F. Caruso et al., INVESTIGATION OF IMMUNE-REACTIONS IN A FLOW-INJECTION SYSTEM USING SURFACE-PLASMON RESONANCE, Colloids and surfaces. A, Physicochemical and engineering aspects, 103(3), 1995, pp. 147-157
The effect of flow rate on the binding of polyclonal sheep immunoglobu
lin G (IgG) and anti-sheep IgG to gold, and to immobilized anti-IgG an
d IgG receptor layers, respectively, has been studied using the optica
l technique of surface plasmon resonance at a gold-solution interface.
The kinetics of protein binding were investigated by monitoring the r
eflectivity of light at a fixed angle close to the plasmon resonance.
Fixed-angle reflectivity data are presented in terms of protein surfac
e coverage. IgG and anti-IgG effective thicknesses were determined by
fitting the full angle-dependent reflectivity data to Fresnel theory.
The effective thicknesses of IgG and anti-IgC bound to the gold surfac
e and to each other (as pre-adsorbed layers) were found to be independ
ent of the flow rate of protein solution. Gold-adsorbed IgG and anti-I
gG layers were found to be 63 +/- 15 Angstrom and 68 +/- 10 Angstrom t
hick, respectively, and 91 +/- 11 Angstrom (anti-IgG) and 20 +/- 7 Ang
strom (IgG) thick when subsequently bound to their biospecific partner
. The differences in the thickness values between the gold-adsorbed an
d protein-adsorbed layers are explained in terms of the orientation of
the immobilized proteins and their deformation upon binding to the go
ld surface. Non-specific binding between immobilized sheep IgG layers
and anti-mouse IgG was shown to be negligible.