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

Abstract

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 g/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.

Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle

“…5). Estimates of antibody avidity are dependent on the homogeneity of antibodies present in the sample (42). Given the diversity of antigens within the WCS-PK, it is likely that the estimate of antibody avidity for these complex antigens is less accurate than that for MPB83/MPB70 antigens, as responses to the various antigens in the WCS-PK may vary over the course of infection and in response to PPD injection.…”

Pattern of functional antibody activity against Haemophilus influenzae type b (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine

“…Avidity of anti-PRP IgG has been tested by ELISA-based elution assays (4,(19)(20)(21)(22)(23)(24), which have demonstrated that PRP conjugates elicit high-avidity antibodies, with increased functional activity (4,18).…”

“…IgG, IgM, and IgG subclasses of antibodies against PRP were analyzed by ELISA (3)(4)(5)(6). Briefly, each well of a microplate (Maxisorb; Nunc, USA) was coated with 0.4 µg/ mL PRP-tyramine antigen in 0.1 mL 0.05 M carbonate buffer, pH 9.6, overnight at 4°C.…”

“…However, the immunological response, which insures the improved immunogenicity and priming induced by conjugated vaccines, is still only partially understood. It is known that IgG and IgM antibodies against the capsular polysaccharide polyribosyl ribitol phosphate (PRP) are very important for the protective immunity to Hib, and that the covalent linkage of PRP to any protein is also important for the enhanced immunogenicity of glycoconjugated vaccines (2)(3)(4).…”

Novel Immunogenic Epitopes in the NaPi-IIb Protein: Production of Monospecific Antibodies Using Synthetic Peptides Outlined on Isoform Specific Regions of the Type IIb Sodium-Dependent Phosphate Transporter (NaPi-IIb)

“…The affinity of the anti-peptide antibodies was measured using ELISA as described above with the inclusion of a potassium thiocyanate (KSCN) elution step, according to the methodology previously described by Pullen et al (1986) and Romero-Steiner et al (2005) [53] [54]. After the serum incubation step, 100 µL of KSCN dilutions (0 to 5 M, with intervals of 0.5 M) in PBS were added to each well and incubated for 30 minutes at room temperature.…”