Method for the detection and quantitation of biomarkers
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-9903868-B2
Publication Date
2018-02-27
Expiration Date
2035-05-18
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Abstract
The invention provides a method for detecting the presence or absence of a biomarker in a biological sample at a very low concentration comprising the steps of (a) contacting the biological sample with a capture binding sequence immobilized on a surface, (b) providing a conjugate comprising a detection binding sequence-glucose oxidase, (c) contacting the surface with the detection binding sequence-glucose oxidase conjugate, (d) separating any unbound detection binding sequence-glucose oxidase conjugate from the surface, (e) incubating the resulting surface with a glucose solution and a mixture comprising gold nanoparticles and a gold salt, wherein the gold nanoparticles have an initial particle size of about 5 nm, and (f) observing any change in color of the mixture. The invention also provides a method for diagnosing the presence of a prostate cancer biomarker in a subject and a kit for detecting or quantifying a biomarker in a biological sample.
Core Innovation
The invention provides a method for detecting the presence or absence of a biomarker in a biological sample at very low concentration. The method comprises contacting the biological sample with a capture binding sequence immobilized on a surface, providing a detection binding sequence conjugated to glucose oxidase, contacting the surface with this conjugate, separating any unbound conjugate from the surface, incubating the surface with a glucose solution and a mixture comprising gold nanoparticles (about 5 nm in size) and a gold salt, and then observing any change in color of the mixture. The method is also applied specifically for diagnosing the presence of a prostate cancer biomarker in a subject and includes a kit for detecting or quantifying a biomarker.
The problem being solved is the inability of existing quantitative immunoassays, such as ELISA, to detect cancer biomarkers at clinically relevant low concentrations, especially in the early disease stages where biomarkers are in the fg/mL to pg/mL range. While gold nanoparticle-based colorimetric immunoassays provide high sensitivity with easily distinguishable color changes, existing assays lack the ability to quantify biomarkers across a wide linear detection range. There is thus an unmet need for an ultrasensitive and quantitative immunoassay.
The inventive method achieves ultrasensitive detection and quantitation by utilizing two rounds of signal amplification: first, magnetic beads loaded with many glucose oxidase molecules are conjugated with detection antibodies, and second, the glucose oxidase catalyzes glucose oxidation to produce hydrogen peroxide which induces growth of gold nanoparticles. This changes the solution color from colorless to red, detectable by the naked eye or spectrophotometrically. The method exhibits an attomolar detection limit and a linear detection range from 10 to 105 fg/mL. It is suitable for point-of-care diagnostics due to the easily observable color change.
Claims Coverage
The patent includes three independent claims detailing methods for detecting prostate specific antigen (PSA) using conjugated antibodies and gold nanoparticle-based colorimetric assays.
Detection of PSA using monoclonal anti-human PSA antibodies and glucose oxidase-conjugated nanoparticles
A method comprising contacting a biological sample with a monoclonal primary anti-human PSA antibody immobilized on a surface, providing nanoparticles conjugated with a secondary anti-human PSA antibody and glucose oxidase, contacting the surface with the nanoparticles, separating unbound nanoparticles, incubating the surface with glucose and a mixture of gold nanoparticles (~5 nm) and a gold salt, and observing a change in absorbance at ~530 nm.
Use of dibenzocyclooctyl-conjugated detection binding sequences
The detection binding sequence in the method can be conjugated to dibenzocyclooctyl, enhancing binding specificity and conjugation efficiency.
Diagnosis of PSA presence using magnetic beads conjugated with detection antibody and glucose oxidase
A method comprising contacting a biological sample with a capture antibody immobilized on a surface, providing magnetic beads conjugated with a secondary anti-human PSA antibody and glucose oxidase, contacting the surface with the magnetic beads, separating unbound beads, incubating with glucose and a mixture of gold nanoparticles (~5 nm) and gold salt, and observing a change in absorbance at ~530 nm.
Use of specific monoclonal antibody clones and polystyrene surface
A method specifying the capture antibody as the mouse monoclonal CHYH1 clone immobilized on a polystyrene surface, and the detection antibody as the CHYH2 clone conjugated to magnetic beads with glucose oxidase, following similar incubation and detection steps for PSA.
The claims cover methods that utilize monoclonal antibodies and glucose oxidase conjugated to nanoparticles or magnetic beads to bind PSA, with subsequent colorimetric detection based on growth of gold nanoparticles caused by hydrogen peroxide produced through enzymatic reaction. The inventive features focus on the use of specific antibody conjugations, nanoparticle sizes, and detection methods to attain ultrasensitive PSA detection.
Stated Advantages
The method exhibits a limit of detection in the attomolar range, exceeding commercial ELISA sensitivity by more than four orders of magnitude.
It provides a wide linear detection range from 10 to 105 fg/mL, enabling quantitative biomarker measurement.
The color change from colorless to red is easily distinguishable by the naked eye, suitable for point-of-care diagnostics.
Documented Applications
Detection and quantitation of prostate specific antigen (PSA) as a prostate cancer biomarker in biological samples such as blood and serum.
Diagnosis of prostate cancer in subjects by detecting PSA using the described immunoassay.
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