BIOL 640 1st Edition Lecture 10Outline of Last Lecture I. Complement pathways and regulation. Outline of Current Lecture II.Serology.III. Immunoelectrophoresis.IV. Single radial immunodiffusion.Current LectureImmunology Lecture 11: SerologyDefinition: Serology is the study of Ab present in a given anti-serum. Antiserum contains the liquid fraction of the blood clot and is also straw colored structure with epitope.Quantification implies sensitivity where a small amount can be detected. Increase in sensitivity implies decrease in the amount of material used.Reagents:1. Monoclonal Ab: single known Ab which has ability to recognize one epitope and can exist only in vitro. It can not form precipitate and therefore it is not ideal.2. Polyclonal Ab: can recognize more than one epitope and exists in vivo.3. Conjugate: radio- labeled or fluorescent tagged.Assay based upon:1. Competition: refers to less signal and therefore high volume of sample used.2. Sandwiching: refers to layers for the 1st Ab and then labeling 2nd Ab based upon the 1st Ab.3. Aggregate formation: Agglutination occurs as not molecular but cellular Ag or insoluble Ag formspellet.Precipitation: refers to cross linking of Ag.Ab to form a lattice structure and precipitate forms in solution. It can also form pellets.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Precipitation requires more reactants and is also more time consuming when made in lab and nor ordered.Skip pgs 74-81.1. Interfacial test:(+) reaction, Ab and anti serum match specifics, interphase has a hazy mix.(-) reaction, the tube remains same, no interphase reaction seen.3.Immunoelectrophoresis: is the separation of Ag based upon the isoelectric point or the PI value of protein. Ab migrate based on specificity. The disadvantage is that it can detect only major differences and low affinity is not detected. There can be comparable comparison to control for disease diagnosis. The main disadvantage is that it is not quantitative- ratio based only.4. Single radial immunodiffusion: measures how much Ag is present. Care is exercised to cool the agar in order to avoid denaturation of Ab. Radius calculates the Ag binding with Ab. In other words, the perimeter of the precipitate circle from the center of the well. Experiment is set up tomeasure the different concentration of Ag by plating different plates. Graph shows the equivalence point. The main disadvantage is that it takes several days and not having quick response can not detect infections that need immediate therapy.5. Rocket immunoelectrophoresis: It can measure the ‘rocket’ signal based upon the Ag migration. Low concentration shows less signal and high concentration shows higher signals. Graph is used for calculations and analysis. It is faster and quantitative.II. Agglutination Assays:Titer: Strength of Ab.Titration: serial dilution.Titer= 1/ dilution factor.1. Hazy mix forms, then pellet develops, and then pellet decreases.2. It is not specific.3. The concentration of dilution is based upon previous known concentration.Example: If titer=16, value of the last pellet, then difficult as titers should be between 100-1000 or much higher in