阅读说明：本技术 一种特殊生化复合质控品、制备方法、试剂盒 (Special biochemical composite quality control product, preparation method and kit ) 是由 李娟� 张雪连 于 2021-07-20 设计创作，主要内容包括：本发明涉及生化检测试剂技术领域,特别涉及一种特殊生化复合质控品、制备方法、试剂盒。本发明所述的特殊生化复合质控品包括基质和特殊生化4项,所述的基质包括：80％-40％质量份的组分A和20％-60％质量份的血清；所述组分A包括缓冲液、酪蛋白、糖类、冻干赋型剂、表面活性剂、盐、酶抑制剂、防腐剂；所述特殊生化4项为β-羟丁酸、5’-核苷酸酶、α-L-岩藻糖苷酶和腺苷脱氨酶。本发明的复合质控品的稳定性和互换性较好,在未开瓶2-8℃可稳定储存24个月,复溶后在2-8℃可稳定储存15天,复溶后-20℃可稳定储存30天,在不同系统中测值的一致性较好。(The invention relates to the technical field of biochemical detection reagents, in particular to a special biochemical composite quality control product, a preparation method and a kit. The special biochemical composite quality control product comprises a substrate and 4 special biochemical items, wherein the substrate comprises: 80-40% of component A and 20-60% of serum; the component A comprises buffer solution, casein, saccharides, freeze-drying excipient, surfactant, salt, enzyme inhibitor and preservative; the special biochemical items 4 are beta-hydroxybutyric acid, 5' -nucleotidase, alpha-L-fucosidase and adenosine deaminase. The composite quality control product has good stability and interchangeability, can be stably stored for 24 months at 2-8 ℃ without opening the bottle, can be stably stored for 15 days at 2-8 ℃ after redissolution, can be stably stored for 30 days at-20 ℃ after redissolution, and has good consistency of measured values in different systems.)

1.A special biochemical composite quality control product is characterized by comprising a substrate and 4 special biochemical items, wherein the substrate comprises:

80-40% by volume of component A and 20-60% by volume of serum;

the component A comprises 10mM-100mM buffer solution, 2% -5% of casein, 5% -15% of saccharides, 1% -10% of freeze-drying excipient, 0.01% -0.1% of surfactant, 0.1% -0.9% of salt, 0.1% -0.5% of enzyme inhibitor and 0.1% -0.5% of preservative; the percentages are mass volume percentages of the component A;

the special biochemical items 4 are beta-hydroxybutyric acid, 5' -nucleotidase, alpha-L-fucosidase and adenosine deaminase.

2. The biochemical composite quality control material as claimed in claim 1, wherein the buffer is selected from the group consisting of PB and Na₂HPO₄HEPES, Tris-HCl, MES, citric acid or glycine.

3. The biochemical composite quality control material as claimed in claim 1, wherein the sugar is selected from one or more of sucrose, trehalose, glucose or lactose.

4. The biochemical composite quality control material as claimed in claim 1, wherein the freeze-drying excipient is mannitol.

5. The biochemical composite quality control substance according to claim 1, wherein the surfactant is selected from the group consisting of TW-20, TW-80, Triton X-100, NP40 and SKL.

6. The specific biochemical composite quality control substance according to claim 1, wherein the salt is selected from NaCl or KCl; the enzyme inhibitor is selected from EDTA; the preservative is selected from one or more of P300, sodium azide or K500.

7. The biochemical composite quality control material according to claim 1, wherein the serum is selected from human serum, calf serum, horse serum or sheep serum.

8. The special biochemical composite quality control material according to claim 1, wherein the content of special biochemical 4 items in the special biochemical composite quality control material is 0.1mM-4.5mM of beta-hydroxybutyric acid, 5U/L-300U/L of 5' -nucleotidase, 3U/L-120U/L of alpha-L-fucosidase and 0.1-150U/L of adenosine deaminase.

9. The method for preparing a biochemical composite quality control substance according to any one of claims 1 to 8, comprising the steps of:

adding casein with the formula amount into a buffer solution, adjusting the pH value of the solution to 11.0-13.0, after the casein is completely dissolved, continuously adding sugar, a freeze-drying excipient, a surfactant, salt, an enzyme inhibitor and a preservative with the formula amount, and adjusting the pH value of the solution to 7.0-8.0 to obtain a component A;

mixing the prescribed amount of component A and serum to obtain a matrix;

adding 4 special biochemical items into the matrix to obtain the special biochemical composite quality control product.

10. A kit comprising a specific biochemical composite quality control according to any one of claims 1 to 8.

Technical Field

The invention relates to the technical field of biochemical detection reagents, in particular to a special biochemical composite quality control product, a preparation method and a kit.

Background

The quality control product is a specimen or solution specially used for quality control, and has high requirements on stability and bottle-to-bottle difference. A biogenic quality control sample needs to meet the following criteria, 1. it is homogeneous within a batch to exclude sample-to-sample gaps; 2. the number of individuals in the batch is enlarged as much as possible, and the cost is reduced to the lowest under the condition that the target value and the stability detection are qualified; 3. the material should be stable enough, not subject to any change, and be storable for a long period of time; 4. the general characteristics of the quality control specimen are the same as those of the patient sample.

At present, quality control products aiming at single detection items are mostly available in the market, however, the use of the single quality control product brings certain inconvenience to the operation and the use.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a special biochemical compound quality control product, which comprises 4 special biochemical items of beta-hydroxybutyric acid (beta-Hb), 5 '-nucleotidase (5' -NT), alpha-L-fucosidase (AFU) and Adenosine Deaminase (ADA), and has excellent bottle opening stability and freezing stability and excellent consistency in different systems.

In order to achieve the purpose, the invention specifically adopts the following technical scheme:

a special biochemical composite quality control product comprises a substrate and 4 special biochemicals, wherein the substrate comprises:

80-40% by volume of component A and 20-60% by volume of serum;

the component A comprises 10mM-100mM buffer solution, 2% -5% of casein, 5% -15% of saccharides, 1% -10% of freeze-drying excipient, 0.01% -0.1% of surfactant, 0.1% -0.9% of salt, 0.1% -0.5% of enzyme inhibitor and 0.1% -0.5% of preservative; the percentages are mass volume percentages of the component A;

the special biochemical items 4 are beta-hydroxybutyric acid, 5' -nucleotidase, alpha-L-fucosidase and adenosine deaminase.

The 10mM-100mM buffer solution in the component A refers to the concentration of 10mM-100mM in the component A, taking glycine water solution as an example, for example, 500ml of the component A is prepared, then the concentration of glycine in the component A is 10mM-100mM, namely the amount of glycine added is 5 mmol-50 mmol, the amount of casein added is 10 g-25 g, and the rest is added with water; if the buffer is changed to PB (adding NaH)₂PO₄-2H₂O&Na₂HPO₄-12H₂O), for example to prepare 500ml of component A, then NaH₂PO4-2H₂O and Na₂HPO₄-12H₂The sum of the concentrations of O in component A is from 10mM to 100mM, i.e. addingInto NaH₂PO₄-2H₂O and Na₂HPO₄-12H₂The sum of the amounts of substances O is 5mmol to 50 mmol.

Preferably, the buffer is selected from PB and Na₂HPO₄HEPES, Tris-HCl, MES, citric acid or glycine. PB herein refers to NaH₂PO₄-2H₂O&Na₂HPO₄-12H₂O; HEPES means 4-hydroxyethylpiperazine ethanesulfonic acid; Tris-HCl refers to Tris hydroxymethyl aminomethane hydrochloride; MES refers to 2- (N-morpholine) ethanesulfonic acid.

Preferably, the saccharide is selected from one or more of sucrose, trehalose, glucose or lactose.

Preferably, the freeze-drying excipient is mannitol.

Preferably, the surfactant is selected from TW-20, TW-80, Triton X-100, NP40, or SKL.

Preferably, the salt is selected from NaCl or KCl; the enzyme inhibitor is selected from EDTA; the preservative is selected from one or more of P300, sodium azide or K500. The EDTA refers to ethylenediamine tetraacetic acid, P300 is totally called Proclin300 (manufacturer: Sigma-Aldrich), and K500 is totally called KroVin 500 (manufacturer: Xibao biology).

Preferably, the serum is selected from human serum, calf serum, horse serum or sheep serum.

Preferably, the content of the special biochemical 4 items in the special biochemical compound quality control product is 0.1mM-4.5mM of beta-hydroxybutyric acid, 5U/L-300U/L of 5' -nucleotidase, 3U/L-120U/L of alpha-L-fucosidase and 0-150U/L of adenosine deaminase. The content refers to the concentration of the 4 items corresponding to the special biochemistry in the whole special biochemical composite quality control product.

The preparation method of the special biochemical composite quality control product comprises the following steps:

adding casein with the formula amount into a buffer solution, adjusting the pH value of the solution to 11.0-13.0, after the casein is completely dissolved, continuously adding sugar, a freeze-drying excipient, a surfactant, salt, an enzyme inhibitor and a preservative with the formula amount, and adjusting the pH value of the solution to 7.0-8.0 to obtain a component A;

mixing the prescribed amount of component A and serum to obtain a matrix;

adding 4 special biochemical items into the matrix to obtain the special biochemical composite quality control product.

A kit comprising a specific biochemical composite quality control according to any one of the preceding claims.

The numerical ranges involved in the above technical solutions of the present invention all include the end values.

Advantageous effects

The invention relates to a plurality of special biochemical compound quality control products, which comprise 4 special biochemical items of beta-hydroxybutyric acid (beta-Hb), 5 '-nucleotidase (5' -NT), alpha-L-fucosidase (AFU) and Adenosine Deaminase (ADA);

the composite quality control product has excellent bottle opening stability and freezing stability, can be stably stored for 24 months at 2-8 ℃ without bottle opening (redissolved), can be stably stored for 15 days at 2-8 ℃ after redissolved, can be stably stored for 30 days at-20 ℃ after redissolved, and has better consistency (namely interchangeability) of measured values in different systems (the deviation of the judgment standard with better stability and consistency and the comparison is within +/-10%).

Detailed Description

The following will clearly and completely describe the technical solutions in the specific embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention will be described in detail with reference to examples below to facilitate understanding of the present invention by those skilled in the art.

The following examples used the following sources of materials:

PB from Nanjing chemical reagents Ltd

Casein was purchased from Sigma-Aldrich

Beta-hydroxybutyric acid from TCI

5' -nucleotidase was purchased from Shanghai Lanyuan bioengineering Co., Ltd

alpha-L-fucosidase available from Abies Biotech Ltd, Beijing

Adenosine deaminase was purchased from Abies Biotech Ltd, Beijing

Calf serum from Hangzhou Biotechnology GmbH, Zhejiang

Human serum purchased from Shanghai Xinfan Biotech Ltd

Example 1

Step (1): firstly preparing 1000ml of matrix of the composite quality control product: 50mM PB (0.7800g of NaH) was accurately weighed first₂PO₄-2H₂O and 7.1628g of Na₂HPO₄-12H₂O, the sum of the two substance amounts is 25mmol) is dissolved in 400ml of purified water, 3% casein is accurately weighed and slowly added into the solution after complete dissolution, meanwhile, a magnetic stirrer is used for stirring, 5mol/L sodium hydroxide is used for adjusting the pH of the solution to 12.0, 8% trehalose, 5% mannitol, 0.05% TW-20, 0.5% sodium chloride, 0.3% EDTA and 0.1% P300 are added after the solution is clear and transparent and has no undissolved impurities, 1mol/L hydrochloric acid is used for adjusting the pH to 7.4 after uniform mixing is carried out and has no impurities, finally, the volume is fixed to 500ml for standby, the solution prepared by 50% volume and 50% volume of calf serum are uniformly mixed before preparing special biochemical composite quality control and used as the matrix of the quality control product, namely, 500ml of the component A and 500ml of calf serum are mixed; the percentages in the preceding steps are mass to volume ratios, i.e. the ratio obtained is g/ml, e.g. 500ml for component A, 8% seaThe mass of the trehalose is 40g, and the rest can be analogized.

Step (2): preparing liquid intermediate products (L1-L3) of 3 special biochemical quality control products with concentration, and adding the raw materials of the 4 special biochemical items into a substrate to ensure that the four items of concentration respectively reach the following concentrations:

item	LI	L2	L3
				5’-NT(U/L)	11.47	23.86	39.26
ADA(U/L)	16.01	50.12	86.39
				D3-H(mmol/L)	0.47	0.92	1.59
AFU(U/L)	31.82	53.11	86.45

And (3): and (3) subpackaging the prepared liquid intermediate product into penicillin bottles (1 ml/bottle) and freeze-drying to obtain freeze-dried powder.

The obtained lyophilized powder was subjected to the following experiment:

table 1. a: decap (reconstitution) stability: standing at 2-8 deg.C after redissolution

Table 1. b: freezing and storing stability: after redissolving, the mixture is frozen at-20 DEG C

Table 1. c: consistency of measured values in different systems (data below are all obtained from testing newly reconstituted quality control articles)

The above examples show that the special biochemical composite quality control product of the present invention has good stability of bottle opening (redissolution) and freezing storage, and has good consistency (i.e. interchangeability) of measured values in different systems.

Example 2

The casein added in the example 1 is changed into bovine serum albumin, the dosage is unchanged, the content of the added trehalose is changed into 4%, and other materials, dosage and operation are unchanged, so that the freeze-dried powder is finally obtained. The freeze-dried powder obtained in the embodiment is detected, and the detection result is as follows:

table 2. a: decap (reconstitution) stability: standing at 2-8 deg.C after redissolution

Table 2. b: freezing and storing stability: after redissolving, the mixture is frozen at-20 DEG C

From the above-mentioned test results, it can be seen that the replacement of casein and the low sugar content greatly reduce the bottle opening (reconstitution) and freezing stability of the product.

Example 3

50% of calf serum in example 1 is replaced by 50% of purified water, and other materials, the using amount and the operation are unchanged, so that the freeze-dried powder is finally obtained. The obtained freeze-dried powder of the embodiment is detected, and the consistency detection results of the measured values in different systems are as follows:

table 3.c consistency of measured values in different systems

As can be seen from the above table, the serum in the system of the present invention is indispensable, and once the serum is not contained, the consistency of the measured value of the product in different systems is greatly reduced.

Example 4

Step (1): firstly preparing 1000ml of matrix of the composite quality control product: accurately weighing 10mM Tris-HCl (namely 8mmol Tris-HCl) in 700ml purified water, accurately weighing 2% casein after completely dissolving, slowly adding the 2% casein into the solution, stirring by using a magnetic stirrer, adjusting the pH of the solution to 11.0 by using 5mol/L sodium hydroxide, adding 15% sucrose, 10% mannitol, 0.1% Triton X-100, 0.1% potassium chloride, 0.1% EDTA and 0.5% sodium azide after the solution is clear and transparent and has no undissolved impurities, uniformly mixing the solution without impurities, adjusting the pH to 7.0 by using 1mol/L hydrochloric acid, and finally fixing the volume to 800ml for later use, wherein the 80% prepared solution and 20% human serum are uniformly mixed to be used as a matrix of a quality control product before preparing a special biochemical composite quality control product;

step (2): preparing liquid intermediate products (L1-L3) of 3 special biochemical quality control products with concentration, and adding the raw materials of the 4 special biochemical items into the matrix to ensure that the concentrations of the 4 special biochemical items respectively reach the following concentrations:

and (3): and (3) subpackaging the prepared liquid intermediate product into penicillin bottles (5 ml/bottle) and freeze-drying to obtain freeze-dried powder.

The quality control product obtained in the above way has good bottle opening (redissolution) and freezing stability, and the consistency (namely interchangeability) of measured values in different systems is good.

Table 4. a: decap (reconstitution) stability: standing at 2-8 deg.C after redissolution

Table 4. b: freezing and storing stability: after redissolving, the mixture is frozen at-20 DEG C

Table 4. c: consistency of measured values in different systems (data below are all obtained from testing newly reconstituted quality control articles)

Example 5

Step (1): firstly preparing 1000ml of matrix of the composite quality control product: accurately weighing 100mM glycine (namely 40mmol glycine) in300 ml purified water, accurately weighing 5% casein after completely dissolving, slowly adding the casein into the solution, stirring by using a magnetic stirrer, adjusting the pH of the solution to 13.0 by using 5mol/L sodium hydroxide, adding 5% glucose, 1% mannitol, 0.01% NP40, 0.9% sodium chloride, 0.5% EDTA and 0.1% K500 after the solution is clear and transparent and has no undissolved impurities, adjusting the pH to 7.0 by using 1mol/L hydrochloric acid after uniformly mixing the solution without impurities, and finally fixing the volume to 400ml for later use, wherein the 40% prepared solution and 60% human serum are uniformly mixed to be used as a matrix of a quality control product before preparing a special biochemical composite quality control;

step (2): preparing liquid intermediate products (L1-L3) of 3 special biochemical quality control products with concentration, and adding the raw materials of the 4 special biochemical items into a substrate to ensure that the four items of concentration respectively reach the following concentrations:

and (3): and (3) subpackaging the prepared liquid intermediate product into penicillin bottles (0.5 ml/bottle) and freeze-drying to obtain freeze-dried powder.

The quality control product obtained in the above way has good bottle opening (redissolution) and freezing stability, and the consistency (namely interchangeability) of measured values in different systems is good.

Table 5. a: decap (reconstitution) stability: standing at 2-8 deg.C after redissolution

Table 5. b: freezing and storing stability: after redissolving, the mixture is frozen at-20 DEG C

Table 5. c: consistency of measured values in different systems (data below are all obtained from testing newly reconstituted quality control articles)

As can be seen from the above examples, the special biochemical composite quality control product of the invention is a stable combined whole, and the components supplement each other, so that the special biochemical composite quality control product of the invention has excellent bottle opening stability and freezing stability, and has better consistency.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.