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Immunoassay Labels and Endpoints

• Immunoassays use labels to detect and quantify analytes.
• Fluorescent labels and enzyme labels are common choices.
• Fluorophore-conjugated antibodies enable detection via light emitted after excitation.
• Enzyme-conjugated antibodies produce detectable products by reacting with specific substrates

Learning Objectives

• Understanding the characteristics of horse radish peroxidase (HRP) and alkaline phosphatase (AP) enzymes is key.
• The chromogenic substrates of HRP and AP are crucial.
• Absorption/excitation and emission spectroscopy are important detection methods for fluorescent and colored labels.
• Knowing how fluorescent and time-resolved fluorescent detection methods work is essential.
• The definitions of Beer-Lambert Law and Stokes Shift are necessary for understanding immunoassays.

Enzyme Labels: Advantages and Disadvantages

• Advantages of enzyme labels include:
  • Low detection limits
  • Stable conjugates
  • Diverse product types (visible, colored, fluorescent, or luminescent) from neutral substrates.
• Disadvantages include:
  • Multi-stage processes (antibody-antigen binding, then detection signal generation)
  • larger sizes influencing diffusion rates.

Factors Affecting Enzyme Selection

• Key factors in enzyme selection include:
  • High catalytic turnover rates
  • Simplicity and sensitivity of assay methods.
  • Reactive groups for conjugation
  • Long-term stability
  • Retention of enzyme activity after conjugation

Horseradish Peroxidase (HRP) Details

• HRP is a 44 kDa glycosylated haemoprotein.
• Contains a protoporphyrin prosthetic group.
• 308 amino acids with 4 disulfide bridges.
• Has 6 lysine residues for conjugation.
• Acts as an oxidoreductase, using a variety of hydrogen donors to reduce hydrogen peroxide.
• Generates colored, fluorescent, or luminescent products.
• High catalytic rate.
• Optimum pH range is 4.0-8.0.

Alkaline Phosphatase (AP) Details

• AP is a 140 kDa dimeric glycoprotein.
• Contains multiple free amino groups for conjugation.
• Catalyzes the hydrolysis of phosphate esters in primary alcohols, phenols, and amines.
• Also known as ALP (alkaline phosphatase) or SEAP (secreted alkaline phosphatase)
• Inhibited by orthophosphate, zinc chelators, and borate.
• Optimum pH range is 9.5-10.5

Chromogenic Substrates

• Common HRP substrates are ABTS, OPD, and TMB. All need hydrogen peroxide.
• Common AP substrates are p-nitrophenyl phosphate and indoxyl phosphate.

Detection Methods: Absorption Spectroscopy

• Absorption spectroscopy measures changes in incident light's wavelengths using an excitation monochromator.
• The intensity of transmitted light is detected to get the absorbance.
• Beer-Lambert law relates absorbance to analyte concentration linearly. ( A = εbc)

Detection Methods: Fluorimetry

• Fluorometric ELISAs are more sensitive than colorimetric methods.
• Uses the same enzymes for detecting molecules
• Types of assays include: Direct (labeling directly to molecule) and Indirect (substrate produce the fluorescent products)
• Common fluorophores include Fluorescein isothiocyanate (FITC), Rhodamine, and Europium chelates.

Types of FIAs

• FIAs are fluorometric immunoassays.
• Indirect method uses two antibodies (secondary bonded to labeled fluorophore) to detect antigens
• Direct method uses a primary antibody which is a fluorophore labeled .

Fluorescent Labels

• Fluorescein isothiocyanate (FITC) has short absorbance/emission times (<1ns), and is hydrophobic.
• Short Stokes shift (<30nm).
• Sensitivity of assays is affected by background fluorescence.
• Lanthanide probes, rare earth metals with long fluorescence lifespans, have significant Stokes shift and narrow emission peaks.
• High absorptivity and Long term stability are advantages of this sort of labeling.

Stokes Shift

• Fluorophores absorb and emit light at differing wavelengths.
• The Stokes shift is the difference between excitation and emission wavelengths.
• A large Stokes shift prevents emitted light interference with incident light, thus improving detection.

Time-Resolved Fluorescent Immunoassays

• Uses lanthanide chelates: display large Stokes shift and longer decay times.
• Fluorescence decay measured after a specific delay.
• Short lived: decays in <1ms
• Long lived: longer decay times, with high sensitivity and zero background.
• High signal to noise ratios are attained by this method.

DELFIA

• DELFIA (Delayed Enhanced Lanthanide Fluor免疫測定) is a time-resolved fluorescent immunoassay system.
• Uses lanthanide chelates.

This quiz explores the key concepts related to immunoassay labels and endpoints, focusing on fluorescent and enzyme conjugates. It covers the features of horse radish peroxidase and alkaline phosphatase, as well as essential detection methods including spectroscopy and the Beer-Lambert Law. Test your understanding of these critical analytical techniques.