MOLECULAR RECOGNITION Bioanalytical Methods Classification 1. Biassay: molecular recognition, signal generation and detection in solution or on inert solid phase 2. Biosensor: molecular recognition system + transducer (electronic) 3.. 4.. 5.. 1
Bioassays Immunoassays: antibody-antigen Diagnosis and management of diseases Recognition sites Antibody (Ab): paratope Antigen (Ag): epitope Specificity allows analysis of very complex samples without treatment or separation High affinity leads to high sensitivity and very low detection limit Detection: usually requires use of label to Ab or Ag Fluorophore, luminescent, radioactive, enzyme or electroactive group Immune system molecules The immune system has the capacity to recognize and respond to about 10 7 different antigens Antibodies, T-cell receptors and MHC 2
Immune system molecules Antibodies systems Antibody-tagged cells or material are recognized and disposed of by macrophages Antibody tagged-bacteria can be directly lysed by a set of nonspecific serum proteins (Complement) B-cells can display on their surface a membranebound form of their antibody: binding of antigen triggers synthesis of many copies of same antibody T-cells receptors recognize antigenic determinants only when presented as part of a complex with a third group of proteins the Major Histocompatibility Complex (MHC) Antibodies 20 % of total plasma proteins Immunoglobulins (Ig) Production: Hosts: mice, rabbits, horses etc. injected with antigen In vitro: use cell cultures 3
Paratope Antibodies Structure Four subunits Y-shaped symmetric dimer (L-H)2 Two identical light chains (L) (25 kda) Two identical heavy chains (H) (50 kda) Chains linked by disulfide bonds Antibodies-Structure Light chains Two domains Constant: CL Variable: VL Two types of light chains with no known functional distinction Heavy chains Four domains One variable: VH Three Constant: CH1, CH2, CH3 Five types of heavy chains associated with different functionality IgG, IgM, IgA, IgD and IgE 4
Antibodies-Structure Hypervariable in sequence and length CDR: Complementarity Determining Regions Classes of Antibodies 5
Classes of Antibodies Five different types of H chains define the five classes of Ig and their physiological function: IgA, IgD, IgE, IgG and IgM Two types of light chains Structure of Immunoglobulin Domains Native Ig are difficult to crystallize due to the flexibility of the hinge region Fragment antigen-binding IgG: most common antibody (70%) Fragment Crystallizes easily Pepsin cuts below the hinge reion 6
Immunoglobulin fold All immunolgobulin domains have similar three dimensional structure Two antiparallel beta sheets packed tightly against each other Constant domain 7β strands; 4 form one sheet and three form a second sheet Sheets joined by a disulfide bridge Greek key barrel Short loops between β strands Loops are constant within each class of immunoglobulin (invariant) Loops vary in length and sequence among immunoglobulin chains of different classes 7
Variable domain 9 beta strands The two additional strands contain a hypervariable region, CDR2. provide framework that positions CDR2 close to the other two hypervariable regions. Hypervariable regions Clustered in loop regions at the end of the variable domain Specificity of immunoglobulins is determined by the sequence and size of the hypervariable regions in the variable domains. 8
Monoclonal and Polyclonal Monoclonal: bind to one particular epitope on the antigen Specific and reproducible Most suitable for analytical work Produced from cell cultures Polyclonal: mixture of antibodies produced by a host due to a single antigen Interesting structures to examine 1IGT: Structure of Immunoglobulin,, McPherson et al. 1IGY: Structure of immunoglobulin, McPherson et al. 1MCO: Human Ig with hinge deletion 1MFD: Complex Trissacharide-Antibody 2DYP 7FAB 1MCP: Phosphocholine-Fab 1MAK: VL domain 1MCI: design of peptide ligands 1A8J 1MCD 9
Refined structure of an intact IgG2a monoclonal antibody. Biochemistry v36 pp.1581-1597, 1997 (1IGT) Harris, L.J., Skaletsky, E., McPherson, A. Crystallographic structure of an intact IgG1 monoclonal antibody. J.Mol.Biol. v275 pp.861-872,1998 10
Antigens Two classes Complete - usually large proteins induce immune response by themselves Can have several copies of the same epitope Can be multideterminant: contain several epitopes that bind to different antibodies Incomplete haptens, low molecular weight, not antigenic Need a carrier protein to induce immune response Generally have a single epitope Epitope make only a small area on the antigen very short peptide segments ( equal or less than 18 aa) Continuous (contiguous segment of aa) Discontinuous (conformational): formed by the 3D fold The Antigen-binding site The antigen-binding site is formed by close association of hypervariable regions from both heavy and light chains CL associates with CH1 and VL with VH Association is very tight and extensive Few contacts between VL and CL and VH abd CH1 Fab fragment: elongated molecule with two globular domains: the variable and the constant 11
Antigen-binding site Four beta strands from each of the variable domisn in Fab showing the six hyper-variable regions Antigen-binding site Phosphorylcholine and side groups in Fab Phosphorylcholine: hapten: small molecules that induce specific antibody production when they are attached to a protein that acts as a carrier Binding cavity in a cleft between the light and the heavy chains (15 A wide at the mouth and 12 A deep). Lined by residues from all CDR groups except the short CDR2 from the light chain Choline binds in the interior Phosphate group ins towards the surface 12
Antibody-Antigen Complex Immuno-complex Reversible binding Affinity (between paratope and epitope) depends on intermolecular interactions Magnitude of equilibrium constant: 10 4 to 10 12 L/mol For bioassay, affinity must be 10 8 L/mol or larger Avidity: total interaction for antigens with multiple epitopes with more than one paratope K eq = [ Ab Ag] [ Ab][ Ag] Classification of Immunoassay Limited or excess reagent format Homogeneous or heterogenous format Labelled or unlabelled format 13
Classification of Immunoassay Limited reagent (competitive) For antigen analysis, antibody is the limiting reagent + fixed known amount of labeled antigen + unknown Competitive reactions between AgL and Ag and Ab Excess reagent: antigen is incubated with excess antibody. Second antibody is added that binds to another epitope of the antigen. Sandwich complex is formed Quantification based on labeled antibody after removal of excess reagent Suitable for large analytes with several epitopes Classification of Immunoassay Heterogeneous format Requires separation of the Ab-Ag complex from the unbound fraction before detection Separation by precipitation, coupling of antibody or antigen to solid phase or by chromatographic techniques Homogenous No need for separation Need for selective detection of bound versus free Ab Methods; laser light scattering (dynamic light scattering) 14
Classification of Immunoassay Labels Almost all immunoassay rely on use of labels for signal generation and detection Radioisotopes: 125 I or 14 C Enzyme Fluorophore (fluorencein) Luminescent species Latex or gold particles Detection method depend on label Isotopic counting Colorimetry for enzyme assays Photomultiplier tubes for fluorescence and luminescent labels Turbidity for particle enhanced assays Home Pregnancy Test Target: glycoprotein hormone, human chorionic gonadotropin (hcg) secreted in urine after conception Test strip sandwich format assay Capture antibody is immobilized onto surface of device Tracer antibody, labeled with dye, is impregnated onto surface of device hcg forms a complex with tracer antibody Sandwich formed between immobilized antibody and hcg-tracer antibody complex At high concentration of hcg, the dye color becomes apparent 15
Home Pregnancy Test Enzyme Immunoassays Enzymes are the most commonly used labels in immunoassay Operational principle: enzyme acts as a catalyst for the conversion of a colorless substrate to a colored product. Signal is amplified in that a single enzyme can catalyse several molecules of substrate Most popular enzyme: horseradish peroxidase (HRP) H 2 O 2 + HRP 2 2 DH 2H O + 2D DH reduced hydrogen donor D : oxidized : hydrogen donor DH: a dye that changes color 16
Properties of a good enzyme label Stable under experimental conditions High turnover rate: Turnover rate: number of product molecules produced per unit time Enzyme reaction v = k K M [ ES] k = k = K + k k [ E][ S] + [ S] k2 : turnover K : Michaelis Menten cons tan t M 2 1 1 2 2 M Catalytic activity depends on Turnover rate Michaelis-Menten constant Enzyme and substrate concentration Catalytic activity also depends on ph, temperature, ionic strength and effectors molecules 17
ELISA for HIV-Antibodies HIV antigens are immobilized on a microtitre well surface and well is washed Sample is applied and left to incubate Well is washed A second antibody (an antihuman immunoglobulin) with enzyme label is added which targets HIV antibodies Excess secondary antibody is washed Substrate is added If secondary antibodies were bound (i.e. HIV antibodies were present), then substrate will react Color change, fluorescence, electroactivity, etc. that results is quantified. http://hivinsite.ucsf.edu/insite?page=kb-02-02-01 ELISA http://www.biology.arizona.edu/immunology/activities/ elisa/technique.html 18