Immunolabeling efficiency of protein A-gold complexes.
A systematic study of the adsorption of protein A on colloidal gold particles varying in size from 5-16 nm was performed at different protein concentrations. The number of protein A molecules bound per colloidal particle was evaluated and the Scatchard analysis of the adsorption parameters was applied for each size of the colloid. The binding of protein A to the colloidal gold surface exhibited the same affinity pattern for all of the particle sizes. At low concentrations of stabilizing protein, adsorption took place with high affinity (Kd 1.96-3.3 nM) and the maximum number of protein A molecules attached with this affinity correlated well with the surface of the particle. At higher concentrations of protein A, adsorption exhibited a significantly lower affinity (Kd 530-800 nM), and no saturation was recorded. Competition by albumin did not reveal a preferential removal of the "low-affinity" bound protein A molecules, contradicting the model of successive shells of stabilizing protein around the colloidal particle. The immunolabeling efficiency of conjugates having the same size of gold nucleus but carrying different numbers of protein A molecules was comparatively investigated by quantitative post-embedding immunocytochemistry. Protein A-gold formed with 5-10-nm colloids gave the highest intensity of labeling when carrying the maximum number of protein A molecules that could be adsorbed with high affinity. Overloading as well as underloading these complexes resulted in a significant decrease of their immunoreactivity. The most efficient conjugates were obtained when stabilization was performed with 6 micrograms protein A/ml gold sol of 5 and 10 nm particle diameter, and 15 micrograms protein/ml of 15-nm colloid.
Département d'Anatomie, Université de Montréal, Québec, Canada.
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Ghitescu
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Bendayan
D000818:Animals; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D006046:Gold / Q000378:metabolism* / Q000493:pharmacokinetics; D007150:Immunohistochemistry / Q000379:methods* / Q000592:standards; D008854:Microscopy, Electron / Q000379:methods; D010179:Pancreas / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D013205:Staphylococcal Protein A / Q000378:metabolism* / Q000493:pharmacokinetics
D013205:Staphylococcal Protein A; D006046:Gold