A perfluorooctanoic acid hapten, a preparation method and application thereof

Abstract

The application discloses a perfluorooctanoic acid hapten as well as a preparation method and application thereof, and belongs to the field of food safety immune detection. A hybridoma cell strain KND secreting perfluorooctanoic acid monoclonal antibody is preserved in the China General Microbiological Culture Collection Center (CGMCC) on February 8, 2023, and the preservation number is 45478. The perfluorooctanoic acid monoclonal antibody obtained by using the cell strain can be used for the analysis and detection of perfluorooctanoic acid residues in food safety detection. The perfluorooctanoic acid monoclonal antibody cell strain obtained by the application can be used for immune analysis and detection, and has good detection sensitivity and specificity (IC 50 The application can be used for preparing an immune detection kit of perfluorooctanoic acid and a colloidal gold test strip, and provides a powerful detection method and means for the detection of perfluorooctanoic acid residues in drinking water, food and food packaging.

Description

Technical Field

[0001] This invention relates to the field of food safety immunoassay technology, and in particular to a perfluorooctanoic acid hapten, its preparation method, and its application. Background Technology

[0002] Perfluoroalkyl and polyfluoroalkyl compounds (PFAS) are a class of aliphatic organofluorine compounds that have garnered significant public attention in recent years due to their potential carcinogenicity and lethality. Perfluorooctanoic acid (PFOA), the most widely used perfluorocarboxylic acid compound, is an environmentally persistent substance with a long half-life and high accumulation potential due to its stable chemical structure, high electronegativity, and small fluorine atomic radius. Furthermore, its high water solubility allows it to rapidly migrate and diffuse from soil media into groundwater, leading to groundwater contamination over considerable distances. Current research has confirmed that PFOA poses risks to human health, such as cancer, infertility, low birth weight, and delayed puberty. In particular, diseases affecting lipid production, such as dyslipidemia, show the strongest metabolic correlation with PFOA exposure. Both domestic and international organizations have recognized the importance of controlling these chemicals and have issued regulations on the perfluorooctanoic acid (PFOA) content in products. Regarding the issue of PFOA pollution control, how to detect the type and concentration of PFOA in water pollutants, food, food packaging materials, and drinking water has become an important research topic. Therefore, it is of great significance to establish a rapid detection method for monitoring the content of PFOA in water on which humans depend for survival and in clinical samples.

[0003] Currently, the main methods for detecting perfluorooctanoic acid (PFOA) residues are instrumental analytical methods, including gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). While instrumental methods offer good accuracy and reproducibility, their high requirements for operators and equipment, coupled with cumbersome and time-consuming sample pretreatment procedures, make them unsuitable for rapid monitoring of PFOA content in randomly collected water and food samples, thus limiting their widespread application at the grassroots level. In contrast, immunoassay methods based on specific antigen-antibody reactions, such as enzyme-linked immunosorbent assay (ELISA), have become complementary to large-scale instrumental methods due to their advantages such as high sample throughput, sensitivity, accuracy, simplicity, good specificity, and suitability for sample screening, and have seen rapid development in recent years. The need to establish a simple, reliable, and rapid PFOA detection method to handle large-scale sample testing is of significant application value. This research remains a challenge. ELISA is an extremely efficient, sensitive, and rapid detection method suitable for rapid on-site detection of large numbers of samples, providing a new detection route for PFOA in drinking water, food, and food packaging. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a perfluorooctanoic acid (PFOA) hapten, its preparation method, and its application. This invention involves immunizing mice with the PFOA complete antigen, followed by cell fusion, culture in HAT selective medium, and screening the cell supernatant using ic-ELISA to ultimately obtain a monoclonal antibody hybridoma cell line with high sensitivity to PFOA.

[0005] The first objective of this invention is to provide a perfluorooctanoic acid hapten, the structural formula of which is:

[0006]

[0007] The second objective of this invention is to provide a method for preparing the perfluorooctanoic acid hapten, comprising the following steps:

[0008] Compound 1 was reacted with thionyl chloride to obtain compound 2;

[0009] In an organic solvent, compound 2 and compound 3 are mixed and reacted under alkaline conditions to obtain compound 4;

[0010] Compound 4 was reacted in a trifluoroacetic acid solution to obtain the perfluorooctanoic acid hapten;

[0011] The structural formula of compound 1 is as follows:

[0012]

[0013] The structural formula of compound 2:

[0014]

[0015] The structural formula of compound 3:

[0016] The structural formula of compound 4:

[0017]

[0018] In some embodiments of the present invention, the organic solvent is selected from dichloromethane and DMF.

[0019] In some embodiments of the present invention, the solvent of the trifluoroacetic acid solution is dichloromethane; the concentration of the trifluoroacetic acid solution is 50 wt%, and the volume is 40 mL.

[0020] In some embodiments of the present invention, the reaction conditions of compound 4 in trifluoroacetic acid solution are: ice bath reaction for 2-3 hours.

[0021] In some embodiments of the present invention, alkaline conditions are obtained by adding an alkaline reagent selected from triethylamine.

[0022] In this invention, the specific preparation method of the perfluorooctanoic acid hapten is as follows:

[0023] Compound 1 was dissolved in thionyl chloride and refluxed overnight. The thionyl chloride was then concentrated under reduced pressure to obtain crude product compound 2. Compound 2 was dissolved in dichloromethane, and compound 3 and triethylamine were added under ice bath conditions. The mixture was then reacted at room temperature overnight. The solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 4. Compound 4 was then dissolved in dichloromethane, and 50% TFA / DCM solution was added under ice bath conditions. The mixture was reacted under ice bath conditions for 2-3 hours, diluted with toluene, and distilled under reduced pressure to remove excess TFA, yielding compound 5, which is the perfluorooctanoic acid hapten.

[0024] The third objective of this invention is to provide a perfluorooctanoic acid (PFOA) complete antigen, prepared from the PFOA hapten. The method for preparing the PFOA complete antigen is as follows: the PFOA hapten is coupled to carrier proteins BSA and OVA respectively using the carbodiimide method to prepare immunogen PFOA-BSA and coated PFOA-OVA; the complete antigen and the uncoupled hapten are then separated by dialysis.

[0025] The fourth objective of this invention is to provide a hybridoma cell line KND that secretes a monoclonal antibody against perfluorooctanoic acid (PFOA). This cell line is obtained by immunizing mice with the PFOA hapten through the synthesis of the corresponding complete antigen. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, classified as a monoclonal cell line, with a deposit date of February 8, 2023, and accession number 45478.

[0026] In this invention, the hybridoma cell line is prepared by the following method:

[0027] (1) Immunization of mice: The immunogen perfluorooctanoic acid-BSA was emulsified with an equal amount of Freund's adjuvant and then injected subcutaneously into the back of BALB / c mice; the first immunization used complete Freund's adjuvant, and the subsequent booster immunizations used incomplete Freund's adjuvant. The interval between the first and second booster immunizations was 28 days, and the interval between the booster immunizations was 21 days. The last immunization was performed using perfluorooctanoic acid-BSA complete antigen (without adjuvant). Serum titers and inhibition were detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

[0028] (2) Cell fusion and cell line establishment: Mouse spleen cells and mouse myeloma cells were fused using the polyethylene glycol (PEG 4000) method and cultured in HAT medium. Positive cell pores were detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), and the inhibitory effect of positive cell pores was further determined by ic-ELISA. The positive cell pores with the best inhibitory effect were subcloned three times by limiting dilution method, and the KND cross-drug cell line was finally obtained by screening.

[0029] (3) Identification of hybridoma cell line characteristics: Sensitivity and specificity were determined by ic-ELISA.

[0030] The fifth objective of this invention is to provide a perfluorooctanoic acid monoclonal antibody secreted by the hybridoma cell line KND.

[0031] A sixth objective of this invention is to provide a composition comprising the hybridoma cell line and / or the perfluorooctanoic acid monoclonal antibody.

[0032] A seventh objective of the present invention is to provide a kit containing one or more of the hybridoma cell line, the perfluorooctanoic acid monoclonal antibody, or the composition thereof.

[0033] An eighth objective of the present invention is to provide a test strip containing one or more of the hybridoma cell line, the perfluorooctanoic acid monoclonal antibody, or the composition thereof.

[0034] A ninth objective of this invention is to provide a chip containing one or more of the aforementioned hybridoma cell line, the aforementioned perfluorooctanoic acid monoclonal antibody, and the aforementioned composition.

[0035] The tenth objective of this invention is to provide the application of the hybridoma cell line, the perfluorooctanoic acid monoclonal antibody, the composition, the kit, the test strip, or the chip in the invention for detecting perfluorooctanoic acid.

[0036] The technical solution of the present invention has the following advantages compared with the prior art:

[0037] The monoclonal antibody secreted by the KND tumor cell line provided by this invention exhibits good specificity and detection sensitivity (IC50) for perfluorooctanoic acid (PFOA). 50 With a value of 1.13 ug / mL, it can be used to detect perfluorooctanoic acid (PFOA) residues in drinking water, food, and food packaging, providing raw materials for the immunological detection of PFOA residues in food and having practical application value.

[0038] Biological material deposit: A hybridoma cell line KND secreting a monoclonal antibody against perfluorooctanoic acid has been deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number 45478, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It is classified as a monoclonal cell line and the deposit date is February 8, 2023. Attached Figure Description

[0039] 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...

[0040] Figure 1 This is the standard curve of the inhibition of perfluorooctanoic acid by the monoclonal antibody of this invention. Detailed Implementation

[0041] 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.

[0042] Solution preparation for this invention:

[0043] 1. Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0044] 2. Phosphate Buffered Sodium (PBS): Dissolve 8.0g 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.

[0045] 3. Washing buffer (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol / L pH 7.4 PBS solution;

[0046] 4. PBST: PBS containing 0.05% Tween-20;

[0047] 5. Antibody dilution buffer: Wash buffer containing 0.1% gelatin;

[0048] 6. 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 volume ratio of 1:5 to obtain the TMB colorimetric solution. Mix fresh before use.

[0049] Example 1: Preparation of the hybridoma cell line KND

[0050] 1. Preparation of complete antigen

[0051] (1) The reaction equation for the synthesis of haptens is shown in the figure below:

[0052]

[0053] The derivation steps are briefly described below:

[0054] 1. Preparation of perfluorooctanoic acid hapten:

[0055] Compound 1 was dissolved in thionyl chloride and refluxed overnight. The thionyl chloride was then concentrated under reduced pressure to obtain crude product compound 2. Compound 2 was dissolved in dichloromethane, and compound 3 and triethylamine were added under ice bath conditions. The mixture was then reacted at room temperature overnight. The solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 4. Compound 4 was then dissolved in dichloromethane, and 50 wt% TFA / DCM solution was added under ice bath conditions. The mixture was reacted under ice bath conditions for 2 hours, diluted with toluene, and distilled under reduced pressure to remove excess TFA, yielding compound 5, which is the perfluorooctanoic acid hapten.

[0056] 2. Preparation of coating with original perfluorooctanoic acid-OVA:

[0057] Weigh 8.6 mg of the prepared perfluorooctanoic acid hapten and dissolve it in 300 μL of DMF with slow stirring. Then, add 6.4 mg of N-hydroxysuccinimide and 5.8 mg of 1-ethylcarbodiimide hydrochloride sequentially and react at room temperature for 6-8 h to obtain a mixture. Then, weigh 10 mg of chicken ovalbumin OVA and dissolve it in 2 mL of carbonate buffer. Slowly add the resulting mixture dropwise to the chicken ovalbumin solution and react at room temperature for 14 h with stirring. Dialyze the mixture with 0.01 mol / L phosphate buffered PBS for 3 days to obtain the conjugate perfluorooctanoic acid-OVA, which is stored at -20 °C for later use.

[0058] 3. Immunization of mice: Healthy BALB / c mice aged 6–8 weeks were selected for immunization. Perfluorooctanoic acid (PFOA) complete antigen was emulsified with an equal volume of Freund's adjuvant and administered subcutaneously via the back of the mouse. The first immunization used complete Freund's adjuvant, and subsequent immunizations used incomplete Freund's adjuvant. The interval between the first and second booster immunizations was 28 days, and the interval between multiple booster immunizations was 21 days. Seven days after the third immunization, blood was collected (5 μL of blood from tail amputation + 995 μL of antibody diluent = antiserum). The serum titer and inhibition were measured using ic-ELISA. Mice with high titers and good inhibition were selected for a sprint immunization 21 days after the fifth immunization, administered via intraperitoneal injection. The sprint immunization dose was halved and contained no adjuvant.

[0059] 4. Cell fusion: Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0060] (1) After euthanizing the mice by enucleation and 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 rubber tip of a syringe and passed through a 200-mesh cell sieve to obtain a suspension of spleen cells. The suspension was collected and centrifuged (1200 rpm, 8 min). The spleen cells were 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.

[0061] (2) Collection of murine myeloma SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured 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 was 1-4 × 10⁻⁶. 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.

[0062] (3) Fusion process: 7 min. At min 1, 1 mL of PEG 1500 was added to the cells dropwise, gradually increasing the speed. At min 2, the cells were allowed to stand. At min 3 and min 4, 1 mL of RPMI-1640 medium was added dropwise over 1 min. At min 5 and min 6, 2 mL of RPMI-1640 medium was added dropwise over 1 min. At min 7, 1 mL of RPMI-1640 medium was added dropwise every 10 s. The cells were then incubated at 37°C for 5 min. After centrifugation (800 rpm, 8 min), the supernatant was discarded, and the cells were resuspended in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT. The medium was added to 96-well cell plates at a rate of 200 μL / well and incubated at 37°C in a 5% CO2 incubator.

[0063] 5. Cell Screening and Cell Line Establishment: On day 3 of cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition 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, perfluorooctanoic acid (PFOA) was used as a standard, and its inhibitory effect on positive cells was determined using ic-ELISA. Cell wells showing good inhibition of PFOA standard were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection. This process was repeated three times to obtain the KND cell line.

[0064] 6. Preparation and identification of monoclonal antibodies: 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 × 10⁻⁶ mol / L of the same oil. 6 Hybridoma cells were used, and ascites fluid was collected starting on day seven. The ascites fluid was purified using the caprylic acid-ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The precipitate was dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0065] The IC50 of the monoclonal antibody against perfluorooctanoic acid was determined using an indirect competitive ELISA method. 50 The concentration was 1.13 ug / mL, and its IC50 for perfluorooctanoic acid, etc., was verified. 50 The cross-reactivity rate is shown in Table 1.

[0066] Table 1. IC50 values ​​of monoclonal antibody KND against perfluorooctanoic acid, perfluorohexadecanoic acid, perfluorooctanoic acid, perfluorosedioic acid, and perfluorononanoic acid. 50 and cross-reactivity rate

[0067]

[0068] 7. Antibody Application: Monoclonal antibodies prepared from the hybridoma cell line KND via in vivo ascites fluid were used in a spiking and recovery assay for perfluorooctanoic acid (PFOA). The specific steps are as follows:

[0069] (1) Coating: The original perfluorooctanoic acid-OVA was serially diluted with 0.05M pH 9.6 carbonate buffer starting from 1 μg / mL, 100 μL / well, and reacted at 37℃ for 2 h.

[0070] (2) Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time.

[0071] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0072] (4) Sample addition: The antiserum (antiserum obtained by diluting the blood from the tail of mice with antibody diluent) was serially diluted from 1:1000 and added to each well of the coated sample at 100 μL / well. The mixture was reacted at 37°C for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The mixture was reacted at 37°C for 30 min.

[0073] (5) Color development: Remove the microplate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 min.

[0074] (6) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD450 value of each well using an ELISA reader.

[0075] The IC50 of monoclonal antibody against perfluorooctanoic acid was determined by ic-ELISA. 50 The value was 1.13 ug / mL, indicating good sensitivity to perfluorooctanoic acid (PFOA), and it can be used for PFOA immunoassay.

[0076] The standard curve of KND monoclonal antibody inhibition against perfluorooctanoic acid is as follows: Figure 1 As shown.

[0077] 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 perfluorooctanoic acid hapten, characterized in that, The structural formula of the perfluorooctanoic acid hapten is: .

2. The method for preparing the perfluorooctanoic acid hapten according to claim 1, characterized in that, Includes the following steps: Compound 1 was reacted with thionyl chloride to obtain compound 2; In an organic solvent, compound 2 and compound 3 are mixed and reacted under alkaline conditions to obtain compound 4; Compound 4 was reacted in a trifluoroacetic acid solution to obtain the perfluorooctanoic acid hapten; The structural formula of compound 1 is as follows: ； The structural formula of compound 2: ; The structural formula of compound 3: ; The structural formula of compound 4: 。 3. A perfluorooctanoic acid complete antigen, characterized in that, It is prepared from the perfluorooctanoic acid hapten described in claim 1.

4. A hybridoma cell line KND that secretes a monoclonal antibody against perfluorooctanoic acid (PFOA), obtained by immunizing mice with the PFOA hapten described in claim 1 through the synthesis of the corresponding complete antigen; it has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, classified as a monoclonal cell line, deposited on February 8, 2023, with accession number CGMCC No. 45478.

5. A perfluorooctanoic acid monoclonal antibody, characterized in that, It is secreted by the hybridoma cell line KND as described in claim 4.

6. A composition, characterized in that, The composition contains the hybridoma cell line of claim 4 and / or the perfluorooctanoic acid monoclonal antibody of claim 5.

7. A reagent kit, characterized in that, The kit contains one or more of the hybridoma cell line of claim 4, the perfluorooctanoic acid monoclonal antibody of claim 5, or the composition of claim 6.

8. A test strip, characterized in that, The test strip contains one or more of the hybridoma cell line of claim 4, the perfluorooctanoic acid monoclonal antibody of claim 5, or the composition of claim 6.

9. A chip, characterized in that, The chip contains one or more of the hybridoma cell line of claim 1, the perfluorooctanoic acid monoclonal antibody of claim 5, and the composition of claim 6.

10. The use of the hybridoma cell line of claim 4, the perfluorooctanoic acid monoclonal antibody of claim 5, the composition of claim 6, the kit of claim 7, the test strip of claim 8, or the chip of claim 9 in the detection of perfluorooctanoic acid; the use does not relate to the diagnosis and treatment of diseases.

Images