CHARACTERIZATION OF IMMUNOGLOBULIN-G BOUND TO LATEX-PARTICLES USING SURFACE-PLASMON RESONANCE AND ELECTROPHORETIC MOBILITY

The main objective of this work was the investigation of passive adsor ption and covalent coupling of a polyclonal IgG and a monoclonal prepa ration of IgG against HSA, to a carboxyl latex particle. The functiona l activity of the coupled protein was then assessed by quantitative im munoassays for the antigen. Sensitized particles, with different prote in coverage, were fully characterized using a range of different techn ologies, including electrophoretic mobility (mu(e)), photon correlatio n spectroscopy, and surface plasmon resonance (SPR). The antibody-labe led particles were studied with respect to electrokinetic behavior in pH and ionic strength titration, stability, antibody functionality, an d their performance in immunoaggregation reactions, Important differen ces were observed between the two sets of particle preparations throug hout the series of experiments. The differences could be attributed to the coupling of the IgG molecules to the particles by the two differe nt adsorption protocols. When proteins were chemically bound to the po lymer surface it was necessary to activate the carboxyl groups with a carbodiimide (CDI) moiety that in our case was positively charged. The differences in characteristics between the adsorbed and the coupled a ntibody particles are thought to be due to the fact that in the covale nt coupling protocol some CDI molecules remained linked to the particl es, which altered the average electrical state of the outer layer in c omparison with those samples where antibodies were physically adsorbed . On the other hand, the isoelectric point of the monoclonal antibody was lower (5.4 +/- 0.1) than the pI of the polyclonal antisera (6.9 +/ - 0.9), which could explain why the IgG-latex complexes created with m onoclonal molecules were colloidally more stable at neutral pH than th ose created with the polyclonal antisera. However, no immunoaggregatio n of antibody particles by the presence of antigen was found with the former. The use of SPR demonstrated that the equilibrium constants for the antibody-antigen recognition of the two antibody preparations wer e quite similar (K-A (polyclonal) (IgG) = 2.8 10(8) M-1; K-A (monoclon al) (IgG) = 9.5 10(7) M-1). These observations suggest that the lack o f aggregation mediated by antigen demonstrated by the monoclonal antib ody coupled to the latex particles may be due to this protein recogniz ing only one epitope in the HSA molecule. However, as the repulsive ch arge between antibody-latex particles counteracts the attractive force s between the antigen epitope and the antibody paratope, the greatest immunoaggregation was obtained when using latex particle-antibody comp lex with a low charge density (N) in the external layer. (C) 1998 Acad emic Press.