Antigen – Antibody Reactions DIL – 7 Nov. 5th 2013 Mohammed El-Khateeb

General characteristic of Ab/Ag Reactions Non-covalent interaction (similar to “lock and key” fit of enzyme-substrate) Does not lead to irreversible alteration of Ag or Ab This exact and specific interaction has led to many immunological assays used to: detect Ag or Ab diagnose disease measure magnitude of humoral IR identify molecules of bio and med interest

Ag-Ab Interactions Bonds: Hydrogen Ionic Hydrophobic interactions Van der Waals forces Each bond is weak; many are strong To “hold” they must be close  requiring high amts of complementarity!

Three Distinct Phases of Ag-Ab Reactions Primary phenomenon: Sensitization – binding of antibody to antigen – not visible Secondary Phenomenon : Lattice Formation The Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutination If both antigen and antibody are SOLUBLE reaction will become visible over time, ie, precipitation Tertiary Phenomenon: Reaction not visible, detected by affect of reaction on tissues or cells.

Factors Affecting Measurement of Ag/Ab Reactions Affinity Avidity Ag:Ab ratio Physical form of Ag

Affinity =  attractive and repulsive forces Strength of the reaction between a single antigenic determinant and a single Ab combining site Ab Ag High Affinity Ab Ag Low Affinity Affinity =  attractive and repulsive forces

Avidity Y Y Y Keq = Affinity Avidity Avidity 104 106 1010 Incorporates affinity of multiple binding sites 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 ) Y Keq = 104 Affinity Y 106 Avidity Y 1010 Avidity

Specificity The ability of an individual antibody combining site to react with only one antigenic determinant. The ability of a population of antibody molecules to react with only one antigen.

Antigen Binding Antigen 2 Antigen 1 Antigen 3 Variable Light Heavy

Ccroos Reactionsross Reactivity The ability of an individual Ab combining site to react with more than one antigenic determinant. The ability of a population of Ab molecules to react with more than one Ag Anti-A Ab Ag C Similar epitope Cross reactions Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope

Secondary Manifestations of Ag-Ab Reactions 1. Precipitation 2. Agglutination 3. Complement dependent reactions a. cytolysis b. chemotaxis c. opsonization 4. Neutralization LATUCE FORMATION

Primary Manifestation of Ag-Ab reactions 1. Immunofluorescence (IF) 2. Radioimmunoassay (RIA) 3. Enzyme-linked immunosorbent assay (ELISA)

Factors affecting solubility Size Charge Temperature Solvent ionic strength

Antibody-antigen Interactions Antibodies and antigens can both be multivalent. The flexibility of the hinge region improves the efficiency of antigen binding and cross-linking.

Polyclonal Antibodies Produced by immunizing an animal with the appropriate antigen. The immunized animal’s serum is collected. Antibodies can then be purified from the serum. Since one antigen induces the production of many antibodies the result is a ‘polyclonal’ mixture of antibodies. Polyclonal antibodies are much less expensive than monoclonal antibodies. Problems related to specificity. You can enrich for antibodies produced from a particular antigen, but they won’t be alone unless the serum is subsequently affinity purified.

Monoclonal Antibodies Much more complicated to produce than polyclonal antibodies. Process begins by immunizing an animal (most commonly a mouse) with an antigen. The animal’s spleen is removed. B-cells are fused with myeloma cells resulting in hybridomas. Hybridomas are screened to find those producing antibodies to the antigen with which they were immunized. Each hybridoma cell is derived from one B-cell so the antibodies that a clonal population of hybridoma cells produce are monoclonal antibodies. Monoclonal antibodies recognize one epitope only. Spleen is the site of B-cell production. Hybridomas are ‘immortal’ cell lines.

PRECIPITATION TESTS Lattice Formation

Precipitin Reactions Reaction of soluble antigens with IgG and IgM antibodies Form visible molecular aggregates called LATTICES Precipitation only occurs where the ratio of antigen to antibody is optimal

TYPES OF PRECIPITATION REACTIONS Double Diffusion (Ouchterlony) Counter electrophoresis Radial Immunodiffusion (Mancini) Roquete Technique Immunoelectrophoresis Immunofixation Nephlometry / Turbidimetry

Precipitation reactions in fluids

Precipitin ring test Principles of Immunological Tests Precipitation – formation of an insoluble complex when a specific antibody is reacted with a soluble antigen (usually in a gelatin-like substance)

Ouchterlony Gel Diffusion Both antigen and antibody can diffuse independently. Holes punched in agar. Known antibody or antigen added to center well. Known sample added to outer well. Unknown sample added to outer well next to unknown sample. Wait for bands to form.

Ouchterlony Analysis: 2-Dimensional Precipitation in Agarose

Ouchterlony Immunodiffusion Identity Partial Identity Non-Identity The precipitation appears as a continuous line in the form of an angle between those two wells and the C well. There are no spurs at the angle and this type of reaction is termed a band of identity If Ag A (patient) and Ag A1 (control) share a common element but are not exactly the same (Abs to A), a single spur is formed. This is the line of partial identity. If the material in wells 1 and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines

Countercurrent electrophoresis Method Ag and Ab migrate toward each other by electrophoresis Used only when Ag and Ab have opposite charges Qualitative Rapid Ag Ab - + Ag Ab - + PS

1 2 Single radial immunodiffusion Method Ab in gel Ag in a well Quantitative Interpretation 1. Diameter of ring is proportional to the concentration 2. Highet of the Rocket 1 2 Electroimmunodiffusion

Serum Protein Electrophoresis (SPE) Elevated total protein suggests hypergammaglobulinemia Total protein = Albumin + Globulins Quantitative immunoglobulins (Measures amount of IgM, IgG and IgA ) In myeloma, typically see “reciprocal depression” of uninvolved Igs

The Monoiclonal Gammopathies IgG C IgM IgA IgM IgG IgA

Serum protein electrophoresis Albumin 1 2   + -

Protein Electrophoresis

Monoclonal Gammopathy Normal Pattern Albumin decreased and sharp peak in gamma region Albumin 1 2  

Immunoelectrophoresis Combines serum protein electrophoresis with immunometric detection Electrophoresis provides separation Immunoprecipitation provides detection Two related applications: Immunoelectrophoresis Immunofixation electrophoresis

IMMUNOELECTROPHORESIS

Immunofixation Electrophoresis Immunofixation Electrophoresis (IFE) combines zone electrophoresis with immunoprecipitation. This technique may be used to identify and characterise serum proteins. In IFE, proteins of sample are first separated by electrophoresis on a support (agarose) according to their charge and after that the medium is overlaid with monospesific antiserá reactive with specific protein - antigen. If the antigen is present a characteristic immunoprecipitin band will be formed.

Immunofixation Electrophoresis

IMMUNOFIXATION IgG Lambda IgM Kappa IgG Kappa 1. Normal Plasma 2. Polyclonal Hyperglobulinemia 3. Monoclonal Spike 4. Bence Jones proteins in urine

Western blotting Separates the components according to their molecular weight. The proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current. Probed with Ab & then radiolabeled or enzyme-linked 2nd Ab. A position is visualized by means of an ELISA reaction.

The Western blot procedure [INSERT FIGURE 17.17]

Tests Based on Ag/Ab Reactions Ab’s and Ag’s in aqueous solution form a lattice Precipitin Lattice formation requires: polyvalent Ab’s Ag must be bivalent, polyvalent All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

Agglutination Tests Lattice Formation

Agglutination Visible clumping together of particulate matter by antigen combining with its specific antibody. The clumps will be called agglutinates Performed Slide Tube Tile Micrtitration plates

TYPES OF AGGLUTINATION REACTIONS Direct aggultination Hemagglutination Indirect (Passive) Agglutination Agglutination inhibition

Direct Agglutination Bacterial Antigen - ve + ve

Direct agglutination Figure 18.6 - Overview

Agglutination Reactions Particulate antigens, such as cells, and antibodies Form visible clumps or aggregates Very sensitive and easy to read Direct agglutination tests Detect antibodies against large cellular antigens – RBC’s, bacteria, fungi Antibody titer Indirect agglutination tests Antigens absorbed onto latex spheres

Agglutination / Hemagglutination Definition - tests that have as their endpoint the agglutination of a particulate antigen both Ag and Ab can be determined Qualitative 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 4 Patient 1 2 3 5 6 7 Applications Blood typing Bacterial infections Fourfold rise in titer Practical considerations Easy Semi-quantitative Quantitative test

Passive Agglutination/Hemagglutination Definition - agglutination test done with a soluble antigen coated onto a particle Y +  Applications Measurement of antibodies to soluble antigens

Agglutination/Hemagglutination Inhibition Definition - test based on the inhibition of agglutination due to competition with a soluble Ag Y +  Prior to Test Y +  Test Patient’s sample

Direct Coombs Test / Direct Antiglobin test Indirect Coombs Test / Indirect Antglobin test

Applications These include detection of anti-rhesus factor (Rh) antibodies. Antibodies to the Rh factor generally do not agglutinate red blood cells. Thus, red cells from Rh+ children born to Rh- mothers, who have anti-Rh antibodies, may be coated with these antibodies. To check for this, a direct Coombs test is performed. To see if the mother has anti-Rh antibodies in her serum an Indirect Coombs test is performed.  52

Antigen-Antibody Reactions In Vivo THE GOOD Neutralization of viruses and toxins Opsonization of pathogens Complement lysis of bacteria Prevention of bacterial adherence The Bad Autoimmunity - myasthenia gravis Transfusion reactions The Ugly Allergy

Monoclonal vs. Polyclonal Selection is based on application, time and money

IMMUNOPRECIPITATION

Immune Testing

Assays Based on Complement Lattice formation not required

Complement Fixation Phosphate Buffer containing Ca+ & Mg+ Inactivated Patient Serum Rabbit Complement. SRBC IgM Anti SRBC

Complement Fixation Y Methodology Ag No Ag Ag mixed with test serum to be assayed for Ab Standard amount of complement is added Erythrocytes coated with Abs is added Amount of erythrocyte lysis is determined Ag No Ag Ag Y Patient’s serum Ag Y

+veC veC P.S CC BC

Specimens for Routine Tests

Sensitivity of Various Immunoassays ASSAY Sensitivity(mg Ab/ml) Precipitation reaction in fluid 20-200 Precipitation reaction in Gel (DD) 20-200 Precipitation reaction in Gel (IEP) 20-200 Precipitation reaction in Gel (RID) 10-50 Precipitation reaction in Gel Rocket IE 2 Agglutination Direct 0.3 Passive 0.006 -0.06 Inhibition 0.006 -0.06 Radioimmunoassay 0.0006-0.006 ELISA <0.0001-0.0 ELISA using Chemiluminescence <0.0001-0.01 Immunoflourescence 1.0 Flowcytometry 0.06-0,006