Administrative issues: Recommended text: Goldsby/Kuby Immunology, 6th edition (Note that Innate Immunity is not adequately covered in the 5th edition.) Text book reading assignments are to supplement the lecture. Exam questions will be drawn primarily from lecture material. Discussion sections start next week. The journal article Akira et al, and the relevant problem set questions will be covered. Both are available on the website.
Office Hours: Questions about the lecture material are best addressed during office hours (Tues 11-12). I will be holding extra office hours (date and time TBA) before the first midterm. Email: Please use email only for VERY simple yes/no questions or simple administrative matters. Antigens & Antibodies I Discovery of antibodies Basic Antibody Structure
brief review of protein structure disulfide linked tetramer: 2 heavy and 2 light chains myeloma proteins, Ig domains, and hypervariable regions The antigen binding site of antibodies Antibody isotypes: IgM, IgG, IgD, IgA, IgE The advantages of multivalency effector functions of antibody isotypes In a normal individual, antibodies are extremely heterogeneous.
Myeloma protein: key to determining Ig structure Heterogeneity of antibodies makes sequencing impossible (each B cell clone produces a unique version of antibody). Multiple myeloma: cancer derived from an antibody producing cells (plasma B cell). Myeloma patients have large amounts of one particular
Ig molecule in their serum (and urine) Many patients produce a large amount of one light chain, known as Bence-Jones proteins. When the amino acid sequences of several different Bence-Jones proteins were compared, they were found to consist of two repeating units of ~110 amino acids: one variable and one constant. Antibody
molecules are composed of repeats of a single structural unit known as the immunoglobulin domain Protein homology
Identity or similarity between domains in two or more proteins Most easy to see at the level of primary amino acid sequence (computer programs find it) Sometimes no obvious primary sequence homology but striking structural homology Homology can sometimes predict structure and function All Ig domains have a
similar 3D structure known as an Immunoglobulin Fold. 2 -pleated sheets come together to form a sandwich, held together by disulfide bond and hydrophobic interactions. 3 flexible loops at end:
correspond to hypervariable regions of primary sequence (HV). The Immunoglobulin Fold is a very commonly used structural motif amongst cell surface proteins
Ig domain: Genome Project Champion! The variability of antibodies occurs within 3 discrete regions of the primary sequence: hypervariable regions HV1-3 The hypervariable regions (HV1-3) are separated in primary structure, but come together in the tertiary structure where they form the antigen binding site. Alias Complementary Determining Regions or CDR1-3.
The HV regions form loops at the end of the Ig domain. The intervening framework regions (FR1-4) make up the rest of the structure. The quaternary structure of immunoglobulin
Associations between Ig domains. Interchain disulfide bonds Hinge region allows flexible movement of Fc regions 6 CDR (3 from HC, 3 from LC) combine to make up antigen binding site Antigens & Antibodies I Discovery of antibodies
Basic Antibody Structure brief review of protein structure disulfide linked tetramer: 2 heavy and 2 light chains myeloma proteins. Ig domains and hypervariable regions The antigen binding site of antibodies Antibody isotypes: IgM, IgG, IgD, IgA, IgE The advantages of multivalency effector functions of antibody isotypes
Antigen-antibody interactions regions come in many shapes including: pockets, grooves, or extended flat surfaces. Because the CDR are highly variable, each antibody molecule has a unique antigen binding site with its own dimensions and complementarity. Antibodies that bind to large
proteins antigens Antibodies that bind to small molecules Ig heavy-chain Ig light chain
antigen (which fragment of an antibody is this ?) antigen Epitope- threedimensional face
of an antigen which makes contact with the antibody antigen Epitope- threedimensional face of an antigen which makes contact with
the antibody Conformational Epitopes denatured Native structure B cell epitopes can be sequential (linear) or non sequential (conformational)
Five sequential epitopes in whale myoglobin A non-sequential epitope in hen lysozyme Demonstration of the importance of conformation in
antibody-antigen binding. Epitope and antigen binding site form complementary surfaces Antigens & Antibodies I Discovery of antibodies Basic Antibody Structure brief review of protein structure
disulfide linked tetramer: 2 heavy and 2 light chains myeloma proteins and the primary structure of antibody crystal structure of antibody: the Ig domain The antigen binding site of antibodies Antibody isotypes: IgM, IgG, IgD, IgA, IgE Differences in valency and tissue distribution effector functions of antibody isotypes Heavy and light chains come in different types
Ig isotypes are due to differences in heavychain or light-chain constant region sequences. Heavy chains come in 5 major types that have different tissue distributions and effector functions : Light chains come in two major types: or Antibodies protect by recruiting other effector functions through the
interaction of CH domains with other cells and proteins of the immune system. Different antibody isotypes recruit different effector functions. Receptors that bind to the Fc portion of antibodies are called Fc receptors. Multivalency leads to tighter binding.
Advantage of multivalency Decameric IgM Dimeric IgG What are the virtues of valence?
10 antigen binding Sites/molecule 4 antigen binding Sites/molecule Some classes of Immunoglobulin (IgG, IgD and IgA) have a flexible, proline-rich hinge region. Flexibility of antibody arms allow for more efficient binding to multivalent antigens.
Antibody arms are connected by a flexible hinge Dimeric antigen Immune complexes Distribution of various Ig
isotypes in body fluids serum secretions Relative amount Ig IgM
IgG IgA IgE Antigens & Antibodies I Discovery of antibodies Basic Antibody Structure
brief review of protein structure disulfide linked tetramer: 2 heavy and 2 light chains myeloma proteins and the primary structure of antibody crystal structure of antibody: the Ig domain The antigen binding site of antibodies Antibody isotypes: IgM, IgG, IgD, IgA, IgE differences in valency and tissue distribution effector functions of antibody isotypes
Neutralization Toxins bind to Cellular receptor Endocytosis of Toxin-receptor Complex
Dissociation of toxin to release active chain which poisons cell Antibody protects cell by blocking
binding of toxin Free Ig binds poorly to Fc receptors Aggregation of Ig on bacterial surface promotes aggregation of Fc receptors
No activation of macrophage, No destruction of bacterium Activation of macrophage leading to destruction of bacterium Antibody-Dependent Cellular Toxicity (ADCC)
Antibody binds antigens on the surface of target cells Fc receptors on NK cells recognize bound antibody Cross-linking of Fc
receptors signals the NK cell to kill the target cell Target cell dies by apoptosis and membrane damage IgG
Predominant Ig in serum. 4 subclasses (IgG1-IgG4) Important for opsonization, complement activation, ADCC, Crosses placenta to protect fetus IgM pentameric (decavalent) Pentameric structure held together by Jchain and disulfide bonds.
First Ig produced in response to infection Good at complement activation IgA dimeric (tetravalent) predominant Ig in secretions. Transported across epithelial cells via polyIg receptor. 10g of IgA secreted/day, more than any other Ig!
Found in breast milk, supplies passive immunity to baby. Antibody levels early in life IgE Present in VERY LOW amounts in serum Binds to Fc Receptors present on mast cells and basophils
Levels increase in setting of parasitic infection Can transfer allergy between individuals Antigens & Antibodies I: focus on antibody structure Discovery of antibodies Basic Antibody Structure The antigen binding site of antibodies
Antibody isotypes: IgM, IgG, IgD, IgA, IgE Antigens & Antibodies II: focus on antibody-antigen interactions Definitions A comparison of antigen recognition by B and T cells Factors that determine immunogenicity Quantitating the strength of antibody-antigen interactions: affinity and avidity Cross-reactivity of antibodies
Measuring antibody-antigen binding Antigens & Antibodies II Definitions A comparison of antigen recognition by B and T cells Factors that determine immunogenicity Quantitating the strength of antibody-antigen interactions: affinity and avidity Equilibrium constants equilibrium dialysis
impact of multivalency Cross-reactivity of antibodies Measuring antibody-antigen binding Definitions Antibody: a protein (immunoglobulin) that binds an antigen. Antigen: a substance that is recognized by the immune system Immunogen: a substance that elicits an immune response (not all antigens are immunogenic!)
Epitope: the portion of an antigen antigen that is recognized by the antibody (or TCR). Also called antigenic determinant Hapten: a small molecule that cannot by itself induce an immune response, but can be an antigen.
The antigen receptor of B cells (antibody) binds directly to antigen. Antibody exists in both a transmembrane receptor and secreted form. The antigen receptor of T cells (TCR) binds processed antigen (peptide) on the surface of an antigen presenting cell. TCR exists only as transmembrane form.
Immunogenicity Foreignness-- greater difference from host Size-- bigger is better Complexity- polyglycine is a poor immunogen
Susceptibility to phagocytosis- particles better than soluble material Genotype of host- esp MHC types Route of administration subcu better than IV Dose - not too high, not too low Adjuvants enhance the immunogenicity of antigens by: -triggering the innate immune system (many contain TLR agonists) -slowing release of antigen
-promoting phagocytosis of antigen, others? The first adjuvant Freunds complete adjuvant: emulsified mineral oil and mycobacterial extract. The most effective adjuvants cannot be used in humans due to toxcity (exception: diptheria-pertussis-tetanus combined vaccine (DPT)) Hapten: a small molecule that cannot by itself induce an immune response, but can be an antigen.
NO2 NO2 NO2 NO2 DNP-- 1,3 Dinitrophenol
DNP-- 1,2 Dinitrophenol Even closely related haptens can be distinguished antigenically; antibodies raised against 1,2 DNP may not react with 1,3 DNP. Haptens are not immunogenic unless they are
coupled to a carrier protein. Antigens & Antibodies II Definitions A comparison of antigen recognition by B and T cells Factors that influence immunogenicity Quantitating the strength of antibody-antigen interactions Equilibrium constants
equilibrium dialysis impact of multivalency Cross-reactivity of antibodies Measuring antibody-antigen binding Epitope and antigen binding site form complementary surfaces Quantitating antibody-antigen interactions:
Strength is determined by the sum of multiple non-covalent bonds. Strength of interaction between a single epitope and antigen binding site is called its affinity. Each antibody-antigen interaction has a distinct affinity. Measuring affinity by equilibrium dialysis Once you know the concentration of free and bound ligand at equilibrium for different ligand
concentrations, you can calculate the equilibrium binding constant (K), which provides a quantiative measure of the affinity of the interaction. Note that equilibrium dialysis is based on differential ability of ligand and antibody to pass through membrane. Can only be used when the ligand is small (e.g. a hapten). Equilibrium binding equation Ag + Ab Free antigen
Free antibody k1 k2 Ag-Ab AntigenAntibody
complex [Ab-Ag] Ka = [Ab][Ag] Ka is the association binding constant. k1 or kon is the association rate constant. k2 or koff is the dissociation rate contant.
Equilibrium binding equation Ag + Ab k1 k2 Ag-Ab [Ab-Ag]
Ka = [Ab][Ag] If binding is weak: k2 (off rate) is high, and Ka (association binding constant) will be low (equilibrium shifted to the left). If binding is strong: k2 (off rate) is low, and Ka will be high (equilibrium shifted to the right). Sometimes binding strength is represented by Kd
(dissociation equilibrium constant) = 1/Ka Ag-Ab k2 k1 Ag + Ab
[Ab][Ag] Kd = [Ab-Ag] Kd (dissociation equilibrium constant) = 1/Ka (units are moles/liter) The ligand concentration at which 1/2 of the antibody is binding ligand at equilibrium, is close to the Kd Stronger binding corresponds to lower Kd
The concentration at which 50% of the antibody and ligand are bound at equilibrium, is close to the Kd Kd = [Ab][Ag] [Ab-Ag] If the concentration of total antibody and antigen (bound and free) is 2 x 10 -7M (moles/liter):
For an interaction whose Kd = 10-7M: 50% of antibody and antigen are bound For an interaction whose Kd = 0.5x10-9 95% of antibody and antigen are bound 10-7M = 10-7M x 10-7M 10-7M
0.5 x 10-9M = 10-8M x 10-8M 1.9 x 10-7M Note that for two antibody-ligand pairs with similar on rates (k 1), a lower off rate (k-1) corresponds to tighter binding (higher K a, lower Kd). Note that for two antibody-ligand pairs with similar off rates (k -1), a faster on rate (k1) corresponds to tighter binding (higher K a, lower Kd). Data from equilibrium dialysis can be analyzed using Scatchard Plot:
r= bound ligand / total antibody c= free ligand n= number of binding sites per antibody molecule Slope = -Ka X-intercept = n Note: this only works if the antibody is homogeneous: all antigen binding sites identical, e.g. myeloma protein or a monoclonal antibody. What happens with polyclonal antibody which consists of mixtures of
many different types of antibodies? Polyclonal antisera Can be generated by repeated immunization of animal (rabbit) with antigen (with adjuvant). polyclonal antibodies are a complex mixture of antibodies directed against different epitopes and that differ in their affinity for the antigen.
Polyclonal antibodies vs Monoclonal antibodies Polyclonal antibodies: antibody preparations from immunized animals. Consist of complex mixtures of different antibodies produced by many different B cell clones Monoclonal Antibody: homogeneous antibody preparations produced in the laboratory. Consist of a single type of antigen binding site, produced by a single B cell clone
(later well talk about how these are made). Affinity between two macromolecules (antibody and protein antigen) can measured using a biosensor. -Resonance units are proportional to the degree of binding of soluble ligand to the immobilized receptor. (or soluble antibody to immobilized antigen, as shown here) - Determining the amount of binding at equilibrium with different known concentrations of receptor (antibody) and ligand (protein antigen) allows you to
calculate equilibrium constants (Ka, Kd). -Rate of dissociation and association (koff, kon) can also be calculated. Affinity refers to strength of binding of single epitope to single antigen binding site. But antibodies have 2 or more identical binding sites. Most antigens are multimeric. What is impact of valence on strength of
binding? Avidity (strength of binding) is influenced by both Affinity (Ka of single binding site) and the Valence of the interaction (number of interacting binding sites) Decameric IgM low affinity interactions
can have high avidity if valence is high. IgM tend to bind tightly, but have less specificity. Dimeric IgG Avid binding due to high affinity. Binding of IgG tends to be more
specific. (more perfect fit between antigen binding site and antigen)
In his book "Juiced", Jose Canseco writes about his 16-year career as a Major League ball player, and he says that from his first season, to his last in 2001, he used illegal anabolic steroids and human growth hormone. in...
Arial Times New Roman Wingdings Arial Black Radial Microsoft Visio Drawing CS6361 Project, Part 1 Fall 2006 Agenda Team Roles Vision & Scope Process Use Case Analysis Respond to meeting invitation Preliminary Semiformal Definition Preliminary Semiformal Definition Preliminary Semiformal Definition...
What is a Profession? A definition of a profession: A self-selected self-disciplined group of individuals who hold themselves out to the public as possessing a special skill derived from training and education and are prepared to exercise that skill in...
AP Gov Exam: The Basics. Essential Question: How is the exam structured? Two sections: Multiple Choice = 60 questions in 45 minutes. (50% of Exam) Free Response Questions = 4 F.R.Q.s in 100 minutes (think 25 minutes each) (50% of...
Eq. (13a) is an auxiliary equation, wherein cY is the auxiliary parameter linking Y to the characteristic kernel current density JkCGB for the general barrier. JkCGB is defined by eq. (12b), in which a is the first FN constant ,...
Dell Online Solutions Configurator. From . www.techdata.com, Click on "Tools" and select "Dell Solutions Configurator" *If you don't have online access to the TD site, you will need to set up an admin for your company.
Arial Times New Roman Tahoma Symbol FAA Presentation White Canvas Drawing Adobe Photoshop Image Bitmap Image Microsoft Excel Chart UCSB Bond Structure Characterization Work Effects of Surface Preparation on the Long-Term Durability of Composite Adhesive Bonds Mechanical Testing Investigation Static...
Typical components of this tier are controller and view components, such as servlets and JSPs. MVC pattern often used for UI interaction. Helper objects such as backing beans. 18/02/2016. EIS architectures in more detail.
Ready to download the document? Go ahead and hit continue!