A peptide sample stabilizer

Abstract

The present invention discloses a polypeptide sample stabilizer, which includes, by mass percentage: 1-3% of ethylenediaminetetraacetic acid or ethylenediaminetetraacetate; 1-3% of N-ethylmaleimide; 1-2% of cyclodextrin; 1-3% of leupeptin; 0.02-0.1% of preservative; and the rest is an acidic buffer solution with a pH of 2.0-4.0. The polypeptide sample stabilizer breaks through the limitation of the types of traditional stabilizers, can be applied to the stability of various polypeptide samples, improves the stability of polypeptides in ex vivo blood, makes polypeptides not easy to decompose in vitro (serum / plasma / whole blood), thereby improving the accuracy of polypeptide detection results.

Description

Technical Field

[0001] The invention belongs to the technical field of medical in vitro diagnosis, and specifically relates to a polypeptide sample stabilizer. Background Art

[0002] Peptide is a compound formed by α-amino acids linked together by peptide bonds. It is also an intermediate product of protein hydrolysis. Peptide has important biological functions and is an important physiological regulator of the human body. It can comprehensively regulate the physiological functions of the human body and enhance and exert the physiological activity of the human body.

[0003] In the human metabolic pathway, polypeptides are key intermediates, and their content in body fluids can reflect many human functional states. In the field of in vitro diagnosis, many pathological markers for disease detection are polypeptide antigens, and their increase or decrease in body fluids can directly reflect a certain disease.

[0004] Among them, gastrin-17, which reflects the state of gastric function, is composed of 17 amino acids. It is a gastrointestinal hormone secreted by G cells in the gastric antrum and duodenum. It plays an important role in regulating the function of the digestive tract and maintaining its structural integrity. The level of gastrin is an important indicator of gastric mucosal damage. Natriuretic peptide (BNP), which reflects cardiovascular function, can be used as an auxiliary diagnostic indicator for congestive heart failure and mild heart dysfunction.

[0005] C-peptide is a single-chain polypeptide composed of 31 amino acids (AA 33-63). C-peptide is a byproduct of the cleavage of proinsulin into insulin during insulin biosynthesis. There is evidence that C-peptide, as a substitute for insulin, can prevent the progression of long-term complications of type 1 diabetes.

[0006] There are many other peptide pathological markers. With the continuous development of diagnostic reagents, human serum testing with peptide pathological markers as the core indicator is a non-invasive, painless, safe and economical detection method. Currently, the methods used for peptide detection mainly include enzyme immunoassay, fluorescence chromatography, magnetic particle chemiluminescence, etc.

[0007] Peptides will further change in vitro (serum / plasma / whole blood). There is no definite conclusion on the mechanism of change at present, but studies have shown that the changes of peptides in vitro may be caused by multiple reasons. The more widely accepted theories include the following:

[0008] The first one: endopeptidases and exopeptidases in the sample will further degrade polypeptide antigens.

[0009] The second type: in vitro aggregation of polypeptides also causes changes in the polypeptide structure, making the detection reagent unable to detect.

[0010] The third type: complement proteins in the sample will also degrade polypeptides.

[0011] Regardless of the reason, the peptides in the sample will change rapidly after blood collection. Theoretically, the stable storage time at 2-8°C is only tens of minutes. As the temperature rises, the degradation rate will be faster, which means that the blood collection and testing must be completed in a very short time. In actual work, many hospitals are unable to conduct timely testing in a short time. This also leads to inaccurate peptide test results in clinical practice, which cannot accurately reflect the patient's condition.

[0012] With regard to the instability of polypeptides in ex vivo blood, those skilled in the art have done a lot of research.

[0013] Chinese patent publication number CN115702637A discloses "a protective agent for gastrin 17 blood samples", and proposes using 2-bis(2-hydroxyethyl)amino-2-hydroxymethyl-1,3-propanediol and / or -1,3-bis[(trihydroxymethyl)methylamino]propane as stabilizers to effectively avoid the degradation of polypeptides and greatly extend the storage time of polypeptide blood samples, thereby ensuring the accuracy of the test results. The mechanism by which this patent can inhibit the degradation of gastrin 17 using only one buffer is not clear.

[0014] Chinese patent publication number CN1950398A discloses "peptide stabilization". The patent shows that the use of DMSO can extend the storage time of gastrin in serum or plasma samples from 3 hours to 72 hours. However, DMSO as an organic solvent can cause red blood cells to break, resulting in errors in the test results.

[0015] Existing stabilizers are only aimed at stabilizing gastrin-like polypeptides in blood samples, which is too single-minded and the stabilization effect is not ideal. Summary of the invention

[0016] The purpose of the present invention is to provide a polypeptide sample stabilizer, which breaks through the type limitation of traditional stabilizers and can be applied to the stabilization of various polypeptide samples, improve the stability of polypeptides in ex vivo blood, and make polypeptides less likely to decompose in vitro (serum / plasma / whole blood), thereby improving the accuracy of polypeptide detection results.

[0017] In order to achieve the above object, the technical solution of the present invention is:

[0018] A polypeptide sample stabilizer, characterized in that it comprises, by mass percentage: ethylenediaminetetraacetic acid or ethylenediaminetetraacetate: 1-3%; N-ethylmaleimide: 1-3%; cyclodextrin: 1-2%; leupeptin: 1-3%; preservative 0.02-0.1%; and the rest is an acidic buffer solution with a pH of 2.0-4.0.

[0019] Preferably, the ethylenediaminetetraacetic acid salt is ethylenediaminetetraacetic acid sodium salt or ethylenediaminetetraethyl potassium salt.

[0020] Preferably, the acidic buffer is one or more of glycine-hydrochloric acid, citric acid-sodium citrate, and acetic acid-sodium acetate buffer.

[0021] Preferably, the leupeptin is one or more of its hydrochloride and sulfate.

[0022] Preferably, the preservative is one or more of sodium azide, P300, Bro.

[0023] Research has found that the degradation of polypeptide molecules in vitro is determined by multiple factors, and the main influencing factors are related to polypeptide structure, sample type, storage conditions, etc.

[0024] The acidic buffer in the present invention can make polypeptides with higher isoelectric points carry positive charges, thereby reducing electrostatic adsorption between polypeptides and ensuring the dispersibility of polypeptides. Cyclodextrin Cyclodextrin molecules have a slightly conical hollow cylindrical three-dimensional ring structure, with hydrophilic outer edges and hydrophobic inner cavities, which can provide a stable microenvironment. For polypeptides with strong hydrophobicity, it can effectively avoid the binding of hydrophobic bonds to cause polypeptide aggregation. The acidic system combined with cyclodextrin can effectively solve the inactivation of polypeptides caused by physical and chemical effects (electrostatic adsorption, hydrophobic binding, etc.).

[0025] EDTA, as a metal chelator, can inhibit the activity of multiple metal enzymes. A single enzyme inhibitor can only have a good stabilizing effect on a certain polypeptide. In order to fundamentally improve the stability of polypeptides, the composite addition of EDTA, NEMI, and leupeptin in the present invention, and the combined action of cyclodextrin and acidic buffer, can effectively inhibit the activity of biologically active substances such as endopeptidases and complements in the blood. The synergy of multiple inhibitors can improve the stability of different types of polypeptides from multiple angles. The stabilizer of the present invention can be used in two ways. First, as an independent stabilizer, the stabilizer is added to the blood collection tube containing blood after blood collection and mixed evenly.

[0026] Secondly, during the production process of blood collection tubes, the stabilizer is added to the blood collection tubes as a component of the blood collection tubes, and the blood collection tubes are directly used for blood collection when blood collection is needed.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention can effectively solve the inactivation of polypeptides caused by physical and chemical effects (electrostatic adsorption, hydrophobic binding, etc.) by adding various types of stabilizers and acidic systems with cyclodextrin, thereby fundamentally improving the stability of polypeptides in in vitro samples.

[0029] The obtained stabilizer is highly versatile, feasible, and low-cost. It can detect the stability of polypeptide pathological markers (such as gastrin-17, natriuretic peptide, etc.) in blood samples. It is easy to operate and can be adapted to different methodologies (POCT, enzyme immunoassay, magnetic particle) for detection. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the embodiments.

[0031] The specific ingredients of the stabilizers in the embodiments of the present invention and the comparative examples are shown in Table 1;

[0032] The stabilizer described in the present invention and the stabilizer described in the comparative example were added to blood samples containing different concentrations of polypeptides, respectively. The samples were then placed in the same environment at room temperature (18-25°C). After being placed for different periods of time, the gastrin-17 (G-17) chemiluminescence detection kit, brain natriuretic peptide (BNP) chemiluminescence detection kit, and C-peptide (CP) chemiluminescence detection kit developed by Pinfeng were used to measure the contents of gastrin-17 (G-17), brain natriuretic peptide (BNP), and C-peptide (CP) in the blood samples, and the deviations from the corresponding polypeptides contained in the initial blood were calculated. The results are shown in Table 2.

[0033] It can be seen from the data in Table 2 that the addition of the stabilizer of the present invention can greatly improve the stability of the polypeptide in the in vitro blood sample.

[0034] In comparative example 1, the activity of polypeptides decreased by more than 40% after being stored at room temperature for 4 hours, while in the test group in which the stabilizer of the present invention was added, the polypeptide content in the blood sample did not decrease significantly after the sample was stored for 7 days.

[0035] It can be seen from the data of comparative examples 2 / 3 / 4 / 5 that the addition of a single component can improve the stability of polypeptides to a certain extent, and the role of each component is also different for different types of polypeptides. Compared with the experimental group, the synergistic effect of the composite components of the experimental group can not only significantly improve the stability of the polypeptide, but also improve the versatility of the stabilizer.

[0036] The above description is only an embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, is also included in the patent protection scope of the present invention.

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Claims

1. A polypeptide sample stabilizer, characterized in that: The composition comprises, by mass percentage, ethylenediaminetetraacetic acid or ethylenediaminetetraacetic acid salt: 1-3%; N-ethylmaleimide: 1-3%; cyclodextrin: 1-2%; leupeptin: 1-3%; preservative 0.02-0.1%; the rest is an acidic buffer solution with a pH of 2.0-4.0, and the polypeptide sample is gastrin-17, brain natriuretic peptide or C-peptide; The acidic buffer is glycine-hydrochloric acid, citric acid-sodium citrate or acetic acid-sodium acetate buffer, and the acidic buffer is 0.2 mol / L; The preservative is sodium azide, P300 or Bro.

2. The polypeptide sample stabilizer according to claim 1, characterized in that: The ethylenediaminetetraacetic acid salt is ethylenediaminetetraacetic acid sodium salt or ethylenediaminetetraethyl potassium salt.

3. The polypeptide sample stabilizer according to claim 1, characterized in that: The leupeptin is one or more of its hydrochloride and sulfate.

4. A method for using the polypeptide sample stabilizer according to any one of claims 1 to 3, characterized in that: The components are mixed evenly to obtain a stabilizer for use, and after blood collection, the stabilizer is added into a blood collection tube containing blood and mixed evenly.

5. A method for using the polypeptide sample stabilizer according to any one of claims 1 to 3, characterized in that: During the production process of blood collection tubes, the stabilizer is added into the blood collection tubes as a component of the blood collection tubes, and the blood collection tubes are directly used for blood collection when blood collection is needed.

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