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US-12618858-B2 - Kinetic modulation for magnetic analyte detection

US12618858B2US 12618858 B2US12618858 B2US 12618858B2US-12618858-B2

Abstract

Methods and kits for detection of presence, absence, or amount of an analyte in a biological sample are provided. In the methods and kits, kinetics of the detection is modulated my adjusting concentrations of a reporter binding moiety and a reporter.

Inventors

  • Mounir A. KOUSSA

Assignees

  • VITAL BIOSCIENCES INC.

Dates

Publication Date
20260505
Application Date
20210807

Claims (12)

  1. 1 . A method for detecting the presence, absence, or amount of an analyte in a biological sample, the method comprising: (a) contacting the biological sample with a magnetic conjugate comprising a magnetic particle and a capture moiety comprising a first antibody configured to bind the analyte in the biological sample; (b) contacting the magnetic conjugate and the bound analyte with a reporter binding moiety comprising a second antibody and a biotin tag bound thereto, wherein the capture moiety and the reporter binding moiety bind different portions of the analyte, and wherein the reporter binding moiety is present at a concentration of about 1 nM to about 600 nM; (c) contacting a complex comprising the magnetic conjugate, the analyte, and the reporter binding moiety with a reporter comprising a streptavidin tag binding partner that is configured to bind the biotin tag of the reporter binding moiety, wherein the reporter comprises a particle studded with a plurality of quantum-dots, and wherein the reporter is present at a concentration of about 10 pM to about 140 pM; (d) applying a magnetic field to separate a detectable complex comprising the magnetic conjugate, the analyte, the reporter binding moiety and the reporter bound thereto; and (e) detecting the presence, absence, or amount of the analyte based on detection of a signal generated by the reporter.
  2. 2 . The method of claim 1 , wherein the reporter binding moiety is present at a concentration of about 1 nM to about 10 nM.
  3. 3 . The method of claim 2 , wherein the reporter binding moiety is present at a concentration of about 5 nM.
  4. 4 . The method of claim 1 , wherein the reporter is present at a concentration of about 15 pM to about 25 pM.
  5. 5 . The method of claim 4 , wherein the reporter is present at a concentration of about 20 pM.
  6. 6 . The method of claim 1 , wherein the reporter comprises a metal core and a silica shell, wherein the silica shell is impregnated with one or more quantum dots.
  7. 7 . The method of claim 1 , wherein the analyte is selected human chorionic gonadotropin (hCG), luteinizing hormone (LH)/Lutropin, prostate specific antigen (PSA), herpes simplex virus (HSV) antibodies, estrone-3-glucuronide (E3G), bacteria, hemoglobin A1C, C-reactive protein (CRP), 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.
  8. 8 . The method of claim 1 , wherein the biological sample is selected from whole blood, plasma, serum, bile, saliva, urine, tears, perspiration, cerebrospinal fluid (CSF), semen, mucus, sputum, menstrual blood, menstrual fluid, vaginal mucus, amniotic fluid, synovial fluid, breast milk, ear wax, preejaculate, lochia, rheum, lymph, and pus.
  9. 9 . The method of claim 1 , wherein the analyte comprises an antibody and the capture moiety comprises an antigen configured to bind the antibody.
  10. 10 . The method of claim 9 , wherein the reporter binding moiety comprises a secondary antibody configured to bind the antigen.
  11. 11 . The method of claim 1 , wherein the method detects an amount of antibodies present in the biological sample.
  12. 12 . The method of claim 1 , further comprising pre-treating the biological sample with a magnetic conjugate comprising a magnetic particle and a moiety configured to bind contaminant antibodies and/or non-antibody moieties.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a National Stage Entry of PCT/IB2021/057295, filed Aug. 7, 2021, which claims the benefit of U.S. Provisional Patent Application No. 63/063,029 filed Aug. 7, 2020 entitled “KINETIC MODULATION FOR MAGNETIC ANALYTE DETECTION,” which is incorporated by reference herein in its entirety. TECHNICAL FIELD The present invention relates to, inter alia, methods to detect presence, absence, or amount of an analyte in a biological sample with improved kinetics of the detection. SEQUENCE LISTING This instant application contains a Sequence Listing that has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on Feb. 2, 2023, is named 124436-5012 Sequence Listing.xml and is 6,089 bytes in size. BACKGROUND To diagnose diseases and conditions, monitor and assess treatment progression, and to perform various other healthcare-related tasks, reliable tests are often required to detect and quantify a diverse range of targets, including but not limited to proteins, bacteria, whole cells, viruses, and small molecules. 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 common detection method. However, conventional ELISA may be time-consuming as it involves various incubation and washing steps. Also, parameters for carrying out ELISA assays are highly variable, and traditional ELISA platforms may not provide adequate sensitivity and specificity. Various diagnostic methods have been developed to detect antibodies and antigens, including ELISA, agglutination, precipitation, complement-fixation, fluorescent antibodies, and chemiluminescence. For example, serological tests are diagnostic methods that are used to identify antibodies and antigens in a patient's sample. The knowledge of a serological status of a person regarding a certain infectious disease, autoimmune disease, allergy, etc. is useful for various applications, including diagnosis, selection of treatment, monitoring of treatment, establishing of quarantine, making decisions in forensics, biometric identification, etc. Serological tests can also be applied to determining a person's blood type. Each of the existing approaches has its advantages and drawbacks, and problems that remain to be solved relate to the reliability, speed, and cost of the testing. Also, conventional immunoassays may have long processing times and often less than desirable sensitivity and specificity. The prolonged time required to perform an analyte detection using a conventional approach can be a significant limitation for many clinical applications where it is desired to process samples promptly. Decreased samples analysis times may be critical for epidemiological applications. For example, as the COVID-19 (SARS-COV-2 or 2019-nCOV) pandemic has shown, quick and reliable processing of large number of samples can be a life-saving approach for identifying infected subjects, for contact tracing, and for ultimate return to normal. Accordingly, there exist a need for quick and accurate diagnostic tests for comprehensive analysis of a biological sample. SUMMARY Accordingly, in various aspects, the present invention provides methods for detecting the presence, absence, or amount of an analyte in a biological sample, or kits to effect such methods. The method allows for detection of one or more analytes in a sample with greatly improved kinetics such that an entire assay in accordance with embodiments of the present disclosure can be performed in a range from about 1 minute to about 20 minutes. For comparison, traditional assays require from about 1.5 hours to about 6 hours. In embodiments, a method for detecting the presence, absence, or amount of an analyte in a biological sample is provided. The method comprises (a) contacting the sample with a magnetic conjugate comprising a magnetic particle and a capture moiety configured to bind an analyte in the sample; (b) contacting the magnetic conjugate with a reporter binding moiety having a tag bound thereto, the reporter binding moiety being configured to bind the analyte; (c) contacting the magnetic conjugate with a reporter having a tag binding partner that is configured to bind the tag thereby optionally associating a reporter binding moiety bound to the tag with the reporter, wherein a concentration of the reporter binding moiety is substantially