This research proposal will develop methods to quantitate the affinity of immunoglobulin G for polysaccharide antigens of Streptococcus pneumoniae. Dialyzable oligosaccharides will be prepared and purified for equilibrium dialysis. This technique measures the binding of a labelled dialyzable antigen to IgG separated by a dialysis membrane. Scatchard analysis is used to calculate the affinity, K, of the reaction, (Ag) + (Ab) yields (AgAb). Plotting data according to the Langmuir equation yields a measurement of total antibody content. Methods will be developed to measure phagocytic cell activation by IgG, including chemiluminescence, granule protein release, and also IgG binding to Fc receptors. The IgG used in these experiments will be well characterized IgG oligomers prepared by heat aggregation or by polymerization by dithiobis (succinimidylpropionate) with subsequent purification by gel filtration. The methods for assessing IgG function will be applied to a group of immunoglobulins prepared for intravenous use (IVIgG) to assess the effect of the method of preparation on IgG function. While intravenous immunoglobulins have been advocated for a number of clinical situations, their ability to function to the full extent as native IgG has not been convincingly shown. In addition, clinical results obtained with one preparation may not be extrapolated to another since these IVIgGs are prepared from a variety of enzymatic and chemically modifying methods. This project aims to establish in vitro standards by which different preparations of IVIgG may be compared, with the potential for correlation with clinical efficacy. These methods will also be used to examine the immune response to polysaccharide antigens and test the hypothesis that anti-pneumococcal IgG of subjects immunized with polyvalent pneumococcal polysaccharide is a homogeneous population with respect to affinity. Correlations will be drawn between antigen affinity and opsonic capacity.
| Hetherington, S V; Lepow, M L (1992) Correlation between antibody affinity and serum bactericidal activity in infants. J Infect Dis 165:753-6 |
| Hetherington, S (1990) Solid phase disruption of fluid phase equilibrium in affinity assays with ELISA. J Immunol Methods 131:195-202 |
| Hetherington, S V; Rutkowski, A F (1990) Antibody affinity in infants after immunization with conjugated capsular polysaccharide from Haemophilus influenzae type b. J Infect Dis 162:1185-8 |
| Hetherington, S V (1989) Antibody to the outer membrane proteins is the dominant opsonic antibody in normal human serum against H. influenzae type b. Immunology 67:87-91 |
| Hetherington, S V (1988) The intrinsic affinity constant (K) of anticapsular antibody to oligosaccharides of Haemophilus influenzae type b. J Immunol 140:3966-70 |
