阅读说明：本技术 一种用于测定活化部分凝血活酶时间的体外诊断试剂盒 (In-vitro diagnostic kit for measuring activated partial thromboplastin time ) 是由 郑琳 陈明峰 郑筱雯 余嘉陵 于 2020-06-08 设计创作，主要内容包括：本发明公开了一种用于测定活化部分凝血活酶时间的体外诊断试剂盒,包括APTT检测试剂和用于盛放所述APTT检测试剂的塑料瓶；所述APTT检测试剂由部分凝血活酶试剂和钙盐溶液组成,所述部分凝血活酶试剂由脑磷脂溶液和缓冲液配制而成,所述缓冲液由下述原料制成：活化剂,tris,氯化钠,甘露醇,聚乙二醇,牛血清白蛋白,丙氨酸,石炭酸,proclin300和水。本发明的用于测定活化部分凝血活酶时间的体外诊断试剂盒,使用方便,在保证APTT检测试剂的准确性和灵敏性的基础上,提高APTT检测试剂的稳定性,使该体外诊断试剂盒在临床上广泛应用,使APTT检验实现标准化。(The invention discloses an in-vitro diagnostic kit for measuring activated partial thromboplastin time, which comprises an APTT detection reagent and a plastic bottle for containing the APTT detection reagent; the APTT detection reagent is composed of a partial thromboplastin reagent and a calcium salt solution, the partial thromboplastin reagent is prepared from a cephalin solution and a buffer solution, and the buffer solution is prepared from the following raw materials: activator, tris, sodium chloride, mannitol, polyethylene glycol, bovine serum albumin, alanine, carbolic acid, proclin300 and water. The in-vitro diagnostic kit for measuring the activated partial thromboplastin time is convenient to use, improves the stability of the APTT detection reagent on the basis of ensuring the accuracy and the sensitivity of the APTT detection reagent, enables the in-vitro diagnostic kit to be widely applied clinically, and enables APTT detection to be standardized.)

1. An in vitro diagnostic kit for determining the time of activated partial thromboplastin, comprising an APTT detection reagent and a plastic bottle for containing the APTT detection reagent; the APTT detection reagent is composed of a partial thromboplastin reagent and a calcium salt solution, the partial thromboplastin reagent is prepared from a cephalin solution and a buffer solution, and the buffer solution is prepared from the following raw materials: activator, tris, sodium chloride, mannitol, polyethylene glycol, bovine serum albumin, alanine, carbolic acid, proclin300 and water.

2. The in vitro diagnostic kit for determining activated partial thromboplastin time of claim 1, wherein said buffer is prepared from the following raw materials in weight percent: 0.3-1.0% of an activating agent, 0.1-8% of tris, 0.1-10% of sodium chloride, 1-3% of mannitol, 1-20% of polyethylene glycol, 1-10% of bovine serum albumin, 0.5-10% of alanine, 0.1-2% of carbolic acid, 3000.5-1% of proclin and the balance of water.

3. The in vitro diagnostic kit for determining activated partial thromboplastin time according to claim 1, wherein the calcium salt solution is a calcium chloride solution, and the concentration of calcium chloride in the calcium chloride solution is 15 to 30 mmol/L.

4. The in vitro diagnostic kit for measuring activated partial thromboplastin time according to claim 1, wherein the cephalin solution is prepared from cephalin and physiological saline.

5. The in vitro diagnostic kit for determining activated partial thromboplastin time according to claim 4, wherein said cephalin is rabbit cephalin or bovine cephalin.

6. The in vitro diagnostic kit for measuring activated partial thromboplastin time according to claim 1, wherein the activating agent is any one of kaolin, ellagic acid, and diatomaceous earth.

7. The in vitro diagnostic kit for determining the time to activate partial thromboplastin of claim 1, wherein the degree of polymerization of the polyethylene glycol is 4000-8000.

8. The in vitro diagnostic kit for determining activated partial thromboplastin time of claim 1, wherein the volume ratio of the cephalin solution to the buffer is 1: (5-15).

9. The in vitro diagnostic kit for measuring activated partial thromboplastin time according to claim 1, wherein the partial thromboplastin reagent is prepared by the following method:

weighing 0.5-10 g of cephalin, and dispersing and uniformly mixing with 5-10 mL of normal saline to obtain a cephalin solution;

uniformly mixing an activating agent, tris, sodium chloride, mannitol, polyethylene glycol, bovine serum albumin, alanine, carbolic acid, proclin300 and water to obtain a buffer solution;

and adding the cephalin solution into the buffer solution to obtain a partial thromboplastin reagent.

Technical Field

The invention relates to the field of clinical diagnostic reagents, in particular to an in-vitro diagnostic kit for measuring the activated partial thromboplastin time.

Background

Whether the blood coagulation function is normal or not is related to the physiological health state of a human body, and the evaluation experiment of the blood coagulation function has important significance for diagnosis, treatment and prognosis judgment of various diseases. The coagulation process is complex, the detection indexes are various, and the detection indexes are mainly divided into an extrinsic coagulation pathway and an intrinsic coagulation pathway. The blood coagulation time is an important index for measuring the state of the blood coagulation state, and has important significance for judging the organism hemostasis and the pathological change of the blood coagulation system, the preoperative blood coagulation disease screening and the like. Wherein, Activated Partial Thromboplastin Time (APTT) is an important index for detecting the intrinsic coagulation function in the coagulation time detection. The reasons for prolonging the APTT are often found in the reduction of blood coagulation factors II, V, VIII, IX, XI and XII, fibrinogen deficiency, increased fibrinolytic activity, presence of anticoagulant substances (e.g. increased heparin content in blood and oral anticoagulants). The reasons for the shortened APTT are often found in hypercoagulable states, thrombotic disorders (e.g. myocardial infarction, unstable angina, cerebrovascular disease, pulmonary infarction, deep vein thrombosis, pregnancy-hypertension syndrome and nephrotic syndrome). Therefore, the detection of the APTT can understand the approximate change of the coagulation factors in the endogenous and exogenous coagulation systems in the blood, and if part of the endogenous coagulation factors are deficient, the APTT is prolonged; the method can also be widely used for observing diagnosis and treatment effects of hemorrhagic diseases before operation of the diseases, is used for monitoring the dosage of anticoagulant heparin, and is an important index for monitoring heparin treatment.

At present, a semi-automatic or automatic instrument is used for APTT determination, and the detection principle is that partial thromboplastin is added into blood plasma to be detected, proper calcium ions are added, fibrinogen is changed into insoluble fibrin under the participation of the calcium ions, and therefore coagulation occurs. And measuring the time required for coagulation, namely the Activated Partial Thromboplastin Time (APTT) of the plasma to be measured. Due to the different quality of the used APTT detection reagents, the results of the same patient measured in different hospitals are greatly different, so that the inconsistency of the detection results is caused, and the correct and timely diagnosis of diseases is influenced. Therefore, the quality of the APTT test reagent becomes the key to obtaining accurate results and diagnosis. However, the APTT detection reagent in the current market has large quality change along with the time, so that the detection results in different periods within the validity period are greatly different; some freeze-dried reagents need to be re-dissolved and then used, so that the difference between bottles of the reagents is large, and the measurement results are large.

Therefore, how to develop an APTT (activated partial thromboplastin time) detection reagent with high stability and convenient use becomes a technical problem to be solved in the field.

Disclosure of Invention

The invention mainly aims to provide an in vitro diagnostic kit for measuring the activated partial thromboplastin time, which is convenient to use, improves the stability of an APTT detection reagent on the basis of ensuring the accuracy and the sensitivity of the APTT detection reagent, enables the in vitro diagnostic kit to be widely applied clinically, and enables APTT detection to be standardized.

The technical problem to be solved by the invention is realized by the following technical scheme:

an in vitro diagnostic kit for determining the time of activated partial thromboplastin, comprising an APTT detection reagent and a plastic bottle for containing the APTT detection reagent; the APTT detection reagent is composed of a partial thromboplastin reagent and a calcium salt solution, the partial thromboplastin reagent is prepared from a cephalin solution and a buffer solution, and the buffer solution is prepared from the following raw materials: activator, tris, sodium chloride, mannitol, polyethylene glycol, bovine serum albumin, alanine, carbolic acid, proclin300 and water.

Further, the buffer solution is prepared from the following raw materials in percentage by weight: 0.3-1.0% of an activating agent, 0.1-8% of tris, 0.1-10% of sodium chloride, 1-3% of mannitol, 1-20% of polyethylene glycol, 1-10% of bovine serum albumin, 0.5-10% of alanine, 0.1-2% of carbolic acid, 3000.5-1% of proclin and the balance of water.

Further, the calcium salt solution is a calcium chloride solution, and the concentration of calcium chloride in the calcium chloride solution is 15-30 mmol/L.

Further, the cephalin solution is prepared from cephalin and normal saline.

Further, the cephalin is rabbit cephalin or bovine cephalin.

Further, the activator is any one of kaolin, ellagic acid, and diatomaceous earth.

Further, the polymerization degree of the polyethylene glycol is 4000-8000.

Further, the volume ratio of the cephalin solution to the buffer is 1: (5-15).

Further, the preparation method of the partial thromboplastin reagent comprises the following steps:

weighing 0.5-10 g of cephalin, and dispersing and uniformly mixing with 5-10 mL of normal saline to obtain a cephalin solution;

uniformly mixing an activating agent, tris, sodium chloride, mannitol, polyethylene glycol, bovine serum albumin, alanine, carbolic acid, proclin300 and water to obtain a buffer solution;

and adding the cephalin solution into the buffer solution to obtain a partial thromboplastin reagent.

The invention has the following beneficial effects:

in the invention, the buffer solution prepared by the activator, tris, sodium chloride, mannitol, polyethylene glycol, bovine serum albumin, alanine, carbolic acid and proclin300 is creatively adopted to be matched with the cephalin solution, and the synergistic effect is achieved, so that the problem that the activator and cephalin are easy to precipitate can be effectively solved, and the stability of the APTT detection reagent is improved on the basis of ensuring the accuracy and the sensitivity of the APTT detection reagent.

In the existing in-vitro diagnostic kit for measuring the activated partial thromboplastin time, an APTT detection reagent is usually contained in a glass bottle, the traditional method is changed, and the APTT detection reagent is contained in a plastic bottle, so that the safety of a transported product can be ensured, and the stability of the APTT detection reagent can be unexpectedly improved.

The APTT detection reagent has good stability, can be stable for at least 10 days at 37 ℃, and is stable for at least more than 30 days at 5 +/-3 ℃ after the bottle is opened.

The APTT detection reagent disclosed by the invention is simple in production process, simple in production equipment, free of freeze-drying process and low in production cost.

The APTT detection reagent is of an instant type, does not need to be redissolved, and is convenient to operate.

Detailed Description

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

Unless otherwise defined, terms used in the present specification have the same meaning as those generally understood by those skilled in the art, but in case of conflict, the definitions in the present specification shall control.

The use of "including," "comprising," "containing," "having," or other variations thereof herein, is meant to encompass the non-exclusive inclusion, as such terms are not to be construed. The term "comprising" means that other steps and ingredients can be added that do not affect the end result. The term "comprising" also includes the terms "consisting of …" and "consisting essentially of …". The compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any of the additional or optional ingredients, components, steps, or limitations described herein.

All numbers or expressions referring to quantities of ingredients, process conditions, etc. used in the specification and claims are to be understood as modified in all instances by the term "about". All ranges directed to the same component or property are inclusive of the endpoints, and independently combinable. Because these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range.

As described in the background art, the problems of great change of the APTT detection reagent in quality along with the time and poor stability of the APTT detection reagent in the prior art, which cause great differences of detection results in different periods of time during the validity period.

In order to solve the technical problems, the invention provides an in vitro diagnostic kit for measuring the time of activated partial thromboplastin, which comprises an APTT detection reagent and a plastic bottle for containing the APTT detection reagent.

The APTT detection reagent consists of a partial thromboplastin reagent and a calcium salt solution.

The partial thromboplastin reagent is prepared from a cephalin solution and a buffer solution.

The buffer solution is prepared from the following raw materials: activator, tris, sodium chloride, mannitol, polyethylene glycol, bovine serum albumin, alanine, carbolic acid, proclin300 and water.

The partial thromboplastin reagent contains cephalin and an activator, the activator and the cephalin are easy to precipitate, and are easily influenced by environmental factors in the preparation and storage processes, so that the stability of the APTT detection reagent is not high, the biological activities of the APTT detection reagent in different batches are different, and the consistency of the detection result of a blood sample is influenced. The invention selects a specific buffer solution based on the influence and the matching with other materials from the comprehensive performance consideration of the whole technical scheme, and the buffer solution has good compatibility with the cephalin solution. In the invention, the buffer solution prepared by the activator, tris, sodium chloride, mannitol, polyethylene glycol, bovine serum albumin, alanine, carbolic acid and proclin300 is creatively adopted to be matched with the cephalin solution, and the synergistic effect is achieved, so that the problem that the activator and cephalin are easy to precipitate can be effectively solved, and the stability of the APTT detection reagent is improved on the basis of ensuring the accuracy and the sensitivity of the APTT detection reagent.

More specifically, the buffer solution is prepared from the following raw materials in percentage by weight: 0.3-1.0% of an activating agent, 0.1-8% of tris, 0.1-10% of sodium chloride, 1-3% of mannitol, 1-20% of polyethylene glycol, 1-10% of bovine serum albumin, 0.5-10% of alanine, 0.1-2% of carbolic acid, 3000.5-1% of proclin and the balance of water.

The weight percentage of the activator is 0.3 to 1.0%, for example, 0.3%, 0.5%, 0.6%, 0.8%, or 1%, but is not limited to the values listed, and other values not listed within the range of values are also applicable. The kind of the activator is not particularly limited in the present invention, and it is sufficient to use an activator well known to those skilled in the art for preparing an APTT detection reagent, and preferably, the activator is any one of kaolin, ellagic acid, and diatomaceous earth, and more preferably, the activator is ellagic acid.

The Tris is 2-amino-2- (hydroxymethyl) -1, 3-propanediol. The tris is present in an amount of 0.1 to 8% by weight, for example 0.1%, 1%, 2%, 5% or 8%, but is not limited to the recited values, and other values not recited within the range of values are also applicable.

The sodium chloride is present in an amount of 0.1 to 10% by weight, for example 0.1%, 0.5%, 2%, 5%, 8% or 10%, but the sodium chloride is not limited to the above-mentioned values, and other values not shown in the above-mentioned range are also applicable.

The mannitol is present in an amount of 1 to 3% by weight, for example 1%, 1.5%, 2%, 2.5% or 3%, but is not limited to the recited values, and other values not recited within the above range are also applicable.

The weight percentage of the polyethylene glycol is 1 to 20%, for example, 1%, 5%, 10%, 15%, or 20%, but is not limited to the recited values, and other values not recited in the range of the values are also applicable. The polymerization degree of the polyethylene glycol is 4000-8000, and more preferably, the polymerization degree of the polyethylene glycol is 6000.

The bovine serum albumin is 1 to 10% by weight, for example, 1%, 2%, 5%, 8%, or 10%, but is not limited to the above-mentioned values, and other values not shown in the above-mentioned range are also applicable.

The alanine is present in an amount of 0.5 to 10% by weight, for example 0.5%, 1%, 5%, 8% or 10%, but the alanine is not limited to the recited values, and other values not recited in the above range are also applicable.

The weight percentage of the carbolic acid is 0.1-2%, for example, 0.1%, 0.3%, 0.8%, 1.2%, 1.5%, or 2%, but not limited to the recited values, and other values not recited in the range of the values are also applicable.

The proclin300 may be 0.5 to 1% by weight, for example, 0.5%, 0.6%, 0.7%, 0.8%, or 1%, but is not limited to the above-mentioned values, and other values not shown in the above-mentioned range are also applicable.

The cephalin solution is prepared from cephalin and normal saline. More specifically, the cephalin solution comprises the following components in percentage by weight: 0.5-10% of cephalin and the balance of normal saline.

The cephalin is preferably, but not limited to, rabbit cephalin or bovine cephalin.

The volume ratio of the cephalin solution to the buffer is 1: (5-15), and more preferably, the volume ratio of the cephalin solution to the buffer is 1: 10.

specifically, the preparation method of the partial thromboplastin reagent comprises the following steps:

weighing 0.5-10 g of cephalin, and dispersing and uniformly mixing with 5-10 mL of normal saline to obtain a cephalin solution;

uniformly mixing an activating agent, tris, sodium chloride, mannitol, polyethylene glycol, bovine serum albumin, alanine, carbolic acid, proclin300 and water to obtain a buffer solution;

and adding the cephalin solution into the buffer solution to obtain a partial thromboplastin reagent.

The calcium salt may be a salt that forms calcium ions in the measurement sample used in the measurement of APTT. Examples of the calcium salt include, but are not particularly limited to, calcium chloride, calcium sulfate, calcium nitrite, calcium carbonate, calcium lactate, and calcium tartrate. These calcium salts may be used alone, or 2 or more kinds thereof may be mixed and used. More preferably, the calcium salt solution is a calcium chloride solution, the concentration of calcium chloride in the calcium chloride solution is 15-30mmol/L, and more preferably, the concentration of calcium chloride in the calcium chloride solution is 25 mmol/L.

In the existing in-vitro diagnostic kit for measuring the activated partial thromboplastin time, an APTT detection reagent is usually contained in a glass bottle, the traditional method is changed, and an APTT detection reagent is creatively contained in a plastic bottle, so that the safety of a transported product can be ensured, the product is not easy to damage in the production and transportation processes, and the inventor unexpectedly finds that the stability of the APTT detection reagent can be improved by containing the APTT detection reagent in the plastic bottle.

In the present invention, the material of the plastic bottle is not particularly limited, and various plastic bottles known in the prior art can be used, and the material of the plastic bottle is PET, PVC, HDPE, LDPE, PP, or PS, for example, and more preferably, the material of the plastic bottle is PET.

At present, most of domestic APTT detection reagents are in a freeze-dried powder preparation form, the reagents need to be redissolved before use, the operation is troublesome, and errors occur in the detection result easily caused by inaccurate redissolved liquid volume and the like. In the invention, the APTT detection reagent is of an instant type, does not need to be redissolved and is convenient to operate.

In the present invention, the detection method of the in vitro diagnostic kit for measuring the activated partial thromboplastin time comprises the following steps: incubating a portion of the thromboplastin reagent at 37 ℃; then adding the blood plasma to be detected into the incubated partial thromboplastin reagent, incubating at 37 ℃, then adding a calcium salt solution, and recording the coagulation time, namely the activated partial thromboplastin time.

In order to better understand the technical solutions, the technical solutions will be described in detail with reference to specific examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.