Immunoassay standards and measurement of clinical biomarkers using intra-assay calibration standards
Inventors
Rhyne, Paul • Mapelli, Claudio • Wong, Oitak Allen • Berisha, Flora • Neely, Robert John
Assignees
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Publication Number
US-9482665-B2
Publication Date
2016-11-01
Expiration Date
Abstract
The present invention provides novel compositions and methods for creating quantitative standards to calibrate analytes. These compositions and methods enable the creation of standards and calibrators for analyzing analytes and measuring clinical biomarkers. Also provided are kits comprising the novel compositions for use in assays, for example sandwich immunoassays.
Core Innovation
The invention relates to improved Aβ42 and tau immunoassay reference standards and calibrators. It uses non-aggregating modified peptides or protein constructs that present N- and C-terminal immunoreactive regions connected by linkers or implemented with deletions and substitutions to remove aggregation domains. The modified Aβ42 peptides are described as isolated modified Aβ42 peptides consisting of an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 13, wherein the peptide is a non-aggregating peptide.
The invention addresses the problem that quantitative immunoassay measurements relying on Aβ42 reference standards are affected by aggregation and reduced solubility of full-length aggregated Aβ42. By providing non-aggregating modified Aβ42 peptides with immunoreactive regions arranged to minimize aggregation behavior, the reference standards are intended to support quantitative calibration using immunoassay standard curves. The document describes the use of immunoassay standard curves in sandwich ELISA/MSD formats and standard curve-based quantitation.
The invention further supports multiplexed intra-assay calibration using bead-based suspension arrays, including bead sets carrying different concentrations of reference standards. In the described intra-assay calibration approach, bead sets are used with suspension array formats such as Luminex xMAP, and the calibration approach supports quantitative measurement from multiplex signals. Performance rationale and characterization and validation described include aggregation minimization targets and monomeric behavior assessment, secondary-structure observations, and stability improvements reflected as reduced signal loss versus full-length Aβ42.
Claims Coverage
The partial content identifies one independent claim directed to a non-aggregating isolated modified Aβ42 peptide with specific sequence selections. Dependent claims narrow how the peptide is used as a reference standard in particular immunoassay formats and as part of an immunoassay detection kit structure.
Non-aggregating isolated modified Aβ42 peptide selected from SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 13
An isolated modified Aβ42 peptide consisting of an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 13, wherein the peptide is a non-aggregating peptide.
Reference standard immunoassay using the isolated modified peptide
The isolated, modified peptide is used as a reference standard in an immunoassay.
Immunoassay selected from sandwich, single antibody, double sandwich, or competition formats
The immunoassay that uses the isolated modified peptide is selected from sandwich immunoassay, single antibody assay, double sandwich immunoassay, or competition assay.
Immunoassay kit including the modified peptide, written instructions, and a container
An immunoassay kit for detecting Aβ42 peptide in a sample, including the peptide, written instructions, and a container holding the kit components.
Overall, the claim coverage centers on an isolated non-aggregating modified Aβ42 peptide defined by sequence selection (SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 13), and it extends to use of the peptide as an immunoassay reference standard in specified immunoassay formats, and in an immunoassay detection kit comprising instructions and containerized components.
Stated Advantages
Improved calibration performance for quantitative immunoassay standard curves by using non-aggregating reference standards rather than aggregated behavior.
Stability improvements indicated by reduced signal loss versus full-length Aβ42.
Reduced aggregation behavior and improved solubility and monomeric behavior as characterized by DLS and CD observations.
Documented Applications
Quantitative calibration and measurement in immunoassays for Aβ42 peptide, including sandwich immunoassay and formats such as ELISA/MSD using immunoassay standard curves.
Multiplex intra-assay calibration using bead-based suspension arrays, including Luminex xMAP, with bead sets carrying different concentrations of reference standards for quantitative measurement.
Immunoassay detection kit use for detecting Aβ42 peptide in a sample, including written instructions and kit components.
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