ruminant, this may cause reverse precipitation in the gel
o End-‐point – allows diffusion to proceed to its conclusion o Fixed time – assess diameter of immunoprecipitate at a fixed time and
compare to a curve constructed with known calibrators.
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5. Ouchterlony double diffusion - Used for non-‐quantitative identification of proteins - Wells are cut into agar and test serum (containing antigen) and antisera are
placed in wells o Diffuse towards each other (takes up to 5 days) o Lines of precipitation form at the point where zone of equivalence is
reached - Precipitation occurs because the antigen is multivalent (ie. has several
antigenic determinants per molecule to which antibodies can bind) o Precipitation/cross-‐linking will not occur if excess antigen is present or
if excess antibody is present o Cross-‐linking and lattice formation will only occur when antigen and
antibody concentrations are optimal - Highly specific but poor sensitivity - Reading is somewhat subjective and can be difficult, particularly if there are
multiple lines
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Autoantibody detection – Ouchterlony method
A: appearance of precipitin lines using known reference anti-‐sera (not line of partial identity between RNP/Sm) B: Identification of optimal serum dilution. Optimal dilution results in a line of immunoprecipitation roughly half-‐way between antigen and antibody C: Line of identity with La D: Line of identity with RNP
Counter-‐immunoelectrophoresis (CIEP) - Principle
o Formation of insoluble immune complexes where an antibody encounters the optimal concentration of antigen
- Technique o Buffered agarose gel (containing electrolytes)
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o Current applied across the gel forcing the antigen and antibody together
o Antibodies are relatively neutrally charged compared to other proteins and are carried by the flow of positive ions towards the cathode (-‐ve) (endosmosis)
o Antigen/other proteins are negatively charged and will migrate in the opposite direction towards the anode (+ve)
o If the antigen and antibody are specific for each other, they combine to form a distinct precipitin line
Nephelometry:
• Immune complexes scatter light at angles different to the direction of incident light
• When light passes through a material containing particles there is interference with the passage of light and light is scattered – the Tyndall effect – depends on wavelength of light and particle size
• At higher particle sizes light is scattered primarily in a forward direction – Rayleigh-‐Debye scattering
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• High-‐intensity light source through reaction vessel. Detectors (usually at 31 degrees or less to incident light)
Turbidimetry:
• Measures ‘cloudiness’ (by absorptiometry) resulting from immune complex formation
• Detector aligned with incident light (unlike nephelometry) • Anything that causes the serum to be turbid before the reaction begins or
interferes with the optical properties of the solution will cause difficulties o Lipaemic sera o Haemolysed samples with excess free Hb (form Hb-‐haptoglobin
complexes) End-‐point nephelometry:
• Maximum amount of scatter taken at a set time when reaction has reached equilibrium
• Scatter measured almost immediately and a set time depending on the assay • Values compared to multi-‐value standard curve generated using calibrators • Can be modified by using inert particles such as latex for coupling antigen or
small compounds Rate nephelometry:
• Measures light scatter using peak rate of Ag-‐Ab reaction • Must be performed in the region of antibody excess • Antigen excess check – add extra antigen – if no increase in scatter then need
to dilute specimen and start again • Don’t require a ‘blank’ as value is a rate measurement and therefore not
affected by background signal.
Luke Droney
Interpret assays based on gel diffusion methods Fungal and avian precipitins
• Immunodiffusion is most widely used technique for serodiagnosis of aspergillosis and hypersensitivity pneumonitis
• Antigens used may be extracts or culture filtrates • Lacking in sensitivity and doesn’t provide quantitation • Agarose gel is applied to slides with a template attached • Test and control sera are loaded in the middle lane with antigen on the
outside adjacent rows • Diffusion proceeds over ~48 hours • Gel stained with Coomassie blue, then destained (?) and dried
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Comparison of precipitins to ELISA/WB
