Radial Immunodiffusion (Mancini assay) Exercise 4: Session 2Radial Immunodiffusion (Mancini assay)Sample Data For Radial ImmunodiffusionSolving For The Unknown Using The Standard CurveAssignments Due of (2/20)Exam 1 (2/15)Slide 7Hemocytometer Calculation:Slide 9EpitopeSlide 11AffinitySlide 13AviditySpecificitySlide 16Slide 17Slide 18Slide 19Unique Epitope (non-shared)Identical (same epitopes)Different Identity (no shared epitopes)Radial Immunodiffusion (Mancini assay) Exercise 4: Session 2K339 10:30AM-12:15PM•Diffusion of antigen through an agarose gel containing antibody.•A precipitin ring will form. •The size of the ring is proportional to the antigen concentration.•Used to determine concentrations of specific proteins (low sensitivity).•Radial immunodiffusion is an easy way to quantitate relatively abundant proteins without needing advanced laboratory equipment.•E.g. measurement of serum immunoglobulins in animals.Radial Immunodiffusion (Mancini assay) Dr. Slayback-BarrySample Data For Radial Immunodiffusion0 0.5 1 1.5 2 2.502468101214R² = 1Standard GraphHalo Diameter vs. Rabbit IgG Concentration Rabbit IgG Concentration (mg/ml)Halo Diameter (mm)StandardsIgG mg/ml Halo Diameter (mm)2 121 60.5 30.25 1.50.125 0.750.063 0.375DataSerum Halo Diameter (mm)1:2 111:4 61:8 31:16 21:32 1 1:64 0.5 1:128 0.3 1:256 0.02Solving For The Unknown Using The Standard Curve•Use the equation of a line y = mx + b from the STANDARD CURVE.•Solve for “x” using the equation generated from your standard curve.•“Y” is the halo diameter of your precipitin ring.•Multiple by dilution factor to get total protein.Assignments Due of (2/20)•Exercise 5 Hemagglutination: Session 1 prelab ONLY•Summary of the lab •Summary of the Protocol for Part 1Exam 1 (2/15) •Bring a scientific calculator. NO GRAPHING CALCULATORS.•Bring a pencil to write with.•You must show all your work for full credit.•Partial credit is possible.•Exam 1 is Short Answer not Multiple Choice.•Study Exercises 1-4 from your lab manual•Study all Concepts •Study Dilutions•Study Data Interpretation •i.e. Using the Standard Curve to solve for a set of unknown data•i.e. Shape of the curveReview Exam 1K3394:30 PM-6:20 PMVeronica Garcia Please note: This is not an all inclusive guide to Exam 1.Hemocytometer Calculation:Example: 200 cells in 4 small squares, the concentration is 50 x 106 cells/mL.This was determined as follows: 200 cells/4 squares= 50 cells per square or 50 million cells per mL.0.5mL0.5mL0.5mL 0.5mL0.5mLNeat(1 mL of A )1:21:41:8 1:161:32Part A Dilution Series 0.5mL0.5 mL0.5mL0.5mL0.5mL0.5mL 0.5mL0.5mL0.5mL0.5mL0.5mL1:64BBBBBBEpitope•Antigenic determinant - another name for an epitope.•Small portions of an Ag that actually fit into the combining site.Affinity•The sum of attractive forces and repulsive forces for ONE binding site on an antibody to one epitope on an antigen.•Low affinity antibodies bind antigen weakly and tend to dissociate readily.•High affinity antibodies bind antigen more tightly and tend to remain bound longer.Dr. Slayback-Barry*Antigen (Ag)Antibody generator: any molecule that induces antibody formation•A molecule that can bind to a B or T cell receptor.•A molecule that can elicit a specific immune response (immunogen).Avidity•The combined strength of multivalent Ag-Ab connections.•Multivalent connections are stronger since all of the bonds would have to be broken at once for the antibody to release the antigen.Specificity•Refers to the specific epitope on an antigen. •Lock and key analogy. •The fit is flexible not rigid. AntigenAntibody EpitopeAnti-A AbAg CSimilar epitopeCross reactionsCross 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 AgAnti-A AbAg AAnti-A AbAg BShared epitopeThis slide from: http://www.med.sc.edu:85/ppt-imm/Ag-Ab%20Reactions%202001.pptDouble Immunodiffusion (Ouchterlony technique)•Diffusion of antibody and antigen towards each other in an agarose gel.•A line of precipitate will form if the antibody binds to antigen.•Used to determine if an antigen or antibody is present.Dr. Slayback-BarryPrecipitin reactionsThe interaction of antibody with antigen in solution may cause formation of an insoluble lattice that will precipitate out of solution.This precipitate will only form if:- The antibody is bivalent or polyvalent.- The antibody or antibody mixture can bind to at least two different sites on the antigen (either two different epitopes or two identical epitopes).Monoclonal antibodies are likely to be less efficient at immunoprecipitation than polyclonal antibodies.Kuby 5e Figure 6-4aDr. Slayback-BarryScenario 1: Partial Identity (some shared, some unique epitopes)anti-Xxzyxxz a b a b a a•The spur comes from unshared the “b” epitope on the X antigen.•The solid line comes from the unique “a” epitope of the X antigen that X antibody binds.Unique Epitope (non-shared)xzxyanti-Y•The precipitin line is unique. •No shared epitopes. •Epitope “c” is unique to Y antigen.Yc cIdentical (same epitopes)xzxxanti-Zy a b a bza a•The Z antibody bound to the “a” epitope on the Z antigen and on the X antigen.• No spur is observed because the Z antibody does not bind to the “b” epitope.Different Identity (no shared epitopes) xza a a b a bYc c•Antigens X & Y do not have shared epitopes. The antibodies pass each other and a prominent cross is observed. •Antigens Z & Y do not have shared epitopes. The antibodies pass each other and a prominent cross is also observed.•A spur is observed between antigens X & Z because of the unshared “b” epitope. Epitope “a” is shared.xzxyanti-X +