BIOT6002 Lecture 8 Labels and Endpoints in Immunoassays PDF

Labels and Endpoints in Immunoassays BIOT6002: Lecture 8 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Detail the important characteristics of the enzymes Horse Radish Peroxidase Alkaline Phosphatase List the chromogenic substrates of these enzymes Understand absorption(exc...

Labels and Endpoints in Immunoassays BIOT6002: Lecture 8 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Detail the important characteristics of the enzymes Horse Radish Peroxidase Alkaline Phosphatase List the chromogenic substrates of these enzymes Understand absorption(excitation) and emission spectroscopy. Explain how fluorescent detection and time resolved fluorescent detection works Define the following: Beer Lambert Law Stokes Shift Enzyme Labels Advantages Disadvantages Low detection limits Two incubation stages (Ab/Ag Conjugates are very stable binding & signal generation) Generate visible, coloured, Large labels – slow diffusion fluorescent or luminescent rates products from neutral substrates Factors affecting enzyme selection High catalytic turnover Simple & sensitive assay methods Possession of reactive groups for conjugation Long term stability Retention of activity after conjugation Enzyme Labels 1. Horseradish Peroxidase (HRP) is a 44 kDa glycosylated haemoprotein Consists of protoporphyrin prosthetic group 308 amino acids with 4 disulphide bridges 6 lysine residues available for conjugation Horse Radish Peroxidase (HRP) Oxidoreductase that can use a variety of hydrogen donors to reduce hydrogen peroxide Generates coloured, fluorescent or luminescent derivatives High catalytic rate pH optimum is 4.0-8.0 Enzyme Labels 2. Alkaline Phosphatase (AP) is a 140kDa dimeric glycoprotein containing many free amino groups that can be used for conjugation. Catalyses the hydrolysis of phosphate esters of primary alcohols, phenols and amines. Also known as ALP or SEAP (secreted alkaline phosphatase)  Alkaline Phosphatase Inhibited by orthophosphate, zinc chelators & borate pH optimum 9.5-10.5 Wu et al., 2013 Chemistry and Biochemistry Enzyme Labels: Chromogenic substrates Common HRP substrates ABTS (2,2’-azino-bis(ethylbenzothiazoline-6-sulphonate) OPD (o-phenylenediamine) TMB (3,3’,5,5’-tetramethylbenzidine) All require hydrogen peroxide Common AP substrates P-nitrophenyl phosphate Indoxyl phosphate Detection methods: Absorption spectroscopy Absorption or Excitation: A = εbC Electronic transitions excited by A = absorbance ultraviolet (UV) and visible light. ε = molar absorptivity Measure changes in the wavelength b = length of light path of the incident light using an C = concentration excitation monochromator. The intensity of transmitted light is captured by a detector. Beer-Lambert Law Absorbance is linearly proportional to the concentration of the analyte of interest Detection methods: Fluorimetry Fluorometric EIAs have greater sensitivity than colorimetric assays Use the same enzymes Two types of fluoroimmunoassay (FIA): Direct – employs a fluorophore label directly in Ab or Ag Indirect - Use substrates that produce fluorescent products Examples: Fluorescein isothiocyanate (FITC) Rhodamine (Texas Red, Rhodamine Red etc.) Europium chelates Types of FIAs Fluorescent Labels 350 Fluorescein Isothiocyanate (FITC) Limited application Absorbance/emission time < 1ns Hydrophobic nature 488 Short Stokes Shift (30nm) Sensitivity of fluoroimmunoassays (FIA) effected by high background fluorescence in sample (e.g. Bilirubin, proteins) and materials (cuvettes, 660 tubes) Fluorescent Labels Fluorophores Lanthanide probes No interference with ligand (Ag):Ab Metals (atomic numbers 57–70) reaction Long lifetime of fluorescence Long term stability Large Stokes shift High absorptivity Narrow emission peak. Absorbance/Emission times – < 1 ns Absorbance/Emission times < 1000 μs Shot Stokes Shift Examples Europium Samarium Terbium Stokes Shift Fluorophores absorb light at one wavelength & emit light at longer wavelength. The difference between the excitation & emission ls is the Stokes Shift. If the Stokes Shift is large, it is easier to measure the emitted light without interference from incidence light. Time Resolved Fluorescent Immunoassays Employs intrinsically fluorescent lanthanide chelates. rare earth metals when chelated to organic ligand display large Stokes shift and longer decay times Fluorescence decay (decrease in emission of fluorescence of excited molecules) can be short lived or long lived. Short lived → decrease to zero in < 1ms Long lived - slower decay – high sensitivity (zero background) A delayed fluorescent signal originating from the lanthanide chelate labelled component is measured once the background fluorescence has declined. This results in a high signal-to-noise -ratio because the background fluorescence present is avoided in the measurement. Time Resolved Fluorescent Immunoassays DELFIA automatic immunoassay system Delayed Enhanced Lanthanide FluoroImmunoAssay (DELFIA) is an example of a time resolved Fluorescent. Learning Objectives Detail the important characteristics of the enzymes Horse Radish Peroxidase Alkaline Phosphatase List the chromogenic substrates of these enzymes Understand absorption(excitation) and emission spectroscopy. Explain how fluorescent detection and time resolved fluorescent detection works Define the following: Beer Lambert Law Stokes Shift