Perfluorohexanesulfonic acid hapten, antigen, antibody and preparation method and application thereof

Abstract

The present invention discloses perfluorohexane sulfonic acid hapten, antigen, antibody and their preparation method and application, and relates to perfluorohexane sulfonic acid hapten, antigen, antibody, colloidal gold detection device and their preparation and application for detecting perfluorohexane sulfonic acid. The present invention provides a method for preparing artificial antigen by synthesizing perfluorohexane sulfonic acid hapten and antigen coupling carrier protein, as well as antibodies specific to perfluorohexane sulfonic acid produced by the body of experimental animals after immunization. 50 The perfluorohexane sulfonic acid (PFHS) enzyme-linked immunosorbent assay (ELISA) kit or perfluorohexane sulfonic acid (PFHS) colloidal gold test strip prepared from the ELISA kit has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, and long shelf life. It is suitable for on-site rapid detection of PFHS by food safety, environmental monitoring and other departments at all levels.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and more specifically, to perfluorohexane sulfonic acid hapten, antigen, antibody, and preparation methods and applications thereof. Background Art

[0002] Perfluorohexane sulfonic acid (PFHxS) exhibits remarkable environmental stability. Conventional natural processes such as photolysis, hydrolysis, and microbial degradation cannot effectively remove it, making it highly susceptible to persistent pollution in ecosystems. PFHxS exhibits significant bioaccumulation in organisms and can pose a potential threat to human health through amplification effects within the food chain. Establishing a rapid detection technology system for PFHxS is of great strategic significance for ensuring food safety and strengthening environmental monitoring.

[0003] Currently, mainstream PFHxS detection technologies are still dominated by instrumental analysis, including gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Although these methods offer excellent detection accuracy, they face technical bottlenecks such as high equipment costs, complex sample pretreatment, and lengthy detection cycles, making them difficult to meet the needs of rapid on-site screening.

[0004] To date, there have been no systematic reports on the design of haptens, synthesis of artificial antigens, or development of specific antibodies targeting PFHxS, both domestically and internationally. This technological gap directly restricts the practical application of immunoassays for PFHxS monitoring. Therefore, overcoming the technical bottleneck of preparing PFHxS-specific immune recognition elements, developing monoclonal antibodies with independent intellectual property rights, and subsequently constructing sensitive, rapid, and portable immunoassay systems have become key technical requirements for improving my country's emerging pollutant monitoring network and enhancing its environmental risk prevention and control capabilities. Summary of the Invention

[0005] In order to solve the problems existing in existing detection methods, the present invention provides a method for preparing perfluorohexane sulfonic acid hapten and artificial antigen. The preparation process is more concise and efficient, providing the necessary artificial antigen for future research; and provides a method for preparing perfluorohexane sulfonic acid antibodies. The antibodies have good specificity and detection sensitivity for perfluorohexane sulfonic acid and can be used to establish an immunological detection method for perfluorohexane sulfonic acid, or a rapid detection method using colloidal gold immunochromatographic test strips, laying the foundation for the research, development and promotion of indirect competitive ELISA kits and colloidal gold test strips.

[0006] According to one aspect of the present invention, a perfluorohexanesulfonic acid hapten is provided, the structure of which is shown in formula (I):

[0007] , formula (Ⅰ).

[0008] According to another aspect of the present invention, there is provided a method for preparing perfluorohexanesulfonic acid hapten, comprising the following steps:

[0009] S1. Weigh methyl 4-aminobutyrate hydrochloride, add anhydrous dichloromethane, then add triethylamine, and slowly dropwise add the dichloromethane solution containing perfluorohexylsulfonyl fluoride in an ice-water bath. After the addition is complete, slowly warm the temperature to room temperature and react overnight. After the reaction is complete, add hydrochloric acid and stir thoroughly to acidify the reaction system. Separate the liquids, collect the organic phase, dry it over anhydrous sodium sulfate, and concentrate the organic phase to dryness to obtain the first intermediate shown in formula (II).

[0010] , formula (II);

[0011] S2. The first intermediate obtained in step S1 is placed in a single-necked bottle, methanol is added, and after stirring, sodium hydroxide is added. After the reaction is completed at room temperature, the pH is adjusted to about 4 with dilute hydrochloric acid, and the reaction solution is extracted with ethyl acetate. The organic phase is collected, dried with anhydrous sodium sulfate, and purified by column to obtain the perfluorohexanesulfonic acid hapten represented by formula (I).

[0012] In some embodiments, the mass ratio of the first intermediate to sodium hydroxide in step S2 is 1:(3-5).

[0013] According to another aspect of the present invention, there is provided a perfluorohexane sulfonic acid antigen which is a conjugate of a perfluorohexane sulfonic acid hapten and a carrier protein, wherein the carrier protein is bovine serum albumin, human serum albumin, chicken ovalbumin or lactoferrin.

[0014] According to a fourth aspect of the present invention, there is provided a non-disease diagnosis application of a perfluorohexanesulfonic acid hapten or a perfluorohexanesulfonic acid antigen in the immunological detection of perfluorohexanesulfonic acid.

[0015] According to a fifth aspect of the present invention, there is provided a perfluorohexane sulfonic acid antibody prepared by immunizing an animal with a perfluorohexane sulfonic acid antigen, wherein the perfluorohexane sulfonic acid antibody is a perfluorohexane sulfonic acid polyclonal antibody or a perfluorohexane sulfonic acid monoclonal antibody.

[0016] According to a sixth aspect of the present invention, there is provided a non-disease diagnosis application of a perfluorohexane sulfonic acid antibody in the immunological detection of perfluorohexane sulfonic acid.

[0017] According to a seventh aspect of the present invention, a perfluorohexane sulfonic acid colloidal gold test strip is provided, which comprises a colloidal gold conjugate pad and a nitrocellulose membrane, wherein a detection line and a quality control line are sequentially provided on the nitrocellulose membrane, wherein the detection line is obtained by streaking with a perfluorohexane sulfonic acid antigen, and the quality control line is obtained by streaking with a goat anti-mouse IgG secondary antibody, and the colloidal gold conjugate pad is coated with a perfluorohexane sulfonic acid antibody.

[0018] According to an eighth aspect of the present invention, a perfluorohexane sulfonic acid enzyme-linked immunosorbent assay kit is provided, which comprises a perfluorohexane sulfonic acid antibody, an ELISA plate, a perfluorohexane sulfonic acid standard solution, a goat anti-mouse IgG secondary antibody and a TMB colorimetric solution, wherein the microwells of the ELISA plate are pre-coated with a perfluorohexane sulfonic acid antigen.

[0019] According to a ninth aspect of the present invention, a method for detecting perfluorohexane sulfonic acid in a sample is provided, wherein the method uses a perfluorohexane sulfonic acid antigen and a perfluorohexane sulfonic acid antibody to detect and analyze the content of perfluorohexane sulfonic acid in the sample.

[0020] Beneficial effects of the present invention: The present invention provides a perfluorohexanesulfonic acid hapten, as well as corresponding perfluorohexanesulfonic acid antigens and perfluorohexanesulfonic acid antibodies. Compared with the prior art, the preparation method of the present invention has the advantages of easy availability of chemical reagents, simple operation process, concise and effective synthesis steps, high reaction yield, and low detection cost. The antibody of the present invention is specific to perfluorohexanesulfonic acid and has high titer. The IC-ELISA standard curve for perfluorohexanesulfonic acid antibodies is established, and its IC 50 The value is 1.08 ng / mL, with a linear range of 0.52 ng / mL to 2.3 ng / mL. It has also been applied in immunological testing, and the perfluorohexane sulfonic acid enzyme-linked immunosorbent assay (ELISA) kit or perfluorohexane sulfonic acid colloidal gold test strip prepared from it has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, and long shelf life. It is suitable for on-site rapid detection of perfluorohexane sulfonic acid by food safety and environmental monitoring departments at all levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a mass spectrum of the perfluorohexanesulfonic acid hapten according to one embodiment of the present invention.

[0022] Figure 2 This is an absorption spectrum of lactoferrin, perfluorohexanesulfonic acid hapten, and perfluorohexanesulfonic acid immunogen according to one embodiment of the present invention.

[0023] Figure 3 This is an absorption spectrum of bovine serum albumin, perfluorohexanesulfonic acid hapten, and perfluorohexanesulfonic acid coating agent according to an embodiment of the present invention.

[0024] Figure 4 The figure shows an ELISA standard curve based on perfluorohexanesulfonic acid monoclonal antibody according to one embodiment of the present invention.

[0025] Figure 5 The figure shows a synthetic route of perfluorohexanesulfonic acid hapten according to one embodiment of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail through specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art are all within the scope of the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents available on the conventional market.

[0027] Example 1 Synthesis and Identification of Perfluorohexanesulfonic Acid Hapten

[0028] The preparation method of perfluorohexanesulfonic acid hapten comprises the following steps:

[0029] S1. Weigh 0.5 g of methyl 4-aminobutyrate hydrochloride into a reaction vessel, add 10 mL of anhydrous dichloromethane as the solvent, then add 1 mL of triethylamine. Slowly add 2 mL of a dichloromethane solution containing 1 g of perfluorohexylsulfonyl fluoride dropwise in an ice-water bath. After the addition is complete, slowly warm the mixture to room temperature and allow the reaction to proceed overnight. After completion, add 25 mL of 1 M hydrochloric acid and stir thoroughly to acidify the reaction system. Separate the layers, collect the organic phase, dry it over anhydrous sodium sulfate, and concentrate to dryness to obtain 1.3 g of the first intermediate of formula (II) with a yield of 81%.

[0030] S2. Add 1g of the first intermediate to a clean single-necked bottle, add 3mL of methanol, stir evenly, add 3-5g of sodium hydroxide, and react at room temperature for 4h. After the reaction is complete, adjust the pH of the reaction solution to about 4 with dilute hydrochloric acid, extract the reaction solution with ethyl acetate, collect the organic phase, dry the organic phase with anhydrous sodium sulfate, and purify it by column to obtain 0.82g of perfluorohexanesulfonic acid hapten of formula (I) with a yield of 84%. The reaction process of the above preparation method is as follows: Figure 5 shown.

[0031] The perfluorohexanesulfonic acid hapten was identified by mass spectrometry, and the obtained mass spectrum was as follows: Figure 1 As shown in the mass spectrum, the molecular ion peak of perfluorohexanesulfonic acid hapten is m / z 486.27 [M + H] + and 507.26[M + Na] + , and 485.27[M + H] + It is the highest peak, which is consistent with the molecular weight of the perfluorohexanesulfonic acid hapten of 485.22, indicating that the perfluorohexanesulfonic acid hapten represented by formula (I) was successfully synthesized.

[0032] Example 2 Synthesis of Perfluorohexanesulfonic Acid Immunogen and Perfluorohexanesulfonic Acid Coating

[0033] The preparation method of perfluorohexane sulfonic acid immunogen comprises the following steps:

[0034] Weigh 15 mg of the perfluorohexanesulfonic acid hapten prepared in Example 1 and dissolve it in 0.5 mL of dimethyl sulfoxide (DMSO). Add 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 15 mg of N-hydroxysuccinimide (NHS). Activate at room temperature for 1 h to prepare an activation solution. This activation solution was added dropwise to 30 mg of lactoferrin (LF) (dissolved in 2.0 mL of PBS, pH 7.4) and stirred overnight at 4°C. The solution was dialyzed against PBS for 3 days (changing the solution three times daily) to remove excess reagent. The perfluorohexanesulfonic acid immunogen, i.e., the perfluorohexanesulfonic acid hapten-KLH conjugate, was prepared and aliquoted and stored at -20°C.

[0035] The preparation method of perfluorohexanesulfonic acid coating comprises the following steps:

[0036] Weigh 12 mg of the perfluorohexanesulfonic acid hapten prepared in Example 1 and dissolve it in 1 mL of dimethyl sulfoxide (DMSO). Add 24 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 12 mg of N-hydroxysuccinimide (NHS) and activate for 1 h to prepare an activation solution. This activation solution was added dropwise to 32 mg of bovine serum albumin (BSA) (dissolved in 3.0 mL of carbonate buffer, pH 9.6) and stirred at 4°C for 4 h. Dialyze the solution against PBS for 3 days. After dialysis, centrifuge at 4000 rpm for 5 min. The supernatant was collected to prepare the perfluorohexanesulfonic acid coating material, i.e., the perfluorohexanesulfonic acid hapten-OVA conjugate. Aliquot and store at -20°C.

[0037] Identification: The carrier protein, perfluorohexane sulfonic acid hapten, perfluorohexane sulfonic acid immunogen, and perfluorohexane sulfonic acid half-coating were prepared into a 1.0 mg / mL solution with pH = 7.4, 0.01 mol / L PBS buffer solution, and the solution was adjusted to zero with pH = 7.4, 0.01 mol / L PBS buffer solution. The absorption spectra of the carrier protein, perfluorohexane sulfonic acid hapten, perfluorohexane sulfonic acid immunogen, perfluorohexane sulfonic acid coating and perfluorohexane sulfonic acid were scanned with an ultraviolet spectrophotometer in the wavelength range of 200-400 nm. Figure 2 and Figure 3 As shown. Figure 2 and Figure 3 As can be seen from the figure, the perfluorohexane sulfonic acid hapten was successfully coupled to the carrier proteins LF and BSA.

[0038] Example 3 Preparation and purification of perfluorohexanesulfonic acid monoclonal antibody

[0039] 3.1 Animal immunization: Healthy 6-8 week old BALB / c mice were selected for immunization. The perfluorohexanesulfonic acid immunogen obtained in Example 2 was mixed and emulsified with an equal amount of Freund's adjuvant, and then the BALB / c mice were immunized by multiple subcutaneous injections at the back of the neck (except for sprint immunization). Complete Freund's adjuvant was used for the first immunization at a dose of 100 μg / mouse; after a lapse of 4 weeks, a booster immunization was performed at a dose of 50 μg / mouse, mixed and emulsified with incomplete Freund's adjuvant, and the intervals between subsequent booster immunizations were 3 weeks; for sprint immunization, the dose was halved to 25 μg / mouse, and the complete antigen was diluted with physiological saline and injected intraperitoneally into the mice. After the third immunization, the mice were tail-cut and blood was collected for testing. The titer and IC of the mouse serum were tested by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 , select high titer, IC 50 Mice with low expression were fused.

[0040] 3.2 Cell fusion and cloning: Spleen cells from immunized BALB / c mice were fused with SP2 / 0 myeloma cells at a ratio of 10:1 to obtain a PFHxS monoclonal hybridoma cell line that stably secreted PFHxS monoclonal antibodies.

[0041] 3.3 Cell freezing and thawing: 2×10 5 Cell suspensions of 100 cells / mL can be stored in liquid nitrogen for a long term. When thawing, remove the cryovials and immediately thaw in a 37°C water bath. Centrifuge to remove the cryopreservation solution and transfer to culture flasks for culture.

[0042] Preparation and purification of monoclonal antibodies: Incremental culture method: Perfluorohexanesulfonic acid monoclonal hybridoma cells are placed in a cell culture medium and cultured at 37°C. The resulting culture fluid is purified using the octanoic acid-saturated ammonium sulfate method to obtain perfluorohexanesulfonic acid monoclonal antibodies, which are then stored at -20°C. The cell culture medium comprises RPMI-1640 medium supplemented with calf serum and sodium bicarbonate, such that the mass percentage of calf serum in the cell culture medium is 10% to 15%, and the mass percentage of sodium bicarbonate in the cell culture medium is 0.1% to 0.2%. The pH of the cell culture medium is 7.4.

[0043] Example 4 Identification of Monoclonal Antibodies Against Perfluorohexane Sulfonic Acid

[0044] 4.1 Coating: Dilute the perfluorohexanesulfonic acid coating agent prepared in Example 2 to 1 μg / mL using coating solution (carbonate buffer, CBS, pH 9.6). Add 100 μL to each well and coat at 37°C overnight.

[0045] 4.2 Washing: Spin off the coating solution, add 200 μL of washing solution (PBST, containing 0.05% Tween-20) to each well, let it stand for 3 minutes, then spin dry, and repeat 3 times;

[0046] 4.3 Blocking: Add 200 μL of blocking solution (1% BSA in PBST) to each well and block at 37°C for 3 h. Shake dry and dry in an oven at 37°C for 1 h.

[0047] 4.4 Sample addition: Shake off the blocking solution and add 50 μL of a mixture of serially diluted PFHxS standard (0.01-1000 ng / mL) and 50 μL of antibody (1:1000 dilution) to each well;

[0048] Set up blank control (PBST), negative control (non-immune serum), and positive control (known positive serum), incubate at 37°C for 1 h, wash the plate and pat dry three times;

[0049] 4.5 Add secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) to each well, incubate at 37°C for 30 min, and wash the plate three times;

[0050] 4.6 Color development: Mix equal volumes of TMB substrate buffer A and B to obtain substrate solution, add substrate solution (100 μL / well), and incubate at 37°C for 10 min;

[0051] 4.7 Termination: Add 10% H2SO4 stop solution (50 μL / well) to the ELISA plate to terminate the reaction;

[0052] 4.8 Reading: Read the absorbance (OD) at a wavelength of 450 nm using a microplate reader. Use OriginPro 8.5 to plot the standard inhibition curve and calculate the IC of the perfluorohexanesulfonic acid monoclonal antibody prepared in Example 3. 50 The standard inhibition curve of perfluorohexane sulfonic acid is as follows. Figure 4 shown.

[0053] Depend on Figure 4 It can be seen that the IC value of the perfluorohexanesulfonic acid monoclonal antibody prepared in Example 3 to perfluorohexanesulfonic acid was determined by indirect competitive ELISA. 50 The linear range was 0.52 ng / mL -2.3 ng / mL, indicating that the monoclonal antibody had high sensitivity to PFHxS.

[0054] Example 5 Specificity Experiment of Perfluorohexanesulfonic Acid Monoclonal Antibody

[0055] In this example, the indirect competitive ELISA method was used to select several analogues with similar structures to perfluorohexane sulfonic acid, such as perfluorooctane sulfonic acid, perfluorooctane sulfonyl fluoride, perfluoroalkyl carboxylic acid, perfluoroalkane sulfonic acid, perfluoroalkyl phosphoric acid, perfluorooctanoic acid, and perfluoropolyether, to investigate the specificity of the perfluorohexane sulfonic acid monoclonal antibody prepared in Example 3 against the above analogues. Standard inhibition curves of perfluorohexane sulfonic acid analogues were prepared in sequence, and the IC values ​​of perfluorohexane sulfonic acid analogues were calculated. 50 The specific results are shown in Table 1 and the calculation formula is as follows:

[0056] Cross-reactivity rate = (IC 50 / IC of other compounds 50 )×100%.

[0057] Table 1 IC values ​​of monoclonal antibodies against PFHxS and its analogs 50 and cross-reactivity rate

[0058]

[0059] As can be seen from Table 1, the cross-reactivity rate of the perfluorohexane sulfonic acid monoclonal antibody prepared in Example 3 to perfluorohexane sulfonic acid is 100%, wherein the cross-reactivity rates to seven compounds of the same matrix type, namely perfluorooctane sulfonic acid, perfluorooctane sulfonyl fluoride, perfluoroalkyl carboxylic acid, perfluoroalkane sulfonic acid, perfluoroalkyl phosphoric acid, perfluorooctanoic acid, and perfluoropolyether, are all less than <0.01%, indicating that the perfluorohexane sulfonic acid monoclonal antibody prepared in the present invention has high specificity to perfluorohexane sulfonic acid.

[0060] Example 6 Perfluorohexanesulfonic acid enzyme-linked immunosorbent assay kit

[0061] This embodiment provides a perfluorohexane sulfonic acid enzyme-linked immunosorbent assay kit, which includes the perfluorohexane sulfonic acid monoclonal antibody prepared in Example 3, an ELISA plate, the perfluorohexane sulfonic acid coating source prepared in Example 2, a perfluorohexane sulfonic acid standard solution, a goat anti-mouse IgG secondary antibody, and a TMB colorimetric solution.

[0062] The microwells of the ELISA plate are pre-coated with a perfluorohexanesulfonic acid coating agent. A perfluorohexanesulfonic acid standard solution or the sample to be tested, a perfluorohexanesulfonic acid monoclonal antibody, a goat anti-mouse IgG secondary antibody, and a TMB colorimetric solution are added to generate a perfluorohexanesulfonic acid standard inhibition curve. The perfluorohexanesulfonic acid content in the sample to be tested is determined based on the perfluorohexanesulfonic acid standard inhibition curve and the absorbance of the sample to be tested. Detection of perfluorohexanesulfonic acid can be achieved using methods commonly used in the art.

[0063] Example 7 Perfluorohexanesulfonic acid colloidal gold test strips

[0064] This example provides a perfluorohexanesulfonic acid colloidal gold test strip, comprising a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and a water-absorbing pad. A test line and a quality control line are sequentially provided on the nitrocellulose membrane. The colloidal gold conjugate pad is coated with the perfluorohexanesulfonic acid monoclonal antibody prepared in Example 3. The test line is drawn using the perfluorohexanesulfonic acid-coated primary sample prepared in Example 2, and the quality control line is drawn using a goat anti-mouse IgG secondary antibody. The colloidal gold test strip can be assembled using methods commonly used in the art.

[0065] The PFHxS colloidal gold test strips detect PFHxS using an indirect competitive assay to detect the presence of PFHxS in the sample. If the sample does not contain PFHxS, the test line will remain unchanged, while the control line will display color. If the sample does contain PFHxS, both the test line and the control line will display color. PFHxS detection can be achieved using commonly used methods in the field.

[0066] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A perfluorohexanesulfonic acid hapten, characterized in that Its structure is shown in formula (I): , formula (Ⅰ).

2. The method for preparing the perfluorohexanesulfonic acid hapten according to claim 1, characterized in that: The following steps are involved: S1. Weigh methyl 4-aminobutyrate hydrochloride, add anhydrous dichloromethane, then add triethylamine, and slowly dropwise add the dichloromethane solution containing perfluorohexylsulfonyl fluoride in an ice-water bath. After the addition is complete, slowly warm the temperature to room temperature and react overnight. After the reaction is complete, add hydrochloric acid and stir thoroughly to acidify the reaction system. Separate the liquids, collect the organic phase, dry it over anhydrous sodium sulfate, and concentrate the organic phase to dryness to obtain the first intermediate shown in formula (II). , formula (II); S2. The first intermediate obtained in step S1 is placed in a single-necked bottle, methanol is added, and after stirring, sodium hydroxide is added. After the reaction is completed at room temperature, the pH is adjusted to about 4 with dilute hydrochloric acid, and the reaction solution is extracted with ethyl acetate. The organic phase is collected, dried with anhydrous sodium sulfate, and purified by column to obtain the perfluorohexanesulfonic acid hapten represented by formula (I).

3. The method according to claim 2, characterized in that In step S2, the mass ratio of the first intermediate to sodium hydroxide is 1:(3-5).

4. Perfluorohexanesulfonic acid antigen, characterized in that The perfluorohexane sulfonic acid antigen is a conjugate of the perfluorohexane sulfonic acid hapten according to claim 1 and a carrier protein, and the carrier protein is bovine serum albumin, human serum albumin, chicken ovalbumin or lactoferrin.

5. Use of the perfluorohexanesulfonic acid hapten according to claim 1 or the perfluorohexanesulfonic acid antigen according to claim 4 in immunological detection of perfluorohexanesulfonic acid for non-disease diagnosis.

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