Ethyl glucuronide hapten, artificial antigen, and preparation method and application thereof

Abstract

The application discloses an ethyl glucuronide hapten, an artificial antigen and a preparation method and application thereof. The application provides an ethyl glucuronide hapten structure shown as formula I. The hapten retains an active characteristic group of ethyl glucuronide and selectively protects a sugar ring part, greatly improving the stability of the compound. High regioselective deprotection is realized through a specific alkali reagent and a solvent system, side reactions under traditional strong acid or strong alkali conditions are avoided, and high-purity target objects are directly obtained through one-step purification without complicated intermediate separation. After the ethyl glucuronide hapten is coupled with a carrier protein, an ethyl glucuronide artificial antigen shown as formula II is prepared. The technical scheme has the characteristics of high stability, long storage time, simplicity and rapidness, high sensitivity, strong specificity and wide application range, and has good application prospect and broad development space in rapid and effective detection of alcohol metabolites.

Description

Technical Field

[0001] This invention belongs to the field of alcohol metabolite detection technology, specifically relating to an ethyl glucuronide hapten, an artificial antigen, its preparation method, and its application. Background Technology

[0002] Ethyl glucuronide (EtG), also known as ethyl β-D-glucuronic acid, is a product formed after ethanol is metabolized by enzymes in the body. EtG can be detected through urine, blood, or hair follicles and is commonly used to detect an individual's ethanol intake, especially in situations such as monitoring drunk driving and enforcing alcohol prohibition laws. EtG has high sensitivity for detecting ethanol and can detect it within hours after ethanol ingestion. It can generally detect ethanol intake within the past 72 hours and can also detect alcohol addiction over the past few days or weeks.

[0003] According to the current industry standard GA / T 1633-2019, the main methods for EtG detection include gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). While these methods possess good specificity and sensitivity, they generally suffer from complex operating procedures and high instrument costs, making them unsuitable for high-throughput screening of large-scale samples. In contrast, immunochemical analysis techniques, based on the specific reaction between antigens and antibodies, exhibit significant advantages in both qualitative and quantitative analysis. This method is simple to operate, fast in analysis, low in cost, and has good sensitivity, while also being capable of processing large numbers of samples, effectively overcoming some limitations of traditional physicochemical analysis methods.

[0004] Patent application CN117645639A discloses a method for preparing an ethyl glucuronide hapten, but it does not explore related application platforms. Furthermore, the reagent kit developed in this study suffers from issues with stability, accuracy, and sensitivity, failing to fully meet detection requirements. Therefore, current research on EtG immunoassay is incomplete and still has significant room for improvement.

[0005] The inventors conducted in-depth research to solve the above-mentioned problems and discovered that the EtG hapten derivative and its artificial antigen represented by Formula I of this invention have excellent sensitivity as immunoassays, and the ELISA platform IC 50 The value was 8.2 ng / mL, the sample detection sensitivity of the colloidal gold platform was 200 ng / mL, and the stability was significantly improved. An application platform for colloidal gold detection was also developed, thus completing this invention. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects and shortcomings of existing technologies in terms of stability, accuracy, and sensitivity, and to provide a method for preparing ethyl glucuronide hapten and artificial antigen and their applications.

[0007] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution:

[0008] The first objective of this invention is to provide an ethyl glucuronide hapten, wherein the ethyl glucuronide hapten has the structural formula shown in Formula I:

[0009] .

[0010] A second objective of this invention is to provide a method for preparing the ethyl glucuronide hapten, comprising the following steps:

[0011] .

[0012] Compound 1 was dissolved in 5 times its volume (v / w) of dichloroethane, and 2.0 equivalents of trimethyltin hydroxide were added. The reaction mixture was stirred at 85°C for 16 hours. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure. The residue was extracted with ethyl acetate and saturated brine at a volume ratio of 1:1. The extraction was repeated 3 times. The organic phases were combined and dried overnight with anhydrous sodium sulfate. The dried organic phase was filtered and concentrated. The crude product was purified by preparative high performance liquid chromatography under the following chromatographic conditions: C18 reversed-phase column (10×250 mm, 5 μm), mobile phase of acetonitrile / water (containing 0.1% formic acid) gradient elution (acetonitrile volume ratio linearly increased from 10% to 40%, elution time 30 min), flow rate 5 mL / min. The target peak fraction was collected and lyophilized to obtain hapten I with a purity ≥98%.

[0013] A third objective of this invention is to provide an ethyl glucuronide artificial antigen as shown in Formula II, prepared from the ethyl glucuronide hapten and a carrier protein.

[0014] .

[0015] A method for preparing the ethyl glucuronide artificial antigen according to the present invention, the method comprising the following steps:

[0016] (1) Dissolve the hapten, NHS and EDC shown in Formula I in DMF solution and activate at room temperature for 2-5 hours by stirring.

[0017] (2) Dissolve the carrier protein in an alkaline buffer solution, add the reaction solution from step (1) dropwise to the carrier protein solution, stir and react overnight to obtain the ethyl glucuronide artificial antigen.

[0018] (3) The reaction product was placed in a dialysis bag rinsed with distilled water, 1.5L of 0.01M PBS (pH=7.4) was added, and the mixture was stirred at 400rpm for 3 days at 2~8℃. The dialysis product was centrifuged at 4000rpm for 5min, dispensed into 1.5mL / tube, labeled with antigen, and stored at -20℃ for later use.

[0019] Preferably, the carrier protein is bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HSA), hemocyanin (KLH), or polylysine (PDL).

[0020] This invention also provides an immunoassay method for detecting ethyl glucuronide, which uses an ethyl glucuronide artificial antigen with bovine serum albumin (BSA) as the coating antigen and an ethyl glucuronide artificial antigen with hemocyanin (KLH) as the immunogen to immunize animals to prepare antibodies for detection. The immunoassay method includes, but is not limited to, enzyme immunoassay, immunochromatography, immunosensing, and immunogold immunoassay.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a method for synthesizing ethyl glucuronide hapten and artificial antigen, and their applications. A colloidal gold immunochromatographic test strip developed based on the above hapten and artificial antigen has a limit of detection (LOD) of 200 ng / mL for ethyl glucuronide, and is compatible with the ELISA platform IC50. 50 The concentration is 8.2 ng / mL, with high detection sensitivity, wide linear range and strong specificity. More importantly, the stability has been significantly improved. Attached Figure Description

[0023] Figure 1 This is a synthetic route diagram for the ethyl glucuronide hapten.

[0024] Figure 2 This is a schematic diagram of the 1H NMR spectrum detection results for the ethyl glucuronide hapten.

[0025] Figure 3 This is a schematic diagram of the liquid chromatography-mass spectrometry detection results for the ethyl glucuronide hapten.

[0026] Figure 4 Ultraviolet scans of ethyl glucuronide hapten, bovine serum albumin, and ethyl glucuronide artificial antigen.

[0027] Figure 5 This is a schematic diagram of the SDS-PAGE results for the ethyl glucuronide artificial antigen.

[0028] Figure 6This is a schematic diagram of the results of colloidal gold immunochromatography.

[0029] Figure 7 This is a schematic diagram of the standard curve for indirect competition ELISA. Detailed Implementation

[0030] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent stores.

[0031] Example 1

[0032] A method for preparing an ethyl glucuronide hapten, comprising the following steps:

[0033]

[0034] Compound 1 was dissolved in 5 times its volume (v / w) of dichloroethane, and 2.0 equivalents of trimethyltin hydroxide were added. The reaction mixture was stirred at 85°C for 16 hours. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure. The residue was extracted with ethyl acetate and saturated brine at a volume ratio of 1:1. The extraction was repeated 3 times. The organic phases were combined and dried overnight with anhydrous sodium sulfate. The dried organic phase was filtered and concentrated. The crude product was purified by preparative high performance liquid chromatography under the following chromatographic conditions: C18 reversed-phase column (10×250 mm, 5 μm), mobile phase of acetonitrile / water (containing 0.1% formic acid) gradient elution (acetonitrile volume ratio linearly increased from 10% to 40%, elution time 30 min), flow rate 5 mL / min. The target peak fraction was collected and lyophilized to obtain hapten I with a purity ≥98%.

[0035] The ethyl glucuronide hapten prepared above was identified by NMR and liquid chromatography-mass spectrometry. The results are shown in the figure. Figure 2 and Figure 3 .

[0036] The NMR results are as follows: 1 H NMR (400MHz, CDCl3) δ 5.30-5.27(m, 2H), 5.04-5.00(m, 1H), 4.63(d, J=8.0Hz, 1H), 4.13-4.10(m, 1H) , 3.99-3.93(m, 1H), 3.71-3.59(m, 2H), 2.08-2.04(m, 9H), 1.23(t, J=6.8Hz, 3H) ppm.

[0037] The mass spectrometry result is: [M+Na]+ 371.17.

[0038] Example 2

[0039] A method for preparing an ethyl glucuronide artificial antigen (coating antigen) includes the following steps:

[0040] 1. Dissolve 20 mg of compound I in 0.3 mL of DMF, add 10 mg of EDC to dissolve, then add 6 mg of NHS, and stir at room temperature for 2 h to activate.

[0041] 2. Weigh 20 mg of BSA and dissolve it in 3.0 mL of 0.01 M PBS buffer solution until it is fully dissolved. Add the BSA solution dropwise to the reaction solution from step 1 under ice bath conditions of 2~8℃ and stir the reaction at room temperature for 24 h.

[0042] 3. After the reaction, centrifuge the liquid at 4000 rpm for 5 min, and dialyze with 0.01M PBS at 2-8°C for 2 days (changing the solution twice a day, morning and evening). Centrifuge the dialysate at 4000 rpm for 5 min, aliquot into 1.5 mL tubes, label each tube with the complete antigen, and store at -20°C for later use. The characterization results of the antigen detected by ethyl glucuronide are shown below. Figure 4 and Figure 5 .

[0043] Example 3

[0044] Establishment of a standard curve for indirect competitive ELISA based on EtG monoclonal antibody

[0045] Using the ethyl glucuronide artificial antigen prepared in Example 2 as the coating antigen, the appropriate coating antigen concentration and antibody dilution factor were determined by checkerboard titration. The final coating antigen concentration was determined to be 250 ng / mL, and the antibody concentration to be 1 μg / mL. The indirect competitive ELISA standard curve established based on monoclonal antibodies is shown below. Figure 7 As shown. With the concentration of ethyl glucuronide as the x-axis and the inhibition rate as the y-axis, the IC is obtained by fitting the four-parameter Logistic curve y = (A - D) / [1 + (x / C)^B] + D (r^2 = 0.99957). 50 The concentration was 8.2 ng / mL, indicating high detection sensitivity.

[0046] Example 4

[0047] Preparation of the ethyl glucuronide colloidal gold chromatography detection kit:

[0048] 4.1 Preparation of ethyl glucuronide monoclonal antibody-colloidal gold label

[0049] (1) Preparation of colloidal gold solution

[0050] Dilute a 1% chloroauric acid solution to 0.01% (mass fraction) with double-distilled deionized water. Place 100 mL of the 0.01% chloroauric acid solution in an Erlenmeyer flask and heat to boiling using a constant-temperature electromagnetic stirrer. While maintaining high temperature and continuous stirring, add 1.5 mL of 1% trisodium citrate solution. Continue stirring and heating at a constant speed until the solution turns a clear red color. After cooling to room temperature, restore the original volume with deionized water to obtain a colloidal gold solution, which should be stored at 2–8℃. The prepared colloidal gold solution is pure, clear, and free of precipitates and floating matter; its color is wine-red when observed under sunlight.

[0051] (2) Preparation of ethyl glucuronide monoclonal antibody-colloidal gold label

[0052] Under magnetic stirring, the pH of the colloidal gold solution was adjusted to 8.0 with 0.2 mol / L potassium carbonate solution. Following the standard of adding 20–50 μg of ethyl glucuronide monoclonal antibody per mL of colloidal gold solution, the solution was stirred and mixed for 30 min. After standing for 10 min, 10% bovine serum albumin (BSA) solution was added to achieve a volume percentage of 1% in the colloidal gold solution, and the mixture was allowed to stand for another 10 min. The solution was then centrifuged at 12,000 rpm for 10 min at 2–8 °C. The supernatant was discarded, and the precipitate was resuspended in rehydration buffer at 1 / 10 the volume of the initial colloidal gold solution to prepare the ethyl glucuronide monoclonal antibody-colloidal gold label. This label was stored at 2–8 °C for later use.

[0053] Reconstitution buffer: 0.02 mol / L phosphate buffer with a pH of 7.2, containing 0.1%~0.5% bovine serum albumin by volume, 0.05%~0.2% Tween-20 by mass, 2%~4% trehalose.

[0054] (3) Preparation of reaction membrane

[0055] Ethyl glucuronide-coated precursor (EtG-BSA) was coated onto the reaction membrane to form the detection line (T line), and goat anti-mouse antibody was coated onto the reaction membrane to form the control line (C line).

[0056] Coating process: The ethyl glucuronide coating agent (EtG-BSA) was diluted to a concentration of 0.3 mg / mL with 0.01 mol / L phosphate buffer (pH=7.4), and then coated onto the detection zone (T line) of a nitrocellulose membrane using a coating instrument, with a coating volume of 1.0 μL / cm. The goat anti-mouse antibody was diluted to a concentration of 500 μg / mL with 0.01 mol / L phosphate buffer (pH=7.4), and then coated onto the control zone (C line) of a nitrocellulose membrane using a coating instrument, with a coating volume of 1.0 μL / cm. The coated reaction membranes were dried at 50℃ for 16 h for production use.

[0057] (4) Preparation of sample absorption pad

[0058] The sample absorption pad was soaked in 1% bovine serum albumin (volume fraction) and 0.1 mol / L phosphate buffer (pH=7.2) for 2 h, and then dried at 37℃ for 2 h for later use. Ethyl glucuronide monoclonal antibody-colloidal gold labeling was sprayed onto the sample absorption pad at a concentration of 6 μL / cm².

[0059] 4.2 Assembly of the Ethyl Glucuronide Colloidal Gold Chromatography Detection Kit

[0060] (1) Assembly of test strips

[0061] The sample absorption pad, reaction membrane, and absorbent pad are sequentially attached to the base plate. The base plate is a PVC base plate, the sample absorption pad is absorbent filter paper, the absorbent pad is absorbent filter paper, and the reaction membrane is a nitrocellulose membrane. The end of the sample absorption pad is connected to the beginning of the reaction membrane, and the end of the reaction membrane is connected to the beginning of the absorbent pad. The beginning of the sample absorption pad is aligned with the beginning of the base plate, and the end of the absorbent pad is aligned with the end of the base plate.

[0062] (2) Assembly of the ethyl glucuronide colloidal gold chromatography detection kit

[0063] Assemble the test strips obtained in step (1) into a kit and store it at room temperature.

[0064] Example 5: Detection method for ethyl glucuronide residues

[0065] 5.1 Measurement Procedure

[0066] Pipette 100 μL of the test solution into the sample inlet, and interpret the results after 5 minutes.

[0067] 5.2 Result Judgment

[0068] The results are determined by comparing the color intensity of the control line (C line) and the detection line (T line):

[0069] Positive: When the control area (C line) shows a band and the detection area (T line) does not show color, it is judged as positive, that is, the sample contains ethyl glucuronide, which is indicated by "+";

[0070] Negative: When both the control area and the test area show bands, it is judged as negative, that is, there is no ethyl glucuronide in the sample, indicated by "-";

[0071] Invalid: The test strip is invalid if no band is displayed in the control area (C line).

[0072] Example 6: Sample Testing Case

[0073] 6.1 Limit of Detection Test of the Method

[0074] Negative urine samples were selected, and the ethyl glucuronide colloidal gold chromatography detection kit prepared in Example 4 was used for testing according to the method described in Example 5. The concentrations of ethyl glucuronide in the samples were tested at blank, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL. The results for each matrix sample are shown in Table 1.

[0075] Table 1 Sample Measurement Results

[0076] -: Negative; +: Positive

[0077] The results in Table 1 show that the detection results were negative when 50 ng / mL and 100 ng / mL of ethyl glucuronide were added to the urine matrix, and positive when 200 ng / mL and 500 ng / mL of ethyl glucuronide were added. Therefore, the detection limit for ethyl glucuronide residues in urine prepared in this invention is 200 ng / mL.

[0078] 6.2 Stability Test of the Method

[0079] Urine samples were selected, and the ethyl glucuronide colloidal gold chromatography detection kit prepared in Example 4 was used. The kit was stored at 37°C for 0, 3, 7, 10, and 14 days. The tests were conducted according to the method described in Example 5 to test for 0 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL ethyl glucuronide in the samples. The sample determination results are shown in Table 2.

[0080] Table 2 Sample Measurement Results

[0081] -: Negative; +: Positive

[0082] The results in Table 2 show that the ethyl glucuronide colloidal gold chromatography detection kit prepared in this invention maintains accurate detection results after being stored at 37°C for 14 days.

[0083] The embodiments of the present invention have been described in detail above. For those skilled in the art, there may be changes in the specific implementation methods and application scope based on the ideas of the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. An ethyl glucuronide hapten, characterized in that, The preparation method includes the following steps: Compound 1 was dissolved in dichloroethane, and trimethyltin hydroxide was added with stirring. The mixture was heated to 80–90 °C and reacted for 12–16 hours. TLC monitoring was performed until the starting material was completely converted. After the reaction, the reaction solution was concentrated to dryness under reduced pressure. The residue was extracted with ethyl acetate and saturated brine. The organic phases were combined and dried overnight with anhydrous sodium sulfate. The dried organic phase was filtered and concentrated. The crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following chromatographic conditions: C18 reversed-phase column, acetonitrile / water gradient elution. The target peak fraction was collected and lyophilized to obtain hapten I with a purity ≥98%. Compound 1 is: ; The hapten has the structural formula shown in Formula I: 。 2. The ethyl glucuronide hapten according to claim 1, characterized in that, The preparation method includes the following steps: Compound 1 was dissolved in 4-6 times its volume (v / w) of dichloroethane, and 1-3 equivalents of trimethyltin hydroxide were added. The reaction mixture was stirred at 80-90°C for 15-17 hours. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure. The residue was extracted with ethyl acetate and saturated brine at a volume ratio of 1:

1. The extraction was repeated 3 times. The organic phases were combined and dried overnight with anhydrous sodium sulfate. The dried organic phase was filtered and concentrated. The crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: C18 reversed-phase column, mobile phase of acetonitrile / water, wherein the aqueous solution contained 0.05-0.15% formic acid; gradient elution, after elution, the acetonitrile volume ratio increased linearly from 8-12% to 35-45%, the elution time was 28-32 min, the flow rate was 4-6 mL / min, the target peak fraction was collected, and lyophilized to obtain hapten I with a purity ≥98%.

3. An ethyl glucuronide artificial antigen, characterized in that, It is a conjugate obtained by conjugating an ethyl glucuronide hapten as described in claim 1 or 2 with a carrier protein. 。 4. The ethyl glucuronide artificial antigen according to claim 3, characterized in that, The carrier protein is one or more of bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HSA), hemocyanin (KLH), or polylysine (PDL).

5. A method for preparing the ethyl glucuronide artificial antigen according to claim 3 or 4, characterized in that, The method includes the following steps: (1) Dissolve the hapten, NHS and EDC shown in Formula I in DMF solution and activate at room temperature for 2-5 hours by stirring. (2) Dissolve the carrier protein in an alkaline buffer solution, add the reaction solution from step (1) dropwise to the carrier protein solution, stir and react overnight to obtain the ethyl glucuronide artificial antigen. (3) The reaction product is placed into a dialysis bag rinsed with distilled water, and dialyzed at pH neutral, 2~8℃ with stirring. The dialysis product is centrifuged, dispensed, labeled with antigens, and frozen for later use.

6. The method for preparing the ethyl glucuronide artificial antigen according to claim 5, characterized in that, Step (3) includes filling the reaction product into a dialysis bag rinsed with distilled water, dialyzing with 1.5L 0.01M PBS pH=7.4 at 2~8℃ and stirring at 400rpm for 3 days, centrifuging the dialysis product at 4000rpm for 5min, aliquoting into 1.5mL / tube, labeling the antigen, and storing at -20℃ for later use.

7. The application of an ethyl glucuronide artificial antigen, characterized in that, This includes using the ethyl glucuronide artificial antigen of claim 4 or 5 in any of the following applications: (a) Used for the detection of ethyl glucuronide and its homologues; (b) Immunochromatographic reagent strips for the preparation of ethyl glucuronide; (c) Used to prepare test strips or kits for the detection of ethyl glucuronide.

8. An ethyl glucuronide immunochromatographic reagent strip, the reagent strip comprising a substrate, a sample application component, a detection component, and a liquid aspiration component, characterized in that, The sample application component adsorbs colloidal gold-labeled ethyl glucuronide clonal antibody prepared using ethyl glucuronide artificial antigen. The detection component is fixed on a substrate. A control band and a detection band are provided in the middle of the detection component. The liquid aspiration component and the sample application component are fixed at both ends of the detection component in a partially overlapping manner. The detection band is coated with the ethyl glucuronide artificial antigen as described in claim 6, and the control band is coated with goat anti-mouse IgG.

9. The ethyl glucuronide immunochromatographic reagent strip according to claim 8, characterized in that, The substrate is a PVC liner, and the detection component is a nitrocellulose membrane.

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