Immunoanalysis Lecture 5 PDF

Introduction to Immunoanalysis Immunoassays BIOT6002: Lecture 5 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Explain how Ab-Ag complexes are visualised What are label free assays Define Pre zone, equivalence zone and post zone Understand the basic principles of Immunodi...

Introduction to Immunoanalysis Immunoassays BIOT6002: Lecture 5 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Explain how Ab-Ag complexes are visualised What are label free assays Define Pre zone, equivalence zone and post zone Understand the basic principles of Immunodiffusion assays Provide examples of immunodiffusion assays Describe how to conduct a Latex agglutination assay (LAA) Label Free Assays The molecular complexes generated by Ab-Ag interaction are Visible, or, Detected by their ability to scatter a beam of light Complex formation indicates that both reactants are present. This means that a constant concentration of Ab can be used to measure a specific Ag and vice versa. If the reagent species (Ab or Ag) is coated onto cells or particles, binding to the target Ab or Ag causes precipitation or agglutination of the particles. Label Free Assays - equivalence a) Pro zone b) Zone of equivalence c) Post zone Immunodiffusion assays Ouchterlony immunodiffusion assay (Double diffusion) Ab in the centre punch of the agarose gel Samples containing Ag, and both positive and negative controls are placed in labelled punches Dish is incubated for 24 -72 h Precipitate of Ab-Ag complex is observed as a white line. Mutjaba et al., 2021 Journal of Microbiology & Biology Education Immunodiffusion assays Single Radial Immunodiffusion (sRID) or Mancini Technique Diffusion ~ 20 h The size of the precipitin rings are dependent on: Ag concentration in the sample well Ab concentration in the agarose gel Size of the sample well Volume of the sample Rings of precipitate will form when concentration of Ag lies within zone of equivalence Diameter of precipitation ring is proportional to conc. – linear plot Densitometric quantification Activity –sRID Data 2018 - “The current gold- standard potency test for inactivated influenza vaccines is the sRID assay.” Are there precipitate rings present Immunodiffusion Assays - electroimmunodiffusion Rocket immunodiffusion (Laurell Technique) More rapid detection than with sRID Electrophoresis of an Ag in an agarose gel containing Ab Negatively charged Ag is electrophoresed through a gel towards a positive charge. The “height” of the rocket is proportional to the antigen concentration in the well. Many antigens just don’t have a sufficiently negative charge, so can’t be evaluated using this assay. Immunodiffusion assays- immunoelectrophoresis Investigation of a complex mixture of proteins to determine presence & characteristics immunologically The protein samples are placed in circular wells & separated by electrophoresis. Antiserum is placed in a central slot & individual proteins are precipitated by the Abs during diffusion Crossed immunoelectrophoresis First dimension Proteins separated electrophoretically in an agarose gel Separated proteins are then electrophoresed at right angles to the first direction into gel containing Abs against protein Each protein will give rise to a precipitate, a peak or a rocket The area enclosed by the precipitate is proportional to the amount of Ag Latex Agglutination Assay (LAA) Samples: blood, saliva, urine, cerebrospinal fluid (CSF). LAA is quicker to run than traditional sRID Latex Beads coated with Ag for Ab capture Steps Preparation of standard and samples Serial dilutions Preparation of beads (specific amounts of purified Antisera or Ag) Sample loading Spectrophotometer reading Total Duration – 2 h The Hook effect Hook effect Inhibition of complex formation by high analyte concentration Leads to restricted sensitivity or “lower detection limits”. Learning Objectives Explain how Ab-Ag complexes are visualised What are label free assays Define Pre zone, equivalence zone and post zone Understand the basic principles of Immunodiffusion assays Provide examples of immunodiffusion assays Describe how to conduct a Latex agglutination assay (LAA)