17

Study Notes

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.

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.

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 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.

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.

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.

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 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.