Download - Concept of Ag-Ab immunological technique
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Concept of Ag-Ab immunological technique
• Using the characteristic of high affinity and specificity of antibody, we can detect or quantitative the antigen. Keep in mind that the antibody is protein, can also be recognized as an antigen. The major principle to determine the antigen-antibody interaction is to separate the bound form of antigen-antibody complex from the free form of either antigen or antibody.
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Analytical Techniques Utilizing Antibodies:• flow cytometry• gel electrophoresis
• immunoprecipitation (IP)• immunoblotting
• microscopy• immunofluorescence (IFA)• electron microscopy
• ELISA• antibodies bind proteins with
high specificity and affinity• affinity chromatography• analytical techniques
Antibodies
CNBr
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Western Blotting
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Western Blotting• Protein denature (SDS)• SDS-PAGE gel
electrophoresis• Blotting (transfer)• Blocking (BSA)• Staining with Coomasie or
Ponceau S (checking)• 1° Ab serum (probe)• Washes• 2 ° Ab serum• Washes• Color development
Why do proteins stick to the membrane? Hydrophobic & charge interactions.
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Vertical Gel Electrophoresis
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Prep and Run Samples
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Example of Western Blot Result
Blot interpretation1. Lane 1, HIV+ serum
(positive control) 2. Lane 2, HIV- serum
(negative control) 3. Lane A, Patient A 4. Lane B, Patient B 5. Lane C, Patient C
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Enzyme-Mediated DetectionEnzyme Horseradish Peroxidase
(HRP)Substrate Abbrev. ColorDiaminobenzidine DAB Brown
Enzyme Alkaline Phosphatase (AP)Substrate Abbrev. ColorBromochloroindolylphosphateNitro Blue Tetrazolium
BCIP (AP substrate) NBT (Enhance color)
Purple
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TUGAS:1. Komposisi dan fungsi dari masing-
masing bahan pada transfer buffer2. Methode secara rinci western
blotting
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Immunopreciptation: Identification of protein-protein interactions
bead
protein A
primary antibody
Steps:1. Attach antibody to beads via protein A2. Lyse cells to release antigen and its binding partners3. Mix cell lysate + antibody-coated beads (antibody binds antigen)4. Purify antigen and its binding partners by centrifugation
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Immunoprecipitation• affinity purification based on
isolation of Ag-Ab complexes• analyze by gel electrophoresis• initially based on centrifugation of
large supramolecular complexes• [high] and equal amounts
• isolation of Ag-Ab complexes• fixed S. aureus• protein A-agarose• protein G-agarose
Bacterial proteins that bind IgG (Fc):• protein A (Staphylococcus aureus)• protein G (Streptococcus)
• binds more species and subclasses
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Typical IP Protocol1. Solubilize antigen
• usually non-denaturing• SDS + excess of TX100
2. Mix extract and Ab 3. Add protein G-agarose, etc4. Extensively wash5. Elute with sample buffer6. SDS-PAGE7. Detection
• protein stain• radioactivity
Gagarose
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Radiolabeling of Proteins
• carried out before IP• metabolic (amino acids or
other precursors + cells)• chemically (eg., iodination)• IP and SDS-PAGE• detect by autoradiography
or fluorography following electrophoresis
• also provides information about synthesis, post-translational events, etc.
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Western Blot vs Immunoprecipitation
• Experimental Design• eg., synthesis (IP)
• Ag concentration• IP better for low
abundance proteins• Ag solubility
• Western for insoluble proteins
• Ab recognition• conformational
dependent epitopes• 4o structure
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Basics of Immunohistochemistr
y
03/2005
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Immunofluorescence Microscopy
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What is Immunohistochemistry?
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CELLULAR ANTIGENS
Sensory
AdhesionMetabolic
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Outline of Procedure Fixwholemount, embed and section tissue (or treat as ”” preparation – small specimens only, such as cultured cells)
Wash sections in physiological buffer, e.g. PBS
Incubate with protein solution (BSA or normal serum) to reduce non-specific binding of antibody to specimen (”blocking”- important!)
Incubate with antibody specific to antigen in question (”primary antibody”). Include positive and negative controls (!)
Wash in physiological buffer
Apply suitable detection system (see below)
Mount specimens and analyse microscopically
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Fixing and Sectioning of Tissue Common fixation methods are chemical fixation (e.g. paraformaldehyde) or cryofixation (i.e. rapid freezing). Fixation serves to preserves tissue structure and properties of antigen
Tissues can be embedded in wax or resins for sectioning, or cut in the frozen state in a cryostat.
The method chosen for fixation and sectioning are dependent on the properties of the tissue, antigen and the antibody used in the procedure
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Immunohistochemical Detection Methods
Through fluorescent substances (fluorophores) –”Immunofluorescence technique”
Through enzymatic conversion (often by horseradish peroxidase, HRP) of a soluble substrate (chromogen) into a non-soluble and colourful reaction product - ”Immunoenzyme technique”
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ImmunofluorescenceFluorescence or confocal microscope necessary for analysis of specimens
High structural resolution possible
Advanced image reconstruction (3D) and signal quantification possible
Multiple labelling easy
Limited shelf life of labelled specimens
Method of choice for labelling of live cells
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Direct Immunofluorescence MethodEasy application, only few steps
Not very sensitive
Not very versatile, as primary antibodies need to be directly labelled with fluorophore
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Fluorochrome-conjugatedPrimary Antibody
Antigen expressing Cell
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Indirect Immunofluorescence MethodMore steps involved
More sensitive
More versatile, as only secondary antibodies need to be labelled and different combinations of fluorophores are possible in multiple labelling experiments
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Antigen expressing Cell
Primary Antibody (from species X)
Fluorochrome-conjugatedSecondary Antibody (anti species X)
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Indirect Immunfluorescence Using Biotinylated secondary
Antibody
Biotin (Vitamin B) binds with high affinity to Avidin – thus good linker system
Very high sensitivity
Endogenous biotin may be present in tissue – risk of background
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Antigen expressing Cell
Primary Antibody (from species X)
BiotinylatedSecondary Antibody (anti species X)
Fluorochrome-conjugatedStreptavidin
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Multiple Immunofluorescence
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Multiple Labelling of a Tissue Section
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Live Labelling of Cultured Cells
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Enzymatic detection methodsBrightfield microscope sufficient for analysis of specimens
Suitable for tissue analysis at low magnification
Resolution of subcellular structures not as good as with fluorescence methods, but can be combined with electron microscopy
Unimited shelf life of labelled specimens
Substrate reagents often toxic/carcinogenic
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Immunoezyme Labelling of Tissue Section
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ABC (Avidin Biotin Complex) - Method
Biotin (Vitamin B) binds with high affinity to Avidin – thus good linker system
Extremely high sensitivity
Endogenous biotin my be present in tissue – risk of background
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Antigen expressing Cell
Primary Antibody (species X)
BiotinylatedSecondary Antibody(anti species X)
Enzyme-conjugatedAvidin-Biotin Complex
Substrate-chromogensolution
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Common Problems
Non-specific binding of primary or secondary antibodies to tissue sample; e.g. through ionic or hydrophobic interactions or binding of antibodies to free amino groups: „Background staining“
Cross-reactivity of antibodies with unrelated antigens present in tissue sample
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Good Resources
Dako On-line Immunohistochemistry Handbook(http://www.dakousa.com/ihcbook/hbcontent.htm)
NIH Immunohistochemistry Protocols(http://dir.niehs.nih.gov/dirlep/immuno.html)
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