A hapten for naphthenic acid marker, artificial antigen and preparation method and application thereof

Abstract

This invention discloses a hapten, an artificial antigen, and their preparation method and application for cycloalkanoic acid markers, relating to the field of artificial antigen technology. A hapten for a cycloalkanoic acid marker is a compound represented by Formula I in Figure 1; an artificial antigen for a cycloalkanoic acid marker is a complete antigen prepared by conjugating the hapten of the compound represented by Formula I with a carrier protein. The core of this invention lies in introducing a specific molecular arm onto the cyclopentylpropionic acid molecule to construct a structurally optimized hapten, which is then conjugated with a carrier protein to form an artificial antigen. It is particularly important to note that the prepared artificial antigen is essentially a polypeptide, capable of effectively stimulating the body's immune response and producing antibodies with high affinity and high specificity.

Description

Technical Field

[0001] This invention relates to the field of artificial antigen technology, specifically to a hapten for cycloalkanoic acid markers, an artificial antigen, its preparation method, and its application. Background Technology

[0002] Naphthenic acids (NAAs) are persistent organic pollutants naturally present in crude oil. During crude oil extraction, transportation, and processing, NAs are inevitably released into water and soil environments. Studies have shown that NAs are one of the main toxic components of petroleum pollutants, exhibiting strong ecotoxicity. Monitoring the concentration levels of NAs in aquatic environments can, to some extent, characterize the degree of pollution from oil spills and provide a basis for real-time tracking of the dynamic changes in oil pollution.

[0003] Currently, the detection of cycloalkanoic acid pollutants mainly follows the national standard GB / T 5750.8, selecting several markers such as cyclopentylpropionic acid and employing instrumental analytical methods for determination. Commonly used techniques include gas chromatography-mass spectrometry (GC-MS) and high-resolution liquid chromatography-mass spectrometry (HPLC-MS). While these instrumental methods offer high sensitivity and accuracy, they require expensive equipment, have long detection cycles, are complex to operate, and necessitate specialized technical personnel. They cannot achieve rapid on-site detection and thus fail to meet the needs of routine environmental monitoring and emergency response.

[0004] Immunoassay technology, based on the specific binding of antigens and antibodies, converts the binding signal into measurable light, color, or radioactive signals using labeling agents such as enzymes, chemiluminescent agents, fluorescent dyes, or colloidal gold, thereby achieving qualitative or quantitative detection of the analyte. This represents a potentially effective approach to solving the aforementioned problems. However, cyclopentylpropionic acid, as a small molecule, is not immunogenic in itself and cannot directly immunize animals to produce antibodies. It must be coupled with a carrier protein to form an artificial antigen to induce an immune response. This artificial antigen is essentially a polypeptide that can effectively stimulate the body's immune response.

[0005] Previous studies have identified two key technical challenges in directly conjugating cyclopentylpropionic acid (CPA) to carrier proteins. First, the complex formed after CPA-carrier protein conjugation has low solubility in aqueous solution, resulting in poor antigen presentation efficiency during immunization and extremely low antibody titers, failing to meet practical detection requirements. Second, the small molecular structure of CPA means that its effective antigen recognition sites are often obscured by the spatial structure of the carrier protein after direct conjugation, making them difficult to expose fully. This results in suboptimal specificity and affinity of antibodies produced by immunized animals for CPA.

[0006] Therefore, there is an urgent need to develop a preparation scheme for haptens and artificial antigens that can improve the coupling efficiency of cyclopentylpropionic acid with carrier proteins, increase the solubility of the conjugates, and effectively expose the antigen recognition sites, so as to provide core reagents for establishing a rapid immunoassay method for cycloalkanoic acid pollutants.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] To address the aforementioned technical problems, embodiments of the present invention provide a hapten, an artificial antigen, and a method for preparing the hapten and its application for cycloalkanoic acid markers, thereby resolving the issues raised in the background section.

[0009] A hapten of a cycloalkanoic acid marker, said hapten as Figure 1 The compound shown in Chinese formula I.

[0010] An artificial antigen of a cycloalkanoic acid marker, wherein the artificial antigen is a complete antigen prepared by conjugating the hapten described above with a carrier protein.

[0011] Preferably, the carrier protein is one of bovine serum albumin, ovalbumin, or keyhole hemocyanin.

[0012] For details on the preparation route of haptens, please refer to [link / reference]. Figure 2 The specific steps involved in developing a hapten include: (1) Cyclopentylpropionic acid and tert-butyl 4-aminobutyrate were dissolved in N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The mixture was stirred at room temperature and then washed with dilute hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride in sequence. The mixture was dried and then evaporated by rotary evaporation to obtain compound 2. (2) Dissolve compound 2 in dichloromethane, add trifluoroacetic acid, react at room temperature, remove the solvent by rotary evaporation after the reaction, and repeat the addition of dichloromethane and rotary evaporation several times to obtain compound 3; compound 3 is the hapten.

[0013] A method for preparing an artificial antigen according to the above-described method includes the following steps: (1) Dissolve the above-mentioned hapten in N,N-dimethylformamide, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and activate at room temperature; (2) Dissolve the carrier protein in phosphate buffer solution, and add the activated hapten solution dropwise to the carrier protein solution, and stir the reaction at 2-8℃; (3) The reaction product was placed in a dialysis bag, dialyzed with phosphate buffer solution, and centrifuged to obtain the artificial antigen.

[0014] An antibody for a cycloalkanoic acid marker, said antibody being produced and isolated from experimental animals after immunization with the artificial antigen described above.

[0015] A kit for detecting cycloalkanoic acid markers, the kit comprising antibodies against the cycloalkanoic acid markers described above, the kit being used for qualitative or quantitative detection of cycloalkanoic acid markers.

[0016] The application of the aforementioned artificial antigen in the preparation of naphthenic acid detection products.

[0017] The present invention provides a hapten, an artificial antigen, and a method for preparing the hapten and its application for cycloalkanoic acid markers, which have the following beneficial effects: (1) This invention significantly improves the water solubility and coupling efficiency of haptens: In the prior art, when cyclopentylpropionic acid is directly coupled to a carrier protein, the activated product is extremely difficult to dissolve in water, resulting in low reaction concentration and poor product yield. This invention introduces an aminobutyric acid molecular arm at the carboxyl terminus of cyclopentylpropionic acid to generate a hapten containing an amide bond and a free carboxyl group. This hapten has good aqueous dispersibility after activation and can be efficiently coupled to the carrier protein in a homogeneous system, thereby greatly improving the preparation yield of artificial antigens.

[0018] (2) This invention significantly improves the immunogenicity of antibodies: Immunizing animals with a directly conjugated cyclopentylpropionic acid-carrier protein complex resulted in extremely low antiserum titers, which were insufficient for detection. However, after immunizing mice with the artificial antigen containing a molecular arm according to this invention, the antiserum titer was increased by orders of magnitude by indirect competitive enzyme-linked immunosorbent assay (ELISA). This result indicates that the artificial antigen developed in this invention, as a well-defined polypeptide, can more effectively present the antigenic determinants of haptens to the immune system, stimulating a strong humoral immune response.

[0019] (3) This invention establishes a highly sensitive method for detecting cycloalkanoic acid markers: the standard curve for indirect competitive enzyme-linked immunosorbent assay (ELISA) established using the antibody prepared in this invention exhibits a good S-shape, with the half-maximal inhibitory concentration (ICP) within an appropriate range and a wide linear interval. The spiked recovery rate of this method for cyclopentylpropionic acid in actual seawater samples is close to the ideal level and highly consistent with the results of high-performance liquid chromatography (HPLC), proving that the detection is accurate and reliable and suitable for rapid screening of environmental samples.

[0020] (4) The antibody prepared in this invention has excellent specificity: Cross-reactivity experiments show that the antibody of this invention exhibits strong recognition ability for cyclopentylpropionic acid and its structural analogs such as cyclopentylcarboxylic acid and cyclopentylacetoacetic acid, while the cross-reactivity rate for other analogs with large structural differences is extremely low, and there is almost no cross-reactivity with cyclohexaneacetic acid. This indicates that the antibody can accurately recognize cyclopentylpropionic acid and its structurally related cycloalkanoic acid markers, effectively eliminating interference from irrelevant substances, and providing a core reagent for the specific immunoassay of cycloalkanoic acid pollutants.

[0021] (5) The present invention is easy to operate and suitable for rapid on-site detection: Based on the artificial antigen and antibody immunoassay method of the present invention, there is no need for expensive large instruments and professional operators. The detection cycle is short and sample screening can be completed on-site, overcoming the disadvantages of traditional chromatography-mass spectrometry, such as expensive equipment, long detection time and difficulty in on-site application. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of the hapten in this invention; Figure 2 This is a flowchart illustrating the preparation route of the hapten in this invention; Figure 3 This is a full-wavelength ultraviolet scan spectrum of the hapten and artificial antigen in this invention; Figure 4 This is an SDS-PAGE electrophoresis image of the immunogen (BSA conjugate) in this invention; Figure 5 This is an SDS-PAGE electrophoresis image of the coating agent (OVA conjugate) in this invention; Figure 6 This is the standard curve for the indirect competitive ELISA of cyclopentylpropionic acid in this invention. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] To address the aforementioned technical problems, embodiments of the present invention provide a hapten, an artificial antigen, and a method for preparing the hapten and its application for cycloalkanoic acid markers, thereby resolving the issues raised in the background section.

[0025] Example 1: Preparation and Identification of Cyclopentylpropionic Acid Hapten The preparation route of cyclopentylpropionic acid hapten is as follows: Figure 2 As shown, the specific preparation steps are as follows: Cyclopentylpropionic acid (71 mg, 1.0 eq) was weighed and dissolved in 1.0 mL of N,N-dimethylformamide (DMF). Then, tert-butyl 4-aminobutyrate (76.7 mg, 1.0 eq) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (115 mg, 1.2 eq) were added, and the mixture was stirred at room temperature for 12 hours.

[0026] The solution was then poured into 20 mL of ethyl acetate and washed successively with 10 mL of dilute hydrochloric acid (0.5 M), 10 mL of saturated NaHCO3, and saturated NaCl. An appropriate amount of anhydrous Na2SO4 was then added to the solution for drying, followed by filtration. The resulting liquid was then subjected to rotary evaporation to obtain a yellow oily compound 2.

[0027] Weigh out all of compound 2 and dissolve it in 2 mL of dichloromethane (DCM). Add 2 mL of trifluoroacetic acid (TFA) under ice bath conditions and react at room temperature for 2 hours. Remove the solvent by rotary evaporation of the reacted liquid. Add 10 mL of DCM and remove the solvent by rotary evaporation again. Repeat the above operation 5 times to obtain a deep yellow oily compound 3.

[0028] Identification of compounds. Take 10 mg of cyclopentylpropionic acid (compound 1) and 10 mg of hapten (compound 3), respectively, and dissolve them in 1 mL of DMSO. Use a UV spectrophotometer to first zero the absorption peak of DMSO, and then perform a full wavelength scan. Figure 3 As shown, the absorption peak of the hapten shifts relative to cyclopentylpropionic acid, indicating that the molecular arm connection was successful.

[0029] The hapten was identified by NMR, and the results are as follows: , 7.70 (s, 1H), 3.42 (t, J = 7.1 Hz, 2H), 2.34–2.30 (m, 4H), 1.92 (m, 2H), 1.90–1.41 (m, 12H), containing two pairs of diastereoprotons at 1.90 / 1.645 and 1.73 / 1.63, and 1.41 (m, 1H). It has been identified as... Figure 1 The structure of the hapten.

[0030] Example 2: Preparation and identification of cyclopentylpropionic acid artificial antigen Compound 3 (2 mg, 1.0 eq) was weighed and dissolved in DMF (40 μL), and then EDC·HCl (2.5 mg, 1.2 eq) and NHS (2.8 mg, 1.2 eq) were added and activated at room temperature for 20 minutes.

[0031] Weigh 2 mg of BSA and dissolve it in 4 mL of 0.01 M PBS solution. Stir for 10 minutes to ensure complete dissolution. Measure 10 μL of the solution from step 1 and add the BSA solution dropwise under gentle vortexing conditions in an ice bath. Vortex the solution at 4 °C for 24 hours.

[0032] The reaction product was transferred to a dialysis bag rinsed with distilled water, and dialyzed for 48 hours at 4°C with stirring, using 1.5 L of 0.01 M PBS (pH=7.4), changing the medium every 12 hours. After dialysis, the dialyzed product was transferred to a 14 kDa ultrafiltration tube and centrifuged at 6000 rpm for 30 minutes at 4°C to a final concentration of approximately 2 mg / mL. It was then stored at -20°C for later use.

[0033] Detection. First, the solvent PBS was zeroed using a spectrophotometer by performing a full-wavelength scan. Then, 1 mg / mL BSA and artificial antigen were scanned at full wavelength. Figure 3 As shown, all synthesized artificial antigens exhibit characteristic absorption peaks of the hapten and carrier protein, or their absorption peaks shift relative to the original carrier protein, indicating successful hapten conjugation.

[0034] Secondly, the preparation results of the artificial antigen were detected by SDS-PAGE gel electrophoresis, and the results are as follows: Figure 4 As shown, the apparent migration rate of the conjugated artificial antigen in the PAGE gel changed, indicating successful synthesis. It should be noted that the molecular weight of cyclopentylpropionic acid is 142 Da, the molecular weight of the artificially synthesized hapten is approximately 227.3 Da, and the molecular weight of BSA is 66 kDa. The artificial antigen band is approximately 70 kDa.

[0035] Example 3: Preparation of cyclopentylpropionic acid detection antigen The synthesis method for the coating antigen corresponding to the cyclopentylpropionic acid hapten follows the same steps as the immunogen synthesis, except that the carrier protein used is replaced with ovalbumin (OVA), and ultrafiltration concentration is not required. Thus, an artificial cyclopentylpropionic acid antigen conjugated with ovalbumin is obtained as the coating antigen, aliquoted, and stored at -20°C for later use.

[0036] Detection. The detection method is consistent with that in Example 3. Results are as follows: Figure 3 and Figure 5 As shown.

[0037] Example 4: A cyclopentylpropionic acid antibody, prepared by the following method: Immunogen preparation: Take 50 μL of the artificial antigen protein from Example 2, mix it with 50 μL of Freund's adjuvant in a 2.5 mL syringe, and emulsify with an emulsifier until the emulsion does not spread on the water surface during the experiment. Coating antigen: Prepared in Example 3.

[0038] Immunization: Five 8-week-old female Balb / C mice were selected as immunization animals and injected with immunogen (100 μL / mouse). The initial immunization used Freund's complete adjuvant, followed by booster immunizations using Freund's incomplete adjuvant; the immunization cycle was 10 days, with a total of 5 booster immunizations. A final sprint immunization was performed 3 days after the 5 booster immunizations.

[0039] Animal antibody production detection: Three days after the last booster immunization, blood was collected from the tail vein of mice, centrifuged, and antiserum was obtained. The antibody titer and inhibition rate of the antiserum were examined using indirect ELISA. The specific steps of the indirect ELISA method are as follows: 1) Plate coating: Dilute the coating agent to 5 μg / mL with coating buffer (0.01 mol / L carbonate buffer CB, pH=9.6), then add 100 μL / well to the microplate, incubate at 4℃ for 12 hours, wash twice with 200 μL washing buffer, and pat dry. 2) Blocking: Add blocking solution (2% OVA), 100 μL / well, incubate at 37°C for 1 hour, wash the plate 3 times with 200 μL washing solution, and pat dry; 3) Primary antibody incubation: The antiserum was serially diluted using 0.01 mol / L PBST solution. Add 100 μL of 0.01 mol / L PBST solution to the negative wells and 100 μL of diluted antibody to the positive wells. After addition, incubate at 37°C for 1 hour, wash the plate 6 times with 200 μL of washing buffer, and pat dry. 4) Secondary antibody incubation: Dilute HRP-labeled goat anti-mouse antibody 2000 times with washing buffer, set at 100 μL / well, incubate at 37℃ for 30 min, wash the plate 6 times with 200 μL washing buffer, and pat dry. 5) Color development: Add color development solution (TMB color development solution), 100 μL / well, and incubate accurately at 37℃ for 10 min; 6) Termination: Add stop solution (2 mol / L H2SO4), 50 μL / well, and read the absorbance value at 450 nm wavelength in the microplate reader.

[0040] 7) Result determination: OD 450 The antiserum dilution factor corresponding to 1.0-1.5 was defined as the titer, and the antiserum titers of all mice were obtained. The results are shown in Table 1; the final titer of the anti-cyclopentylpropionic acid specific antibody of the present invention was approximately 1:128000.

[0041] Table 1 Results of the determination of specific antiserum titer against cyclopentylpropionic acid

[0042] Cyclopentylpropionic acid was used as a hapten to conjugate with BSA to obtain an artificial antigen without molecular arms. Steps 1-3 were repeated, and the final detection results are shown in Table 2. The antibody titer is expected to be about 1:150.

[0043] Table 2. Results of antiserum titer assay in mice immunized with molecular arm-less artificial antigens

[0044] Therefore, it is evident that conjugating cyclopentylpropionic acid to a carrier protein alone cannot produce highly specific antibodies. Adding a molecular arm significantly increases the antibody's specificity for recognizing cyclopentylpropionic acid.

[0045] Example 5: Cyclopentylpropionic acid test Determine the concentration of the coating antigen and the antibody dilution concentration. Using the artificial antigen prepared in Example 3 as the coating antigen, determine the appropriate concentration of the coating antigen and the antibody dilution factor by checkerboard titration. Dilute the coating antigen to different concentrations for coating, and serially dilute the antibody. Add 50 μL each of cyclopentylpropionic acid standard solution and antibody of different concentrations to each well of the coated ELISA plate, incubate at 37°C for 30 min, wash 6 times with washing buffer, blot dry the liquid in the wells, add 1:5000 diluted ELISA-labeled secondary antibody, incubate at 37°C for 30 min, wash 6 times with washing buffer, blot dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop color at 37°C in the dark for 10 min; add 50 μL of stop solution (2M H2SO4) to stop the reaction, and read the absorbance at 450 nm using an ELISA reader. The next step of plotting the inhibition curve was to select an antibody concentration between 1.0 and 1.5 A450nm and an antibody dilution factor between 1.0 and 1.5. Finally, it was determined that the antibody was diluted 60,000 times and the original antibody concentration was 6 μg / mL.

[0046] Establishment of the competitive standard curve. Based on the original coating concentration and antibody dilution concentration determined in section 1, 50 μL of antibody (diluted 60,000 times) and a series of 50 μL of cyclopentylpropionic acid standards at different concentrations were added to each well of an ELISA plate with an original coating concentration of 6 μg / mL. The plate was incubated at 37°C for 30 min, washed 6 times with washing buffer, and the liquid in the wells was blotted dry. A 1:5000 dilution of enzyme-labeled secondary antibody was added, and the plate was incubated at 37°C for 40 min. The plate was washed 6 times with washing buffer, and the liquid in the wells was blotted dry. 100 μL of TMB substrate solution was added, and the plate was incubated at 37°C in the dark for 10 min. The reaction was terminated by adding 50 μL of stop solution (2M H₂SO₄). The absorbance was read at 450 nm using an ELISA reader. A standard curve was established with the concentration of cyclopentylpropionic acid standards on the x-axis and the relative binding rate (B / B₀) on the y-axis. Figure 6 The indirect competition standard curve is shown in the figure. The results show that the standard curve is S-shaped, indicating good linear correlation, and IC... 50 The linear range is approximately 28.7 ng / mL, with a range of 5.2–250 ng / mL.

[0047] Example 6: Environmental Sample Testing Cyclopentylpropionic acid was added to seawater at concentrations of 0, 10, 100, and 1000 µg / L, respectively. The concentration of cyclopentylpropionic acid in the seawater was detected using high-performance liquid chromatography (HPLC) and the indirect competitive ELISA method (ic-ELISA) described in step 2 of Example 5, respectively. The results are shown in Table 3. The results indicate that this method can accurately detect cycloalkanoic acid markers in real environmental samples, with high recovery rate, good repeatability, and strong stability, making it suitable for rapid screening of real environmental samples.

[0048] Table 3 Environmental Sample Testing Data

[0049] Example 7: Specific detection of cyclopentylpropionic acid antibody The cross-reactivity of the cyclopentylpropionic acid antibody prepared in Example 4 with cyclopentylpropionic acid structural analogs such as cyclopentylcarboxylic acid and 2-cyclopentylpropionic acid, as well as other analogs in cycloalkyl acids such as cyclohexaneacetic acid, was detected. The specific method was the same as in Example 5, and the results are shown in Table 4.

[0050] Experimental results show that cyclopentylpropionic acid has a high affinity for its structural analog cyclopentylcarboxylic acid, but no cross-reactivity with other structural analogs. This antibody exhibits good recognition ability for cyclopentylpropionic acid and structurally similar cycloalkyl acid markers, with extremely low cross-reactivity with other structurally unrelated substances, high specificity, and can effectively avoid interference, making it suitable for the specific detection of cycloalkyl acid contaminants.

[0051] Table 4 Cross-reactivity results

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A hapten of a naphthenic acid marker, characterized in that, The hapten is a compound represented by Formula I: 。 2. An artificial antigen of a naphthenic acid marker, characterized in that, The artificial antigen is a complete antigen prepared by coupling the hapten of claim 1 with a carrier protein.

3. Artificial antigen according to claim 2, characterized in that, The carrier protein is one of bovine serum albumin, ovalbumin, or keyhole hemocyanin.

4. A method for preparing a hapten according to claim 1, characterized in that, The preparation route of the hapten is as follows: 。 5. The method for preparing a hapten according to claim 4, characterized in that, Includes the following steps: (1) Cyclopentylpropionic acid and tert-butyl 4-aminobutyrate were dissolved in N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The mixture was stirred at room temperature and then washed with dilute hydrochloric acid, saturated sodium bicarbonate and saturated sodium chloride in sequence. The mixture was dried and then evaporated by rotary evaporation to obtain compound 2. (2) Dissolve compound 2 in dichloromethane, add trifluoroacetic acid, react at room temperature, remove the solvent by rotary evaporation after the reaction, and repeat the addition of dichloromethane and rotary evaporation several times to obtain compound 3; compound 3 is the hapten.

6. A method for preparing an artificial antigen according to claim 2, characterized in that, Includes the following steps: (1) Dissolve the hapten of claim 1 in N,N-dimethylformamide, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and activate at room temperature; (2) Dissolve the carrier protein in phosphate buffer solution, and add the activated hapten solution dropwise to the carrier protein solution, and stir the reaction at 2-8℃; (3) The reaction product was placed in a dialysis bag, dialyzed with phosphate buffer solution, and centrifuged to obtain the artificial antigen.

7. An antibody for a cycloalkanoic acid marker, characterized in that, The antibody is produced and isolated from experimental animals after immunizing them with the artificial antigen described in claim 2.

8. A kit for detecting cycloalkanoic acid markers, characterized in that, The kit includes an antibody for the cycloalkanoic acid marker as described in claim 7, and the kit can be used for the qualitative or quantitative detection of the cycloalkanoic acid marker.

9. The application of the artificial antigen as described in claim 2 in the preparation of naphthenic acid detection products.

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