Preparation method of polyethylene glycol terephthalate artificial antigen

Abstract

The invention discloses a preparation method and application of a polyethylene glycol terephthalate (PET) artificial antigen and an egg yolk antibody (IgY) thereof. The preparation method comprises the following steps: preparing a PET-BSA artificial antigen by adopting an EDC coupling method, emulsifying the successfully coupled artificial antigen, injecting the emulsified artificial antigen into a healthy laying hen body for immunization, and extracting an IgY antibody from an immunized egg. The titer of the prepared IgY antibody reaches up to 1: 8000, and the IgY antibody has the advantages of high detection efficiency, strong specificity and reliable detection result when being used for detecting the PET content in an actual sample.

Description

Technical Field

[0001] The present invention relates to the preparation and application of specific egg yolk antibodies in the field of antibody technology, and in particular to a method for preparing polyethylene terephthalate artificial antigens, and a method for preparing anti-PET avian polyclonal antibodies and applications thereof. Background Art

[0002] Nanoplastics (MNPs) refer to microplastics with a diameter of less than 100 nm. Their potential accumulation in the environment and their impact on human health are the focus of current scientific research. Nanoplastics may enter the human body through the food chain, drinking water, air and other pathways. Studies have shown that exposure to microplastics and nanoplastics may exacerbate inflammation and affect distal organs, increasing the risk of non-communicable diseases such as cancer, diabetes, cardiovascular disease and chronic lung disease. Polyethylene terephthalate (PET) is a thermoplastic polyester with excellent physical and mechanical properties and electrical insulation. Therefore, PET is widely used in the food packaging industry and daily necessities, and is used unrestrainedly by humans, resulting in the accumulation of PET in the human body. This in turn has adverse effects on the human body, including potential inflammatory responses, endocrine disruption and cell damage, causing neurodegenerative diseases, reproductive problems, heart disease, cancer and autoimmune diseases. Given the important impact of PET on life systems and human health, it is of great significance to develop detection technologies that can identify PET. In the face of the challenges of PET, antibody development has become particularly urgent. Traditional antibody preparation methods (such as mouse or rabbit antibody preparation) may make it difficult to obtain specific antibodies against PET. Yolk antibodies (IgY) have gradually become an important tool in biomedical research due to their relatively simple preparation process, high yield, no mammalian pathogen contamination and low cross-reactivity. Therefore, the development of an efficient method for preparing yolk antibodies for PET is of great significance for further detailed evaluation of the long-term effects of nanoplastics on human health.

[0003] This patent provides a method for preparing a polyethylene terephthalate (PET) artificial antigen, a method for preparing an anti-PET avian polyclonal antibody, an indirect competitive enzyme-linked immunosorbent assay method for detecting the polyethylene terephthalate (PET) content, and an ic-ELISA detection kit for detecting the polyethylene terephthalate (PET) content. Summary of the invention

[0004] The first object of the present invention is to provide a method for preparing polyethylene terephthalate (PET) artificial antigen; The second object of the present invention is to provide a method for preparing an anti-PET avian polyclonal antibody; The third object of the present invention is to establish an immunoassay method for detecting polyethylene terephthalate; The fourth object of the present invention is to provide an ic-ELISA detection kit for detecting the content of polyethylene terephthalate (PET).

[0005] Specifically, the first object of the present invention is to provide a method for preparing a PET artificial antigen, the method comprising the following steps: (1) Dissolve 10 mg of BSA protein in 200 μL of ddH2O to prepare a 10 mg / mL BSA solution. (2) Add 2 mg of 80 nm carboxyl-modified PET into 450 μL MES (2-morpholineethanesulfonic acid) buffer to dissolve it; It should be noted that, except for PET with a particle size of 80 nm, PET with other particle sizes and other forms can be used for coupling and are within the scope of protection of this patent.

[0006] (3) Add 200 μL of BSA solution to 450 μL of PET solution to obtain a mixed solution of PET and BSA or OVA; (4) Add the PET-BSA or PET-OVA mixed solution into a 1.5 mL centrifuge tube containing EDC (10 mg) and incubate on a vortex roller at room temperature at 10 rpm / min for 3 h; (5) The conjugate was purified by dialyzing against 0.01 mol / L PBS (pH 7.2) to remove unreacted cross-linking agent to obtain PET-BSA artificial antigen.

[0007] It should be noted that the reaction ratio of the compound to the carrier protein is between 5:1 and 50:1. In addition to BSA, other carrier proteins, such as OVA, KLH, HAS, BTG, MSA, RSA or PDL, etc., are all within the scope of protection of this patent, that is, PET-OVA, PET-KLH, PET-HAS, PET-BTG, PET-MSA, PETRSA, PET-PDL artificial antigens are prepared respectively. A second object of the present invention is to provide a method for preparing an anti-PET avian polyclonal antibody, the method comprising the following steps: (1) PET-BSA was used as the immune antigen and mixed with Freund's complete adjuvant at a ratio of 1:1 and then emulsified for the primary immunization; then Freund's incomplete adjuvant was mixed with PET-BSA at a ratio of 1:1 and then emulsified for the booster immunization; (2) preparing polyclonal antibodies against PET artificial antigens, wherein the immune antigen obtained in the first object of the invention is injected into laying hens, and the eggs produced by the poultry are collected, marked, and stored in an environment of 4°C; (3) Extracting IgY, wherein the yolk and egg white are separated from the eggs collected as described above, and then IgY polyclonal antibodies are extracted.

[0008] It should be noted that the method for preparing immunoglobulin includes: using PET-BSA immune antigen to perform initial immunization injection on 21-week-old laying hens, with an immunization dose of 300 μg / hen, and performing three booster immunization injections 14 days after the initial immunization, with an interval of 14 days between each booster immunization and an immunization dose of 100-250 μg / hen.

[0009] It should be noted that the method for extracting IgY using the PEG6000 precipitation method includes the following steps: (1) Take the eggs obtained after four immunizations, separate the yolks, place them in a 50 mL round-bottom centrifuge tube, add 2 volumes of 0.01 mol / L PBS (pH 7.2) to dilute and mix thoroughly; after mixing thoroughly, add 3.5% (w / v) powdered PEG6000 while stirring; (2) Place the thoroughly mixed solution on a vortex roller and shake at 80-100 rpm / min for 30 min, then let it stand for 10 min and centrifuge at 10,000 rpm / min for 20 min at 4 °C. (3) After centrifugation, filter the clear liquid with filter paper and collect the filtrate into a new 50 mL centrifuge tube. Discard the filter paper precipitate. Add powdered PEG6000 to the supernatant to a final concentration of 8.5% (w / v). Vortex and mix thoroughly to completely dissolve PEG6000. (4) Place the mixture on a vortex roller at room temperature, shake at 80-200 rpm / min for 30 min, let stand at room temperature for 10 min, centrifuge at 10,000 rpm / min for 20 min at 4 °C, and collect the precipitate; (5) Resuspend the precipitate in 10 mL PBS (pH 7.2). After complete resuspending, add 12% (w / v) PEG6000 while stirring. Shake at 80 rpm / min for 30 min, let stand at room temperature for 10 min, centrifuge at 10,000 rpm / min for 20 min at 4 °C, and collect the precipitate. (6) Dissolve the precipitate with 2 mL of PBS and mix well. Place the mixture into a dialysis bag and dialyze it against PBS (pH 7.2) at 4 °C overnight. After dialysis, divide the IgY antibody solution into centrifuge tubes and store them at -80 °C.

[0010] It should be noted that this patent is not limited to PBS (pH 7.2). Buffer solutions of carbonate, Tris-HCl or other salts with a pH value in the range of 7-8 can be used for dialysis and are within the scope of protection of this patent.

[0011] The third object of the present invention is to establish an indirect competitive enzyme-linked immunosorbent assay method for detecting PET content, the method comprising the following steps: (1) Antigen coating: Prepare the PET-OVA conjugate with 0.01 mol / L PBS (pH 7.2) buffer to a concentration of 2 μg / mL, add 100 μL / well to a 96-well ELISA plate, and coat overnight at 4 °C. (2) Blocking: Discard the coating solution in the ELISA plate, add 200 μL PBS-T washing solution (0.01 mol / L PBS solution containing 0.05% Tween-20) to each well, shake it slightly horizontally, discard the washing solution and pat it dry, and repeat the washing three times; after washing the ELISA plate, add 200 μL of 5% skimmed milk powder solution (prepared by adding 1.0 g skimmed milk powder to 20 mL PBS buffer) to each well and incubate at 37 °C for 2 h; It should be noted that, in addition to skimmed milk powder, common blocking agents such as casein, gelatin, polyethylene glycol (PEG) and the like can be used for blocking and are all within the scope of protection of this patent.

[0012] (3) Competition: After washing the ELISA plate, prepare PET standards at concentrations of 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, and 640 μg / mL using 0.01 mol / L PBS (pH 7.2). Take 50 μL of each concentration of the standard and mix it with 50 μL of anti-PET polyclonal IgY (1:8000). After slight horizontal shaking for 10 seconds, incubate at 4 °C for 40 min. (4) Incubation with secondary antibody: After washing the ELISA plate, dilute the horseradish peroxidase (HRP)-labeled rabbit anti-chicken secondary antibody to 1:5000 with 0.01 mol / L PBS (pH 7.2) containing 2% skim milk powder. Add 100 μL of the diluted secondary antibody to each well and incubate at 37 °C for 40 min. It should be noted that: in addition to HRP enzyme-labeled rabbit anti-chicken secondary antibodies, other HRP-labeled mouse anti-chicken, horse anti-chicken, goat anti-chicken enzyme-labeled secondary antibodies can be applied to the established ELISA method, but are not limited to these HRP enzyme-labeled secondary antibodies. The biotin-labeled antibody and streptavidin-labeled horseradish peroxide (biotin / SA-HRP) system are all within the scope of protection of this patent.

[0013] (5) Color development: After washing the ELISA plate, mix 3,3',5,5'-tetramethylbenzidine (TMB) color development solution A and solution B in a ratio of 1:1, mix well and add to each well, adding 100 μL to each well for color development; (6) Detection: First, add 2 mol / L H2SO4 stop solution, 50 μL per well, to terminate the enzyme reaction; then use a full-wavelength microplate reader to measure the absorbance at a wavelength of 450 nm; (7) Calculation and determination: The average absorbance (B) of each standard divided by the absorbance at 0 μg / mL (B0) multiplied by 100% can be obtained to obtain the percentage of the corresponding absorbance of each standard, i.e., RLU; a curve is drawn with the ratio of the absorbance of each standard as the ordinate and the Log value of PET at different concentrations as the abscissa to obtain the competitive inhibition rate curve of the indirect competitive enzyme-linked immunosorbent assay, and the linear range and half inhibition rate IC of the curve can be obtained. 50 In a specific embodiment of the present invention, the PET content in the sample to be tested is calculated based on a standard curve.

[0014] The fourth object of the present invention is to provide an ic-ELISA detection kit for detecting PET content, wherein the kit comprises: a coated antigen, a primary antibody, an enzyme-labeled secondary antibody, a PET standard, a signal reagent, and the like.

[0015] In a specific embodiment of the present invention, the coated antigen is an artificial antigen obtained by coupling purified PET to a carrier protein.

[0016] In another specific embodiment of the present invention, the primary antibody is an anti-PET polyclonal IgY antibody.

[0017] In another specific embodiment of the present invention, preferably, the enzyme-labeled secondary antibody is rabbit anti-chicken IgG-HRP.

[0018] The beneficial effects of the present invention are: The present invention provides a method and application of a PET-BSA artificial antigen and its egg yolk antibody. The prepared egg yolk antibody can be used to detect and monitor PET and related nanoplastics. Compared with the prior art, the anti-PET antibody developed by the present invention has higher sensitivity and better detection effect. The method and steps for preparing the antibody are simple and convenient, and a large amount of PET-specific antibodies can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : PET artificial antigen UV scanning identification image; Figure 2 : PET artificial antigen SDS-PAGE electrophoresis; Figure 3:SDS-PAGE electrophoresis of PET-specific IgY antibody; Figure 4 : Inhibition rate curve of indirect competitive enzyme-linked immunosorbent assay. DETAILED DESCRIPTION

[0020] The above contents of the present invention are further described in detail below through embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1: Preparation of a polyethylene terephthalate artificial antigen (1) Dissolve 10 mg of BSA or OVA protein in 200 μL of ddH2O to prepare a 10 mg / mL BSA or OVA solution. It should be noted that the reaction ratio of the compound to the carrier protein is between 5:1 and 50:1. In addition to BSA or OVA, other carrier proteins, such as KLH, HAS, BTG, MSA, RSA or PDL, etc., are all within the scope of protection of this patent.

[0022] The compound has a degree of polymerization of 1.92×10 5 , molecular weight 3.69×10 7 g / mol microspheres.

[0023] (2) Add 2 mg of 80 nm carboxyl-modified PET into 450 μL MES (2-morpholineethanesulfonic acid) buffer to dissolve it; It should be noted that, except for PET with a particle size of 80 nm, PET with other particle sizes and other forms can be used for coupling and are within the scope of protection of this patent.

[0024] (3) Add 200 μL of BSA or OVA solution to 450 μL of PET solution to obtain a mixed solution of PET and BSA or OVA; (4) Add the PET-BSA or PET-OVA mixed solution into a 1.5 mL centrifuge tube containing EDC (10 mg) and incubate on a vortex roller at room temperature at 10 rpm / min for 3 h; (5) The conjugate was purified by dialyzing against 0.01 mol / L PBS (pH 7.2) to remove unreacted cross-linking agent to obtain PET-BSA or PET-OVA artificial antigen.

[0025] It should be noted that this patent is not limited to PBS (pH 7.2). Buffer solutions of carbonate, Tris-HCl or other salts with a pH value in the range of 7-8 are all within the scope of protection of this patent.

[0026] Example 2: A method for preparing an anti-PET avian polyclonal antibody (1) preparing PET-BSA or PET-OVA artificial antigens by the EDC coupling method, that is, the antigens are prepared by the method of Example 1; (2) emulsifying the antigen prepared in step (1): mixing the antigen with Freund's complete adjuvant at a ratio of 1:1 and emulsifying the mixture as the primary immunization antigen; and mixing Freund's incomplete adjuvant with the antigen at a ratio of 1:1 and emulsifying the mixture as the booster immunization antigen; (3) preparing anti-PET avian polyclonal antibodies, wherein PET-BSA immune antigens are regularly injected into laying hens, and eggs produced by the laying hens are collected, marked, and stored in an environment of 4°C; It should be noted that this patent is not limited to IgY antibodies produced by PET as an antigen. All antibodies obtained by immunizing animals with the PET artificial antigen invented by this patent, such as IgG, IgA, IgM, IgD, IgE, etc., are within the scope of protection of this patent.

[0027] (4) Extracting IgY, wherein the yolk and egg white are separated from the collected eggs, and then the IgY polyclonal antibody is extracted from the immunized eggs using a PEG6000 precipitation method.

[0028] It should be noted that the method for preparing immunoglobulin is as follows: PET-BSA immune antigen is used to perform the initial immunization injection on 21-week-old laying hens, with an immunization dose of 300 μg / hen. 14 days after the initial immunization, three booster immunization injections are performed, with an interval of 14 days between each booster immunization and an immunization dose of 100-250 μg / hen.

[0029] It should be noted that the method for extracting IgY using the PEG6000 precipitation method includes the following steps: (1) Take the eggs obtained after four immunizations, separate the yolks, place them in a 50 mL round-bottom centrifuge tube, add 2 volumes of 0.01 mol / L PBS (pH 7.2) to dilute and mix thoroughly; after mixing thoroughly, add 3.5% (w / v) powdered PEG6000 while stirring; (2) Place the thoroughly mixed solution on a vortex roller and shake at 80-100 rpm / min for 30 min, then let it stand for 10 min and centrifuge at 10,000 rpm / min for 20 min at 4 °C. (3) After centrifugation, filter the clear liquid with filter paper and collect the filtrate into a new 50 mL centrifuge tube. Discard the filter paper precipitate. Add powdered PEG6000 to the supernatant to a final concentration of 8.5% (w / v). Vortex and mix thoroughly to completely dissolve PEG6000. (4) Place the mixture on a vortex roller at room temperature, shake at 80-200 rpm / min for 30 min, let stand at room temperature for 10 min, centrifuge at 10,000 rpm / min for 20 min at 4 °C, and collect the precipitate; (5) Resuspend the precipitate in 10 mL PBS (pH 7.2). After complete resuspending, add 12% (w / v) PEG6000 while stirring. Shake at 80 rpm / min for 30 min, let stand at room temperature for 10 min, centrifuge at 10,000 rpm / min for 20 min at 4 °C, and collect the precipitate. (6) Dissolve the precipitate with 2 mL of PBS and mix well. Place the mixture into a dialysis bag and dialyze it against PBS (pH 7.2) at 4 °C overnight. After dialysis, divide the IgY antibody solution into centrifuge tubes and store them at -80 °C.

[0030] It should be noted that this patent is not limited to PBS (pH 7.2). Buffer solutions of carbonate, Tris-HCl or other salts with a pH value in the range of 7-8 can be used for dialysis and are within the scope of protection of this patent.

[0031] Example 3: Establishment of indirect competitive enzyme-linked immunosorbent assay (1) Antigen coating: Prepare the PET-OVA conjugate with 0.01 mol / L PBS (pH 7.2) buffer to a concentration of 2 μg / mL, add 100 μL / well to a 96-well ELISA plate, and coat overnight at 4 °C. (2) Blocking: Discard the coating solution in the ELISA plate, add 200 μL of PBST washing solution (0.01 mol / L PBS solution containing 0.05% Tween-20) to each well, slightly shake horizontally, discard the washing solution and pat dry, and repeat the washing three times; after washing the ELISA plate, add 200 μL of 5% skimmed milk powder solution (prepared by adding 1.0 g skimmed milk powder to 20 mL PBS buffer) to each well and incubate at 37 °C for 2 h; It should be noted that, in addition to skimmed milk powder, common blocking agents such as casein, gelatin, polyethylene glycol (PEG) and the like can be used for blocking and are all within the scope of protection of this patent.

[0032] (3) Competition: After washing the ELISA plate, prepare PET standards at concentrations of 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, and 640 μg / mL using 0.01 mol / L PBS (pH 7.2). Take 50 μL of each concentration of the standard and mix it with 50 μL of anti-PET polyclonal IgY (1:8000). Incubate at 4 °C for 40 min after slight horizontal shaking. (4) Incubation with secondary antibody: After washing the ELISA plate, dilute the horseradish peroxidase (HRP)-labeled rabbit anti-chicken secondary antibody to 1:5000 with 0.01 mol / L PBS (pH 7.2) containing 2% skim milk powder. Add 100 μL of the diluted secondary antibody to each well and incubate at 37 °C for 40 min. It should be noted that: in addition to HRP enzyme-labeled rabbit anti-chicken secondary antibodies, other HRP-labeled mouse anti-chicken, horse anti-chicken, goat anti-chicken enzyme-labeled secondary antibodies can be applied to the established ELISA method, but are not limited to these HRP enzyme-labeled secondary antibodies. The biotin-labeled antibody and streptavidin-labeled horseradish peroxide (biotin / SA-HRP) system are all within the scope of protection of this patent.

[0033] (5) Color development: After washing the ELISA plate, mix 3,3',5,5'-tetramethylbenzidine (TMB) color development solution A and solution B in a ratio of 1:1, mix well and add to each well, adding 100 μL to each well for color development; (6) Detection: First, add 2 mol / L H2SO4 stop solution, 50 μL per well, to terminate the enzyme reaction; then use a full-wavelength microplate reader to measure the absorbance at a wavelength of 450 nm; (7) Data processing and sample determination: The average absorbance (B) of each standard divided by the absorbance at 0 μg / mL (B0) multiplied by 100% can be obtained to obtain the percentage of the corresponding absorbance of each standard, that is, RLU / RLU0; a curve is drawn with the ratio of the absorbance of each standard as the ordinate and the Log value of PET at different concentrations as the abscissa to obtain the competitive inhibition rate curve of the indirect competitive enzyme-linked immunosorbent assay, and the monitoring range of the curve is 16.22 μg / mL to 204.17 μg / mL, and the half inhibition rate IC 50 The minimum detection limit IC is 57.68 μg / mL. 10 The results are shown in Figure 4 .

[0034] Example 4: A method for detecting using an ic-ELISA detection kit, comprising the following steps: (1) Take out the ELISA plate coated with the detection antigen from the kit and set up 3 wells for positive control and 3 wells for negative control; (2) Add 50 μL of diluted sample to the sample well, add 50 μL of positive and negative control to the positive and negative control wells, and then add 50 μL of primary antibody to each of the above wells and incubate at 4 °C for 40 min; (3) Discard the liquid in each well into a waste container and wash three times with PBST. Discard the liquid in the well after each wash. After the last wash, pat the remaining wash liquid in the well dry on absorbent paper. Add 100 μL of enzyme-labeled secondary antibody to each well, incubate at 37°C for 40 min, discard the liquid in the well, and wash three times with 0.01 mol / L PBST. (4) After washing the ELISA plate, mix 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution A and solution B in a 1:1 ratio, mix well and add to each well, adding 100 μL to each well for color development; (6) After color development, add 2 mol / L H2SO4 stop solution, 50 μL per well, to terminate the enzyme reaction; then use a full-wavelength microplate reader to measure the absorbance at 450 nm. (7) Calculation and determination: The average absorbance (B) of each standard divided by the absorbance at 0 μg / mL (B0) multiplied by 100% can be obtained to obtain the percentage of the corresponding absorbance of each standard, i.e., RLU; a curve is drawn with the ratio of the absorbance of each standard as the ordinate and the Log value of PET at different concentrations as the abscissa to obtain the competitive inhibition rate curve of the indirect competitive enzyme-linked immunosorbent assay, and the linear range and half inhibition rate IC of the curve can be obtained. 50 , and then the PET content in the sample to be tested can be calculated.

Claims

1. A method for preparing a polyethylene terephthalate artificial antigen, characterized in that: The following steps are included: (1) Dissolve 10 mg of BSA protein in 200 μL of ddH2O to prepare a 10 mg / mL BSA solution. (2) Add 2 mg of 80 nm carboxyl-modified PET into 450 μL MES (2-morpholineethanesulfonic acid) buffer to dissolve it; (3) Add 200 μL of BSA solution to 450 μL of PET solution to obtain a mixed solution of PET and BSA or OVA; (4) Add the PET-BSA or PET-OVA mixed solution into a 1.5 mL centrifuge tube containing EDC (10 mg) and incubate on a vortex roller at room temperature at 10 rpm / min for 3 h; (5) The conjugate was purified by dialyzing against 0.01 mol / L PBS (pH 7.2) to remove unreacted cross-linking agent to obtain PET-BSA artificial antigen.

2. The method for preparing the polyethylene terephthalate artificial antigen according to claim 1, characterized in that: In step (1), one of OVA, KLH, HAS, BTG, MSA, RSA or PDL proteins is used to prepare PET-OVA, PET-KLH, PET-HAS, PET-BTG, PET-MSA, PETRSA or PET-PDL artificial antigens.

3. A method for preparing an anti-PET avian polyclonal antibody, characterized in that: The steps include: (1) The artificial antigen PET-BSA prepared according to claim 1 or 2 or the artificial antigens PET-OVA, PET-KLH, PET-HAS, PET-BTG, PET-MSA, PETRSA, PET-PDL are used as the immunization antigen and mixed with Freund's complete adjuvant at a ratio of 1:1 and then emulsified for primary immunization; then Freund's incomplete adjuvant and PET-BSA are mixed at a ratio of 1:1 and then emulsified for booster immunization; (2) Preparing polyclonal antibodies against PET artificial antigens, wherein the immune antigen prepared according to claim 1 or 2 is injected into laying hens, and the eggs produced by the poultry are collected, marked, and stored in an environment of 4°C; (3) Extracting IgY, wherein the yolk and egg white are separated from the eggs collected as described above, and then IgY polyclonal antibodies are extracted.

4. The method for preparing the anti-PET avian polyclonal antibody according to claim 3, characterized in that: The method for preparing immunoglobulin includes: using PET-BSA immune antigen to perform initial immunization injection on 21-week-old laying hens, with an immunization dose of 300 μg / hen, and performing three booster immunization injections 14 days after the initial immunization, with an interval of 14 days between each booster immunization and an immunization dose of 100-250 μg / hen.

5. The method for preparing the anti-PET avian polyclonal antibody according to claim 3, characterized in that: IgY was extracted using the PEG6000 precipitation method, which included the following steps: (1) Take the eggs obtained after four immunizations, separate the yolks, place them in a 50 mL round-bottom centrifuge tube, add 2 volumes of 0.01 mol / L PBS (pH 7.2) to dilute and mix thoroughly; after mixing thoroughly, add 3.5% (w / v) powdered PEG6000 while stirring; (2) Place the thoroughly mixed solution on a vortex roller and shake at 80-100 rpm / min for 30 min, then let it stand for 10 min and centrifuge at 10,000 rpm / min for 20 min at 4 °C. (3) After centrifugation, filter the clear liquid with filter paper and collect the filtrate into a new 50 mL centrifuge tube. Discard the filter paper precipitate. Add powdered PEG6000 to the supernatant to a final concentration of 8.5% (w / v). Vortex and mix thoroughly to completely dissolve PEG6000. (4) Place the mixture on a vortex roller at room temperature, shake at 80-200 rpm / min for 30 min, let stand at room temperature for 10 min, centrifuge at 10,000 rpm / min for 20 min at 4 °C, and collect the precipitate; (5) Resuspend the precipitate in 10 mL PBS (pH 7.2). After complete resuspending, add 12% (w / v) PEG6000 while stirring. Shake at 80 rpm / min for 30 min, let stand at room temperature for 10 min, centrifuge at 10,000 rpm / min for 20 min at 4 °C, and collect the precipitate. (6) Dissolve the precipitate with 2 mL of PBS and mix well. Place the mixture into a dialysis bag and dialyze it against PBS (pH 7.2) at 4 °C overnight. After dialysis, divide the IgY antibody solution into centrifuge tubes and store them at -80 °C.

6. An immunoassay method for detecting polyethylene terephthalate, characterized in that The following steps are involved: (1) Antigen coating: The PET-OVA conjugate prepared in claim 2 was prepared with 0.01 mol / L PBS (pH 7.2) buffer to a concentration of 2 μg / mL, and 100 μL / well was added to a 96-well ELISA plate for coating at 4°C overnight; (2) Blocking: Discard the coating solution in the ELISA plate, add 200 μL PBST washing solution (0.01 mol / L PBS solution containing 0.05% Tween-20) to each well, shake it slightly horizontally, discard the washing solution and pat it dry. Repeat the washing three times, add 200 μL of 5% skim milk powder solution (prepared by adding 1.0 g skim milk powder to 20 mL PBS buffer) to each well, and incubate at 37 °C for 2 h; (3) Competition: After washing the ELISA plate, prepare PET standards at concentrations of 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, and 640 μg / mL using 0.01 mol / L PBS (pH 7.2). Take 50 μL of each concentration of the standard and mix it with 50 μL of anti-PET polyclonal IgY (1:8000). Incubate at 4 °C for 40 min after slight horizontal shaking. (4) Incubation with secondary antibody: After washing the ELISA plate, dilute the horseradish peroxidase (HRP)-labeled rabbit anti-chicken secondary antibody to 1:5000 with 0.01 mol / L PBS (pH 7.2) containing 2% skim milk powder. Add 100 μL of the diluted secondary antibody to each well and incubate at 37 °C for 40 min. (5) Color development: After washing the ELISA plate, mix 3,3',5,5'-tetramethylbenzidine (TMB) color development solution A and solution B in a ratio of 1:1, mix well and add to each well, adding 100 μL to each well for color development; (6) Detection: First, add 2 mol / L H2SO4 stop solution, 50 μL per well, to terminate the enzyme reaction, and then use a full-wavelength microplate reader to measure the absorbance at a wavelength of 450 nm; (7) Data processing and sample determination: The average absorbance (B) of each standard divided by the absorbance at 0 μg / mL (B0) multiplied by 100% can be obtained to obtain the percentage of the corresponding absorbance of each standard, i.e., RLU; a curve is drawn with the ratio of the absorbance of each standard as the ordinate and the Log value of PET at different concentrations as the abscissa to obtain the competitive inhibition rate curve of the indirect competitive enzyme-linked immunosorbent assay, and the monitoring range of the curve is 16.22 μg / mL to 204.17 μg / mL, and the half inhibition rate IC is 2.17 μg / mL. 50 The minimum detection limit IC is 57.68 μg / mL. 10 It is 10.65 μg / mL.

7. An ic-ELISA test kit for detecting the content of polyethylene terephthalate (PET), characterized in that The following steps are involved: (1) Take out the ELISA plate coated with the detection antigen from the kit and set up 3 wells for positive control and 3 wells for negative control; (2) Add 50 μL of diluted sample to the sample well, add 50 μL of positive and negative control to the positive and negative control wells, and then add 50 μL of primary antibody to each of the above wells and incubate at 4 °C for 40 min; (3) Discard the liquid in each well into a waste container and wash three times with PBST. Discard the liquid in the well after each wash. After the last wash, pat the remaining wash liquid in the well dry on absorbent paper. Add 100 μL of enzyme-labeled secondary antibody to each well, incubate at 37°C for 40 min, discard the liquid in the well, and wash three times with 0.01 mol / L PBST. (4) After washing the ELISA plate, mix 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution A and solution B in a 1:1 ratio, mix well and add to each well, adding 100 μL to each well for color development; (6) After color development, add 2 mol / L H2SO4 stop solution, 50 μL per well, to terminate the enzyme reaction; then use a full-wavelength microplate reader to measure the absorbance at 450 nm. (7) Calculation and determination: The average absorbance (B) of each standard divided by the absorbance at 0 μg / mL (B0) multiplied by 100% can be obtained to obtain the percentage of the corresponding absorbance of each standard, i.e., RLU; a curve is drawn with the ratio of the absorbance of each standard as the ordinate and the Log value of PET at different concentrations as the abscissa to obtain the competitive inhibition rate curve of the indirect competitive enzyme-linked immunosorbent assay, and the linear range and half inhibition rate IC of the curve can be obtained. 50 , and then the PET content in the sample to be tested can be calculated.

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