Calibrators or quality controls for an exosome membrane marker detection reagent based on engineered phage

Abstract

The application discloses an exosome membrane marker detection reagent calibration or quality control product based on an engineered bacteriophage, wherein the engineered bacteriophage is a bacteriophage simultaneously displaying two or more exosome membrane markers, the bacteriophage comprises any one of M13, T4, T7 or lambda bacteriophage, and the exosome membrane markers comprise any two or more of exosome transmembrane transport proteins, exosome specific proteins or disease related markers. The exosome membrane marker detection reagent calibration or quality control product based on the engineered bacteriophage is obtained by diluting the engineered bacteriophage by a diluent in a certain proportion to obtain a low high-value calibration or quality control product, and the problems of high cell culture extraction cost, large batch difference and poor stability during use when biological extraction exosomes are used as the calibration or quality control product are solved, and the problems of calibration traceable value source and quality control of the exosome membrane marker in vitro detection kit are solved.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a calibrator or quality control product for a reagent for detecting exosome membrane markers based on engineered bacteriophages. Background Technology

[0002] Exosomes are extracellular vesicles with a lipid bilayer structure of 30nm-150nm released from cells. The exosome membrane surface contains lipids and various proteins, including cytoskeletal proteins, transmembrane transport proteins (CD9, CD63, CD81, and CD82), and heat shock proteins involved in protein folding (HSP70 and HSP90). Among these, proteins such as Alix, TSG101, and CD63 can serve as biomarkers for exosome identification. Exosomes can be detected in various bodily fluids, including blood, saliva, amniotic fluid, urine, and tears. In recent years, they have gradually developed into biomarkers for liquid biopsies, aiding in the diagnosis of various diseases. Researchers have found that the level of programmed death-1-ligand 1 (PD-L1) on peripheral blood exosome membranes is more correlated with tumor progression and prognosis of tumor immunotherapy, and that detecting PD-L1 on exosomes can assist in tumor immunotherapy. Detection of prostate-specific membrane antigen (PSMA) on exosome membranes plays an important role in the early diagnosis of prostate cancer. Exosomes in amniotic fluid also have significant diagnostic value, including exosomes secreted by syncytiotrophoblast extracellular vesicles (STBEVs) and markers on the STBEV membrane such as placental alkaline phosphatase. Phosphatase (PLAP) and other proteins 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 diagnostic tool for Parkinson's disease.

[0003] The design and development of exosome membrane biomarker detection kits are of significant clinical importance. Conventional double-antibody sandwich assays typically use two different specific antibodies that recognize different antigenic determinants of the same antigen. However, exosome membrane biomarker detection kits require two different antibodies: an exosome-specific antibody and an exosome membrane biomarker-specific antibody. These antibodies recognize the antigenic determinants of two different membrane biomarkers on the exosome membrane surface, forming a sandwich complex for detection. Meanwhile, calibrators and quality control materials are crucial for metrological transfer and quality control in in vitro diagnostic clinical testing. Developing exosomes directly used as calibrators and quality control materials for detection kits is challenging. Currently, large-scale exosome production generally relies on engineered cell culture methods to obtain exosomes containing specific exosome membrane biomarkers. Cell culture methods are not only expensive, but different cell donors, tissue sources, and culture batches can cause batch-to-batch variability in exosomes. Laboratory storage conditions for exosomes are typically -80°C for long-term storage; as part of an assay kit, their transportation and storage also fall short of these requirements.

[0004] The preparation of low-cost, batch-to-batch variability, and stable exosome calibrators and quality control products is a significant challenge. Phage display techniques (PDT) use modified bacteriophages as vectors to insert a foreign gene fragment into the phage coat protein region. With phage passage, the foreign peptide or protein is expressed and displayed on the phage surface. After affinity enrichment screening, phages displaying specific peptides or proteins are obtained. Common phage display systems include single-stranded filamentous phage (M13) display systems, T4 phage display systems, T7 phage display systems, and λ phage display systems. Phage display technology is characterized by its ease of operation, rapid production, and high efficiency. Therefore, phage display technology can be used to insert two exosome membrane markers into the phage coat protein region, and phages that can simultaneously express both exosome membrane markers can be screened to mimic exosomes as calibrators and quality control products for detection reagents. Summary of the Invention

[0005] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the present invention provides a calibrator or quality control material for exosome membrane marker detection reagents based on engineered bacteriophages. It solves the problems of high cell culture extraction cost, large batch-to-batch variation, and poor stability during use when using biologically extracted exosomes as calibrators and quality control materials for detection reagents. It also solves the problem of calibration traceability and quality control of exosome membrane marker in vitro detection kits.

[0006] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

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

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

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

[0010] This invention provides a method for preparing liquid calibrators or quality control products for exosome membrane marker detection reagents using engineered bacteriophages. The bacteriophages are diluted proportionally using a liquid preservation buffer to obtain low- and high-value calibrators or quality control products.

[0011] The diluents mentioned above include liquid preservation buffer and lyophilized product preservation buffer. The liquid preservation buffer contains 0.5%-3% w / v BSA or its equivalent, 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 basal buffer, and 0.1% v / v Proclin 300 preservative, with a pH of 5.0-8.0.

[0012] The aforementioned base buffers include one of the following: 10-200 mM Tris, PBS, citrate, and MES buffer.

[0013] This invention provides a method for preparing lyophilized calibrators or quality control samples for exosome membrane marker detection reagents using engineered bacteriophages. The method involves diluting the bacteriophages in a proportional manner using a lyophilized preservation buffer to obtain low- and high-value calibrators or quality control samples, which are then freeze-dried.

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

[0015] The aforementioned base buffers include one of the following: 10-200 mM Tris, PBS, citrate, and MES buffer.

[0016] This invention also provides a lyophilization method for the above-mentioned engineered phage lyophilized calibrators or quality control samples, wherein the phage solution diluted with the lyophilized sample diluent is dispensed into lyophilization bottles in 0.3 mL-2.0 mL increments, and then lyophilized according to the following procedure:

[0017] Pre-cool the freeze dryer to -50℃ and maintain the temperature for 120-240 minutes;

[0018] The temperature is gradually increased in stages to -40℃, -30℃, -20℃, -10℃, 0℃, 10℃, and 25℃, with each stage taking about 10 minutes. The temperature is then maintained for 60-480 minutes, and the vacuum level is kept below 60Pa.

[0019] Beneficial effects: The calibrators or quality control materials for the exosome membrane marker detection reagent based on engineered bacteriophages provided by this invention have the following advantages:

[0020] (1) The phage display technology was pioneered and applied to the field of in vitro diagnostics. Two exosome membrane markers were simultaneously and relatively independently displayed on the surface of engineered phages in large quantities, which ensured the biological activity of the two exosome membrane markers and served as equivalent simulations of exosomes as calibrators or quality control products for exosome membrane marker detection reagents.

[0021] (2) The constructed engineered phages were diluted directly with liquid preservation solution or diluted with lyophilization preservation solution and then lyophilized to prepare calibrators or quality control products. Compared with exosomes extracted from cell culture, this method can not only significantly reduce raw material costs and reduce batch-to-batch differences, but also has good real-time, accelerated and transport stability.

[0022] (3) The calibrators or quality control materials for exosome membrane biomarker detection reagents based on engineered bacteriophages of this invention have the advantages of low cost, small batch-to-batch variation, and good stability, solving the problems of value transfer and quality control difficulties in exosome membrane biomarker detection kits. The technical solution of this invention has strong universality and can be extended to a variety of exosome biomarkers and detection scenarios. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of exosome membrane markers.

[0024] Figure 2 This is a schematic diagram of the structure of the exosome PLAP-engineered bacteriophage.

[0025] Figure 3 A flowchart and detection diagram for the preparation process of PD-L1 & CD63 engineered phages.

[0026] Figure 4A flowchart and detection diagram for the preparation process of PLAP & CD81 engineered phages. Detailed Implementation

[0027] This invention discloses a calibrator or quality control for an exosome membrane biomarker detection reagent based on engineered bacteriophages. The engineered bacteriophage is one that simultaneously displays two or more exosome membrane biomarkers, including any one of M13, T4, T7, or λ bacteriophages. The exosome membrane biomarkers include at least two of exosome transmembrane transport proteins, exosome-specific proteins, or disease-related biomarkers. This calibrator or quality control for an exosome membrane biomarker detection reagent based on engineered bacteriophages is prepared by diluting the engineered bacteriophages proportionally with a diluent to obtain low- and high-value calibrators or quality control. This solves the problems of high cell culture extraction costs, large batch-to-batch variations, and poor stability during use when using biologically extracted exosomes as calibrators or quality control for detection reagents. It also solves the challenges of calibrating traceability and quality control in exosome membrane biomarker in vitro detection kits.

[0028] This invention provides an engineered bacteriophage, such as Figure 2 As shown, the engineered bacteriophage is a bacteriophage that simultaneously displays two or more exosomal membrane markers, among which,

[0029] The bacteriophages include, but are not limited to, any one of M13, T4, T7 or λ bacteriophages;

[0030] The exosome membrane markers include at least two of the following: exosome transmembrane transport proteins, exosome-specific proteins, or disease-related markers. For example... Figure 1 As shown.

[0031] Optionally, in some embodiments of the present invention, the exosome 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 biomarkers include at least one of the following: tumor-related disease biomarkers such as PD-L1 and PSMA; neurodegenerative disease-related biomarkers such as α-synuclein, Aβ protein, phosphorylated Tau protein, NfL, and GFAP; and preeclampsia-related biomarkers such as PLAP.

[0034] Optionally, in some embodiments of the present invention, the engineered phage is a phage that simultaneously displays two exosome 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, and screening, separating and purifying engineered phages that simultaneously display two or more exosome membrane markers.

[0036] A second aspect of the present invention provides a calibrator or quality control sample for detecting exosome membrane markers based on engineered phages, wherein the engineered phages are diluted proportionally with a diluent to obtain low- and high-value calibrators or quality control samples, wherein the engineered phages are any of the engineered phages described above.

[0037] Optionally, in some embodiments of the present invention, the diluent comprises a liquid preservation buffer containing 0.5%-3% w / v BSA or its equivalent, 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 basal buffer, and 0.1% v / v Proclin 300 preservative, with a pH of 5.0-8.0. The basal buffer comprises any one of 10-200 mM 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-to-high value reagent, the reagent is lyophilized, and the diluent includes a lyophilized product preservation buffer.

[0039] Optionally, in some embodiments of the present invention, the lyophilized preservation buffer contains 2%-8% w / v BSA or its equivalent, 0.5%-5% w / v trehalose, 3%-10% w / v mannitol, 0.05-0.25% v / v Tween-20, 100-200mM basal buffer, and 0.1% v / v Proclin 300 preservative, with a pH of 5.0-8.0. The basal buffer includes any one of 10-200mM 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-dried product diluent into freeze-drying bottles, and performing freeze-drying according to the following freeze-drying procedure:

[0041] Pre-cool the freeze dryer to -50°C and maintain the temperature for 120-240 minutes; and / or

[0042] The temperature is gradually increased in stages to -40℃, -30℃, -20℃, -10℃, 0℃, 10℃, and 25℃, with each stage taking about 10 minutes. The temperature is then maintained for 60-480 minutes, and the vacuum level is kept below 60Pa.

[0043] A third aspect of the present invention provides a kit comprising calibrators or quality control products for detecting exosome membrane markers based on engineered phages as described above.

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

[0045] The fifth aspect of the present invention provides a detection method using calibrators or quality control products for exosome membrane marker detection reagents based on engineered bacteriophages as described above, or kits as described above, or detection devices as described above.

[0046] Example

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments. The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the present invention as described in detail in the claims.

[0048] Calibrators and quality control materials for PD-L1 & CD63 detection reagents extracted from exosomes of control cell line 1

[0049] (I) Extraction of PD-L1 & CD63 from cell line exosomes

[0050] The control group underwent exosome extraction via cell culture, specifically as follows: The PD-L1-overexpressing human lung adenocarcinoma cell line H1975 was cultured at 37℃ and 5% CO2, and the cell culture supernatant was collected. The cell pellet was removed by centrifugation at 300g, 4℃ for 10 min, and the supernatant was collected again. The cell debris pellet was removed by centrifugation at 2000g, 4℃ for 10 min, and the supernatant was collected again. The supernatant was then collected by centrifugation at 10000g, 4℃ for 30 min, followed by ultracentrifugation at 120000g, 4℃ for 70 min to remove the supernatant. The bottom pellet was resuspended in PBS to obtain the control group exosomes.

[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 lyophilization preservation 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 pH 7.40. The engineered phage purified in step one was diluted to quality control 1 (1.00 ± 0.20) AU / mL and quality control 2 (20.00 ± 4.00) AU / mL, aliquoted into lyophilization vials, and lyophilized according to the following procedure:

[0053] Turn on the freeze dryer, cool it to -50℃, transfer the packaged quality control samples into the freeze dryer, and keep it at a constant temperature for 180 minutes;

[0054] Heat to -40℃ for 10 minutes, hold at that temperature for 120 minutes, and maintain a vacuum level below 10Pa.

[0055] Heat to -30℃ for 10 minutes, hold at that temperature for 120 minutes, and maintain a vacuum of less than 10 Pa.

[0056] Heat to -20℃ for 10 minutes, hold at that temperature for 120 minutes, and maintain a vacuum of less than 10 Pa.

[0057] The temperature was raised to -10℃ for 10 minutes, held at that temperature for 120 minutes, and the vacuum level was maintained below 10 Pa.

[0058] Heat to 0℃ for 10 minutes, hold at that temperature for 360 minutes, and maintain a vacuum of less than 10 Pa.

[0059] Heat to 10℃ for 10 minutes, maintain the temperature for 360 minutes, and keep the vacuum below 10Pa.

[0060] Heat to 25°C for 10 minutes, maintain the temperature for 360 minutes, and keep the vacuum below 10 Pa.

[0061] (III) Evaluation of cell line exosomes PD-L1 & CD63

[0062] 1. Titer and inter-batch variation of cell line exosomes PD-L1 & CD63

[0063] Using an exosome PD-L1 chemiluminescence kit, the potency and batch-to-batch differences of exosome PD-L1 & CD63 extracted from three batches of cells cultured in the control group were detected. The original concentrations were recalculated based on the dilution ratios. The results are shown in Table 1. A batch-to-batch difference of cell-extracted exosomes exceeding 30% can lead to significant batch-to-batch variations in the production process of in vitro diagnostic products.

[0064] Table 1. Titer assay of exosomes from PD-L1 engineered bacteriophages and cell line exosomes

[0065]

[0066]

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

[0068] The accelerated stability at 37℃ of exosomes extracted from cell lines and formulated into quality control products was tested using the exosome PD-L1 chemiluminescence kit. The results are shown in Table 2. The results indicate that the accelerated thermal stability of exosomes extracted from cell lines after 8 days is poor, with an acceleration deviation within -25%, which does not meet the requirements for accelerated stability of in vitro diagnostic products.

[0069] Table 2. Stability data of cell line exosome antigens extracted after 8 days of accelerated processing.

[0070]

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

[0072] Using the exosome PD-L1 chemiluminescence reagent kit, calibrators and quality control samples were placed at a high temperature of 37°C for 1, 3, and 5 days for testing, simulating transport conditions in the laboratory. On the day of sampling, the kit was dropped from a height of 1.5m three times in a drop test to assess the transport stability of the quality control samples prepared from exosomes extracted from cell lines. The results are shown in Table 3. The results indicate that exosome antigens extracted from cell lines exhibit poor stability after 5 days of transport at 37°C, with a deviation within -15%, which does not meet the transport stability requirements for in vitro diagnostic products.

[0073] Table 3. Transport stability data of freeze-dried exosomes extracted from cell lines.

[0074]

[0075] 4. Real-time stability of cell line exosomes PD-L1 & CD63

[0076] The long-term stability of quality control products prepared from exosomes extracted from cell lines at 4°C was tested using an exosome PD-L1 chemiluminescence assay kit. The results are shown in Table 4. The lyophilized quality control products from cell line exosomes could only be stored at 4°C for 6 months. The results indicate that the real-time deviation of exosomes PD-L1 & CD63 exceeded 10% after 12 months, failing to 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 samples

[0078]

[0079] Example 1: Calibrators and quality control materials for engineered phage exosome PD-L1 & CD63 detection reagents

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

[0081] (I) Construction of engineered bacteriophages

[0082] Exosomal PD-L1 & CD63 engineered phages were constructed using a phage display system, preferably the M13 phage display system. The steps are as follows:

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

[0084] 2. Using the positive clones obtained in step 1) as primers, a template was designed to insert the exosome transmembrane protein CD63 gene sequence SEQ (Gene ID: 967) between the signal peptide and the first amino acid of the PVIII protein. PCR amplification was performed, followed by ligation and transformation into competent *E. coli* cells. The cells were cultured overnight at 37°C, and 96 single clones were selected. 3. The 96 groups of *E. coli* cells selected in step 2) were cultured overnight, and the bacterial cultures were eluted and separated. The positive clones with the highest expression levels were selected. 4. The final positive clones obtained in step 3) were expanded and cultured overnight at 37°C. The engineered phages capable of simultaneously expressing both CD63 and PD-L1, two exosome membrane markers, were purified.

[0085] (II) Preparation of calibrators for the detection reagent of engineered bacteriophage exosomes PD-L1 & CD63

[0086] In this embodiment, the calibrators are prepared using a liquid preservation solution 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, 100mM PBS buffer, and 0.1% v / v Proclin 300 preservative, with a pH of 7.40. The engineered phage purified in step one is 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 materials for the detection reagent of engineered bacteriophage exosomes PD-L1 & CD63

[0088] In this embodiment, the quality control samples used a lyophilization preservation solution formulation 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, with a pH of 7.40. The engineered phage purified in step one was diluted to quality control sample 1 (1.00 ± 0.20) AU / mL and quality control sample 2 (20.00 ± 4.00) AU / mL, aliquoted into lyophilization bottles, and freeze-dried according to the following procedure:

[0089] Turn on the freeze dryer, cool it to -50℃, transfer the packaged quality control samples into the freeze dryer, and keep it at a constant temperature for 180 minutes;

[0090] Heat to -40℃ for 10 minutes, hold at that temperature for 120 minutes, and maintain a vacuum level below 10Pa.

[0091] Heat to -30℃ for 10 minutes, hold at that temperature for 120 minutes, and maintain a vacuum of less than 10 Pa.

[0092] Heat to -20℃ for 10 minutes, hold at that temperature for 120 minutes, and maintain a vacuum of less than 10 Pa.

[0093] The temperature was raised to -10℃ for 10 minutes, held at that temperature for 120 minutes, and the vacuum level was maintained below 10 Pa.

[0094] Heat to 0℃ for 10 minutes, hold at that temperature for 360 minutes, and maintain a vacuum of less than 10 Pa.

[0095] Heat to 10℃ for 10 minutes, maintain the temperature for 360 minutes, and keep the vacuum below 10Pa.

[0096] Heat to 25°C for 10 minutes, maintain the temperature for 360 minutes, and keep the vacuum below 10 Pa.

[0097] (iv) Evaluation of PD-L1 & CD63 engineered phages

[0098] 1. Screening of engineered bacteriophages using an exosome PD-L1 chemiluminescence reagent kit

[0099] Using the exosome PD-L1 chemiluminescence kit, the phages cultured and purified from the 96 plaques in Example 1 were tested. 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 exosome PD-L1 & CD63 engineered phage.

[0100] Table 5. Luminescence values ​​of 96 monoclonal bacteriophages

[0101]

[0102] 2. Titer and batch-to-batch variation of PD-L1 & CD63 engineered phages

[0103] Using the exosome PD-L1 chemiluminescence kit, three batches of engineered phages (F05-LOT1, F05-LOT2, and F05-LOT3) expressing the PD-L1 & CD63 engineered phage F05 of this invention were tested. The original concentrations were recalculated based on the dilution ratios, and the results are shown in Table 6. The batch-to-batch variation of engineered phage F05 was within 5%. These results indicate that the batch-to-batch variation of the PD-L1 & CD63 engineered phage F05 of this invention is significantly smaller than that of exosomes extracted from the control cell line, thus meeting the batch-to-batch variation requirements for in vitro diagnostic products.

[0104] Table 6. Titer and batch-to-batch variation data of PD-L1 & CD63 engineered bacteriophage F05

[0105]

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

[0107] The accelerated stability at 37°C of calibrators and quality control samples prepared from the PD-L1 & CD63 engineered phage F05 of this invention was tested using an exosome PD-L1 chemiluminescence kit. The results are shown in Tables 7-8. The engineered phage F05 of this invention exhibits good thermal stability; whether in liquid or lyophilized form, the accelerated stability deviation of the calibrators and quality control samples is within -5% after 8 days. These results indicate that the accelerated stability of engineered phage F05 is significantly better than that of exosomes extracted from control cell lines, meeting the requirements for accelerated stability in in vitro diagnostic products.

[0108] Table 7 Accelerated 8-day stability data for engineered bacteriophage F05 liquid calibrators

[0109]

[0110]

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

[0112]

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

[0114] Using the exosome PD-L1 chemiluminescence reagent kit, calibrators and quality control samples were placed at a high temperature of 37°C for 1, 3, and 5 days under simulated laboratory transport conditions. On the day of sampling, the kit was dropped from a height of 1.5m three times in a drop test to assess the transport stability of the calibrators and quality control samples prepared with the PD-L1 & CD63 engineered phage F05 of this invention. The results are shown in Tables 9 and 10. The engineered phage F05 of this invention exhibits good transport stability; whether in liquid or lyophilized form, after 5 days of transport at 37°C, the deviation of the calibrators and quality control samples is within -5%. These results indicate that the transport stability of the PD-L1 & CD63 engineered phage of this invention is better than that of exosomes extracted from the control cell line, and it can meet 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 freeze-dried quality control samples of engineered bacteriophage F05

[0119]

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

[0121] Using the exosome PD-L1 chemiluminescence reagent kit, the long-term stability of the calibrators and quality control samples prepared from the PD-L1 & CD63 engineered phage F05 of this invention was compared with that of the quality control sample prepared from exosomes extracted from cell lines at 4°C. The results are shown in Table 11. The liquid calibrator of engineered phage F05 could be stored at 4°C for 15 months, and the lyophilized quality control sample of phage F05 could be stored at 4°C for 24 months. The results indicate that the real-time stability of the PD-L1 & CD63 engineered phage of this invention is better than that of exosomes extracted from the control cell lines, and can meet the requirements of long-term real-time stability for in vitro diagnostic products.

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

[0123]

[0124]

[0125] Calibrator and quality control materials for exosome PLAP & CD81 detection reagents extracted from plasma in control group 2.

[0126] (I) Extraction of exosomes from serum: PLAP & CD81 extraction

[0127] The control group underwent plasma exosome extraction using a precipitation method, as follows: Pregnant woman serum stored at -80℃ was thawed at room temperature, centrifuged at 2000g for 30 min at 4℃ to remove blood cell debris, and the supernatant was filtered through a 0.22μm filter. The filtrate was collected. 1 / 4 volume of 40% PEG6000 was added, vortexed, and incubated overnight at 4℃. The mixture was then centrifuged at 10000g for 60 min at 4℃, and the supernatant was removed. The bottom precipitate was resuspended in PBS to obtain the control group exosomes.

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

[0129] The calibrators and quality control samples in this control group were prepared using a lyophilization preservation 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, at pH 8.00. The exosome PLAP engineered phage was diluted to quality control 1 (1.00 ± 0.20) AU / mL and quality control 2 (10.00 ± 2.00) AU / mL, aliquoted into lyophilization vials, and freeze-dried according to the lyophilization procedure in Example 1 below.

[0130] Turn on the freeze dryer, cool it to -50℃, transfer the packaged quality control samples into the freeze dryer, and keep it at a constant temperature for 180 minutes;

[0131] Heat to -40℃ for 10 minutes, hold at that temperature for 120 minutes, and maintain a vacuum level below 10Pa.

[0132] Heat to -30℃ for 10 minutes, hold at that temperature for 120 minutes, and maintain a vacuum of less than 10 Pa.

[0133] Heat to -20℃ for 10 minutes, hold at that temperature for 120 minutes, and maintain a vacuum of less than 10 Pa.

[0134] The temperature was raised to -10℃ for 10 minutes, held at that temperature for 120 minutes, and the vacuum level was maintained below 10 Pa.

[0135] Heat to 0℃ for 10 minutes, hold at that temperature for 360 minutes, and maintain a vacuum of less than 10 Pa.

[0136] Heat to 10℃ for 10 minutes, maintain the temperature for 360 minutes, and keep the vacuum below 10Pa.

[0137] Heat to 25°C for 10 minutes, maintain the temperature for 360 minutes, and keep the vacuum below 10 Pa.

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

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

[0140] The potency and batch-to-batch variation of exosomes extracted from three batches of pregnant women's serum were tested using an exosome PLAP chemiluminescence assay kit. The original concentrations were recalculated based on the dilution ratios. The results are shown in Table 12. The batch-to-batch variation of serum exosome PLAP & CD81 exceeded 30%. The results indicate that the large batch-to-batch variation of serum-extracted exosome PLAP & CD81 does not meet the requirements of in vitro diagnostic products.

[0141] Table 12 Serum-extracted exosome PLAP & CD81 titers and inter-batch variation

[0142] 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-calculation concentration 232.3 311.2 184.6 Batch variation 33.96％ -20.55％

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

[0144] The accelerated stability at 37°C of the quality control product prepared from serum-extracted exosomes PLAP & CD81 was tested using an exosome PLAP chemiluminescence assay kit. The results are shown in Table 13. The accelerated stability of serum-extracted exosomes after 8 days was approximately -25%. The results indicate that the accelerated stability of serum-extracted exosomes PLAP & CD81 is poor and cannot meet the requirements for accelerated stability in in vitro diagnostic products.

[0145] Table 13 Accelerated stability data of serum-extracted exosome PLAP & CD81 lyophilized quality control products

[0146]

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

[0148] Using an exosome PLAP chemiluminescence reagent kit, calibrators or quality control samples were placed at a high temperature of 37°C for 1, 3, and 5 days to simulate transport conditions in the laboratory. On the day of sampling, the kit was dropped from a height of 1.5m three times in a drop test to assess the transport stability of the quality control sample prepared from serum-extracted exosome PLAP & CD81. The results are shown in Table 14. The deviation reached -20% on the 5th day of simulated transport. These results indicate that serum-extracted exosome PLAP & CD81 has poor transport stability and cannot meet the transport stability requirements of in vitro diagnostic products.

[0149] Table 14. Simulated transport stability data of serum-extracted exosome PLAP & CD81 lyophilized quality control products.

[0150]

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

[0152] The real-time stability of the quality control product prepared from serum-extracted exosome PLAP & CD81 was tested using an exosome PLAP chemiluminescence assay kit. The results are shown in Table 15. The stability of the serum-extracted exosome PLAP & CD81 quality control product was only 6 months. These results indicate that the real-time stability of serum-extracted exosome 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 exosome PLAP & CD81 lyophilized quality control products

[0154]

[0155]

[0156] Example 2: Calibrators and quality control materials for engineered phage in vitro oosome PLAP detection reagent

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

[0158] (I) Construction of engineered bacteriophages

[0159] The PLAAP & CD81 engineered phages are constructed using a phage display system, preferably the T4 phage display system. The steps are as follows:

[0160] 1. Preparation of PLAP-SOC fusion protein: The PLAP-pSOC recombinant plasmid was constructed, transformed into E. coli BL21(DE3) competent cells to express the target protein, and purified by dialysis to obtain the PLAP-SOC fusion protein.

[0161] 2. Preparation of CD81-HOC fusion protein: The CD81-pHOC recombinant plasmid was constructed, transformed into E. coli BL21(DE3) competent cells to express the target protein, and purified by dialysis to obtain the CD81-HOC fusion protein.

[0162] 3.HOC - SOC - Preparation of T4 phage: Culture E. coli to the logarithmic phase, then take the E. coli bacterial culture and HOC...- SOC - After mixing the T4 phage, it was cultured with shaking at 37°C for 6 hours. The supernatant was collected by centrifugation, filtered through a 0.22 μm filter membrane, and then centrifuged at high speed to remove the supernatant, yielding HOC. - SOC - T4 phage.

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

[0164] (II) Preparation of calibrators for the detection reagent of engineered phage exosomes PLAAP

[0165] In this embodiment, the calibrators were prepared using a liquid preservation solution 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, with a pH of 8.00. The exosome 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 materials for the detection reagent of engineered phage exosome PLAAP

[0167] In this embodiment, the quality control samples used a lyophilization preservation solution formulation 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, with a pH of 8.00. The exosome PLAP engineered phage was diluted to quality control sample 1 (1.00 ± 0.20) AU / mL and quality control sample 2 (10.00 ± 2.00) AU / mL, aliquoted into lyophilization bottles, and freeze-dried according to the lyophilization procedure in Example 1 below.

[0168] (iv) Evaluation of PLAP & CD81 engineered phages

[0169] 1. Titer and batch-to-batch variation of PLAP & CD81 engineered phages

[0170] The titer and batch-to-batch variation of the three batches of PLAP&CD81 engineered phages of this invention were tested using an exosome PLAP chemiluminescence assay kit. The original concentrations were recalculated based on the dilution ratios. The results are shown in Table 16. The batch-to-batch variation of the PLAP&CD81 engineered phages was within 5%. These results indicate that the batch-to-batch variation of the PLAP&CD81 engineered phages of this invention is significantly smaller than that of the control group serum-extracted PLAP&CD81, thus meeting the requirements for batch-to-batch variation in in vitro diagnostic products.

[0171] Table 16 PLAP & CD81 engineered phage titer and batch-to-batch variation test

[0172]

[0173] 2. Accelerated stability of exosome-PLAP engineered phages

[0174] The accelerated stability at 37°C of the calibrators and control samples prepared with the PLAP&CD81 engineered phage of this invention was tested using the PLAP&CD81 chemiluminescence kit. The results are shown in Tables 17 and 18. Whether in liquid or lyophilized state, the PLAP&CD81 engineered phage calibrators and control samples showed a deviation of 4% after 8 days of accelerated stability testing. These results indicate that the PLAP&CD81 engineered phage has good thermal stability, superior to the control group serum-extracted PLAP&CD81 control samples, and can meet the accelerated stability requirements of 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 samples

[0179]

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

[0181] Using the exosome PLAP chemiluminescence reagent kit, calibrators and quality control samples were placed at a high temperature of 37°C for 1, 3, and 5 days under simulated laboratory transport conditions. On the day of sampling, the kit was dropped from a height of 1.5m three times in a drop test to assess the transport stability of the calibrators and quality control samples prepared with the PLAP&CD81 engineered phage of this invention. The results are shown in Tables 19 and 20. Whether in liquid or lyophilized form, the PLAP&CD81 engineered phage of this invention exhibited a deviation of less than 5% in calibrators and quality control samples after 5 days of transport at 37°C. These results demonstrate that the PLAP&CD81 engineered phage of this invention possesses excellent transport stability, superior to the simulated transport stability of the control group serum-extracted exosome PLAP&CD81, and meets the transport stability requirements of in vitro diagnostic products.

[0182] Table 19 Transport stability data of PLAP & CD81 engineered bacteriophage liquid calibrators

[0183]

[0184] Table 20. Transport stability data of PLAP&CD81 engineered phage freeze-dried quality control samples.

[0185]

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

[0187] The long-term stability of calibrators and quality control samples prepared with PLAP&CD81 engineered phage was tested at 4°C using an exosome PLAP chemiluminescence assay kit. The results are shown in Table 21. The liquid calibrator of PLAP&CD81 engineered phage could be stored at 4°C for 15 months, and the lyophilized quality control sample could be stored at 4°C for 24 months. These results indicate that the long-term stability of PLAP&CD81 engineered phage is significantly better than that of the control group serum-extracted exosomes PLAP&CD81, meeting the requirements for long-term real-time stability in in vitro diagnostic products.

[0188] Table 21 Real-time stability data for calibrators and quality control samples

[0189]

[0190]

[0191] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A calibrator or quality control sample for a double-antibody sandwich immunoassay kit based on exosome membrane markers from engineered bacteriophages, characterized in that, The engineered phage was diluted with a diluent to obtain low- and high-value calibrators or quality control samples. The engineered phage is a phage that simultaneously displays two or more exosomal membrane markers. During phage replication and proliferation, the exosomal membrane marker proteins are expressed and displayed on the phage surface. The exosome membrane markers are exosome transmembrane transporter CD63 and tumor-associated disease marker PD-L1, or exosome transmembrane transporter CD81 and pregnancy-associated epilepsy marker PLAP. The diluent includes a liquid preservation buffer containing 0.5%-3% w / v BSA, 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 basal buffer, and 0.1% v / v Proclin 300 preservative, with a pH of 5.0-8.

0. The basal buffer includes any one of 10-200 mM Tris, PBS, citrate, and MES buffer.

2. The calibrator or quality control material for the exosome membrane marker double antibody sandwich immunoassay kit based on engineered phages according to claim 1, characterized in that, The liquid preservation buffer contains 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, with a pH of 7.

40.

3. A reagent kit, characterized in that, The calibrator or quality control material includes the double antibody sandwich immunoassay kit for exosome membrane markers based on engineered phages as described in any one of claims 1 or 2.

4. A detection device, characterized in that, The calibrator or quality control material includes the double antibody sandwich immunoassay kit for exosome membrane markers based on engineered phages as described in any one of claims 1 or 2.

5. A calibrator or quality control sample for a double-antibody sandwich immunoassay kit for exosome membrane markers based on engineered bacteriophages, characterized in that, After diluting the engineered phage according to the specified ratio to obtain low and high value calibrators or quality control samples, the calibrators or quality control samples are lyophilized. The engineered phage is a phage that simultaneously displays two or more exosome membrane markers. The exosome membrane marker proteins are expressed and displayed on the surface of the phage during replication and proliferation. The exosome membrane markers are exosome transmembrane transporter CD63 and tumor-associated disease marker PD-L1, or exosome transmembrane transporter CD81 and pregnancy-associated epilepsy marker PLAP. The diluent includes a lyophilization preservation buffer containing 2%-8% w / v BSA, 0.5%-5% w / v trehalose, 3%-10% w / v mannitol, 0.05%-0.25% v / v Tween-20, 100-200 mM basal buffer, and 0.1% v / v Proclin 300 preservative, with a pH of 5.0-8.

0. The basal buffer includes any one of 10-200 mM Tris, PBS, citric acid, and MES buffer.

6. The calibrator or quality control material for the exosome membrane marker double antibody sandwich immunoassay kit based on engineered phages according to claim 5, characterized in that, The lyophilized preservation buffer contains 5.0% w / v BSA, 2.0% w / v trehalose, 5.0% w / v mannitol, 0.05% w / v Tween-20, 100 mM PBS buffer, and 0.1% w / v Proclin 300 preservative, with a pH of 7.

40.

7. The calibrator or quality control material for the exosome membrane marker double antibody sandwich immunoassay kit based on engineered phages according to claim 5, characterized in that, The freeze-drying method includes aliquoting the phage solution diluted with the freeze-dried product diluent into freeze-drying bottles at a rate of 0.3 mL to 2.0 mL, and then freeze-drying according to the following procedure: Pre-cool the freeze dryer to -50℃ and maintain the temperature for 120-240 minutes; The temperature is gradually increased in stages to -40℃, -30℃, -20℃, -10℃, 0℃, 10℃, and 25℃, with each stage lasting 10 minutes. The temperature is then maintained for 60-480 minutes, while the vacuum level is kept below 60Pa.

8. A reagent kit, characterized in that, The calibrator or quality control material includes the double antibody sandwich immunoassay kit for exosome membrane markers based on engineered phages as described in any one of claims 5-7.

9. A detection device, characterized in that, The calibrator or quality control material includes the double antibody sandwich immunoassay kit for exosome membrane markers based on engineered phages as described in any one of claims 5-7.

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