Establishment method and application of hexestrol ic-ELISA (Enzyme Linked Immunosorbent Assay)

Abstract

The invention relates to an establishment method of hexestrol ic-ELISA (Enzyme-Linked Immunosorbent Assay). The establishment method comprises the following steps: (1) preparing HES-BSA (Human Estilbestrol-Bovine (2) preparing anti-HES (Hydroxyethyl Sulfonate) mAbs by using the HES-BSA; and (3) coating an ELISA plate by using a bicarbonate buffer solution and HES-OVA, incubating and sealing holes, adding anti-HES mAbs and the diluted free HES standard substance into a PBS solution, incubating, and adding GaM IgG-HRP, so as to obtain the hexestrol ic-ELISA. According to the establishment method of the hexestrol ic-ELISA provided by the invention, carboxyl is introduced, the hapten HES and the carrier protein are coupled to generate a complete antigen, and the anti-HES mAb is produced through HES-BSA immune animals and a hybridoma technology, so that the prepared hexestrol ic-ELISA is high in sensitivity and high in reliability, and the ic-ELISA is wide in application prospect when being applied to non-diagnostic purpose detection of the hexestrol.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, and in particular to a method for establishing hexaneestrol ic-ELISA and application thereof. Background Art

[0002] Hexestrol (HES) is a synthetic nonsteroidal estrogen used clinically to treat prostate cancer, amenorrhea, uterine dysgenesis, functional uterine bleeding and menopausal syndrome. However, HES has been banned for use in humans due to its adverse effects on human health and reproductive system, and can also be transferred from treated animals and the environment to humans. Since HES can increase the weight of animals by promoting protein synthesis and inhibiting fat accumulation, the agricultural sector is still illegally using HES as a growth hormone to increase the growth rate of animal husbandry and aquaculture, which may lead to HES exposure in the environment. Therefore, humans are not only directly exposed to HES in the environment, it also enters the human body through the food chain, interferes with normal physiological processes, and causes serious health problems such as cancer, endometriosis, fetal malformations and metabolic disorders.

[0003] To date, many methods for detecting HES have been established, including gas chromatography coupled with mass spectrometry (GC-MS), liquid chromatography coupled with mass spectrometry (LC-MS), and high-performance liquid chromatography (HPLC). Although these methods are highly sensitive and selective, they are time-consuming, costly, and require professional technicians. Therefore, it is necessary to develop simpler methods to determine HES. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention proposes a method for establishing hexane estradiol ic-ELISA to solve the problems of the prior art such as long time consumption, high cost, and need for professional technicians.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] A method for establishing hexestrol ic-ELISA comprises the following steps:

[0007] 1) Preparation of HES-BSA and HES-OVA;

[0008] 2) preparing anti-HES mAbs using the HES-BSA prepared in step 1);

[0009] 3) Establishment of hexane estradiol ic-ELISA: Coat the ELISA plate with bicarbonate buffer and HES-OVA prepared in step 1), incubate, seal the wells, add anti-HES mAbs prepared in step 2) and diluted free HES standard in PBS solution, incubate, add GaM IgG-HRP to obtain hexane estradiol ic-ELISA.

[0010] Furthermore, the preparation process of HES-BSA and HES-OVA in step 1) is as follows: under N2 protection, 1100 mg HES and 3.8 g K2CO3-Al2O3 carrier reagent are added to 40.0 mL anhydrous acetone solution, and then 0.4 mL ethyl 4-bromobutyrate is added, and the mixture is stirred and heated overnight under light-proof conditions; the upper layer liquid is rotary evaporated to 10 mL, separated, extracted with methanol, dried, concentrated, and saponified and hydrolyzed to obtain the modified active HES (HES-MCPE); 70.0 mg HES-MCPE, 24.0 mg NHS, and 40 mg EDC are added to 2.0 mL DMF solution, and the mixture is fully reacted at room temperature for 5 h to obtain liquid No. I; 132.0 mg BSA is added to 4.0 mL The PBS solution was dissolved to obtain solution II. At 4°C, solution I was added dropwise to solution II to obtain the final solution. The final solution was reacted for 24 hours at room temperature, dialyzed for 3 days, and centrifuged to obtain HES-BSA. The chemical equations for the synthesis process of HES-MCPE (a) and the synthesis process of HES-BSA by the active ester method (b) are as follows:

[0011] @a@

[0012]

[0013] @b@

[0014]

[0015] Furthermore, the preparation process of HES-OVA in the step 1) is as follows: 70.0 mg HES-MCPE, 24.0 mg NHS, and 40.0 mg EDC are successively added to 2.0 mL DMF solution, and the mixture is fully reacted at room temperature for 5 hours to obtain solution III; 89.5 mg OVA is added to 4.0 mL PBS solution to dissolve to obtain solution IV, and solution III is added dropwise to solution IV at 4° C. to obtain a final solution; the final solution is reacted for 24 hours at room temperature, dialyzed for 3 days, and centrifuged to obtain HES-OVA.

[0016] Furthermore, the preparation process of the anti-HES mAbs is as follows: 80.0 mg HES-BSA and 150.0 μL FCA are completely emulsified, and three BALB / c mice are immunized. After 35 days, 80.0 mg HES-BSA and 150.0 μL Freund's incomplete adjuvant are used for the second to fifth immunizations of mice, and the interval between each immunization is 21 days; blood is collected from the tail vein of the mice, and blood is measured, and the mice with the strongest sensitivity are selected for hybridoma preparation; mouse spleen cells are fused with myeloma cells, and ascites is produced in paraffin-induced BALB / c mice after culture medium screening, identification, limiting dilution subcloning, and ascites is produced in paraffin-induced BALB / c mice, and anti-HES mAbs are obtained after centrifugation and purification.

[0017] Furthermore, the hexaneestrol ic-ELISA was constructed by the above-mentioned establishment method.

[0018] Furthermore, the establishment method of the hexane estradiol ic-ELISA is used in the detection of hexane estradiol for non-diagnostic purposes.

[0019] Furthermore, the ic-ELISA is used to detect HES in multiple environmental samples.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention mainly introduces a carboxyl group (-COOH) on the hydroxyl group of HES to transform the hapten HES, and couples it with a carrier protein to generate a complete antigen (HES-BSA), and produces anti-HES antibodies (anti-HES mAb) by immunizing animals with HES-BSA and using hybridoma technology. By establishing an indirect competitive ELISA method for detecting hexane estradiol residues, it is expected to enrich the residual screening data of hexane estradiol in multiple environmental samples and provide a new detection method for monitoring hexane estradiol in multiple environmental samples.

[0022] 2. The hexane estradiol ic-ELISA of the present invention is used to detect HES in multiple environmental samples. Experimental verification shows that the IC value of the hexane estradiol ic-ELISA constructed by the establishment method of the present invention is 50 The value is 1.64ng / mL, the detection limit is 0.44ng / mL, the sensitivity is high, and the practicality is strong. For example, the ic-ELISA method detects fish samples, and the recovery rate is 84.0%-92.9% at different addition concentrations, and the CV value is less than 9.1%. To evaluate the precision of the method, the recovery rate is 84.8%-90.9% at different addition concentrations, and the CV value is less than 7.9%. The cross-reaction rate with diethylstilbestrol is 7.94%, and there is no cross-reaction with other hormone analogs. These results confirm the reliability and repeatability of the established ic-ELISA method for detecting HES in fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation of the present invention, the following will briefly introduce the drawings required for use in the specific implementation. In all the drawings, each element or part is not necessarily drawn according to the actual scale.

[0024] Figure 1 UV scanning spectra of HES, HES-BSA and BSA.

[0025] Figure 2 It is the SDS-PAGE spectrum of Marker, BSA and HES-BSA.

[0026] Figure 3 The HES standard curve detected by test strips; wherein the x-axis represents the logarithmic concentration, and B / B0 represents the corresponding measured value when each concentration measured value is divided by 0 ng / mL. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings, the purpose of which is to better understand the content of the present invention rather than to limit the protection scope of the present invention.

[0028] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0029] Reagent Source

[0030] HES, RPMI-1640, hypoxanthine aminopterin thymidine (HAT), mouse mAb isotype analysis kit, and hypoxanthine thymidine (HT) were purchased from Sigma (St Louis, MO, USA).

[0031] PEG1500 (Mannheim, Germany).

[0032] 1-(3-(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) (Fluka, China).

[0033] BSA, OVA, Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) were purchased from BDH (VWR International Ltd).

[0034] ELISA plates and culture plates were purchased from Nunc (Roskilde, Denmark).

[0035] 4-bromobutyric acid ethyl ester, goat anti-mouse IgG antibody (GaM IgG-HRP), N,N-dimethylformamide (DMF) (Shanghai).

[0036] Seven-week-old female BALB / c mice were obtained from the SPF-grade standard experimental animal center of Zhengzhou University.

[0037] GS-NS0 myeloma cells were obtained from the Institute of Animal Health, UK.

[0038] All other reagents in the present invention are of analytical grade or higher.

[0039] Example 1 Preparation and identification of immunogens and coated antigens

[0040] Preparation of HES-BSA and HES-OVA. (1) Under the protection of N2, 1100 mg of hexaneestrol and 3.8 g of K2CO3-Al2O3 carrier reagent were added to 40.0 mL of anhydrous acetone solution, and then 0.4 mL of ethyl 4-bromobutyrate was added. The mixture was stirred and heated overnight under light-proof conditions. The upper layer was evaporated to 10.0 mL by rotary evaporation. The upper layer was separated by thin layer chromatography and extracted with methanol. After a series of operations such as drying, concentration, saponification and hydrolysis, the modified active HES (HESmono-caroxyl-propyl-ethyl, HES-MCPE) was obtained. (2) 70.0 mg of HES-MCPE, 24.0 mg of NHS and 40 mg of EDC were added to 2.0 mL of DMF solution. At room temperature, the mixture was fully reacted for 5 h (Solution I). 132.0 mg of BSA was added to 4.0 mL of PBS solution and fully dissolved (Solution II). At 4°C, solution I was added dropwise to solution II to obtain the final solution. At room temperature, the final solution was allowed to react for 24 hours, and then transferred to a dialysis bag with a pipette, dialyzed for 3 days with 1×PBS buffer, and centrifuged to obtain the complete antigen (HES-BSA). HES-OVA was prepared according to the same technical route. 70.0 mg HES-MCPE, 24.0 mg NHS, and 40.0 mg EDC were added to 2.0 mL DMF solution, and the reaction was fully carried out at room temperature for 5 hours to obtain solution III; 89.5 mg OVA was added to 4.0 mL PBS solution to dissolve solution IV, and solution III was added dropwise to solution IV at 4°C to obtain the final solution; the final solution was allowed to react for 24 hours at room temperature, dialyzed for 3 days, and centrifuged to obtain HES-OVA.

[0041] Identification of HES complete antigen: (1) UV scanning. Within the wavelength range, use a UV spectrophotometer to scan BSA and HES-BSA solutions (200-350nm) respectively, and observe whether the peak of HES-BSA is offset compared with the peak of BSA to determine whether HES and BSA are successfully coupled. (2) SDS-PAGE. After gel preparation and electrophoresis target treatment, add Marker, BSA and HES-BSA to the electrophoresis lane, and determine whether HES-BSA is successfully coupled based on the migration rate of the electrophoresis target. After UV scanning ( Figure 1 ) and SDS-PAGE( Figure 2 ) proved that the coupling was successful.

[0042] Example 2 Preparation of anti-HES mAbs

[0043] 80.0mg HES-BSA and 150.0μL Freund's complete adjuvant (FCA) were completely emulsified with an emulsifier and then immunized three BALB / c mice (multiple injections at the back subcutaneously). After 35 days, 80.0mg HES-BSA and 150.0μL Freund's incomplete adjuvant (FIA) were used for the second to fifth immunizations, and the interval between each immunization was 21 days. After the immunization procedure was completed, blood was collected from the tail vein of the mice, the characteristics of the mouse serum were determined, and the mice with the strongest sensitivity were selected for hybridoma preparation. The mouse splenocytes were fused with myeloma cells, screened and identified with a selective medium (HAT), subcloned by the limiting dilution method, and ascites was produced in paraffin-induced BALB / c mice. Monoclonal antibodies (mAbs) were obtained by centrifugation and purification.

[0044] Example 3 Establishment of indirect competitive ELISA assay method

[0045] The indirect competitive ELISA operation steps are as follows: (1) 96-well ELISA plates were coated with 0.05 mol / L bicarbonate buffer (pH 9.6) and HES-OVA, incubated at 37°C for 2 h, and then sealed with 5% skim milk at 37°C for 1 h. (2) Free HES standards and mAbs diluted in 1× PBS solution were added and incubated at 37°C for 30 min. (3) GaM IgG-HRP was added to each well and incubated at 37°C for 30 min. (4) TMB (3,30,5,5-tetramethylbenzidine) colorimetric solution was used for color development, and 2 mol / L H2SO4 was used to stop the color development. (5) The optical density (OD) was measured at 450 nm using a microplate reader. In steps (1) to (3), TBST [TBS solution containing 0.05% Tween 20 (v / v), pH 7.4] is used as a washing buffer to remove unbound antigens or antibodies.

[0046] Example 4 Sample pretreatment and performance evaluation of ic-ELISA

[0047] The fish samples purchased from the market were identified as negative by HPLC-MS. 6.0 g of negative fish meat (carp, HES-free) was homogenized, mixed with 12.0 ml of methanol, vortexed vigorously for 1 min, shaken for 10 min, and centrifuged at 4000 g for 5 min. The supernatant (1200.0 μL) was evaporated at 60 °C under a gentle N2 flow. The resulting residue was resuspended in 600 μL of a mixed solution [PBS and methanol (4:1, V / V)] as a negative sample.

[0048] According to the determined conditions, the HES standard was diluted to a series of concentrations (0, 1.0, 2.0, 4.0, 8.0, and 16.0 ng / mL), the calibration curve of ic-ELISA was established, and the IC 50 Value, see Figure 3 .

[0049] The sensitivity of ic-ELISA was determined by adding 2.0, 5.0 and 50.0 ng / mL HES to negative fish samples. Each sample was tested 6 times. The test results are shown in Table 1:

[0050] Table 1 Recovery rate of HES in fish detected by ic-ELISA method

[0051]

[0052] In order to evaluate the precision, 4.0, 10.0 and 30.0 ng / mL of HES were added to the fish samples and measured 6 times by ic-ELISA. The precision was expressed as CV. The test results are shown in Table 2:

[0053] Table 2 Recovery and precision of ic-ELISA method for detecting HES added to fish samples

[0054]

[0055] The cross-reactivity (CR) of the monoclonal antibody was measured using a panel of structural analogs of HES (e.g., DES, dienstilbestrol, BPA, E2, E3, and Prog). The CR value was calculated as follows: CR% = IC of HES 50 / Competitor IC 50 ×100%, the test results are shown in Table 3:

[0056] Table 3 Cross-reaction rate of ic-ELISA

[0057]

[0058] The data in Tables 1, 2 and 3 show that an IC-ELISA for detecting HES residues was established using high affinity and specific mAbs 2E6 against HES. The performance evaluation results of the method showed that the IC 50 The value was 1.64ng / mL, and the detection limit was 0.44ng / mL, indicating that the method had high sensitivity. At the same time, the detection ability of the method for fish samples was evaluated. At different addition concentrations, the recovery rate was 84.0%-92.9%, and the CV value was lower than 9.1%. To evaluate the precision of the method, at different addition concentrations, the recovery rate was 84.8%-90.9%, and the CV value was lower than 7.9%. The cross-reaction rate with diethylstilbestrol was 7.94%, and there was no cross-reaction with other hormone analogs. These results confirmed the reliability and repeatability of the established ic-ELISA method for detecting HES in fish.

[0059] The above embodiments are only used for illustration rather than limitation of the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for establishing hexaneestrol ic-ELISA, characterized in that: The following steps are involved: 1) Preparation of HES-BSA and HES-OVA; 2) preparing anti-HES mAbs using the HES-BSA prepared in step 1); 3) Establishment of hexane estradiol ic-ELISA: Coat the ELISA plate with bicarbonate buffer and HES-OVA prepared in step 1), incubate, seal the wells, add anti-HES mAbs prepared in step 2) and diluted free HES standard in PBS solution, incubate, add GaM IgG-HRP to obtain hexane estradiol ic-ELISA.

2. The method according to claim 1, characterized in that The preparation method of HES-BSA in the step 1) is as follows: under N2 protection, 1100 mg HES and 3.8 g K2CO3-Al2O3 carrier reagent are added to 40.0 mL anhydrous acetone solution, and then 0.4 mL ethyl 4-bromobutyrate is added, and the mixture is stirred and heated overnight under light-proof conditions; the upper layer liquid is rotary evaporated to 10 mL, separated, extracted with methanol, dried, concentrated, and saponified and hydrolyzed to obtain the modified active HES; 70.0 mg HES-MCPE, 24.0 mg NHS, and 40.0 mg EDC are added to 2.0 mL DMF solution, and the mixture is fully reacted at room temperature for 5 hours to obtain liquid No. I; 132.0 mg BSA is added to 4.0 mL PBS solution to dissolve to obtain liquid No. II, and liquid No. I is added dropwise to liquid No. II at 4°C to obtain the final liquid; the final liquid is reacted for 24 hours at room temperature, dialyzed for 3 days, and centrifuged to obtain HES-BSA.

3. The method according to claim 1, characterized in that The preparation method of HES-OVA in step 1) is as follows: 70.0 mg HES-MCPE, 24.0 mg NHS and 40.0 mg EDC are successively added to 2.0 mL DMF solution, and the mixture is fully reacted at room temperature for 5 hours to obtain liquid III; 89.5 mg OVA is added to 4.0 mL PBS solution to dissolve the mixture to obtain liquid IV; at 4° C., liquid III is added dropwise to liquid V to obtain a final solution; the final solution is reacted at room temperature for 24 hours, dialyzed for 3 days, and centrifuged to obtain HES-OVA.

4. The method according to claim 1, characterized in that: Step 2) The preparation method of the anti-HES mAbs is as follows: 80.0 mg HES-BSA and 150.0 μL FCA are completely emulsified and immunized into 3 BALB / c mice. After 35 days, 80.0 mg HES-BSA and 150.0 μL Freund's incomplete adjuvant are used for the 2nd to 5th immunization of mice, and the interval between each immunization is 21 days; blood is collected from the tail vein of the mice, and the blood is measured, and the mice with the strongest sensitivity are selected for hybridoma preparation; mouse spleen cells are fused with myeloma cells, and ascites is produced in paraffin-induced BALB / c mice by screening, identification, limiting dilution method, and subcloning, and anti-HES mAbs are obtained by centrifugation and purification.

5. Use of the method according to any one of claims 1 to 4 in the detection of hexylestradiol for non-diagnostic purposes.

6. The application according to claim 5, characterized in that: The ic-ELISA was used to detect HES in multiple environmental samples.

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