Abstract
Ab avidity is a measure of the overall strength of Ab–Ag interactions and hence is important for understanding the functional efficiency of Abs. In vaccine evaluations, Ab avidity measurements can provide insights into immune correlates of protection and generate hypotheses regarding mechanisms of protection to improve vaccine design to achieve higher levels of efficacy. The commonly used Ab avidity assays require the use of chaotropic reagents to measure avidity index. In this study, using real-time detection of Ab–Ag binding by biolayer interferometry (BLI) technique, we have developed a qualified assay for measuring avidity of vaccine-induced Abs specific for Plasmodium falciparum circumsporozoite protein (CSP) Ags. Human mAb derived from plasmablasts of recipients of RTS,S/AS01 (RTS,S), the most advanced malaria vaccine candidate, were used in the assay development to measure Ag-specific binding responses and rate constants of association and dissociation. The optimized BLI binding assay demonstrated 1) good precision (percentage of coefficient of variation <20), 2) high specificity, 3) a lower limit of detection and quantitation in the 0.3–3.3 nM range, and 4) a range of linearity up to 50–100 nM for the CSP Ags tested. Analysis of polyclonal sera of malaria vaccinees demonstrated the suitability of this method to distinguish among vaccinees and rank Ab responses by avidity. These results demonstrate that precise, specific, and sensitive BLI measurements of Ab avidity in polyclonal sera from malaria vaccinees can map Ab response heterogeneity and potentially help to determine the role of Ab avidity as an immune correlate of protection for vaccines.
Highlights
In this study, using realtime detection of Ab–Ag binding by biolayer interferometry (BLI) technique, we have developed a qualified assay for measuring avidity of vaccine-induced Abs specific for Plasmodium falciparum circumsporozoite protein (CSP) Ags
Experiments performed to address the parameters required for assay qualification demonstrated that the BLI avidity assay can be performed with good precision, specificity, limits of detection (LOD), and limits of quantitation (LOQ) in the 0.3–3.3 nM range, and a range of linearity up to 50–100 nM for the CSP Ags tested
The enhanced avidity of AB236 was due to a 17.7-fold increase in association rate of binding to full-length CSP (CSP-FL) than to PF16, whereas a 9.7-fold decrease in dissociation rate accounted for the stronger avidity of AB334 for CSP-FL
Summary
The assay was optimized further to enhance its sensitivity for measuring CSP Ag-specific binding responses and the dissociation rates of polyclonal serum Abs. Experiments performed to address the parameters required for assay qualification demonstrated that the BLI avidity assay can be performed with good precision (coefficient of variation [CV] ,20%), specificity, limits of detection (LOD), and limits of quantitation (LOQ) in the 0.3–3.3 nM range, and a range of linearity up to 50–100 nM for the CSP Ags tested. System suitability assessment performed as a part of the assay qualification showed that the BLI assay is capable of measuring vaccine-induced polyclonal serum Ab binding responses to CSP Ags and dissociation rates with good repeatability (CV ,20%).
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