Labelling of colloidal gold with protein A. A quantitative study

Area of Science:

• Bioconjugation Chemistry
• Immunocytochemistry Reagents
• Nanoparticle Surface Chemistry

Background:

• Protein A-gold complexes are widely used secondary reagents in immunocytochemistry for antigen detection.
• Limited quantitative data exists regarding protein A adsorption onto gold nanoparticles, optimal preparation conditions, and complex stability.

Purpose of the Study:

• To quantitatively investigate the adsorption isotherm of protein A onto gold nanoparticles.
• To determine optimal conditions for preparing stable and effective protein A-gold complexes.
• To assess the stability and binding characteristics of these complexes.

Main Methods:

• Quantitative adsorption isotherm studies of 125I-labeled protein A onto 11.2 nm gold particles.
• Experiments conducted using gold sols buffered across a pH range of 4-7.
• Assessment of complex stability and binding to immobilized IgG.

Main Results:

• Adsorption was pH-independent at low protein A coverage.
• Highest protein A coverage was achieved at pH 5.1 (isoelectric point of protein A).
• Complexes were stable with up to 12 protein A molecules per particle, showing maximum binding to IgG with at least 9 molecules; high coverage (26 molecules/particle) led to protein A loss.

Conclusions:

• Optimal preparation of protein A-gold complexes involves buffering at pH 5.1 for maximal binding and stability.
• The number of adsorbed protein A molecules significantly impacts complex stability and binding efficiency.
• These findings provide crucial parameters for the reliable preparation of immunocytochemistry reagents.