Method for detecting environmental estrogen

Abstract

The invention relates to the technical field of environmental monitoring and analysis, in particular to a detection method of environmental estrogen. According to the detection method, a recombinant human estrogen receptor GST-ER alpha-LBD is used as a biological recognition molecule, and an anti-GST antibody and a corresponding enzyme-labeled secondary antibody are used as reporter molecules; an estradiol derivative-bovine serum albumin conjugate with a proper connecting arm is coated on a microwell plate, and the equivalent concentration of environmental estrogen is detected in an indirect competition mode. The method for detecting the equivalent concentration of the environmental estrogen is accurate, reliable, sensitive, rapid, green and safe, the detection limit of the estradiol can be as low as 0.072 microgram / L, and the equivalent concentration of the environmental estrogen can be detected.

Description

Technical Field

[0001] This invention relates to the field of environmental monitoring and analysis technology, and specifically to a method for detecting environmental estrogens. Background Technology

[0002] Environmental estrogens are a class of substances that can exert estrogenic effects by binding to estrogen receptors and regulating estrogen signaling pathways. These substances have diverse chemical structures and are widely sourced, including not only natural estrogens produced by humans, animals, plants, and fungi, but also a large number of synthetic chemicals, such as pharmaceuticals, personal care products, and agrochemicals.

[0003] The release of natural estrogens is difficult to completely avoid, and synthetic chemicals with estrogenic activity are also difficult to effectively identify and control. Furthermore, traditional wastewater treatment processes cannot completely remove the estrogenic active components contained in these substances, leading to widespread residues of environmental estrogens in various environmental substrates. Events of feminization in fish caused by environmental estrogens occur frequently, and their persistent presence in the environment poses a serious threat to ecosystem stability and human health. Therefore, accurately identifying and quantifying the estrogen equivalent concentration in environmental samples is of great significance for environmental monitoring and risk assessment.

[0004] However, traditional instrument-based analytical methods (chromatography, mass spectrometry) cannot quantify all substances with estrogenic effects. While biologically based analytical methods such as uterine weight gain assays, cell proliferation assays, and yeast two-hybrid assays can reflect the estrogenic effect level of samples to some extent, they suffer from limitations such as poor experimental repeatability and long experimental cycles.

[0005] In contrast, receptor-based detection methods can specifically identify estrogen contaminants, enabling the identification of these substances and the detection of their equivalent concentrations. Currently used receptor binding assays often rely on radiolabeled ligands competing with the analyte for receptor binding, which can easily lead to radioactive contamination and pose certain safety risks. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting environmental estrogens, in order to solve the problem that existing detection methods mostly rely on radiolabeled ligands, which can easily lead to radioactive pollution.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: An estradiol derivative-bovine serum albumin conjugate, wherein the estradiol derivative-bovine serum albumin conjugate is prepared according to the following method: Take an estradiol derivative containing a carboxyl group. The structural formula of the estradiol derivative containing a carboxyl group is: , Where n represents the number of carbon atoms, and its values ​​include 1, 3, 5, and 7; corresponding to estradiol four-carbon linker derivatives, estradiol six-carbon linker derivatives, estradiol eight-carbon linker derivatives, and estradiol ten-carbon linker derivatives, respectively.

[0008] EDC and NHS were added to the above-mentioned estradiol derivative containing carboxyl groups, and the pH was adjusted to 6.0 with hydrochloric acid. The mixture was stirred at room temperature for 2 hours to activate the carboxyl groups of the estradiol derivative. The activated solution was then added dropwise to a BSA solution dissolved in 0.05 mol / L carbonate buffer solution and stirred at room temperature for 4 hours. Finally, small molecules that were not bound to BSA were removed by ultrafiltration or dialysis to obtain the estradiol derivative-bovine serum albumin conjugate.

[0009] Furthermore, the molar ratio of the estradiol derivative containing a carboxyl group to EDC and NHS is 1:6:4; the molar ratio of the estradiol derivative containing a carboxyl group to BSA is 10:1 to 80:1.

[0010] The present invention further provides an ELRBA kit for detecting estrogen equivalent concentration in environmental samples. The kit includes an enzyme-labeled plate, estradiol derivative-bovine serum albumin conjugate, coating buffer, washing buffer, blocking buffer, binding buffer, GST-ERα-LBD, estradiol standard stock solution, antibody dilution buffer, anti-GST antibody, enzyme-labeled secondary antibody, chromogenic solution, and stop solution.

[0011] Further, the ELISA plate is a 96-well high-binding-capacity ELISA plate; the coating buffer is 0.05 mol / L carbonate buffer, pH=9.6; the blocking solution includes 1-5% BSA (pH=7.4, 9.6) and 2-5% skim milk powder (pH=7.4, 9.6), preferably 5% BSA (pH=7.4); the binding buffer is 0.02 mol / L PBS with 0.05% EDTA; the estradiol standard stock solution is 1000 μg / mL estradiol methanol solution; the washing buffer and antibody dilution solution is 0.01 mol / L PBS with 0.05% Tween-20, pH=7.4; the anti-GST antibody includes rabbit anti-GST polyclonal antibody and rabbit anti-GST recombinant antibody, preferably rabbit anti-GST recombinant antibody; the ELISA-labeled secondary antibody is goat anti-rabbit IgG-HPR; the chromogenic solution is a TMB two-component chromogenic solution, namely solution A and solution B; the stop solution is 1 mol / L H2SO4.

[0012] Further, the concentration of the estradiol derivative-bovine serum albumin conjugate is 0.25~2 μg / mL, preferably 1 μg / mL; the concentration of the GST-ERα-LBD is 0.25~4 μg / mL, preferably 0.5 μg / mL; the dilution factor of the anti-GST antibody is 1:1000~1:4000, preferably 1:1000; the dilution factor of the enzyme-labeled secondary antibody can be 1:3000~1:10000, preferably 1:3000.

[0013] This invention also provides a method for using the above-mentioned ELRBA kit for detecting estrogen equivalent concentrations in environmental samples, as detailed below: (1) Coating: Add estradiol derivative-bovine serum albumin conjugate diluted with coating buffer to a 96-well high-binding enzyme plate at a concentration of 1 μg / mL, 100 μL per well, coat overnight at 4℃, and then wash the wells with washing buffer. Repeat 3 times. (2) Blocking: Add blocking solution, 200 μL per well, incubate at 37°C for 2 h, then wash the wells with washing buffer, repeat 3 times; (3) Competition: Add 50 μL of estradiol standard solution or sample solution to each well; then add 50 μL of GST-ERα-LBD diluted with binding buffer at a concentration of 0.5 μg / mL to each well; set up negative control and blank control wells at the same time; set up two parallel experiments, incubate at 4 ℃ for 60 min, and wash the wells with washing buffer, repeat 3 times; (4) Add antibody dilution buffer to the anti-GST antibody at a dilution ratio of 1:1000, 100 μL per well, incubate at room temperature for 60 min, and then wash the wells with washing buffer. Repeat 3 times. (5) Add the enzyme-labeled secondary antibody diluted with antibody dilution buffer at a dilution ratio of 1:3000, 100 μL per well, incubate at room temperature for 60 min, and then wash the wells with washing buffer. Repeat 3 times. (6) Add colorimetric reagents. Add 50 μL TMB colorimetric reagent A and 50 μL TMB colorimetric reagent B to each well and develop the color at room temperature for about 15 min. (7) Add stop solution, 50 μL per well, and stop the reaction at room temperature. The color in the well changes from blue to yellow. After stabilization, record the absorbance value at 450 nm using an ELISA reader. Analyze the absorbance values ​​of all wells after subtracting the absorbance values ​​of the negative control wells.

[0014] The beneficial effects of this invention are: This invention achieves the detection of equivalent concentrations of endocrine disruptors in samples through indirect competition. The process is highly specific and accurate, while avoiding the radioactive pollution problems caused by traditional methods where radiolabeled ligands compete with the test compound for binding receptors. It is more accurate, reliable, sensitive, rapid, and safe. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the principle of the method for detecting environmental estrogens in this invention. Figure 2 This is a standard curve of estradiol concentration versus absorbance value in the method of this invention. Detailed Implementation

[0016] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention.

[0017] The method of this invention uses recombinant human estrogen receptor GST-ERα-LBD as the recognition molecule, and anti-GST antibody and corresponding enzyme-labeled secondary antibody as reporter molecules. An estradiol derivative-bovine serum albumin conjugate with suitable linker arms is coated onto the surface of a microplate and then blocked. The immobilized derivative-bovine serum albumin conjugate competitively binds to GST-ERα-LBD and estrogen-active substances in the sample. The GST-ERα-LBD bound to the microplate surface can further bind to the anti-GST antibody and the corresponding enzyme-labeled secondary antibody, ultimately catalyzing the development of the chromogenic substrate. The magnitude of the chromogenic signal is inversely proportional to the equivalent concentration of environmental estrogen.

[0018] Example 1 In this embodiment, recombinant human estrogen receptor protein GST-ERα-LBD (GST tag-estrogen receptor α-ligand binding domain) was prepared.

[0019] 1. Expression of recombinant human estrogen receptor protein The plasmid expressing GST-ERα-LBD was transformed into E. coli BL21 competent cells. After recovery, the cells were plated on LB agar plates containing 50 mg / mL ampicillin. After incubation at 37°C for 16 h, single colonies were picked and transferred to LB agar containing 50 mg / mL ampicillin. The cells were then incubated at 37°C with shaking for 12 h. 20 mL of the bacterial culture was transferred to 150 mL of LB agar containing 50 mg / mL ampicillin. After incubation at 37°C with shaking for 1 h, the culture temperature was gradually decreased to 15°C over 3 h. IPTG was added to a final concentration of 1 mmol / L for low-temperature induction for 18 h. The cells were aliquoted into 50 mL tubes, centrifuged to obtain bacterial cells, and stored at -20°C.

[0020] 2. Extraction and purification of recombinant human estrogen receptor protein The bacterial cells were resuspended in 5 mL of lysis buffer containing 1 mmol / L PMSF (phenylmethylsulfonyl fluoride, a protease inhibitor), and sonicated under ice conditions. The supernatant was obtained by ultracentrifugation at 4 °C. The supernatant was added to 200 μL of GST agarose purification resin (commercially available; GST agarose purification resin is a resin that specifically binds to GST-tagged proteins and is modified with GSH), and the mixture was shaken at 4 °C for 2 h. The supernatant was discarded by centrifugation, and the GST-ERα-LBD bound to the surface of the purification resin was eluted with elution buffer containing GSH. The supernatant was collected, aliquoted, and stored at -20 °C for later use.

[0021] In this embodiment, GST is the glutathione S-transferase tag, and GSH is glutathione. GSH and GST can bind specifically, so the GSH-containing purification resin is used to specifically capture the recombinant estrogen receptor protein containing the GST tag in the solution, and then the receptor protein solution is obtained by elution with a GSH-containing elution buffer (the binding sites of GSH and GST are different from the binding sites of recombinant human estrogen receptor and environmental estrogens).

[0022] Example 2 In this embodiment, the estradiol derivative-bovine serum albumin conjugate of the present invention was prepared and a standard curve was established.

[0023] 1. Synthesis of estradiol 8-carbon linker derivative-bovine serum albumin conjugate In this embodiment, the estradiol derivative is an estradiol eight-carbon linker derivative containing a carboxyl group, with the following structural formula: , where n=5.

[0024] The estradiol derivative was prepared by activating the carboxyl group of the 8-carbon linker with EDC [1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride] and NHS (N-hydroxysuccinimide), followed by coupling with an amino group from bovine serum albumin. The specific steps are as follows: (1) Dissolve 10 mM estradiol octane linker derivative containing carboxyl group, 40 mM EDC and 60 mM NHS in aqueous solution, adjust pH to 6.0 with hydrochloric acid, stir at room temperature for 2 h to activate the carboxyl group of estradiol octane linker derivative. (2) Weigh 5 mg of bovine serum albumin powder and dissolve it in 1 mL of 0.05 mol / L carbonate buffer solution to obtain a concentration of 5 mg / mL; (3) The activated estradiol octane derivative was added dropwise to the bovine serum albumin solution at a molar ratio of 20:1 to the estradiol octane linker derivative containing a carboxyl group. The mixture was stirred at room temperature for 4 hours. (4) The solution after the reaction was ultrafiltered using a 30kDa ultrafiltration tube and washed three times with 0.01mol / L PBS buffer solution to remove the carboxyl estradiol octane linker derivatives and other small molecules that were not bound to bovine serum albumin. The ultrafiltered solution was collected and stored at -20℃.

[0025] 2. Establishment of the estradiol concentration-absorbance standard curve First, using a methanol standard stock solution of estradiol (1000 μg / mL), a gradient standard solution of estradiol was prepared with ultrapure water, with concentrations of 33.3 μg / L, 11.1 μg / L, 3.7 μg / L, 1.23 μg / L, 0.41 μg / L, 0.14 μg / L, 0.045 μg / L, 0.015 μg / L, 0.005 μg / L, and 0.0015 μg / L.

[0026] Then, testing is performed, and the specific steps are as follows: (1) Coating: Add estradiol octane linker derivative-bovine serum albumin conjugate diluted with coating buffer (0.05 mol / L carbonate buffer) to a 96-well high-binding microplate at a concentration of 1 μg / mL, 100 μL per well. Coat overnight at 4°C and then wash the wells with washing buffer (0.01 mol / L PBS with 0.05% Tween-20, pH=7.4, the same below). Repeat 3 times. (2) Blocking: Add 5% BSA blocking solution, pH 7.4, 200 μL per well, incubate at 37℃ for 2 h, then wash the wells with washing buffer, repeat 3 times; (3) Competition: Add 50 μL of estradiol gradient standard solution to each well; then add 50 μL of GST-ERα-LBD (prepared in Example 1) diluted with binding buffer (0.02 mol / L PBS with 0.05% EDTA) to each well at a concentration of 0.5 μg / mL; set up negative control and blank control wells at the same time; set up two parallel experiments, incubate at 4℃ for 60 min, and wash the wells with washing buffer, repeat 3 times; (4) Add the rabbit anti-GST recombinant antibody diluted with antibody dilution buffer (0.01 mol / L PBS with 0.05% Tween-20, pH=7.4) at a dilution ratio of 1:1000, 100 μL per well, incubate at room temperature for 60 min, and then wash the wells with washing buffer. Repeat 3 times. (5) Add the goat anti-rabbit IgG-HRP diluted with antibody diluent at a ratio of 1:3000, 100 μL per well, incubate at room temperature for 60 min, and then wash the wells with washing buffer. Repeat 3 times. (6) Add colorimetric solution (TMB two-component colorimetric solution). Add 50 μL of TMB colorimetric solution A and 50 μL of TMB colorimetric solution B to each well and develop the color at room temperature for about 15 min. (7) Add stop solution (1 mol / L H2SO4), 50 μL per well, and stop the reaction at room temperature. The color in the well changes from blue to yellow. After stabilization, record the absorbance value at 450 nm using an ELISA reader. Analyze the absorbance values ​​of all wells after subtracting the absorbance values ​​of the negative control wells.

[0027] In this example, the relationship between estradiol concentration and absorbance value is fitted using a logistic equation. Figure 2 ), Correlation coefficient R 2 The value is 0.99, so the detection limit of the ELRBA kit for estradiol is 0.072 μg / L, and the linear range is 0.12–0.79 μg / L. Therefore, based on the standard curve and the absorbance value of the sample solution, the equivalent concentration of environmental estrogens in the sample solution can be obtained.

[0028] This invention is not limited to the preferred embodiments described above. Anyone can derive other forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. An estradiol derivative-bovine serum albumin conjugate, characterized in that: The carboxyl group of the estradiol derivative containing carboxyl group is coupled with the amino group in the bovine serum albumin to obtain.

2. The conjugate of estradiol derivative-bovine serum albumin according to claim 1, wherein The estradiol derivative containing carboxyl group has the following structural formula: ， The value of n includes 1, 3, 5 and 7.

3. The method of preparing the estradiol derivative-bovine serum albumin conjugate according to claim 1 or 2, characterized by: The estradiol derivative containing carboxyl group is activated by an activating agent, and then reacts with the bovine serum albumin.

4. The method for preparing a conjugate of estradiol derivative-bovine serum albumin according to claim 3, wherein the step of reacting the estradiol derivative with the bovine serum albumin is carried out in the presence of a coupling agent. The activating agent includes EDC and NHS.

5. A method for detecting an environmental estrogen, characterized by: The estradiol derivative-bovine serum albumin conjugate is used to detect the equivalent concentration of environmental estrogen in a sample by means of indirect competition between the estradiol derivative-bovine serum albumin conjugate and the environmental estrogen in the sample for binding to the estrogen receptor.

6. The method of claim 5, wherein the environmental estrogen is selected from the group consisting of bisphenol A, bisphenol F, bisphenol S, and combinations thereof. The estrogen receptor is recombinant human estrogen receptor GST-ERa-LBD, GST is glutathione S-transferase tag, ERa is estrogen receptor, and LBD is ligand binding domain.

7. The method of claim 6, wherein the environmental estrogen is selected from the group consisting of bisphenol A, bisphenol F, bisphenol S, and combinations thereof. The estradiol derivative-bovine serum albumin conjugate is coated on an enzyme-labeled plate, blocked by a blocking solution, and then added with a gradient standard solution of estradiol and a sample solution, followed by addition of the estrogen receptor, washing with a washing buffer, and then added with a labeled antibody and a corresponding enzyme-labeled secondary antibody, followed by color development with a color developing solution, termination of the reaction, and recording of the absorbance value; an absorbance value curve of the gradient standard solution of estradiol is established, and then the equivalent concentration of environmental estrogen in the sample solution is determined according to the absorbance value of the sample solution.

8. The method of claim 7, wherein the environmental estrogen is selected from the group consisting of bisphenol A, bisphenol F, bisphenol S, and combinations thereof. The labeled antibody is an anti-GST antibody, including rabbit anti-GST polyclonal antibody and rabbit anti-GST recombinant antibody; and the enzyme-labeled secondary antibody includes goat anti-rabbit IgG-HPR.

9. An environmental estrogen detection kit, characterized by: The estradiol derivative-bovine serum albumin conjugate is used to detect the equivalent concentration of environmental estrogen in a sample by means of indirect competition between the estradiol derivative-bovine serum albumin conjugate and the environmental estrogen in the sample for binding to the estrogen receptor.

10. An environmental estrogen detection kit according to claim 9, characterized by: The kit further includes an enzyme-labeled plate, a coating buffer, a washing buffer, a blocking solution, a binding buffer, GST-ERa-LBD, an estradiol standard stock solution, an antibody diluent, an anti-GST antibody, an enzyme-labeled secondary antibody, a color developing solution and a termination solution; in the GST-ERa-LBD, GST is glutathione S-transferase tag, ERa is estrogen receptor, and LBD is ligand binding domain.

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