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WO2018140719A1 - Immunodosage à base de particules magnétiques et ses procédés d'utilisation - Google Patents

Immunodosage à base de particules magnétiques et ses procédés d'utilisation Download PDF

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Publication number
WO2018140719A1
WO2018140719A1 PCT/US2018/015440 US2018015440W WO2018140719A1 WO 2018140719 A1 WO2018140719 A1 WO 2018140719A1 US 2018015440 W US2018015440 W US 2018015440W WO 2018140719 A1 WO2018140719 A1 WO 2018140719A1
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WO
WIPO (PCT)
Prior art keywords
reporter
sample
analyte
interest
biomarker
Prior art date
Application number
PCT/US2018/015440
Other languages
English (en)
Inventor
Andrew Ward
Arun Richard CHANDRASEKARAN
Daniel Chen
Christopher Blanchard
Padric GARDEN
Bradley DEMARCO
Joshua Forman
Mounir A. KOUSSA
Lisa CALDWELL
Original Assignee
Confer Health, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Confer Health, Inc. filed Critical Confer Health, Inc.
Priority to CN201880014031.9A priority Critical patent/CN110462383A/zh
Priority to US16/479,827 priority patent/US20210389313A1/en
Priority to EP18744906.1A priority patent/EP3574309A4/fr
Priority to CA3051475A priority patent/CA3051475A1/fr
Publication of WO2018140719A1 publication Critical patent/WO2018140719A1/fr
Priority to US16/750,629 priority patent/US20200158721A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • the invention described herein relates generally to improved methods, assays, and kits for detecting analytes in biological samples.
  • Analyte detection has various clinical and non-clinical applications in industries ranging from medicine and biological research to environmental science and beyond.
  • Traditional methods for analyte detection involve assays such as enzyme-linked immunosorbent assays (ELISA), mass spectrometry, and high pressure liquid chromatography (HPLC). While HPLC and mass spectrometry may be used to detect analytes on the basis of charge and/or size, ELISA may be used to detect an analyte based on antigens on the analyte that are recognizable by capture and detection agents (e.g., antibodies, aptamers, etc.). In particular, ELISA assay has become a relatively common detection method utilized in the life sciences.
  • capture and detection agents e.g., antibodies, aptamers, etc.
  • Immunoassays kits including such assays, and methods of using the same to detect the presence, absence, or level of an analyte of interest in a sample are described herein.
  • the invention relates to systems and methods for detecting analytes, e.g. antigens from biological samples, with improved sensitivity than presently available methods, for instance relying on antibody-based detection of an antigen of interest, e.g. one that is useful for correlating with an individual's health state, with magnetic-mediated separation.
  • analytes e.g. antigens from biological samples
  • antibody-based detection of an antigen of interest e.g. one that is useful for correlating with an individual's health state
  • magnetic-mediated separation e.g.
  • the immunoassays, kits, and methods described herein provide considerable advantages over immunoassays in the field. Indeed, in some embodiments, the invention described herein includes a lack of sample preparation that is not found in known immunoassays. Current methods in the field require significant sample preparation.
  • samples may be combined with the necessary reagents, which include a magnetic conjugate and a reporter, or reporter conjugate, in an analysis chamber and a magnetic field may be applied as a "pull down" step, followed by visualization and/or quantification of the reporter.
  • necessary reagents which include a magnetic conjugate and a reporter, or reporter conjugate
  • the invention includes a method for detecting the presence, absence, or level of an analyte of interest in a sample.
  • the sample may be a bodily fluid, as defined herein.
  • the methods of the invention may include the step of adding sample to an analysis chamber.
  • adding sample to the analysis chamber may include delivering the sample to a sample collector (e.g., an absorbent or wicking material) in fluid communication with the analysis chamber.
  • the sample collector may then feed the sample into the analysis chamber.
  • the methods of the invention may include contacting the sample with a magnetic conjugate comprising a magnetic particle and a capture moiety configured to bind the analyte of interest in the sample.
  • the magnetic conjugate may be disposed at the sample collector and the step of contacting the sample with the magnetic conjugate may occur at the sample collector.
  • the magnetic conjugate may be imbedded in a portion of the sample collector before a sample is added to the sample collector.
  • the methods of the invention may include contacting the sample in the analysis chamber with a magnetic conjugate.
  • the capture moiety is an antibody, an antigen-binding fragment, an antigen, a receptor, a ligand, an aptamer, an aptamer receptor, a nucleic acid, or a small molecule.
  • the capture moiety is a capture antibody.
  • the magnetic conjugate comprises a magnetic particle and a capture antibody.
  • the methods described herein may include the step of contacting the sample with a reporter or a reporter conjugate comprising a reporter and a reporter binding moiety configured to bind the analyte of interest in the sample.
  • the reporter or reporter conjugate may be disposed at the sample collector and the step of contacting the sample with the reporter or reporter conjugate may occur at the sample collector.
  • the reporter or reporter conjugate may be imbedded in a portion of the sample collector before a sample is added to the sample collector.
  • the methods described herein may include the step of contacting the sample in the analysis chamber with a reporter or a reporter conjugate.
  • the reporter binding moiety is an antibody, an antigen-binding fragment, an antigen, a receptor, a ligand, an aptamer, an aptamer receptor, a nucleic acid, or a small molecule.
  • the reporter binding moiety is a reporter antibody.
  • the reporter conjugate comprises a reporter and a reporter antibody.
  • the methods described herein may include the step of binding the analyte of interest with the capture antibody and the reporter antibody.
  • the methods described herein may include the step of separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber.
  • the methods described herein may include the step of detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • the reporter may include a metal core (e.g., a metal microparticle or metal nanoparticle) and may include a silica shell.
  • the reporter includes a metal core and has a silica shell.
  • the reporter includes a plurality of quantum dots.
  • the reporter includes a metal core with a silica shell and the silica shell is impregnated with a plurality of quantum dots.
  • the reporter includes a metal core and the metal core may comprise, or another metal as described herein.
  • the reporter described herein may be a fluorescent reporter, a phosphorescent reporter, or a colorimetric reporter such as a colored particle that may be configured to measure absorbance or scattering of light (or, for example, the presence/absence of a certain color by colorimetric analysis).
  • the methods described herein may further include the step of concentrating the analyte of interest in the sample by applying a magnetic field to the analysis chamber after contacting the sample with the magnetic conjugate; and then reducing the volume of the sample in the analysis chamber.
  • the method described herein may further include the step of deactivating the magnetic field before contacting the sample with the reporter conjugate.
  • the methods described herein may further include the step of concentrating the analyte of interest in the sample by applying a magnetic field to the analysis chamber after contacting the sample with the magnetic conjugate; removing a volume of the sample from the analysis chamber; and adding a volume of buffer and/or an additional volume of the sample to the analysis chamber.
  • the methods described herein further include the step of deactivating the magnetic field before contacting the sample with the reporter conjugate.
  • the reporter antibody described herein is labeled with biotin. In some embodiments, the reporter described herein is functionalized with streptavidin. In some embodiments, the reporter antibody described herein is labeled with streptavidin. In some embodiments, the reporter described herein is functionalized with biotin.
  • the analyte of interest described herein may be any of the analytes and/or biomarkers described herein.
  • the analyte of interest may be selected from human chorionic gonadotropin (hCG), luteinizing hormone (LH)ZLutropin, prostate specific antigen (PSA), herpes simplex virus (HSV) antibodies, estrone-3-glucuronide (E3G), bacteria, hemoglobin AIC, C-reactive protein, an inflammation biomarker, troponin, lyme disease antigen, lyme disease antibodies, an LDL biomarker, an HDL biomarker, a total cholesterol biomarker, thyroid stimulating hormone, a hepatitis C virus biomarker, a rhino virus biomarker, an influenza virus biomarker, a liver function biomarker, estrogen, progesterone, lactic acid, and combinations thereof.
  • hCG human chorionic gonadotropin
  • LH luteinizing hormone
  • PSA prostate specific antigen
  • the bacteria may be Streptococcus- A, Chlamydia, and/or Gonorrhea.
  • the inflammation biomarker may be CRP, SAA, and/or MP8.
  • the liver function biomarker may be ALT and/or AST.
  • the analyte of interest may be selected from the group consisting of an ovulation biomarker, a pregnancy biomarker, a strep throat biomarker, a prostate cancer biomarker, a herpes biomarker, a diabetes biomarker, an inflammation biomarker, a heart attack biomarker, a chlamydia biomarker, a bacteria biomarker, a lyme disease biomarker, a cholesterol biomarker, a hypothydroidism biomarker, a hepatitis C biomarker, a rhino virus biomarker, an influenza biomarker, a liver function biomarker, a fertility biomarker, a muscle fatigue biomarker, and combinations thereof.
  • an ovulation biomarker a pregnancy biomarker, a strep throat biomarker, a prostate cancer biomarker, a herpes biomarker, a diabetes biomarker, an inflammation biomarker, a heart attack biomarker, a
  • an ovulation biomarker may be derived from a urine, blood, or serum based sample.
  • a pregnancy biomarker may be derived from a urine or blood based sample.
  • a strep throat biomarker may be derived from a saliva based sample.
  • a saliva based sample may be an aliquot of saliva, a cheek swab, or a throat swab.
  • a prostate cancer biomarker may be derived from a blood, serum, or urine based sample.
  • a herpes biomarker may be derived from a blood or saliva derived sample.
  • the methods described herein may detect the presence, absence, or level of an analyte of interest in a sample.
  • the methods described hrein may include the steps of contacting a sample with a magnetic conjugate comprising a magnetic particle and a capture moiety configured to bind the analyte of interest in the sample; contacting the sample with a reporter conjugate comprising a reporter and a reporter binding moiety configured to bind the analyte of interest in the sample; binding the analyte of interest with the capture moiety and the reporter binding moiety; separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber; and/or detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • the methods described hrein may include the steps of contacting the sample with a reporter conjugate comprising a reporter and a reporter binding moiety configured to bind the analyte of interest in the sample; contacting a sample with a magnetic conjugate comprising a magnetic particle and a capture moiety configured to bind the analyte of interest in the sample; binding the analyte of interest with the capture moiety and the reporter binding moiety; separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber; and/or detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • the methods described herein may include the steps of contacting a sample with a magnetic conjugate comprising a magnetic particle and a capture moiety configured to bind the analyte of interest in the sample; binding the analyte of interest with the capture moiety; separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber to pull down the magnetic conjugates with analyte of interest associated therewith; contacting the sample with a reporter conjugate comprising a reporter and a reporter binding moiety configured to bind the analyte of interest in the sample; binding the analyte of interest with the reporter binding moiety; separating the analyte of interest with reporter binding moiety bound thereto from the sample by applying a magnetic field to the analysis chamber; and/or detecting the presence, absence, or level of the analyte of interest by detecting the reporter with a light source and photodetector.
  • the methods described herein may include the steps of contacting a sample with a reporter conjugate comprising a reporter and a reporter binding moiety configured to bind the analyte of interest in the sample; binding the analyte of interest with the reporter binding moiety; contacting the sample with a magnetic particle-labeled analyte configured to bind the reporter conjugate in the absence of the analyte of interest in the sample; separating the magnetic particle-labeled analyte from the sample by applying a magnetic field to the sampe; and/or detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • the methods described herein may include the steps of contacting a sample with a magnetic conjugate comprising a magnetic particle and a capture moiety configured to bind the analyte of interest in the sample; binding the analyte of interest with the capture moiety; contacting the sample with a reporter binding moiety comprising a biotin label configured to bind the analyte of interest in the sample; contacting the sample with a reporter comprising a streptavidin label configured to bind the biotin label; separating the analyte of interest from the sample by applying a magnetic field to the sample; and/or detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • the methods described herein may include the steps of contacting a sample with a magnetic conjugate comprising a magnetic particle and a capture moiety configured to bind the analyte of interest in the sample; binding the analyte of interest with the capture moiety; contacting the sample with a reporter binding moiety comprising a streptavidn label configured to bind the analyte of interest in the sample; contacting the sample with a reporter comprising a biotin label configured to bind the streptavidin label; separating the analyte of interest from the sample by applying a magnetic field to the sample; and/or detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • the methods described herein may include the steps of adding sample to an analysis chamber; contacting the sample with a magnetic conjugate comprising a magnetic particle and a capture antibody configured to bind the analyte of interest in the sample; binding the analyte of interest with the capture antibody; separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber to pull down the magnetic conjugates with analyte of interest associated therewith; contacting the sample with a reporter conjugate comprising a reporter and a reporter antibody configured to bind the analyte of interest in the sample; binding the analyte of interest with the reporter antibody; and detecting the presence, absence, or level of the analyte of interest by detecting the reporter with a light source and photodetector.
  • the methods described herein may be performed on a negative sample (i.e., a sample that does not include the analyte of interest) and thereby determine the absence of the analyte of interest in the sample.
  • a negative sample i.e., a sample that does not include the analyte of interest
  • the methods described herein may include the steps of adding sample to an analysis chamber; contacting the sample with a magnetic conjugate comprising a magnetic particle and a capture antibody configured to bind the analyte of interest in the sample; binding the analyte of interest with the capture antibody; contacting the sample with a reporter- labeled analyte configured to bind the magnetic conjugate in the absence of the analyte of interest in the sample; separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber; and detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • the methods described herein may include the steps of adding sample to an analysis chamber; contacting the sample with a reporter conjugate comprising a reporter and a reporter antibody configured to bind the analyte of interest in the sample; binding the analyte of interest with the reporter antibody; contacting the sample with a magnetic particle- labeled analyte configured to bind the reporter conjugate in the absence of the analyte of interest in the sample; separating the magnetic particle-labeled analyte from the sample by applying a magnetic field to the sample; and detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • the methods described herein may include the steps of adding sample to an analysis chamber; contacting the sample with a magnetic conjugate comprising a magnetic particle and a capture antibody configured to bind the analyte of interest in the sample; binding the analyte of interest with the capture antibody; contacting the sample with a reporter antibody comprising a biotin label configured to bind the analyte of interest in the sample; contacting the sample with a reporter comprising a streptavidin label configured to bind the biotin label; separating the analyte of interest from the sample by applying a magnetic field to the sample; and detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • contacting step e.g., contacting the sample with a magnetic conjugate, a reporter antibody, a reporter-labeled conjugate, and/or a reporter conjugate
  • contacting step may include incubating the sample, which may contain an analyte, with the respective magnetic conjugate, reporter antibody, reporter-labeled conjugate, and/or reporter conjugate for a selected period of time and a selected temperature.
  • FIGS. 1 A to ID illustrate exemplary features of a sandwich mode for an immunoassay described herein where antigen is present or absent.
  • FIG. 1 A to ID illustrate exemplary features of a sandwich mode for an immunoassay described herein where antigen is present or absent.
  • FIG. 1A illustrates exemplary parameters for the sandwich mode: (a) analyte of interest (e.g., hCG); (b) Antibody #1 that is specific to the analyte of interest (e.g., MoLogic Anti-hCG 4F9); (c) Antibody #2 that is specific to the analyte of interest (e.g., Medix Anti-hCG 5011); (d) Antibodies 1 and 2 are able to simultaneously bind to the analyte as shown here; (e) fluorescent reporter; and (f) magnetic particle. In the presence of analyte, a complex will form that is both labeled and can be attracted to a magnet.
  • FIG. IB illustrates the addition of magnetic conjugate and reporter conjugate.
  • FIG. 1C illustrates the magnetic pulldown.
  • FIG. ID illustrates the detection of the analyte.
  • FIGS. 2 A to 2F illustrate exemplary features of a separate addition mode for an immunoassay described herein where antigen is present or absent.
  • FIG. 2A illustrates exemplary parameters for the separate addition mode: (a) analyte of interest (hCG); (b) Antibody #1 that is specific to the analyte of interest (e.g., MoLogic Anti-hCG 4F9); (c) Antibody #2 that is specific to the analyte of interest (e.g., Medix Anti-hCG 5011); (d) Antibodies 1 and 2 are able to simultaneously bind to the analyte as shown here; (e) fluorescent reporter; and (f) magnetic particle.
  • FIG. 2B illustrates the addition of magnetic conjugate.
  • FIG. 2C illustrates a magnetic pulldown.
  • FIG. 2D illustrates the addition of reporter conjugate.
  • FIG. 2E illustrates a second magnetic pulldown.
  • FIG. 2F illustrates the detection of the analyte.
  • FIGS. 3A to 3E illustrate exemplary features of a competitive mode for an immunoassay described herein where antigen is present or absent.
  • FIG. 3 A illustrates exemplary parameters for the competitive mode: (a) Analyte of interest; (b) Antibody that is specific to the analyte of interest; (c) fluorescent reporter; and (d) magnetic particle.
  • the binding site on the antibody remains available, thus allowing the formulation of a complex between a magnetic particle and a reporter.
  • a free antigen can block the binding site on the antibody, thus preventing formation of a complex with the magnet particle and a reporter.
  • FIG. 3B illustrates the addition of magnetic conjugate.
  • FIG. 3C illustrates the addition of reporter-labeled analyte.
  • FIG. 3D illustrates a magnetic pulldown.
  • FIG. 3E illustrates the detection of the analyte.
  • FIG. 4A to 4G illustrate exemplary features of a tertiary mode for an immunoassay described herein where antigen is present or absent.
  • FIG. 4A illustrates exemplary parameters for the tertiary mode: (a) Analyte of interest; (b) Antibody #1 that is specific to the analyte of interest and labeled with a biotin; (c) Antibody #2 that is specific to the analyte of interest; (d) Antibodies 1 and 2 are able to simultaneously bind to the analyte as shown here; (e) fluorescent reporter functionalized with streptavidin; and (f) magnetic particle. In the presence of analyte, a complex will form with a magnetic particle labeled biotin.
  • FIG. 4B illustrates the addition of magnetic conjugate.
  • FIG. 4C illustrates a magnetic pulldown.
  • FIG. 4D illustrates the addition of a biotin labeled antibody.
  • FIG. 4E illustrates the addition of streptavidin labeled reporter.
  • FIG. 4F illustrates a second magnetic pulldown.
  • FIG. 4G illustrates the detection of the analyte.
  • FIG. 5 illustrates the sensitivity of an assay of the invention for detecting Human Chorionic Gonadotropin (hCG).
  • HCG STAT cobas e 601 module, cobas e 602 module.
  • 1 fM 10 "15 moles/liter.
  • FIG. 7 illustrates the sensitivity of an assay of the invention for detecting Prostate Specific Antigen (PSA).
  • PSA Prostate Specific Antigen
  • FIGS. 8 A to 8D illustrate exemplary features of a bacteria detecting mode for an immunoassay described herein where bacteria is present or absent.
  • FIG. 8A illustrates exemplary parameter for the bacteria detecting mode: (a) Bacterium of interest; (b) Antibody #1 that is specific to an analyte on the surface of the bacterium; (c) Antibody #2 (may be the same as Antibody #1) that is specific to an analyte on the surface of the bacterium; (d) fluorescent reporter; and (e) magnetic particle. Many labeled antibodies can bind to a single bacterium at once resulting in a bacterium that can be attracted to a magnet and is fluorescently labeled.
  • FIG. 8B illustrates the addition of magnetic conjugate and reporter conjugate.
  • FIG. 8C illustrates a magnetic pulldown.
  • FIG. 8D illustrates the detection of the analyte.
  • FIG. 9 illustrates the sensitivity of an assay of the invention for detecting Group A Strep.
  • FIG. 10 illustrates the detection of E3G in an exemplary competitive mode immunoassay of the invention.
  • FIG. 11 illustrates a correlation between measurements of PSA for an immunoassay described herein compared to those performed in a clinical laboratory.
  • FIG. 12 illustrates a typical test of hCG in urine samples as measured by an immunoassay described herein.
  • FIG. 13 illustrates a plot quantifying the results from FIG. 12.
  • FIG. 14 illustrates a stair-step curve showing the fraction positive as a function of days post ovulation.
  • An immunoassay as described herein i.e., Confer Magneto
  • First Response is shown in pink.
  • Confer Magneto is the top curve
  • First Response is the bottom curve.
  • FIG. 15 illustrates a summary of the pregnancy data showing the percent of samples that read positive for a given number of days before First Response.
  • FIG. 16 illustrates a cis excitation method using a dichroic mirror arrangement with a 405 nm laser excitation source.
  • Dichroic Mirror Thor Labs, DMLP490R, 25 mm x 36 mm Longpass Dichroic Mirror, 490 nm Cutoff.
  • Filter Thor Labs FGL610 Filter.
  • FIGS. 17A and 17B illustrate the results of a protocol for analyzing serum samples to detect C-reactive protein.
  • FIG. 17A illustrates a CRP concentration series in buffer and
  • FIG. 17B illustrates a CRP concentration series in spiked serum.
  • the invention is based, in part, on the discovery that analytes of interest, e.g. for health-related applications, can be detected in a sensitive and efficient manner using magnetic separation.
  • the invention provides a method for detecting the presence, absence or level of an analyte in a solution, comprising placing a labeled binding agent under conditions that allow for binding of the binding agent to the analyte; placing a particle comprising a binding agent under conditions that allow for binding of the binding agent to the analyte; applying a magnetic field with sufficient strength to separate resultant complexes comprising the analyte and label from the solution; and detecting the label in the complexes.
  • the amount of detected label is compared in the presence or absence of the magnetic field.
  • the detection of the label e.g. using any of the techniques described herein
  • the detection of the amount of label is indicative of the amount of analyte.
  • the amount of detected label is proportional to the amount of analyte.
  • no (or minimal) detection of the label is indicative of the absence (or substantial absence) of the analyte.
  • the information regarding presence, absence, or level of an analyte of interest in a sample directs a healthcare or health-related lifestyle decision.
  • the invention described herein is directed, in part, to improved immunoassays for detecting analytes for detecting analytes of interest in samples, which may include samples of bodily fluids.
  • the invention includes immunoassays that may be used to detect the presence, absence, or level of an analyte of interest in a sample.
  • the immunoassays described herein may include an analysis chamber that may contain a sample, a magnetic conjugate, a reporter or a reporter conjugate, a magnet, a light source, and/or a photodetector.
  • the immunoassays described herein may include a sample collector associated with, or otherwise in fluid communication with, the analysis chamber.
  • the sample collector may include as an absorbent and/or wicking material that may absorb the sample and then feed the sample into the analysis chamber.
  • one or more of the magnetic conjugate, reporter, and reporter conjugate may be disposed at the sample collector such that when a sample is added to the sample collector, the sample may contact the magnetic conjugate, reporter, reporter conjugate, or a combination thereof.
  • one or more of the magnetic conjugate, reporter, and reporter conjugate may be imbedded in a portion of the sample collector.
  • the sample collector includes a sponge, foam, or membrane, such as a polyurethane sponge or foam, or another adsorbent and/or absorbent material.
  • the sample collector may include a cellulose, nitro-cellulose, and/or polyvinyl difluoride (PVDF) membrane, sponge, or foam.
  • the sample collector may be a Porex adsorbent, which may be PE/PET based.
  • the Porex adsorbent may include Porex conjugate release layer that may be sintered PE based.
  • the magnet may be a permanent magnet that may be separated from the analysis chamber in order to apply a magnetic field to the analysis chamber.
  • the magnet may be an electromagnet that may be activated or deactivated in order to apply a magnetic field to the analysis chamber.
  • the light source connected to the analysis chamber and may be configured to transmit light through a portion of the analysis chamber.
  • the analysis chamber may be one chamber, or two chambers, or three chambers, or four chambers. In some embodiments, the analysis chamber may be one or more chambers, or two or more chambers, or three or more chambers, or four or more chambers. In some embodiments, the analysis chamber may include a plurality of chambers. In some embodiments, the plurality of chambers may be in fluid communication. In some embodiments, the sample, reporter or reporter conjugate, and magnetic conjugate may be mixed in a first chamber. In certain embodiments, the magnetic field may be applied in a second chamber and the light source connected to the analysis chamber may be configured to transmit light through the second chamber. In some embodiments, the method steps described herein may each be performed in separate chambers of the analysis chamber. In some embodiments, the analysis chamber may be one chamber and all method steps may be performed in the same chamber.
  • a photodetector may be connected to the analysis chamber (e.g., facing, in line with, or opposite the light source) and may be configured to detect light transmitted through the analysis chamber by the light source and thereby measure transmittance and/or absorbance of the light.
  • the photodetector may be connected to the analysis chamber, orthogonal to the light source (orthogonal illumination), and may be configured to detect fluorescence and/or phosphorescence of a reporter or reporter conjugate in a portion of the analysis chamber.
  • the photodetector may be connected to the analysis chamber, opposite to the light source (trans illumination), and may be configured to detect fluorescence and/or phosphorescence of a reporter or reporter conjugate in a portion of the analysis chamber.
  • the photodetector may be connected to the analysis chamber, in line with the light source (e.g., by way of a dichroic mirror as shown in FIG. 16 (cis illumination)), and may be configured to detect fluorescence and/or phosphorescence of a reporter or reporter conjugate in a portion of the analysis chamber.
  • a photodetector may include one or more photomultiplier tube detectors and photodiode detectors.
  • the term “photomultiplier” or “photomultiplier tube” refers to optical detection components that convert incident photons into electrons via the photoelectric effect and secondary electron emission.
  • the term photomultiplier tube is meant to include devices that contain separate dynodes for current multiplication as well as those devices that contain one or more channel electron multipliers.
  • the term “optical detector” or “photodetector” refers to a device that generates an output signal when irradiated with optical energy. Thus, in its broadest sense the term optical detector system is taken to mean a device for converting energy from one form to another for the purpose of measurement of a physical quantity or for information transfer.
  • Optical detectors include but are not limited to photomultipliers and photodiodes.
  • the term "photodiode” refers to a solid-state light detector type including, but not limited to PN, PIN, APD, CMOS, and CCD.
  • the photodetector may include one or more of a PN based detector, a PIN based detector, an APD based detector, a CMOS based detector, and a CCD based detector.
  • the analysis chamber comprises a photodetector as described herein.
  • the analysis chamber comprises one or more of a PN based detector, a PIN based detector, an APD based detector, a CMOS based detector, and a CCD based detector.
  • the magnetic conjugate described herein may include a magnetic particle and a capture antibody associated therewith.
  • the magnetic particle may be bound to the capture antibody.
  • the reporter conjugate described herein may include a reporter and a reporter antibody.
  • the reporter may be bound to the reporter antibody.
  • the capture moiety and the reporter binding moiety may be the same or different.
  • the capture moiety and/or the reporter binding moiety may be an antibody, an antigen-binding fragment, an antigen, a receptor, a ligand, an aptamer, an aptamer receptor, a nucleic acid, or a small molecule.
  • the capture moiety may be a capture antibody.
  • the reporter binding moiety may be a reporter antibody.
  • the capture antibody and/or the reporter antibody may be selected from the group consisting of antibodies for the analytes of interest described herein.
  • the capture antibody and/or the reporter antibody may be selected from the group consisting of an anti-hCG antibody, anti-LH antibody, anti-PSA antibody, anti-HSV antibody, anti-E3G antibody, an anti -bacterial cell surface protein antibody, an anti-hemoglobin AIC antibody, an anti-C-reactive protein antibody, an anti -inflammation biomarker antibody, an anti-troponin antibody, an anti-lyme disease antibody, an anti-LDL biomarker antibody, an anti- HDL biomarker antibody, an anti-total cholesterol biomarker antibody, an anti-estrogen antibody, an anti-progesterone antibody, an anti-thyroid stimulation hormone antibody, an anti- hepatitis C virus biomarker antibody, an anti-rhino virus biomarker antibody, an anti-influenza biomarker antibody, an anti-liver
  • the capture antibody and/or the reporter antibody may be an antibody that may bind an analyte of interest selected from the group consisting of human chorionic gonadotropin (hCG), luteinizing hormone (LH)/Lutropin, prostate specific antigen (PSA), herpes simplex virus (HSV) antibodies, estrone-3-glucuronide (E3G), bacteria, hemoglobin AIC, C-reactive protein, an inflammation biomarker, troponin, lyme disease antigen, lyme disease antibodies, an LDL biomarker, an HDL biomarker, a total cholesterol biomarker, thyroid stimulating hormone, a hepatitis C virus biomarker, a rhino virus biomarker, an influenza virus biomarker, a liver function biomarker, estrogen, progesterone, lactic acid, and combinations thereof.
  • hCG human chorionic gonadotropin
  • LH luteinizing hormone
  • PSA prostate specific antigen
  • HSV herpes simplex virus
  • E3G
  • the bacteria may be Streptococcus-A, Chlamydia, and/or Gonorrhea.
  • the inflammation biomarker may be CRP, SAA, and/or MP8.
  • the liver function biomarker may be ALT and/or AST.
  • the analyte of interest may be selected from the group consisting of an ovulation biomarker, a pregnancy biomarker, a strep throat biomarker, a prostate cancer biomarker, a herpes biomarker, a diabetes biomarker, an inflammation biomarker, a heart attack biomarker, a chlamydia biomarker, a bacteria biomarker, a lyme disease biomarker, a cholesterol biomarker, a hypothydroidism biomarker, a hepatitis C biomarker, a rhino virus biomarker, an influenza biomarker, a liver function biomarker, a fertility biomarker, a muscle fatigue biomarker, and combinations thereof.
  • an ovulation biomarker a pregnancy biomarker, a strep throat biomarker, a prostate cancer biomarker, a herpes biomarker, a diabetes biomarker, an inflammation biomarker, a heart attack biomarker, a
  • the binding partners described herein may include, without limitation, antibodies including but not limited to single chain antibodies, antigen-binding antibody fragments, antigens (to be used to bind to their antibodies, for example), receptors, ligands, aptamers, aptamer receptors, nucleic acids, small molecules, and the like.
  • the capture antibody and/or the reporter antibody may be selected from the group consisting of INN-hCG-2, INN-hCG-2, 5008-SP5, 5008-SP5, and 5011 SPRN-1, or functional variants thereof.
  • the capture antibody and/or the reporter antibody may be one or more antibodies described in Table 1.
  • Table 1 Listed in Table 1 are exemplary antibody pairs that can be used to detect hCG (e.g., selecting one or two of the following to make a pair):
  • an embodiment of the invention pertains to the use or one, or two of INN-hCG-2, INN-hCG-22, 5008-SP5, 5014-SPTN5, and 5011 SPRN-1, or functional fragments thereof.
  • the capture antibody and/or the reporter antibody may be Fitzgerald 10-L15A and 10-L15B, or functional variants thereof.
  • the capture antibody and/or the reporter antibody may be anti- PSA 5001 (Medix), anti-PsA 5012 (Medix), or functional variants thereof.
  • the capture antibody and/or the reporter antibody may be polyclonal antibodies targeting the Strep- A antigen or monoclonal Strep- A 2601 SPTN-5 or 2603 SPTN-5 antibodies, or functional variants thereof manufactured by Biospacific.
  • the analyte to be detected may be virtually any analyte provided that binding partners specific for the analyte are available.
  • the analyte can be bound by at least two binding partners simultaneously.
  • the analyte is bound by the binding partners at the same epitope or at different epitopes.
  • the analytes may be or may comprise nucleic acids, peptides or proteins, carbohydrates, lipids, or any combination thereof.
  • the invention described herein contemplates the detection of Human chorionic gonadotropin (hCG), for example, as part of a pregnancy test.
  • hCG Human chorionic gonadotropin
  • Fully intact hCG includes a dimer formed between two hCG subunits, alpha-hCG and beta-hCG.
  • the hCG is detected using antibodies, for example, a pair of antibodies that recognize one or more epitopes on alpha-hCG and beta-hCG.
  • the hCG is detected using antibodies that recognize beta-hCG.
  • any known antibodies directed against alpha-hCG or beta-hCG may be utilized in the invention described herein.
  • the antibodies include INN-hCG-2, INN-hCG-2, 5008-SP5, 5008- SP5, and 5011 SPRN-1, or functional variants thereof.
  • the methods described herein can detect hCG earlier and with greater accuracy than conventional pregnancy tests on the market such as those pregnancy tests developed by First Response (as used herein, "First Response” refers to an over the counter chromatographic immunoassay for the qualitative detection of human chorionic gonadotropin (hCG)).
  • First Response refers to an over the counter chromatographic immunoassay for the qualitative detection of human chorionic gonadotropin (hCG)).
  • the invention described herein contemplates the detection of luteinizing hormone (LH)/Lutropin, for example, as part of a test for identifying ovulation.
  • LH luteinizing hormone
  • Exemplary antibodies that recognize LH include, but are not limited to, Fitzgerald 10-L15A and 10-L15B, or functional variants thereof.
  • the invention described herein further contemplates the detection of estrone-3-glucuronide (E3G) as another biomarker for identifying ovulation.
  • E3G estrone-3-glucuronide
  • the methods described herein can detect LH or E3G earlier and with greater accuracy than conventional ovulation tests on the market such as the ClearBlue Digital Ovulation Test (an over the counter LH test) or other ovulation tests developed by ClearBlue.
  • the methods described herein are particularly suited for predicting ovulation in women with polycystic ovary syndrome (PCOS) who cannot use the ovulation tests currently on the market due to their high LH baseline.
  • PCOS polycystic ovary syndrome
  • the invention contemplates the detection of Prostate Specific Antigen (PSA).
  • PSA Prostate Specific Antigen
  • Exemplary antibodies that recognize PSA that may be used in methods described herein include, but are not limited to, anti-PSA 5001 (Medix), anti-PsA 5012 (Medix), or functional variants thereof.
  • the invention contemplates the detection of Herpes Simplex Virus (HSV) or antibodies against HSV present in the blood or serum.
  • HSV Herpes Simplex Virus
  • the methods described herein relate to the detection of HSV-1 (oral herpes) or antibodies against HSV-1 present in the blood or serum.
  • the methods described herein relate to the detection of HSV-2 (genital herpes) or antibodies against HSV-2.
  • HSV-1 or HSV-2 antigen may be used as binding partners to detect the presence of antibodies against HSV-1 or HSV-2 in the blood or serum.
  • the invention contemplates the detection of Streptococcus-A (Strep-A).
  • Exemplary antibodies that may be utilized for the detection of Strep-A include, but are not limited to, polyclonal antibodies targeting the Strep-A antigen or monoclonal Strep-A 2601 SPTN-5 or 2603 SPTN-5 antibodies, or functional variants thereof manufactured by Biospacific.
  • the methods described herein can detect Strep-A antigen earlier and with greater accuracy than conventional tests such as the QuickVue Dipstick Strep A test (an immunofluorescence test to detect Group A Streptococcal antigens from throat swabs of symptomatic patient).
  • the methods described herein are at least about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, or at least about 100 times more sensitive than other rapid tests currently on the market, e.g., the QuickVue Dipstick Strep A test.
  • the methods described herein have enhanced specificity to Strep-A compared to other Streptococcus bacteria such as Strep-B, Strep-C or Strep-G.
  • the invention contemplates the detection of various infections, including gonorrhea and chlamydia.
  • exemplary antigens that may be detected using antibodies in the methods described herein include, but are not limited to, chlamydial LPS KDO-tri saccharide, chlamydial major outer membrane protein, all antigens of Neisseria gonorrhea including any major outer membrane protein.
  • the invention contemplates the detection of various diseases or conditions including diabetes and inflammation.
  • Exemplary antigens that may be detected using antibodies in the methods described herein include, but are not limited to, Hemoglobin AIC and C-reactive protein. Additional antigens that may be detected by methods described herein include any known antigen that may be detected by ELISA or sandwich ELISA immunoassays currently on the market.
  • the methods described herein may encompass the use of one antibody (e.g., a capture antibody or a reporter antibody).
  • the invention described herein contemplates the use of multiple antibodies, such as, for example, one or more capture antibodies and one or more reporter antibodies.
  • the invention described herein may utilize at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 350, about 400, about 450, or about 500, about 750, about 1000, about 1250, about 1500, about 1750, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000 antibodies.
  • the invention described herein may utilize at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, or about 250, or about 500, or about 750, or about 1000, or about 1250, or about 1500, or about 1750, or about 2000, or about 3000, or about 4000, or about 5000, or about 6000, or about 7000, or about 8000, or about 9000, or about 10000 antibody pairs.
  • the invention described herein may utilize multivalent antibodies.
  • the invention may involve coupling of bi-valent or trivalent single- chain variable fragment antibodies (e.g., each of which can contain about 4 or about 6 analyte, or more, binding sites, respectively).
  • methods described herein may involve chemically forming aggregates of multiple antibodies. This could be performed with a variety of multifunctional linkers.
  • the present methods minimize false positive signals.
  • the present methods reduce signals by controlling solution pH.
  • the solution pH is controlled by the use of appropriate buffers, which can be specific for the antibodies used.
  • Tris/Borate/EDTA buffer and/or buffers with EDTA are utilized.
  • Various buffers may be utilized in the invention.
  • Exemplary buffers that may be utilized for running gels in the invention include, but are not limited to, single buffers systems such as Sodium Borate, Sodium Acetate, Sodium Citrate, Lithium Borate, Tris/ Acetic Acid/EDTA, Tris/ Acetic Acid, Tris-Acetate, Tris Acetate EDTA, Tris/TAPS/EDTA Buffer, Bis-Tris/HCl buffer, Tris-Acetate SDS, MOPS, MOPS/Tris/SDS/EDTA, MOPS/Tris/EDTA, MOPS/Tris/SDS, MOPS/Tris, MES, MES/Tri s/ SD S/EDT A, MES/Tris/EDTA, MES/Tris/SDS, MES/Tris, Tris-glycine, or dual buffer systems such as Tris EDTA on one side and Boric Acid on the other side of the gel.
  • single buffers systems such as Sodium Borate, Sodium Acetate, Sodium Citrate, Lithium Borate, Tris/
  • Additional exemplary buffers that may be utilized for stabilizing pH include, but are not limited to, Sodium Borate, Sodium Acetate, Sodium Citrate, Lithium Borate, Tris-HCl, TAPS, Tris/Acetic Acid/EDTA, Tris- Acetate, Tris Acetate EDTA, Tris/TAPS/EDTA Buffer, Ammonium Bicarbonate, Sodium Bicarbonate, Phosphate buffer, Guanidine Hydrochloride, Guanidine Thiocyanate, Bis-Tris/HCl buffer, Tris-Acetate SDS, MOPS, MOPS/Tris/EDTA, MOPS/Tris/SDS, MOPS/Tris, MES, ME S/Tri s/SD S/EDT A, MES/Tris/SDS, MES/Tris, and Tris-glycine.
  • passivating agents such as Tween, BSA, poly ethylene glycol, or casein are used. Additional exemplary passivating agents that may be utilized in the invention include, but are not limited to, Glycerol, Sucrose, Glucose, TritonX, SDS, LDS, Sigmacoat, DNA oligos, Fish Gelatin, Whole sera, Polyvinyl alcohol, polyvinylpyrrolidone, salmon-sperm DNA, Silanes, and Silica.
  • Methods described herein include methods for detecting the presence, absence, or level of an analyte of interest in a sample.
  • the methods described herein may include the steps of: a. contacting the sample with a magnetic conjugate comprising a magnetic particle and a capture antibody configured to bind the analyte of interest in the sample; b. contacting the sample with a reporter conjugate comprising a reporter and a reporter antibody configured to bind the analyte of interest in the sample; c. binding the analyte of interest with the capture antibody and the reporter antibody; d. separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber to pull down the magnetic conjugates with analyte of interest associated therewith; and e. detecting the presence, absence, or level of the analyte of interest by detecting the reporter with a light source and photodetector.
  • the methods described herein may include the steps of: a. contacting the sample with a reporter conjugate comprising a reporter and a reporter antibody configured to bind the analyte of interest in the sample; b. contacting the sample with a magnetic conjugate comprising a magnetic particle and a capture antibody configured to bind the analyte of interest in the sample; c. binding the analyte of interest with the capture antibody and the reporter antibody; d. separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber to pull down the magnetic conjugates with analyte of interest associated therewith; and e. detecting the presence, absence, or level of the analyte of interest by detecting the reporter with a light source and photodetector.
  • the methods described herein may include the steps of: a. contacting a sample with a magnetic conjugate comprising a magnetic particle and a capture antibody configured to bind the analyte of interest in the sample; b. binding the analyte of interest with the capture antibody; c. separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber to pull down the magnetic conjugates with analyte of interest associated there with; d. contacting the sample with a reporter conjugate comprising a reporter and a reporter antibody configured to bind the analyte of interest in the sample; e. binding the analyte of interest with the reporter antibody; f.
  • the methods described herein may include the steps of: a. contacting a sample with a magnetic conjugate comprising a magnetic particle and a capture antibody configured to bind the analyte of interest in the sample; b. binding the analyte of interest with the capture antibody; c. contacting the sample with a reporter-labeled analyte configured to bind the magnetic conjugate in the absence of the analyte of interest in the sample; d. separating the analyte of interest from the sample by applying a magnetic field to the analysis chamber to pull down the magnetic conjugates with analyte of interest associated there with; and e. detecting the presence, absence, or level of the analyte of interest by detecting the reporter with a light source and photodetector.
  • the methods described herein may include the steps of: a. contacting a sample with a reporter conjugate comprising a reporter and a reporter antibody configured to bind the analyte of interest in the sample; b. binding the analyte of interest with the reporter antibody; c. contacting the sample with a magnetic particle-labeled analyte configured to bind the reporter conjugate in the absence of the analyte of interest in the sample; d. separating the magnetic particle-labeled analyte from the sample by applying a magnetic field to the sample; and e. detecting the presence, absence, or level of the analyte of interest by detecting the reporter with a light source and photodetector.
  • the methods described herein may include the steps of: a. contacting a sample with a magnetic conjugate comprising a magnetic particle and a capture antibody configured to bind the analyte of interest in the sample; b. binding the analyte of interest with the capture antibody; c. contacting the sample with a reporter antibody comprising a biotin label configured to bind the analyte of interest in the sample; d. contacting the sample with a reporter comprising a streptavidin label configured to bind the biotin label; e. separating the analyte of interest from the sample by applying a magnetic field to the sample; and f. detecting the presence, absence, or level of the analyte of interest by detecting the reporter.
  • the magnetic conjugate (including a capture antibody) may be added simultaneously with a reporter or reporter conjugate.
  • the magnetic conjugate (including a capture antibody) and a reporter or reporter conjugate may be added separately.
  • the methods of invention may include the step of adding sample to an analysis chamber.
  • adding sample to the analysis chamber may include delivering the sample to a sample collector (e.g., an absorbent and/or wicking material) in fluid communication with the analysis chamber.
  • the sample collector may then feed the sample into the analysis chamber.
  • one or more of the magnetic conjugate, reporter, and reporter conjugate may be disposed at the sample collector and the steps of contacting the sample with the magnetic conjugate, reporter, or reporter conjugate may occur at the sample collector.
  • one or more of the magnetic conjugate, reporter, and reporter conjugate may be imbedded in a portion of the sample collector before a sample is added to the sample collector.
  • the methods described herein may include contacting the sample in the analysis chamber with a magnetic conjugate, reporter, and/or reporter conjugate.
  • the reporter may be a fluorescent reporter, a phosphorescent reporter, or a colorimetric reporter such as a colored particle that may be configured to measure absorbance or scattering of light (or, for example, the presence/absence of a certain color by colorimetric analysis).
  • the methods described herein may further include the step of concentrating the analyte of interest in the sample by applying a magnetic field to the analysis chamber after contacting the sample with the magnetic conjugate; and then reducing the volume of the sample in the analysis chamber. In some embodiments, the methods described herein may further include the step of deactivating the magnetic field before contacting the sample with the reporter conjugate.
  • reducing the volume of the sample in the analysis chamber may be performed by, for example, syphoning of a fraction of the volume or by removing the entire sample and resuspending in a new lesser volume.
  • the methods described herein may further include the steps of concentrating the analyte of interest in the sample by applying a magnetic field to the analysis chamber after contacting the sample with the magnetic conjugate; removing a volume of the sample from the analysis chamber; and adding a volume of buffer and/or an additional volume of the sample to the analysis chamber.
  • the methods described herein may include the step of deactivating the magnetic field before contacting the sample with the reporter conjugate.
  • the methods described herein may include the step of adding a volume of buffer and/or additional volumes of sample to the analysis chamber. [00105] In some embodiments, the methods described herein may include the step of removing volumes of sample from the analysis chamber after a pull down of the magnetic conjugate (i.e., application of a magnetic field) and before or after contacting the sample with a reporter or reporter conjugate.
  • the reporter antibody is labeled with biotin and the reporter is functionalized with streptavidin. In some embodiments of the methods described herein the reporter antibody is functionalized with streptavidin and the reporter is labeled with biotin.
  • contacting step e.g., contacting the sample with a magnetic conjugate, a reporter antibody, a reporter-labeled conjugate, and/or a reporter conjugate
  • contacting step may include incubating the sample, which may contain an analyte, with the respective magnetic conjugate, reporter antibody, reporter-labeled conjugate, and/or reporter conjugate for a selected period of time and a selected temperature.
  • the analyte of interest may be any analyte of interest described herein.
  • the analyte of interest may be selected from the group consisting of human chorionic gonadotropin (hCG), luteinizing hormone (LH)/Lutropin, prostate specific antigen (PSA), herpes simplex virus (HSV) antibodies, estrone- 3-glucuronide (E3G), bacteria, hemoglobin AIC, C-reactive protein, an inflammation biomarker, troponin, lyme disease antigen, lyme disease antibodies, an LDL biomarker, an HDL biomarker, a total cholesterol biomarker, thyroid stimulating hormone, a hepatitis C virus biomarker, a rhino virus biomarker, an influenza virus biomarker, a liver function biomarker, estrogen, progesterone, lactic acid, and combinations thereof.
  • hCG human chorionic gonadotropin
  • LH luteinizing hormone
  • PSA prostate specific antigen
  • HSV herpe
  • the bacteria may be Streptococcus-A, Chlamydia, and/or Gonorrhea.
  • the inflammation biomarker may be CRP, SAA, and/or MP8.
  • the liver function biomarker may be ALT and/or AST.
  • the analyte of interest may be selected from the group consisting of an ovulation biomarker, a pregnancy biomarker, a strep throat biomarker, a prostate cancer biomarker, a herpes biomarker, a diabetes biomarker, an inflammation biomarker, a heart attack biomarker, a chlamydia biomarker, a bacteria biomarker, a lyme disease biomarker, a cholesterol biomarker, a hypothydroidism biomarker, a hepatitis C biomarker, a rhino virus biomarker, an influenza biomarker, a liver function biomarker, a fertility biomarker, a muscle fatigue biomarker, and combinations thereof.
  • an ovulation biomarker a pregnancy biomarker, a strep throat biomarker, a prostate cancer biomarker, a herpes biomarker, a diabetes biomarker, an inflammation biomarker, a heart attack biomarker, a
  • an ovulation biomarker may be derived from a urine, blood, or serum based sample.
  • a pregnancy biomarker may be derived from a urine or blood based sample.
  • a strep throat biomarker may be derived from a saliva based sample.
  • a prostate cancer biomarker may be derived from a blood, serum, or urine based sample.
  • a herpes biomarker may be derived from a blood or saliva derived sample.
  • the analyte of interest may be selected from the group consisting of hCG, C-reactive protein, LH, PSA, HSV, E3G, a bacterium (e.g., Strep A), and combinations thereof.
  • the sample may be a bodily fluid as described herein.
  • the methods described herein may include obtaining a bodily fluid sample from a patient.
  • the methods described herein encompass a sandwich method, a separate addition method, a competitive method, and a tertiary method.
  • the sandwich method described herein may be well suited for processing small fluid sample volumes in an immunoassay format.
  • the separate addition method described herein may enable processing of larger fluid volumes with improved sensitivity.
  • the competitive assay method may be useful for assaying in which a user cannot find both a capture antibody and a reporter antibody that may bind to the analyte simultaneously, e.g., where the analyte is a small molecule.
  • the tertiary assay method may provide three binding events to enhance the kinetics of a system.
  • the tertiary binding motif can be applied through a sandwich method, separate addition method, or competitive assay method formats.
  • immunoassays, methods, and kits described herein allow for personal base lining for an analyte of interest.
  • the immunoassays, methods, and kits described herein allow for a determination of a normal analyte range for each individual user.
  • the user is alerted if there is any deviation from the individual's personal normal analyte range.
  • the analyte that may be detected is any biomarker for a biological event.
  • the biological events may include a disease event (i.e., disease biomarker), an inflammation event (i.e., an inflammation biomarker), a reproduction event (i.e., a reproduction biomarker), and/or an aging event (i.e., an aging biomarker).
  • the invention relates to the detection of a biomarker for a biological event using the systems and methods described herein.
  • a method of pregnancy detection using the systems and methods described herein.
  • a method of ovulation detection using the systems and methods described herein.
  • a method of prostate health detection e.g. detecting the presence a cancer, or likelihood of developing the same
  • a method of herpes detection using the systems and methods described herein.
  • the methods described herein include various detection techniques, e.g. for reporter signal.
  • detection techniques may involve a microscope, a spectrophotometer, a fluorometer, a tube luminometer or plate luminometer, x-ray film, magnetic fields, a scintillator, a fluorescence activated cell sorting (FACS) apparatus, a microfluidics apparatus, a bead-based apparatus or the like.
  • FACS fluorescence activated cell sorting
  • the magnetic particle is a paramagnetic particle.
  • the paramagnetic particle is a nanoparticle or a microparticle.
  • the paramagnetic particle is a bead, such as a nanobead or a microbead.
  • the paramagnetic particle is, in various embodiments, a magnetic nano- or microbead, which allows the particle to be held and/or manipulated by magnets.
  • the paramagnetic particle is a metallic nanoparticles coated with a thin (ca. 2 nm) graphene-like carbon layer.
  • the paramagnetic particle is coated, e.g. streptavidin- or PEG-coated.
  • magnétique particles examples include DYNABEADs (THERMOFISHER), MACS beads (MILTENYI BIOTEC), TURBOBEADS (TURBOBEADS), ABSOLUTE MAG STREPTAVIDIN MAGNETIC PARTICLES (CREATIVE DIAGNOSTICS), and GOLD NANOPARTICLES (SIGMAALDRICH).
  • the magnetic particles described herein may include a biocompatible coating that may be activated with amine groups or carboxyl groups to facilitate amid coupling. In some embodiments, the magnetic particles described herein may be activated with amine groups or carboxyl groups to facilitate amid coupling.
  • the reporter particles described herein may include a biocompatible coating that may be activated with amine groups or carboxyl groups to facilitate amid coupling. In some embodiments, the reporter particles described herein may be activated with amine groups or carboxyl groups to facilitate amid coupling.
  • the particles described herein may be be microparticles (e.g. microbeads), which are about 0.5 micrometers to about 500 micrometers in diameter (e.g. about 0.5 micrometers, or about 1 micrometer, or about 10 micrometers, or about 50 micrometers or about 100 micrometers or about 250 micrometers or about 500 micrometers).
  • the particles described herein may be nanoparticles (e.g. nanobeads), which are smaller than 1 micrometer in diameter (e.g. about 5 to about 500 nanometers, e.g. about 5 nanometers, or about 10 nanometers, or about 50 nanometers, or about 100 nanometers, or about 250 nanometers, or about 500 nanometers).
  • the nanoparticles e.g. nanobeads
  • nanobeads have a mean particle diameter of 25-500 nm+/-5 nm, 25-500 nm+/-10 nm, 25-500 nm+/-15 nm, 25-500 nm+/-20 nm, 25-500 nm+/-25 nm, 25-500 nm+/-30 nm, 25-500 nm+/-35 nm, 25-500 nm+/-40 nm, 25-500 nm+/-45 nm, or 25-500 nm+/-50 nm.
  • the nanoparticles e.g. nanobeads
  • the nanoparticles have a mean particle diameter of about 20 to about 200 nm.
  • the microparticles are about 0.5 micrometers to about 500 micrometers in diameter (e.g. about 0.5 micrometers, or about 1 micrometer, or about 10 micrometers, or about 50 micrometers or about 100 micrometers or about 250 micrometers or about 500 micrometers).
  • the nanoparticles are smaller than 1 micrometer in diameter (e.g. about 5 to about 500 nanometers, e.g. about 5 nanometers, or about 10 nanometers, or about 50 nanometers, or about 100 nanometers, or about 250 nanometers, or about 500 nanometers). In some embodiments, the nanoparticles (e.g.
  • nanobeads have a mean particle diameter of 25-500 nm+/-5 nm, 25-500 nm+/-10 nm, 25-500 nm+/-15 nm, 25-500 nm+/- 20 nm, 25-500 nm+/-25 nm, 25-500 nm+/-30 nm, 25-500 nm+/-35 nm, 25-500 nm+/-40 nm, 25- 500 nm+/-45 nm, or 25-500 nm+/-50 nm.
  • the nanoparticles e.g. nanobeads
  • the nanoparticles have a mean particle diameter of about 20 to about 200 nm.
  • the magnetic particle may be a magnetic nanoparticle (e.g. nanobead) that is composed of oxides, such as ferrites, maghemite, magnetite, or iron oxide, optionally modified by surfactants, silica, silicones or phosphoric acid derivatives.
  • the nanoparticle e.g. nanobead
  • the nanoparticle is composed of ferrites with a shell (e.g. a silica shell, optionally modified).
  • the magnetic nanoparticle (e.g. nanobead) is metallic (e.g. iron, cobalt, etc.).
  • the magnetic nanoparticle (e.g. nanobead) is metallic with a shell (e.g. of gentle oxidation, surfactants, polymers and precious metals (e.g. of gold, graphene, etc.)).
  • a particle described herein may be a nanoparticle (e.g. nanobead) that comprises one or more quantum dots.
  • the nanoparticle comprises a metal core and one or more quantum dots.
  • the nanoparticle comprises a metal core that may be studded with one or more quantum dots.
  • the nanoparticle comprises a metal core that may be studded with a plurality of quantum dots.
  • Quantum dots are discrete nanoparticles that have properties similar to bulk semiconductors such that when exposed to electromagnetic energy they in turn emit energy. Quantum dots can be engineered to be sensitive to energy in the infrared region, the visible spectrum, and even ultraviolet range through changes in size and composition. Further, they can be designed to be either photoluminescent or photovoltaic, producing either light or energy, respectively.
  • the reporter may be a nanoparticle (e.g. nanobead), which may comprise one or more quantum dots.
  • the reporter comprises a metal core and one or more quantum dots.
  • the reporter comprises a metal core that may be studded with one or more quantum dots.
  • the reporter comprises a metal core that may be studded with a plurality of quantum dots.
  • the reporter may comprise one or more quantum dots.
  • the reporter conjugate may comprise one or more quantum dots.
  • the reporter and/or the reporter conjugate may comprise a plurality of quantum dots.
  • quantum dots e.g. produced by colloidal methods, include, but are not limited to, cadmium-selenide (CdSe), cadmium- sulfide (CdS), indium-arsenide (InAs), and indium-phosphide (InP) cadmium -tellurium-sulfide (CdTeS).
  • CdSe cadmium-selenide
  • CdS cadmium- sulfide
  • InAs indium-arsenide
  • InP indium-phosphide
  • the number of atoms that comprise a quantum dot can range from 100 to 100,000, typically with a diameter ranging from 2 to 20 nm (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 1 1.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5 nm).
  • particle materials include, but are not limited to, carbon, colloidal gold, germanium, indium arsenide, indium antimonide, gallium arsenide, gallium nitride, cadmium/selenium/telluride, lead, lead oxide, lead sulfide, lead selenide, indium gallium phosphide, silicon, colloidal silver, mercury cadmium telluride, iron, iron oxide, cobalt, graphene, lanthanum, cerium, strontium carbonate, manganese, manganese oxide, nickel oxide, platinum, lithium, lithium titanate, tantalum, copper, palladium, molybdenum, boron carbide, silicon carbide, titanium carbide, tungsten oxide, aluminum, niobium, thulium, aluminum nitride, tin, aluminum oxide, tin oxide, antimony, dysprosium, paseodynium, antinmony oxide, erb
  • the magnetic field is applied using an external magnet.
  • the magnet is a permanent magnet (e.g. neodymium iron boron (NdFeB), samarium cobalt (SmCo), alnico, and ceramic or ferrite magnets).
  • the magnet is a temporary magnet.
  • the magnet is an electromagnet.
  • the detection of the label is undertaken near the magnetic field. In various embodiments, the detection of the label is undertaken away from the magnetic field as in, for example, performed in a chamber that is separate from a chamber in which a magnetic pull down step is performed.
  • kits that include an immunoassay as described herein for practicing one or more methods described herein.
  • the invention provides a pregnancy detection kit, which includes an immunoassay described herein, and involves the detection of hCG (e.g., ⁇ -hCG).
  • hCG e.g., ⁇ -hCG
  • the invention provides an ovulation detection kit, which includes an immunoassay described herein, and involves the detection of LH.
  • the invention provides a pregnancy detection kit, which includes an immunoassay described herein, and involves the detection of E3G.
  • the invention provides a kit for detecting PSA, which includes an immunoassay described herein.
  • the invention provides a kit for detecting antibodies against HSV (e.g., HSV-1 and/or HSV-2), which includes an immunoassay described herein.
  • the kits described herein are specific for HSV-2 antibodies over HSV-1 antibodies.
  • the invention provides a kit for detecting Strep-A, which includes an immunoassay described herein.
  • the invention provides a kit for detecting infection by, for example, gonorrhea and chlamydia, which includes an immunoassay described herein.
  • the invention provides a kit for detecting a disease or condition, such as, but not limited to, inflammation and diabetes, which includes an immunoassay described herein.
  • the term "about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication.
  • the language “about 50%” covers the range of 45% to 55%.
  • something is "decreased" if a read-out of activity and/or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%), at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%), at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation.
  • activity is decreased and some downstream read-outs will decrease but others can increase.
  • activity is "increased" if a read-out of activity and/or effect is increased by a significant amount, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%), at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3 -fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8- fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.
  • compositional percentages are by weight of the total composition, unless otherwise specified.
  • the word "include,” and its variants is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • sample may refer to a solution, suspension, mixture, or undiluted amount of bodily fluid that may or may not include an analyte of interest.
  • a sample as used herein, may include water and/or a buffer.
  • the buffers described herein may be added to reduce or eliminate hook effects, which are present in most immunoassay platforms in the art.
  • a "large volume" of sample may be a 20 ⁇ _, or greater volume of sample or 20 ⁇ _, to 500 ⁇ _, of sample.
  • a "small volume” of sample may be less than 20 ⁇ _, of sample or 1 ⁇ _, to 15 ⁇ _, of sample.
  • the term "bodily fluid” may refer to any fluid that can be isolated from the body of an individual and includes, but is not limited to whole blood, plasma, serum, bile, saliva, urine, tears, perspiration, cerebrospinal fluid (CSF), semen, swabbed samples (e.g. cheek swabs, throat swabs, etc.), mucus, sputum, menstrual blood, menstrual fluid, vaginal mucus, amniotic fluid, synovial fluid, breast milk, ear wax, preejaculate, lochia, Rheum, lymph, pus, and the like.
  • CSF cerebrospinal fluid
  • bodily fluid may more particularly refer to whole blood, serum, urine, saliva, swabbed samples, mucus, or semen. In certain embodiments, bodily fluid may more particularly refer to whole blood, serum, urine, or saliva. In some embodiments, the bodily fluid may include an analyte of interest (e.g., a biomarker).
  • an analyte of interest e.g., a biomarker
  • the term "analyte of interest” or “target analyte” or “analyte” may be used interchangeably and refer to an antigen and/or a biomarker for a biological event, including any of the biomarkers described herein.
  • the biological events may include a disease event (e.g., disease biomarker), an inflammation event (e.g., an inflammation biomarker), a reproduction event (e.g., a reproduction biomarker), and/or an aging event (e.g., an aging biomarker).
  • Disease biomarkers may include one or more disease biomarkers related to or associated with the onset of disease, the offset of disease, and/or the presence of a disease state in a patient.
  • Disease biomarkers may include one or more of a viral biomarker, a bacterial biomarker, a cancer biomarker, or a symptom biomarker.
  • Viral biomarkers may include, but are not limited to biomarkers for common cold (e.g. rhinovirus), influenza, herpes, Zika, and/or HIV.
  • viral biomarkers may include herpes simplex virus (HSV), one or more rhinovirus proteins, one or more influenza A/B/C proteins, one or more HSF-1/2 proteins, and/or one or more HIV virus proteins.
  • HSV herpes simplex virus
  • Bacterial biomarkers may include, but are not limited to, biomarkers for strep throat (i.e., Streptococcus-A (Strep-A)), biomarkers for chlamydia, and/or biomarkers for gonorrhea.
  • bacterial biomarkers may include, but are not limited to, one or more streptococcus proteins, one or more chlamydia trachomatis proteins, and/or one or more neisseria gonorrhoeae proteins.
  • Symptom biomarkers may include, but are not limited to, biomarkers for coughing, wheezing, runny nose, nausea, cramps, tightness of the chest, light-headedness, sore throat, and/or chest pain.
  • Disease biomarkers may also include, but are not limited to, biomarkers for cardiac distress and/or diabetes.
  • disease biomarkers may include troponin, CRP, and/or hale.
  • Cancer biomarkers may include biomarkers for prostate cancer, breast cancer, colorectal cancer, gastric cancer, GIST, leukemia/lymphoma, lung cancer, melanoma, and or pancreatic cancer.
  • prostate cancer biomarkers may include PSA.
  • breast cancer biomarkers may include one or more of ER/PR and HER-2/neu.
  • colorectal cancer biomarkers may include one or more of EGFR, KRAS, and UGT1A1.
  • gastric cancer biomarkers may include HER-2/neu.
  • GIST biomarkers may include c-KIT.
  • leukemia/lymphoma biomarkers may include one or more of CD20 antigen, CD30, FIP1L1-PDGRF alpha, PDGFR, PML/RAR alpha, TPMT, and UGT1A1.
  • lung cancer biomarkers may include one or more of ALK, EGFR, and KRAS.
  • melanoma biomarkers may include BRAF.
  • Inflammatory biomarkers which may include anti-inflammatory biomarkers, may include one or more inflammatory biomarkers described in U.S. Patent Application Publication No. 2010/0275282, the entirety of which is incorporated herein by reference.
  • Reproduction biomarkers may include biomarkers for ovulation, fertilization, implantation, and/or embryo development.
  • reproduction biomarkers may include ⁇ -human Chorionic Gonadotropin ( ⁇ -hCG or hCG), hyperglycosylated hCG, luteinizing hormone (LH), estrone-3- glucuronide (E3G), early pregnancy factor (EPF), and/or pre implantation factor.
  • Aging biomarkers or age-related biomarkers include one or more biomarkers described in U.S. Patent Application Publication No. 2008/0124752, the entirety of which is incorporated herein by reference. Additional antigens/biomarkers of interest include, but are not limited to, any known antigens/biomarkers associated with SARS, Hand foot and mouth disease, cardiac biomarkers, thyroid hormone, obesity biomarkers, biomarkers relating to bleeding disorders such as vWF, Factor 8, Factor 10, fifths disease, cold, flu, Ebola, E coli, listeria, and salmonella.
  • magnetic particle refers to any particle having at least some magnetic characteristic, e.g., ferromagnetic, paramagnetic, and superparamagnetic property.
  • a magnetic particle may include magnetic materials such as iron, nickel, and cobalt, as well as metal oxides such as Fe 3 0 4 , Mn 2 0 3 , Cr 2 0 3 , CoO, NiO, and CoMnP.
  • the magnetic particle contains, or fully consists of, a polymeric magnetic material.
  • Polymeric magnetic material includes for example, material in which the magnetic material is mixed with polymeric material and magnetic material that is coated with polymeric material.
  • the magnetic material is only one component of the microparticle whose remainder consists of a polymeric material to which the magnetically responsive material is affixed (see coded particles below).
  • a magnetic particle may be a magnetic nanoparticle or magnetic microparticle, as described herein.
  • the term “capture moiety” may refer to antibodies, single chain antibodies, antigen-binding antibody fragments, antigens, receptors, ligands, aptamers, aptamer receptors, nucleic acids, or small molecules that are conjugated or bound, or that may be conjugated or bound, to a magnetic particle that is selected to bind a target analyte.
  • the capture moiety is a capture antibody.
  • the term “capture antibody” may refer to an antibody conjugated or bound, or that may be conjugated or bound, to a magnetic particle that is selected to bind a target analyte.
  • the term “reporter binding moiety” may refer to antibodies, single chain antibodies, antigen-binding antibody fragments, antigens, receptors, ligands, aptamers, aptamer receptors, nucleic acids, or small molecules that are conjugated or bound, or that may be conjugated or bound, to a reporter that is selected to bind a target analyte.
  • the reporter binding moiety is a reporter antibody.
  • reporter antibody may refer to an antibody conjugated or bound, or that may be conjugated or bound, to a reporter that is selected to bind a target analyte.
  • the "capture antibody” and “reporter antibody” may both bind different portions of the target analyte.
  • the terms “reporter” and “label” may be used interchangeably, and may generally refer to a signal generating compound and/or detectable label or a core (e.g., a metal core) with one or more signal generating compounds and/or detectable labels connected to the core.
  • the reporter may be a fluorescent reporter, a phosphorescent reporter, or colorimetric reporter such as a colored particle for measuring absorbance and/or scattering of light (or, for example, the presence absence of a certain color through colorimetric analysis).
  • any suitable detectable label as is known in the art can be used.
  • the detectable label can be a radioactive label (such as H, I, S, C, P, and 33 P), an enzymatic label (such as horseradish peroxidase, alkaline phosphatase, glucose 6- phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5 -fluorescein, 6-carboxyfluorescein, 3'6- carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, and the
  • the label includes without limitation fluorophores, chromophores, radioisotopes, magnetic particles, gold particles, enzyme substrates, and the like.
  • the label is a chemiluminescent or fluorescent protein, such as, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2, GFPuv4, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), citrine and red fluorescent protein from discosoma (dsRED), luciferase, umbelliferone, rhodamine, fluorescein, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, and the like.
  • GFP green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • Renilla Reniformis green fluorescent protein GFPmut2, GFPuv4
  • yellow fluorescent protein YFP
  • the label is a non-protein organic fluorophore of any of the following families: xanthene derivatives, such as fluorescein, rhodamine, Oregon green, eosin, and Texas red; cyanine derivatives, such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine; squaraine derivatives and ring- substituted squaraines, including Seta, SeTau, and Square dyes; naphthalene derivatives (dansyl and prodan derivatives); coumarin derivatives; oxadiazole derivatives, such as pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole; anthracene derivatives, such as anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange; pyrene derivatives, such as cascade blue, etc.
  • xanthene derivatives such as
  • the label includes without limitation enzymatic labels, e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose 6-phosphate dehydrogenase, and the like.
  • the reporter may be a quantum dot as described herein. In some embodiments, the reporter may comprise a quantum dot as described herein. In some embodiments, the reporter may include a metal core (i.e., gold core) with a silica shell, wherein the silica shell is impregnated with a plurality (e.g., 100-600) quantum dots.
  • a metal core i.e., gold core
  • a silica shell wherein the silica shell is impregnated with a plurality (e.g., 100-600) quantum dots.
  • the words "preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.
  • a covalent coupling procedure was developed to couple antibodies to fluorescently labeled reporter particles.
  • the reporter particles described are metal core quantum dot studded particles (Nanocompsix)), which have a carboxyl surface coating.
  • This procedure describes an exemplary protocol for covalently coupling antibodies to magnetic capture particles (i.e., magnetic beads).
  • the magnetic beads used in this protocol are nanoparticles with a superparamagnetic Fe 2 0 3 core and a biocompatible outer coating.
  • the surface is activated with carboxyl groups.
  • Protein Preparation [00197] Using a 7K Zeba Desalting Column, buffer exchange the antibody of interest into MB Coupling Buffer (i.e., PBS, 0.01% Tween-20, pH 7.4); if the concentration of antibody is greater than 1 mg/mL dilute the antibody solution to 1 mg/mL using MB coupling buffer before using the Zeba Column. Measure IgG Concentration.
  • MB Coupling Buffer i.e., PBS, 0.01% Tween-20, pH 7.4
  • Antibody Conjugation Using a P200 set to 150, transfer the 150 ⁇ L ⁇ activated Magbeads into a new Eppendorf lo-bind tube.
  • This procedure describes an exemplary protocol for covalently coupling a biomarker (i.e., estrone-3-glucoronide (E3G) to magnetic capture particles (i.e., magnetic beads).
  • a biomarker i.e., estrone-3-glucoronide (E3G)
  • magnetic capture particles i.e., magnetic beads
  • the magnetic beads used were nanoparticles with a superparamagnetic Fe 2 0 3 core and a biocompatible outer coating.
  • the surface is activated with amine groups.
  • An immunoassay as described herein is prepared for use in sandwich mode, which is an ideal setup for processing small fluid volumes.
  • a sandwich mode for an immunoassay described herein may be used.
  • a reporter conjugate including a reporter gold core particles with a silica shell impregnated with 100-600 quantum dots (nanoComposix)
  • an antibody #1 and a magnetic conjugate including an antibody #2 and a magnetic particle may be used detect an hCG analyte of interest in an analysis chamber (FIG. 1 A).
  • the magnetic conjugate and the reporter conjugate may be added to the assay chamber and mixed with the sample containing the analyte of interest (hCG) (FIG. IB).
  • a magnetic field may be applied (a "pulldown") by a magnet to separate the analyte of interest from the sample (FIG. 1C).
  • Light may then be transmitted through a portion of the analysis chamber to cause the reporter to fluoresce (FIG. ID). Such fluorescence may be detected by the detector.
  • An immunoassay as described herein is prepared for use in separate addition mode, which is a preferred setup for processing large volumes of samples and allowing for concentration of the analyte. This leads to greatly improved sensitivity.
  • a separate addition mode for an immunoassay described herein may be used.
  • a reporter conjugate including a reporter gold core particles with a silica shell impregnated with 100-600 quantum dots (nanoComposix)
  • an antibody #1 and a magnetic conjugate including an antibody #2 and a magnetic particle may be used to detect an hCG analyte of interest in an analysis chamber (FIG. 2A).
  • the magnetic conjugate may be added to the assay chamber and mixed with the sample containing the analyte of interest (hCG) (FIG. 2B).
  • a pulldown may be performed and, optionally, a volume of sample may be removed and an additional volume of sample may then be added (FIG. 2C).
  • the magnetic field may be deactivated and the magnetic conjugate may again be mixed with the sample. This process may be repeated to concentrate the analyte of interest.
  • the reporter conjugate may be added and mixed with the sample to bind to the analyte of interest (FIG. 2D).
  • An additional pulldown may be performed to separate the analyte of interest from the sample (FIG. 2E).
  • Light may then be transmitted through a portion of the analysis chamber to cause the reporter to fluoresce (FIG. 2F). Such fluorescence may be detected by the detector.
  • An immunoassay as described herein is prepared for use in competitive mode, which is a preferred setup where the analyte of interest is too small for binding by two antibodies. This may also be preferred where only one antibody exists for binding the analyte of interest.
  • FIGS. 3A to 3E a competitive mode for an immunoassay described herein may be used.
  • a reporter-labeled analyte and a magnetic conjugate including an antibody and a magnetic particle may be used to detect an analyte of interest in an analysis chamber (FIG. 3 A).
  • the magnetic conjugate may be added to the assay chamber and mixed with the sample containing the analyte of interest (FIG. 3B).
  • the reporter-labeled analyte may then be added and mixed with the sample (FIG. 3C).
  • the reporter-labeled analyte does not bind to the magnetic conjugate. Indeed, the binding site on the magnetic conjugate's antibody is occupied with the analyte of interest from the sample. However, if the analyte of interest is not present in the sample, the magnetic conjugate will bind to the reporter-labeled analyte (FIG. 3C).
  • a magnetic particle may be used with an analyte of interest bound thereto.
  • the magnetic particle bears the analyte of interest at the start of the assay rather than the reporter as in Example 3.
  • the reporter conjugate includes an antibody and a reporter that may be used to detect the analyte of interest in the sample in an analysis chamber.
  • the reporter conjugate is added to the analysis chamber and mixed with the sample containing the analyte of interest.
  • the magnetic particles with analyte bound thereto are added to the analysis chamber and mixed with the sample.
  • a pulldown is then performed to separate the magnetic particles with analyte bound thereto from the sample.
  • the sample is then removed.
  • Light may then be transmitted through the sample to cause the reporter to fluoresce. Such fluorescence may be detected by the detector.
  • the reporter conjugate In the absence of analyte, the reporter conjugate will be pulled down and will not be present in the sample, resulting in no fluorescence.
  • An immunoassay as described herein is prepared for use in tertiary mode, makes use of three binding events to enhance the kinetics of the system.
  • the tertiary binding motif can be applied to the sandwich mode (Example 1), the separate addition (Example 2), and the competitive assay modes (Example 3 and 4).
  • a tertiary mode for an immunoassay described herein may be used.
  • a reporter conjugate including a reporter (fluorescent quantum dot functionalized with streptavidin) and an antibody #1 (labeled with a biotin) and a magnetic conjugate including an antibody #2 and a magnetic particle may be used detect an hCG analyte of interest in an analysis chamber (FIG. 4A).
  • the magnetic conjugate may be added to the assay chamber and mixed with the sample containing the analyte of interest (FIG. 4B).
  • a magnetic field may be applied (a "pulldown") by a magnet to separate the analyte of interest from the sample (FIG. 4C). Volumes of sample may be removed and analyte concentration steps may be performed as described in Example 2.
  • the magnetic field may be deactivated and antibody #1 may be added to the analysis chamber, which may then bind to the analyte of interest that is also bound to the magnetic conjugate (FIG. 4D).
  • the reporter may then be added to the analysis chamber, which may then bind to antibody #1 through the streptavidin-biotin binding interaction (FIG. 4E).
  • a pulldown may then be performed to again separate the analyte from the sample (FIG. 4F).
  • Light may then be transmitted through a portion of the analysis chamber to cause the reporter to fluoresce (FIG. 4G). Such fluorescence may be detected by the detector.
  • Example 6 Demonstration of an Assay for Detecting Human Chorionic Gonadotropin
  • An immunoassay was provided for detecting human Chorionic Gonadotropin (hCG) in a urine sample using the protocol of Example 15. The assay exhibited femtomolar scale sensitivity as shown in FIG. 5.
  • An immunoassay was provided to detect leukinizing Hormone (LH) in a urine sample.
  • the assay exhibited femtomolar scale sensitivity as shown in FIG. 6.
  • the magnetic beads in this assay are magnetic nanoparticles coated with an antibody that binds LH (Medix 5304).
  • the reporter in this assay is a fluorescently labeled nanoparticle, which has been coated with an antibody that binds LH (Medix 5304).
  • TBS to the First replicate tube, and then remove the ⁇ . of TBS by over-pipetting.
  • An immunoassay was provided to detect prostate specific antigen (PSA) in a serum/whole blood sample.
  • PSA prostate specific antigen
  • the assay exhibited femtomolar scale sensitivity as shown in FIG. 7.
  • the magnetic beads in this assay are magnetic nanoparticles coated with an antibody that binds PSA (Medix Anti-h PSA 8311).
  • a solution of magnetic beads was prepared mixing and sonicating the magnetic beads for 10s.
  • a 200 pM magnetic bead solution includes 2.5 ⁇ of magnetic beads and 22.5 ⁇ of Chon Block.
  • the reporter in this assay is a fluorescently labeled nanoparticle, which has been coated with an antibody that binds PSA (Medix Anti-h PSA 8301) [00341] PSA Stripping Antibody Solution
  • PSA 8301 ab Stock 5.3 mg/mL (35.35 ⁇ ). For 6.66 ⁇ , 5.65 ⁇ , of Stock 8301 Antibody and 24.35 ⁇ _, Chon Block were combined. For 660 nM, add 10 ⁇ _, of 6.66 ⁇ of the solution and 90 ⁇ _, of Chon Block.
  • Standard serum preparation included adding 20 ⁇ _, of serum to 80 ⁇ _, of Chon Block.
  • PSA stripped serum was prepared by adding 20 ⁇ _, of serum to 80 ⁇ _, of serum stripping solution.
  • Standard whole blood preparation included combining 76 ⁇ _, of whole blood, 19 ⁇ _, of Chon Block, and 4.75 ⁇ , of 10% Triton X-100.
  • PSA stripped whole blood was prepared by combining 76 ⁇ _, of whole blood, 19 ⁇ _, of whole blood stripping solution, and 4.75 ⁇ _, of 10% Triton X-100.
  • Incubation was performed by first adding 25 ⁇ _, of magnetic beads and 9 ⁇ _, of reporters to 100 ⁇ _, of prepared serum, and then incubating the sample for 30 minutes.
  • Incubation was also performed by first adding 25 ⁇ _, of magnetic beads and 9 ⁇ _, of reporters to 100 ⁇ _, of whole blood, and then incubating the sample for 30 minutes.
  • a pull down was performed by exposing the incubated solutions to a magnet for 5 minutes.
  • An immunoassay as described herein is prepared for use in detecting bacteria in a sample.
  • a sandwich mode for an immunoassay described herein may be used to detect bacteria (e.g., Strep A) in a sample.
  • bacteria e.g., Strep A
  • a reporter conjugate including a reporter gold core particles with a silica shell impregnated with 100-600 quantum dots (nanoComposix)
  • an antibody #1 and a magnetic conjugate including an antibody #2 and a magnetic particle may be used detect a bacteria analyte of interest in an analysis chamber (FIG. 8A).
  • the magnetic conjugate and the reporter conjugate may be added to the assay chamber and mixed with the sample containing the analyte of interest (bacteria) (FIG. 8B).
  • a magnetic field may be applied (a "pulldown") by a magnet to separate the analyte of interest from the sample (FIG. 8C).
  • Light may then be transmitted through a portion of the analysis chamber to cause the reporter to fluoresce (FIG. 8D). Such fluorescence may be detected by the detector.
  • a bacteria immunoassay was provided as in Example 9, which was used to detect the presence of Strep A in a sample including throat, cheek, and saliva samples from 26 subjects with Strep throat and 15 subjects suspected to not be infected with Group A strep.
  • the assay exhibited 200 organism scale sensitivity as shown in FIG. 9.
  • the present assay showed a sensitivity of 93 % ND a specificity of 100% as compared to the Quidel Clinical grade test, which is 92% sensitive and 98% specific.
  • the magnetic beads in this assay are magnetic nanoparticles coated with an antibody that binds to Strep A (Biospacific G47091041).
  • a 300 pM solution of magnetic beads was prepared by adding 4 ⁇ _, of magnetic beads (2.89 nM) to a flask, pulling them down with a magnet, then resuspending in 38.5 ⁇ _, of Chon Block
  • the reporter used in this assay is a fluorescently labeled nanoparticle, which has been coated with an antibody that binds to Strep A (Biospacific G47091041).
  • a 250 pM solution of reporters was prepared by combining 4.5 ⁇ _, of reporters (2.2 nM) and 35.5 ⁇ _, of Chon Block.
  • Biospacific G47091041 (lm/mL) was prepared as a stock solution at 6.67 ⁇ and 1 ⁇ , 500 nM, and 100 nM solutions were prepared in PBS.
  • a subject's tonsils are swabbed.
  • 150 ⁇ _, of PBS is then added to a squeezable tube.
  • the swab is then submerged in the PBS solution. After 1 minute, the swab is pulled midway up the tube and pinched with fingers to extract the liquid from the swab.
  • a strep solution is prepared by combining 16 ⁇ _, of swab processed mix and 2 ⁇ _, of Chon Block.
  • a blocked strep solution is prepared by combining 16 ⁇ _, of swab processed mix and 2 ⁇ _, of 500 nM Strep blocking mixture.
  • Strep Incubation 16 ⁇ _, of Strep Solution is combined with 3 ⁇ _, of magnetic beads and 3 ⁇ _, of reporters. Incubation proceeds for 10 minutes.
  • Blocked Incubation 16 ⁇ _, of Blocked Strep Solution is combined with 3 ⁇ _, of magnetic beads and 3 ⁇ _, of reporters. Incubation proceeds for 10 minutes.
  • the resuspended magnetic beads/reporters could then be analyzed by fluorescence spectroscopy to detect Strep A in a sample.
  • the difference in signal between signal of the strep incubation tube and the blocked incubation tube will give a measure of Group A Streptococcus that removes the matrix effects from swabs of different individuals.
  • E3G can be detected in a sample analyzed by a competitive immunoassay described herein.
  • E3G Coupled magnetic beads and Anti-E3G Coupled Quantum Dots can be used. Buffers may be used in the analysis chamber, which may include chonblock and PBS.
  • the Quantum Dot working solution may include 1 ⁇ _, of Stock Anti-E3G Quantum Dots and 6 ⁇ , of Chonblock.
  • the protocol may be as follows: a. Mix 6.25 ⁇ _, of urine with 1.25 ⁇ _, Quantum Dot Working Solution; b. Incubate 5 minutes; c. Add 5 ⁇ _, of E3G Coupled Magnetic Beads; d. Incubate 30 minutes; e. Place on Magnet stand for 1 minute; f. Remove Supernatant; g. Rinse twice with 50 ⁇ _, of PBS; h. Resuspend in 27 ⁇ _, PBS; and i. Pipette 20 ⁇ _, into well plate.
  • the pipetted material may then be imaged as shown in FIG. 10 where 44 ⁇ and 200 nM E3G were detected in two samples as compared to blanks.
  • Example 12 Clinical Study Measuring Correlation between Measurements of Described Immunoassays and Measurements Performed in a Clinical Laboratory
  • Frozen Sample Protocol Subjects collecting frozen samples were provided with urine cups, labels, Ziploc bags, and a sample log so that they could collect daily samples. The subjects would collect urine in the urine cup each morning, log the time of urination, label the cup, bag it, and immediately put it in their freezer. Upon completion of their cycle (either by getting a positive pregnancy test result using one of the provided tests, or having their period) the samples would be picked up from the subject's home by study staff who would retrieve the samples and transport them to the lab in a cooler filled with ice-packs. Samples were immediately racked and stored in our laboratory freezers (for details see materials and methods).
  • Fresh Sample Protocol Subjects collecting fresh samples were provided with a single urine cup and a small thermos. The subjects would collect urine each morning and place the urine cup in the thermos. They would then send a text message to the clinical research coordinator who would bring the sample in for analysis within 2 hours of urine collection.
  • Sample Testing Paradigm Samples were tested to find the first day on which First Response yielded a positive result (i.e. First Response reads negative on the day before, but positive on that day's urine). This day was termed the First Response Day. The immunoassay platform was then tested on the First Response Day, and at least three days prior. Additionally, two baseline days (days before the participant ovulates ensuring that she cannot be pregnant) were tested.
  • FIG. 12 A typical test looks as shown in FIG. 12 were the first and second rows are duplicate samples. A darker circle indicates a higher concentration of hCG in the sample. As would be expected, the baseline days show very low signal, while the days preceding the first response yes day increase in intensity. The image from FIG. 12 is quantified and plotted in FIG. 13, where error bars represent 1 standard deviation.
  • FIG. 14 shows a stair-step curve illustrating the fraction positive as a function of days post ovulation (day of LH spike) where the Confer Magneto is the immunoassay described herein.
  • a probability is calculated by assuming each day' s signal is described by a Gaussian probability with the mean equal to the measured signal and the standard deviation estimates for each respective day.
  • the probability for the baseline day signal, P BL (S), is given by:
  • PCD ⁇ BL P BL (S) P CD (S')dSdS'
  • hCG 2 Phase Binding Protocol - 7 minute protocol.
  • Reaction Buffer 5mM potassium phosphate, pH 7.4, 0.5% 20K MW PEG.
  • Method provides 4.4 ⁇ _, of Prepped Magnetic Beads.
  • Phase 1 Binding Magnetic Bead Analyte Binding a. Mix the following in a 0.5 mL Protein Lo Bind eppendorf tube: i. 4 ⁇ _, of Prepped Magnetic Beads ii. 25 ⁇ _, of Urine b. Incubate at room temperature for 1 minute on the bench. c. Place the tube on the magnet stand and allow it to sit for 45 seconds. d. Remove the supernatant with a P200 pipette (or appropriate pipette that will remove the volume if scaled up). e. Add 4 ⁇ _, of Chon Block to the pellet and mix with a pipette to re-suspend.
  • Method provides 4 ⁇ _, of Phase 1 Binding Mix.
  • Phase 2 Binding Quantum Dot Binding a. Mix the following in a 0.5mL Protein Lo Bind eppendorf tube: i. 4 ⁇ _, of Phase 1 Binding Mix ii. 1 iL of 2.5x dilute 4F9 Quantum Dots b. Incubate at room temperature for 5 minutes. [00411] Method provides 5 ⁇ L ⁇ of Phase 2 Binding Mix. [00412] Pulldown and Rinse a. Place the tube containing 5 ⁇ ⁇ of Phase 2 Binding Mix on magnet stand and allow it to sit for 20 seconds b. Keep the tube on the stand for the remaining 5 steps i.
  • Method provides 10 ⁇ _, of analyte bound to both a magnetic particle and a fluorescent Quantum Dot ready to be imaged.
  • E3G estrone-3-glucoronide
  • the magnetic beads in this assay are magnetic nanoparticles coated with E3G according to the foregoing protocol.
  • the reporter in this assay is a fluorescently labeled nanopoartice, which has been coated with an antibody (Absolute Antibody 4115) that binds to E3G.
  • volume of ChonBlock ( ⁇ ,) ⁇ Volume of Magnetic Beads) x (50 X Dilution Factor)— (Volume of Magnetic Beads)
  • Sonicate reporters by holding the tube in the sonicator for 10s. Mix reporters using a PI 00 set to 100.
  • Eppendorf Protein Lo-bind tubes pre-labeled with the subject number, cycle date and replicate number (1,2,3), using a P10 pipette set to 3 ⁇ ⁇ .
  • TBS tris buffered saline
  • HCG human chorionic gonadotropin
  • the magnetic beads in this assay are magnetic nanoparticles coated with an antibody (Scripps GC099) that binds HCG.
  • the reporter in this assay is a fluorescently labeled nanoparticle, which has been coated with an antibody (Scripps GC099) that binds HCG.
  • TBS to the First replicate tube, and then remove the ⁇ . of TBS by over-pipetting.
  • the magnetic beads in this assay are magnetic nanoparticles coated with an antibody (Medix 5304) that binds LH.
  • the reporter in this assay is a fluorescently labeled nanoparticle, which has been coated with an antibody (Medix 5304) that binds LH.
  • Eppendorf Protein Lo-bind tubes pre-labeled with the subject number, cycle date and replicate number (1,2,3), using a P10 pipette set to 3 ⁇ ⁇ .
  • TBS to the First replicate tube, and then remove the ⁇ . of TBS by over-pipetting.
  • CRP C-reactive protein
  • the magnetic beads in this assay are magnetic nanoparticles coated with an antibody that binds CRP (anti-CRP-C2).
  • the reporter in this assay is a fluorescently labeled nanoparticle, which has been coated with an antibody (anti-CRP-C6).
  • the concentration of a reporter solution for this assay was 440 pM, which was diluted with Chon Block from a stock solution of 2.2 nM.
  • Serum was prepared by pulling down 20 ⁇ ⁇ of the aforementioned magnetic beads and then resuspending in 10 ⁇ ⁇ of serum, then incubating the same for 10 minutes.
  • Spike normal serum samples to prepare spiked samples according to the following: a. 10 mg/L: 8 ⁇ . of 2.5 mg/L CRP dilution + 2 L Normal Serum (2 mg/L Final); b. 5 mg/L: 8 of 1.25 mg/L CRP dilution + 2 ⁇ , Normal Serum (1 mg/L Final); c. 2 mg/L: 8 of 0.5 mg/L CRP dilution + 2 yL Normal Serum (0.4 mg/L Final); and d. 0 mg/L: 8 of Chon Block + 2 ⁇ L ⁇ Normal Serum (0 mg/L Final). [00542] Assay Protocol
  • FIGS. 17A and 17B show the CRP concentration series in buffered solution (FIG. 17 A) and the CRP concentration in the spiked serum (FIG. 17B).

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Abstract

L'invention concerne, en partie, des procédés, des dosages et des kits améliorés pour détecter des analytes dans des échantillons biologiques à l'aide de particules magnétiques. Le procédé comprend des étapes de mise en contact d'un échantillon avec un conjugué magnétique constitué d'une particule magnétique et d'un fragment de capture conçu pour se lier à l'analyte d'intérêt dans l'échantillon ; mise en contact de l'échantillon avec un conjugué rapporteur constitué d'un rapporteur et d'un fragment se liant au rapporteur conçu pour se lier à l'analyte d'intérêt dans l'échantillon ; liaison de l'analyte d'intérêt avec le fragment de capture et le fragment se liant au rapporteur ; séparation de l'analyte d'intérêt de l'échantillon par application d'un champ magnétique à la chambre d'analyse ; et détection de la présence, de l'absence ou du niveau de l'analyte d'intérêt par détection du rapporteur.
PCT/US2018/015440 2017-01-26 2018-01-26 Immunodosage à base de particules magnétiques et ses procédés d'utilisation WO2018140719A1 (fr)

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CN201880014031.9A CN110462383A (zh) 2017-01-26 2018-01-26 基于磁性颗粒的免疫测定及其使用方法
US16/479,827 US20210389313A1 (en) 2017-01-26 2018-01-26 Magnetic particle-based immunoassay and methods of using the same
EP18744906.1A EP3574309A4 (fr) 2017-01-26 2018-01-26 Immunodosage à base de particules magnétiques et ses procédés d'utilisation
CA3051475A CA3051475A1 (fr) 2017-01-26 2018-01-26 Immunodosage a base de particules magnetiques et ses procedes d'utilisation
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US20220146509A1 (en) * 2018-12-28 2022-05-12 Proteometech Inc. Immunochromatography strip for pregnancy diagnosis with multiple test lines, and pregnancy diagnosis kit comprising same
US20210055290A1 (en) * 2019-08-22 2021-02-25 The Hong Kong University Of Science And Technology System for single-molecule isolation for cell populations and single cells, and methods and uses thereof
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WO2022029731A1 (fr) * 2020-08-07 2022-02-10 Vital Biosciences Inc. Détection d'analyte multiplexée
WO2022117978A1 (fr) * 2020-12-02 2022-06-09 Paraytec Ltd Test de diagnostic
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WO2024175169A1 (fr) * 2023-02-20 2024-08-29 STILLER, Olaf Procédé permettant de détecter directement la présence d'une substance de test
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