concept of ag-ab immunological technique using the characteristic of high affinity and specificity...
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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.
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
Western Blotting
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.
Vertical Gel Electrophoresis
Prep and Run Samples
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
Enzyme-Mediated Detection
Enzyme Horseradish Peroxidase (HRP)
Substrate Abbrev. ColorDiaminobenzidine DAB Brown
Enzyme Alkaline Phosphatase (AP)
Substrate Abbrev. ColorBromochloroindolylphosphateNitro Blue Tetrazolium
BCIP (AP substrate) NBT (Enhance color)
Purple
TUGAS:1. Komposisi dan fungsi dari masing-
masing bahan pada transfer buffer2. Methode secara rinci western
blotting
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
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
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
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.
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
Basics of Immunohistochemistr
y
03/2005
Immunofluorescence Microscopy
What is Immunohistochemistry?
What is Immunohistochemistry?
CELLULAR ANTIGENS
Sensory
AdhesionMetabolic
Outline of Procedure 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
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
Fixing and Sectioning of Tissue 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
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
Immunohistochemical Detection Methods
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”
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”
ImmunofluorescenceImmunofluorescence
Fluorescence 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
Fluorescence 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
Direct Immunofluorescence MethodDirect Immunofluorescence Method
Easy application, only few steps
Not very sensitive
Not very versatile, as primary antibodies need to be directly labelled with fluorophore
Easy application, only few steps
Not very sensitive
Not very versatile, as primary antibodies need to be directly labelled with fluorophore
Fluorochrome-conjugatedPrimary Antibody
Antigen expressing Cell
Indirect Immunofluorescence MethodIndirect Immunofluorescence Method
More 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
More 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
Antigen expressing Cell
Primary Antibody (from species X)
Fluorochrome-conjugatedSecondary Antibody (anti species X)
Indirect Immunfluorescence Using Biotinylated secondary
Antibody
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
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
Antigen expressing Cell
Primary Antibody (from species X)
BiotinylatedSecondary Antibody (anti species X)
Fluorochrome-conjugatedStreptavidin
Multiple ImmunofluorescenceMultiple Immunofluorescence
Multiple Labelling of a Tissue SectionMultiple Labelling of a Tissue Section
Live Labelling of Cultured CellsLive Labelling of Cultured Cells
Enzymatic detection methodsEnzymatic 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
Brightfield 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
Immunoezyme Labelling of Tissue Section
Immunoezyme Labelling of Tissue Section
ABC (Avidin Biotin Complex) - Method
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
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
Antigen expressing Cell
Primary Antibody (species X)
BiotinylatedSecondary Antibody(anti species X)
Enzyme-conjugatedAvidin-Biotin Complex
Substrate-chromogensolution
Common Problems 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
Good Resources 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)