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

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 interpretation 1.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) SubstrateAbbrev.Color DiaminobenzidineDABBrown Enzyme Alkaline Phosphatase (AP) SubstrateAbbrev.Color Bromochloroindolylphosphate Nitro Blue Tetrazolium BCIP (AP substrate) NBT (Enhance color) Purple

TUGAS: 1.Komposisi dan fungsi dari masing- masing bahan pada transfer buffer 2.Methode secara rinci western blotting

Immunopreciptation: Identification of protein-protein interactions bead protein A primary antibody Steps: 1. Attach antibody to beads via protein A 2. Lyse cells to release antigen and its binding partners 3. 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 Protocol 1. Solubilize antigen usually non-denaturing SDS + excess of TX Mix extract and Ab 3. Add protein G-agarose, etc 4. Extensively wash 5. Elute with sample buffer 6. SDS-PAGE 7. Detection protein stain radioactivity G agarose

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 4 o structure

Basics of Immunohistochemistry 03/2005

Immunofluorescence Microscopy

What is Immunohistochemistry?

CELLULAR ANTIGENS Sensory Adhesion Metabolic

Outline of Procedure 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 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 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”

Immunofluorescence 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 Method Easy application, only few steps Not very sensitive Not very versatile, as primary antibodies need to be directly labelled with fluorophore

Fluorochrome-conjugated Primary Antibody Antigen expressing Cell

Indirect 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

Antigen expressing Cell Primary Antibody (from species X) Fluorochrome-conjugated Secondary Antibody (anti species X)

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

Antigen expressing Cell Primary Antibody (from species X) Biotinylated Secondary Antibody (anti species X) Fluorochrome-conjugated Streptavidin

Multiple Immunofluorescence

Multiple Labelling of a Tissue Section

Live Labelling of Cultured Cells

Enzymatic detection methods 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

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

Antigen expressing Cell Primary Antibody (species X) Biotinylated Secondary Antibody (anti species X) Enzyme-conjugated Avidin-Biotin Complex Substrate-chromogen solution

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 Dako On-line Immunohistochemistry Handbook ( NIH Immunohistochemistry Protocols (