LABORATORY METHODS BASED ON ANTIGEN-ANTIBODY INTERACTIONS I 8th SEMINAR LABORATORY METHODS BASED ON ANTIGEN-ANTIBODY INTERACTIONS I

THE SENSITIVITY OF IMMUNOASSAYS Sensitive methods: precise expensive usually used for verification Less sensitive methods: give semiquantitative results cheap usually used for screening

IMMUNOAFFINITY CHROMATOGRAPHY Separation/purification of antigens or antibodies from a mixture

AFFINITY PURIFICATION OF ANTIBODIES USING AN ANTIGEN-SORBENT COLUMN polymer beads covalently bound antigen affinity purified antibody : monoclonal antibodies which can be ordered from catalogues are also purified using this technique

STEPS OF PURIFICATION Addition of antibodies to be purified Binding Washing Elution

PURIFICATION OF ANTIGENS Loading the antigen mixture column Binding Washing Elution polymer bead fixed antigen-specific Abs on the surface of the bead Purified antigens

ELISA Enzyme Linked Immune Sorbent Assay ELISA plate well

Antigen/antibody adsorbed to solid surface Antibody conjugated with Enzyme Linked Immune Sorbent enzyme Antigen/antibody adsorbed to solid surface Antibody conjugated with enzyme

ENZYME ACTIVITY IN ELISA IS DIRECTLY PROPORTIONAL TO THE AMOUNT OF IMMUNECOMPLEX PRESENT Enzyme activity is measured by the color reaction due to conversion of substrate Similar principle applies to many other antibody-based detection methods

BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Direct method Indirect method Label Secondary antibodies Label Primary antibodies Antigen

BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Enzyme/anti-enzyme system PAP – peroxidase / anti-peroxidase APAAP – alkaline phosphatase / anti- alkaline phosphatase Enzyme Enzyme-specific antibody, same isotype as the primary antibody Secondary antibody Primary antibody Antigen

BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity ) Basic ABC Avidin-biotin enzyme complexes Avidin-enzyme complexes Biotin-enzyme complex Avidin is a tetrameric or dimeric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein (approximately 1.8 mg per egg). The tetrameric protein contains four identical subunits (homotetramer), each of which can bind to biotin (Vitamin B7, vitamin H) with a high degree of affinity and specificity. The dissociation constant of avidin is measured to be KD ≈ 10−15 M, making it one of the strongest known non-covalent bonds. The natural function of avidin in eggs is not known, although it has been postulated to be made in the ovaduct as a bacterial growth-inhibitor, by binding biotin the bacteria need. As evidence for this, streptavidin, a loosely related protein with equal biotin affinity and a very similar binding site, is made by certain strains of Streptomyces bacteria, and is thought to serve to inhibit the growth of competing bacteria, in the manner of an antibiotic. (Source: wikipedia) Avidin Biotinylated antibody Antigen

STEPS OF COMBINED/’SANDWICH’ ELISA For antigens present at low concentration in complex biological samples Coating with Ag-specific „capture” antibody Removal of excess enzyme Removal of unbound material Blocking free plastic surface with inert protein Removal of unbound protein Removal of unbound material Addition of antigen- containing solution Addition of biotinylated antibody specific to a different epitope on target protein Addition of avidin-conjugated enzyme Addition of substrate

STEPS OF BASIC INDIRECT ELISA Detection of antigen or specific antibody Removal of excess antigen Removal of excess protein Adsorption of antigen (coating) Removal of excess antibody Removal of excess antibody Saturation of uncovered surface area with proteins Addition of Ag-specific antibodies Addition of Secondary Ab conjugated with enzyme Addition of chromogenic substrate

You should also dilute the unknown sample EQUAL ABSORBANCE = EQUAL CONCENTRATION OD You should also dilute the unknown sample The sample with unknown concentration This region could indicate the concentration According to OD: it could be anyone ? 500 250 125 62 31 16 7.8 3.9 1.9 1000 0.97 0.49 0.24 0.12 0.061 0.030 0.015 0.007 0.004 concentration

PRACTICAL USE OF ELISA TECHNICS Sandwich ELISA Indirect ELISA Measuring the amount of a given antigen (molecule) cytokines, hormones, drugs viral/bacterial antigens – diagnosis of infection tumor antigens – diagnosis of tumors / screening / follow-up Measuring the amount of antigen- specific antibodies pathogen-specific antibodies – diagnosis of infection* isotype of antibodies – time course, monoclonal antibodies autoantibodies – diagnosis of autoimmune disorders *Viral antigens cannot be detected efficiently in a lot of cases (latency), but you can efficiently detect the antibodies that were produced by the body in response to the viral infection. These antibodies can serve as diagnostic markers.

WESTERN BLOT (IMMUNOBLOT) Identification of defined components from protein mixtures by antigen specific antibodies Anode(+) Steps: Sample preparation (cells, tissues) Gel electrophoresis Blotting Labeling (by primary and secondary antibodies) Detection Cathode(-)

WESTERN BLOT (IMMUNOBLOT) Lysis of sample Loading Gel-electrophoresis Blotting Labeling Primary antibody binds to its epitope in the protein, then the labeled secondary antibody binds to the primary antibody Standard Protein sample SDS-PAGE Membrane X-ray film

IMMUNOPRECIPITATION Isolation and concentration of a particular protein from a protein mixture Detection of protein associations (e.g. members of receptor signalization) The bacterial protein A/G is a recombinant fusion protein, which can strongly bind to Fc regions of antibodies. http://en.wikipedia.org/wiki/Immunoprecipitation

CHROMATIN IMMUNOPRECIPITATION (ChIP) Identification of molecules (mainly transcription factors) binding to a specific site of the DNA Provides information about the link between signaling pathways and gene activation

IMMUNOHISTOCHEMISTRY Labeled antibodies added to fixed tissue sections detect the distribution of the chosen antigen within the tissue or within the cells of a particular tissue Immunofluorescence Fluorescent dye coupled to antibody FITC – fluorescein isothiocyanate (green) PE – phycoerythrin (orange) Immunoenzyme method enzyme-coupled antibody P – peroxidase AP – alkaline phosphatase (Substrates converted into an insoluble compound)

IMMUNOHISTOCHEMISTRY Fixation Sectioning Tissue sample Section before staining Freezing

IMMUNOHISTOCHEMISTRY Enzyme Secondary antibody X Avidin Primary antibody Biotin Slide Cells Tissue sample

Classical histochemistry Acute bronchopneumonia (hematoxylin-eozin staining) Only few cell types could be identified

Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma)

Human epithelial type 2 (HEp-2) cells, considered to originate from a human laryngeal carcinoma, allow recognition of over 30 different nuclear and cytoplasmic patterns that are given by upwards of 50 different autoantibodies are associated with various autoimmune conditions. Used autoantibody: anti-Sm Image source: http://asc.dental.ufl.edu/ Antinuclear (ANA) autoantibodies from the serum of a SLE patient can be visualized in cell culture (HEp-2) by indirect fluorescent labeling (immunofluorescence)

A fixed and permeabilized skin fibroblast Mitochondria F-actin Nucleus Fixed and permeabilized pulmonary artery endothelial cell Peroxisomes Mitochondria Nuclei