In-tube additive of cfDNA vacuum blood collection tube and blood collection tube

Abstract

The invention provides an in-tube additive for a cfDNA vacuum blood collection tube and the blood collection tube. The in-tube additive comprises an anticoagulant, a preservative, a cell protective agent, an enzyme inhibitor, a metabolic inhibitor and an antioxidant, the enzyme inhibitor comprises a nuclease inhibitor; the nuclease inhibitor comprises an anti-deoxyribonuclease antibody; and the anti-deoxyribonuclease antibody comprises at least one of an anti-DNASE1 antibody and an anti-DNASE1L3 antibody. According to the in-tube additive of the cfDNA vacuum blood collection tube, cfDNA can be stably stored, and the hemolysis phenomenon in the blood storage process can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to an in-tube additive for a cfDNA vacuum blood collection tube and the blood collection tube. Background Art

[0002] cfDNA is a short fragment of DNA that exists freely in the blood circulation. By analyzing cfDNA in the blood, non-invasive detection of various diseases can be achieved, providing an important means for monitoring disease development, treatment effects and recurrence.

[0003] Current methods for preserving cfDNA rely primarily on the use of specialized blood collection tubes containing large amounts of anticoagulants and various proprietary chemical preservatives. These additives can chelate divalent cations (such as Mg) required for nuclease activity. 2+ and Ca 2+ ) to inhibit nuclease activity and preserve cfDNA. However, this broad-spectrum inhibition may affect the activity of other enzymes or interfere with enzymatic reactions required for downstream analysis, such as PCR amplification, hindering the development of cfDNA sequencing technology. Furthermore, these blood collection tubes are prone to hemolysis during blood storage. The high-molecular-weight genomic DNA released by lysed blood cells can contaminate cfDNA samples and interfere with downstream cfDNA analysis.

[0004] Therefore, there is an urgent need for a method that can stably preserve cfDNA and effectively improve the hemolysis phenomenon during blood storage. Summary of the Invention

[0005] The present invention provides an in-tube additive for a cfDNA vacuum blood collection tube and a blood collection tube, which can stably preserve cfDNA and effectively improve hemolysis during blood storage.

[0006] The embodiment of the present invention provides an in-tube additive for a cfDNA vacuum blood collection tube, including an anticoagulant, a preservative, a cell protectant, an enzyme inhibitor, a metabolic inhibitor, and an antioxidant;

[0007] The enzyme inhibitors include nuclease inhibitors;

[0008] The nuclease inhibitor includes an anti-deoxyribonuclease antibody; the anti-deoxyribonuclease antibody includes at least one of an anti-DNASE1 antibody and an anti-DNASE1L3 antibody.

[0009] In some embodiments of the present invention, the mass-to-volume ratio of the anti-DNase antibody to the volume of the additive in the tube is 0.1 ng / ml to 10 ng / ml.

[0010] In some embodiments of the present invention, the anti-deoxyribonuclease antibody comprises an anti-DNASE1 antibody and an anti-DNASE1L3 antibody, and the mass ratio of the anti-DNASE1 antibody to the anti-DNASE1L3 antibody is (0.2-5):1.

[0011] In some embodiments of the present invention, the anti-DNase antibody comprises at least one of a polyclonal antibody, a monoclonal antibody, and a genetically engineered antibody.

[0012] In some embodiments of the present invention, based on the total volume of the in-tube additives, the mass volume ratio of the anticoagulant in the in-tube additives is 0.8 mg / ml to 5 mg / ml;

[0013] In some embodiments of the present invention, based on the total volume of the in-tube additive, the mass volume ratio of the preservative in the in-tube additive is 10 mg / ml to 500 mg / ml;

[0014] And / or, the mass volume ratio of the cell protectant in the in-tube additive is 1 mg / ml to 100 mg / ml;

[0015] and / or, the enzyme inhibitor has a mass volume ratio in the in-tube additive of 1 mg / ml to 610 mg / ml;

[0016] And / or, the mass volume ratio of the metabolic inhibitor in the in-tube additive is 1 mg / ml to 50 mg / ml;

[0017] And / or, the antioxidant accounts for a mass volume ratio of 5 mg / ml to 100 mg / ml in the in-tube additive.

[0018] In some embodiments of the present invention, the enzyme inhibitor further comprises a protease inhibitor, and the protease inhibitor comprises one or more of genistein, aprotinin, sodium orthovanadate, leupeptin, phenylmethylsulfonyl fluoride, serine protease inhibitors, cysteine protease inhibitors, aspartic acid protease inhibitors and metalloproteinase inhibitors;

[0019] And / or, the anticoagulant includes one or more of ethylenediaminetetraacetic acid potassium salt, ethylenediaminetetraacetic acid sodium salt, ethylenediamine succinate sodium salt, sodium citrate, and heparin salt;

[0020] And / or, the preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, isopropyl parahydroxybenzoate, butyl parahydroxybenzoate, isobutyl parahydroxybenzoate, sodium methyl parahydroxybenzoate, sodium ethyl parahydroxybenzoate, sodium propyl parahydroxybenzoate, sodium isobutyl parahydroxybenzoate, sodium isopropyl parahydroxybenzoate, sodium butyl parahydroxybenzoate, diazolidinyl urea, imidazolidinyl urea, Diazolidinyl urea, iodopropynyl butylcarbamate, sorbic acid, potassium sorbate, cetyltrimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride, diisobutylethoxyethyldimethylbenzylammonium chloride, diisobutylphenoxyethoxyethyldimethylbenzylammonium chloride, N-alkyl-N,N-dimethylbenzyl ammonium chloride, ammonium bromide, saccharin ammonium, trimethylammonium chloride, sodium aluminum chloroglycolate, triethyl citrate, tricetylmethylammonium chloride, 3,4,4'-trichlorocarbonanilide, L-lysine hexadecylamide, DMDM hydantoin, sodium hydroxymethylaminoacetate, benzoic acid, propionic acid, salicylic acid, 2,4-hexadienoic acid, 2-hydroxybiphenyl, 3-acetyl-methyl-2,4-(3H)pyrandione, formic acid, undecylenic acid, 5-amino-1,3-bis(2-ethylhexyl)-5-methylhexahydropyrimidine, 2- One or more of bromo-2-nitro-1,3-propanediol, 2,4-dichlorobenzyl alcohol, N-(4-chlorophenyl)-N'-(3,4-dichlorophenyl)urea, 2,4,4'-trichloro-2'-hydroxy-diphenyl ether, poly(hexamethylene biguanide)-hydrochloride, 1,2-dibromo-2,4-dicyanobutane, 4,4-dimethyl-1,3-oxazolidine, sodium benzoate, methylisothiazolinone, ethanol, and methanol;

[0021] and / or, the cell protectant comprises one or more of serine, cysteine, glutamine, glycine, tyrosine, aspartic acid, asparagine, D-mannitol, glucose, and rhamnose;

[0022] and / or, the metabolic inhibitor comprises one or more of auronic acid, glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 3-phospho-2-phosphoglycerate, phosphoric acid, pyruvic acid, gluconic acid, sodium fluoride, potassium fluoride, and aluminum fluoride;

[0023] And / or, the antioxidant includes at least one of glutathione and N-acetylcysteine.

[0024] In some embodiments of the present invention, the volume ratio of the in-tube additive to the blood sample is 1:(5-10).

[0025] An embodiment of the present invention also provides a blood collection tube, comprising a test tube, a rubber stopper, and the in-tube additive of the cfDNA vacuum blood collection tube as described above, wherein the rubber stopper is arranged at the tube mouth of the test tube, the in-tube additive is added to the test tube, the inside of the test tube is in a vacuum state, and a label is affixed to the outer wall of the test tube.

[0026] In some embodiments of the present invention, the test tube is a glass tube or a plastic tube;

[0027] The glass tube is made of low borosilicate material;

[0028] The plastic tube is made of polypropylene, polyethylene terephthalate or polyethylene;

[0029] The outside of the rubber plug is also provided with a cap, and the rubber plug is made of halogenated butyl rubber;

[0030] The cap is made of polyethylene or high-density polyethylene.

[0031] An embodiment of the present invention provides an in-tube additive and a cfDNA vacuum blood collection tube, which, by using anti-deoxyribonuclease antibodies as their enzyme inhibitor components, allows cfDNA to exist stably after blood collection and can effectively improve hemolysis for subsequent downstream analysis of cfDNA. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0033] Currently, cfDNA preservation methods have problems such as limited long-term stability of cfDNA preservation and the use of common additives that affect downstream analysis. In addition, current conventional preservation methods also cause hemolysis problems, which seriously affect related downstream tests.

[0034] After long-term research, the inventors found that the main cause of cfDNA degradation in blood collection tubes is the activity of deoxyribonucleases (DNases). Using inhibitory antibodies as blood collection tube additives to target these DNases provides a more specific and potentially more effective cfDNA preservation method compared to broad-based chemical inhibitors.

[0035] Based on this, an embodiment of the present invention provides an in-tube additive for a cfDNA vacuum blood collection tube, including an anticoagulant, a preservative, a cell protectant, an enzyme inhibitor, a metabolic inhibitor, and an antioxidant; the enzyme inhibitor includes a nuclease inhibitor; and the nuclease inhibitor includes an anti-deoxyribonuclease antibody.

[0036] The embodiment of the present invention can effectively improve the long-term stability of cfDNA storage and reduce the impact of hemolysis on subsequent cfDNA testing by using the above-mentioned in-tube additives of the cfDNA vacuum blood collection tube.

[0037] In detail, when the nuclease inhibitor includes anti-deoxyribonuclease antibodies, the activity of ribonucleases can be effectively inhibited. Moreover, since anti-deoxyribonuclease antibodies have high specificity and only target deoxyribonucleases, the use of the above-mentioned anti-deoxyribonuclease antibodies can effectively reduce the use of anticoagulants and chemical preservatives, and effectively improve the impact on downstream analysis of cfDNA. At the same time, when the additives in the tube of the cfDNA vacuum blood collection tube contain the above-mentioned components, the blood is less hemolyzed during the storage process, thereby effectively improving the impact of hemolysis on subsequent tests.

[0038] It should be clarified that the additives in the tube of the above-mentioned cfDNA vacuum blood collection tube also include a solvent, which can be pure water without RNase activity. The embodiment of the present invention does not specifically limit the content of the solvent, and it can be selected according to actual conditions. For example, based on the total volume of the additives in the tube, the content of pure water without RNase activity can be 20µl / ml.

[0039] In a specific embodiment, the components of the additives in the tube of the above-mentioned cfDNA vacuum blood collection tube can be tested for protein components (such as anti-deoxyribonuclease antibodies) of the additives in the tube by ELISA or Western blot; and the non-protein components of the additives in the tube (such as preservatives, cell protectants, etc.) can be tested by high performance liquid chromatography (HPLC) or atomic absorption spectroscopy (AAS).

[0040] The inventors have also discovered that DNaseI and DNase1L3, two deoxyribonucleases, have a more significant impact on cfDNA degradation. Given the important role of DNaseI and DNase1L3 in cfDNA degradation, in some embodiments of the present invention, the anti-DNase antibodies include at least one of anti-DNASE1 and anti-DNASE1L3 antibodies. When the in-tube additives of the cfDNA vacuum blood collection tubes of the present invention include these anti-DNase antibodies, the long-term stability of the cfDNA storage can be further improved.

[0041] In some embodiments of the present invention, when the mass-to-volume ratio of the anti-DNase antibody to the volume of the in-tube additive is between 0.1 ng / ml and 10 ng / ml, the long-term stability of the in-tube additive in cfDNA vacuum blood collection tubes can be further improved. For example, the mass-to-volume ratio of the anti-DNase antibody to the volume of the in-tube additive is in the range of 0.1 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, or any two thereof.

[0042] In some embodiments of the present invention, the anti-DNASE1 antibodies include anti-DNASE1 antibodies and anti-DNASE1L3 antibodies. When the mass-to-volume ratio of the anti-DNASE1 antibodies to the anti-DNASE1L3 antibodies is (0.2-5:1), the long-term stability of the additives in cfDNA vacuum blood collection tubes can be further improved during the storage of cfDNA. For example, the mass-to-volume ratio of the anti-DNASE1 antibodies to the anti-DNASE1L3 antibodies is 0.2:1, 1:1, 2:1, 3:1, 4:1, 5:1, or any combination thereof.

[0043] In some embodiments of the present invention, the anti-DNase antibody includes at least one of a polyclonal antibody, a monoclonal antibody, and a genetically engineered antibody. The anti-DNase antibody has better targeting performance and is more conducive to improving the long-term stability of cfDNA storage.

[0044] The inventors also found that the mass-volume ratio of each component of the additives in the cfDNA vacuum blood collection tube can better improve the long-term stability of cfDNA storage and reduce the impact of hemolysis on subsequent cfDNA testing.

[0045] In some embodiments, the mass volume ratio of the anticoagulant in the in-tube additive is 0.8 mg / ml to 5 mg / ml, based on the total volume of the in-tube additive. For example, the mass volume ratio of the anticoagulant in the in-tube additive is 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, or any two thereof.

[0046] In some embodiments, the mass volume ratio of the preservative in the in-tube additive is 10 mg / ml to 500 mg / ml, based on the total volume of the in-tube additive. For example, the mass volume ratio of the preservative in the in-tube additive is 10 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, 500 mg / ml, or any two thereof.

[0047] In some embodiments, the mass volume ratio of the cell protectant in the in-tube additive is 1 mg / ml to 100 mg / ml, based on the total volume of the in-tube additive. For example, the mass volume ratio of the cell protectant in the in-tube additive is 1 mg / ml, 10 mg / ml, 20 mg / ml, 40 mg / ml, 60 mg / ml, 80 mg / ml, 100 mg / ml, or a range consisting of any two thereof.

[0048] In some embodiments, the mass volume ratio of the enzyme inhibitor in the in-line additive is 1 mg / ml to 610 mg / ml, based on the total volume of the in-line additive. For example, the mass volume ratio of the enzyme inhibitor in the in-line additive is, for example, 1 mg / ml, 10 mg / ml, 20 mg / ml, 40 mg / ml, 60 mg / ml, 80 mg / ml, 100 mg / ml, 610 mg / ml, or a range consisting of any two thereof.

[0049] In some embodiments, the mass volume ratio of the metabolic inhibitor in the in-tube additive is 1 mg / ml to 50 mg / ml, based on the total volume of the in-tube additive. For example, the mass volume ratio of the metabolic inhibitor in the in-tube additive is, for example, 1 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, or any two thereof.

[0050] In some embodiments, the blood collection tubes of the present invention may further include an osmotic pressure regulator. The mass-volume ratio of the osmotic pressure regulator to the total volume of the additives in the tube is 50 mg / ml to 100 mg / ml. For example, the mass-volume ratio of the osmotic pressure regulator to the additives in the tube is 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, or any combination thereof.

[0051] In addition, the embodiments of the present invention further improve the long-term stability of cfDNA preserved by the additives in the cfDNA vacuum blood collection tube and the hemolysis problem by further controlling the types of enzyme inhibitors, anticoagulants, preservatives, cell protectants, metabolic inhibitors, and antioxidants.

[0052] In some embodiments of the present invention, the enzyme inhibitor further comprises a protease inhibitor, and the protease inhibitor comprises one or more of aprotinin, sodium orthovanadate, leupeptin, phenylmethylsulfonyl fluoride, serine protease inhibitors, cysteine protease inhibitors, aspartic acid protease inhibitors and metalloproteinase inhibitors.

[0053] In some embodiments, the anticoagulant includes one or more of ethylenediaminetetraacetic acid potassium salt (EDTA-3K), ethylenediaminetetraacetic acid sodium salt, ethylenediamine succinate sodium salt (EDDHA-Na), sodium citrate, and heparin salts. Specifically, the heparin salt includes one or more of heparin sodium, heparin lithium, heparin calcium, and heparinamine.

[0054] In some embodiments, preservatives include methyl paraben, ethyl paraben, propyl paraben, isopropyl paraben, butyl paraben, isobutyl paraben, sodium methyl paraben, sodium ethyl paraben, sodium propyl paraben, sodium isobutyl paraben, sodium isopropyl paraben, sodium butyl paraben, diimidazolidinyl urea, imidazolidinyl urea, diazolidinyl urea, iodopropynyl butylcarbamate, sorbic acid, potassium sorbate, cetyltrimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride, diisobutylethoxyethyldimethylbenzyl ammonium chloride, diisobutylphenoxyethoxyethyldimethylbenzyl ammonium chloride, N-alkyl-N,N-dimethylbenzyl ammonium chloride, ammonium bromide, saccharin ammonium, trimethylammonium chloride, sodium aluminum chloroglycolate, trisodium citrate Ethyl ester, tricetyl methyl ammonium chloride, 3,4,4'-trichlorocarbonanilide, L-lysine hexadecylamide, DMDM hydantoin, sodium hydroxymethylaminoacetate, benzoic acid, propionic acid, salicylic acid, 2,4-hexadienoic acid, 2-hydroxybiphenyl, 3-acetyl-methyl-2,4-(3H)pyrandione, formic acid, undecylenic acid, 5-amino-1,3-bis(2-ethylhexyl)-5-methylhexahydropyrimidine, 2 -bromo-2-nitro-1,3-propanediol, 2,4-dichlorobenzyl alcohol, N-(4-chlorophenyl)-N'-(3,4-dichlorophenyl) urea, 2,4,4'-trichloro-2'-hydroxy-diphenyl ether, poly(hexamethylene biguanide)-hydrochloride, 1,2-dibromo-2,4-dicyanobutane, 4,4-dimethyl-1,3-oxazolidine, sodium benzoate, methylisothiazolinone, ethanol, and methanol. One or more of the following:

[0055] In some embodiments, the cytoprotectant comprises one or more of serine, cysteine, glutamine, glycine, tyrosine, aspartic acid, asparagine, D-mannitol, glucose, and rhamnose.

[0056] In some embodiments, the metabolic inhibitor comprises one or more of aureotric acid, glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 3-phospho-2-phosphoglycerate, phosphoric acid, pyruvic acid, gluconic acid, sodium fluoride, potassium fluoride, and aluminum fluoride.

[0057] In some embodiments, the osmotic pressure adjusting agent comprises sodium chloride.

[0058] In some embodiments, the antioxidant comprises at least one of glutathione and N-acetylcysteine.

[0059] In some embodiments of the present invention, by controlling the volume ratio of the additive to the blood sample within the tube to 1:(5-10), the long-term stability of the cfDNA stored by the additive in the cfDNA vacuum blood collection tube can be further improved while also addressing hemolysis issues. For example, the volume ratio of the additive to the blood sample within the tube can be controlled to be 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any combination thereof.

[0060] Embodiments of the present invention also provide a blood collection tube comprising a test tube, a rubber stopper, and the aforementioned intra-tube additive for the cfDNA vacuum blood collection tube. The rubber stopper is positioned at the mouth of the test tube, the intra-tube additive is added to the test tube, the test tube is vacuum-sealed, and a label is affixed to the outer wall of the test tube. The blood collection tube of the embodiment of the present invention has advantages corresponding to the intra-tube additive for the aforementioned cfDNA vacuum blood collection tube, which are not further described here.

[0061] In some embodiments, the test tube is a glass tube or a plastic tube; the glass tube is made of low-borosilicate material; the plastic tube is made of polypropylene, polyethylene terephthalate, or polyethylene; the rubber stopper is provided with a cap made of halogenated butyl rubber; and the cap is made of polyethylene or high-density polyethylene. The blood collection tubes of the present invention can further improve the long-term stability of cfDNA stored in the cfDNA vacuum blood collection tubes and address hemolysis issues.

[0062] The technical solution of the present invention is further described below with reference to specific embodiments.

[0063] Example 1

[0064] The additives in the cfDNA vacuum blood collection tube of this embodiment have the following components:

[0065] EDTA-3K (anticoagulant) 0.5 mg / ml, EDDHA–Na (anticoagulant) 0.3 mg / ml, DNASE1 Polyclonal Antibody (anti-DNASE1 polyclonal antibody) 0.2 ng / ml, DNASE1L3 Polyclonal Antibody (anti-DNASE1L3 polyclonal antibody) 0.98 ng / ml, rhamnose (cytoprotectant) 10 mg / ml, glucose (cytoprotectant) 30 mg / ml, glutamine (cytoprotectant) 5 mg / ml, glycine (cytoprotectant) 5 mg / ml, sodium orthovanadate (protease inhibitor) 600 mg / ml, genistein (protease inhibitor) 5 mg / ml, aprotinin (protease inhibitor) 5 mg / ml, aluminum fluoride (metabolic inhibitor) 5 mg / ml, glutathione (antioxidant) 30 mg / ml, diazolidinyl urea (preservative) 200 mg / ml, RNase-free purified water 20 µl / ml.

[0066] The above-mentioned in-tube additives are placed in a glass vacuum blood collection tube (the glass tube is made of low borosilicate material, the rubber stopper is made of halogenated butyl rubber material, and the cap is made of polyethylene), and the volume ratio of the in-tube additives to the blood sample is 1:8.

[0067] Example 2

[0068] This embodiment is basically the same as Example 1, except that the anti-deoxyribonuclease antibody in this embodiment is DNASE1 Polyclonal Antibody.

[0069] Example 3

[0070] This embodiment is basically the same as Example 1, except that the anti-deoxyribonuclease antibody in this embodiment is DNASE1L3 Polyclonal Antibody.

[0071] Example 4

[0072] This embodiment is basically the same as Example 1, except that the mass-to-volume ratio of the anti-DNase antibody in this embodiment to the volume of the additive in the tube is 0.1 ng / ml.

[0073] Example 5

[0074] This embodiment is basically the same as Example 1, except that the mass-to-volume ratio of the anti-DNase antibody in this embodiment to the volume of the additive in the tube is 10 ng / ml.

[0075] Example 6

[0076] This embodiment is basically the same as Example 1, except that the mass-to-volume ratio of the anti-DNase antibody in the volume of the additive in the tube in this embodiment is 0.5 ng / ml.

[0077] Example 7

[0078] This embodiment is basically the same as Example 1, except that the mass-to-volume ratio of the anti-DNase antibody in this embodiment to the volume of the additive in the tube is 12 ng / ml.

[0079] Example 8

[0080] This embodiment is basically the same as Example 1, except that the mass ratio of DNASE1PolyclonalAntibody to DNASE1L3PolyclonalAntibody in this embodiment is 0.2:1.

[0081] Example 9

[0082] This example is basically the same as Example 1, except that the mass ratio of DNASE1 Polyclonal Antibody to DNASE1L3 Polyclonal Antibody in this example is 5:1.

[0083] Example 10

[0084] This embodiment is basically the same as Example 1, except that the mass ratio of DNASE1 Polyclonal Antibody to DNASE1L3 Polyclonal Antibody in this embodiment is 0.1:1.

[0085] Example 11

[0086] This example is basically the same as Example 1, except that the mass ratio of DNASE1 Polyclonal Antibody to DNASE1L3 Polyclonal Antibody in this example is 6:1.

[0087] Example 12

[0088] The additives in the cfDNA vacuum blood collection tube of this embodiment have the following components:

[0089] EDTA-3K (anticoagulant) 2 mg / ml, EDDHA–Na (anticoagulant) 3 mg / ml, DNASE1 Polyclonal Antibody (anti-DNAse antibody) 8 ng / ml, DNASE1L3 Polyclonal Antibody (anti-DNAse antibody) 2 ng / ml, rhamnose (cytoprotectant) 50 mg / ml, glucose (cytoprotectant) 10 mg / ml, glutamine (cytoprotectant) 10 mg / ml, glycine (cytoprotectant) 30 mg / ml, sodium orthovanadate (protease inhibitor) 90 mg / ml, aprotinin (protease inhibitor) 10 mg / ml, aluminum fluoride (metabolic inhibitor) 50 mg / ml, glutathione (antioxidant) 100 mg / ml, diazolidinyl urea (preservative) 500 mg / ml, RNase-free purified water 20 µl / ml.

[0090] Example 13

[0091] The additives in the cfDNA vacuum blood collection tube of this embodiment have the following components:

[0092] Sodium citrate (anticoagulant) 0.5 mg / ml, sodium heparin (anticoagulant) 0.5 mg / ml, DNASE1 polyclonal antibody (anti-DNAse antibody) 0.05 ng / ml, DNASE1L3 polyclonal antibody (anti-DNAse antibody) 0.05 ng / ml, rhamnose (cytoprotectant) 0.4 mg / ml, glucose (cytoprotectant) 0.4 mg / ml, glutamine (cytoprotectant) 0.1 mg / ml, glycine (cytoprotectant) 0.1 mg / ml, sodium orthovanadate (protease inhibitor) 0.5 mg / ml, aprotinin (protease inhibitor) 0.5 mg / ml, aluminum fluoride (metabolic inhibitor) 1 mg / ml, glutathione (antioxidant) 5 mg / ml, diazolidinyl urea (preservative) 10 mg / ml, RNase-free purified water 20 µl / ml.

[0093] Example 14

[0094] This embodiment is basically the same as embodiment 1, except that the volume ratio of the additive in the tube to the blood sample in this embodiment is 1:10.

[0095] Example 15

[0096] This embodiment is basically the same as embodiment 1, except that the volume ratio of the additive in the tube to the blood sample in this embodiment is 1:5.

[0097] Comparative Example 1

[0098] The difference between this comparative example and Example 1 is that the in-tube additive in this comparative example does not include anti-DNase.

[0099] Comparative Example 2

[0100] The blood collection tubes used in this comparative example are non-invasive vacuum blood collection tubes manufactured by Streck (Streck Company).

[0101] Comparative Example 3

[0102] The blood collection tube in this comparative example is an EDTA blood collection tube (BD Vacutainer).

[0103] Comparative Example 4

[0104] This comparative example is basically the same as Example 1, except that the anti-deoxyribonuclease antibody in this comparative example is an anti-DNASE2 monoclonal antibody 1.18 ng / ml.

[0105] Test example

[0106] Five male volunteers aged 25-30 years (after a physical examination showing normal blood test results, no signs of tumors, and no signs of inflammation) were recruited. 180 ml of blood was collected from each of the volunteers (placed in vacuum blood collection tubes as described in the Example and Comparative Examples). The blood was stored in an incubator at 20°C until the start of the experiment.

[0107] On day 14, each sample was tested as follows:

[0108] Hemolysis testing was performed by measuring the OD value at 414 nm. Three groups of samples were tested for each example and comparative example, and the average of the three samples was calculated as the hemolysis status of the blood collection tube. The results are shown in Table 1 (OD value, hemolysis status).

[0109] Following the kit's instructions, the Kaishuo Bioplasma Free Nucleic Acid DNA (cfDNA) Extraction Kit was used to extract cfDNA from the samples in the above examples and comparative examples. Fluorescence quantification and capillary electrophoresis were used to calculate the total cfDNA content. Three groups of samples were tested for each example and comparative example, and the average of the three samples was calculated as the total cfDNA content in the blood collection tube. The results are shown in Table 1 (Total cfDNA Content).

[0110]

[0111] As can be seen from the table, compared to the comparative examples, the present embodiments utilize anti-DNASE1 antibodies (including at least one of anti-DNASE1 and anti-DNASE1L3 antibodies) as their enzyme inhibitor component, allowing cfDNA to remain stable after blood collection and effectively improving hemolysis for subsequent downstream analysis of cfDNA. Compared to Examples 6 and 7, Example 1 further improves both cfDNA stability and hemolysis by controlling the mass-to-volume ratio of the anti-DNASE1 antibodies to the volume of the in-tube additive. Compared to Examples 10 and 11, Example 1 further improves both cfDNA stability and hemolysis by controlling the mass ratio of the anti-DNASE1 to anti-DNASE1L3 antibodies.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An additive for a cfDNA vacuum blood collection tube, characterized in that: Including anticoagulants, preservatives, cytoprotectants, enzyme inhibitors, metabolic inhibitors, antioxidants; The enzyme inhibitors include nuclease inhibitors; The nuclease inhibitors include anti-deoxyribonuclease antibodies; The anti-deoxyribonuclease antibody includes at least one of an anti-DNASE1 antibody and an anti-DNASE1L3 antibody.

2. The in-tube additive for cfDNA vacuum blood collection tube according to claim 1, characterized in that: The mass volume ratio of the anti-deoxyribonuclease antibody to the volume of the additive in the tube is 0.1 ng / ml to 10 ng / ml.

3. The in-tube additive for the cfDNA vacuum blood collection tube according to claim 1 or 2, characterized in that: The mass ratio of the anti-DNASE1 antibody to the anti-DNASE1L3 antibody is (0.2-5):

1.

4. The in-tube additive for the cfDNA vacuum blood collection tube according to claim 1 or 2, characterized in that: The anti-DNase antibody includes at least one of a polyclonal antibody, a monoclonal antibody, and a genetically engineered antibody.

5. The in-tube additive for the cfDNA vacuum blood collection tube according to claim 1 or 2, characterized in that: Based on the total volume of the additives in the tube, the mass volume ratio of the anticoagulant in the additives in the tube is 0.8 mg / ml to 5 mg / ml.

6. The in-tube additive for the cfDNA vacuum blood collection tube according to claim 1 or 2, characterized in that: Based on the total volume of the additives in the tube, the mass volume ratio of the preservative in the additives in the tube is 10 mg / ml to 500 mg / ml; And / or, the mass volume ratio of the cell protectant in the in-tube additive is 1 mg / ml to 100 mg / ml; and / or, the enzyme inhibitor has a mass volume ratio in the in-tube additive of 1 mg / ml to 610 mg / ml; And / or, the mass volume ratio of the metabolic inhibitor in the in-tube additive is 1 mg / ml to 50 mg / ml; And / or, the antioxidant accounts for a mass volume ratio of 5 mg / ml to 100 mg / ml in the in-tube additive.

7. The in-tube additive for the cfDNA vacuum blood collection tube according to claim 1 or 2, characterized in that: The enzyme inhibitors further include protease inhibitors, which include one or more of genistein, aprotinin, sodium orthovanadate, leupeptin, phenylmethylsulfonyl fluoride, serine protease inhibitors, cysteine protease inhibitors, aspartic acid protease inhibitors and metalloproteinase inhibitors; And / or, the anticoagulant includes one or more of ethylenediaminetetraacetic acid potassium salt, ethylenediaminetetraacetic acid sodium salt, ethylenediamine succinate sodium salt, sodium citrate, and heparin salt; And / or, the preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, isopropyl parahydroxybenzoate, butyl parahydroxybenzoate, isobutyl parahydroxybenzoate, sodium methyl parahydroxybenzoate, sodium ethyl parahydroxybenzoate, sodium propyl parahydroxybenzoate, sodium isobutyl parahydroxybenzoate, sodium isopropyl parahydroxybenzoate, sodium butyl parahydroxybenzoate, diazolidinyl urea, imidazolidinyl urea, Diazolidinyl urea, iodopropynyl butylcarbamate, sorbic acid, potassium sorbate, cetyltrimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride, diisobutylethoxyethyldimethylbenzylammonium chloride, diisobutylphenoxyethoxyethyldimethylbenzylammonium chloride, N-alkyl-N,N-dimethylbenzyl ammonium chloride, ammonium bromide, saccharin ammonium, trimethylammonium chloride, sodium aluminum chloroglycolate, triethyl citrate, tricetylmethylammonium chloride, 3,4,4'-trichlorocarbonanilide, L-lysine hexadecylamide, DMDM hydantoin, sodium hydroxymethylaminoacetate, benzoic acid, propionic acid, salicylic acid, 2,4-hexadienoic acid, 2-hydroxybiphenyl, 3-acetyl-methyl-2,4-(3H)pyrandione, formic acid, undecylenic acid, 5-amino-1,3-bis(2-ethylhexyl)-5-methylhexahydropyrimidine, 2- One or more of bromo-2-nitro-1,3-propanediol, 2,4-dichlorobenzyl alcohol, N-(4-chlorophenyl)-N'-(3,4-dichlorophenyl)urea, 2,4,4'-trichloro-2'-hydroxy-diphenyl ether, poly(hexamethylene biguanide)-hydrochloride, 1,2-dibromo-2,4-dicyanobutane, 4,4-dimethyl-1,3-oxazolidine, sodium benzoate, methylisothiazolinone, ethanol, and methanol; and / or, the cell protectant comprises one or more of serine, cysteine, glutamine, glycine, tyrosine, aspartic acid, asparagine, D-mannitol, glucose, and rhamnose; and / or, the metabolic inhibitor comprises one or more of auronic acid, glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 3-phospho-2-phosphoglycerate, phosphoric acid, pyruvic acid, gluconic acid, sodium fluoride, potassium fluoride, and aluminum fluoride; And / or, the antioxidant includes at least one of glutathione and N-acetylcysteine.

8. The in-tube additive for cfDNA vacuum blood collection tube according to claim 1 or 2, characterized in that: The volume ratio of the additive in the tube to the blood sample is 1:(5-10).

9. A blood collection tube, characterized in that: The invention comprises a test tube, a rubber stopper and an in-tube additive for a cfDNA vacuum blood collection tube according to any one of claims 1 to 8, wherein the rubber stopper is arranged at the tube mouth of the test tube, the in-tube additive is added to the test tube, the inside of the test tube is in a vacuum state, and a label is affixed to the outer wall of the test tube.

10. The blood collection tube according to claim 9, characterized in that: The test tube is a glass tube or a plastic tube; The glass tube is made of low borosilicate material; The plastic tube is made of polypropylene, polyethylene terephthalate or polyethylene; The outside of the rubber plug is also provided with a cap, and the rubber plug is made of halogenated butyl rubber; The cap is made of polyethylene or high-density polyethylene.

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