Calibration product or quality control product for exosome membrane marker detection reagent based on engineering bacteriophage

Abstract

The invention discloses a calibrator or a quality control product for an exosome membrane marker detection reagent based on an engineering bacteriophage, the engineering bacteriophage is a bacteriophage capable of simultaneously displaying more than two exosome membrane markers, and the bacteriophage comprises any one of M13, T4, T7 or lambda bacteriophage; the exosome membrane marker comprises at least two of an exosome transmembrane transporter, an exosome specific protein or a disease-related marker. The invention relates to a calibrator or a quality control product for an exosome membrane marker detection reagent based on engineering bacteriophage, which is a low-high-value calibrator or quality control product obtained by diluting the engineering bacteriophage by using a diluent in proportion, and solves the problems of high cell culture and extraction cost, large batch difference, low cost and the like when an exosome is biologically extracted as the calibrator or quality control product for the detection reagent. The problems of poor stability in the use process and the like are solved, and the problems of calibration of a traceable value source and quality control of an exosome membrane marker in-vitro detection kit are solved.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and specifically relates to a calibrator or quality control product for an exosome membrane marker detection reagent based on an engineered bacteriophage. Background Art

[0002] Exosomes are 30-150nm extracellular vesicles with a lipid bilayer structure released by cells. The exosome membrane surface contains lipids and a variety of proteins, including cytoskeletal proteins, transmembrane transport proteins (CD9, CD63, CD81, and CD82), and heat shock proteins involved in protein folding (HSP70 and HSP90). Proteins such as Alix, TSG101, and CD63 can be used as biomarkers for exosome identification. Exosomes can be detected in a variety of body fluids, including blood, saliva, amniotic fluid, urine, and tears. In recent years, they have gradually developed into biomarkers for liquid biopsies, assisting in the diagnosis of various diseases. Some researchers have found that the level of programmed death-1-ligand 1 (PD-L1) on the membrane of peripheral blood exosomes is more correlated with the progression of tumors and the prognosis of tumor immunotherapy. Detection of PD-L1 on exosomes can assist tumor immunotherapy. Detection of prostate-specific membrane antigen (PSMA) on the membrane of exosomes plays an important role in the early diagnosis of prostate cancer. Exosomes in the amniotic fluid of pregnant women also have important diagnostic value. Exosomes secreted by syncytiotrophoblast extracellular vesicles (STBEV) of the placenta, and markers on the membrane of STBEV such as placental alkaline phosphatase (Placental Alkaline Phosphatase) are also important for the diagnosis of prostate cancer. Phosphatase (PLAP) can be used to diagnose and monitor pathological conditions such as preeclampsia and gestational diabetes; the detection of related proteins of neuronal-derived exosomes in peripheral blood is of great significance for the early diagnosis of neurodegenerative diseases. For example, α-synuclein in neuronal-derived exosomes can be used as an auxiliary diagnosis for Parkinson's disease.

[0003] The design and development of detection kits for exosome membrane markers is of great clinical significance. Conventional double-antibody sandwich assays typically utilize two different antibodies that recognize different antigenic determinants of the same antigen. However, detection kits for exosome membrane markers require the use of two different antibodies—an exosome-specific antibody and an exosome membrane marker-specific antibody—to form a sandwich complex for detection. Calibrators and quality control materials are crucial for quantitative measurement and quality control in in vitro diagnostic clinical testing. However, the development of exosomes for direct use as calibrators and quality control materials for assays is challenging. Currently, large-scale exosome production typically utilizes engineered cell culture methods to obtain exosomes containing specific exosome membrane markers. Cell culture methods are not only expensive, but also subject to batch-to-batch variability due to differences in cell donors, tissue origin, and culture batches. Exosomes are typically stored at -80°C for long periods in the laboratory, making their transportation and storage as part of a test kit challenging.

[0004] The challenge of preparing low-cost, low-batch-to-batch variability, and stable exosome calibrators and quality controls is currently a major challenge. Phage display techniques (PDT) use modified bacteriophages as vectors to insert an exogenous gene fragment into the phage coat protein region. As the phages are passaged, the exogenous peptide or protein is expressed and displayed on the phage surface. Through affinity enrichment screening, phages displaying the specific peptide or protein are ultimately obtained. Common phage display systems include the single-stranded filamentous phage (M13) display system, the T4 phage display system, the T7 phage display system, and the λ phage display system. Phage display technology offers advantages such as ease of operation, rapid production, and increased efficiency. Therefore, phage display technology can be used to insert two exosome membrane markers into the phage coat protein region. Phages expressing both exosome membrane markers can then be screened to mimic exosomes and serve as calibrators and quality controls for detection reagents. Summary of the Invention

[0005] Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides a calibrator or quality control product for an exosome membrane marker detection reagent based on an engineered bacteriophage, which solves the problems of high cell culture extraction cost, large batch-to-batch variability, and poor stability during use when biologically extracting exosomes as calibrators and quality control products for detection reagents. It also solves the difficulties of sourcing calibration traceability values ​​and quality control for in vitro detection kits for exosome membrane markers.

[0006] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] The present invention provides a method for preparing an engineered phage displaying two exosome membrane markers, comprising the following steps: inserting the two exosome membrane markers into the phage display region respectively, and screening, isolating and purifying the engineered phage that can simultaneously display the two exosome membrane markers.

[0008] The aforementioned phages include, but are not limited to, any one of M13, T4, T7 or λ phage.

[0009] The above-mentioned exosome membrane markers include, but are not limited to, exosome transmembrane transport proteins such as CD9, CD63, CD81, Alix, etc., exosome-specific proteins such as TSG101, etc., and also include, but are not limited to, disease-related markers, such as tumor-related disease markers such as PD-L1, PSMA, etc., neurodegenerative disease-related markers such as α-synuclein, Aβ protein, phosphorylated Tau protein, NfL, GFAP, etc., and eclampsia-related markers such as PLAP, etc.

[0010] The present invention provides a method for preparing liquid calibrators or quality control products for exosome membrane marker detection reagents using engineered bacteriophages, wherein the phages are diluted proportionally using a liquid storage buffer to obtain low and high value calibrators or quality control products, respectively.

[0011] The above-mentioned diluent includes a liquid storage buffer and a lyophilized product storage buffer, wherein the liquid storage buffer contains 0.5%-3% w / v BSA or its equivalent substitute, 0.5%-5% w / v sucrose, 0.5%-5% w / v sorbitol, 2.5%-10% v / v glycerol, 0.05-0.25% v / v Tween-20, 100-200mM basic buffer and 0.1% v / v Proclin300 preservative, with a pH of 5.0-8.0.

[0012] The above-mentioned basic buffer comprises one of 10-200 mM Tris, PBS, citric acid and MES buffer.

[0013] The present invention provides a method for preparing freeze-dried calibrators or quality control products for exosome membrane marker detection reagents using engineered bacteriophages, wherein the phages are diluted proportionally using a freeze-drying storage buffer to obtain low-value and high-value calibrators or quality control products, respectively, and then freeze-dried.

[0014] The lyophilization storage buffer contains 2%-8% w / v BSA or its equivalent substitute, 0.5%-5% w / v trehalose, 3%-10% w / v mannitol, 0.05-0.25% v / v Tween-20, 100-200 mM basic buffer, and 0.1% v / v Proclin 300 preservative is added, with a pH of 5.0-8.0.

[0015] The above-mentioned basic buffer comprises one of 10-200 mM Tris, PBS, citric acid and MES buffer.

[0016] The present invention also provides a freeze-drying method for the above-mentioned engineered phage freeze-dried calibrator or quality control product, wherein 0.3 mL to 2.0 mL of the phage solution diluted with the freeze-dried product diluent is dispensed into freeze-drying bottles and freeze-dried according to the following freeze-drying procedure:

[0017] Precool the freeze dryer to -50°C and keep the temperature constant for 120-240 minutes;

[0018] The temperature is raised in stages to -40°C, -30°C, -20°C, -10°C, 0°C, 10°C, and 25°C. The heating time for each stage is about 10 minutes. The temperature is kept constant for 60-480 minutes, and the vacuum degree is maintained below 60Pa.

[0019] Beneficial effects: The calibrator or quality control product for the exosome membrane marker detection reagent based on engineered bacteriophage provided by the present invention has the following advantages:

[0020] (1) The phage display technology was pioneered in the application of in vitro diagnostics. Two exosome membrane markers were displayed relatively independently and in large quantities on the surface of the engineered phage, ensuring the biological activity of the two exosome membrane markers. The equivalent simulated exosomes were used as calibrators or quality control products for exosome membrane marker detection reagents.

[0021] (2) The engineered phages constructed are diluted directly with liquid preservation solution or diluted with freeze-dried preservation solution and then freeze-dried to prepare calibration products or quality control products. Compared with exosomes extracted from cell culture, this method can not only significantly reduce the cost of raw materials and reduce batch differences, but also has good real-time, accelerated and transportation stability.

[0022] (3) The present invention provides a calibrator or quality control product for exosome membrane marker detection reagents based on engineered bacteriophages, which has the advantages of low cost, small batch-to-batch variability, and good stability. It solves the problems of difficult value transfer and quality control in exosome membrane marker detection kits. The technical solution of the present invention is highly universal and can be expanded to a variety of exosome markers and detection scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of exosome membrane markers.

[0024] Figure 2 Schematic diagram of the exosome PLAP-engineered phage structure.

[0025] Figure 3 This is the process flow chart and detection schematic diagram for PD-L1&CD63 engineered phage preparation.

[0026] Figure 4This is the process flow chart and detection schematic diagram for PLAP&CD81 engineered phage preparation. DETAILED DESCRIPTION

[0027] The present invention discloses a calibrator or quality control product for an exosome membrane marker detection reagent based on an engineered bacteriophage. The engineered bacteriophage is a bacteriophage that simultaneously displays two or more exosome membrane markers, the bacteriophage including any one of M13, T4, T7, or λ phage; the exosome membrane markers include at least two of an exosome transmembrane transporter, an exosome-specific protein, or a disease-related marker. A calibrator or quality control product for an exosome membrane marker detection reagent based on an engineered bacteriophage is prepared by proportionally diluting the engineered bacteriophage with a diluent to obtain a low-value or high-value calibrator or quality control product. This solves the problems of high cell culture extraction costs, large batch-to-batch variability, and poor stability during use when biologically extracting exosomes as calibrators or quality control products for detection reagents, and addresses the difficulties of sourcing calibration traceability values ​​and quality control for in vitro detection kits for exosome membrane markers.

[0028] In one aspect, the present invention provides an engineered phage, such as Figure 2 As shown, the engineered phage is a phage that simultaneously displays two or more exosome membrane markers, wherein,

[0029] The bacteriophage includes but is not limited to any one of M13, T4, T7 or λ phage;

[0030] The exosome membrane markers include at least two of exosome transmembrane transporters, exosome-specific proteins, or disease-related markers. Figure 1 shown.

[0031] Optionally, in some embodiments of the present invention, the exosomal transmembrane transporter includes at least one of CD9, CD63, CD81, and Alix.

[0032] Optionally, in some embodiments of the present invention, the exosome-specific protein includes TSG101.

[0033] Optionally, in some embodiments of the present invention, the disease-related markers include at least one of tumor-related disease markers such as PD-L1, PSMA, etc., neurodegenerative disease-related markers such as α-synuclein, Aβ protein, phosphorylated Tau protein, NfL, GFAP, etc., and pregnancy eclampsia-related markers such as PLAP.

[0034] Optionally, in some embodiments of the present invention, the engineered phage is a phage that simultaneously displays two exosomal membrane markers.

[0035] Optionally, in some embodiments of the present invention, the method includes: inserting two or more exosome membrane markers into the phage display region respectively, and screening, isolating and purifying engineered phages that simultaneously display the two or more exosome membrane markers.

[0036] In a second aspect, the present invention provides a calibrator or quality control for an exosome membrane marker detection reagent based on an engineered bacteriophage, wherein the engineered bacteriophage is diluted proportionally with a diluent to obtain a low-value or high-value calibrator or quality control, wherein the engineered bacteriophage is any of the engineered bacteriophages described above.

[0037] Optionally, in some embodiments of the present invention, the diluent comprises a liquid storage buffer, the liquid storage buffer containing 0.5%-3% w / v BSA or its equivalent substitute, 0.5%-5% w / v sucrose, 0.5%-5% w / v sorbitol, 2.5%-10% v / v glycerol, 0.05-0.25% v / v Tween-20, 100-200mM basic buffer and 0.1% v / v Proclin 300 preservative, pH 5.0-8.0, the basic buffer comprising any one of 10-200mM Tris, PBS, citric acid and MES buffer.

[0038] Optionally, in some embodiments of the present invention, after the engineered phage is diluted proportionally with a diluent to obtain a low-high value reagent, the reagent is lyophilized, and the diluent includes a lyophilized product storage buffer.

[0039] Optionally, in some embodiments of the present invention, the lyophilization storage buffer contains 2%-8% w / v BSA or its equivalent substitute, 0.5%-5% w / v trehalose, 3%-10% w / v mannitol, 0.05-0.25% v / v Tween-20, 100-200 mM basic buffer, and 0.1% v / v Proclin 300 preservative is added, with a pH of 5.0-8.0, and the basic buffer includes any one of 10-200 mM Tris, PBS, citric acid and MES buffer.

[0040] Optionally, in some embodiments of the present invention, the freeze-drying method includes dispensing 0.3 mL to 2.0 mL of the phage solution diluted with the freeze-drying product diluent into freeze-drying vials, and freeze-drying according to the following freeze-drying procedure:

[0041] Precool the freeze dryer to -50°C and keep the temperature constant for 120-240 minutes; and / or

[0042] The temperature is raised in stages to -40°C, -30°C, -20°C, -10°C, 0°C, 10°C, and 25°C. The heating time for each stage is about 10 minutes. The temperature is kept constant for 60-480 minutes, and the vacuum degree is maintained below 60Pa.

[0043] A third aspect of the present invention provides a kit comprising a calibrator or quality control product for an engineered phage-based exosome membrane marker detection reagent as described above.

[0044] A fourth aspect of the present invention provides a detection device comprising any of the above-described calibrators or quality control products for detecting exosome membrane markers based on engineered phages.

[0045] A fifth aspect of the present invention provides a detection method using any of the above-described calibrators or quality control products for the engineered phage-based exosome membrane marker detection reagent, or the above-described kit, or the above-described detection device.

[0046] Example

[0047] The present invention will be further described below in conjunction with the accompanying drawings and examples. The present invention can be better understood based on the following examples. However, it is readily understood by those skilled in the art that the specific material proportions, process conditions, and results described in the examples are merely illustrative of the present invention and should not, and will not, limit the present invention described in detail in the claims.

[0048] Control group 1: Calibrators and quality control products for extracting exosomes from cell lines for PD-L1 & CD63 detection reagents

[0049] (I) Extraction of exosome PD-L1 & CD63 from cell lines

[0050] In the control group, exosomes were extracted by cell culture. The specific method was as follows: human lung adenocarcinoma cell line H1975, which highly expresses PD-L1, was cultured at 37°C and 5% CO2. The cell culture supernatant was collected. The cell pellet was removed by centrifugation at 300g and 4°C for 10 minutes, and the supernatant was collected. The cell pellet was removed by centrifugation at 2000g and 4°C for 10 minutes, and the cell debris pellet was removed by centrifugation at 10,000g and 4°C for 30 minutes. The supernatant was collected and then ultracentrifuged at 120,000g and 4°C for 70 minutes. The supernatant was removed and the bottom pellet was resuspended in PBS to obtain exosomes from the control group.

[0051] (II) Preparation of calibrators and quality control materials for cell line exosome PD-L1 & CD63 detection reagents

[0052] The calibrators and quality controls in this control group were prepared using a freeze-dried storage solution containing 5.0% w / v BSA, 2.0% w / v trehalose, 5.0 w / v mannitol, 0.05% v / v Tween-20, 100 mM PBS buffer, and 0.1% v / v Proclin 300 preservative at a pH of 7.40. The engineered phage purified in step 1 was diluted to a concentration of (1.00 ± 0.20) AU / mL for quality control 1 and (20.00 ± 4.00) AU / mL for quality control 2, and the diluted phages were dispensed into freeze-dried bottles and freeze-dried according to the following freeze-drying procedure:

[0053] Open the freeze dryer, cool to -50°C, transfer the subpackaged quality control products into the freeze dryer, and keep the temperature constant for 180 minutes;

[0054] Heat to -40°C for 10 minutes and maintain constant temperature for 120 minutes; maintain vacuum below 10Pa;

[0055] Heat to -30°C for 10 minutes, maintain constant temperature for 120 minutes, and maintain vacuum below 10 Pa;

[0056] Heat to -20°C for 10 minutes, maintain constant temperature for 120 minutes, and maintain vacuum below 10 Pa;

[0057] Heat to -10°C for 10 minutes, maintain constant temperature for 120 minutes, and maintain vacuum below 10 Pa;

[0058] Heat to 0°C for 10 minutes, maintain constant temperature for 360 minutes, and maintain vacuum below 10Pa;

[0059] Heat to 10°C for 10 minutes, maintain constant temperature for 360 minutes, and maintain vacuum below 10 Pa;

[0060] The temperature was raised to 25°C for 10 minutes, and the temperature was kept constant for 360 minutes, while the vacuum degree was maintained below 10 Pa.

[0061] (III) Evaluation of exosome PD-L1 & CD63 in cell lines

[0062] 1. Potency and inter-batch variation of exosome PD-L1 & CD63 in cell lines

[0063] The exosomal PD-L1 chemiluminescence kit was used to detect the titer and inter-batch variation of exosome PD-L1 and CD63 extracted from three batches of cells cultured in the control group. The original concentration was calculated based on the dilution ratio. The results are shown in Table 1. The inter-batch variation of cell-extracted exosomes exceeded 30%, which will lead to large inter-batch variation in the production process of in vitro diagnostic products.

[0064] Table 1. Exosomal PD-L1 engineered phage and cell line exosome titer test

[0065]

[0066]

[0067] 2. Accelerated stability of exosomes PD-L1 & CD63 in cell lines

[0068] The 37°C accelerated stability of a quality control product prepared from exosomes extracted from cell line cultures was tested using an exosome PD-L1 chemiluminescence kit. The results are shown in Table 2. The results showed that the 8-day accelerated thermal stability of exosomes extracted from cell line cultures was poor, with an accelerated deviation of less than -25%, which does not meet the accelerated stability requirements for in vitro diagnostic products.

[0069] Table 2 Accelerated 8-day stability data of exosome antigens extracted from cell lines

[0070]

[0071] 3. Simulated transport stability of exosomes PD-L1 & CD63 in cell lines

[0072] Using an exosomal PD-L1 chemiluminescent kit, we simulated shipping conditions in the laboratory by storing calibrators and quality control samples at 37°C for 1, 3, and 5 days. On the day of sampling, we performed a drop experiment in which the kit was dropped from a height of 1.5 meters three times to test the shipping stability of quality control samples prepared with exosomes extracted from cell line cultures. The results are shown in Table 3. The results showed that exosome antigens extracted from cell line cultures exhibited poor stability after 5 days of transportation at 37°C, with a deviation within -15%, which does not meet the shipping stability requirements for in vitro diagnostic products.

[0073] Table 3 Transport stability data of freeze-dried quality control products of cell line extracted exosomes

[0074]

[0075] 4. Real-time stability of exosome PD-L1 & CD63 in cell lines

[0076] The long-term stability of a quality control product prepared from exosomes extracted from cell line cultures at 4°C was tested using an exosome chemiluminescence kit. The results are shown in Table 4. The freeze-dried cell line exosome quality control product can only be stored at 4°C for 6 months. The results showed that the real-time deviation of cell line exosome PD-L1 and CD63 exceeded 10% at 12 months, which does not meet the long-term real-time stability requirements for in vitro diagnostic products.

[0077] Table 4 Real-time stability data of calibrators and quality control products

[0078]

[0079] Example 1 Calibrators and quality control products for engineered bacteriophage exosome PD-L1 & CD63 detection reagents

[0080] like Figure 3 Shown is the process flow chart and detection schematic diagram of PD-L1 & CD63 engineered phage preparation. The specific process is as follows.

[0081] (1) Construction of engineered phage

[0082] Use the phage display system to construct exosomal PD-L1 & CD63 engineered phage. Here, the M13 phage display system is preferably used to construct exosomal PD-L1 & CD63 engineered phage. The steps are as follows:

[0083] 1. Phage modification of PD-L1: Insert the PD-L1 gene sequence SEQ (Gene ID: 29126) between the signal peptide (Sg III) and the first amino acid sequence of the PIII protein. PCR amplification was performed, and the cells were transformed into competent E. coli cells. The cells were cultured overnight at 37°C. Plaques were picked, and positive clones with the correct sequence inserted were selected for amplification.

[0084] 2. Using the positive clone obtained in step 1) as a template for primer design, insert the exosomal transmembrane protein CD63 gene sequence SEQ (Gene ID: 967) between the signal peptide and the first amino acid sequence of the PVIII protein. PCR amplify, ligate and transform into E. coli competent cells, culture at 37°C overnight, and pick 96 single clones. 3. Culture the 96 groups of E. coli selected in step 2) overnight, elute and separate the 96 groups of bacterial liquid, and select the positive clone with the highest expression level. 4. In step 3), expand the culture of the final positive clone obtained by screening, culture at 37°C overnight, and purify the engineered phage that can simultaneously express two exosomal membrane markers, CD63 and PD-L1.

[0085] (II) Preparation of calibrators for engineered bacteriophage exosome PD-L1 & CD63 detection reagents

[0086] In this example, the calibrators were prepared using a liquid preservative formulation containing 1.0% w / v BSA, 2.0% w / v sucrose, 2.5% v / v sorbitol, 5.0% v / v glycerol, 0.05% v / v Tween-20, 100 mM PBS buffer, and 0.1% v / v Proclin 300 preservative, at a pH of 7.40. The engineered phage purified in step 1 was diluted to calibrator 1 (1.00 ± 0.20) AU / mL and calibrator 2 (20.00 ± 4.00) AU / mL.

[0087] (III) Preparation of quality control products for engineered bacteriophage exosome PD-L1 & CD63 detection reagents

[0088] In this example, the quality control samples were prepared using a freeze-dried storage solution containing 5.0% w / v BSA, 2.0% w / v trehalose, 5.0 w / v mannitol, 0.05% v / v Tween-20, 100 mM PBS buffer, and 0.1% v / v Proclin 300 preservative at a pH of 7.40. The engineered phage purified in step 1 was diluted to a concentration of (1.00 ± 0.20) AU / mL for quality control sample 1 and (20.00 ± 4.00) AU / mL for quality control sample 2. The diluted solutions were dispensed into freeze-dried bottles and freeze-dried according to the following freeze-drying procedure:

[0089] Open the freeze dryer, cool to -50°C, transfer the subpackaged quality control products into the freeze dryer, and keep the temperature constant for 180 minutes;

[0090] Heat to -40°C for 10 minutes and maintain constant temperature for 120 minutes; maintain vacuum below 10Pa;

[0091] Heat to -30°C for 10 minutes, maintain constant temperature for 120 minutes, and maintain vacuum below 10 Pa;

[0092] Heat to -20°C for 10 minutes, maintain constant temperature for 120 minutes, and maintain vacuum below 10 Pa;

[0093] Heat to -10°C for 10 minutes, maintain constant temperature for 120 minutes, and maintain vacuum below 10 Pa;

[0094] Heat to 0°C for 10 minutes, maintain constant temperature for 360 minutes, and maintain vacuum below 10Pa;

[0095] Heat to 10°C for 10 minutes, maintain constant temperature for 360 minutes, and maintain vacuum below 10 Pa;

[0096] The temperature was raised to 25°C for 10 minutes, and the temperature was kept constant for 360 minutes, while the vacuum degree was maintained below 10 Pa.

[0097] (IV) Evaluation of PD-L1 & CD63 engineered phage

[0098] 1. Screening of engineered phages using the exosome PD-L1 chemiluminescence kit

[0099] The 96 phages purified from the plaque cultures in Example 1 were tested using an exosomal PD-L1 chemiluminescence kit. The results are shown in Table 5. It was found that several positive clones, such as F05, B08, and C10, had the highest luminescence values. Phage F05 was selected as the exosomal PD-L1 & CD63 engineered phage.

[0100] Table 5 Luminescence values ​​of 96 monoclonal phages

[0101]

[0102] 2. Potency and batch variation of PD-L1 & CD63 engineered phage

[0103] Three batches of engineered phage (F05-LOT1, F05-LOT2, and F05-LOT3) expressing the PD-L1 & CD63 engineered phage F05 of the present invention were tested using an exosomal PD-L1 chemiluminescence kit. The original concentrations were calculated based on the dilution ratios. The results, shown in Table 6, showed that the inter-batch variability of engineered phage F05 was within 5%. These results demonstrate that the inter-batch variability of the PD-L1 & CD63 engineered phage F05 of the present invention was significantly smaller than that of exosomes extracted from the control cell line, meeting the inter-batch variability requirements for in vitro diagnostic products.

[0104] Table 6 PD-L1 & CD63 engineered phage F05 titer and inter-batch difference data

[0105]

[0106] 3. Accelerated stability of PD-L1 & CD63 engineered phage

[0107] The 37°C accelerated stability of calibrators and quality controls formulated with the PD-L1 & CD63 engineered phage F05 of the present invention was tested using an exosomal PD-L1 chemiluminescence kit. The results, shown in Tables 7-8, demonstrate excellent thermal stability. Whether in liquid or lyophilized form, the calibrators and quality controls exhibited an 8-day accelerated stability deviation within -5%. These results demonstrate that the accelerated stability of engineered phage F05 significantly outperforms that of exosomes extracted from cell lines in the control group, meeting the accelerated stability requirements for in vitro diagnostic products.

[0108] Table 7 Accelerated 8-day stability data of engineered bacteriophage F05 liquid calibrator

[0109]

[0110]

[0111] Table 8 Accelerated 8-day stability data of engineered bacteriophage F05 freeze-dried quality control

[0112]

[0113] 4. Simulated transport stability of PD-L1 & CD63 engineered phage

[0114] Using an exosomal PD-L1 chemiluminescent kit, calibrators and quality controls were stored at 37°C for 1, 3, and 5 days, respectively, under laboratory conditions simulating transport conditions. On the same day of sampling, the kit was dropped from a height of 1.5 meters three times in a oscillatory drop experiment to test the transport stability of calibrators and quality controls formulated with the PD-L1 and CD63 engineered phage F05 of the present invention. The results, as shown in Tables 9 and 10, show that the engineered phage F05 of the present invention exhibits excellent transport stability. Whether in liquid or lyophilized form, the deviation between the calibrators and quality controls remained within -5% after five days of transport at 37°C. These results demonstrate that the transport stability of the PD-L1 and CD63 engineered phage F05 of the present invention is superior to that of exosomes extracted from cell lines in the control group, meeting the transport stability requirements of in vitro diagnostic products.

[0115] Table 9 Transport stability data of engineered bacteriophage F05 liquid calibrator

[0116]

[0117]

[0118] Table 10 Transport stability data of engineered bacteriophage F05 freeze-dried quality control

[0119]

[0120] 5. Real-time stability of PD-L1 & CD63 engineered phage

[0121] Using an exosomal PD-L1 chemiluminescent kit, the long-term stability of the calibrators and quality controls prepared with the PD-L1 & CD63 engineered phage F05 of the present invention was compared with that of the quality control prepared with exosomes extracted from cell lines at 4°C. The results are shown in Table 11. The engineered phage F05 liquid calibrator can be stored at 4°C for 15 months, and the phage F05 freeze-dried quality control can be stored at 4°C for 24 months. These results demonstrate that the real-time stability of the PD-L1 & CD63 engineered phage of the present invention is superior to that of the control group of cell line-extracted exosomes, meeting the long-term real-time stability requirements of in vitro diagnostic products.

[0122] Table 11 Real-time stability data of engineered bacteriophage F05 calibrators and quality control products

[0123]

[0124]

[0125] Control group 2 plasma extracted exosomes PLAP&CD81 detection reagent calibrator and quality control

[0126] (1) Extraction of exosomes PLAP and CD81 from serum

[0127] In the control group, plasma exosomes were extracted using a precipitation method. The specific method was as follows: maternal serum stored at -80°C was reconstituted at room temperature and centrifuged at 2000g for 30 minutes at 4°C to remove blood cell debris. The supernatant was filtered through a 0.22μm filter and the filtrate was collected. One-quarter volume of 40% PEG6000 was added, vortexed to mix, and incubated at 4°C overnight. The mixture was centrifuged at 10000g for 60 minutes at 4°C, and the supernatant was removed. The pellet was resuspended in PBS to obtain the exosomes from the control group.

[0128] (II) Preparation of calibrators and quality control materials for serum-extracted exosome PLAP & CD81 detection reagents

[0129] The calibrators and quality controls in this control group were prepared using a freeze-dried storage solution containing 5.0% w / v BSA, 5.0% w / v trehalose, 8.0 w / v mannitol, 0.05% v / v Tween-20, 50 mM Tris buffer and 0.1% v / v Proclin 300 preservative, pH 8.00. The exosome PLAP engineered phage was diluted to quality control product 1 (1.00 ± 0.20) AU / mL and quality control product 2 (10.00 ± 2.00) AU / mL, and then dispensed into freeze-dried bottles and freeze-dried according to the freeze-drying procedure of Example 1 below.

[0130] Open the freeze dryer, cool to -50°C, transfer the subpackaged quality control products into the freeze dryer, and keep the temperature constant for 180 minutes;

[0131] Heat to -40°C for 10 minutes and maintain constant temperature for 120 minutes; maintain vacuum below 10Pa;

[0132] Heat to -30°C for 10 minutes, maintain constant temperature for 120 minutes, and maintain vacuum below 10 Pa;

[0133] Heat to -20°C for 10 minutes, maintain constant temperature for 120 minutes, and maintain vacuum below 10 Pa;

[0134] Heat to -10°C for 10 minutes, maintain constant temperature for 120 minutes, and maintain vacuum below 10 Pa;

[0135] Heat to 0°C for 10 minutes, maintain constant temperature for 360 minutes, and maintain vacuum below 10Pa;

[0136] Heat to 10°C for 10 minutes, maintain constant temperature for 360 minutes, and maintain vacuum below 10 Pa;

[0137] The temperature was raised to 25°C for 10 minutes, and the temperature was kept constant for 360 minutes, while the vacuum degree was maintained below 10 Pa.

[0138] (III) Evaluation of serum-extracted exosomes PLAP and CD81

[0139] 1. The titer and inter-batch variation of serum-extracted exosomes PLAP and CD81

[0140] The exosome PLAP chemiluminescence kit was used to test the titer and inter-batch variability of exosomes extracted from three batches of maternal serum. The original concentration was calculated based on the dilution ratio. The results, shown in Table 12, showed inter-batch variability of serum exosome PLAP and CD81 exceeding 30%. This indicates that the inter-batch variability of serum-exosome PLAP and CD81 is large and cannot meet the requirements of in vitro diagnostic products.

[0141] Table 12. Titer and inter-batch difference of serum-extracted exosomes PLAP & CD81

[0142] Dilution ratio Serum-extracted exosomes-LOT1 Serum-extracted exosomes-LOT2 Serum-extracted exosomes-LOT3 1 / 1000 0.242 0.296 0.181 1 / 100 2.36 3.24 1.76 1 / 10 21.89 31.36 19.67 Back-calculated concentration 232.3 311.2 184.6 Batch-to-batch variation 33.96％ -20.55％

[0143] 2. Accelerated stability of serum-extracted exosomes PLAP & CD81

[0144] The 37°C accelerated stability of a quality control prepared with exosomes PLAP and CD81 extracted from the serum of the control group was tested using an exosome PLAP chemiluminescence kit. The results, shown in Table 13, showed an 8-day accelerated stability deviation of approximately -25%. This indicates that the accelerated stability of serum-extracted exosomes PLAP and CD81 is poor and does not meet the accelerated stability requirements for in vitro diagnostic products.

[0145] Table 13 Accelerated stability data of serum-extracted exosomes PLAP & CD81 freeze-dried quality control products

[0146]

[0147] 3. Simulated transport stability of serum-extracted exosomes PLAP & CD81

[0148] Using an exosome PLAP chemiluminescence kit, we simulated transport conditions in the laboratory. Calibrators or quality control samples were stored at 37°C for 1, 3, and 5 days. On the day of sampling, the kit was dropped from a height of 1.5 meters three times to test the transport stability of a quality control prepared with serum-extracted exosome PLAP & CD81. The results, shown in Table 14, showed a deviation of -20% on the fifth day of simulated transport. These results indicate that serum-extracted exosome PLAP & CD81 has poor transport stability and does not meet the transport stability requirements for in vitro diagnostic products.

[0149] Table 14 Simulated transportation stability data of serum-extracted exosomes PLAP&CD81 freeze-dried quality control products

[0150]

[0151] 4. Real-time stability of serum-extracted exosomes PLAP & CD81

[0152] The real-time stability of a quality control prepared with serum-extracted exosomes PLAP & CD81 was tested using an exosome PLAP chemiluminescence kit. The results, shown in Table 15, show that the serum-extracted exosome PLAP & CD81 quality control was only stable for 6 months. This indicates that the real-time stability of serum-extracted exosomes PLAP & CD81 is poor and cannot meet the long-term real-time stability requirements of in vitro diagnostic products.

[0153] Table 15 Real-time stability data of serum-extracted exosomes PLAP&CD81 freeze-dried quality control products

[0154]

[0155]

[0156] Example 2 Calibrators and quality control products for engineered bacteriophage exosome PLAP detection reagent

[0157] like Figure 4 Shown is the process flow chart and detection schematic diagram of PLAP&CD81 engineered phage preparation process. The specific steps are as follows.

[0158] (1) Construction of engineered phage

[0159] The PLAP&CD81 engineered phage is constructed using a phage display system. Here, the T4 phage display system is preferably used to construct the PLAP&CD81 engineered phage. The steps are as follows:

[0160] 1. Preparation of PLAP-SOC fusion protein: construct PLAP-pSOC recombinant plasmid, transform into expression vector E. coli BL21 (DE3) competent cells to express target protein, and dialysis purification to obtain PLAP-SOC fusion protein.

[0161] 2. Preparation of CD81-HOC fusion protein: Construct CD81-pHOC recombinant plasmid, transform it into expression vector E. coli BL21 (DE3) competent cells to express target protein, and dialysis purification is used to obtain CD81-HOC fusion protein.

[0162] 3.HOC - SOC - Preparation of T4 phage: Culture E. coli to the logarithmic phase, take E. coli liquid and HOC- SOC - After the T4 phage was mixed, it was shaken and cultured at 37 ° C for 6 hours, and the supernatant was obtained by centrifugation. After filtering with a 0.22 μm filter membrane, the supernatant was removed by high-speed centrifugation to obtain HOC. - SOC - T4 bacteriophage.

[0163] 4. Preparation of placental exosomes CD81-PLAP engineered phage: Step 3) HOC - SOC - The T4 phage was mixed evenly with the PLAP-SOC fusion protein from step 1 and the CD81-HOC fusion protein from step 2, respectively, incubated, and purified to obtain the CD81-PLAP-T4 engineered phage.

[0164] (II) Preparation of calibrators for PLAP detection reagents for engineered bacteriophage exosomes

[0165] In this example, the calibrators were prepared using a liquid preservative formulation containing 2.0% w / v BSA, 3.0% w / v sucrose, 2.5% v / v sorbitol, 10.0% v / v glycerol, 0.05% v / v Tween-20, 50 mM Tris buffer, and 0.1% v / v Proclin 300 preservative, pH 8.00. The exosomal PLAP engineered phage was diluted to calibrator 1 (1.00 ± 0.20) AU / mL and calibrator 2 (10.00 ± 2.00) AU / mL.

[0166] (III) Preparation of quality control products for PLAP detection reagents of engineered bacteriophage exosomes

[0167] In this example, the quality control product adopts a freeze-dried preservation solution formula containing 5.0% w / v BSA, 5.0% w / v trehalose, 8.0w / v mannitol, 0.05% v / v Tween-20, 50mM Tris buffer and 0.1% v / v Proclin 300 preservative, pH 8.00, and the exosome PLAP engineered phage is diluted to quality control product 1 (1.00±0.20) AU / mL and quality control product 2 (10.00±2.00) AU / mL, and then divided into freeze-dried bottles and freeze-dried according to the freeze-drying procedure of Example 1 below.

[0168] (IV) Evaluation of PLAP&CD81 engineered phage

[0169] 1. Titer and batch variation of PLAP & CD81 engineered phage

[0170] Using an exosome PLAP chemiluminescence kit, the titer and inter-batch variability of three batches of the PLAP & CD81 engineered phage of the present invention were tested. The original concentration was calculated based on the dilution ratio. The results, shown in Table 16, showed that the inter-batch variability of the PLAP & CD81 engineered phage was within 5%. These results demonstrate that the inter-batch variability of the PLAP & CD81 engineered phage of the present invention is significantly lower than that of the exosome PLAP & CD81 extracted from serum in the control group, meeting the inter-batch variability requirements for in vitro diagnostic products.

[0171] Table 16 PLAP & CD81 engineered phage titer and batch difference test

[0172]

[0173] 2. Exosome PLAP-engineered phage accelerates stability

[0174] The PLAPCD81 engineered phage calibrators and controls formulated with the exosome PLAP chemiluminescence kit were tested for their 37°C accelerated stability. The results, shown in Tables 17 and 18, show that the PLAP&CD81 engineered phage calibrators and controls, whether in liquid or lyophilized form, exhibited an 8-day accelerated stability deviation of 4%. These results demonstrate that the PLAP&CD81 engineered phage exhibits excellent thermal stability, surpassing the serum-extracted PLAP&CD81 control, meeting the accelerated stability requirements for in vitro diagnostic products.

[0175] Table 17 Accelerated 8-day stability data of PLAP & CD81 engineered phage liquid calibrators

[0176]

[0177]

[0178] Table 18 Accelerated 8-day stability data of PLAP&CD81 engineered phage freeze-dried quality control products

[0179]

[0180] 3. PLAP & CD81 engineered phage engineered phage simulated transport stability

[0181] Using an exosome PLAP chemiluminescence kit, calibrators and quality controls were stored at 37°C for 1, 3, and 5 days, respectively, under laboratory conditions simulating transport conditions. On the same day of sampling, the kit was dropped from a height of 1.5 meters three times in a oscillatory drop experiment to test the transport stability of calibrators and quality controls formulated with the PLAP & CD81 engineered phage of the present invention. The results, shown in Tables 19 and 20, show that the PLAP & CD81 engineered phage of the present invention, whether in liquid or lyophilized form, maintained a deviation of less than 5% for calibrators and quality controls after 5 days of transport at 37°C. These results demonstrate that the PLAP & CD81 engineered phage of the present invention exhibits excellent transport stability, surpassing the simulated transport stability of the serum-extracted exosome PLAP & CD81 control group, and meeting the transport stability requirements of in vitro diagnostic products.

[0182] Table 19 PLAP & CD81 engineered phage liquid calibrators transport stability data

[0183]

[0184] Table 20 PLAP&CD81 engineered phage freeze-dried quality control product transportation stability data

[0185]

[0186] 5. Real-time stability of PLAP & CD81 engineered phage

[0187] The long-term stability of PLAP&CD81 engineered phage-derived calibrators and quality controls at 4°C was tested using an exosome PLAP chemiluminescence kit. The results, shown in Table 21, indicate that the PLAP&CD81 engineered phage liquid calibrator can be stored at 4°C for 15 months, while the lyophilized quality control can be stored at 4°C for 24 months. These results demonstrate that the long-term stability of PLAP&CD81 engineered phage is significantly superior to that of the serum-derived exosome PLAP&CD81 control, meeting the long-term, real-time stability requirements for in vitro diagnostic products.

[0188] Table 21 Real-time stability data of calibrators and quality control products

[0189]

[0190]

[0191] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An engineered bacteriophage, characterized in that: The engineered phage is a phage that displays two or more exosome membrane markers simultaneously, among which, The bacteriophage includes but is not limited to any one of M13, T4, T7 or λ phage; The exosome membrane markers include at least two of exosome transmembrane transport proteins, exosome-specific proteins, or disease-related markers.

2. The engineered bacteriophage according to claim 1, characterized in that The exosome transmembrane transporter comprises at least one of CD9, CD63, CD81 or Alix; The exosome-specific proteins include TSG101; The disease-related markers include but are not limited to tumor-related disease markers, neurodegenerative disease-related markers or pregnancy eclampsia-related markers, wherein the tumor-related disease markers include but are not limited to PD-L1 or PSMA, the neurodegenerative disease-related markers include but are not limited to α-synuclein, Aβ protein, phosphorylated Tau protein, NfL or GFAP, and the pregnancy eclampsia-related markers include but are not limited to PLAP.

3. The method for preparing an engineered phage according to claim 1 or 2, wherein: The method comprises: inserting two or more exosome membrane markers into the phage display region respectively, and screening, separating and purifying an engineered phage that simultaneously displays the two or more exosome membrane markers.

4. A calibrator or quality control product for an exosome membrane marker detection reagent based on an engineered bacteriophage, characterized in that: The engineered phage is diluted proportionally with a diluent to obtain a low-value or high-value calibrator or quality control product, wherein the engineered phage is the engineered phage according to claim 1 or 2.

5. The calibrator or quality control product for the detection reagent of exosome membrane markers based on engineered bacteriophage according to claim 4, characterized in that: The diluent includes a liquid storage buffer containing 0.5%-3% w / v BSA or its equivalent substitute, 0.5%-5% w / v sucrose, 0.5%-5% w / v sorbitol, 2.5%-10% v / v glycerol, 0.05-0.25% v / v Tween-20, 100-200 mM basic buffer and 0.1% v / v Proclin 300 preservative, with a pH of 5.0-8.0, and the basic buffer includes any one of 10-200 mM Tris, PBS, citric acid and MES buffer.

6. The calibrator or quality control product for the detection reagent of exosome membrane markers based on engineered bacteriophage according to claim 4, characterized in that: After the engineered phage is diluted proportionally with a diluent to obtain a low-value and high-value reagent, the calibrator or quality control product is lyophilized, and the diluent includes a lyophilized product storage buffer, the lyophilized storage buffer contains 2%-8% w / v BSA or its equivalent substitute, 0.5%-5% w / v trehalose, 3%-10% w / v mannitol, 0.05-0.25% v / v Tween-20, 100-200 mM basic buffer, and 0.1% v / v Proclin 300 preservative is added, with a pH of 5.0-8.0, and the basic buffer includes any one of 10-200 mM Tris, PBS, citric acid and MES buffer.

7. The calibrator or quality control product for the detection reagent of exosome membrane markers based on engineered bacteriophage according to claim 6, characterized in that: The freeze-drying method comprises dispensing 0.3 mL to 2.0 mL of the phage solution diluted with the freeze-drying product diluent into freeze-drying bottles, and freeze-drying according to the following freeze-drying procedure: Precool the freeze dryer to -50°C and keep the temperature constant for 120-240 minutes; The temperature is raised in stages to -40°C, -30°C, -20°C, -10°C, 0°C, 10°C, and 25°C. The heating time for each stage is about 10 minutes. The temperature is kept constant for 60-480 minutes, and the vacuum degree is maintained below 60Pa.

8. A kit, characterized in that A calibrator or quality control product for an exosome membrane marker detection reagent based on an engineered phage according to any one of claims 4 to 7.

9. A detection device, characterized in that: A calibrator or quality control product for an exosome membrane marker detection reagent based on an engineered phage according to any one of claims 4 to 7.

10. A detection method, characterized in that: Use the calibrator or quality control product for the engineered phage-based exosome membrane marker detection reagent according to any one of claims 4 to 7, or the kit according to claim 8, or the detection device according to claim 9.

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