Immunoanalysis Lecture 3 PDF

Introduction to Immunoanalysis BIOT6002: Lecture 3 Lecturer: Caroline Browne Learning Objectives Describe what happens during the primary and secondary immune response Define Plasma Serum Antiserum Clone Polyclonal Antibody (PAbs) Immunogen...

Introduction to Immunoanalysis BIOT6002: Lecture 3 Lecturer: Caroline Browne Learning Objectives Describe what happens during the primary and secondary immune response Define Plasma Serum Antiserum Clone Polyclonal Antibody (PAbs) Immunogen Adjuvant Understand how PAbs are generated Serum, antiserum and cloning Ab locations Within B cells (plasma cells) – cytoplasmic membrane bound structures E.g. Golgi complex and Endoplasmic reticulum On B cells –expressed as integral membrane proteins Attached to the cell surface of other immune effector cells Plasma (fluid potion of blood), mucosal secretions and interstitial fluid When blood or plasma forms a clot, antibodies are left in the residual fluid, known as serum. The Abs within the serum that bind to a particular antigen is referred to as antiserum Antibody cloning refers to the process of producing identical copies or clones of a specific antibody Adaptive (humoral) Immune Response Primary immune response Occurs when host is first exposed to an Ag or immunogen IgG levels appear after lag period ~ 7 days, rise rapidly & decrease gradually Secondary immune response Occurs after second & subsequent exposure to Ag. IgG lag period is shorter, response & duration longer. Note - Healthy adult human (70 Kg weight) produces 3 g of antibodies per day (IgA accounts for two thirds of this Ab pool). Immune response – cell types Immune Response - Immunoglobulins Polyclonal Antibodies (PAbs) Produced when several different plasma cell lines “clones” secrete Ab molecules. Each clone secretes Abs against a different epitope. Heterogeneous antiserum Contains different types of Ab molecules directed against different epitopes on the antigen molecule Group Activity – generating and purifying Abs Generation of Abs Purification of Abs Emil Adolph von Behring (1854–1917) and Shibasaburo Kitasato (1852–1931) Polyclonal Antibody Production – host Laboratory - small volume serum required - rabbit, guinea pig, chicken Large scale - large volume serum – sheep, pig, donkey, horse, goat Antiserum - serum extracted from host animal after immunization – IgG secondary immune response Antisera is then used in immunological assays Polyclonal Antibody Production - adjuvants Immunogen Protein, polysaccharide, lipid or nucleic acid, conjugate (e.g. hapten + BSA), micro-organism, mammalian cell. Adjuvant Promotes a sustained immune response by forming a depot of Ag which is released slowly over time e.g. Freunds adjuvant (mineral oil + surfactant) Adjuvants enhance to production of Abs Polyclonal Antibody Production Polyclonal Antibody Production Standard immunization protocols designed to maximise response of immune system Factors to be considered: Species & strain of animal Form & dose of immunogen Choice of adjuvant Route, number & timing of initial & booster immunizations Polyclonal Antibody Purification 1. High titre Abs in serum – dilution to required concentration 2. Pure Ab or Ab conjugate preparation required – initial purification e.g. Ammonium sulphate precipitation: 40% saturated ammonium sulphate Precipitate recovered by centrifugation Dialysis to remove excess precipitant Purification of polyclonal IgG Immunoaffinity column chromatography Matrix : cyanogen bromide activated Sepharose 4B bound with antigen Antiserum applied to column Specific Ab binds to attached Ag Unbound material washed through with buffer Elution buffer applied ( e.g. glycine buffer pH3) Bound Ab eluted (dissociation of Ab:Ag bond) Immunoaffinity Chromatography - Antigen Antibodies are immobilised on Sepharose matrix by binding to Antigen at the FAb or F(ab)2 region. Protein purification - immobilised antibodies are eluted from a mixed protein solution and dissociated from the antigen by changing the pH. Immunoaffinity Chromatography – Ab Antibodies are immobilised on Sepharose matrix by binding to Protein A through IgG Fc region. Protein purification - immobilised antibodies are eluted from a mixed protein solution and dissociated from the antigen by changing the pH. Learning Objectives Describe what happens during the primary and secondary immune response Define Plasma Serum Antiserum Clone Polyclonal Antibody (PAbs) Immunogen Adjuvant Understand how PAbs are generated