EP-4739992-A1 - DETECTION OF TARGET MOLECULES IN DRIED BIOLOGIC MATRICES

Abstract

Methods for detection of target molecules in dried biologic matrices in proteomic based assays are provided. Such methods have a wide utility in proteomic applications for research and development, diagnostics and therapeutics by providing methods for the preparation of dried biologic matrices for detection of target molecules in a proteomic assay and to methods of detecting one or more target molecules from dried biologic matrices in a proteomic assay.

Inventors

• SMITH, Weston J.
• DROLET, Daniel W.

Assignees

• SomaLogic Operating Co., Inc.

Dates

Publication Date

20260513

Application Date

20240703

Claims (20)

1. 1. A method of preparing a biological sample for a multiplex assay comprising: depositing the biological sample comprising a plurality of target molecules onto a collection device; drying the biological sample on the collection device for a period of time to stabilize the dried sample prior to any temperature fluctuation; wherein the biological sample is dried for at least 4 hours at about room temperature or below room temperature so that target molecules in the dried biological sample are detectable in the multiplex assay.
2. 2. The method of claim 1, wherein the biological sample is dried at about room temperature, 4°C-8°C, or -20°C.
3. 3. The method of claim 1 or claim 2, wherein the dried biological sample is stored at about room temperature or below room temperature prior to detection in the multiplex assay.
4. 4. The method of any one of claims 1-3, wherein the dried biological sample is stored at about 4°C-8°C, or -20°C.
5. 5. The method of any one of claims 1-4, wherein the biological sample is dried for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, one day, at least two days, or at least three days.
6. 6. The method of any one of claims 1-5, wherein the biological sample is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal washes, nasal aspirate, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, bronchial brushing, synovial fluidjoint aspirate, organ secretions, cells, a cellular extract, and cerebrospinal fluid.
7. 7. The method of any one of claims 1-5, wherein the biological sample is selected from plasma, serum, urine, and whole blood.
8. 8. The method of any one of claims 1-7, wherein the plurality of target molecules are selected from a protein, a peptide, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a bacteria, a metabolite, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, a cell and a tissue.
9. 9. The method of any one of claims 1-8, wherein the dried biological sample is homogenized by the drying process.
10. 10. The method of any one of claims 1-9, wherein the plurality of target molecules are extractable from the collection device after the biological sample is dried and detectable in the multiplex assay.
11. 11. A method of detecting a plurality of target molecules comprising: extracting target molecules in a dried biological sample from a collection device; diluting the extracted target molecules into a first dilution and a second dilution; contacting the first dilution with a first capture reagent to form a first capture reagent affinity complex with its target molecule if the target molecule is present in the first dilution; contacting the second dilution with a second capture reagent to form a second capture reagent affinity complex with its target molecule if the target molecule is present in the second dilution; incubating the first and second dilution samples separately to allow capture reagent affinity complex formation; wherein each of the first capture reagent affinity complex and the second capture reagent affinity complex are immobilized on separate first solid supports; releasing and capturing the first capture reagent affinity complex on a second solid support; after releasing the first capture reagent affinity complex, releasing and capturing the second capture reagent affinity complex on the second solid support; and detecting for the presence of or determining the level of the first capture reagent, second capture reagent of the first or second capture reagent affinity complexes, or the presence or amount of the first or second capture reagent affinity complexes.
12. 12. The method of claim 11, wherein the target molecules are extracted from the collection device in a formulation for at least 5 minutes.
13. 13. The method of claim 11 or claim 12, wherein the target molecules are extracted from the collection device in a formulation for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, at least 150 minutes, at least 160 minutes, at least 170 minutes, at least 180 minutes, at least 190 minutes, or at least 200 minutes.
14. 14. The method of claim 12 or claim 13, wherein the formulation comprises a buffering agent, one or more salts, a chelating agent, a protease inhibitor, a non-ionic surfactant, and an oligonucleotide.
15. 15. The method of claim 14, wherein the one or more salts are each independently selected from a sodium salt, a potassium salt and a magnesium salt.
16. 16. The method of claim 14, wherein, the one or more salts comprise a sodium salt, a potassium salt and a magnesium salt.
17. 17. The method of claims 15 or 16, wherein the sodium salt is NaCl, the potassium salt is KC1 and the magnesium salt is MgCh.
18. 18. The method of claim 17, wherein NaCl in the formulation is at a concentration of from about 10 mM to about 500 mM, or from about 50 mM to about 250 mM, or from about 100 mM to about 200 mM, or from about 75-125 mM, or about 100 mM.
19. 19. The method of claim 17 or claim 18, wherein the KC1 in the formulation is at a concentration of from about 0.5 mM to about 30 mM, or from about 1 mM to about 20 mM, or from about 2 mM to about 15 mM, or from about 4 mM to about 10 mM, or about 5 mM.
20. 20. The method of any one of claims 17-19, wherein the MgCh in the formulation is at a concentration of from about 0.5 mM to about 30 mM, or from about 1 mM to about 20 mM, or from about 2 mM to about 15 mM, or from about 4 mM to about 10 mM, or about 8 mM.

Description

DETECTION OF TARGET MOLECULES IN DRIED BIOLOGIC MATRICES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of priority of US Provisional Patent Application Nos. 63/525,341, filed July 6, 2023, and 63/530,113, filed August 1, 2023, each of which is incorporated by reference herein in its entirety for any purpose. FIELD [0002] The present disclosure relates generally to the field of proteomic assays, and methods for detection of target molecules in dried biologic matrices. Such methods have a wide utility in proteomic applications for research and development, diagnostics, and therapeutics. Specifically, methods are provided for the preparation of dried biologic matrices for detection of target molecules in a proteomic assay and to methods of detecting target molecules from dried biologic matrices in a proteomic assay. BACKGROUND [0003] Assays directed to the detection and quantification of physiologically significant molecules in biological samples and other sample types are important tools in scientific research and in the health care field. For example, multiplex array assays employ surface bound probes to detect target molecules in a sample. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies, affibodies, aptamers or other molecules (collectively biopolymers) capable of binding with target molecules from the sample. These binding interactions are the basis for many of the methods and devices used in a variety of different fields, e.g., genomics, transcriptomics and proteomics. [0004] Assays using solution-based samples typically require maintaining the solution-based samples at -80°C prior to the assay, including shipping and storage. In addition, frozen solutionbased samples that include cells, such as whole blood, may have lysis of the cells with the freeze-thaw sample processing, the samples may have insufficient homogenization of the cells. Improper storage of solution-based samples and incomplete homogenization of cells within solution has the potential to introduce pre-analytical variation on the target molecules withing the solution-based samples. [0005] This disclosure describes methods to eliminate the need for -80°C shipping and storage of samples prior to the assay. This disclosure describes methods to eliminate or reduce incomplete homogenization of cells within solution. This disclosure describes methods for detecting target molecules within the samples extracted from dried biologic matrices. SUMMARY [0006] The foregoing and other objects, features, and advantages of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. [0007] Certain non-limiting exemplary embodiments are as follows: [0008] In some embodiments, methods of preparing a biological sample for a multiplex assay are disclosed. The methods comprise depositing the biological sample comprising a plurality of target molecules onto a collection device, drying the biological sample on the collection device for a period of time to stabilize the dried sample prior to any temperature fluctuation, wherein the biological sample is dried for at least 4 hours at about room temperature or below room temperature so that target molecules in the dried biological sample are detectable in the multiplex assay. [0009] In some embodiments, the biological sample is dried at about room temperature, 4°C- 8°C, or -20°C. In some embodiments, the dried biological sample is stored at about room temperature or below room temperature prior to detection in the multiplex assay. In some embodiments, the dried biological sample is stored at about 4°C-8°C, or -20°C. [0010] In some embodiments, the biological sample is dried for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, one day, at least two days, or at least three days. [0011] In some embodiments, the biological sample is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal washes, nasal aspirate, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, bronchial brushing, synovial fluidjoint aspirate, organ secretions, cells, a cellular extract, and cerebrospinal fluid. In some embodiments, the biological sample is selected from plasma, serum, urine, and whole blood. In some embodiments, the plurality of target molecules are selected from a protein, a peptide, a carbohydrate, a polysaccharide, a glycoprotein, a hormone, a receptor, an antigen, an antibody, a virus, a bacteria, a metabolite, a cofactor, an inhibitor, a drug, a dye, a nutrient, a growth factor, a cell and a tissue. [0012] In some embodiments, the dried biological sample is h