B cell Antigen Receptors, Signaling and Antigen-Antibody interactions

Azad K. Kaushik, DVM, MVSc, DSc (Paris)

Azad Kaushik 2013 (Illustrations from Kuby, 7th Edition)

Receptor-ligand interactions
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Receptor-ligand binding occurs via multiple non-covalent bonds " Each individual bond may be weak " Many such bonds occur between receptors and ligands, providing great cumulative bond strength

Antibody affinity: is the total sum of non-covalent attractive and repulsive forces between a single antigencombining site and a single epitope present on an antigen

Receptor-ligand interactions

high affinity = high specificity

Receptor-antigen interactions are usually multivalent " Multivalency increases avidity of the interactions ! Individual interactions have an affinity!a strength of that individual binding ! Avidity is the combined strength of multiple bindings ! As such, an interaction may have weak affinity, but high overall avidity
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Affinity of antigen-antibody interactions

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Equilibrium dialysis can be used to measure antibody affinity for antigen " Older method; inexpensive association
Ka (K1)

Ag + Ab ! Ag.Ab
Kd (K-1)

disassociation

Affinity of antigen-antibody interactions: SPR ! Surface plasmon resonance (SPR) " Since mid-1990s; more sensitive " Measures EM waves at interface of a metal and a solvent ! Nature of the wave is sensitive to adsorption of molecules to the metal surface ! SPR detects changes in reflectance at the surface of sensor when it binds Ab
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Affinity of antigen-antibody interactions: SPR ! A plot of resonant angle versus time is a sensorgram " The plot rises until all sites capable of binding Ab have done so, then plateaus " Data from these plots can be used to calculate association rate constants " Washing the chamber, collecting new readings can determine dissociation rate constants

Signaling pathways
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Binding of antigen to receptor induces: " An internal signaling cascade, which: ! Leads to cellular alterations in: " Motility " Adhesive properties " Transcriptional programming These cascades are behind the various cellular changes that take place during an immune response against an antigen Often, the same players/proteins are used in different cell types!triggering receptors may be different
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Signaling pathways

Signaling pathways
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Antigen-mediated receptor clustering initiates signaling in B and T cells " Receptor dimerization is often a result " Multimerization can also occur " Clustered receptors are localized in lipid rafts

Immunoprecipitation-based techniques
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Immunoprecipitation can be performed in solution " Formation of such large immune complexes (precipitate) ! Only occurs when Ag/Ab concentrations are roughly equal!leading to formation of large complexes " Can be used to purify antigen molecules or to remove antigens from a solution

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Immunoprecipitation-based techniques
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Immunoprecipitation of soluble antigens can be performed in gel matrices " Ouchterlony method ! Ag placed in center well, serum samples placed in outer wells ! Immune complexes form visible precipitin line

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Immunoprecipitation-based techniques
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Immunoprecipitation of soluble antigens can be performed in gel matrices " More sensitive methods available

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Agglutination reactions

Hemagglutination reactions can be used to detect any Ag conjugated to the surface of red blood cells " Antibodies that can bind a surface Ag will cross-link the RBCs, forming a large clump that doesnt settle ! Routinely used in blood typing

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Agglutination reactions

Hemagglutination inhibition reactions are used to detect the presence of viruses or anti-viral Ab " Some viruses can interact with RBC molecules and induce hemagglutination (e.g., influenza)

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Agglutination reactions
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Bacterial agglutination can be used to detect antibodies to bacteria ! The visible pellet is made of bacteria cross-linked by antibacterial Ab " Can provide quantitative information about serum antibacterial Ab concentrations ! Can be diagnostic (e.g., typhoid fever titer rise against S. typhi) ! Can be used to type bacteria with a panel of typing antisera

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Assays based on molecule bound to solidphase: RIA

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Radioimmunoassays (RIAs) used to measure concentrations of biologically relevant proteins/ hormones in body fluids Replaced by enzyme-based assays

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Assays based on molecule bound to solid-phase: RIA

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Known amount of radiolabeled molecule added to control wells ! Std. curve created by adding increasing amounts of unlabeled molecule to the wells " Unlabeled molecule competes with labeled; less binding of hot molecule ! Amount of bound radiolabeled molecule is measured by washing off unbound molecule, measuring radioactivity ! Measurement of molecule in samples is achieved in the same way as the standard curve, then compared against it
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Assays based on molecule bound to solid-phase: ELISA

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ELISA assays use antibodies or antigens covalently bound to enzymes " Indirect ELISA ! Detects presence/concentration of Ab in a sample

if Ab present will bind

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Assays based on molecule bound to solid-phase: ELISA

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Sandwich ELISA
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Measures Ag presence and levels

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Assays based on molecule bound to solid-phase: ELISA

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Competitive ELISA ! Measures amount of Ag in a sample ! More Ag in original sample, lower the final signal " Similar to concept of RIAs

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Assays based on molecule bound to solid-phase: Western Blot

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Western blotting can identify a specific protein in a complex protein mixture ! Separation on polyacrylamide gel by electrophoresis ! Identification by enzymetagged specific Ab binding to Ag, followed by substrate exposure

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Microscopic visualization of cells and subcellular structures Immunoelectron microscopy uses gold beads to visualize antibody-bound antigens " Ab molecules linked to electron-dense particles ! Colloidal gold particles " After Ab are allowed to bind, electron microscopy is used " Dark dots indicate spots where Ab have bound ! Different sized gold particles can be linked to different Ab to differentiate structures

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Immunofluorescence
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Fluorescence can be used to visualize cells and molecules " Some dyes absorb light of one wavelength and emit it at a longer

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Flow cytometry Cells are stained with fluorescently tagged mAb " Cells passed through laser beam ! Light striking the cells scatters " Detectors the color of the scattered light and turn it into signals " Signals are read and analyzed by computer ! Can be used to sort cell

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