بسم الله الرحمن الرحيم

Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

Antigen Antibody Reaction Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: elgreatlyem@hotmail.com

Clinical Immunology & Serology Practice (Code: MLIS 201) TEACHING OBJECTIVES: 1. To discuss the factors affecting Ag-Ab reactions 2. To define Affinity & Avidity 3. To know different methods of Ag-Ab reactions 4. To describe the basics and types of precipitation reactions 6. To describe the basics and types of aggulination reactions 7. To describe the basics and types of immuno-flourescent techniques 8- To describe the basics and types of ELISA

Serology The science that deals with the properties and reactions of sera, especially blood serum. Usually refers to the diagnostic identification of Ag or Ab in the serum. Serological assays depends on Antigen-antibody interactions

Nature of antigen-antibody interaction i.e epitope-paratope binding forces

Binding of the epitope with the paratope (antigen binding site) POOR FIT GOOD FIT Antigen binding site Antigen binding site epitope epitope Study Guide What constitutes a good fit in an antigen binding site? How does antibody affinity relate to fit? What are the forces that are attractive and repulsive? What is actually "fitting" into the combining site? antigen determinant high attraction low repulsion high repulsion low attraction

Factors Affecting binding of Ag/Ab Affinity Avidity Law of Mass Action Physical form of Ag Ag:Ab ratio

1-Affinity Antibody affinity is the strength of the binding between a single antigenic determinant and a single combining site on the antibody. The higher the affinity of the antibody for the antigen, the more stable will be the interaction. 2-Avidity Avidity is a measure of the overall strength of binding of multivalent Ag (i.e. antigen with many antigenic determinants) and multivalent antibodies. Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.

2-Avidity (cont.) The overall strength of binding between a multivalent Antigen and multivalent Antibodies Y Keq = 104 Affinity Y 106 Avidity Y 1010 Avidity

Specificity Cross Reactivity The ability of an individual Ab binding site to react with only one antigenic determinant. The ability of a population of antibody molecules to react with only one antigen. Cross Reactivity The ability of an individual Ab binding site to react with more than one antigenic determinant. The ability of a population of Ab molecules to react with more than one Ag

Similar epitope Shared epitope Anti-A Ab Ag A Anti-A Ab Ag C Similar epitope Cross reactions Anti-A Ab Ag B Shared epitope Specific Reaction

Antigen antibody tests All tests based on Ag/Ab reactions can be used to detect either Ag or Ab Cannot be visualized, thus Detection of antigen/antibody reactions is difficult Various laboratory methods have been developed to make this reaction visible eg. Precipitation, agglutination, labeled reagents (ELISA, RIA, IF), …etc.

Methods for detection of Ag-Ab Reactions Precipitation reactions (Ag is soluble) precipitin. Agglutination reactions (Ag is particles) clumping. Complement fixation reactions. Labelling methods: a- Immuno-fluorescence reactions. b- ELISA. c- Flow cytometry Methods for detection of Ag-Ab Reactions

Methods for detection of Ag-Ab Reactions 1- Precipitation reactions

Precipitation Serological Tests

Precipitation techniques Precipitation reactions Involve soluble antigens with antibodies cross-linked and form lattice that Eventually develops into a visible precipitate. In immunodiffusion, antigens and antibodies diffuse through a gel until they reach the zone of equivalence. In immunoelectrophoresis, diffusion is combined with electrophoresis.

Precipitation reactions Polyclonal Ab can form lattices, or large aggregates with polyvalent Ag. Monoclonal antibody can link only two molecules (epitopes) of antigen and no precipitate is formed. ( Lattices or large aggregates ) ( no precipitate is formed)

Precipitation curve The plot of the amount of antibody precipitated versus increasing antigen concentrations (at constant total Ab) reveals 3 zones: Zone of antibody excess: Precipitation is inhibited. Ab not bound to Ag can be detected in the supernatant; 2. Zone equivalence: Maximal precipitation in which antibody and antigen form large insoluble complexes. No Ab or Ag can be detected in the supernatant 3. Zone of antigen excess: Precipitation is inhibited. Ag not bound to Ab can be detected in the supernatant

Precipitation curve

Precipitation techniques 1. Tube precipitation test 2. Gel diffusion Single radial Double 3. Immuno-electrophoresis, immuno-fixation

Precipitation techniques 1- Tube precipitation test Ag & Ab molecules diffuse until they reach equivalence zone Visible Precipitation (In gel) PPT in Fluid PPT in Gel

Precipitation techniques: 2. Gel Immunodiffusion Techniques Radial Immunodiffusion A single diffusion technique where Ab is put into a gel and Ag is measured by the size of a precipitin ring formed when it diffused out in all directions from a well cut into the gel Ouchterlony Double Diffusion Both Ab and Ag diffuse from wells into a gel medium

Radial Immunodiffusion A quantitative technique based upon the reaction between an Ag placed in a well diffuses into an agar containing the Ab. During diffusion period the Ag & Ab continue to react until the zone of equivalence is reached with formation of a well-defined ring of precipitation around the Ag well which is proportional to the Ag concentration.

Ouchterlony Double Diffusion Both Ab and Ag diffuse independently from wells into a gel medium

Electrophoresis Techniques Electrophoresis separates molecules according to differences in their electrical charge. Rocket Immunoelectrophoresis Countercurrent Immunoelectrophoresis Immunoelectrophoresis Immunofixation Electrophoresis

3. Immuno-electrophoresis Ag molecultes are separated according to differences in their electrical charge by electrophoresis Ab is placed in a trough cut in the agar Ab diffuse towards Ags precipitin arcs Ag - + Ag Ab Ag Ab Interpretation- Precipitin arc represent individual antigens

Measurement of Precipitation Turbidimetry Passing light through a cloudy solution of Ag-Ab complexes. Net decrease in light intensity correlates to the concentration of the Ag Nephelometry Measuring light scattered at a particular angle after being passed through a solution of Ag-Ab complexes. Amount of light scattered correlates to the concentration of the Ag

Methods for detection of Ag-Ab Reactions 1- Precipitation Reactions 2- Agglutination Reactions

Agglutination Serological Tests

Agglutination Principle evaluation: Qualitative (y/n), Particulate antigen + antibody –> clumping The clumps will be called agglutinates Lattice formation (antigen binds with Fab sites of 2 antibodies forming bridges between antigens) Agglutination Curve follow the same roles as in precipitation curve evaluation: Qualitative (y/n), Semi-quantitative (serum titration)

Agglutination Tests Types of Agglutination Reactions Direct agglutination Tests Indirect agglutination Tests Agglutination Inhibition Tests

(I) Direct Agglutination Tests Used to determine antibody titer against particulate antigens (Bacteria, Fungi or RBCs) Particulate antigen reacts directly with antibodies. Applications Blood group typing Direct Coomb’s Test The diagnosis of certain diseases (Bacterial infection Eg. Syphilis) The identification of some viruses.

a) Blood Grouping Y +  Y Anti-RBC RBC Agglutinate

b) Direct Coombs (Antiglobulin)Test detects the presence of antibodies on red blood cell Simply add a second antibody ( Antiglobulin antibody, Coomb’ Reagent) directed against the immunoglobulin coating the red cells. This anti-immunoglobulin antibodies cross link the red blood cells and result in agglutination.

C) Latex Test Ag or Ab are not part of particulate matter but are attached to it (ANTIBODIES OR ANTIGENS ARE ATTACHED TO PLASTIC BEADS) EASY TO DO Eg. Latex test for RF & CRP positive negative

(II) indirect agglutination (Coomb’s)test To detect anti- RBC’s antibodies in a serum sample. This test is done by incubating RBC’s with the serum sample washing out any unbound antibodies then adding a second anti-immunoglobulin reagent (Coomb’s Reagent) to cross link the cells resulting in agglutination.

Hemagglutination-Inhibition Some viruses (eg. Measles virus) agglutinate RBCs in vitro. Anti-virus Ab will bind the virus and inhibit hemagglutination.

Applications of agglutination tests in Serology Determination of blood group antigens. Diagnosis of cases with Rh incombatability (erythoblastosis fetalis) Diagnostics of autoimmune hemolytic diseases Latex test for detection of Ags or Abs (eg. RF, CRP) To assess bacterial infections (e.g. Typhoid Fever, Syphilis)

Agglutination vs. Precipitation Soluble Ag & Ab Ag must have at least two determinants Ag xs results in postzone reaction Ab xs results in prozone reactions Reaction time: hours to days Test results: qualitative, semi-quantitative or quantitative Agglutination Insoluble or particulate Ag or Ab Ag must have at least two determinants Ag xs results in postzone reaction Ab xs results in prozone reactions Reaction time: minutes to hours Test results: qualitative or semi-quantitative

Methods for detection of Ag-Ab Reactions 1- Precipitation Reactions 2- Agglutination Reactions 3- Labelling methods: a- Immuno-fluorescence b- ELISA. c- Flow cytometry

Immunofluorescence: Methods Direct Indirect Fix specimen on slide Add Flourescein labeled antibody specific for the desired antigen Look for fluorescence Fix specimen on slide Add antibody specific for the desired antigen Add second Flourescein labeled antibody Look for fluorescence

Immunofluorescence: Interpretation Fluorescence = patient has the antigen

Immunologic Lab Tests Outline Agglutination reactions DAT IAT Immunofluorescence ELISA

Enzyme-Linked Immunosorbent Assay: Purpose Detection of antibodies in patient specimen Examples: home pregnancy tests HIV tests tests for some coagulation factors, cytokines, and autoantibodies

ELISA: Method Fix the target (AG or Ab) to wells of microtiter plate Add patient specimen to well coated with ligand Add AHG with enzyme attached Add substrate Measure color change

ELISA: Interpretation Color change = patient has the antibody

ELISA: Variations detects antigen (not antibody) Sandwich immunoassay detects antigen (not antibody) coat well with antibody rest is like ELISA

Radioimmunoassay detects antibody or antigen Label (detector) is a radioactive substance otherwise like ELISA or sandwich immunoassay

Immunologic Lab Tests Outline Agglutination reactions DAT IAT Immunofluorescence ELISA Western blot

Western Blot: Purpose Detection of antibodies in patient specimen Most common example: HIV test

Western Blot: Method Make a protein suspension of the target you’re looking for (e.g., HIV) Electrophorese the suspension onto a little gel strip Apply the patient’s specimen (containing antibodies) to the strip Add AHG that has an enzyme attached Add substrate and look for bands

Western Blot: Interpretation Bands on strip = patient has antibodies to corresponding proteins

Enough bands = patient is “positive”

Immunologic Lab Tests Outline Agglutination reactions DAT IAT Immunofluorescence ELISA Western blot Flow cytometry

Flow Cytometry: Purpose Examples: diagnosis of leukemia and lymphoma determination of CD4/CD8 counts in patients with HIV

Immunologic Lab Tests Outline Agglutination reactions DAT IAT Immunofluorescence ELISA Western blot Flow cytometry

Study Questions: Complete: The Factors Affecting binding of Ag/Ab are ………………………. Compare between: Agglutination & Precipitation.

Assignment Write on the following subjects: 1. Factors affecting Ag-Ab reaction أميرة رفعت – اميرة صلاح – ايمان محمود – ايمن شكرى – داليا ناصر 2. Precipitation reactions دعاء عبد الله – رغدة رشدى – رنا ابراهيم – سمر عبد الحميد – سمية جمال

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