Hybridoma cell strain secreting monoclone antibody of psoralen and isopsoralen

Abstract

The present application relates to a hybridoma cell strain secreting psoralen and isopsoralen monoclonal antibody. The hybridoma cell strain of the present application is preserved in China General Microbiological Culture Collection Center on April 18, 2024, the preservation address is No. 1, Xibaixili, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 45909. The psoralen and isopsoralen monoclonal antibody secreted by the hybridoma cell strain of the present application has good sensitivity and specificity to psoralen and isopsoralen, so that the psoralen and isopsoralen can be efficiently and rapidly detected.

Description

Technical Field

[0001] This invention relates to the field of immunochemical technology, and in particular to a hybridoma cell line that secretes monoclonal antibodies against psoralen and isopsoralen. Background Technology

[0002] Psoralen (PSO) and isopsoralen (IPSO) are derived from the fruit of the legume *Psoralea corylifolia* and are two basic furanocoumarin isomers. They are widely found not only in traditional Chinese medicines such as *Adenophora stricta*, *Saposhnikovia divaricata*, and *Angelica pubescens*, but also in citrus fruits. Psoralen and isopsoralen possess multiple pharmacological effects, including anti-osteoporosis, neuroprotection, anti-tumor activity, estrogen-like properties, anti-inflammation, and promotion of melanin synthesis. They can be used to treat diseases such as rheumatoid arthritis, leukemia, and Alzheimer's disease, and are therefore widely used in medicine, cosmetics, and other fields.

[0003] However, increasing research indicates that psoralen and isopsoralen can cause hepatotoxicity by affecting liver regeneration, bile acid balance, oxidative stress, and mitochondrial dysfunction. Furthermore, psoralen has photosensitizing properties, causing redness and increased pigmentation at the radiation site. Inhalation, skin contact, and ingestion of isopsoralen are harmful to humans. Due to their widespread use, there is growing concern about their potential adverse effects and hazards on human health. To safeguard human health, it is necessary to establish immunoassay methods for detecting psoralen and isopsoralen residues.

[0004] Currently, there are few methods for detecting psoralen and isopsoralen. Commonly used methods are mainly instrumental analysis methods, including high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS). Instrumental analysis methods offer high sensitivity, accuracy, low detection limits, low false positive rates, and high specificity. However, sample pretreatment is complex, requires specialized personnel, and the equipment is expensive, making it difficult to meet the requirements of rapid on-site detection. Compared to instrumental analysis, immunological detection methods have simpler pretreatment, offering advantages such as convenience, speed, and ease of operation. They can also improve detection sensitivity and reduce costs, giving them a unique advantage and great development potential for large-scale sample screening and rapid on-site detection. Currently, the preparation of monoclonal antibodies for psoralen and isopsoralen has not been detected. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem that the lack of an immunoassay method for psoralen and isopsoralen in the prior art leads to low detection sensitivity and high detection cost for psoralen and isopsoralen.

[0006] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes monoclonal antibodies against psoralen and isopsoralen. This hybridoma cell line has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 18, 2024, with accession number CGMCC NO.45909. The monoclonal antibodies secreted by this hybridoma cell line exhibit good sensitivity and specificity for psoralen and isopsoralen, thus enabling rapid and efficient detection of these two substances.

[0007] The first objective of this invention is to provide a hybridoma cell line that secretes monoclonal antibodies against psoralen and isopsoralen. The hybridoma cell line was deposited on April 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.45909.

[0008] Furthermore, the hybridoma cell line is obtained by immunizing animals with an immunogen derived from a hapten conjugated with a carrier protein, wherein the structural formula of the hapten is shown in Formula I:

[0009]

[0010] Furthermore, the carrier protein includes bovine serum albumin.

[0011] Furthermore, the hapten was prepared by reacting bergamot with ethyl 6-bromohexanoate, and the synthetic route is shown below:

[0012]

[0013] Further, the preparation steps of the hapten are as follows: 10 mg of bergamot is dissolved in 300 mL of DMF, excess K₂CO₃ (68 mg), 27 μL of ethyl 6-bromohexanoate, and 1 mg of KI are added. The mixture is refluxed at 60 °C for 24 h, allowed to stand overnight, and then... The hapten was dissolved in NaOH-methanol solution at 80℃ for 1 hour, dried under nitrogen, dissolved in 2 ml of double-distilled water, and the pH was adjusted to 3.5 with hydrochloric acid. It was then extracted three times with 3 ml of ethyl acetate, the aqueous phase was discarded, and the hapten was dried under nitrogen to obtain the hapten.

[0014] A second objective of this invention is to provide the application of the above-mentioned hybridoma cell line in the detection of psoralen and isopsoralen.

[0015] A third objective of this invention is to provide monoclonal antibodies against psoralen and isopsoralen secreted by the aforementioned hybridoma cell line.

[0016] A fourth objective of this invention is to provide the application of the above-mentioned monoclonal antibodies against psoralen and isopsoralen in the detection of psoralen and isopsoralen.

[0017] The fifth objective of this invention is to provide a detection product for psoralen and isopsoralen, characterized in that it comprises monoclonal antibodies against the aforementioned psoralen and isopsoralen.

[0018] Furthermore, the test product also includes psoralen and isopsoralen-coated antigens.

[0019] Furthermore, the psoralen and isopsoralen are coated with a hapten-coupled carrier protein, wherein the carrier protein includes ovalbumin.

[0020] The beneficial effects of this invention are:

[0021] The monoclonal antibodies against psoralen and isopsoralen secreted by the hybridoma cell line described in this invention exhibit good detection sensitivity (IC50 of PSO) for psoralen and isopsoralen. 50 The value was 0.62 μg / mL, and the IC50 of IPSO was... 50 With a value of 0.54 μg / mL and high specificity, it can achieve specific detection of psoralen and isopsoralen, and the detection cost is low, providing raw materials for the immunoassay of psoralen and isopsoralen.

[0022] Preservation of biological materials

[0023] The monoclonal cell line CXJ was deposited on April 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45909, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0024] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0025] Figure 1 These are the standard inhibition curves for psoralen and isopsoralen monoclonal antibodies;

[0026] Figure 2 This is a synthetic route diagram for haptens;

[0027] Figure 3 This is the mass spectrum of the hapten. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0029] In the following examples, the solutions are prepared as follows:

[0030] Carbonate buffer (CBS): Weigh 1.59g of Na2CO3 and 2.93g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000mL. Store at 4℃ for later use.

[0031] Phosphate-buffered saline (PBS): Dissolve 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, and 2.9g Na2HPO4·12H2O in 800mL of pure water. Adjust the pH to 7.2-7.4 with NaOH or HCl and bring the volume to 1000mL.

[0032] PBST: PBS containing 0.05% Tween 20.

[0033] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution. Mix fresh before use.

[0034] Example 1: Preparation of haptens and complete antigens

[0035] (1) Preparation of hapten

[0036] In this embodiment, the hapten BER-6C was prepared by derivatizing a carboxyl group from the hydroxyl group on the benzene ring of bergamot. The specific steps were as follows: 10 mg of bergamot was weighed and dissolved in 300 mL of DMF, excess K₂CO₃ (68 mg), 27 μL of ethyl 6-bromohexanoate, and 1 mg of KI were added. The mixture was refluxed at 60 °C for 24 h, allowed to stand overnight, and then... The product hapten BER-6C was obtained by alkaline hydrolysis in NaOH-methanol solution at 80℃ for 1 h, followed by drying under nitrogen. The solution was dissolved in 2 ml of double-distilled water, and the pH was adjusted to 3.5 with hydrochloric acid. The solution was then extracted three times with 3 ml of ethyl acetate. The aqueous phase was discarded, and the product was dried with anhydrous magnesium sulfate and then dried under nitrogen.

[0037] To verify whether the obtained product is the hapten needed in this invention, the product was subjected to mass spectrometry detection, and the detection results are as follows: Figure 3As shown, the relative molecular mass of BER-6C is 316. The LC-MS mass spectrum of BER-6C in negative ion mode shows that the derivative exhibits a significant absorption peak at 4.93 min. Combined with the mass spectrum, it can be seen that the molecular ion peak with a mass-to-charge ratio (m / z) of 315 at this time is consistent with the molecular weight of the hapten BER-6C, thus indicating that the hapten derivatization was successful.

[0038] (2) Preparation of immunogen BER-6C-BSA

[0039] The hapten BER-6C was conjugated with bovine serum albumin (BSA) using the EDC method to obtain the immunogen BER-6C-BSA. The specific steps were as follows: 0.76 mg of hapten BER-6C (the molar ratio of BER-6C to BSA was 40:1) was weighed and dissolved in 300 μL of N,N-dimethylformamide (DMF). First, 0.9 mg of N-hydroxysuccinimide (NHS) was added, and the mixture was reacted at room temperature for 10 min. Then, 1.5 mg of 1-ethyl-carbodiimide hydrochloride (EDC) was added, and the reaction was continued at room temperature for 4 h (this is called solution A). 4 mg of BSA was dissolved in 2 mL of 0.01 M carbonate buffer (CB, pH = 9.0) (this is called solution B). Solution A was then slowly added dropwise to solution B, and the conjugation was allowed to proceed overnight. The mixture was then dialyzed with 0.01 M PBS to remove unreacted small molecule hapten molecules, yielding the complete antigen BER-6C-BSA.

[0040] (3) Preparation of coating original BER-6C-OVA

[0041] The hapten BER-6C was conjugated with ovalbumin (OVA) using the EDC method to obtain the encapsulated BER-6C-OVA. The specific steps were as follows: Weigh 1.5 mg of the hapten BER-6C (BER-6C to OVA molar ratio of 60:1), dissolve it in 300 μL of DMF, add 1.8 mg of NHS, react at room temperature for 10 min, then add 3 mg of EDC, and continue the reaction at room temperature for 4 h (this is called solution A). Take 4 mg of OVA, dissolve it in 2 mL of 0.01 M carbonate buffer (CB, pH = 9.0) (this is called solution B), and then slowly add solution A dropwise to solution B, conjugating overnight. Then dialyze with 0.01 M PBS solution to remove unreacted small molecule hapten, obtaining the encapsulated BER-6C-OVA.

[0042] Example 2: Preparation of hybridoma cell lines

[0043] (1) Animal Immunization

[0044] Immunogen BER-6C-BSA was emulsified with an equal amount of Freund's adjuvant and then administered to BALB / c mice via subcutaneous injection at multiple sites on the back of the neck (except for sprint immunization).

[0045] The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse. Multiple booster immunizations used incomplete Freund's adjuvant at half the dose (50 μg / mouse). The final sprint immunization did not use adjuvant; it was directly diluted with physiological saline and injected intraperitoneally at a dose halved again (25 μg / mouse). The interval between the initial and second booster immunizations was one month, between multiple booster immunizations was 21 days, and between the final sprint immunization and the last booster immunization was 18-21 days. The immunization efficacy in mice was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

[0046] (2) Cell fusion

[0047] Three days after the sprint immunization, cell fusion was performed using the standard polyethylene glycol (PEG, molecular weight 4000) method, with the following specific steps:

[0048] a. Blood was collected from the eyeballs of mice. After euthanizing the mice by cervical dislocation, the mice were immediately disinfected in 75% alcohol and soaked for about 5 minutes. The spleen of the mice was removed under aseptic conditions. The spleen cells were moderately ground with the tip of a syringe and passed through a 200-mesh cell sieve to obtain a spleen cell suspension. The suspension was collected, centrifuged (1200 rpm, 8 min), and washed three times with RPMI-1640 medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and set aside for later use.

[0049] b. Collection of SP2 / 0 cells: 7-10 days before fusion, culture SP2 / 0 tumor cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The required number of SP2 / 0 tumor cells before fusion should reach 1-4 × 10⁶ cells / year. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0050] c. Fusion process (7 min): Minute 1: Add 1 mL of PEG 4000 to the cells dropwise, gradually increasing the speed. Minute 2: Let stand. Minutes 3 and 4: Add 1 mL of RPMI-1640 medium dropwise over 1 min. Minutes 5 and 6: Add 2 mL of RPMI-1640 medium dropwise over 1 min. Minute 7: Add 1 mL of RPMI-1640 medium dropwise every 10 s. Then incubate at 37°C for 5 min, centrifuge (800 rpm, 10 min), discard the supernatant, gently break up the cells, and add RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT to a 96-well cell plate at a rate of 200 μL / well. Incubate at 37°C in a 5% CO2 incubator.

[0051] (3) Cell screening and cell line establishment

[0052] On day 3 after cell fusion, the fused cells were partially replaced with HAT medium; on day 5, the medium was completely replaced with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on day 7, the cell supernatant was collected for screening. Screening consisted of two steps: first, positive cell wells were selected using ic-ELISA; second, psoralen and isopsoralen were used as standards, and their inhibitory effect on positive cells was determined using ic-ELISA. Cell wells showing good inhibition of psoralen and isopsoralen standards were selected for subcloning using limiting dilution, and the same method was used for detection seven days later. Subcloning was performed three times using the above method to finally obtain cell lines containing psoralen and isopsoralen monoclonal antibodies.

[0053] The ic-ELISA detection procedure is as follows:

[0054] Coating: Dilute the original BER-6C-OVA to 0.1 μg / mL with CBS, coat the microplate, 100 μL / well, and react at 37℃ for 2 h;

[0055] Washing: Discard the solution in the plate and wash 3 times with PBST for 3 min each time, 200 μL / well, and spin dry;

[0056] Blocking: Add 200 μL / well blocking solution, react at 37℃ for 2 h, and wash.

[0057] Sample loading: First, add 50 μL of serially diluted psoralen and isopsoralen standard working solutions (standard working solution concentrations are 0, 0.02, 0.06, 0.17, 0.5, 1.5, 4.5, and 13.5 μg / mL) to each well. Dilute the antibody to 0.1 μg / mL with PBS, 50 μL / well, and incubate at 37°C for 30 min. Wash thoroughly. Then, add 100 μL / well of HRP-goat anti-mouse IgG diluted 1:3000, and incubate at 37°C for 30 min. Wash thoroughly.

[0058] Color development: Add 100 μL of TMB color development solution to each well and react at 37°C in the dark for 15 min;

[0059] Termination and Measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0060] Example 3: Preparation of Monoclonal Antibodies

[0061] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6Ascites fluid was collected from psoralen and isopsoralen hybridoma cells starting on day 7. The ascites fluid was then purified using the caprylic acid-saturated ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded after centrifugation. Next, an equal volume of saturated ammonium sulfate solution was used to precipitate IgG-type monoclonal antibodies. After centrifugation and discarding the supernatant, the antibodies were dissolved in 0.01M PBS solution (pH = 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0062] Example 4: Sensitivity and Specificity Detection of Monoclonal Antibodies

[0063] IC50 of psoralen and isopsoralen monoclonal antibodies was determined by ic-ELISA. 50 The values ​​were 0.62 μg / mL and 0.54 μg / mL, respectively, indicating that this monoclonal antibody has good sensitivity to psoralen and isopsoralen and can be used for the immunoassay detection of psoralen and isopsoralen.

[0064] The cross-reactivity results of psoralen and isopsoralen monoclonal antibodies against psoralen, isopsoralen, imperatorin, angelicin, coumarin, and dicoumarin are shown in Table 1.

[0065] Table 1. Cross-reactivity results of psoralen, isopsoralen and their analogues

[0066]

[0067]

[0068] The monoclonal antibodies against psoralen and isopsoralen show good specificity for psoralen and isopsoralen, and have low cross-reactivity with other analogues, all less than 10%. This indicates that the antibodies have strong specificity and will not recognize psoralen and isopsoralen analogues, thus avoiding false positive results when testing samples.

[0069] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line secreting a monoclonal antibody to psoralen and isopsoralen, characterized in that, The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center on April 18, 2024, the address is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 45909.

2. A psoralen and isopsoralen monoclonal antibody secreted by the hybridoma cell strain of claim 1.

3. A psoralen and isopsoralen detection product, characterized by The psoralen and isopsoralen monoclonal antibody of claim 2 is contained.

4. The test product according to claim 3, characterized in that The detection product further comprises a psoralen and isopsoralen coating agent.

5. The detection product according to claim 4, characterized in that, The psoralen and isopsoralen coating agent is prepared from a hapten coupled carrier protein, wherein the carrier protein comprises ovalbumin.

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