JPWO2020198011A5

JPWO2020198011A5 -

Info

Publication number: JPWO2020198011A5
Application number: JP2021552175A
Authority: JP
Publication date: 2023-03-23

Claims

A method for preparing a plurality of biological samples for protein detection, comprising:
a plurality of test samples by performing buffer exchange on the plurality of biological samples with a formulation comprising a buffer, one or more salts, a chelating agent, and a nonionic surfactant; and
adjusting the total protein concentration of the plurality of test samples to produce a plurality of adjusted test samples, wherein the total protein concentration of each adjusted test sample is approximately the same, and wherein the method comprises: The above method, which does not comprise concentrating the total protein of the plurality of biological samples prior to performing buffer exchange.

A method for preparing a biological sample for detecting a protein, comprising:
Generating a test sample by performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a nonionic surfactant. and adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is from about 70 μg/mL to about 100 μg/mL. , said method.

3. The method of claim 1 or 2, comprising quantifying the protein concentration of said test sample or said plurality of test samples and/or said biological sample or said plurality of biological samples.

A method for preparing a biological sample for detecting a protein, comprising:
generating a test sample by performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a nonionic surfactant; ,
quantifying the total protein concentration of the test sample;
and adjusting the total protein concentration of the test sample to produce an adjusted test sample, wherein the total protein concentration in the adjusted test sample is from about 70 μg/mL to about 100 μg/mL. Method.

5. The method of claims 2-4 comprising generating a plurality of test samples by performing buffer exchange on a plurality of biological samples, wherein the total protein concentration of each adjusted test sample is approximately the same. A method according to any one of paragraphs.

A method of preparing a biological sample for detecting a protein, comprising:
generating a test sample by performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a nonionic surfactant; ,
quantifying the protein concentration of the test sample;
adjusting the total protein concentration of the test sample if the total protein concentration of the test sample is not within the range of about 70 μg/mL to about 100 μg/mL to produce an adjusted test sample; total protein concentration in the adjusted test sample is from about 70 μg/mL to about 100 μg/mL;
if the total protein concentration of said test sample is not within the range of about 70 μg/mL to about 100 μg/mL; if not within the range of about 70 μg/mL to about 95 μg/mL; not within the range of about 70 μg/mL to about 85 μg/mL; not within the range of about 70 μg/mL to about 80 μg/mL; or about 70 μg/mL to about 75 μg/mL If not, then optionally comprising adjusting the total protein concentration of the test sample to be within said range to produce an adjusted test sample.

A method according to any one of claims 2 to 7 , wherein said method does not comprise enriching said biological sample.

A test sample comprising a buffer-exchanged biological sample and one or more protein capture reagents , wherein said one or more protein capture reagents are aptamers, and wherein said test sample has a total protein concentration of about 70 μg/mL. - about 100 μg/mL, and wherein the buffer-exchanged biological sample comprises a buffer, one or more salts, a chelating agent, and a nonionic surfactant.

The method of any one of claims 1-7 or the test sample of claim 8 , wherein said one or more salts are each independently selected from sodium, potassium and magnesium salts.

The method of any one of claims 1-7 and 9 or the test sample of claim 8 or 9 , wherein said one or more salts comprise sodium, potassium and magnesium salts.

The method of any one of claims 1-7, 9 and 10 or claim 9 or 10 , wherein said sodium salt is NaCl, said potassium salt is KCl and said magnesium salt is _MgCl2 . Test sample as described.

12. Any one of claims 1-7 and 11, wherein the NaCl in the test sample is at a concentration of about 10 mM to about 500 mM, or about 50 mM to about 250 mM, or about 100 mM to about 200 mM, or about 102 mM. 12. A method according to claim 11 or a test sample according to claim 11 .

Claims 1-, wherein said KCl in said test sample is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM. 13. A method according to any one of claims 7, 11 and 12 or a test sample according to claim 11 or 12 .

Clause ₁ , wherein said MgCl2 in said test sample is at a concentration of about 0.5 mM to about 30 mM, or about 1 mM to about 20 mM, or about 2 mM to about 15 mM, or about 4 mM to about 10 mM, or about 5 mM. A method according to any one of claims 7 and 11-13 or a test sample according to any one of claims 11-13 .

The buffer is HEPES, MES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, colamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO , EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS and CABS, claims 1-7 and 9-14. or the test sample according to any one of claims 8-14 .

the buffer in the test sample is at a concentration of about 4 mM to about 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or 35 mM to about 50 mM, or about 40 mM , a method according to any one of claims 1-7 and 9-15 or a test sample according to any one of claims 8-15 .

The method of any one of claims 1-7 and 9-16 or any one of claims 8-16 , wherein the chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS and ALA. Test sample described in section.

18. Any one of claims 1-7 and 9-17, wherein the chelating agent in the test sample is at a concentration of about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM. A method as described or a test sample as described in any one of claims 8-17 .

The nonionic surfactants include polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-40). -80 ) .

The nonionic surfactant is about 0.01% to about 1% of the test sample, or about 0.02% to about 0.5% of the test sample, or about 0.03% of the test sample from about 0.1%, or from about 0.04% to about 0.08% of said test sample, or from about 0.05% of said test sample (volume to volume), claims 1-7 and 9- 20. A method according to any one of claims 19 or a method according to any one of claims 8-19 .

21. The method of any one of claims 1-7 and 9-20, wherein the total protein concentration of the test sample is quantified by a bicinchoninic acid (BCA) assay, wherein the BCA assay is optionally a micro BCA assay. A method or test sample according to any one of claims 8-20 .

22. The method of claim 21 or claim 21 , wherein said assay comprises a bicinchoninic acid reagent or a bicinchonic acid reagent, and optionally alkaline tartaric acid-carbonate buffer and/or copper sulfate solution. The test sample described in .

23. Any of claims 1-7 and 9-22, wherein the test sample comprises 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 _mM MgCl2, 1 mM EDTA, and 0.05% Tween-20. A method according to claim 1 or a test sample according to any one of claims 8-22 .

24. Any one of claims 1-7 and 9-23, wherein the test sample has a pH of about pH 5 to about pH 9, or about pH 6 to about pH 8, or about pH 7 to about pH 7.9, or about pH 7.5. A method according to claim or a test sample according to any one of claims 8-23 .

Said at least one aptamer comprises a 5-modified pyrimidine, optionally said 5-modified pyrimidine comprises a hydrophobic moiety at position 5 of said pyrimidine, optionally said hydrophobic moiety is a naphthyl moiety, a phenyl moiety, a tyrosyl moiety. , an indole moiety, and a morpholino moiety .

The method of any one of claims 1-7 and 9-24 or the test sample of any one of claims 8-25, wherein said biological sample is a urine sample.

The method of any one of claims 1-7 and 9-24 or the test sample of any one of claims 8-25, wherein said biological sample is a serum sample.

The method of any one of claims 1-7, 9-24, 26 and 27 or the test sample of any one of claims 8-27, wherein said buffer exchange is not performed by ultrafiltration. .

Claims 1-7, 9-24, wherein gel filtration chromatography was used for performing said buffer exchange on said biological sample for the purpose of producing said buffer-exchanged biological sample and a method according to any one of claims 26-28 or a test sample according to any one of claims 8-28 .

A method for detecting a target protein in a test sample prepared by the method of any one of claims 1-7, 9-24 and 26-28, comprising:
a) contacting the test sample with at least one capture reagent, wherein the at least one capture reagent is capable of binding to the target protein to form a complex;
b) incubating the test sample with the at least one capture reagent under conditions that allow formation of the complex;
c) quantifying the level of said target protein in said test sample by measuring the level of said at least one capture reagent, said complex, or said protein, wherein said at least one capture reagent or said said quantifying, wherein the level of the complex is a proxy for the level of said target protein;
wherein the test sample is produced by performing buffer exchange on the biological sample with a formulation comprising a buffer, a salt, a chelating agent, and a nonionic surfactant;
The above method, wherein the total protein concentration of the test sample is greater than or equal to about 70 μg/mL and less than about 100 μg/mL.

30. The method of any one of claims 1-7, 9-24 and 26-29 or claims , comprising measuring the protein concentration of the biological sample prior to performing the buffer exchange. A test sample according to any one of paragraphs 8-28 .

32. The method of claim 31 , wherein the protein concentration of said biological sample is measured by an assay selected from the group consisting of a fluorescence readout assay, a bicinchoninic acid (BCA) assay, a micro BCA assay, a Lowry assay, and an ELISA assay. Method.

33. The method of claim 32 , wherein said assay is a BCA assay or micro BCA assay.

33. The method of claim 32 , wherein said assay comprises a bicinchoninic acid or bicinchonic acid reagent, and optionally alkaline tartaric acid-carbonate buffer and/or copper sulfate solution.

33. The method of claim 32 , wherein said assay is a fluorescence readout assay.

36. The method of claim 35 , wherein said fluorescence readout assay comprises a reagent selected from merocyanine dyes and epicocconone.

A method for preparing a biological sample for detecting a protein, comprising:
Generating a test sample by performing buffer exchange on the biological sample with a formulation comprising a buffer, one or more salts, a chelating agent, and a nonionic surfactant. and quantifying the total protein concentration of said biological sample or said test sample;
adjusting the total protein concentration of the biological sample or the test sample, wherein the method does not comprise concentrating the total protein of the biological sample prior to performing the buffer exchange;
Optionally, (i) said total protein concentration is adjusted prior to performing said buffer exchange, or (ii) said total protein concentration is adjusted after performing said buffer exchange. Method.

38. The method of claim 37 , wherein the total protein concentration of said test sample is adjusted from about 2 μg/mL to about 60 μg/mL after performing said buffer exchange.

39. The method of claim 37 or 38 , comprising generating a plurality of test samples by performing buffer exchange on a plurality of biological samples, wherein the total protein concentration of each test sample is about the same or the same. the method of.

total protein concentration of said biological sample is from about 2 μg/mL to about 50 μg/mL, or from about 2 μg/mL to about 45 μg/mL, or from about 4 μg/mL to about 40 μg/mL, or from about 8 μg/mL to about 40 μg/mL, or about 10 μg/mL to about 40 μg/mL, or about 10 μg/mL to about 30 μg/mL, or less than 40 μg/mL, or less than 30 μg/mL, or less than 20 μg/mL, or about 15 μg/mL; or from about 10 μg/mL to about 20 μg/mL, said method comprising adjusting the total protein concentration of said biological sample to said range . The method described in .

41. The method of any one of claims 37-40 , wherein said one or more salts are each independently selected from sodium, potassium, and magnesium salts.

42. The method of claim 41 , wherein said sodium salt is NaCl, said potassium salt is KCl and said magnesium salt is _MgCl2 .

The buffer is HEPES, MES, bis-trismethane, ADA, ACES, bis-trispropane, PIPES, MOPSO, colamine chloride, MOPS, BES, TES, DIPSO, MOB, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO , EPS, HEPPS, tricine, tris, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, and CABS, and the buffer in the formulation is selected from about 4 mM to about 43. A concentration of 400 mM, or about 10 mM to about 300 mM, or about 20 mM to about 200 mM, or about 30 mM to about 100 mM, or about 35 mM to about 50 mM, or about 40 mM. the method of.

The chelating agent is selected from EDTA, EGTA, DTPA, BAPTA, DMPS, and ALA, and the chelating agent in the formulation is about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM. 44. The method of any one of claims 37-43 , wherein the concentration is

The nonionic surfactants include polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (40) sorbitan monolaurate (Tween-40), and polyoxyethylene (80) sorbitan monolaurate (Tween-40). -80), and the nonionic surfactant is from about 0.01% to about 1% of the formulation, or from about 0.02% to about 0.5% of the formulation, or from about 0.03% to about 0.1%, or about 0.04% to about 0.08% of the formulation, or about 0.05% or about 0.1% (volume to volume) of the formulation; The method of any one of claims 37-44 .

wherein the total protein concentration of said test sample or said biological sample is quantified by an assay selected from the group consisting of a fluorescence readout assay, a Bradford assay, a bicinchoninic acid (BCA) assay, a Lowry assay, and an ELISA assay. Item 46. The method of any one of Items 37-45 .

47. The method of claim 46 , wherein said fluorescence readout assay comprises reagents selected from merocyanine dyes and epicocconone.

wherein the formulation comprises 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl ₂ , 1 mM EDTA, and 0.05% Tween-20, and the pH of the formulation is from about pH 5 to about pH 9, or 48. The method of any one of claims 37-47 , wherein the pH is about pH 6 to about pH 8, or about pH 7 to about pH 7.9, or about pH 7.5.

The method of any one of claims 37-48 , wherein said buffer exchange is performed by gel filtration chromatography.

The method of any one of claims 37-49 , wherein said biological sample is a urine sample.