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Data Handling - Part 4

• This lecture covers data handling, focusing on signal generation methods.

Revision - Labels for Signal Generation

• Signal generation in assays typically involves a multi-step process involving labeled components.
• A capture antibody (1) is used to bind to a target antigen.
• A primary antibody (2), specific for the antigen, binds to the antigen.
• A secondary antibody (3) then binds to the primary antibody. This secondary antibody is often enzyme-labeled.
• Enzyme-labeled detection antibody (3) binds to the target.
• Finally, a substrate reacts with the enzyme to produce a measurable signal (4).
• Fluorochromes are often used for signal generation.

Detection Methods: Absorption Spectroscopy

• Absorption spectroscopy measures the absorption of light as it passes through a solution.
• Electronic transitions are excited by ultraviolet (UV) or visible light.
• A monochromator is used to adjust the wavelength of light.
• The intensity of the transmitted light is measured by a detector.
• Beer-Lambert Law: Absorbance (A) is directly proportional to the concentration (C), the molar absorptivity (ɛ), and the path length (b). (A = εbc)
• Measurements are taken using a standard curve for quantification.

Enzyme Labels

• Horse Radish Peroxidase (HRP)

  • Generates colored, fluorescent, or luminescent products.
  • Uses a variety of hydrogen donors to reduce hydrogen peroxide.
  • Has a high catalytic rate.
  • Optimum pH range is 4.0-8.0.
  • Commonly used to detect HRP substrates such as ABTS, OPD or TMB.

• Alkaline Phosphatase

  • Generates colored, fluorescent, or luminescent signals.
  • Catalyses the hydrolysis of phosphate esters of primary alcohols, phenols or amines.
  • Inhibited by orthophosphate, zinc chelators & borate
  • Optimum pH range is 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)

• Common AP substrates

  • P-nitrophenyl phosphate
  • Indoxyl phosphate All common HRP substrates require peroxide for the reaction to occur.

Fluoroimmunoassay (FIA)

• Direct FIA: A fluorophore is directly attached to the antigen or antibody.
• Indirect FIA: The use of substrates producing fluorescent products is used as an alternative signal detection method.
• This method is based on binding reactions.

Stokes Shift

• Fluorophores absorb light at one wavelength and emit light at a longer wavelength.
• The difference between the excitation and emission wavelengths is the Stokes shift.
• A larger Stokes shift allows the emitted light to be easily measured without interference from incident light.

Fluorescent Labels

• Fluorophores: Molecules that absorb and emit light.
• Lanthanide Probes
  • Use metals from atomic numbers 57-70 for signal generation.
  • Exhibit long fluorescence lifetimes.
  • Have a large Stokes shift, with narrow emission peak.
  • Absorbance/emission times less than 1000 μs.

Time Resolved Fluorescent Immunoassays

• Time-resolved fluorescence measurements can improve signal-to-noise (S/N) ratios by measuring the fluorescence signal at longer delays after excitation.
• Improves sensitivity and specificity by generating signals at different delay times.

Competitive Assay: %B/Bo calculation

• Competitive assays measure the signal decrease with increasing analyte concentrations.
• The blank has the highest baseline signal strength.

This quiz covers the essential concepts of data handling, focusing specifically on signal generation methods used in assays. It delves into the multi-step process of using labeled components like antibodies and detection methods, especially absorption spectroscopy.