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)