IP Library › Granted Patent US 12,345,641
Granted Patent B2
US 12,345,641 · App. 17/232,647 · Granted Jul 1, 2025

Fluorescence quenching immunoassay

Inventors: Jennifer E. Gagner (Portland, ME); Kiamars Hajizadeh (Yarmouth, ME); Evan M. Peck (Gorham, ME); James R. Salter (Marietta, GA); Paul J. Travers (Scarborough, ME); Christopher P. Turmel (Westbrook, ME); Yingzi Wu (Johns Creek, GA); Hongzhi Xie (Falmouth, ME); Murthy V. S. N. Yerramilli (Scarborough, ME)
Assignee: IDEZZ Laboratories, Inc.
G01N21/6428C07D311/82G01N33/542G01N2021/6432G01N2021/6441G01N2470/12
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Quick Facts
Patent No.
US 12,345,641
App. No.
17/232,647
Granted
Jul 1, 2025
Kind
B2
Abstract

The present invention relates to a method and reagents for determining the presence of or the amount of an analyte in a sample.

Claims (54)

1. A method for determining the presence of or the amount of an analyte in a sample comprising:

(i) providing a sample suspected of containing an analyte;

(ii) contacting the sample with a fluorescent tracer and a binding partner to provide an assay composition;

wherein the binding partner is specific for the analyte and the fluorescent tracer;

(iii) irradiating the assay composition with light at a first wavelength; and

(iv) measuring the intensity of light emitted at a second wavelength,

wherein

(a) the intensity of the light emitted at the second wavelength is greater if the fluorescent tracer is not bound to the binding partner than if the fluorescent tracer is bound to the binding partner,

(b) the light at the first wavelength is not linearly polarized, and

(c) the fluorescent tracer is selected from the group consisting of:

wherein T is a bond or a linking group and E is an epitopic moiety capable of specifically binding to the binding partner; and

wherein the intensity of the light emitted at the second wavelength is directly proportional to the concentration of the analyte in the sample.

2. The method of claim 1 , wherein the analyte is a macromolecule.

3. The method of claim 2 , wherein the analyte is a protein.

4. The method of claim 3 , wherein (i) the epitopic moiety is an amino acid chain and wherein the amino acid sequence of the amino acid chain comprises the amino acid sequence of an epitope on the protein that is responsible for complexing with the binding partner, and (ii) the binding partner is optionally conjugated to a quencher.

5. The method of claim 4 , wherein the quencher is selected from the group consisting of

wherein the wavy line indicates the linkage point to the binding partner.

6. The method of claim 4 , wherein the binding partner is an antibody.

7. The method of claim 4 , wherein the protein is selected from the group consisting of canine CysB protein and canine NT-proBNP.

8. The method of claim 7 , wherein the protein is canine CysB protein.

9. The method of claim 8 , wherein the binding partner is an anti-cystatin-B antibody and the fluorescent tracer is fluorescein attached to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, and SEQ ID NO:7.

10. The method of claim 1 , wherein the amino acid sequence is attached to the 4′-position of the fluorescein with a —CH 2 —linker.

11. The method of claim 9 , wherein the anti-cystatin-B antibody is conjugated to the quencher.

12. The method of claim 1 , wherein the analyte is an antigen and the binding partner is an antibody.

13. The method of claim 12 , wherein the antigen is SDMA and the antibody is an antibody for SDMA.

14. The method of claim 13 , wherein the fluorescent tracer is selected from the group consisting of:

wherein each X is —H—, or each X is —F.

15. The method of claim 14 , wherein the antibody for SDMA is conjugated to a quencher.

16. The method of claim 15 , wherein the quencher is selected from the group consisting of CY3, CY5, IRDyeQC1, BHQ1, and BHQ10.

17. The method of claim 1 , comprising:

(A) providing a slide, wherein the slide comprises sequentially:

(i) a support layer selected from the group consisting of glass, polystyrene, polyesters, polycarbonates, cellulose derivatives, polyethylene terephthalate, and mixtures thereof, wherein the support layer is optically clear to the first wavelength and the second wavelength;

(ii) a primer layer comprising polyurethane;

(ii an indicator layer comprising the fluorescent tracer and the binding partner dispersed in a second polymer, wherein the second polymer is selected from the group consisting of cellulose, cellulose derivatives, polysaccharides, gelatin, gelatin derivatives, polyvinyl alcohol, polyvinyl pyrrolidone, acrylamide polymers, polyurethane, alginate, xantham, and mixtures thereof,

(iv) a filtering layer comprising polyurethane and cellulose; and

(v) a spreading layer comprising a mixture of cellulose and a hydrophilic polymer, wherein the hydrophilic polymer is selected from the group consisting of polyacrylic acid, polyvinylpyrrolidone, polythylene glycol, polyethylene oxide, polyvinyl alcohol, polyacrylamides, and polethylenimines,

(B) depositing the sample on the spreading layer of the slide;

(C) allowing the sample to diffuse from the spreading layer into the indicator layer so that the sample can contact the fluorescent tracer and the binding partner;

(D) irradiating the support layer of the slide with the light that is not polarized at a first wavelength; and

(E) measuring the intensity of the light emitted at the second wavelength from the support layer of the slide.

18. The method of claim 17 , wherein the analyte is SDMA and the antibody is an antibody for SDMA.

19. A method for determining the presence of or the amount of an analyte in a sample comprising:

(i) providing a sample suspected of containing an analyte;

(ii) contacting the sample with a fluorescent tracer and a binding partner to provide an assay composition;

wherein the binding partner is specific for the analyte and the fluorescent tracer;

(iii) irradiating the assay composition with light at a first wavelength; and

(iv) measuring the intensity of light emitted at a second wavelength,

wherein

(a) the intensity of the light emitted at the second wavelength is greater if the fluorescent tracer is not bound to the binding partner than if the fluorescent tracer is bound to the binding partner,

(b) the light at the first wavelength is not linearly polarized,

(c) the fluorescent tracer is selected from the group consisting of:

wherein T is a bond or a linking group and E is an epitopic moiety capable of specifically binding to the binding partner;

(d) the intensity of the light emitted at the second wavelength is directly proportional to the concentration of the analyte in the sample; and

(e) wherein the analyte is selected from the group consisting of SDMA, melamine, T4, bile acid, biotin, thyroxine, cystatin-B, NT-proBNP, antibiotics, sulfadimethoxine, cortisol, and progesterone.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 17, 2021
From: GAGNER, JENNIFER E.; PECK, EVAN M.; SALTER, JAMES ROBERT; TRAVERS, PAUL JAMES; WU, YINGZI; XIE, HONGZHI; YERRAMILLI, MURTHY V.S.N.; HAJIZADEH, KIAMARS; TURMEL, CHRISTOPHER P.
To: IDEXX LABORATORIES, INC.
Reel/Frame 056256/0443 →
Continuity (3)
Provisional Application 63152365 · Feb 23, 2021
Provisional Application 63011403 · Apr 17, 2020
Related Publication 20220082501A1 · Mar 17, 2022
References Cited (118)
US 3092465A · Adams et al. · 1963 [cited by applicant]
US 3715192A · Wenz et al. · 1973 [cited by applicant]
US 3992158A · Przybylowicz et al. · 1976 [cited by applicant]
US 3996345A · Ullman et al. · 1976 [cited by applicant]
US 3998943A · Ullman · 1976 [cited by applicant]
US 4042335A · Clement · 1977 [cited by applicant]
US 4050898A · Goffe et al. · 1977 [cited by applicant]
US 4066403A · Bruschi · 1978 [cited by applicant]
US 4144306A · Figureas · 1979 [cited by applicant]
US 4160016A · Ullman · 1979 [cited by applicant]
US 4174384A · Ullman et al. · 1979 [cited by applicant]
US 4258001A · Pierce et al. · 1981 [cited by applicant]
US 4261968A · Ullman et al. · 1981 [cited by applicant]
US 4292272A · Kitajima et al. · 1981 [cited by applicant]
US 4323536A · Columbus · 1982 [cited by applicant]
US 4459358A · Berke · 1984 [cited by applicant]
US 4614823A · Kirkemo et al. · 1986 [cited by applicant]
US 4868132A · Byrnes et al. · 1989 [cited by applicant]
US 5371241A · Brush · 1994 [cited by applicant]
US 5427960A · Wang et al. · 1995 [cited by applicant]
US 5489668A · Scott · 1996 [cited by applicant]
US 7179658B2 · Cheng et al. · 2007 [cited by applicant]
US 7371562B2 · Cunningham et al. · 2008 [cited by applicant]
US 7939164B2 · Koops et al. · 2011 [cited by applicant]
US 8012720B2 · Fujita et al. · 2011 [cited by applicant]
US 8158259B2 · Ramamurthy et al. · 2012 [cited by applicant]
US 8309369B2 · Tan et al. · 2012 [cited by applicant]
US 8415172B2 · Zuk · 2013 [cited by applicant]
US 8481690B2 · Murthy et al. · 2013 [cited by applicant]
US 8492139B2 · Tan et al. · 2013 [cited by applicant]
US 8608984B1 · Taranekar et al. · 2013 [cited by applicant]
US 8628973B2 · Woloszczuk et al. · 2014 [cited by applicant]
US 8629412B2 · Sakai · 2014 [cited by applicant]
US 8778699B2 · Yerramilli et al. · 2014 [cited by applicant]
US 8877898B2 · Markiv et al. · 2014 [cited by applicant]
US 9005901B2 · Gayda et al. · 2015 [cited by applicant]
US 9005984B2 · Woloszczuk et al. · 2015 [cited by applicant]
US 9034655B2 · Patsenker et al. · 2015 [cited by applicant]
US 9128055B2 · Sekino et al. · 2015 [cited by applicant]
US 9157910B2 · Dowell et al. · 2015 [cited by applicant]
US 9427181B2 · Emken et al. · 2016 [cited by applicant]
US 9427182B2 · Emken et al. · 2016 [cited by applicant]
US 9434789B2 · Zuk · 2016 [cited by applicant]
US 9566353B2 · Markiv et al. · 2017 [cited by applicant]
US 9591979B2 · Taranekar et al. · 2017 [cited by applicant]
US 9605068B2 · Woloszczuk et al. · 2017 [cited by applicant]
US 9612238B2 · Lasmezas et al. · 2017 [cited by applicant]
US 9709562B2 · Jakubowicz et al. · 2017 [cited by applicant]
US 9933428B2 · Chan et al. · 2018 [cited by applicant]
US 9970927B2 · Yerramilli et al. · 2018 [cited by applicant]
US 10064573B2 · Emken et al. · 2018 [cited by applicant]
US 10073090B2 · Dowell et al. · 2018 [cited by applicant]
US 10131178B2 · Pudleiner et al. · 2018 [cited by applicant]
US 10321864B2 · Zhu · 2019 [cited by applicant]
US 10379046B2 · Katzlinger et al. · 2019 [cited by applicant]
US 10379116B2 · Tan et al. · 2019 [cited by applicant]
US 10393665B2 · Ahuja et al. · 2019 [cited by applicant]
US 10408713B2 · Borch et al. · 2019 [cited by applicant]
US 10509029B2 · Tamura · 2019 [cited by applicant]
US 10570500B2 · Kim · 2020 [cited by applicant]
US 10571396B2 · Schramm et al. · 2020 [cited by applicant]
US 10725052B2 · Yerramilli et al. · 2020 [cited by applicant]
US 10942179B2 · Dowell et al. · 2021 [cited by applicant]
US 10948640B2 · Ockenfuss et al. · 2021 [cited by applicant]
US 11175229B1 · Wang et al. · 2021 [cited by applicant]
US 20110053076A1 · Sweeney et al. · 2011 [cited by applicant]
US 20130130285A1 · Atkinson et al. · 2013 [cited by applicant]
US 20130224884A1 · Briand et al. · 2013 [cited by applicant]
US 20130280740A1 · Yerramilli et al. · 2013 [cited by applicant]
US 20140011811A1 · Dudley et al. · 2014 [cited by applicant]
US 20160025639A1 · Jakubowicz · 2016 [cited by examiner]
US 20180335423A1 · Yerramilli et al. · 2018 [cited by applicant]
US 20180353113A1 · Emken et al. · 2018 [cited by applicant]
US 20190120856A1 · Xie et al. · 2019 [cited by applicant]
US 20190219569A1 · Sun et al. · 2019 [cited by applicant]
US 20190370447A1 · Houck et al. · 2019 [cited by applicant]
US 20200181486A1 · Iwamoto et al. · 2020 [cited by applicant]
US 20210055222A1 · Katzlinger · 2021 [cited by applicant]
US 20210102919A1 · Foerderung et al. · 2021 [cited by applicant]
US 20210247555A1 · Ockenfuss et al. · 2021 [cited by applicant]
US 20220034879A1 · Elztov · 2022 [cited by applicant]
CA 2799536C · 2011 [cited by applicant]
CN 110596396A · 2022 [cited by applicant]
EP 0002963B1 · 1983 [cited by applicant]
EP 0310940A2 · 1989 [cited by applicant]
EP 0884591A1 · 1998 [cited by applicant]
EP 2364845A1 · 2011 [cited by applicant]
EP 2364846A1 · 2011 [cited by applicant]
EP 3441766A1 · 2019 [cited by examiner]
WO 2005123105A1 · 2005 [cited by applicant]
WO WO2019079707A1 · 2019 [cited by examiner]
WO 2021069902A1 · 2021 [cited by applicant]
Wei et al. Use of Synthetic Peptides as Tracer Antigens in Fluorescence Polarization Immunoassays of High Molecular Weight Analytes. Anal. Chem. 1993, vol. 65, pp. 3372-3377. (Year: 1993). [cited by examiner]
Feldman et al. Cystatin B as a tissue and urinary biomarker of bladder cancer recurrence and disease progression. Clin Cancer Res. Feb. 1, 2009; 15(3): 1024-1031. (Year: 2009). [cited by examiner]
Nanda et al. Labeling a Protein with Fluorophores Using NHS Ester Derivitization. Methods in Enzymology, 2014, vol. 536; pp. 87-93. (Year: 2014). [cited by examiner]
Ullman et al. Fluorescent Excitation Transfer Immunoassay a general method for determination of antigens. The journal of Biological Chemistry, 1976, vol. 251, No. 14, Issue of Jul. 25, pp. 4172-4178. (Year: 1976). [cited by examiner]
International Search Report and Written Opinion issued in International Application No. PCT/US2021/27684 as filed on Apr. 16, 2021, and mailed on Nov. 26, 2021. [cited by applicant]
Results of structure search for 2-(2,4,5, 7-tetrabromo-3-hydroxy-6-oxoxanthen-9-yl}benzoic acid provided with the International Search Report. [cited by applicant]
B. Kannan, et al., Printed Paper Sensors for Serum Lactate Dehydrogenase using Pullulan-Based Inks to Immobilize Reagents, Anal. Chem.,87, 9288-9293, (2015). [cited by applicant]
S. Jahanshahi-Anbuhi et al., Pullulan Encapsulation of Labile Biomolecules to Give Stable Bioassay Tablets, Angew. Chem. Int. Ed., 53, 6155-6158, (2014). [cited by applicant]
S. Jahanshahi-Anbuhi et al., Simple and Ultrastable All-inclusive Pullulan Tablets for Challenging Bioassays, Chem. Sci., 7, 2342, (2016). [cited by applicant]
A. Schou-Pederson et al., Comparison of Three Sample Preparation Procedures for the Quantification of L-Arginine, Asymmetric Dimethylarginine, and Symmetric Dimethylarginine in Human Plasma Using HPLC-FLD, J. Anal. Meth… [cited by applicant]
C. Tan et al., A New One Step Antigen Heterologous Homogenous Fluorescence Immunoassay for Progesterone Detection in Serum, Talanta, 134, 508-13, (2015). [cited by applicant]
M. Jolly et al., Fluorescence Polarization Immunoassay I. Monitoring Aminoglycoside Antibiotics in Serum and Plasma, Clin. Chem., 27(7):1190-1197, (1981). [cited by applicant]
J. Kada et al., Accurate Measurement of Avadin and Streptavadin in Biofluids with a New, Optimized Biotin Flourescein Congugate, Biochimicha et Biophysica Acta 1427, 33-43, (1999). [cited by applicant]
J. Kada et al., Rapid Estimation of Avadin and Streptavadin by Fluoresence Quenching of Fluoresence Polarization, iochimicha et Biophysica Acta 1427, 44-48, (1999). [cited by applicant]
E. Shaw et al., Estimation of Serum Gentamicin by Quenching Fluoroimmunoassay, J. Cin. Path., 30, 526-531, (1977). [cited by applicant]
M. Shipchandler et al., 4′-[Aminomethyl]fluorescein and Its N-Alkyl Derivatives: Useful Reagents in Immunodiagnostic Techniques, Anal. Bichem., 162, 89-101, (1987). [cited by applicant]
E. Ullman et al., Fluorescent Excitation Transfer Immunoassay, A General Method for Determination of Antigens, J. Bio. Chem., 251(14), 4172-4178, (1976). [cited by applicant]
I. Hemmilia, Fluoroimmunoassays and Immunofluorometric Assays, Clin. Chem., 31(3), 359-70, (1985). [cited by applicant]
Mikaela Nichkova, Competitive Quenching Fluorescence Immunoassay for Chlorophenols Based on Laser-Induced Fluorescence Detection in Microdroplets, Analytical Chemistry, American Chemical Society, US, vol. 75, No. 1, Jan… [cited by applicant]
Na Young Ji, Development of a fluorescent microsphere immunoassay for cystatin B (CSTB) in serum of patients with hepatocellular carcinoma, Clinical Chemistry and Laboratory Medicine, De Gruyter, De, vol. 49, No. 1, Jan… [cited by applicant]
Supplementary Partial European Search Report, May 22, 2024. [cited by applicant]
Kobayashi, Y. et al., Fluorescence Quenching Immunoassay of Serum Cortisol, Steroids, Jan. 10, 2003, pp. 177-183, vol. 36, No. 2. [cited by applicant]
Wei, A. P. et al., Use of Synthetic Peptides as Tracer Antigens in Fluorescence Polarization Immunoassays of High Molecular Weight Analytes, Analytical Chemistry, Dec. 1, 1993, pp. 3372-3377, vol. 65, No. 23. [cited by applicant]
Ji, N. Y. et al., Development of a Fluorescent Microsphere Immunoassay for Cystatin B (CSTB) in serum of patients with hepatocellular carcinoma. Clin Chem Lab Med, Oct. 20, 2010, pp. 151-155, vol. 49, No. 1. [cited by applicant]
Tianjin Yian Biotechnology Co Ltd, CN110596396A English Translation, 0Protein detection method, test strip and kit, Jul. 19, 2022, page one. [cited by applicant]
Slade, Daniel J. et al., Chemical and biological methods to detect posttranslational modifications of arginine, Biopolymers, Feb. 2014, pp. 133-143, vol. 101, No. 2. [cited by applicant]