Immunoassays Lecture 6 PDF

Introduction to Immunoanalysis Immunoassays BIOT6002: Lecture 6 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Describe the characteristics of Homogenous assay Heterogenous assay Outline the basic principles of a Non-competitive assay Competitive assay Understand...

Introduction to Immunoanalysis Immunoassays BIOT6002: Lecture 6 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Describe the characteristics of Homogenous assay Heterogenous assay Outline the basic principles of a Non-competitive assay Competitive assay Understand the term conjugation List the components of an immunoassay that can be conjugated to Ab and Ag Immunoassay Immunochemical methods Exploit the properties of the Ab:Ag complexes Use Ab as reagents to detect and quantify Ag A variety of compounds can be quantified by immunoassays, ranging in size from small to large proteins. Immunoassay design Considerations: Careful selection of Abs with appropriate binding strength & specificity Ab should have high affinity and high specificity Selection of label & detection method for measurement of Ab:Ag binding. Labelled Ab-Ab complex prepared by covalent attachment (conjugation) of the label. Enzyme-substrate interactions (signal) Signal detection Direct labelling Indirect labelling Homogenous v’s Heterogenous assays Homogenous immunoassays No separation required before analysis Reagents added at the same time, no wash/blocking steps required Can be competitive or non-competitive Heterogenous immunoassays Separation required prior to analysis Multiple steps of filtration, washing, and/or blocking required. Can be competitive or non-competitive Most common type of assay Heterogenous Assay Characteristics Can be competitive or non-competitive Covalent or non-covalent (adsorption) of immuno-reagent to solid phase agarose, polystyrene beads, micro-titre plates, magnetic particles Considerations: Immunoreaction or Binding of Ab to Ag has no effect on signal activity Enzyme on conjugate retains enzymatic activity after immunoreaction Non-specific adsorption Ab/Ag binding can change depending on solid phase Edge effects on micro-titre plates (thermal conductivity of plastic) Non-competitive immunoassays In a non-competitive or immunometric assay An excess amount of Ab is used to capture analyte from the sample A labelled second Ab is added and binds to the first Ab:Ag complex to form a sandwich The complex formed is then measured by spectrophotometry The signal generated is directly proportional to the concentration of Ag present Non-competitive techniques are more sensitive & reproducible than competitive assays Non-competitive immunoassays Unlabelled capture Ab is immobilised in solid phase Sample (analyte) added. Capture Ab binds specifically targeted Ag in analyte Detection Ab is added. Linked with an enzyme, e.g. horseradish peroxidase Binds specifically to another region of the Ag forming a “sandwich”. Addition of enzyme substrate to produce signal Colorimetric change detected by a spectrophotometer. Wash step Post binding (capture and secondary Ab binding), a wash buffer is used to remove unbound analyte and excess Ab. Non-competitive Immunoassays ADVANTAGES LIMITATIONS Excellent detection in complex Requires carefully matched Ab sample types (plasma, serum) pairs High specificity and sensitivity Difficult to test very small antigens Heterogenous Competitive Immunoassay Competitive Immunoassays: Use either labelled Ag or Ab Both the labeled Ag and the unlabeled specimen Ag (or test sample analyte) compete for a limited amount of antibody. Competitive assays are less sensitive than reagent excess assays Heterogenous Competitive Immunoassay Less label measured in the assay means more of the unlabelled (test sample) Ag is present. Concentration of bound label is plotted against analyte concentration, generating an inverse standard curve Homogenous Competitive Immunoassay In the one step competitive assay both the labeled Ag reagent (Ag*) and the unlabeled Ag (or test sample analyte) compete for a limited amount of antibody.  Competitive Immunoassay Capture Ab is immobilised on solid phase Analyte (Ag) and Enzyme labelled Analyte Ag* compete for a limited amount of antibody binding sites. Washing removes all unbound material Addition of enzyme substrate to produce signal Activity - Comparison of ELISA procedures Labelling & Detection techniques Conjugation is the process of linking either Ag or Ab to another molecule to enable detection & quantitation Ab or Ag can be conjugated to: Enzymes e.g. Alkaline phosphatase (AP), horse radish peroxidase (HRP) Fluorescent labels e.g. fluorescein isothiocyanate Biotin / Avidin Polystyrene beads e.g. latex Magnetic beads Isotopic labels e.g. 3H, 125I or 14C Learning Objectives Describe the characteristics of Homogenous assay Heterogenous assay Outline the basic principles of a Non-competitive assay Competitive assay Understand the term conjugation List the components of an immunoassay that can be conjugated to Ab and Ag