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Publication Number
US-11428686-B2
Publication Date
2022-08-30
Expiration Date
Abstract
Provided herein is technology relating to methods for assaying a cell-containing body sample and particularly, but not exclusively, to treating a sample under conditions to cause cell lysis, preferably by means of a detergent; and subjecting the lysed sample to conditions causing the cleavage of nucleic acid molecules. The technology additionally relates to using nucleic acid cleavage conditions to enhance a membrane assay, a device for carrying out such an assay, and a kit for use in the assay.
Core Innovation
The invention relates to membrane flow-through or immunoconcentration assays using a chamber accepting a cell-containing body fluid, in particular whole blood, in which detergent-mediated cell lysis is followed by nucleic acid cleavage to prevent unexpected rate-determining membrane blockage and high background signals caused by cellular nucleic acid from lysed white cells. It addresses that membrane assays can be adversely affected by DNA originating from lysed cells and by the resultant nonspecific background.
The core approach is to provide the device with a nuclease together with a small-pore membrane of pore size no greater than 10 μm carrying an immobilized specific binding agent for a non-nucleic acid analyte, while also including a second binding agent specific for the same analyte and conjugated to a signal generating moiety. By cleaving DNA under nuclease-mediated DNA degradation conditions, the assay flow is improved and non-specific binding and background are reduced, supporting faster and quantitatively accurate membrane-based assays for analytes such as C-reactive protein (CRP).
The document further includes nuclease formulations in dried form, including freeze-dried or lyosphere bead formats, to maintain nuclease functionality in the assay device or kit. Corresponding assay device and kit configurations are described as incorporating chamber(s), small-pore membrane(s), lysis agent, nuclease cleavage chemistry, binders, and signal-generating moieties, with example results indicating corrected CRP results and improved assay performance metrics.
Claims Coverage
The partial content provided includes one independent claim, covering an assay device with four inventive features: a chamber for cell-containing whole blood, a small-pore membrane, a nuclease in the device, and a second signal-generating conjugated binding agent specific for the same non-nucleic acid analyte.
Assay device for cell-containing whole blood with a small-pore membrane
A chamber for accepting a cell-containing body fluid; and at least one membrane of pore size no greater than 10 μm.
Nuclease included in the assay device
The device contains a nuclease.
Immobilized specific binding agent for a non-nucleic acid analyte on the membrane
The membrane comprises an immobilized specific binding agent that is specific for a non-nucleic acid analyte of a whole blood sample.
Second signal-generating conjugated binding agent
The device further comprises a second binding agent that is specific for said non-nucleic acid analyte and wherein said second binding agent is conjugated to a signal generating moiety.
Overall, the claim coverage focuses on integrating a nuclease into a membrane immunoconcentration/flow assay device that uses a small-pore membrane and whole-blood compatible binding chemistry, while employing two binding agents specific for a non-nucleic acid analyte.
Stated Advantages
Prevents unexpected rate-determining membrane blockage caused by cellular nucleic acids from lysed white cells.
Reduces high background signals caused by nucleic acids from lysed cells.
Improves flow through the membrane-based assay.
Reduces non-specific binding/background.
Enables faster and quantitatively accurate CRP membrane assays.
Improves assay performance metrics, including corrected CRP results.
Documented Applications
Membrane flow-through/immunoconcentration assays on cell-containing samples, notably whole blood.
Point-of-care diagnostic assays for C-reactive protein (CRP), including use in cartridge-based formats for automated analysers.
Assay device and kit configurations incorporating chamber(s), small-pore membrane, lysis agent, nuclease cleavage chemistry, binders, and signal-generating moieties.
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