Immunoanalysis Lecture Notes (PDF)

Introduction to Immunoanalysis BIOT6002: Lecture 2 Lecturer: Caroline Browne Learning Objectives List the different classes of Abs Describe the function of different Ab classes Label the components of an Ab VL and CL VH and CH Fab Fc Hinge Understand how an Ab (IgG)...

Introduction to Immunoanalysis BIOT6002: Lecture 2 Lecturer: Caroline Browne Learning Objectives List the different classes of Abs Describe the function of different Ab classes Label the components of an Ab VL and CL VH and CH Fab Fc Hinge Understand how an Ab (IgG) is cleaved by digestion enzymes  Immune cells https://www.genome.gov/genetics-glossary/Lymphocyte Antibody Classes IgG Major Ig in normal serum (70-75%) Single IgG molecule Major Ab of secondary immune response Immobilization and agglutination of pathogens for phagocytosis IgM 10% of Ig pool Major Ab of primary immune response – 1st Ig in the human foetus Frequent in immune response to antigenically complex infectious organisms Binds C1 of complement leading to opsonization Antibody Classes IgA 15-20% of Ig pool Predominant Ig in seromucous secretions e.g. saliva, milk Protection against respiratory and gastrointestinal infections IgD less than 1% of pool, mostly found on membranes of immature B cells Enhance mucosal homeostasis and immune surveillance Binds basophils and mast cells IgE commonly associated with allergic diseases e.g. asthma responds to parasitic worm infections Stimulates eosinophils and the release of histamine from mast cells and basophils Group Activity – Which Ab to deploy Respiratory illness - Influenza, COVID-19 or RSV Seasonal allergy to tree pollen Colostrum - breast feeding neonates Irritable bowel syndrome (mucosal lining affected by pathogenic microbe) (Note: More than one Ig can be assigned) Types of Ab - immunoglobulins (Ig) Antibody Properties Structure IgA Found in mucous, saliva, tears, and breast milk. Protects against pathogens IgD Found on immature B cells. Activates basophils and mast cells. IgE Protects against parasitic worms. Responsible for allergic reactions. IgG Secreted by plasma cells in the blood. Able to cross the placenta into the foetus. IgM May be attached to the surface of a B cell or secreted into the blood. Responsible for early stages of immunity. Group Activity – Which Ab to deploy Respiratory illness - Influenza, COVID-19 or RSV – IgG, IgA Seasonal allergy to tree pollen - IgE Colostrum (breast milk) for neonates – IgA, IgM and IgG Irritable bowel syndrome (mucosal lining affected by pathogenic microbe) – IgD, IgA IgG - Predominantly used in immunoassays 2 x Light chains 1 constant and 1 variable region (CL and VL) 2 x Heavy chains 3 constant and 1 variable region (CH and VH) Chains are linked by disulphide bonds Hinge region of H chain confers flexibility ability to bind adjacent identical antigens Fab – antibody fragment Fc – crystallizable fragment Digestion of Antibody Fragments Papain digests Ab (IgG) at hinge region results in 2 Fab (MW = 50,000) + Fc (MW = 50,000) fragments Pepsin digests IgG below inter H chain disulfhydryl link results in F(ab’)2 (MW =100,000) + Fc (MW=50,000) fragments Ab Fragment Applications Linker region - Ab to solid phase in immunoassays Anchors IgG at Fc region - enhances binding of Ag to exposed Fab regions Ab Fragment Applications Fab fragments – small size Reduced tendency to bind non-specifically Lower reactivity to factors found in serum Immunoaffinity chromatography purification of IgG in pharmaceutical preparations (e.g. monoclonal Abs) Fc region of IgG binds to Protein A (from Staphylococcus aureus) and to Protein G (Streptococcal bacteria). CHIP (chromatin immunoprecipitation) Abs Fragment Applications Novel diagnostic and therapeutic development Some Fabs can be as small as 3kDa in size. Can be tagged with radioisotopes, enzymes, and fluorescent molecules to tag or target hard to reach tissues (tumours etc.) Xenaki et al., Front. Immunol., 12 October 2017 https://doi.org/10.3389/fimmu.2017.01287 Advantages of Antibodies in Immunoassays Stable, soluble & abundant Standard procedure for preparation, selection, isolation coupling (conjugation) & immobilisation Available as a mixture with related specificities (polyclonal) or with a single binding specificity (monoclonal) Amenable to modification Binding to Ag & non-antigenic haptens Learning Objectives List the different classes of Abs Define the function of different Ab classes Describe the components of an Ab VL and CL VH and CH Fab Fc Hinge Understand how an Ab (IgG) is cleaved by digestion enzymes