BIOT6002 Lecture 17 Data Handling 5 PDF

Data Handling - Part 4 BIOT6002: Lecture 16 Lecturer: Dr. Caroline A. Browne Revision – labels for signal generation Detection methods: Absorption spectroscopy Absorption or Excitation: A = εbC Electronic transitions excited by...

Data Handling - Part 4 BIOT6002: Lecture 16 Lecturer: Dr. Caroline A. Browne Revision – labels for signal generation 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 Enzyme labels Horse radish peroxidase (HRP) Generates coloured, fluorescent or luminescent derivatives Oxidoreductase that can use a variety of hydrogen donors to reduce hydrogen peroxide High catalytic rate pH optimum is 4.0-8.0 Enzyme labels Alkaline Phosphatase Generates coloured, fluorescent or luminescent derivatives Catalyses the hydrolysis of phosphate esters of primary alcohols, phenols and amines. Inhibited by orthophosphate, zinc chelators & borate pH optimum 9.5-10.5 Common enzyme 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 Fluoroimmunoassay (FIA) Direct – employs a fluorophore label directly in Ab or Ag Indirect - Use substrates that produce fluorescent products Stokes Shift Fluorophores absorb light at one wavelength & emit light at longer wavelength. The difference between the excitation & emission is the Stokes Shift. If the Stokes Shift is large, it is easier to measure the emitted light without interference from incidence light. 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 Time Resolved Fluorescent Immunoassays Competitive assay: %B/Bo calculation The signal in a competitive assay can be generated by an enzyme linked assay or indirect and direct FIA. The blank has the highest signal – the most binding of the labelled Ab to the known concentration of Ag in the assay or vice versa. With increasing analyte (Ag of interest) added to the assay, there is a decrease in signal. The signal is inversely proportional to the concentration of the analyte. %B/B0 (Absorbance of sample or standard/Absorbance of the blank) * 100