Dichlorophenac haptens, artificial antigens, and methods of making and using the same

Abstract

The application discloses a diclofenac hapten, an artificial antigen and a preparation method and application thereof, and provides a diclofenac hapten structure as shown in formula I, and then the diclofenac hapten is coupled with a carrier protein to prepare a diclofenac artificial antigen as shown in formula II. The length of the spacer arm introduced in the application is 4 carbon atoms, the molecular skeleton is lengthened, the key characteristic groups are fully exposed, the antibody recognition efficiency is improved, the detection system is simple to operate, low in cost and short in time consumption.

Description

Technical Field

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

[0002] Diclofenac (DCF) is a representative nonsteroidal anti-inflammatory drug (NSAID) of the phenylacetic acid class. It possesses analgesic, anti-inflammatory, and antipyretic effects and is widely used in the treatment of rheumatoid arthritis, osteoarthritis, and postoperative pain in humans, as well as in dairy cows and other lactating animals for anti-inflammatory and antipyretic purposes. The widespread use of DCF has led to abnormally high levels of it in dairy products, functional foods, and environmental water bodies. Long-term or excessive use of DCF can easily cause nausea, gastrointestinal ulcer bleeding, allergic rashes, and in some cases, even anaphylactic shock or acute kidney injury. Therefore, there is an urgent need to establish a sensitive, reliable, and suitable method for rapid on-site screening of diclofenac.

[0003] Current immunoassay protocols for DCF generally suffer from deficiencies in hapten design: CN103267842A and CN109735503A directly utilize the carboxyl group already present on the diclofenac molecule, or even directly use the original diclofenac drug as a hapten to conjugate with bovine serum albumin (BSA) or ovalbumin (OVA) to prepare artificial antigens; CN119224291A and CN118858613A, although called "hapten-carrier protein conjugates," are essentially still diclofenac (sodium) activated by EDC / NHS and then directly conjugated to the carrier protein. In the above protocols, there is a lack of spacer arm structure between the hapten and the carrier protein, and the characteristic epitopes of diclofenac are closely attached to the surface of the carrier protein and partially obscured, limiting the conformational space that the antibody can recognize, thus restricting the affinity and detection sensitivity of the resulting antibody.

[0004] In terms of application scenarios, existing solutions are mainly aimed at screening for illegal additives in health foods and traditional Chinese medicines (such as CN103267842A, CN111562385A, CN108267603A), or trace residue detection in tap water, fruits and vegetables (such as CN118858613A), and animal tissues such as chicken, chicken liver, feed, and pork (such as CN109735503A, CN119224291A). There are few colloidal gold immunochromatographic detection solutions that are suitable for dairy products, which are high in protein and fat and have a complex matrix, and that take into account both sensitivity and matrix tolerance.

[0005] Furthermore, existing immunoassay protocols struggle to balance sensitivity and ease of operation: protocols such as fluorescent biosensors (CN118858613A) and enzyme-linked immunosorbent assay kits (CN119224291A) achieve low limits of detection, but rely on nanomaterial synthesis, enzyme signal amplification, and fluorescence / ELISA reader readings, involving numerous steps and demanding requirements for instruments and personnel, making them unsuitable for rapid on-site screening; while protocols such as colloidal gold and quantum dot immunochromatography (CN103267842A, CN108267603A, etc.), which are relatively simple to operate, generally have limits of detection in the 2.0 ng / mL range, indicating significant room for improvement in sensitivity. 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 diclofenac 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 a diclofenac hapten, wherein the diclofenac 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 diclofenac hapten, comprising the following steps:

[0011] .

[0012] Starting with diclofenac, the carboxyl group was activated by NHS and EDC, and then condensed with methyl 4-aminobutyrate. The methyl ester protecting group was then removed by alkaline hydrolysis to obtain hapten I.

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

[0014] .

[0015] The inventors discovered that by introducing a spacer arm of appropriate length at the carboxyl terminus of diclofenac, they designed and synthesized a novel hapten, improving the spatial conformation of the hapten-carrier protein conjugate. This effectively exposed the characteristic structure of the drug, demonstrating extremely high sensitivity and good accuracy when applied to immunoassay. (ELISA platform IC) 50 The value was 0.1 ng / mL, the detection sensitivity of the colloidal gold platform was 0.07 ng / mL, and the stability was significantly improved.

[0016] A method for preparing diclofenac artificial antigen according to the present invention, the method comprising the following steps:

[0017] (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.

[0018] (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 diclofenac artificial antigen.

[0019] (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 2 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.

[0020] Preferably, the carrier protein is bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HSA), hemocyanin (KLH), or polylysine (PDL).

[0021] This invention also provides an immunoassay method for detecting diclofenac, which uses an artificial diclofenac antigen with bovine serum albumin (BSA) as the carrier protein as the coating antigen and an artificial diclofenac antigen with hemocyanin (KLH) as the carrier protein 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.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a method for synthesizing diclofenac 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 0.07 ng / mL for diclofenac, and is compatible with the ELISA platform IC50. 50 With a concentration of 0.1 ng / mL, it has high detection sensitivity, simple operation, low equipment dependence, low cost, short processing time, wide linear range and strong specificity, and is suitable for rapid on-site detection of diclofenac in complex matrices such as dairy products. Attached Figure Description

[0024] Figure 1 This is a synthetic route diagram of the diclofenac hapten of the present invention.

[0025] Figure 2 This is a schematic diagram of the 1H NMR spectrum detection results of the diclofenac hapten of the present invention.

[0026] Figure 3 This is a schematic diagram of the liquid chromatography-mass spectrometry detection results of the diclofenac hapten of the present invention.

[0027] Figure 4 This is an ultraviolet scan image of the diclofenac hapten, bovine serum albumin, and diclofenac artificial antigen of the present invention.

[0028] Figure 5 This is a schematic diagram of the SDS-PAGE results of the diclofenac artificial antigen of the present invention.

[0029] Figure 6 This is a schematic diagram of the colloidal gold immunochromatographic results of the present invention.

[0030] Figure 7 This is a schematic diagram of the indirect competitive ELISA standard curve of the present invention. Detailed Implementation

[0031] 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.

[0032] Example 1

[0033] A method for preparing diclofenac hapten, comprising the following steps:

[0034] .

[0035] (1) Weigh 100 mg (0.34 mmol, 1.0 eq) of diclofenac, 130 mg (0.68 mmol, 2.0 eq) of EDC hydrochloride, and 78 mg (0.68 mmol, 2.0 eq) of NHS, place them in a 10 mL Erlenmeyer flask, add 2 mL of DMF, and activate by stirring at room temperature. Separately, dissolve 52 mg of methyl 4-aminobutyrate hydrochloride in 0.5 mL of DMF, add 50 μL of triethylamine (0.34 mmol, 1.0 eq) to neutralize, filter to remove the white precipitate, and slowly add the filtrate dropwise to the above activation solution and continue stirring overnight. After the reaction is complete, wash with ethyl acetate and saturated brine to remove DMF, concentrate by rotary evaporation, and obtain diclofenac-4-aminobutyrate intermediate.

[0036] (2) The above intermediate was added to 0.5 mL of 1M sodium hydroxide solution and 1 mL of anhydrous ethanol, and reacted at 65°C for 4 h. After the reaction was complete, dilute hydrochloric acid was added and the mixture was extracted with ethyl acetate and concentrated by rotation to obtain hapten I.

[0037] The diclofenac hapten prepared above was identified by NMR and liquid chromatography-mass spectrometry. The results are shown in the figure. Figure 2 and Figure 3 .

[0038] The NMR results are as follows: 1 H NMR (400MHz, DMSO-d6) δ 12.05 (s, 1H), 8.39 (s, 2H), 7.50 (d, J=4.0Hz, 2H), 7.30-7.19 (m, 2H), 7.05-7.00 (m, 1H), 6.95-6.8 3(m, 1H), 6.31-6.25(m, 1H), 3.56(s, 2H), 3.20-3.05(m, 2H), 2.33-2.20(m, 2H), 1.70-1.57(m, 2H) ppm.

[0039] The mass spectrometry result was: [MH]+ 379.15.

[0040] Example 2

[0041] A method for preparing diclofenac artificial antigen (coating antigen) includes the following steps:

[0042] 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.

[0043] 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.

[0044] 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 the artificial antigens, and store at -20°C for later use. The characterization results of diclofenac antigen detection are shown below. Figure 4 and Figure 5 .

[0045] Example 3

[0046] Establishment of a standard curve for indirect competitive ELISA based on DCF monoclonal antibodies

[0047] Using the diclofenac 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 optimal values ​​for highest inhibition rate, lowest background value, and IC50 were considered. 50The optimal coating concentration was determined to be 250 ng / mL, corresponding to a working antibody concentration of 1 μg / mL. The indirect competitive ELISA standard curve established based on monoclonal antibodies is shown below. Figure 7 As shown. With diclofenac concentration on the x-axis and inhibition rate on the y-axis, the half-maximal inhibitory concentration (IC50) was obtained by fitting a four-parameter Logistic model: y = (A - D) / [1 + (x / C)^B] + D (r^2 = 0.9969). 50 The concentration is 0.1 ng / mL, indicating high sensitivity for surface detection.

[0048] Example 4

[0049] Preparation of the diclofenac colloidal gold chromatography detection kit:

[0050] 4.1 Preparation of diclofenac monoclonal antibody-colloidal gold label

[0051] (1) Preparation of colloidal gold solution

[0052] Dilute a 1% chloroauric acid solution to 0.01% (mass fraction) with double-distilled deionized water. Measure 100 mL of this diluted solution into an Erlenmeyer flask and heat to boiling using a thermostatic magnetic stirrer. While maintaining boiling and continuous stirring, quickly 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. Allow it to cool naturally to room temperature and then reconstitute the original volume with deionized water to obtain a colloidal gold solution. Store at 2–8°C. The resulting colloidal gold solution should be clear and transparent, free of precipitates and floating matter, and appear wine-red under sunlight.

[0053] (2) Preparation of diclofenac monoclonal antibody-colloidal gold label

[0054] Place the colloidal gold solution on a magnetic stirrer and adjust its pH to 8.0 with 0.2 mol / L potassium carbonate solution. Add 20-50 μg of diclofenac monoclonal antibody per mL of colloidal gold solution while stirring. After the addition is complete, continue stirring and mixing for 30 min, then let it stand at room temperature for 10 min. Then add 10% bovine serum albumin (BSA) solution to achieve a final volume fraction of 1%, and let it stand for another 10 min for blocking. Centrifuge the above system at 12000 rpm for 10 min at 2-8℃, discard the supernatant, and resuspend the precipitate with 1 / 10 of the initial colloidal gold solution volume in reconstitution buffer to obtain the diclofenac monoclonal antibody-colloidal gold label, which can be stored at 2-8℃ for later use.

[0055] 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.

[0056] (3) Preparation of reaction membrane

[0057] Diclofenac-coated antigen (DCF-BSA) and goat anti-mouse antibody were coated onto the reaction membrane to form a detection line (T line) and a control line (C line), respectively.

[0058] Coating process: Diclofenac coating agent (DCF-BSA) was diluted to 0.3 mg / mL with 0.01 mol / L, pH 7.4 phosphate buffer and streaked onto the detection zone (T line) of the nitrocellulose membrane using a streaking apparatus at a volume of 1.0 μL / cm. Goat anti-mouse antibody was diluted to 500 μg / mL with 0.01 mol / L, pH 7.4 phosphate buffer and streaked onto the control zone (C line) of the same membrane at a volume of 1.0 μL / cm. The coated membranes were then dried at 50 °C for 16 h for later use.

[0059] (4) Preparation of sample absorption pad

[0060] Sample pad preparation: First, soak the sample pad in 0.1 mol / L phosphate buffer solution containing 1% (volume fraction) bovine serum albumin at pH 7.2 for 2 h, and then dry it at 37℃ for 2 h for later use.

[0061] (5) Preparation of colloidal gold micropores

[0062] Add 6 μL of diclofenac monoclonal antibody-colloidal gold label to the microwells.

[0063] 4.2 Assembly of the Diclofenac Colloidal Gold Chromatography Detection Kit

[0064] (1) Assembly of test strips

[0065] Attach the sample pad, reaction membrane, and absorbent pad to the base plate in the following order: the base plate is a PVC base plate, the sample pad is filter paper, the reaction membrane is a nitrocellulose membrane, and the absorbent pad is absorbent filter paper. During assembly, overlap the end of the sample pad with the beginning of the reaction membrane, and the end of the reaction membrane with the beginning of the absorbent pad. Align the beginning of the sample pad with the beginning of the base plate, and align the end of the absorbent pad with the end of the base plate.

[0066] (2) Assembly of the diclofenac colloidal gold chromatography detection kit

[0067] The test strips obtained in step (1) are further assembled into a test kit and stored at room temperature for later use.

[0068] Example 5: Detection method for diclofenac residues

[0069] 5.1 Preparation of the test solution

[0070] In a preferred embodiment of the present invention, the pretreatment of dairy samples is carried out according to the following steps:

[0071] (1) Take 2-5 mL of dairy product sample and centrifuge at 8000-10000 rpm for 5-10 minutes;

[0072] (2) Aspirate the middle layer of skim whey and transfer it to a new centrifuge tube;

[0073] (3) Add 2 to 5 times the volume of PBS buffer (0.01 M, pH 7.4) to dilute, mix well, and obtain the target test solution;

[0074] 5.2 Measurement Procedure

[0075] Pipette 100 μL of the test solution into the colloidal gold micropore, dissolve it, and then drop it into the sample port. After 5 min, interpret the result.

[0076] 5.3 Result Judgment

[0077] The results are determined by comparing the color intensity of the control line (C line) and the detection line (T line):

[0078] Positive: Using the colloidal gold competitive method, if the control area (C line) shows normal color development, but the detection area (T line) does not show color development, or the color intensity of the T line is significantly weaker than that of the negative control (i.e., the color becomes lighter), it is judged as positive, indicating that the sample contains diclofenac. The result is indicated by "+".

[0079] Negative: When both the control area and the test area show bands, it is judged as negative, that is, there is no diclofenac in the sample, indicated by "-";

[0080] Invalid: The test strip is invalid if no band is displayed in the control area (C line).

[0081] Example 6: Sample Testing Case

[0082] 6.1 Limit of Detection Test of the Method

[0083] Negative milk samples were selected, and the diclofenac colloidal gold chromatography detection kit prepared in Example 4 was used for testing according to the method described in Example 5. The diclofenac concentrations in the samples were tested, including blank, 0.035 ng / mL diclofenac, 0.07 ng / mL diclofenac, 0.14 ng / mL diclofenac, and 0.28 ng / mL diclofenac. The results are shown in Table 1 and [Table data missing]. Figure 6 .

[0084] Table 1 Sample Measurement Results

[0085] -: Negative; +: Positive

[0086] The results in Table 1 show that the diclofenac concentration in the milk matrix was 0.035 ng / mL, and the results were negative; the concentrations were 0.07 ng / mL and above (0.07, 0.14, and 0.28 ng / mL), and the results were positive. Therefore, the detection limit for diclofenac residues in milk prepared in this invention using the colloidal gold chromatography kit and method is 0.07 ng / mL.

[0087] 6.2 Stability Test of the Method

[0088] Using milk samples as a matrix, the diclofenac colloidal gold chromatography detection kit prepared in Example 4 was used. After the kit was placed at 37°C for 0, 3, 7, 10, and 14 days, the detection was performed according to the method described in Example 5. The interpretation results of the kit for five diclofenac concentration gradients of 0, 0.035, 0.07, 0.14, and 0.28 ng / mL were examined. The results are shown in Table 2.

[0089] Table 2 Sample Measurement Results

[0090] -: Negative; +: Positive

[0091] The results in Table 2 show that the diclofenac colloidal gold chromatography detection kit prepared in this invention remains accurate and reliable after being placed at 37°C for 14 days, indicating that the kit has good stability.

[0092] Example 7: Experiment on the effect of spacer arm length on detection sensitivity

[0093] To fully illustrate the impact of spacer arm length on detection sensitivity, we further designed hapten structures with different spacer arm lengths and conducted systematic comparative experiments. Diclofenac haptens with 2 and 6 carbon atoms in their spacer arms were synthesized and conjugated with bovine serum albumin (BSA) to prepare artificial antigens. The inhibitory effect of the antibodies against diclofenac at different spacer arm lengths was determined using an indirect competitive ELISA method. The experimental results are shown in Table 3.

[0094]

[0095] Table 3 IC under different spacer arm lengths 50 Value and detection limit

[0096] The above results indicate that the length of the spacer arm has a significant impact on the spatial conformation of the hapten and the antibody recognition efficiency; both excessively short and excessively long spacers are detrimental to efficient antibody recognition. This application selects a spacer arm length of 4 carbon atoms (as shown in Formula II) as the optimal solution. At this length, the characteristic groups of the hapten are fully exposed, resulting in the highest antibody recognition efficiency and optimal detection sensitivity.

[0097] 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. A diclofenac hapten, characterized in that, The preparation method includes the following steps: Diclofenac, EDC, and NHS were placed in an Erlenmeyer flask, and DMF was added for activation and stirring. After the reaction was complete, methyl 4-aminobutyrate hydrochloride was weighed and dissolved in DMF. Triethylamine was added for neutralization, and a white precipitate appeared. The precipitate was removed by filtration, and the solution was added dropwise to the activation solution and the reaction was continued overnight with stirring. Finally, ethyl acetate and saturated saline were added to wash away DMF. After rotary evaporation and concentration, sodium hydroxide solution and anhydrous ethanol were added, and the reaction was carried out to obtain hapten I. This hapten has the structural formula shown in Formula I: 。 2. The diclofenac hapten according to claim 1, characterized in that, The preparation method includes stirring the reaction overnight, then adding ethyl acetate and saturated saline to wash away DMF, followed by rotary evaporation and concentration, then adding 1M sodium hydroxide solution and anhydrous ethanol, and reacting at 65°C for 4 h to obtain hapten I.

3. A diclofenac artificial antigen, which is a conjugate obtained by conjugating the diclofenac hapten of claim 1 with a carrier protein. 。 4. The diclofenac 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 diclofenac 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 diclofenac artificial antigen; (3) The reaction product is placed into a dialysis bag rinsed with distilled water, and dialyzed at pH neutral and 2-8℃ with stirring. The dialysis product is centrifuged, dispensed, labeled with antigens, and frozen for later use.

6. The method for preparing a diclofenac 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 2 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. An application of a diclofenac artificial antigen, comprising using the diclofenac artificial antigen of claim 3 or 4 in any of the following applications: (a) Used for the detection of diclofenac and its homologues; (b) Immunochromatographic reagent strips used to prepare diclofenac; (c) Used to prepare diclofenac test strips or kits.

8. A diclofenac 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 a colloidal gold-labeled diclofenac clonal antibody prepared using a diclofenac 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 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 diclofenac artificial antigen as described in claim 3 or 4, and the control band is coated with goat anti-mouse IgG.

9. A diclofenac 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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