Structure of Antibodies

Structure of Antibodies
STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala
Antibodies A.K.A Immunoglobulins & gamma globulins Structure variable hypervariable constant STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala Figure 21.13a

Antibody Structure Antibodies Are Made Up Of: 2 Light Chains (identical) ~25 KDa 2 Heavy Chains (identical) ~50 KDa Each Light Chain Bound To Heavy Chain By Disulfide (H-L) Heavy Chain Bound to Heavy Chain (H-H) First 100 a/a Of Amino Terminal Vary of Both H and L Chain Are Variable Referred To As VL , VH, CH And CL CDR (Complementarity Determining Regions) Are What Bind Ag Remaining Regions Are Very Similar Within Same Class STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Antibodies Constant (C) region defines antibody class determines chemical & cellular interactions determines how class functions to eliminate antigens STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Antibody Classes Antibody Classes: IgM, IgG, IgA, IgD, IgE (Ig = immunoglobulin) STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Antibody Classes IgG: the most abundant circulating Ig. The dominant circulating Ig of the primary & the secondary response. Crosses the placenta. Complement binding (Monomer). IgA: the Ig of secretions. Helps prevent antigen penetration of membranes (Dimer). IgD: the Ig of B-cell activation. Found on B-cell surface (Monomer). STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Antibody Classes IgM: occurs as a monomer & a pentamer Occurs on the B-cell surface (Monomer). The Ig of early primary plasma cell response, circulating antibody; a potent agglutinator. Complement binding (Pentamer). STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Antibody Classes IgE: the Ig associated with allergies. Stem binds to mast cells & basophils. Receptor binding results in histamine release & inflammation. Found mostly in mucosa of respiratory & GI tract (Monomer). STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Antigen-Antibody interactions
Characterized as: Non-covalent interaction (similar to “lock and key” fit of enzyme-substrate) Do not lead to irreversible alteration of Ag or Ab This exact and specific interaction has led to many immunological assays that are used to: detect Ag or Ab diagnose disease measure magnitude of humoral IR identify molecules of biological and medical interest STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Introduction Ag – Ab reactions are one of the most specific noncovalent biochemical reactions known The forces that hold the reactants together are: - van der Waals forces - Electrostatic forces - Hydrophobic forces They can be represented by the simple formula: Ag + Ab ↔ AgAb The reaction is driven to the right but it is reversible STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Strength of Reaction The strength of the reaction (how far it is driven to the right) is referred to as affinity Antibody affinity - A quantitative measure of binding strength - Combined strength of the noncovalent interactions between a binding site on an Ab & monovalent Ag - Affinity varies broadly among immunoglobulins STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Strength of Reaction Antibody avidity - Avidity is often used to describe the collective affinity of multiple binding sites on an antibody molecule - True strength of the Ab -Ag interaction within biological systems - The interaction at one site will increase the possibility of reaction at a second site - High avidity can compensate for low affinity (secreted pentameric IgM has a higher avidity than IgG ) STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

STAGES OF Ag - Ab REACTIONS
Primary reactions Vs secondary reactions: Small Ag - Ab complexes Vs large complexes (The Lattice hypothesis) Development of macroscopic manifestations reactions (e.g. immunoprecipitation) Ag – Ab reactions involving IgM are confined to the blood stream, while those of lower molecular weight (IgG and IgE) can leave the vasculature and enter tissues Time required is hours to days for precipitin formation leading to irreversible immunoprecipitates STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

ZONES OF PRECIPITIN FORMATION
Precipitin Curve STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Precipitation Precipitation can take place in capillary tubes, test tubes, and in gel STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Precipitation in gel - Double diffusion - Single (radial) diffusion - Combination of diffusion in gel and electrophoresis STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

SINGLE VS. DOUBLE DIFFUSION
Single diffusion Supporting medium (gel) contains one reactant at a uniform concentration Only the unknowns move through the medium Double diffusion Gel is inert (contains no reactants) Both Ag and Ab travel through the medium STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

The region of equivalence STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

RADIAL IMMUNODIFFUSION
Ab uniformly distributed in gel; Ag diffuses outward from a well (single diffusion) Ag- Ab complexes form as concentric rings around the well at zone of equivalence At a set time, ring diameters are measured [Ag] is directly proportional to the ring d2 Unknown value is determined by comparing to a 3-standard curve STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Samples Standards Precipitin Rings A B C a b c Standard Curve STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Fahey method (kinetic) Read at 18 hours Plot [std] vs. ring diameter on semi-log paper Mancini method (endpoint) Read at 48 or 72 hours Plot [std] vs. ring diameter squared on graph paper Results reliable only if the ring size is within the range of the standards; if greater than highest std, dilute and repeat test Used to measure IgM, IgG, C4,C3,transferrin, CRP, others STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

OUCHTERLONY DOUBLE DIFFUSION
Ag & Ab placed in wells cut into an agarose gel (both reactants diffuse) Precipitin line (or arc) indicates Ab has specificity for Ag Position of precipitin between wells depends on MW and concentration of reactants 3 possible patterns of reaction: identity, non-identity, partial identity STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

OUCHTERLONY DOUBLE DIFFUSION
Ouchterlony Plates Precipitin Patterns STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Agglutination Titer Zeta potential Types of Agglutination - Direct agglutination or hemagglutination - Indirect (passive) agglutination or hemagglutination - Agglutination or hemagglutination inhibition The Coombs test - Direct - Indirect STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Agglutination Reactions

Agglutination Qualitative slide agglutination - identification of bacteria with antisera directed against O, H, K antigens STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Agglutination Latex agglutination Coagglutination STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Agglutination Tube agglutination tests: - Gruber-Widal: typhoid fever (S. typhi) - Weil-Felix: typhus (Rickettsia) - Wright: brucellosis Identify and titrate antibodies in the patient’s serum. Titre: is defined as the reciprocal of the highest dilution of serum showing agglutination. STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

1:200 1:400 1:100 STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala Titer

Agglutination inhibition

Hemagglutination Inhibition Test
To Detect Antibodies (Rubella) - Serum (Ab)+ HA +RBCs= No Hemagglutination = Positive Test - Serum (No Ab)+ HA + RBCs =Hemagglutination =Negative Test To Detect Antigen (HBsAg) - Serum (HBsAg) +Anti HBsAG + HBsAg coated RBCs = No Hemagglutination = Positive Test - Serum (No HBsAg)+ Anti HBsAG + HBsAg coated RBCs = Hemagglutination =Negative Test STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Use of Labels in Ag – Ab Reactions
Immunoassays - Radioimmunoassay (RIA) - Enzyme Immunoassys (EIA) Immunofluorescence (IF) - Direct IF - Indirect IF Flow cytometry and Cell Sorting (FACS) STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Immunologic Tests 4) Radioimmunoassay (RIA)– a very sensitive test; used for measuring hormones, serum proteins, drugs, etc. at low concentrations (≤ 0.001ug/ml) measures “competitive binding” of radiolabelled Ag + unlabelled (test) Ag to high affinity Ab STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

ELISA STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

ELISA tests Depend on enzyme conjugated to 2 Ab reacting with a specific substrate to produce a color reaction. Variations of ELISA’s: Allows for qualitative or quantitative testing. Each one can be used for qualitative detection of Ag or Ab Also, a standard curve based on known concentrations of Ag/Ab can be prepared and an unknown concentration can be determined Indirect ELISA Sandwich ELISA Competitive ELISA STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Direct and indirect Immunofluorescence

Immunoprecipitation Provides a quick and sensitive test for finding proteins/Ag’s especially in low concentrations Binds Ab to synthetic bead support  centrifuged Or 2° Ab with bead or magnetic bead and collect by magnetism STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Distribution of selected markers on some leukemia
cell types → Immunophenotyping using “flow cytometry & mAb” STES, Sinhgad Institute of Pharmaceutical Sciences, Lonavala

Structure of Antibodies

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