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Clinical and Diagnostic Laboratory Immunology, September 2005, p. 1029-1035, Vol. 12, No. 9
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.9.1029-1035.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Avidity Determinations for Haemophilus influenzae Type b Anti-Polyribosylribitol Phosphate Antibodies

Sandra Romero-Steiner,^1,* Patricia F. Holder,^1, Patricia Gomez de Leon,² Willie Spear,^1, Thomas W. Hennessy,³ and George M. Carlone¹

Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia,¹ School of Medicine, Universidad Nacional Autónoma de México, Mexico City, Mexico,² Arctic Investigations Program, Centers for Disease Control and Prevention, Anchorage, Alaska³

Received 24 March 2005/ Returned for modification 28 April 2005/ Accepted 17 June 2005

Determination of antibody avidity measurements can be difficult in human serum depending on the population evaluated. We evaluated three approaches for the determination of antibody avidity for immunoglobulin G (IgG). These approaches were (i) elution of bound antibody with increasing concentrations of a chaotropic agent using a single serum dilution, (ii) binding interference of multiple serum dilutions by a single concentration of a chaotrope, and (iii) elution of multiple serum dilutions by a single concentration of a chaotrope. Parameters that affect the determination of avidity measurements and their limitations were evaluated with pre- and post-Haemophilus influenzae type b conjugate vaccination sera (n = 89). We determined that elution of low-avidity antibodies present in multiple dilutions of the serum sample by a single concentration of a chaotrope (0.15 M sodium thiocyanate [NaSCN]) was optimal for the determination of avidity measurements throughout a wide range of IgG concentrations (0.94 to 304.6 µg/ml). The percent reduction in concentration as determined by the elution assay with 0.15 M NaSCN correlated highly (r = 0.84) with weighted averages obtained by an elution assay with multiple solutions of NaSCN. The correlation (r = 0.57) between elution and binding interference, when a single concentration of a chaotrope was used, was lower than the correlation between the two elution methods (r = 0.84). We found that the serum dilution, the heterogeneity of the antibody population, and the concentration of the chaotrope were the primary variables affecting avidity determinations. In this study, we present multiple analysis methods depending on the methodology used. We also present the factors that affect the analysis of avidity determinations given the polyclonal nature of human sera. This experimental approach should benefit the evaluation of similar antibodies induced by other bacterial polysaccharide vaccines.

S.R.-S. and P.F.H. contributed equally to this study.

Present address: Emory University, Department of Pulmonary Medicine, Room 215, Whitehead Building, 615 Michael St., Atlanta, GA 30322.