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WO2024243119A2 - Produits et méthodes de conservation et de détection de biomolécules dans des échantillons de salive humaine - Google Patents

Produits et méthodes de conservation et de détection de biomolécules dans des échantillons de salive humaine Download PDF

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Publication number
WO2024243119A2
WO2024243119A2 PCT/US2024/030180 US2024030180W WO2024243119A2 WO 2024243119 A2 WO2024243119 A2 WO 2024243119A2 US 2024030180 W US2024030180 W US 2024030180W WO 2024243119 A2 WO2024243119 A2 WO 2024243119A2
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composition
mixture
agent
amount equal
still
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WO2024243119A3 (fr
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David Vu
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Spectrum Solutions LLC
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Spectrum Solutions LLC
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    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • the present disclosure relates to preserving biomolecules, particularly proteins, antibodies, lipoproteins, enzymes, hormones, steroid hormones, protein hormones, peptide hormones, and so forth. Specifically, the present disclosure relates to compositions and methods for preserving biomolecules in a biological sample, and particularly to compositions and methods for preserving human proteins, antibodies, lipoproteins, enzymes, hormones, hormone proteins, and so forth in a human saliva sample, for further analysis.
  • biomolecules such as proteins, antibodies, lipoproteins, enzymes, hormones, steroid hormones, protein hormones, peptide hormones, and so forth.
  • Biological samples including blood, bone marrow, tissue samples, biopsies, saliva, and other solid and fluid samples, may contain one or more types of cellular and/or cell-free biomolecules in a wide range of concentrations. With proper biochemical handling techniques and reagents, these biomolecules can be obtained, extracted, and/or isolated from the samples for detection, analysis, quantification, diagnostics, and/or further use in therapeutic or other medical or research use.
  • Extracted antibodies, hormones, and other biomolecules can be used for a variety of analytical purposes, including detection, quantification, and/or diagnosis of infection, immune response, disease, or other medical condition(s). Extraction of antibodies, hormones, and other biomolecules from saliva can be particularly useful, as saliva sample collection is relatively non-invasive.
  • Antibodies can be directed against or have specificity for one or more viral strains, including strains of: coronavirus (Coronaviridae), such as the severe acute respiratory syndrome (or SARS)-associated coronavirus SARS-CoV (e.g., SARS-CoV-2, which is known to have caused the coronavirus disease of 2019 (COVID-19), as well as the UK and/or South African variant(s) thereof, etc.), the Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), and others; filovirus (Filoviridae), which is known to cause severe viral
  • coronavirus coronaviridae
  • SARS severe acute respiratory syndrome
  • SARS-associated coronavirus SARS-CoV e.g., SARS-CoV-2, which is known to have caused the coronavirus disease of 2019 (COVID-19), as well as the UK and/or South African variant(s) thereof, etc.
  • MERS Middle East respiratory syndrome
  • MERS-CoV Middle
  • VHF hemorrhagic fever
  • Cuevavirus including Cuevavirus, Marburgvirus, and Ebolavirus, and species/subtypes thereof (e.g., Zaire ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, formerly Cote d’Irete ebolavirus ⁇ , Bundibugyo ebolavirus), Reston ebolavirus), and Bombali ebolavirus),' Lentivirus, such as human immunodeficiency viruses (HIV), adenovirus (Adenoviridae), Flavivirus, such as Dengue, West Nile viruses, and Zika virus; and so forth.
  • Antibodies or other protein markers against or associated with bacterial, inflammatory, autoimmune, and other diseases have also gained some attention.
  • hormones such as cortisol, estradiol, progesterone, and others, peptide hormones, such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGF- alpha), and cytokines, such as interleukin-8 and leptin, may also be detected and/or quantified in biological samples, including saliva.
  • EGF epidermal growth factor
  • TGF- alpha transforming growth factor-alpha
  • cytokines such as interleukin-8 and leptin
  • Antibody-, hormone-, and other biomolecule-containing biological samples including saliva samples, often need to be properly processed for specific types of analysis.
  • Analytical techniques such as mass spectrometry, immunoassays, and others, may require specific processing or pre-processing steps that depend on the specific platform to be used.
  • the antibody-containing biological samples may need to be processed in order to stabilize the sample or proteins thereof. Stabilizing solutions may need to be added to antibodycontaining biological samples to ensure survival of a portion of the antibody portions until analysis thereof can be performed.
  • Existing protein-stabilizing solutions may not be optimal for certain types of biological samples and/or certain analytical techniques or devices for performing the same. For instance, a stabilizing solution formulated for optimal or suitable analysis in a certain mass spectrometers, may not be optimal or suitable for analysis in immunoassays, and vice versa. In some cases, improper formulation may produce or lead to analytical artifacts and/or high background signal (or noise).
  • Existing protein-stabilizing solutions may also be deficient in preserving antibodies, specifically, or for controlling microbial (e.g., bacterial, fungal) growth or life. Biological sample, such as saliva, often include and/or become contaminated with one or more microbes (e.g., bacteria, fungi, etc.).
  • microbes contain proteins and nucleic acids that may interfere with or be detected along with the antibodies and other proteins in the biological sample.
  • Preservation solutions may inadvertently stabilize bacterial or fungal proteins or even permit the growth of the microorganisms, thereby enriching the sample for interfering proteins.
  • the biological sample may contain nucleic acid of the subject,
  • SUBSTITUTE SHEET (RULE 26) host, or source of the biological sample (e.g., human) that may interfere with or be detected along with the proteins or antibodies in the biological sample.
  • Embodiments of the present disclosure solve one or more of the foregoing or other problems in the art with one or more biomolecule preservation, stabilization, and/or preparation compositions, kits comprising the same, and methods of manufacturing and using the same.
  • some embodiments of the present disclosure include compositions for preserving, stabilizing, and/or preparing proteins, antibodies, lipoproteins, enzymes, hormones, hormone proteins, and so forth, in a biological sample, such as saliva, blood (e.g., capillary blood), etc.
  • the composition(s) of the present disclosure can be suitable for use in a variety of analytical techniques and devices.
  • the composition can yield high amounts of protein (e.g., antibodies) for subsequent analysis.
  • the composition can yield high amounts of human antibodies (or immunoglobulins) or human hormones (e.g., Cortisol, Estradiol, Testosterone, DHEA, Progesterone, etc.), preferably and/or optionally with low amounts of microbial (e.g., bacterial, fungal) proteins or other contaminants, for subsequent analysis.
  • the composition can comprise a liquid mixture, solution, or water-based (e.g., aqueous) liquid suitable for use in the stabilization of protein (e.g., antibodies, Ig), hormone, etc. and/or prevention of bacterial and/or fungal contamination and/or for long term storage.
  • embodiments (or composition, kit, or method, etc.) of the present disclosure can be suitable for use of the in the detection, analysis, quantification, and/or measurement of antibodies against viral strains, including strains of: coronavirus (Coronaviridae), such as the severe acute respiratory syndrome (or SARS)-associated coronavirus SARS-CoV (e.g., SARS-CoV-2, which is known to have caused the coronavirus disease of 2019 (COVID-19), as well as the UK and/or South African variant(s) thereof, etc.), the Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), and others; filovirus (Filoviridae), which is known to cause severe viral hemorrhagic fever (VHF), including Cuevavirus, Marburgvirus, and Ebolavirus, and species/subtypes thereof (e.g., Zaire
  • SUBSTITUTE SHEET (RULE 26) ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, formerly Cote d’Ilude ebolavirus), Bundibugyo ebolavirus), Reston ebolavirus), and Bombali ebolavirus),' Lentivirus, such as human immunodeficiency viruses (HIV), adenovirus (Adenoviridae), Flavivirus, such as Dengue, West Nile viruses, and Zika virus; and so forth, as well as antibodies or other protein markers against or associated with bacterial, inflammatory, autoimmune, and other diseases (e.g., Celiac disease, Lyme disease, etc.) have also gained some attention.
  • Lentivirus such as human immunodeficiency viruses (HIV), adenovirus (Adenoviridae), Flavivirus, such as Dengue, West Nile viruses, and Zika virus; and so forth, as well as antibodies or other protein markers against or associated with bacterial, inflammatory,
  • embodiments (or composition, kit, or method, etc.) of the present disclosure can be used in connection with detection, analysis, quantification, and/or measurement of human antibodies against the novel coronavirus leading to COVID-19 and its variants, and other human or mammalian virus, pathogens, or infectious diseases, particularly in human saliva, and specifically in expectorated human saliva.
  • Embodiments of the present disclosure can, therefore, include, (human) antibody preservation compositions, methods, kits, etc. as set forth herein.
  • embodiments (or composition, kit, or method, etc.) of the present disclosure can be suitable for use of the in the detection, analysis, quantification, and/or measurement of (human) biomolecule analytes, such as proteins, hormones, steroid hormones, protein hormones, peptide hormones, lipoproteins, enzymes, and so forth, particularly inhuman saliva or blood, and specifically in expectorated human saliva or capillaiy blood.
  • biomolecules include Cortisol, Estradiol, Testosterone, DHEA, Progesterone, and so forth.
  • Embodiments of the present disclosure can, therefore, include, (human) biomolecule analyte preservation compositions, methods, kits, etc. as set forth herein.
  • compositions and methods can preserve or stabilize the biomolecule(s) against degradation and/or loss.
  • the compositions and methods can preserve or stabilize the biomolecule(s) against degradation and/or loss (1) for a period of time, such as (at least, or between) 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 28 days, and/or 30 days, and/or at room temperature or without (constant) refrigeration or freezing.
  • the compositions and methods can provide and/ or result in high detectable amounts of the biomolecule (from a (human and/or expectorated) saliva sample).
  • the compositions and methods can preserve the biomolecule(s) in a manner consistent and/or compatible with post-preservation, qualitative and/or quantitative testing, analysis, and/or measurement of the same.
  • SUBSTITUTE SHEET (RULE 26) “enzymes,” or other biomolecule, specifically, may, unless clearly indicated to the contrary, serve as a reference to any other or all biomolecules, generally, as described herein.
  • embodiments of the present disclosure can be used in connection with antibody preservation, detection, and/or analysis from expectorated saliva samples, such as expectorated human saliva samples.
  • antibody detection, quantification, etc. can be more effective using expectorated saliva in accordance with embodiments of the present disclosure. It will be appreciated, however, that nasal, oral, pharyngeal, etc. swabs are also contemplated herein.
  • a composition for antibody detection, preservation and/or analysis includes (1) water (e.g., DNAse free water), (2) a chelating agent, such as ethylenediaminetetraacetic acid (EDTA) (e.g., EDTA dihydrate, EDTA disodium salt, or EDTA disodium salt dihydrate), (3) a buffering agent, such L-histidine (e.g., L-histidine monohydrochloride monohydrate), (4) a biocidal and/or biostatic agent (e.g., against bacteria, virus, fungi, and/or yeast), such as benzalkonium chloride (e.g., a 50% solution), cetylpyridinium chloride, sodium azide, 5-chloro-2-methyl-4-thiazoline-3-ketone (EMIT), 2- methyl-4-thiazoline-3-ketone (MIT), a mixture of CMIT and MIT (e.g., a ProCiinTM, such as Pro
  • EDTA ethylenediaminet
  • the pH of the composition can be (or be adjusted to) a pH of around 6 (e.g., pH 6 +/- ⁇ 1).
  • the composition can include a visual indicator, dye or coloring agent, preferably bromophenol blue or FD&C blue #1.
  • the carrier or water can be included quantum satis (q.s.) 100%, wt/wt, or in an amount of about 95%-99%, wt/wt, about 96%-99%, wt/wt, about 97%-99%, wt/wt, or about 98%-99%, wt/wt.
  • the water can be included in the composition in an amount of about 98.5%-99%, wt/wt.
  • the chelating agent or EDTA such as EDTA disodium dihydrate
  • the chelating agent or EDTA can be included in an amount of about 0.001%-l%, wt/wt, about 0.005%-0.05%, wt/wt, about 0.01%-0.10%, wt/wt, about 0.02%-0.10%, wt/wt, about 0.025%-0.06%, wt/wt, about 0.04%-0.05%, wt/wt, or in an amount of about 0.04086%-0.04994% (or about 0.0409%- 0.0499%), wt/wt.
  • the EDTA (disodium dihydrate) can be included in an amount of about 0.0454% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt. It will be appreciated that anhydrous, monohydrate, trihydrate, etc. or other forms of EDTA may also be suitable for use in certain embodiments, and that suitable and
  • SUBSTITUTE SHEET (RULE 26) preferred ranges or amounts may be adjusted for equivalents.
  • alternative chelating agents as known in the art and/or as disclosed and described herein, can be used in additional or alternative embodiments.
  • the concentration of chelating agent (e.g., EDTA) in the composition can be sufficient to provide a final concentration of about 0.03243% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, about 0.01513% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, or about 0.01%-0.04%, wt/wt, or about 0.0125 %-0.035%, wt/wt, chelating agent in a preserved saliva sample (i.e., a mixture of saliva and preservation solution) or a preserved blood sample (i.e., a mixture of blood and preservation solution).
  • a preserved saliva sample i.e., a mixture of saliva and preservation solution
  • a preserved blood sample i.e., a mixture of blood and preservation solution
  • the concentration of chelating agent in the composition can be about 0.0454% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt.
  • the buffering agent or L-histidine such as L-histidine hydrochloride, L-histidine monohydrate, or L-histidine monohydrochloride monohydrate
  • the buffering agent or L-histidine can be included in an amount of about 0.1 %-3%, wt/wt, about 0.70%-1.40%, wt/wt, about 0.70%- 1.30%, wt/wt, about 0.80%-1.30%, wt/wt, about 0.90%-1.25%, wt/wt, about 0.95%-1.25%, wt/wt, or about 0.9819%-1.2001%, wt/wt.
  • the L-histidine can be included in an amount of about 1.091% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt. It will be appreciated that other forms of L-histidine may also be suitable for use in certain embodiments, and that suitable and preferred ranges or amounts may be adjusted for equivalents. Those skilled in the art will appreciate that alternative buffering agents, as known in the art and/or as disclosed and described herein, can be used in additional or alternative embodiments.
  • the concentration of buffering agent (e.g., L-histidine) in the composition can be sufficient to provide a final concentration of about 0.77929% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, about 0.36367% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, or about 0. l%-0.
  • buffering agent e.g., L-histidine
  • a preserved saliva sample i.e., a mixture of saliva and preservation solution
  • a preserved blood sample i.e., a mixture of blood and preservation solution
  • concentration of buffering agent in the composition for a 2: 1 mixture of saliva: preservation solution (composition of the present disclosure) or a 1:2.5 mixture of (capillary) blood: preservation solution (composition of the present disclosure), the concentration of buffering agent in the composition (before
  • SUBSTITUTE SHEET (RULE 26) mixture, or 2: 1 dilution for saliva or 1:2.5 dilution for blood) can be about 1.091% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt.
  • the biocidal and/or biostatic agent can be or comprise benzalkonium chloride.
  • the benzalkonium chloride can be included in the composition in an amount of about 0.005%-0.2%, wt/wt.
  • the benzalkonium chloride can be included in the composition in an amount of about 0.03% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, or in an amount of about 0.09% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%).
  • the benzalkonium chloride can be included in the composition in an amount of about 0.01%, wt/wt, to about 0.06%, wt/wt, or in an amount of about 0.06%, wt/wt, to about 0.12%, wt/wt. In some embodiments, the benzalkonium chloride can be included in the composition in an amount of about 0.024%- 0.036%, wt/wt, (or, about 0.027%-0.033%, wt/wt), or in an amount of about 0.072%-0.108%, wt/wt, (or, about 0.081%-0.099%, wt/wt). In some embodiments, the benzalkonium chloride can be added to the composition as a 50% solution.
  • the concentration of benzalkonium chloride in the composition can be sufficient to provide a final concentration of about 0.03%, wt/wt, or about 0.027%-0.033%, wt/wt, or about 0.024%-0.036%, wt/wt, benzalkonium chloride in a preserved saliva sample (i.e., a mixture of saliva and preservation solution) or a preserved blood sample (i.e., a mixture of blood and preservation solution).
  • a preserved saliva sample i.e., a mixture of saliva and preservation solution
  • a preserved blood sample i.e., a mixture of blood and preservation solution
  • the concentration of benzalkonium chloride in the composition can be about 0.09%, wt/wt, about 0.081%-0.099%, wt/wt, or about 0.072%- 0.108%, wt/wt.
  • the concentration of benzalkonium chloride in the composition (before mixture, or 1: 1 dilution) can be about 0.06%, wt/wt, about 0.054%- 0.066%, wt/wt, or about 0.048%-0.072%, wt/wt.
  • the concentration of benzalkonium chloride in the composition can be sufficient to provide a final concentration of about 0.01%, wt/wt, of about 0.009%-0.011%, wt/wt, or about 0.08%-0.012%, wt/wt.
  • the concentration of benzalkonium chloride in the composition can be about 0.02%, wt/wt, about 0.018%-0.022%, wt/wt, or about 0.016%-0.024%, wt/wt.
  • concentration of benzalkonium chloride in the composition before mixture, or 2: 1 dilution
  • SUBSTITUTE SHEET (RULE 26) with saliva) can be about 0.03%, wt/wt, about 0.027%-0.033%, wt/wt, or about 0.024%- 0.036%, wt/wt.
  • the biocidal and/or biostatic agent can be or comprise 5-chloro- 2-methyl-4-thiazoline-3-ketone (CMIT), 2-methyl-4-thiazoline-3 -ketone (MIT), or a mixture of CMIT and MIT (e.g., a ProCiinTM, such as ProCiinTM 300).
  • CMIT 5-chloro- 2-methyl-4-thiazoline-3-ketone
  • MIT 2-methyl-4-thiazoline-3 -ketone
  • a mixture of CMIT and MIT e.g., a ProCiinTM, such as ProCiinTM 300.
  • the biocidal and/or biostatic agent can be or comprise a mixture of CMIT and MIT (e.g., a ProCiinTM, such as ProCiinTM 300).
  • the CMIT/MIT mixture (ProCiinTM), such as ProClinTM 300 can be included in the composition in an amount of about 0.01%-l%, wt/wt, preferably about 0.05%-0.5%, wt/wt, more preferably about 0.1%-0.2%, wt/wt, still more preferably about 0. 12%-0.
  • the CMIT/MIT mixture can be included in the composition in an amount of about 0.15% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt.
  • the CMIT/MIT mixture such as ProClinTM 300
  • the CMIT/MIT mixture can be included in the composition in an amount of about 0.3% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt.
  • the ProClinTM can be included in the composition in an amount of about 0.1%, wt/wt, to about 0.5%, wt/wt, or in an amount of about 0.2%, wt/wt, to about 0.4%, wt/wt.
  • the ProClinTM can be included in the composition in an amount of about 0.25%-0.35%, wt/wt, (or, about 0.27%-0.33%, wt/wt), or in an amount of about 0.285%-0.315%, wt/wt, (or, about 0.29%-0.31%, wt/wt).
  • the concentration of biocidal/biostatic agent in the composition can be sufficient to provide a final concentration of about 0.10714% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, or about 0.05000% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, biocidal/biostatic agent in a preserved saliva sample (i.e., a mixture of saliva and preservation solution).
  • a preserved saliva sample i.e., a mixture of saliva and preservation solution.
  • the concentration of biocidal/biostatic agent in the composition can be about 0.150% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, or about 0. 1 %-0.2%, wt/wt, biocidal/biostatic agent, or about 0. 1%-1%, wt/wt, or about 0.3% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, 0.25%-
  • SUBSTITUTE SHEET (RULE 26) 0.35%, wt/wt, (or, about 0.27%-0.33%, wt/wt), or about 0.285%-0.315%, wt/wt, (or, about 0.29%-0.31%, wt/wt).
  • biocidal and/or biostatic agent(s) as known in the art and/or as disclosed and described herein, can be used in additional or alternative embodiments.
  • the pH adjusting agent can be or comprise and acid (e.g., hydrochloric acid) or a base (sodium hydroxide).
  • the pH adjusting agent can be included in the composition q.s. to pH about 6.0, or pH 5-7, pH 5-6.9, pH 5-6.8, pH 5-6.5, pH 5. 1-6.9, pH 5.2-6.8, or pH S.3-6.7, pH 5.4-6.6, pH 5.5-6.5, pH S.6-6.4, pH S.7-6.3, pH 5.8-6.2, or pH 5.9- 6.1.
  • the pH adjusting agent can be included in the composition an amount of about 0.01%- 0.2%, wt/wt, or in an amount of about 0.01%-0.
  • the pH adjusting agent can be included in an amount of about 0.080%, wt/wt.
  • acid or basic pH adjusting agents can be used, as needed, to raise or lower the pH of the (otherwise final) composition to a target pH.
  • the necessary amount can be presented in terms of ”qs” or “q.s ”, as defined herein.
  • the necessary may vary depending on the amount of water and/or the amount, concentration, and/or strength of buffer present in the (otherwise final) composition.
  • the composition need not include an added acid or base.
  • the water and/or buffering agent can be sufficient to bring the composition to pH about 6.0, or pH 5-7, pH 5-6.9, pH 5-6.8, pH 5-6.5, pH 5.1-6.9, pH 5.2-6.8, or pH 5.3-6.7, pH 5.4-6.6, pH 5.5-6.5, pH 5.6-6.4, pH 5.7-6.3, pH 5.8-6.2, or pH 5.9-6. 1 (or any value or range of values therebetween).
  • the composition (without acid and/or base) can have a pH of about 6.0, or pH 5-7, pH 5-6.9, pH 5- 6.8, pH 5-6.5, pH 5. 1-6.9, pH 5.2-6.8, or pH S.3-6.7, pH 5.4-6.6, pH 5.5-6.5, pH 5.6-6.4, pH 5.7-6.3, pH 5.8-6.2, or pH 5.9-6.1.
  • the composition can optionally further comprise a visual indicator, coloring agent, or dye.
  • the visual indicator, coloring agent, or dye can be or comprise bromophenol blue or FD&C blue #1.
  • the bromophenol blue, FD&C blue #1, or other suitable dye, coloring agent, or visual indicator can be included in the composition at -0.0004% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, or -0.0009% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt.
  • the composition and/or mixture can be (substantially) devoid of (additional or any) antimicrobial(s) (e.g., bactericidal and/or bacteriostatic) agent(s) (e.g., besides or other than the benzalkonium chloride, cetylpyridinium chloride, sodium azide, 5-chloro-2-methyl-4-thiazoline-3-ketone (CMIT), 2-methyl-4-thiazoline-3-ketone (MIT), or mixture of CMIT and MIT (e.g., a ProClinTM, such as ProCiinTM 300)).
  • antimicrobial(s) e.g., bactericidal and/or bacteriostatic
  • antimicrobial(s) e.g., bactericidal and/or bacteriostatic agent(s)
  • CMIT 5-chloro-2-methyl-4-thiazoline-3-ketone
  • MIT 2-methyl-4-thiazoline-3-ketone
  • mixture of CMIT and MIT
  • One or more embodiments can be (substantially) devoid of (additional or any) ribonuclease inhibitor(s), or inhibitor(s) of ribonuclease).
  • One or more embodiments can be (substantially) devoid of (any) a protease(s) and/or protease inhibitor(s).
  • the compositions of the present disclosure can be formulated for ⁇ 2:1 (or between about 7.5:1 and about 1 :2) dilution with saliva or -1 :2.5 (or between about 10: 1 and about 5:3) dilution with blood.
  • the composition can be provided (e.g., in a collection device or kit) in an amount of about 0.5mL, or about 0.2mL to about l.OmL, or any volume therebetween.
  • the composition can be formulated so as to be combined or combinable with about l.OmL saliva (e.g., expectorated saliva), or about 0.5-1.5mL or about 0.7-1.3mL saliva, or any volume therebetween. In certain embodiments, the composition can be formulated so as to be combined or combinable with about 0.2mL blood (e.g., capillary blood), or about 0.1-0.3mL or about 0.16-0.24mL blood, or any volume therebetween.
  • saliva e.g., expectorated saliva
  • 0.2mL blood e.g., capillary blood
  • 0.1-0.3mL or about 0.16-0.24mL blood or any volume therebetween.
  • Table 1 provides illustrative example of a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of biomolecule analyte(s), preferably antibodies, hormones, etc., listing exemplary ingredients and their respective illustrative target concentrations and illustrative ( ⁇ 10%) minimum and maximum amounts.
  • the target pH of the illustrative composition in Table 1 can be about pH 6. Adjustments to amounts of sodium hydroxide to achieve (q.s.) pH 6 are contemplated herein.
  • Table 1.1 provides illustrative example of a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of biomolecule analyte(s), preferably antibodies, hormones, etc., listing exemplary ingredients and their respective amounts.
  • Table 1.1 provides illustrative example of a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of biomolecule analyte(s), preferably antibodies, hormones, etc., listing exemplary ingredients and their respective amounts.
  • Table 1.2 below, provides illustrative example of a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably protein (antibody, hormone, etc.), listing exemplary ingredients and their respective amounts.
  • Table 2 provides alternative illustrative example of a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their respective amounts.
  • analyte(s) e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their respective amounts.
  • Table 2.1 provides alternative illustrative example of a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their respective amounts.
  • analyte(s) e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their respective amounts.
  • the illustrative concentrations of ingredients/components listed in Table 1 and Table 2, above may be adjusted, as necessary, in order to provide a target final concentration when combined with a particular amount (volume) of saliva or other protein-containing sample.
  • the exemplary composition (ingredient ranges or amounts) listed in Tables 1-2.1 are formulated such that 0.5mL of the composition may be combined with ImL of saliva (i.e., a dilution ratio of 2 parts saliva to 1 part preservation solution).
  • the illustrative composition is suitable for
  • the illustrative composition may be concentrated or diluted, proportionally, depending on the amount of composition intended to be added to a particular amount (volume) of saliva or other protein-containing sample. For example, 0.25mL of a 2x concentrated embodiment of the composition of Table 1 may be suitable for combining with ImL of saliva, and so forth.
  • Table 3 below, provides additional illustrative examples of embodiments of the present disclosure.
  • Table 3 [0044] The amounts listed in Table 3 may be adjusted by ⁇ 1%, ⁇ 2%, ⁇ 5%, ⁇ 10%, ⁇ 15%, or
  • Table 3.1 below, provides additional illustrative examples of embodiments of the present disclosure.
  • Table 3.1 [0046] The amounts listed in Table 3.1 may be adjusted by ⁇ 1%, ⁇ 2%, ⁇ 5%, ⁇ 10%, ⁇ 15%, or
  • Table 3.2 below, provides additional illustrative examples of embodiments of the present disclosure.
  • the amounts listed in Table 3.2 may be adjusted by ⁇ 1%, ⁇ 2%, ⁇ 5%, ⁇ 10%, ⁇ 15%, or ⁇ 20%, or any percentage value or range of values therebetween.
  • Table 3.5 below, provides additional illustrative examples of embodiments of the present disclosure.
  • the amounts listed in Table 3.5 may be adjusted by ⁇ 1%, ⁇ 2%, ⁇ 5%, ⁇ 10%, ⁇ 15%, or ⁇ 20%, or any percentage value or range of values therebetween.
  • Table 3.6 below, provides additional illustrative examples of embodiments of the present disclosure.
  • Table 4 provides illustrative examples of a mixture comprising a biological sample (e.g., a saliva sample) and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a biological sample e.g., a saliva sample
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • Table 4.1 below, provides illustrative examples of a mixture comprising a biological sample (e.g., a saliva sample) and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a biological sample e.g., a saliva sample
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • Table 4.2 below, provides illustrative examples of a mixture comprising a biological sample (e.g., a saliva sample) and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a biological sample e.g., a saliva sample
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • Table 4.3 provides illustrative examples of a mixture comprising a biological sample (e.g., a saliva sample) and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a biological sample e.g., a saliva sample
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • Table 4.4 provides illustrative examples of a mixture comprising a biological sample (e.g., a saliva sample) and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a biological sample e.g., a saliva sample
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • Table 4.5 provides illustrative examples of a mixture comprising a biological sample (e.g., a saliva sample) and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a biological sample e.g., a saliva sample
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of protein analyte(s) (e.g., antibody, hormone, etc.), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • Table 5 provides illustrative example of a mixture comprising a biological sample (e.g., a saliva sample) and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a biological sample e.g., a saliva sample
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a biological sample e.g., a saliva sample
  • a composition of the present disclosure e.
  • Table 5.1 provides illustrative example of a mixture comprising a biological sample (e.g., a saliva sample) and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a biological sample e.g., a saliva sample
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth)
  • analyte(s) e.g., for use in preservation, detection, and/or analysis of analyte(s)
  • multi-omic analysis e
  • Table 5.2 below, provides illustrative example of a mixture comprising a saliva sample and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • Table 5.3 below, provides illustrative example of a mixture comprising a blood sample and a composition of the present disclosure (e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • a composition of the present disclosure e.g., for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth), listing exemplary ingredients and their final amounts, as diluted in exemplary use.
  • exemplary' mixtures can also be suitable for use in preservation, detection, and/or analysis of analyte(s), preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth).
  • analyte(s) preferably for multi-omic analysis (e.g., nucleic acid (DNA, RNA), protein (antibody, hormone, etc.), and so forth).
  • the composition (preferably for use in preserving protein analyte(s) (e.g., antibody), preferably in an ex vivo saliva sample, comprises 0.70%-1.30%, w/w, of a buffering agent; 0.01%-0. 15%, w/w; of an antibacterial agent; 0.02%-0.10%, w/w, of a chelating agent; a carrier qs to 100%; and pH ⁇ 5 to ⁇ 7.
  • a buffering agent 0.01%-0. 15%, w/w
  • an antibacterial agent 0.02%-0.10%, w/w, of a chelating agent
  • a carrier qs to 100%
  • the composition can have apH 5.1-6.9, preferably pH 5.2-6.8, more preferably pH 5.3-
  • the buffering agent can be or comprise L-histidine, preferably L-histidine hydrochloride or L-histidine monohydrochloride monohydrate.
  • the chelating agent can be or comprise ethyenediaminetetraacetic acid (EDTA), preferably EDTA disodium salt or EDTA disodium salt dihydrate.
  • EDTA ethyenediaminetetraacetic acid
  • the carrier can be or comprise an aqueous carrier, preferably comprising water, more preferably filtered, purified, distilled, and/or deionized water.
  • the composition can comprise, for example, 0.9819%-1.2001 %, w/w, of the buffering agent; 0.027%-0.033%, w/w; of the antibacterial agent; and/or 0.04086%-0.04994, w/w, of the chelating agent.
  • the composition can comprise, for example, about: 1.091%, w/w, of the buffering agent; 0.030%, w/w; of the antibacterial agent; and/or 0.0454, w/w, of the chelating agent.
  • the composition can further comprise a pH adjusting agent, preferably comprising an acid or a base, preferably hydrochloric acid or sodium hydroxide.
  • the pH adjusting agent can preferably comprise sodium hydroxide, preferably at 0.01%-0.2%, w/w, more preferably 0.072%-0.088, w/w, still more preferably 0.080%, w/w.
  • the ex vivo saliva sample can be or comprise (human or mammalian) saliva, preferably expectorated human saliva.
  • the protein can be or comprise an antibody or immunoglobulin.
  • the protein can be or comprise a peptide, hormone, or enzyme.
  • the composition is or can be: (i) substantially free or devoid of a or any mucolytic agent or reducing agent; (ii) substantially free or devoid of additional or any antimicrobial agent(s), bactericidal agent(s), and/or bacteriostatic agent(s) besides or other than the biocidal/biostatic agent (e.g, benzalkonium chloride and/or ProCiinTM (or, optionally, cetylpyridinium chloride)); (iii) substantially free or devoid of additional or any ribonuclease inhibitor(s) or inhibitor(s) of ribonuclease, the composition preferably substantially devoid of heparin, heparan sulfate, oligo (vinylsulfonic acid), poly(vinylsulfonic acid), oligo(vinylphosphonic acid), and/or poly(vinylsulfonic acid), or salt(s) thereof ); (iv) substantially free or devoid of
  • Embodiments may also or alternatively show passing result with good log reductions of all organisms in USP 51, antimicrobial effectiveness testing (e.g., post-dilution).
  • Some embodiments include a method of stabilizing protein or protein analyte(s) of interest (e.g., antibodies, hormones, enzymes, peptides, etc.).
  • the method can include providing a biological sample containing the protein or protein analyte(s) of interest and combining a composition of the present disclosure with the biological sample.
  • the method can also include one more processing steps (e.g., as known in the art or developed in connection with the present invention).
  • benzalkonium chloride, ProCiinTM, or other biocide may be (partially or completely) removed from the mixture prior to assaying the mixture for the protein analyte(s) of interest.
  • An embodiment of the present disclosure includes a method of stabilizing protein (e.g., antibody or immunoglobulin).
  • An embodiment comprises contacting a biological sample containing the protein with a composition of the present disclosure.
  • the biological sample comprises human (or mammalian) saliva.
  • compositions according to the present disclosure can be effective at preserving nucleic acid (e.g., DNA, RNA, etc.) for detection, in addition to proteins.
  • nucleic acid e.g., DNA, RNA, etc.
  • compositions with a lower final concentration of benzalkonium chloride or ProCiinTM in the final, diluted mixture of saliva and preservation fluid, e.g., -0.01%, wt/wt, or -0.1%, wt/wt
  • compositions with a higher final concentration of benzalkonium chloride or ProCiinTM in the final, diluted mixture of saliva and preservation fluid, -0.03%, wt/wt, or -0.3%, wt/wt), which may be more suited for protein-only, or antibody, preservation and detection).
  • kits can comprise a sample collection apparatus and a protein preservation composition.
  • the sample collection apparatus can comprise a solution compartment.
  • the protein preservation composition can be disposed in the solution compartment.
  • An embodiment of the present disclosure includes a kit comprising a composition of the present disclosure disposed in a portion of a sample collection apparatus.
  • Some embodiments include a method of manufacturing a composition of the present disclosure.
  • the method can include combining components of the present disclosure.
  • the method can also include other manufacturing steps known in the art.
  • An embodiment of the present disclosure includes a method of manufacturing a protein stabilization composition.
  • SUBSTITUTE SHEET (RULE 26) embodiment comprises obtaining a earner and adding to the carrier components or ingredients of a composition of the present disclosure.
  • embodiments of the present disclosure can be used in connection with protein preservation, detection, and/or analysis, as well as human nucleic acid (DNA and/or RNA) preservation, detection, and/or analysis, particularly from saliva samples, such as human or non-human animal (mammal) saliva samples.
  • DNA and/or RNA human nucleic acid
  • saliva samples such as human or non-human animal (mammal) saliva samples.
  • отно ⁇ ии such as antibodies against viral strains, including strains of: coronavirus (Coronaviridae), such as the severe acute respiratory syndrome (or SARS)-associated coronavirus SARS-CoV (e.g., SARS-CoV-2, which is known to have caused the coronavirus disease of 2019 (COVID- 19), as well as the UK and/or South African variant(s) thereof, etc.), the Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), and others; filovirus (Filoviridae), which is known to cause severe viral hemorrhagic fever (VHF), including Cuevavirus, Marburgvirus, and Ebolavirus, and species/subtypes thereof (e.g., Zaire ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, formerly Cote d’Irium ebolavirus), Bundibugyo ebola
  • coronaviridae coronaviridae
  • Embodiments of the present disclosure can, therefore, include, protein (e.g., antibody) preservation compositions, methods, kits, etc. as set forth herein.
  • the compositions and methods can preserve human antibodies against degradation and/or loss.
  • the compositions and methods can provide and/or result in high yield amounts of antibodies.
  • the compositions and methods can preserve antibodies in a manner consistent and/or compatible with post- preservation, qualitative and/or quantitative testing, analysis, and/or measurement of the same.
  • embodiments of the present disclosure can be used in connection with human or non-human animal (mammal) saliva samples. In some embodiments, rather than a nasal, oral, pharyngeal, etc.
  • swab (as used in connection with typical antibody detection methods), embodiments of the present disclosure can be used in connection with antibody preservation, detection, and/or analysis from expectorated saliva samples, such as expectorated human saliva samples. It will be appreciated, however, that biological samples of or collected from nasal, oral, pharyngeal, etc. swab is/are also contemplated herein. In at least one embodiment, protein yield, detection, quantification, etc. can be more effective using
  • SUBSTITUTE SHEET (RULE 26) expectorated saliva in accordance with embodiments of the present disclosure, including, for example, protein preservation composition(s) and/or methodologies.
  • Figure 1 illustrates preservation of HIV antigen/antibody in saliva using composition(s) of the present disclosure, demonstrating stability out to 14 days at room temperature.
  • Figure 2 illustrates high levels of correlation for antibody concentration on day 2 (typical delay time between sample collection and arrival at processing/analysis lab) at room temperature, and day 4 (typical delay time between sample collection and sample processing/analysis at the lab) at room temperature for COVID-19 IgG and IgA.
  • Figure 3 illustrates the exemplary data, comparing the non-colored solution to the colored (blue dyed) solution, and demonstrating that the addition of blue dye did not interfere with the assay.
  • Figure 4 illustrates the stability of microRNA in the illustrative solutions with and without colorant for 2 participants out to 4 days.
  • the dashed line indicates the lower limit of what may be considered an “adequate” sample.
  • the volume of microRNA went “below” the “adequate” limit at 1 hour to being acceptable at 24 hours.
  • inventive compositions may, in fact, help to liberate RNA from proteins and mucins that had bound and sequestered the material.
  • Figure 5 illustrates the recovery of cytokines (MIG) from saliva using an illustrative inventive solution (“Spectrum SPD”) — utilizing an illustrative collection device — against competitors “A” and “B”.
  • MIG cytokines
  • Figure 6 illustrates the recovery of cytokines (INF-y) from saliva using an illustrative inventive solution (“Spectrum SPD”) — utilizing an illustrative collection device — against competitors “A” and “B”.
  • Figure 7 illustrates room temperature stability of the cytokines of Figures 5 and 6 at room temperature.
  • the transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP ⁇ 2111.03.
  • the term “consisting essentially of’ when used in a claim of this disclosure is not intended to be interpreted to be equivalent to “comprising.”
  • SARS-CoV-2 refers to severe acute respiratory syndrome coronavirus 2. SARS-CoV-2 is the virus that causes COVID- 19.
  • CPE Cytopathic effect, i.e., structural changes in a host cell resulting from viral infection. CPE occurs when the infecting virus causes lysis (dissolution) of the host cell or when the cell dies without lysis because of its inability to reproduce.
  • sample refers to an animal; a tissue or organ from an animal; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; a solution containing one or more molecules derived from a cell, cellular material, or viral material (e.g. a polypeptide or nucleic acid); or a solution containing a naturally or non- naturally occurring nucleic acid, which is or can be assayed as described herein.
  • a sample may also be any bodily fluid or excretion that contains one or more cells, cell components, or nucleic acids, including, but not limited to cellular, nuclear, or cell-free nucleic acids.
  • bodily fluid is meant a naturally occurring fluid, including without limitation a liquid, semi-solid, aerated liquid, liquid-gas mixture, and so forth, from an animal (e.g., human or non-human animal or mammal).
  • Such bodily fluids can include, but are not limited to, saliva, sputum, serum, plasma, blood, urine, mucus, perspiration, tears or other ophthalmic fluids, otic fluids, puss (e.g., from a blister or sore), gastric fluids or juices, fecal fluids, pancreatic fluids or juices, semen, products of lactation or mensuration, spinal fluid, fluid bone marrow, or lymph.
  • ProClinTM is meant a microstatic (biostatic) preservative.
  • ProClinTM products contain active substances like EMIT and MIT which inhibit the Krebs cycle at four different check points, the enzymes pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase, and NADH dehydrogenase.
  • the inhibition of Krebs cycle leads to effective broad-spectrum biocidal activity against bacteria, fungi, and yeast.
  • the active components in ProClin preservative are two isothiazolones: 2-methyl-4-isothiazolin-3-one and 5-chloro-2- methyl-4- isothiazolin-3-one.
  • ProClinTM preservatives are water-soluble preparations of biocides that are generally used to increase the shelflife of reagents.
  • ProClinTM 300 for example, comprises of 3% of 5-chloro-2-methyl-4-isothiazohn-3-one (CMIT) and 2- methyl-4-isothiazloin-3-one (MIT) in a salt-free glycol carrier containing an alkly carboxylate stabilizer. It has a recommended working pH range of 2.5 - 8.5.
  • ProClin biocides are immediately microstatic upon contact with microbial organisms as a result of their ability to quickly penetrate cell membranes and inhibit specific enzymes crucial to cellular respiration.
  • Sputum is meant that mucoid matter contained in or discharged from the nasal or buccal cavity of a mammal. Sputum, as used herein, generally includes saliva and discharges from the respiratory passages, including the lungs.
  • saliva is meant the secretion, or combination of secretions, from any of the salivary glands, including the parotid, submaxillary, and sublingual glands, optionally mixed with the secretion from the buccal glands.
  • mucin any bodily fluid containing mucin.
  • mucoprotein any mucoprotein that raises the viscosity of the medium surrounding the cells that secrete it.
  • the term “about,” with regard to a value means +/-10% of the stated value or amount represented thereby.
  • the term “about” is used in connection with a percent concentration or composition of a component or ingredient (e.g., in a mixture, such as a fluid or liquid mixture, aqueous mixture, solution, etc., optionally or preferably measured as a w/w percent, w/v percent, v/v percent, etc.).
  • the term “about” and/or the term “+/-10%” implies and/or includes +/-10% of the stated numeric value, as opposed to +/-10 percentage points of the recited percent.
  • the term “about” and/or the term “+/-10%” implies and/or includes a recited range from 18g to 22g (i.e., from 18% w/w to 22% w/w), not a range of 10% w/w to 30% w/w.
  • the terms “approximately” and “substantially” represent or imply an (or any) amount close to the stated amount (e.g., that still performs a desired function or achieves a (desired or expected) result).
  • the terms “approximately” and “substantially” may refer to an amount that is within, or less than, 10%, 5%, 1%, 0. 1%, 0.01%, or other percent of a stated amount.
  • substantially devoid means (1) an undetectable or unquantifiable amount, (2) less than or below an amount generally considered by those skilled in the art to reflect a detectable or quantifiable amount, and/or (3) less than or below an amount generally considered by those skilled in the art to be functional
  • SUBSTITUTE SHEET (RULE 26) or able to achieve a (desired or expected) result (e.g., less than 10%, 5%, 1%, 0.1%, 0.01%, or other percent).
  • Quantum satis is meant the amount that is enough. Accordingly, a component or ingredient “qs 100%,” “provided at qs 100%,” or “qs to 100%” indicates that the component or ingredient is provided or included in an amount sufficient to complete the composition or to bring the total (of all components, whether recited or not) to 100%. It is noted, however, that a (final) component or ingredient “qs 100%,” “provided at qs 100%,” or “qs to 100%” does not indicate that the mixture consists of, consists essentially of, or only contains the components listed or recited immediately before the “qs 100%” component. In other words, “qs 100%,” and similar terms, is meant to be an open-ended expression indicating the source of the remainder, whatever that remainder may be.
  • alcohol is meant a water-miscible organic compound containing a hydroxyl group, including water-miscible mixtures of hydroxyl-containing organic compounds.
  • aqueous is meant a medium or matter that contains 30% or more water (by volume or by weight).
  • aqueous solution is meant a solution or suspension that contains 30% or more water by volume.
  • denaturing agent is meant a substance that alters the natural state of that to which it is added.
  • chaotropic agent is meant a molecule that exerts chaotropic activity.
  • molecules that exert chaotropic activity may disrupt the hydrogen-bonding network between water molecules, thereby affecting the stability of the native state of other molecules (in the solution), mainly macromolecules (proteins, nucleic acids) by weakening the hydrophobic effect.
  • molecules that exert chaotropic activity may have protein-denaturing activity (or be protein denaturants).
  • antimicrobial agent is meant a substance or group of substances which reduces the rate of growth of an organism compared to the rate of growth of the organism in their absence. A reduction in the rate of growth of an organism may be by at least 5%, more desirably, by at least 10%, even more desirably, by at least 20%, 50%, or 75%, and most desirably, by 90% or more.
  • the definition also extends to substances which affect the viability, virulence, or pathogenicity of an organism.
  • An antimicrobial agent can be natural (e.g., derived from bacteria or other source), synthetic, or recombinant. An antimicrobial agent can be bacteriostatic, bactericidal or both. An antimicrobial agent is bacteriostatic if it inhibits cell
  • An antimicrobial agent is bactericidal if it causes cell death. Cell death is commonly detected by the absence of cell growth in liquid growth medium (e.g., absence of turbidity) or on a solid surface (e.g., absence of colony formation on agar).
  • liquid growth medium e.g., absence of turbidity
  • a solid surface e.g., absence of colony formation on agar.
  • bacteriostatic substances are not bactericidal at any concentration.
  • composition includes products, formulations, and mixtures, as well as devices, apparatus, assemblies, kits, and so forth.
  • method includes processes, procedures, steps, and so forth.
  • a “feature” of the present disclosure or embodiment disclosed herein refers to a property, component, ingredient, element, part, portion, (method) step, or other aspect of the subject matter at hand.
  • the words “can” and “may” are used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must).
  • the terms “including,” “having,” “involving,” “containing,” “characterized by,” variants thereof (e.g., “includes,” “has,” and “involves,” “contains,” etc.), and similar terms as used herein, including the claims, shall be inclusive and/or open-ended, shall have the same meaning as the word “comprising” and variants thereof (e.g, “comprise” and “comprises”), and do not exclude additional, un-recited elements or method steps, illustratively.
  • SUBSTITUTE SHEET (RULE 26) contemplates and specifically discloses one, as well as two or more proteins. Similarly, use of a plural referent does not necessarily require a plurality of such referents, but contemplates, includes, and specifically discloses one, as well as two or more of such referents, unless the context clearly dictates otherwise.
  • embodiments of the present disclosure can comprise one or more combinations of two or more of the features described herein.
  • feature(s) and similar terms can include, for example, compositions, ingredients, components, elements, members, parts, portions, systems, methods, configurations, parameters, properties, and so forth.
  • Embodiments can include any of the features, options, and/or possibilities set out elsewhere in the present disclosure, including in other aspects or embodiments of the present disclosure. It is also noted that each of the foregoing, following, and/or other features described herein represents a distinct embodiment of the present disclosure.
  • SUBSTITUTE SHEET (RULE 26) about 10 units or between 0 and 10 units includes, illustratively, a specific disclosure of: (i) a measurement of 9 units, 5 units, 1 units, or any other value between 0 and 10 units, including 0 units and/or 10 units; and/or (ii) a measurement between 9 units and 1 units, between 8 units and 2 units, between 6 units and 4 units, and/or any other range of values between 0 and 10 units.
  • Embodiments of the present disclosure permit non-invasive saliva specimen collection for protein (antibody) preservation and analysis.
  • Embodiments of the present disclosure are herein shown to be effective in the collection of saliva samples, preservation of protein (e.g., antibody) for molecular analysis, and safe transportation of the biosample to laboratory for molecular testing.
  • Embodiments further provide high quality analytical results, including high purity, high yield, and/or low artifact results.
  • compositions can render sputum or saliva as a viable source of protein for purification and analysis.
  • the compositions provide the advantageous properties of chemical stabilization of protein. Chemical stabilization of the protein in a saliva sample can be achieved through the use of buffers, chelating agents, and antimicrobial agents, with pH adjusting agents used, as necessary, to target a suitable pH.
  • compositions of the present disclosure when mixed with a biological sample, e.g., saliva, can preserve the proteins in the sample, at room temperature, under ambient conditions, for extended periods of time. Samples can also be refrigerated, but freezing of the samples before protein recovery and purification is not required.
  • a biological sample e.g., saliva
  • the composition can include a carrier.
  • the carrier can be a liquid carrier or solvent, more preferably an aqueous carrier or solvent, still more preferably water.
  • the carrier can be or comprise purified, filtered (e.g., 0.2 micron filtered), distilled, and/or deionized water.
  • the composition can include a carrier.
  • the carrier can be or comprise water, such as filtered water, purified water, distilled water, or deionized water.
  • the composition can include a carrier qs to 100%. In some embodiments, the composition can include 95-99%, preferably 96-99%, more preferably 97- 99%, still more preferably 98.758%.
  • the composition can include one or more buffering agents (or buffers, pH buffers, etc.).
  • buffering agents include, but are not limited to: L-Histidine, such as L- Histidine monohydrochloride, L-Histidine monohydrate, or L-Histidine monohydrochloride monohydrate; tris(hydroxymethyl)aminomethane (also known as Tris; Tris base, 2-Amino-2- (hydroxymethyl)- 1,3 -propanediol, THAM, Trometamol) or a suitable formulation thereof (e.g., tris(hydroxymethyl)aminomethane hydrochloride, or Tris-HCl, ); Trizma® base (e.g., Tris 40% (w/w) stock solution in water); HEPES; BES; MOPS; HEPES; TAE; TBE; phosphate buffer; sodium borate buffer; sodium cacodylate buffer; and so forth.
  • the buffering agent can be or comprise L-Histidine,
  • the buffering agent can be in, have, comprise, or be provided in a dry, solid, powdered, anhydrous, and/or granular form.
  • the buffering agent can have a purity of at least, up to, and/or about 90%, 95%, 96%, 97%, 98%,
  • the buffenng agent can comprise or be (provided) in the form of a stock solution (e.g., in water) having any suitable concentration.
  • the buffering agent can have a purity substantially corresponding to the concentration of the buffering agent in solution (as measured by a suitable material assay, such as CoA).
  • the buffering agent can be included in the composition at about 1.091% ( ⁇ 10%), w/w, or in a range of about 0.7% to about 1.4%, preferably about 0.75% to about 1.35%, more preferably about 0.8% to about 1.3%, still more preferably about 0.85% to about 1.25%, still more preferably about 0.9% to about 1.2%, still more preferably about 0.95% to about 1.2%, still more preferably about 0.98% to about 1.2%, w/w, still more preferably about 0.9819% to about 1.2001 %, w/ w.
  • the composition can include a chelating agent (or chelator).
  • the chelating agent can be or comprise ethyenediaminetetraacetic acid (EDTA) or suitable salt and/or hydrate thereof. More preferably, the chelating agent can be or comprise, or be provided as EDTA disodium salt. Still more preferably, the chelating agent can be or comprise, or be provided as EDTA disodium (salt) dihydrate.
  • the chelating agent can be or comprise ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'- tetraacetic acid (EGTA), nitrilotriacetic acid (NT A), an ethylenediamine (or 1,2- diaminoethane), and so forth.
  • the chelating agent comprises, includes, or is provide with a counter ion (e.g., sodium).
  • the chelating agent comprises, includes, or is provide as a hydrate (e.g., dihydrate).
  • the composition can include one or more chelating agents.
  • the chelating agent of the composition can be selected from the group consisting of ethylenediamine tetraacetic acid (EDTA), cyclohexane diaminetetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTP A), tetraazacyclododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetetraacetic acid (TETA), desferri oximine, nitrilotriacetic acid (NT A), an ethylenediamine (or 1,2- diaminoethane), or respective chelator analogs, salts, and/or hydrates thereof.
  • the chelating agent can be or comprise EDTA (e.g., as EDTA disodium salt, preferably as EDTA disodium (salt) dihydrate).
  • the chelating agent comprises, includes, or is provide with a counter ion (e.g., sodium).
  • the chelating agent comprises, includes, or is provide as a hydrate (e.g., dihydrate).
  • the chelating agent can be in, have, comprise, or be provided in a dry, solid, powdered, anhydrous, and/or granular form.
  • the chelating agent can have a purity of at least, up to, and/ or about 90%, 95 %, 96%, 97%, 98%, 99%, 99.1 %, 99.2%. 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (as measured by a suitable material assay, such as CoA).
  • the chelating agent can comprise or be (provided) in the form of a stock solution (e.g., in water) having any suitable concentration.
  • the chelating agent can have a purity substantially corresponding to the concentration of the chelating agent in solution (as measured by a suitable material assay, such as CoA).
  • the chelating agent e.g., EDTA
  • the chelating agent can be included in the composition at about -0.0454% ( ⁇ 10%), w/w, or in a range of about 0.01% to about 0.10%, w/w, preferably about 0.015% to about 0.09%, w/w, more preferably about 0.02% to about 0.08%, w/w, still more preferably about 0.025% to about 0.07%, w/w, still more preferably about 0.03% to about 0.06%, w/w, still more preferably about 0.035% to about 0.05%, w/w, still more preferably about 0.04% to about 0.05%, w/w, still more preferably about 0.04086% to about 0.04994%, w/w.
  • the composition can include (about) 0.0454%, w/w, EDTA (e.g., disodium salt dihydrate, or anhydrous).
  • the composition can include a biocide or antimicrobial (e.g., antibacterial) agent.
  • a biocide or antimicrobial agent e.g., antibacterial
  • the biocide, antimicrobial or antibacterial agent can be or comprise benzalkonium chloride (e.g., 50% solution, or equivalent) and/or a ProClinTM.
  • the antimicrobial or antibacterial agent e.g., benzalkonium chloride
  • the antimicrobial or antibacterial agent can be included in the composition at a concentration of about 0.03% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, about 0.09% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, or between about 0.03%-0.09%, wt/wt.
  • the antimicrobial or antibacterial agent e.g., benzalkonium chloride
  • the antimicrobial or antibacterial agent can be included in the composition at a concentration of about 0.01-0.15% or about 0.027-0.033%, wt/wt.
  • the antimicrobial or antibacterial agent e.g., benzalkonium chloride
  • the antimicrobial or antibacterial agent e.g., ProClinTM
  • the antimicrobial or antibacterial agent can be included in the composition at a concentration of about 0.3% ( ⁇ 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), wt/wt, or between about 0.1%-0.9%, wt/wt.
  • the antimicrobial or antibacterial agent e.g., ProClinTM
  • SUBSTITUTE SHEET (RULE 26) concentration of about 0.25%-0.35%, wt/wt, (or, about 0.27%-0.33%, wt/wt).
  • the antimicrobial or antibacterial agent e.g., ProClinTM
  • the antimicrobial or antibacterial agent can be included in the composition at a concentration of about 0.285%-0.315%, wt/wt, (or, about 0.29%-0.31%, wt/wt).
  • ProClinTM preservatives are water-soluble formulations of biocides that are among the most effective biocides in the IVD industry, used in over 1,000 FDA registered IVD kits from industry-leading diagnostic manufacturers. At low working concentrations, ProClinTM products help extend the shelflife of IVD reagents by effectively and immediately inhibiting a broad spectrum of microbes. Unlike other biocides, ProClinTM preservatives present reduced health hazards, toxicology problems, and disposal issues at recommended usage levels. ProClinTM biocides attack the Krebs cycle at four key points: the enzymes pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase, and NADH dehydrogenase. Because all bacteria and fungi possess at least part of the Krebs cycle, they are broad spectrum in their activity.
  • cetylpyridinium chloride or sodium azide may be used in the place of benzalkonium chloride and/or ProClinTM.
  • the composition or mixture can be devoid of a biocide, such as benzalkonium chloride, cetylpyridinium chloride, and/or ProClinTM. pH adjusting agents - acids and bases
  • the composition can include an acid or a base.
  • the acid can be or comprise hydrochloric acid (HC1) and/or the base can comprise sodium hydroxide.
  • the base sodium hydroxide
  • the base can be in, have, comprise, or be provided in a dry . solid, pellet, powdered, anhydrous, and/or granular form.
  • the base can have a purity of at least, up to, and/or about 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%.
  • the base can comprise or be (provided) in the form of a stock solution (e.g., in water) having any suitable concentration (e.g., about 10%, 15%, 20%, 25%, 30%, 32%, 35%, 37%, 38%, 40%, or 45%, w/w, aqueous solution (e.g., in water).
  • a stock solution e.g., in water
  • any suitable concentration e.g., about 10%, 15%, 20%, 25%, 30%, 32%, 35%, 37%, 38%, 40%, or 45%, w/w
  • aqueous solution e.g., in water
  • the composition can include acid (e.g., hydrochloric acid or other source of H+) or base (e.g., sodium hydroxide or other source of -OH) qs to pH about 6.0, or pH 5-7, pH 5. 1-6.9, pH 5.2-6.8, or pH 5.3-6.7, pH 5.4-6.6, pH 5 5-6.5, pH 5.6-6.4, pH 5.7-6.3, pH 5.8-6.2, or pH 5.9-6.1 (or any value or range of values therebetween).
  • acid e.g., hydrochloric acid or other source of H+
  • base e.g., sodium hydroxide or other source of -OH
  • strong acids are hydrochloric acid (HC1), hydroiodic acid (HI), hydrobromic acid (HBr), perchloric acid (HCIO4), nitric acid (HNO3) and sulfuric acid (H2SO4).
  • HC1 hydrochloric acid
  • HI hydroiodic acid
  • HBr hydrobromic acid
  • HCIO4 perchloric acid
  • HNO3 nitric acid
  • sulfuric acid H2SO4
  • HC1 hydrochloric acid
  • H2CO3 hydroiodic acid
  • HNO3 nitric acid
  • sulfuric acid H2SO4
  • Ka acid dissociation constant
  • pKa logarithmic constant
  • Two key factors that contribute to the ease of deprotonation
  • the w/w amount of each base necessary to bring the pH of the composition to a desired level is different.
  • (about) 0.08% sodium hydroxide (pellets), w/w (in water) may be sufficient to bring certain embodiments of the present disclosure to pH (about) 6.0
  • different amount(s) of different base(s) may be necessary or sufficient to bring certain embodiments of the present disclosure to pH (about) 6.0.
  • acids are suitable in one or more embodiments of the present disclosure.
  • the composition need not include an added acid or base.
  • the water and/or buffering agent can be sufficient to bring the composition to pH about 6.0, or pH 5-7, pH 5. 1-6.9, pH 5.2-6.8, or pH 5.3-6.7, pH 5.4-6.6, pH 5.5-6.5, pH 5.6-6.4, pH 5.7-6.3, pH 5.8-6.2, or pH 5.9-6.1 (or any value or range of values therebetween).
  • At least one embodiment can include a visual indicator.
  • the visual indicator can be or comprise a coloring agent. More preferably, the visual indicator can be or comprise a dye or colored dye. Still more preferably, the visual indicator can be or comprise a blue dye. Most preferably, the visual indicator can be or comprise bromophenol blue or FD&C
  • the composition can include a visual indicator, preferably a coloring agent, more preferably a colored dye, still more preferably a blue dye, still more preferably bromophenol blue or FD&C blue #1.
  • the visual indicator can be in, have, comprise, or be provided in a dry, solid, powdered, anhydrous, and/or granular form.
  • the visual indicator can have a purity of at least, up to, and/or about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (as measured by a suitable material assay, such as CoA).
  • the visual indicator can comprise or be (provided) in the form of a stock (solution (e.g., in water)) having any suitable concentration (e.g., about 0.01%, 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.175%, 0.2%, 0.25%, 0.3%, or 0.5%, w/w, aqueous solution (e.g., in water).
  • stock solution can be made using the dry, solid, powdered, anhydrous, and/or granular material.
  • the visual indicator can have a purity substantially corresponding to the concentration of the acid in solution (e.g., about 0.2%, w/w) (as measured by a suitable material assay, such as CoA).
  • the composition can include a visible (or visibly suitable) amount of a visual indicator, preferably a coloring agent, more preferably a colored dye, still more preferably a blue dye, still more preferably bromophenol blue or FD&C blue #1.
  • the visual indicator e.g., bromophenol blue
  • the composition can include any visually suitable amount, such as about 0.00037% w/w, about 0.0004%, w/w, or in a range of about 0.0001% to about 0.0008%, preferably about 0.0002% to about 0.0006%, more preferably about 0.0003% to about 0.0005%, w/w, still more preferably about 0.00035% to about 0.00045%, w/w.
  • the visual indicator (e.g., FD&C blue #1) can be included in the composition in any visually suitable amount, such as about 0.0009% w/w, or in a range of about 0.0001% to about 0.0018%, preferably about 0.0005% to about 0.0015%, more preferably about 0.0006% to about 0.0012%, w/w, still more preferably about 0.0007% to about 0.0011%, w/w, or still more preferably about 0.0008% to about 0.001%, w/w, or about 0.00081% to about 0.0099%, w/w.
  • the composition can include (about) 0.0004% w/w of bromophenol blue or FD&C blue #1.
  • the visual indicator e.g., bromophenol blue or FD&C blue #1
  • the concentrate can be an aqueous or water-based concentrate in some embodiments.
  • the composition can include 0.01-2.5%, w/w, of a 0.01-5%, w/w (in water) visual indicator concentrate.
  • the composition can include 0.05-1%, w/w, of a 0.05-1%, w/w (in water) visual indicator concentrate. More preferably, the composition can include 0.075-0.5%, w/w, of a 0.075-0.5%, w/w (in water) visual indicator concentrate. Still more preferably, the composition can include 0.1-0.25%, w/w, of a 0.1-0.25%, w/w (in water) visual indicator concentrate. Still more preferably, the composition can include (about) 0.185% w/w of (about) 0.2% w/w (in water) visual indicator concentrate.
  • the visual indicator e.g., bromophenol blue or FD&C blue #1
  • the visual indicator can be included in the composition at about 0.185%, w/w, of a -0.2% stock (aqueous) solution, or equivalent thereof.
  • the composition can include (about) 0.185%, w/w or (about) 0.2%, w/w (in water) bromophenol blue or FD&C blue #l concentrate.
  • Embodiments of the present disclosure may include a surfactant or detergent.
  • the antibacterial agent benzalkonium chloride (alkyldimethylbenzylammonium chloride) is a type of cationic surfactant; an organic salt classified as a quaternary ammonium compound.
  • Surfactants, or detergents in high amounts, can degrade or interfere with testing results. It was discovered that benzalkonium chloride, in certain concentration ranges, was effective as an antibacterial agent, without disrupting, degrading, or interfering with the testing results of protein analytes.
  • benzalkonium chloride may be partially or entirely removed from the preserved sample (through a pre-processing step) prior to assaying the sample for the protein analyte(s).
  • Some embodiments can be (substantially) free or devoid of surfactant(s) or detergent(s) (other than benzalkonium chloride, if present).
  • Illustrative surfactant(s) or detergent(s) include a lauroyl sarcosinate (e.g., sodium lauroyl sarcosinate (SLS)), sodium dodecyl sulfate (SDS), polysorbates (TweenTM), lauryl dimethyl amine oxide, cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol (Triton XI 00TM), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethyl ammonium bromide (HTAB), polyoxyl 10 lauryl ether, Bile salts (sodium deoxycholate, sodium cholate), polyoxyl castor oil (C
  • Embodiments of the present disclosure can be (substantially) free or devoid of alcohol. Without being bound to any particular theory, alcohol may deform or denature protein analyte(s) of interest. Alternative embodiments may include an amount of alcohol that does not deform or denature protein analyte(s) of interest.
  • Embodiments of the present disclosure can be (substantially) free or devoid of one or more mucolytic agent or reducing agent.
  • mucolytic agents or reducing agents may be destructive to disulfide bridges in protein analyte(s) of interest (e.g., antibodies, hormones, etc.).
  • Illustrative mucolytic agent or reducing agent include an acetylcysteine (i.e., N-acetylcysteine (NAC), including N-acetyl-L-cysteine, N-acetyl-D-cysteine, and racemic N-acetylcysteine or a (racemic) mixture of N-acetyl-L- cysteine andN-acetyl-D-cysteine), ascorbic acid, dithionite, erythiorbate, cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, dierythritol, a resin-supported thiol, a resin-supported phosphine, vitamin E, and/or trolox, or salts thereof, sodium citrate, potassium citrate, potassium iodide, ammonium chloride, guaiphenesin (or guaifenesin), Tolu balsam, Vasaka
  • Some embodiments include a ribonuclease inhibitor, or inhibitor of ribonuclease, such as heparin, heparan sulfate, oligo (vinylsulfonic acid), poly(vinylsulfonic acid), oligo(vinylphosphonic acid), and poly(vinylsulfonic acid), or salts thereof.
  • the composition does not include a ribonuclease inhibitor or inhibitor of ribonuclease, or is (substantially) devoid of one or more (e.g., any) ribonuclease inhibitor or inhibitor of ribonuclease.
  • At least one embodiment is (substantially) devoid of one or more (any) ribonuclease inhibitor, or inhibitor of ribonuclease.
  • One or more embodiments are (substantially) devoid of any ribonuclease inhibitor, or inhibitor of ribonuclease (e.g., other than a chaotropic agent, such as guanidine thiocyanate).
  • Embodiments of the present disclosure can be (substantially) free or devoid of (a or any) protease(s) and/or protease inhibitor(s).
  • a protease or proteolytic enzyme, peptidase or proteinase is a type of enzyme that breaks one or more peptide bonds through hydrolysis, thereby converting proteins into smaller protein fragments
  • SUBSTITUTE SHEET (RULE 26) (or peptides) or individual protein subunits (or amino acids).
  • One or more embodiments may or can include a protease inhibitor.
  • Embodiments of the present disclosure can be (substantially) free or devoid of (any) protein denaturants.
  • Figure 1 illustrates preservation of HIV antigen/antibody in saliva using composition(s) of the present disclosure, demonstrating stability out to 14 days at room temperature.
  • Figure 2 illustrates high levels of correlation for antibody concentration on day 2 (typical delay time between sample collection and arrival at processing/analysis lab) at room temperature, and day 4 (typical delay time between sample collection and sample processing/analysis at the lab) at room temperature for COVID-19 IgG and IgA.
  • Figure 3 illustrates the exemplary data, comparing the non-colored solution to the colored (blue dyed) solution, and demonstrating that the addition of blue dye did not interfere with the assay.
  • Figure 4 illustrates the stability of microRNA in the illustrative solutions with and without colorant for 2 participants out to 4 days.
  • the dashed line indicates the lower limit of what may be considered an “adequate” sample.
  • the volume of microRNA went “below” the “adequate” limit at 1 hour to being acceptable at 24 hours.
  • inventive compositions may, in fact, help to liberate RNA from proteins and mucins that had bound and sequestered the material.
  • Figure 5 illustrates the recovery of cytokines (MIG) from saliva using an illustrative inventive solution (“Spectrum SPD”) — utilizing an illustrative collection device — against competitors “A” and “B”.
  • MIG cytokines
  • Figure 6 illustrates the recovery of cytokines (INF-y) from saliva using an illustrative inventive solution (“Spectrum SPD”) — utilizing an illustrative collection device — against competitors “A” and “B”.
  • Figure 7 illustrates room temperature stability of the cytokines of Figures 5 and 6 at room temperature.
  • Table 6 presents several statistics relevant to the figure, with exemplary statistics bolded (e.g., a slope of 1 is equivalent to perfect stability in this context):
  • Some embodiments of the present disclosure include obtaining, providing, and/or collecting a biological sample (e.g., from a subject, such as a human subject).
  • the biological sample can be or comprise (human) saliva.
  • the biological sample can be or comprise expectorated (human) saliva.
  • the (human) sample can be collected aseptically (to avoid (microbial) contamination).
  • the sample can be collected into a sample collection apparatus or sample container thereof.
  • the sample collection apparatus or container can be part of a kit and/or can include a composition of the present disclosure.
  • Embodiments can include contacting the sample with a composition of the present disclosure.
  • the AlimetrixTM SLV-Cortisol Assay is a competitive enzyme immunoassay for the in vitro quantitative measurement of active free cortisol (hydrocortisone and hydroxycorticosterone) in saliva.
  • the assay may be used to screen for levels of cortisol production by the adrenal glands in patents presenting with symptoms or signs of elevated or inadequate cortisol.
  • saliva was self-collected by the patient using the SpectrumTM Solutions SMD Saliva Collection Device in the morning (before 10 AM) and at bedtime and shipped to AlimetrixTM (a Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. ⁇ 263a certified high-complexity laboratory) at room temperature for testing.
  • AlimetrixTM a Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. ⁇ 263a certified high-complexity laboratory
  • the saliva specimens were processed per SOP: 87167.475 (see Alimetrix TM MediaLab document 87167.475 Processing Patient Samples for Salivary Hormone Assays).
  • the processed salivary specimens were added to microtiter wells coated with (mouse) monoclonal anti -cortisol antibodies directed towards the antigenic site of the cortisol hormone molecule.
  • Endogenous cortisol hormones present in the patient sample compete with a fixed input amount of horseradish peroxidase enzyme conjugate of cortisol for binding to the coated antibody.
  • the unbound conjugate was washed off and a substrate solution (Tetramethylbenzidine) was added to the microtiter wells to produce a blue color.
  • the intensity of the color is inversely proportional to the concentration of cortisol hormone present in the sample (i.e., the more intense the color developed the more bound peroxidase conjugates there are in the sample).
  • the limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) were determined consistent with the guidelines in CLSI document EP17-A2 [2] using two reagent lots of SpectrumTM Solutions SMD Stabilization Solution.
  • the LoD for the SLV- Cortisol assay is 0.843 ng/mL based on the proportions of false positives (a) ⁇ 5% and the false negatives ( ⁇ ) ⁇ 5%; using 240 determinations, with 120 blank and 120 low level samples; and an LoB of 0.314 ng/mL.
  • the LoQ for the SLV-Cortisol assay is 1.059 ng/mL based on 102 determinations; a total error (TE) goal of ⁇ 20% calculated using the RMS error model; and a
  • Table 9 illustrates the Overall Precision Across Instruments Estimated via 2-Way Nested ANOVA Analysis (with replication).
  • Table 10 illustrates the Precision Estimates of Each Instrument via 1-Way ANOVA Analysis.
  • Table 11 illustrates the Precision Estimates Across Reagent Lots via 2-Way Nested ANOVA Analysis (with replication).
  • Table 12 illustrates the Concordance Results of Positive and Negative Controls.
  • SUBSTITUTE SHEET (RULE 26) 1) comprised a patient-sample pool spiked with cortisol ( ⁇ 10% v/v) at the upper reportable limit, resulting in a final concentration greater than the upper limit of the measuring interval (>ULMI).
  • the high concentration patient-sample pool was diluted sequentially with a low concentration patient sample-pool to create concentration levels spanning the measuring range of the assay.
  • the high concentration pool was sequentially diluted with specimen diluent (Matrix 2).
  • the data was modeled with linear and best-fit non-linear polynomial curves, and the difference between the estimated concentrations from both curves was calculated to obtain the % Bias.
  • Analytical specificity was determined by assessing the cross-reactivity and interference of potentially interfering agents as outlined in CLSI document EP07-A2. Positive and negative cortisol salivary specimens spiked with high or low concentrations of interfering substances were tested in duplicate and analyzed. Percent interference and recovery was estimated for each specimen in the presence of the interfering agents.
  • Table 15 illustrates the Estimated cross-reactivity and interference of potentially interfering agents spiked at high concentration (per CLSI EP07-A2) into positive and negative cortisol salivary specimens.
  • i i
  • SUBSTITUTE SHEET (RULE 26) titer) or high (high-titer) amounts of cortisol into negative cortisol saliva sample pools.
  • a negative specimen pool was prepared by spiking PBS instead of cortisol into negative cortisol sample pools.
  • SMD stabilization solution was added to each contrived sample to simulate the matrix of actual test specimens.
  • the contrived samples were partitioned into 3 sets. 2 were subjected to either a low temperature (Table 17) or a high temperature (Table 18) excursion to emulate transport conditions, while the last was left at RT and used as the transport stability control. On completion of the 3-day transport excursions, each sample set was aliquoted and split into two groups (RT and CL).
  • Table 17 illustrates Low Temperature Excursion. *The temperature profile was designed following estimated temperature ranges provided by shipping courier (FedEx). Per courier, the lower cargo and bulk compartments of their aircrafts are exposed to temperatures between 18C and -18C at high altitudes.
  • Table 18 illustrates High Temperature Excursion. *The temperature profile was designed following estimated temperature ranges provided by shipping courier (FedEx). According to courier, temperature ranges aboard most wide- body aircraft main cargo compartments vary between 18°C and 32°C. Samples are exposed to “worst case” stress conditions by extending temperature to 40°C.
  • Table 19 illustrates Estimated variance of samples after transport simulation. *%CV of negative samples cannot be calculated, because the measured means are ⁇ LoQ of the assay.
  • B is the total number of results in the dataset
  • Xi+1 is the upper rank value bracketing the Rank Position
  • Xi-1 is the lower rank value bracketing the Rank Position.
  • Table 20 Summarizes Table of Rank Positions and LoB from Blank Sample Test Results of the SLV-Cortisol Assay using the Classical Non-parametric Approach per CLSI document EP17-A2.
  • LOD was calculated according to the classical method using four charcoal stripped salivary specimens spiked with low amounts of cortisol. The samples were run thrice in replicates of five across two SMD stabilization solution lots for a total of 120 determinations (60 determinations per lot). Calculations were based on the equations outlined below:
  • SDL is the standard deviation across all low-level samples
  • Cp is a multiplier to give the 95th percentile of a normal distribution
  • L is the total number of all low level sample results across all reagent lots
  • J is the number of low level samples.
  • the 1.645 value represents the 95th percentile from the normal distribution for a Type II error ([3) of 5%.
  • Table 21 illustrates Standard Deviation and LoD Calculations of Low Level Cortisol Sample Test Results of the SLV-Cortisol Assay using the Classical Non-parametric Approach per CLSI document EP17-A2. The highest calculated LoD of the two reagent lots was selected as the LoD of the assay.
  • SD is the standard deviation of all observed results at a particular measurand concentration level and Bias is the difference between the mean observed result (x) and the reference value at that concentration level (R).
  • Table 22 illustrates Observed Means and SDs for Low Level Salivary Cortisol Samples used to Evaluate LoQ.
  • Table 23 illustrates TE and Bias Calculations for Low Level Cortisol Salivary Samples. The percentages were obtained by dividing the calculated TE and Bias by the reference value and multiplying by 100. The lowest reference concentration level that satisfies the TE and Bias goals was selected as the LoQ, and was associated with the reagent lot that gave the highest.
  • V error Vrun, Vday
  • MSi is the mean square of component i and Nrep and Nrun are the number of replicates and number runs respectively.
  • Standard deviation of each component was calculated by taking the square root of its corresponding variance component.
  • %CVs were obtained by dividing standard deviations by the grand mean of all measurement results for a given sample and multiplying by 100.
  • Table 24 presents a Two-way Nested ANOVA Summary
  • DF degree of freedom
  • N total number of results per sample
  • n number of components
  • SS sum of squares
  • MS mean squares
  • site instrument or reagent lot variation
  • o variance
  • EMS expected value of the mean square.
  • Table 25 presents a One-way ANOVA Summary i
  • DF degrees of freedom
  • DFtotal total degrees of freedom
  • MS mean squares
  • SS sum of squares
  • SStotal total sum of squares
  • Variance components used to calculate between-site and within-site precisions are calculated from the ANOVA table A2-1 using equations A2.1 and A2.2 and the following equations: [00215] Where MSi is the mean square of component i and and are the number of runs and number of replicates respectively.
  • Standard deviation of each component was calculated by taking the square root of its corresponding variance component similar to the single-site precision approach with two additional components:
  • SDR is the repeatability standard deviation of the variation
  • SDWL IS the within-site precision standard deviation
  • SDREP is the reproducibility of the variation under surveillance (i.e. reagent lot or instrument).
  • SDR is the between-day standard deviation of the variation
  • SDR is the repeatability standard deviation variance that corresponds directly with the within-day variance component
  • SDWL here represents the within-instrument standard deviation
  • Table 26 presents a 1-Way ANOVA Summary of Variance Components for RLT on each instrument.
  • Table 27 presents a 1-Way ANOVA Summary of Variance Components for RMT on each instrument. > i i 1
  • Table 28 presents a 1-Way ANOVA Summary of Variance Components for RHT on each instrument.
  • Table 29 presents a 2-Way Nested ANOVA Summary of Variance Components Across All Instruments.
  • Table 30 presents a 2-Way Nested ANOVA Summary of Variance Components Across All Reagent Lots.
  • Table 31 presents an Estimation of repeatability for patient-sample pool matrix dilutions.
  • Table 32 presents an Estimation of repeatability for a high cone, patient-sample pool diluted with diluent.
  • Table 33 presents Results of regression analysis for patient-sample pool matrix dilutions.
  • the third-order regression model is shown to have a lower Standard Error (Syx) than the second-order model.
  • Table 34 presents Results of regression analysis for a high cone, patient-sample pool diluted with diluent.
  • the second order regression model is shown to have the lower Standard Error (Syx) than the third-order model.
  • Table 35 presents Comparison of storage conditions of negative saliva samples after
  • ND Not detected. Estimated concentrations are interpreted as “ND” if ⁇ LoD. [00233] Table 36 presents Comparison of storage conditions of low-titer saliva samples after
  • Table 37 presents Comparison of storage conditions of high-titer saliva samples after
  • Table 38 presents a Comparison of transport conditions of negative saliva samples after a 3-day high (HI) and low (LO) temperature excursion.
  • ND Not detected. Estimated concentrations are interpreted as “ND” if ⁇ LoD.
  • Table 39 presents a Comparison of transport conditions of low-titer saliva samples after a 3-day high (HI) and low (LO) temperature excursion.
  • Table 40 presents a Comparison of transport conditions of high-titer saliva samples after a 3-day high (HI) and low (LO) temperature excursion.
  • Table 40 [00239] Table 41 presents Stability of negative samples after 14-days storage at room temperature (RT).
  • Table 42 presents Stability of negative samples after 14-days storage at 2-8C (CL).
  • Table 43 presents Stability of low-titer samples after 14-days storage at room temperature (RT).
  • Table 43 [00242] Table 44 presents Stability of low-titer samples after 14-days storage at 2-8C (CL).
  • Table 45 presents Stability of high-titer samples after 14-days storage at room temperature (RT).
  • Table 46 presents Stability of high-titer samples after 14-days storage at 2-8C (CL).
  • compositions, kits, method, etc. may include, incorporate, or otherwise comprise features (e.g., properties, components, ingredients, elements, parts, portions, steps, etc.) described in other embodiments disclosed and/or described herein. Accordingly, the various features of one embodiment can be compatible with, combined with, included in, and/or incorporated into other embodiments of the present disclosure. Disclosure of certain features relative to one embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. Rather, it will be appreciated that other embodiments can also include said features without necessarily departing from the scope of the present disclosure. Moreover, unless a feature is described as requiring another features in combination therewith, any feature described herein may be combined with any other feature of a same or different embodiment disclosed herein.

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Abstract

L'invention concerne des compositions de conservation de biomolécules, des mélanges et des kits les comprenant, et des méthodes d'utilisation associées, en particulier pour la conservation de la biomolécule, telle qu'un anticorps ou une hormone, à partir de salive humaine ou de sang. Les compositions comprennent un support, un agent tampon, un agent chélatant, un agent antimicrobien et éventuellement un agent d'ajustement du pH. Des exemples comprennent de l'eau, de la L-histidine, de l'EDTA, un ProClin™ (ou du chlorure de cétylpyridinium) et, éventuellement, un acide ou une base, tel que l'hydroxyde de sodium. La composition ou le mélange peut avoir un pH d'environ 6. Un colorant coloré en tant qu'indicateur visuel peut être utilisé. Les kits comprennent un appareil de collecte d'échantillon biologique et la composition, éventuellement disposée dans une partie de l'appareil. Les méthodes d'utilisation comprennent la fourniture d'un échantillon biologique, de préférence de la salive, qui comprend une protéine et la mise en contact de l'échantillon biologique avec la composition. La détection d'anticorps est démontrée.
PCT/US2024/030180 2023-05-19 2024-05-20 Produits et méthodes de conservation et de détection de biomolécules dans des échantillons de salive humaine Pending WO2024243119A2 (fr)

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US4734434A (en) * 1986-03-11 1988-03-29 Rorer Pharmaceutical Corporation Method for the treatment of pruritus and composition for the use therein
FR2797689B1 (fr) * 1999-08-16 2003-06-27 Pasteur Sanofi Diagnostics Utilisation de composes synthetiques pour des immunodosages
US8703490B2 (en) * 2008-06-05 2014-04-22 Ventana Medical Systems, Inc. Compositions comprising nanomaterials and method for using such compositions for histochemical processes
US20130034515A1 (en) * 2011-08-03 2013-02-07 Melaleuca, Inc. Hair care compositions
WO2014152211A1 (fr) * 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation et administration de compositions de nucléosides, de nucléotides, et d'acides nucléiques modifiés
WO2019140163A1 (fr) * 2018-01-12 2019-07-18 Fmc Corporation Compositions herbicides comportant une préparation de péthoxamide en suspension pour capsule
US20220272987A1 (en) * 2019-04-12 2022-09-01 Jeffrey H. ROBBINS Composition including effervescent agents, biostimulant, nutrient, and pesticide
US20230033248A1 (en) * 2021-07-23 2023-02-02 Amir Reza Salar Parvini Compositions and methods for enhanced decontamination

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