Method for determination of members of the S100 family of calcium binding proteins by immunoturbidimetry
Inventors
ARMBRUSTER, Franz-Paul • GRIMMLER, Matthias • Schu, Pia • Becker, Tobias • Walzer, Felix
Assignees
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Publication Number
US-11714082-B2
Publication Date
2023-08-01
Expiration Date
Abstract
A method for measuring the presence of calprotectin (S100A8/A) heterodimer in a biological sample using a particle-enhanced turbidimetric immunoassay (PETIA) based on monoclonal antibodies. The method can be adapted on automated standard analyzers and provides a reliable clinical measurement of calprotectin in faecal samples and extracts. The method is comparable to commerical two-site sandwich ELISA. The disclosed method counters spontaneous agglutination caused by calcium ions and low-molecular weight calcium-binding S100 proteins as observed with conventional PETIAs. The method can be used for measuring the presence of human calprotectin in stool, urine, serum, plasma, synovial liquid and other body liquids. Metrological traceability and high commutability with conventional immunoassays (ELISA) has been shown despite of different measurement principles used.
Core Innovation
The invention relates to an in vitro particle-enhanced turbidimetric immunoassay (PETIA) and a test kit for quantitative measurement of calprotectin (S100A8/A9) in a biological sample of a patient. The assay uses latex particles carrying immobilized monoclonal antibodies that bind S100A8 and/or S100A9 or calprotectin (S100A8/A9), and the latex particle format supports acquisition of an optical property of the mixture to obtain a signal indicative of the content of calprotectin (S100A8/A9).
A central issue addressed is the solubilized presence of essentially heterodimeric calprotectin (S100A8/A9) in the biological sample when S100 family calcium-binding proteins undergo Ca2+-driven oligomerization/agglutination. The method extracts and solubilizes the sample in a first aqueous reagent-component having an organic buffer with a pH in the range 5.0 to 6.0 and an osmolality of at least 150 mosmol/kg, and the buffer is described as co-coordinating and sequestering calcium and zinc ions, thereby supporting a defined molecular state of calprotectin for subsequent immunoreaction.
The reagent composition and particle reaction are designed to counter unwanted latex and protein interactions. The first reagent-component includes salt and protease-treated serum albumin, and further includes an anionic surfactant such as sodium dodecyl sulfate, with optional beta-aldoses through hexoses and related carbohydrates to achieve an osmolality target. The second reagent-component provides latex particles of 150 to 350 nm diameter carrying immobilized monoclonal antibodies or fragments, and the mixture is incubated before determining the optical property used for calibrated quantification and clinical assessment.
Claims Coverage
The document provides one independent claim describing a method for measuring calprotectin (S100A8/A9) in a patient biological sample using a specified PETIA test kit. The claim includes two main inventive feature groups within the method steps.
Two-component particle-enhanced turbidimetric immunoassay test kit for calprotectin measurement
A test kit comprising a first aqueous reagent-component and a second reagent-component, wherein the first reagent-component contains an organic buffer with pH 5.0 to 6.0, salt of sodium, potassium or lithium, protease-treated serum albumin, and sodium dodecyl sulfate, optionally with beta-aldoses through hexoses or related carbohydrates to achieve an osmolality, and the second reagent-component comprises latex particles of 150 to 350 nm carrying immobilized monoclonal antibodies binding S100A8 and/or S100A9 or calprotectin (S100A8/A9).
Calcium and zinc co-coordinating and sequestering extraction to obtain essentially heterodimeric calprotectin
Extracting and solubilizing the biological sample in the first aqueous reagent-component having pH 5.0 to 6.0 and osmolality of at least 150 mosmol/kg, where the organic buffer co-coordinates and sequesters calcium and zinc ions, followed by homogenizing and extracting the matrix and removal of particulate material to obtain a sample solution with a solubilized presence of essentially heterodimeric calprotectin (S100A8/A9).
Latex nanoparticle antibody-antigen particle-bound reaction and optical quantification
Mixing the extracted sample solution with the second reagent-component to obtain a particle-bound antibody-antigen reaction with calprotectin (S100A8/A9) in a defined molecular state, incubating the mixture, acquiring an optical property of the mixture, determining a signal indicative of the content of calprotectin (S100A8/A9) based on the optical property, and relating the content to a calibrated control to assess the clinical condition of the patient.
The inventive coverage centers on a two-component PETIA test kit combining an acidic calcium/zinc co-coordinating and sequestering extraction reagent with latex particles carrying immobilized monoclonal antibodies, together with mixing, incubating, optical-property acquisition, calibration-based determination, and clinical assessment for calprotectin (S100A8/A9) in a patient sample.
Stated Advantages
Improved commutability versus ELISA.
Reduced negative absorbance and interference in stool.
Reduced nonspecific aggregation and enhanced metrological traceability.
Documented Applications
Quantitative measurement of calprotectin (S100A8/A9) in biological samples, notably faecal extracts, for assessing the clinical condition of a patient.
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