Highly sensitive immunoassay for rapid quantification of meningococcal capsular polysaccharide antigens
Inventors
MANOHAR, Kale Sameer • KESHAV, Sharma Pankaj • JAGDISHPRASAD, Gairola Sunil
Assignees
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Publication Number
US-10365272-B2
Publication Date
2019-07-30
Expiration Date
Abstract
The present disclosure relates to the field of immunoassays for Gram negative bacteria, in particular N. meningitidis. The disclosure provides a simple and affordable immunoassay to quantitate polysaccharides in meningococcal vaccines for the evaluation of antigen content and lot-to-lot manufacturing consistency. The inventors have found a Sandwich ELISA that can be applicable for the quantitation and identification of N. meningitidis serogroup X polysaccharide in a multivalent meningococcal polysaccharide-protein conjugate vaccine as well as in a multivalent meningococcal plain polysaccharide vaccine. Said assay employs a polyclonal antibody as capture antibody and a novel monoclonal antibody against serogroup X polysaccharide as detection antibody. Further the assay is rapid, robust and reproducible.
Core Innovation
The invention relates to a rapid, robust sandwich immunological assay for quantification of Neisseria meningitidis serogroup X capsular polysaccharide in a sample. The method uses a solid support coated with a capture polyclonal antibody, followed by application of a sample containing Neisseria meningitidis serogroup X capsular polysaccharide-TT conjugate and unconjugated Neisseria meningitidis serogroup X capsular polysaccharide. Unbound sample is removed, and then a detection monoclonal antibody is applied to the solid support for immunodetection.
The detection monoclonal antibody recognizes non-overlapping epitopes relative to the capture polyclonal antibody, supporting sandwich format quantification. After removing unbound detection monoclonal antibody, the method applies a labeled secondary antibody with a label and detects the label to quantify serogroup X capsular polysaccharide. The assay accommodates samples that are lyophilized and resuspended, or samples that are liquid and directly diluted, using phosphate buffer saline having pH 7.2 ± 0.2 comprising KCl at a concentration from 75 mM to 140 mM.
The assay is claimed to provide specificity and robustness in the presence of sucrose and glucose excipients and with respect to serogroup cross-reactivity. The assay does not show interference due to presence of sucrose and glucose, and the serogroup X monoclonal detection antibody does not cross-react with serogroups A/C/W/Y. The solid support can be selected from materials including polyvinyl chloride, polystyrene, agarose, or sepharose, and performance metrics include reported LOD and LOQ for serogroup X.
Claims Coverage
Independent claim clm-00001 provides the core coverage of a solid-phase sandwich immunological quantification of Neisseria meningitidis serogroup X capsular polysaccharide using a capture polyclonal antibody and a detection monoclonal antibody, followed by a labeled secondary antibody and label detection, together with explicit constraints on sample preparation and no interference from sucrose and glucose. Dependent claims refine the coverage with additional specificity and quantitative/performance limitations and with optional choices for assay components and labeling modality.
Solid support coated with capture polyclonal antibody for sandwich immunological quantification
Providing a solid support coated with a capture polyclonal antibody, and applying a sample to the solid support for sandwich immunological assay of Neisseria meningitidis serogroup X capsular polysaccharide, including removal of unbound sample and unbound detection monoclonal antibody prior to secondary detection.
Sample preparation in phosphate buffer saline (pH 7.2±0.2) with KCl (75 mM to 140 mM) and lyophilized/liquid options
Providing a sample in which lyophilized sample is resuspended in phosphate buffer saline having pH 7.2±0.2 comprising KCl at a concentration from 75 mM to 140 mM followed by further dilution, or wherein the sample is liquid and directly diluted with phosphate buffer saline having pH 7.2±0.2 comprising KCl at a concentration from 75 mM to 140 mM.
Detection using a detection monoclonal antibody followed by labeled secondary antibody and label detection
Providing a detection monoclonal antibody, applying the detection monoclonal antibody to the solid support, applying a labeled secondary antibody with a label, and detecting the label to quantify serogroup X capsular polysaccharide.
No interference due to sucrose and glucose excipients
Wherein the assay does not show any interference due to presence of sucrose and glucose excipients.
No cross-reactivity with serogroup polysaccharides A, C, W, and Y
The method wherein the assay does not show cross-reactivity of the detection monoclonal antibody with N. meningitidis serogroup polysaccharides A, C, W, and Y.
LOD constraint for serogroup X (0.4 ng/mL)
The method wherein the limit of detection (LOD) is 0.4 ng/ml for N. meningitidis X.
Specific solid-support materials (PVC, polystyrene, agarose, sepharose)
The method wherein the solid support is chosen from polyvinyl chloride (PVC), polystyrene, agarose, or sepharose.
Label selection for secondary antibody (enzyme, fluorescent, radioactive, or biotin)
The method characterized by selecting a label from the group of an enzyme label, fluorescent label, a radioactive label and biotin.
Across the independent-method core and its refinements, the claim set centers on a sandwich immunological quantification of Neisseria meningitidis serogroup X capsular polysaccharide using a capture polyclonal antibody and a detection monoclonal antibody on a solid support, with sample dilution/resuspension in PBS (pH 7.2±0.2) containing KCl (75–140 mM), labeled secondary antibody detection, and explicit claims that the assay has no interference from sucrose and glucose and no cross-reactivity with serogroups A/C/W/Y, alongside a reported LOD constraint and optional choices for solid support and label modality.
Stated Advantages
Assay does not show any interference due to presence of sucrose and glucose excipients.
Serogroup X detection using the monoclonal antibody does not cross-react with serogroups A/C/W/Y.
Provides quantified performance with a reported limit of detection (LOD) for serogroup X of 0.4 ng/mL.
Rapid assay characterization is described, including a total turnaround of about 1 day.
Documented Applications
Quantification of Neisseria meningitidis serogroup X capsular polysaccharide in samples including multivalent polysaccharide-protein conjugate and plain polysaccharide contexts, including pentavalent formulations containing A-TT, X-TT, C-CRM197, W-CRM197, and Y-CRM197.
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