A tumor marker multiple quality control product

Abstract

The present invention provides a tumor marker multiple quality control product, which belongs to the intersection of synthetic biology and in vitro diagnostic reagents. The tumor marker multiple quality control product provided by the present invention can include up to 25 tumor markers including recombinant gastrin releasing peptide precursor and recombinant gastrin 17. It has the advantages of covering a wide range of projects, stable performance, stable and simple preparation process, small difference between bottles, and low production cost. It is particularly suitable for daily quality control monitoring by testing institutions.

Description

Technical Field

[0001] The present invention relates to the intersection of synthetic biology and in vitro diagnostic reagents, and in particular to a tumor marker multiple quality control product. Background Art

[0002] Tumor marker detection is an important cancer detection method, which is of great value in early diagnosis, differential diagnosis, and efficacy monitoring of cancer. By detecting specific tumor markers, cancer can be detected when there are no symptoms or mild symptoms, thereby improving the success rate of treatment and reducing the mortality rate.

[0003] In the detection of tumor markers, most tumor markers lack international standards due to their complex structures, so the test results obtained by different detection methods or test products from different manufacturers may be inconsistent. In the quality management system of tumor marker detection, the use of quality control products for internal quality control and participation in inter-laboratory quality evaluation is an important measure to ensure the quality of laboratory test results, reduce errors, and make the results between different laboratories more comparable. At present, most quality control products for tumor marker detection are single quality control products with test kits, which are time-consuming and labor-intensive in clinical practice, inefficient, and the superimposed cost of single matching quality control products is relatively high. The Chinese invention patent with publication number CN101105495A discloses a method for preparing tumor marker material control products, but there are only 15 tumor markers involved, and the technology does not include the most important and main marker of small cell lung cancer (SCLC), gastrin-releasing peptide precursor, and the three most important markers for evaluating gastric function, pepsinogen I, pepsinogen II, and gastrin 17, which cannot meet clinical needs well.

[0004] At present, most of the composite quality control products on the market lack gastrin releasing peptide precursor and gastrin 17. The reason is that gastrin releasing peptide precursor is not as stable in serum as plasma. The activity of gastrin releasing peptide precursor decreases by 20% when stored at 2~8℃ in serum for 24 hours, while the activity remains almost unchanged when stored at 2~8℃ in plasma for 24 hours. It can be stable for at least 1 month at -20℃. The instability of gastrin releasing peptide precursor in serum greatly limits its application in quality control products with serum as the matrix. The high stability of gastrin 17 in serum or plasma has always been a major problem in the industry. Studies have shown that the activity of gastrin 17 decreases significantly within 2~8 hours in serum, whether at room temperature or 2~8℃. This also leads to the inability of gastrin 17 antigen to be stably used in quality control with serum or plasma as the matrix. A Chinese invention patent with publication number CN114609396A discloses a method for improving the stability of gastrin 17: inactivate the mixed serum at 56°C, add 1-5% blood coagulant, and filter. However, the operation process is relatively complicated, the cost of mixed serum is relatively high, and the technology involves a single project, which is difficult to promote on a large scale in clinical practice. Summary of the invention

[0005] Based on this, the technical problem to be solved by the present invention is how to improve the stability of gastrin-releasing peptide precursor and / or gastrin 17 in quality control products based on human serum.

[0006] In order to solve the above technical problems, the present invention provides a tumor marker multiple quality control product, wherein the tumor marker multiple quality control product comprises a tumor marker, wherein the tumor marker is a recombinant antigen gastrin releasing peptide precursor and / or a recombinant antigen gastrin 17, wherein:

[0007] The amino acid sequence of the recombinant antigen gastrin releasing peptide precursor is TagA-(STGESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKD)n, wherein TagA is a Sumo or Flag tag, n is a repeating fragment, 5≤n≤15;

[0008] The amino acid sequence of the recombinant antigen gastrin 17 is TagB-(QGPWLEEEEEAYGWMDF)m, wherein TagB is a Trx or MBP tag, and m is a repeating fragment, 10≤m≤30.

[0009] The gastrin releasing peptide precursor and gastrin 17 are inherently unstable. Based on the gastrin releasing peptide precursor of the amino acid sequence shown in SEQ ID No.1, the present invention adds a Sumo tag or a Flag tag at the 5' end, and repeats the fragment multiple times. Based on the gastrin 17 of the amino acid sequence shown in SEQ ID No.2, a Trx tag or an MBP tag is added at the 5' end, and the fragment is repeated multiple times. The stability of the recombinant antigen gastrin releasing peptide precursor and the recombinant antigen gastrin 17 obtained by recombinant expression in serum is greatly improved. Adding expression tags and repeated fragments can protect gastrin releasing peptide precursor and gastrin 17 from the influence of substances such as proteases and metal ions in serum to a certain extent, thereby improving the stability of gastrin releasing peptide precursor and gastrin 17 in the serum matrix.

[0010] In the present invention, SEQ ID No. 1: STGESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKD.

[0011] In the present invention, SEQ ID No. 2: QGPWLEEEEEAYGWMDF.

[0012] In some preferred embodiments, m is preferably 5 or 10 or 15, and most preferably 10.

[0013] In some preferred embodiments, n is preferably 10 or 20 or 30, most preferably 20.

[0014] Preferably, the concentration of the recombinant antigen gastrin-releasing peptide precursor is 3-2000 pg / mL, and the concentration of the recombinant antigen gastrin 17 is 0.2-300 pmol / mL.

[0015] Preferably, the tumor markers also include any one or more of alpha-fetoprotein, carcinoembryonic antigen, human chorionic gonadotropin, β-human chorionic gonadotropin, prostate-specific antigen, free prostate-specific antigen, carbohydrate antigen 125, carbohydrate antigen 153, carbohydrate antigen 19-9, carbohydrate antigen 72-4, neuron-specific enolase, cytokeratin 19 fragment, ferritin, carbohydrate antigen 50, carbohydrate antigen 242, squamous cell carcinoma antigen, human epididymis protein 4, β2 microglobulin, thyroglobulin, Sangtec-100 protein, calcitonin, pepsinogen I, and pepsinogen II.

[0016] The tumor marker multiple quality control products provided by the present invention can support the quality control detection of 25 common clinical and special tumor marker items, which can greatly improve the efficiency of laboratory testing.

[0017] Preferably, the concentration of alpha-fetoprotein is 1.5~800IU / mL, the concentration of carcinoembryonic antigen is 1~900ng / mL, the concentration of human chorionic gonadotropin is 0.2~1000mIU / mL, the concentration of β-human chorionic gonadotropin is 0.1~100mIU / mL, the concentration of prostate-specific antigen is 0.01~100ng / mL, the concentration of free prostate-specific antigen is 0.01~50ng / mL, the concentration of carbohydrate antigen 125 is 2~1000U / mL, the concentration of carbohydrate antigen 153 is 3~300U / mL, the concentration of carbohydrate antigen 19-9 is 2~800U / mL, the concentration of carbohydrate antigen 72-4 is 0.5~300U / mL, the concentration of neuron-specific enolase is 0.1~300ng / mL, and the concentration of cell culture medium is 0. The concentration of cytokeratin 19 fragment is 0.5~500 ng / mL, the concentration of ferritin is 0.5~2000 ng / mL, the concentration of carbohydrate antigen 50 is 1~180 U / mL, the concentration of carbohydrate antigen 242 is 1~150 U / mL, the concentration of squamous cell carcinoma antigen is 0.1~100 ng / mL, the concentration of human epididymis protein 4 is 15~1000 pmol / L, the concentration of β2 microglobulin is 0.1~8 mg / L, the concentration of thyroglobulin is 0.05~500 ng / mL, the concentration of Sangtec-100 protein is 0.005~35 ng / mL, the concentration of calcitonin is 1.0~300 pg / mL, the concentration of pepsinogen I is 3~160 ng / mL, and the concentration of pepsinogen II is 2~70 ng / mL.

[0018] Preferably, the tumor marker multiple quality control product further comprises a human serum matrix, a buffer, a stabilizing substance and a preservative, and the pH of the tumor marker multiple quality control product is 6.8-8.0.

[0019] In some preferred embodiments, the human serum matrix content is ≥ 95%.

[0020] In some preferred embodiments, the content of the buffer is 1% to 5%.

[0021] In some preferred embodiments, the content of the stabilizing substance is 15-50 g / L.

[0022] In some preferred embodiments, the content of the preservative is 0.1-1% by mass.

[0023] Preferably, the buffer is selected from any one or more of MOPS buffer, HEPES buffer, PIPES buffer, Tris-HCl buffer, and phosphate buffer, and the preservative is selected from any one or more of PC150, PC300, PC950, MIT, and BND-10.

[0024] Preferably, the stabilizing substance comprises marine gelatin, and the marine gelatin is selected from any one or more of cod bone gelatin, squid skin gelatin, and mackerel gelatin.

[0025] Gelatin is a water-soluble, high-molecular-weight polypeptide derived from partial hydrolysis of collagen. In the multiple quality control products of tumor markers provided by the present invention, marine biological gelatin plays an important role in the composite quality control stability of the quality control products and can improve the stability of the quality control products.

[0026] Preferably, the stabilizing substance further comprises any one or more of aprotinin, pepstatin, trypsin inhibitor, thiol protease inhibitor, leupeptin, bovine serum albumin, sucrose and trehalose.

[0027] Preferably, the tumor marker multi-quality control product is in the form of a liquid or a lyophilized powder.

[0028] Preferably, the dosage form of the tumor marker multiple quality control product is a lyophilized powder, and the tumor marker multiple quality control product also includes mannitol.

[0029] The present invention has the following beneficial effects:

[0030] 1. The tumor marker multiple quality control products provided by the present invention can be used for the composite quality control of up to 25 common or special tumor marker items, which can meet the needs of clinical testing, provide great convenience for clinical testing, and greatly improve the detection efficiency. The tumor marker multiple quality control products provided by the present invention have extremely high stability and can ensure the quality of laboratory test results;

[0031] 2. The tumor marker multi-quality control products provided by the present invention improve the stability of the quality control products in multiple aspects, including: using human serum matrix and adding buffer to effectively improve the freeze-drying yield and stability of the quality control products, adding stabilizing substances such as marine gelatin to further improve the stability of the quality control products, when the dosage form of the quality control products is a freeze-dried powder, additionally adding mannitol as a freeze-drying excipient to give the freeze-dried products a stable form and improve the stability of the freeze-dried products, and at the same time, the addition of preservatives improves the antibacterial ability of the quality control products, and is convenient for daily use and storage of the quality control products;

[0032] 3. The tumor marker multiple quality control products provided by the present invention are constructed by molecular biological means to express long-fragmented gastrin-releasing peptide precursor and gastrin 17 antigen with labels. After freeze-drying and re-dissolving in serum matrix, they can be stable for 14 days at 2-8°C and for 1 month at -20°C, solving the industry difficulty of gastrin-releasing peptide precursor and gastrin 17 being unstable in serum;

[0033] 4. The dosage form of the tumor marker multiple quality control product provided by the present invention can be a liquid dosage form or a lyophilized powder. Even if it is a liquid dosage form, it can still be stored for 2 years at -20°C and has excellent stability;

[0034] 5. The tumor marker multiple quality control products provided by the present invention can cover the quality control detection of up to 25 common or special tumor markers, covering a wide range of projects, stable performance, stable and simple preparation process, small differences between bottles, low production cost, and convenient for daily use and quality management of testing institutions. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0036] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0037] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0038] Terminology: In this article, unless otherwise specified, progastrin-releasing peptide and PRO-GRP represent the same substance, gastrin 17 and G17 represent the same substance, alpha-fetoprotein and AFP represent the same substance, carcinoembryonic antigen and CEA represent the same substance, human chorionic gonadotropin and HCG represent the same substance, β-human chorionic gonadotropin and β-HCG represent the same substance, prostate-specific antigen and PSA represent the same substance, free prostate-specific antigen and F-PSA represent the same substance, carbohydrate antigen 125 and CA125 represent the same substance, carbohydrate antigen 153 and CA153 represent the same substance, carbohydrate antigen 19-9 and CA19-9 represent the same substance, carbohydrate antigen 72-4 and CA72-4 represent the same substance. The same substance is represented by the table, neuron-specific enolase and NSE represent the same substance, cytokeratin 19 fragment and CYFRA21-1 represent the same substance, ferritin and Fer represent the same substance, carbohydrate antigen 50 and CA50 represent the same substance, carbohydrate antigen 242 and CA242 represent the same substance, squamous cell carcinoma antigen and SCCA represent the same substance, human epididymis protein 4 and HE-4 represent the same substance, β2-microglobulin and β2-MG represent the same substance, thyroglobulin and TG represent the same substance, Sangtec-100 protein and S100 represent the same substance, calcitonin and CT represent the same substance, pepsinogen I and PGⅠ represent the same substance, pepsinogen II and PGⅡ represent the same substance.

[0039] As described in the background technology, in the prior art, PRO-GRP and G17 have poor stability in quality control products based on human serum, which also leads to the fact that PRO-GRP and G17 are almost not included in the current tumor marker multi-quality control products. Based on this, a specific embodiment of the present invention provides a tumor marker multi-quality control product including tumor markers, and the tumor marker multi-quality control product includes recombinant antigen gastrin releasing peptide precursor and / or recombinant antigen gastrin 17.

[0040] In a specific embodiment of the present invention, the amino acid sequence of the recombinant antigen gastrin releasing peptide precursor is TagA-(STGESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKD)n, wherein TagA is a Sumo or Flag tag, n is a repeating fragment, 5≤n≤15; the amino acid sequence of the recombinant antigen gastrin 17 is TagB-(QGPWLEEEEEAYGWMDF)m, wherein TagB is a Trx or MBP tag, m is a repeating fragment, 10≤m≤30.

[0041] In the above embodiment, the recombinant antigen gastrin releasing peptide precursor and the recombinant antigen gastrin 17 are long fragments with labels, and the Sumo or Flag label and the repeating fragment can greatly increase the stability of the gastrin releasing peptide precursor in the serum matrix, and the Trx or MBP label and the repeating fragment can greatly increase the stability of gastrin 17 in the serum matrix.

[0042] In the above embodiment, the concentration of the recombinant antigen gastrin releasing peptide precursor is 3-2000 pg / mL, and the concentration of the recombinant antigen gastrin 17 is 0.2-300 pmol / mL.

[0043] In some embodiments, the tumor marker multiple quality control products also include 23 other tumor markers, specifically including 13 common clinical tumor markers including alpha-fetoprotein, carcinoembryonic antigen, human chorionic gonadotropin, β-human chorionic gonadotropin, prostate-specific antigen, free prostate-specific antigen, carbohydrate antigen 125, carbohydrate antigen 153, carbohydrate antigen 19-9, carbohydrate antigen 72-4, neuron-specific enolase, cytokeratin 19 fragment, ferritin, and 10 clinical special tumor markers including carbohydrate antigen 50, carbohydrate antigen 242, squamous cell carcinoma antigen, human epididymis protein 4, β2-microglobulin, thyroglobulin, Sangtec-100 protein, calcitonin, pepsinogen I, and pepsinogen II.

[0044] In the above embodiment, the concentration of alpha-fetoprotein is 1.5~800IU / mL, the concentration of carcinoembryonic antigen is 1~900ng / mL, the concentration of human chorionic gonadotropin is 0.2~1000mIU / mL, the concentration of β-human chorionic gonadotropin is 0.1~100mIU / mL, the concentration of prostate-specific antigen is 0.01~100ng / mL, the concentration of free prostate-specific antigen is 0.01~50ng / mL, the concentration of carbohydrate antigen 125 is 2~1000U / mL, the concentration of carbohydrate antigen 153 is 3~300U / mL, the concentration of carbohydrate antigen 19-9 is 2~800U / mL, the concentration of carbohydrate antigen 72-4 is 0.5~300U / mL, and the concentration of neuron-specific enolase is 0.1~300ng / mL. , the concentration of cytokeratin 19 fragment is 0.5~500ng / mL, the concentration of ferritin is 0.5~2000ng / mL, the concentration of carbohydrate antigen 50 is 1~180U / mL, the concentration of carbohydrate antigen 242 is 1~150U / mL, the concentration of squamous cell carcinoma antigen is 0.1~100ng / mL, the concentration of human epididymis protein 4 is 15~1000pmol / L, the concentration of β2 microglobulin is 0.1~8mg / L, the concentration of thyroglobulin is 0.05~500ng / mL, the concentration of Sangtec-100 protein is 0.005~35ng / mL, the concentration of calcitonin is 1.0~300pg / mL, the concentration of pepsinogen I is 3~160ng / mL, and the concentration of pepsinogen II is 2~70ng / mL.

[0045] The multiple quality control products for tumor markers provided in the above-mentioned embodiment include gastrin-releasing peptide precursor and gastrin, and may additionally include 23 common or special clinical tumor markers, and can simultaneously realize quality control detection of up to 25 tumor markers, covering a wide range of complete items, with stable performance, stable and simple preparation process, small difference between bottles, low production cost, and convenient for daily use and quality management of testing institutions.

[0046] The above-mentioned tumor marker multiple quality control products also include human serum matrix, buffer, stabilizer and preservative in terms of ingredients, and the pH of the tumor marker multiple quality control products is 6.8~8.0.

[0047] In the above-mentioned embodiment, the human serum matrix can be made from finished human plasma which is readily available from clinical sources and has sufficient clinical sources. After simple process treatment, the organic components of human plasma are basically consistent with those of serum, which not only ensures the production of multiple quality control products of tumor markers, but also effectively avoids the occurrence of matrix effect problems.

[0048] The buffer is selected from any one or more of MOPS buffer, HEPES buffer, PIPES buffer, Tris-HCl buffer and phosphate buffer.

[0049] In the multiple quality control products of tumor markers provided in the above embodiment, the present invention uses the "buffer + human serum matrix" system as the matrix of the multiple quality control products of tumor markers, and the system can effectively improve the freeze-drying yield and stability of the multiple quality control products of tumor markers. In terms of volume, the proportion of human serum matrix is ​​≥95%, and the proportion of buffer is 1~5%. In the above embodiment, if the buffer is a MOPS buffer, the final concentration of the MOPS buffer is 10~50mM, if the buffer is a HEPES buffer, the final concentration of the HEPES buffer is 10~50mM, if the buffer is a PIPES buffer, the final concentration of the PIPES buffer is 5~25mM, and if the buffer is a phosphate buffer, the phosphate buffer is preferably Dulbecco's phosphate buffer.

[0050] The preservative is selected from any one or more of PC150, PC300, PC950, MIT, and BND-10.

[0051] The stabilizing substance comprises marine biological gelatin, and the marine biological gelatin is selected from any one or more of cod bone gelatin, squid skin gelatin and mackerel gelatin.

[0052] Marine biological gelatin plays an important role in the composite quality control stability of quality control products and can improve the stability of most tumor markers in the above-mentioned embodiments.

[0053] The stabilizing substance also includes any one or more of aprotinin, pepstatin, trypsin inhibitor, thiol protease inhibitor, leupeptin, bovine serum albumin, sucrose, and trehalose.

[0054] In some specific embodiments, the dosage form of the tumor marker multiple quality control product is a liquid or a lyophilized powder. If the dosage form of the tumor marker multiple quality control product is a lyophilized powder, the tumor marker multiple quality control product also includes a lyophilized excipient mannitol, and the added amount of mannitol is 10~50g / L.

[0055] The technical solution of the present invention is further described below through specific embodiments.

[0056] Example 1

[0057] Validation of PRO-GRP and G17 stability improvements.

[0058] This embodiment provides a tumor marker multiple quality control product, the pH of the tumor marker multiple quality control product liquid is 7.4, and includes the following components:

[0059] 99% human serum by volume;

[0060] 1% volume HEPES buffer, final HEPES concentration 20 mM;

[0061] 10 g / L mannitol;

[0062] 0.3% by mass of PC300;

[0063] The stabilizing substances include: 10 g / L sucrose, 5 g / L cod bone gelatin, and 2 g / L trypsin inhibitor;

[0064] Tumor markers included: 80 IU / mL alpha-fetoprotein, 50 ng / mL carcinoembryonic antigen, 60 mIU / mL human chorionic gonadotropin, 10 mIU / mL beta human chorionic gonadotropin, 10 ng / mL prostate-specific antigen, 5 ng / mL free prostate-specific antigen, 100 U / mL carbohydrate antigen 125, 120 U / mL carbohydrate antigen 153, 80 U / mL carbohydrate antigen 50, 100 U / mL carbohydrate antigen 19-9, 110 U / mL carbohydrate antigen 72-4, 120 U / mL carbohydrate antigen 242, and 1 5ng / mL squamous cell carcinoma antigen, 20ng / mL neuron-specific enolase, 50ng / mL cytokeratin 19 fragment, 60pmol / L human epididymal protein 4, 700ng / mL ferritin, 8mg / L β2 microglobulin, 40ng / mL thyroglobulin, 15ng / mL Sangtec-100 protein, 100pg / mL calcitonin, 1000pg / mL gastrin-releasing peptide precursor, 100ng / mL pepsinogen I, 50ng / mL pepsinogen II, 250pmol / L gastrin 17.

[0065] Among them, the amino acid sequence of gastrin releasing peptide precursor is TagA-(STGESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKD)n, TagA is a Sumo or Flag tag, n is a repeating fragment, 5≤n≤15; the amino acid sequence of gastrin 17 is TagB-(QGPWLEEEEEAYGWMDF)m, TagB is a Trx or MBP tag, m is a repeating fragment, 10≤m≤30.

[0066] PRO-GRP and G17 were treated as variables according to the formulations shown in Table 1.

[0067] Table 1

[0068]

[0069] The above-mentioned tumor marker multiple quality control liquid was divided into freezing bottles, and freeze-dried using a freeze dryer to obtain tumor marker multiple quality control freeze-dried powder.

[0070] The stability of PRO-GRP and G17, the quality control products of the above tumor markers, was verified, and all the detection reagents used were from Mekang Biotechnology Co., Ltd.

[0071] 2~8℃ reconstitution stability verification test: The lyophilized powder of multiple quality control products of tumor markers prepared in this example was taken, and placed at 2~8℃ after reconstitution. The relative deviation of stability was detected on the 0th day and the 14th day. The results are shown in Table 2.

[0072] Table 2

[0073]

[0074] -20°C reconstitution stability test: The lyophilized powder of multiple quality control products of tumor markers prepared in this example was taken, reconstituted and placed at -20°C, and the relative deviation of stability was tested on the 0th day and the 30th day. The results are shown in Table 3.

[0075] Table 3

[0076]

[0077] It can be seen from Tables 2 and 3 that after the multiple quality control products of tumor markers prepared in this embodiment are reconstituted, the pro-GRP and G17 in Formula 1 and Formula 5 are stored at 2-8°C for 14 days, and the deviation compared with the day 0 is more than 60%, which is a significant decrease, while for Formulas 2 to 4, the deviation is within 30%. After reconstitution and storage at -20°C, pro-GRP and G17 in Formula 1 and Formula 5 have a significant downward trend within 30 days, while Formulas 2 to 4 are relatively stable. The above data show that pro-GRP and G17 in Formula 3 have the best stability. It can be seen that the composite quality control products of tumor markers in serum matrix provided in this embodiment have good stability after reconstitution and can better meet clinical testing needs.

[0078] Example 2

[0079] Stability verification of multiple quality control products for tumor markers in different systems.

[0080] The matrix used in the tumor marker multi-quality control liquid provided in this example is 97% by volume of human serum matrix and 3% by volume of buffer;

[0081] The stabilizers were all a combination of cod bone gelatin + 1g / L leupeptin + 30g / L sucrose with a final concentration of 10g / L;

[0082] Tumor markers include: alpha-fetoprotein 10 IU / mL, carcinoembryonic antigen 5 ng / mL, human chorionic gonadotropin 10 mIU / mL, beta human chorionic gonadotropin 2.5 mIU / mL, prostate-specific antigen 4.5 ng / mL, free prostate-specific antigen 1.5 ng / mL, carbohydrate antigen 125 20 U / mL, carbohydrate antigen 153 35 U / mL, carbohydrate antigen 50 15 U / mL, carbohydrate antigen 19-9 25 U / mL, carbohydrate antigen 72-4 10 U / mL, carbohydrate antigen 242 12 U / mL, squamous cell carcinoma antigen 3 ng / mL, neuron-specific enolase 6 ng / mL, cytokeratin 19 fragment 8 ng / mL, human epididymis protein 4 15 pmol / L, iron Protein 70ng / mL, β2 microglobulin 2mg / L, thyroglobulin 15ng / mL, Sangtec-100 protein 0.08ng / mL, calcitonin 5pg / mL, gastrin releasing peptide precursor 60pg / mL, pepsinogen I 3ng / mL, pepsinogen II 3ng / mL, gastrin 17 30pmol / L, the amino acid sequence of PRO-GRP is: Flag-(STGESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKD)10; the amino acid sequence of G17 is Trx-(QGPWLEEEEEAYGWMDF)20.

[0083] Buffer, pH, mannitol and preservatives were treated with variables according to the formula shown in Table 4.

[0084] Table 4

[0085]

[0086] The above-mentioned tumor marker multiple quality control liquid was divided into freezing bottles, and freeze-dried using a freeze dryer to obtain tumor marker multiple quality control freeze-dried powder.

[0087] The stability of multiple quality control products of the above-mentioned tumor markers was verified, and all the detection reagents used were from Mekang Biotechnology Co., Ltd.

[0088] 2~8℃ reconstitution stability verification test: The lyophilized powder of multiple quality control products of tumor markers prepared in this example was taken, and placed at 2~8℃ after reconstitution. The relative deviation of stability was detected on the 0th day and the 14th day. The results are shown in Table 5.

[0089] Table 5

[0090]

[0091] -20°C reconstitution stability test: The lyophilized powder of multiple quality control products of tumor markers prepared in this example was taken, reconstituted and placed at -20°C, and taken out on the 0th day and the 30th day to detect the relative deviation of stability. The results are shown in Table 6.

[0092] Table 6

[0093]

[0094] It can be seen from Tables 5 and 6 that the quality control products of the 7 formulations in this embodiment have a stability deviation of 25 items at 2-8°C on the 14th day and the 0th day within 8% after reconstitution; the stability deviation at -20°C for 30 days is within 7%. It can be seen that the tumor marker multiple quality control products provided in this embodiment have good stability after reconstitution and can meet the needs of clinical testing.

[0095] Example 3

[0096] Stability tests of multiple quality control products of tumor markers with different stabilizer formulations.

[0097] This embodiment provides a tumor marker multiple quality control product, the pH of the tumor marker multiple quality control product liquid is 7.4, and includes the following components:

[0098] 99% human serum by volume;

[0099] 1% volume HEPES buffer, final HEPES concentration 20 mM;

[0100] 10 g / L mannitol;

[0101] 0.3% by mass of PC300;

[0102] Tumor markers included: alpha-fetoprotein 40 IU / mL, carcinoembryonic antigen 25 ng / mL, human chorionic gonadotropin 30 mIU / mL, beta human chorionic gonadotropin 6 mIU / mL, prostate-specific antigen 5 ng / mL, free prostate-specific antigen 2.5 ng / mL, carbohydrate antigen 125 50 U / mL, carbohydrate antigen 153 70 U / mL, carbohydrate antigen 50 50 U / mL, carbohydrate antigen 19-9 60 U / mL, carbohydrate antigen 72-4 75 U / mL, carbohydrate antigen 242 60 U / mL, squamous cell carcinoma antigen 7.5 ng / mL, neuron-specific enolase 10 ng / mL, cytokeratin 19 fragment 30 ng / mL, human epididymal protein 4 30pmol / L, ferritin 350ng / mL, β2-microglobulin 4mg / L, thyroglobulin 20ng / mL, Sangtec-100 protein 8ng / mL, calcitonin 50pg / mL, gastrin release precursor peptide 400pg / mL, pepsinogen I 30ng / mL, pepsinogen II 25ng / mL, gastrin 17 125pmol / L, among which the amino acid sequence of PRO-GRP is: Flag-(STGESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKD)10; the amino acid sequence of G17 is Trx-(QGPWLEEEEEAYGWMDF)20.

[0103] The stable material system was treated with variables according to the formula shown in Table 7.

[0104] Table 7

[0105]

[0106] The above-mentioned tumor marker multiple quality control liquid was divided into freezing bottles, and freeze-dried using a freeze dryer to obtain tumor marker multiple quality control freeze-dried powder.

[0107] The stability of multiple quality control products of the above-mentioned tumor markers was verified, and all the detection reagents used were from Mekang Biotechnology Co., Ltd.

[0108] 2~8℃ reconstitution stability verification test: The lyophilized powder of multiple quality control products of tumor markers prepared in this example was taken, and placed at 2~8℃ after reconstitution. The relative deviation of stability was detected on the 0th day and the 14th day. The results are shown in Table 8.

[0109] Table 8

[0110]

[0111] -20°C reconstitution stability test: The lyophilized powder of multiple quality control products of tumor markers prepared in this example was taken, reconstituted and placed at -20°C, and the relative deviation of stability was tested on the 0th day and the 30th day. The results are shown in Table 9.

[0112] Table 9

[0113]

[0114] As can be seen from Tables 8 and 9, the quality control products of the six different formulations in this embodiment have a stability deviation of 25 items at 2-8°C on the 14th day and the 0th day after reconstitution within 7.5%; the stability deviation at -20°C for 30 days is within 7%. In general, the tumor marker composite quality control products of the above formulations have good stability after reconstitution and can meet the needs of clinical testing.

[0115] Example 4

[0116] Stability testing of multiple quality control products of tumor markers in different dosage forms.

[0117] This embodiment provides a tumor marker multiple quality control product, which is divided into a liquid group and a freeze-dried group. The pH of the tumor marker multiple quality control product is 7.4, and includes the following components:

[0118] 98% human serum by volume;

[0119] 2% volume HEPES buffer, final HEPES concentration 20 mM;

[0120] 0.3% by mass of PC300;

[0121] The lyophilized group received an additional 10 g / L of mannitol, while the liquid group did not receive mannitol;

[0122] Stabilizing substances include: 5 g / L squid skin gelatin, 1 g / L trypsin inhibitor and 20 g / L sucrose;

[0123] Tumor markers included: alpha-fetoprotein 40 IU / mL, carcinoembryonic antigen 25 ng / mL, human chorionic gonadotropin 30 mIU / mL, beta human chorionic gonadotropin 6 mIU / mL, prostate-specific antigen 5 ng / mL, free prostate-specific antigen 2.5 ng / mL, carbohydrate antigen 125 50 U / mL, carbohydrate antigen 153 70 U / mL, carbohydrate antigen 50 50 U / mL, carbohydrate antigen 19-9 60 U / mL, carbohydrate antigen 72-4 75 U / mL, carbohydrate antigen 242 60 U / mL, squamous cell carcinoma antigen 7.5 ng / mL, neuron-specific enolase 10 ng / mL, cytokeratin 19 fragment 30 ng / mL, human epididymal protein 4 30pmol / L, ferritin 350ng / mL, β2-microglobulin 4mg / L, thyroglobulin 20ng / mL, Sangtec-100 protein 8ng / mL, calcitonin 50pg / mL, gastrin release precursor peptide 400pg / mL, pepsinogen I 30ng / mL, pepsinogen II 25ng / mL, gastrin 17 125pmol / L, among which the amino acid sequence of PRO-GRP is: Flag-(STGESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKD)10; the amino acid sequence of G17 is Trx-(QGPWLEEEEEAYGWMDF)20.

[0124] The lyophilized components were packaged in cryovials and stored at 2-8°C after freeze drying, and the liquid components were packaged in cryovials and stored at -20°C.

[0125] Stability test: On day 0, 1 month, 3 months, 6 months, 12 months, 18 months and 24 months, one bottle of the lyophilized group was reconstituted for stability test; on day 0, 1 month, 3 months, 6 months, 12 months and 18 months, one bottle of the liquid group was reconstituted for testing. The test results were compared with those on day 0 to calculate the relative deviation and significance analysis. The stability test results of the lyophilized group are shown in Table 10, and the experimental results of the liquid group are shown in Table 11.

[0126] Table 10

[0127]

[0128] Table 11

[0129]

[0130] It can be seen from the data in Tables 10 and 11 that the multiple quality control products of tumor markers provided in this embodiment can be stable for 2 years in the freeze-dried powder state and can be stable for 18 months in the liquid frozen state, and can provide long-term stable quality control products for clinical use.

[0131] It can be seen from the experimental results of Examples 1 to 4 that the tumor marker multi-quality control products provided by the present invention have excellent stability and are particularly suitable for daily quality control monitoring by testing institutions.

[0132] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A tumor marker multiple quality control product, characterized in that: The tumor marker multiple quality control products include tumor markers, and the tumor markers are recombinant antigen gastrin releasing peptide precursor and / or recombinant antigen gastrin 17, wherein: The amino acid sequence of the recombinant antigen gastrin releasing peptide precursor is TagA-(STGESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKD)n, wherein TagA is a Sumo or Flag tag, n is a repeating fragment, 5≤n≤15; The amino acid sequence of the recombinant antigen gastrin 17 is TagB-(QGPWLEEEEEAYGWMDF)m, wherein TagB is a Trx or MBP tag, and m is a repeating fragment, 10≤m≤30.

2. The tumor marker multiple quality control product according to claim 1, characterized in that: The concentration of the recombinant antigen gastrin releasing peptide precursor is 3-2000 pg / mL, and the concentration of the recombinant antigen gastrin 17 is 0.2-300 pmol / mL.

3. The tumor marker multiple quality control product according to claim 1, characterized in that: The tumor markers also include any one or more of alpha-fetoprotein, carcinoembryonic antigen, human chorionic gonadotropin, β-human chorionic gonadotropin, prostate-specific antigen, free prostate-specific antigen, carbohydrate antigen 125, carbohydrate antigen 153, carbohydrate antigen 19-9, carbohydrate antigen 72-4, neuron-specific enolase, cytokeratin 19 fragment, ferritin, carbohydrate antigen 50, carbohydrate antigen 242, squamous cell carcinoma antigen, human epididymis protein 4, β2 microglobulin, thyroglobulin, Sangtec-100 protein, calcitonin, pepsinogen I, and pepsinogen II.

4. The tumor marker multiple quality control product according to claim 3, characterized in that: The concentration of alpha-fetoprotein is 1.5-800 IU / mL, the concentration of carcinoembryonic antigen is 1-900 ng / mL, the concentration of human chorionic gonadotropin is 0.2-1000 mIU / mL, the concentration of β-human chorionic gonadotropin is 0.1-100 mIU / mL, the concentration of prostate-specific antigen is 0.01-100 ng / mL, the concentration of free prostate-specific antigen is 0.01-50 ng / mL, the concentration of carbohydrate antigen 125 is 2-1000 U / mL, the concentration of carbohydrate antigen 153 is 3-300 U / mL, the concentration of carbohydrate antigen 19-9 is 2-800 U / mL, the concentration of carbohydrate antigen 72-4 is 0.5-300 U / mL, the concentration of neuron-specific enolase is 0.1-300 ng / mL, the concentration of cytokeratin is 0. The concentration of protein 19 fragment is 0.5~500ng / mL, the concentration of ferritin is 0.5~2000ng / mL, the concentration of carbohydrate antigen 50 is 1~180U / mL, the concentration of carbohydrate antigen 242 is 1~150U / mL, the concentration of squamous cell carcinoma antigen is 0.1~100ng / mL, the concentration of human epididymis protein 4 is 15~1000pmol / L, the concentration of β2 microglobulin is 0.1~8mg / L, the concentration of thyroglobulin is 0.05~500ng / mL, the concentration of Sangtec-100 protein is 0.005~35ng / mL, the concentration of calcitonin is 1.0~300pg / mL, the concentration of pepsinogen I is 3~160ng / mL, and the concentration of pepsinogen II is 2~70ng / mL.

5. The tumor marker multiple quality control product according to claim 1, characterized in that: The tumor marker multiple quality control products also include human serum matrix, buffer, stabilizing substance and preservative, and the pH of the tumor marker multiple quality control products is 6.8~8.

0.

6. The tumor marker multiple quality control product according to claim 5, characterized in that: The buffer is selected from any one or more of MOPS buffer, HEPES buffer, PIPES buffer, Tris-HCl buffer, and phosphate buffer, and the preservative is selected from any one or more of PC150, PC300, PC950, MIT, and BND-10.

7. The tumor marker multiple quality control product according to claim 5, characterized in that: The stabilizing substance comprises marine gelatin, and the marine gelatin is selected from any one or more of cod bone gelatin, squid skin gelatin, and mackerel gelatin.

8. The tumor marker multiple quality control product according to claim 7, characterized in that: The stabilizing substance further comprises any one or more of aprotinin, pepsin inhibitor, trypsin inhibitor, thiol protease inhibitor, leupeptin, bovine serum albumin, sucrose, and trehalose.

9. The tumor marker multiple quality control product according to claim 1, characterized in that: The dosage form of the tumor marker multiple quality control product is liquid or lyophilized powder.

10. The tumor marker multiple quality control product according to claim 9, characterized in that: The dosage form of the tumor marker multiple quality control product is a lyophilized powder, and the tumor marker multiple quality control product also includes mannitol.

Images