Antigen antibody reaction

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Slide1:

Antigen-Antibody interactions Batch: Regular Duration: 90 mins For Queries, Contact support.biotecnika.org

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Characterized as : Non-covalent interactions (similar to “lock and key” fit of enzyme-substrate) between Ag and Ab In the variable region of the antibody molecule particularly the CDRs 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 biological and medical interest For Queries, Contact support.biotecnika.org

Ag-Ab interactions:

Ag-Ab interactions Non-covalent interactions involved Bonds: Hydrogen Ionic Hydrophobic interactions Van der Waals forces Each bond is weak; many together make it strong To “hold” they must be close  requiring high amts of complementarity! For Queries, Contact support.biotecnika.org

Measuring affinity of Ab to Ag:

Measuring affinity of Ab to Ag Association between CDR and monovalent Ag can be expressed as: Ag + Ab ⇆ Ag-Ab ; k 1 = forward (assoc) rate constant whereby k 1 /k -1 = K a k -1 = reverse (dissoc) rate constant the assocn or equilibrium constant K a = [Ag-Ab] value of K a depends on k 1; [Ag] [Ab] for small haptens, k 1 is high for large protein Ag’s, k 1 is lower Affinity of Antibody is interaction between single epitope on antigen and one antibody binding site For Queries, Contact support.biotecnika.org

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For small proteins : Ag-Ab complex is very stable K a (association constant is high ) K d (dissociation constant is low ) For large proteins : Ag-Ab complex is less stable K a (association constant is low ) K d (dissociation constant is high ) Dissociation constant (K d ) is inverse of association constant For polyclonal antibodies Ka is not constant as heterogenous antibodies of different affinity are present For Queries, Contact support.biotecnika.org

Ka determined by equilibrium dialysis:

K a determined by equilibrium dialysis For Queries, Contact support.biotecnika.org

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Scatchard Plot If all antibodies have same affinity then there will be a straight line with a slope of –K a- monoclonal ab For heterogenous antibody is a curved line with constantly changing slope showing the heterogeneity of antibodies- Polyclonal antibodies For Queries, Contact support.biotecnika.org

Slide8:

Scatchard plots are based on repeated equilibrium dialyses with a constant concentration of Ab and varying concentration of ligand. ( # 1 Ab has a higher affinity than Ab # 2 ) ( all Ab have the same affinity ) (equals moles of bound ligand / mole Ab ) (The binding sites per Ab ) ( c is the conc. Of free ligand )

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Scatchard plots yield a curved line whose slope is constantly changing, if Ab preparation is polyclonal. >>> antiserum # 3 has a higher affinity than #4

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Antibody affinity is a quantitative measure of binding strength combined strength of the noncovalent interactions between a binding site on an Ab & monovalent Ag Antibody avidity 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 ) For Queries, Contact support.biotecnika.org

Cross-reactivity:

Cross-reactivity Sometimes, Ab can “cross-react” with unrelated Ag …. (can occur if Ag’s share an identical/similar epitope) Affinity is usually less Often seen with polysaccharide Ag’s e.g. ABO Blood groups – glycoproteins -persons lacking one or both of the blood (AB) Ag’s will have serum Ab’s vs.the missing Ag’s -these Ab’s produced from cross-reactive Ag’s!! -provides basis for blood typing tests -necessitates compatible blood types during transfusions, etc. Other cross-reactions: 1) Streptococcus pyogenes : Ab produced against its wall protein crossreacts with myocardial and skeletal muscle proteins. 2) Vaccinia virus For Queries, Contact support.biotecnika.org

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ABO blood types - The antibodies are induced by exposure to cross-reacting microbial antigens present on common intestine bacteria. - ABO blood-group antigens have subtle differences in the terminal residues of the sugars on glyco-proteins in RBC. - Providing the basis for blood typing test in blood transfusion For Queries, Contact support.biotecnika.org

Immunologic tests::

Immunologic tests : Precipitation Rxns : -Ab’s and Ag’s in aqueous soln’s form a lattice => Precipitin Lattice formation requires : 1) Bivalent Ab’s 2) Ag must be bivalent, polyvalent Precipitation rxns, once popular, have been replaced by faster, more sensitive tests

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Immunologic tests::

Immunologic tests : Precipitation rxns in gels Visible precipitin lines in Immunodiffusion - To determine the Antigen concentration For Queries, Contact support.biotecnika.org

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2. Immunoelectrophoresis : electrophoresis w/ double diffusion An Ag mixture is 1 st separated by charge Then, “troughs” are cut ∥ to direction of electric field and antisera is added to trough Ag’s and Ab’s diffuse towards each other to produce precipitin bands Used to detect : a) presence/absence of specific proteins or antibody classes b) immunodeficiency or immune proliferative disorder For Queries, Contact support.biotecnika.org

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Immunoelectrophoresis an antigen mixture is first electrophoresed to separate its components by charge diffusion & producing lines of precipitation For Queries, Contact support.biotecnika.org

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3) Agglutination reactions : visible clumping because of interaction b/w Ag and Ab Antibodies that produce this reaction are called Agglutinins Several types exist: a) Hemagglutination of RBC’s: used in Blood typing b) Bacterial Agglutination: Diagnosis of Infection c) Passive Agglutination: with soluble antigens d) Agglutination Inhibition: highly sensitive for small quantity antigens For Queries, Contact support.biotecnika.org

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FIGURE 6-7 a) Demonstration of hemagglutination using Ab against sheep red blood cells (SRBCs): a constant number of SRBCs plus serial two-fold dilutions of anti-SRBC serum + + + ( control) visible clumping by interaction between Ab & a particulate antigen suchas RBC, latex beads. -depend on the crosslinking of polyvalent antigens, similar to precipitation rxns (lgM is a good agglutinin) -provide a way to type bacteria with a panel of typing antisera. -routinely performed to type RBCs for blood transfusion. For Queries, Contact support.biotecnika.org

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Agglutination Inhibition -The original home pregnancy test kit employed hapten inhibition ( agglutination inhibition ) to determine the presence or absence of h uman c horionic g onadotropin (HCG) >>> The kits currently on the market use ELISA-based assays . -Also used to determine the use of illegal drugs, & immunity (Ab) to virus (rubella).

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4) ELISA tests: depends on enzyme conjugated to 2 ⁰ Ab reacting with a specific substrate to produce a color rxn . Most sensitive of tests for Ag/Ab Variations of ELISA’s : Allows for qualitative or quantitative testing of antigen and antibody Each one can be used for qualitative detection of Ag or Ab Also, a standard curve based on known [C]’s of Ag/Ab can be prepped and an unknown [C} can be ascertained Indirect ELISA: determines Ab Sandwich ELISA: detects Ag concentration Competitive ELISA: measuring Ag For Queries, Contact support.biotecnika.org

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The ELISPOT assay, a modification of the ELISA assay to determine quantitatively the # of cells in a population that are producing specific Ab or cytokine. -> precipitates & forms a spot only on the areas of the well where cytokine-secreting cells had been deposited.

6) Western Blotting:

Used to identify specific protein in mixture Proteins are separated on SDS-PAGE Proteins then transferred to membrane Membrane flooded w/ radio-labelled or enz-linked poly/monoclonal Ab’s specific for protein 6) Western Blotting

7) Immunoprecipitation:

7) Immunoprecipitation Provides a quick and sensitive test for finding proteins/Ag’s Especially in low [C]’s Binds Ab to synthetic bead support  centrifuged Or 2 ° Ab w/ bead or magnetic bead -> collect by magnetism For Queries, Contact support.biotecnika.org

8) Immunofluorescence:

8) Immunofluorescence Provides a quick method for the detection of pathogens and lymphocytes Ab’s are conjugated with a fluorescent dye (fluorescein, rhodamine, phycoerythrin) If Ab’s bind to specific Ag’s, they can be illuminated w/ UV light and emit bright colors There are currently 2 methods employed: Direct staining Indirect staining For Queries, Contact support.biotecnika.org

Direct and indirect Immunofluorescence:

Direct and indirect Immunofluorescence Fluorochromes Fluorescein (490 →517 nm) Rhodamine (515 →546 nm) Phycoerythrin: absorb light of one wavelength & emit fluorescence at a longer wavelength than fluorescein For Queries, Contact support.biotecnika.org

Slide29:

Flow Cytometer or Flow associated Cell Sorter (FACS) Capable of sorting cells for molecular analysis Most common application in determination of kind and number of WBCs in blood. We can find by FACS: -Cells expressing particular target antigen -Percentage of cells -Antigen expressing particular population -Size of Cells (by analysis of light scattering properties)

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Able to count cells as it passes the laser beam and fluorescence emitted by cell is recorded Important tool for detection Classification of leukaemia's For identification of T cell Populations in HIV CD4 and CD8 antigen ratio in Patient blood using FACS Anti-A Ab fluorescence Anti-B Ab fluorescence

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Distribution of selected markers on some leukemic cell types (leukemia can wise at any maturational stage of any one of the hematopoietic lineages) → Immuno phenotyping: the determination of the profile of selected cell- surface markers displayed by the leukemic cell, using “flow cytometry & mAb” [: a cute l ymphocytic l eukemia] [: c hronic l ymphocytic l eukemia]

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