Practical immunology Immunological tools

Immunological methods: Antibodies as tools Specific recognition of macromolecules Cell types: surface molecules (CD markers) Pathogens Antibodies Antibodies as labels Antibodies as traps Antibodies as competitors Assays: Detection Localization Quantitation

A brief history of antibodies 1891: The term “Antikörper” coined by Paul Ehrlich Substance in the blood that confers immunity "if two substances give rise to two different antikörper, then they themselves must be different” “Lock-and-Key” theory 1920’s: Heidelberger and Avery identified antibodies as proteins 1940’s: Linus Pauling confirms lock-and-key theory 1948:Immunoprecipitation: use of antibodies for detection 1956: Glick and Chang-Bursa of Fabricius: Antibodies come from B cells 1976: Hozumi and Tonegawa, antibody gene rearrangement

Useful characteristics of antibodies Specificity: Ability to recognize individual epitopes Cross-reactivity: Ability to bind to more than one epitope Shared epitopes between different antigens Strength of binding: Affinity: Strength of binding between Fab and epitope Avidity: Overall strength of binding of serum and antigen

Antibody specificity Good: recognize and identify related antigens Bad: Confuse one antigen with a related antigen

Affinity and Avidity Property of the antibody binding site Property of the valency

Antibody types Polyclonal antibodies: Monoclonal antibodies: Isolated from immune serum Many different idiotypes Recognize many different epitopes Strong avidity Highly sensitive Cross reactive: Less specific Monoclonal antibodies: Single idiotype Single epitope Highly specific Lower avidity

Generation of antibodies as reagents Polyclonal: Purify antigen Inject into rabbit or goat (or other species) Collect serum Purify immunoglobulin Specificity depends on purity of antigen Monoclonal: Immunize a mouse Culture spleen cells fused with myeloma cells Select clones based on antibody specificity Specificity to a single epitope

http://www.udel.edu/biology/Wags/histopage/illuspage/ilst/lymphspleenthymusppt.htm

Immunological assays Diagnostic: Research: Assay for antigen Assay for antibody Research: Identify cells Identify cell products Isolate cells or macromolecules Assay cell function

Examples Research techniques Diagnostic assays Hemaggluination ELISA Hemagglutination inhibition Enzyme-linked Immunosorbant Assay (ELISA) Radioimmunoassay (RIA) Immunofluorescent assay (IFA) ELISA Immunohistochemistry Flow cytometry Cell sorting ELIspot Immunoblot Immunoprecipitation Mixed lymphocyte response Chromium release assay

Types of assays Precipitation tests: Detection based on precipitation of complexes Colorimetric tests: Fluorescent Enzyme-linked

Precipitation reactions: Antibody-antigen complexes precipitate out at equivalence Utility: Detection (visual precipitate) Quantification Dilution Diffusion images from http://pathmicro.med.sc.edu/mayer/

“Prozone”

Precipitin tests: Immunodiffusion Double immunodiffusion: Well or trough punched in an agarose gel Antibody and antigen placed in separate well Precipitate forms if antibody recognizes antigen Identification of antigen or antigen mix

Radial immunodiffusion Antibody incorporated into gel Antigen added to wells Zone of precipitation is proportional to concentration Identification and quantification Precipitation at antigen/antibody equivalence

Electrophoresis Differential charge of antibody and antigen Improves sensitivity of radial immunodiffusion Antigen precipitates at equivalence Height of “rocket” is proportional to antigen concentration Electric current

Quantification by titration Principle: Quantitation of antibody or antigen by serial dilution Single concentration of antigen (or antibody ) Dilutions of antibody (or antigen) Titer = the dilution at which precipitation occurs Reflects but does not equal concentration Assays: Hemagglutination ELISA

Antigen of interest bound to red blood cells Easy to see Bind to antigens Antigen of interest bound to red blood cells Agglutination Sample Antibody Titer a 1:32 b Undetectable c 1:16 d 1:128 e 1:256 f g 1:8 h No agglutination Prozone

Colorimetric tests: Enzyme-Linked Immunosorbant Assay ELISA Principles: Specific interaction between antibody and antigen Solid-phase Sensitive detection Description: Antigen bound to a surface Enzyme-bound antibody Colorimetric reaction Semi-quantitative Generate titer

Variations on ELISA Indirect ELISA Capture (Sandwich) ELISA ELIspot Competitive ELISA Radioimmunoassay Immunoblot (“Western” blot) Diagnostic kits

Primary goat anti-X Labeled Secondary rabbit anti-goat (labeled) Unlabeled Labeled

Competitive ELISA

“Western blot”

Immunochemistry and Immunofluorescence Solid-phase antigen: In tissue section Cytology Detect presence and distribution of: Surface marker Intracellular product Extracellular product Metabolic product

Immunofluorescence Direct Indirect

Cell surface markers: Red: Bcl-6, germinal center B cells Green: IRF-4: Plasma cells Spleen red:Bcl-6 germinal center B cells green: IRF-4 plasma cells (dark) B cells (light green)

B cells: CD45R Plasma cells: IRF-4

Immunochemistry of enzymatic reactions Activated Caspase 3

Immunochemistry Kit: “Vectastain ABC”

Enzyme-linked and related assays Target antigen Detection Variants ELISA Bound to well Chromogen Direct, Indirect, Sandwich ELIspot Product of cultured cells B cell or T cell products Immuno-histochemistry In tissue Chromogen, Fluorochrome Direct or indirect Competitive ELISA Soluble   Immunoblot Gel separated proteins Chromogen, Fluorochrome, Radioactive isotope

Fluorescence-activated cell sorting (FACS): Antibodies as traps Fluorescence-activated cell sorting (FACS): Quantitation Isolation Plate-isolation of cell populations (“panning”) Magnetic bead isolation of cells and macromolecules

B cells: red T cells: green Macrophages: cyan Cells coated with fluorescent antibody B cells: red T cells: green Macrophages: cyan

Lymphocyte assays Lymphocytes as tools: Assays: Easy to isolate Survive well in culture Assays: ELIspot: B and T helper cell function Lymphocyte blastogenesis (stimulation) test: T helper cells Chromium release assay: Cytotoxicity

(blastogenesis) Magnetic bead isolation Subculture and cloning

That’s all, folks!! that’s all folks