CN111718321A - Dioxin antibody and preparation method and application thereof - Google Patents
Dioxin antibody and preparation method and application thereof Download PDFInfo
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- CN111718321A CN111718321A CN202010558461.6A CN202010558461A CN111718321A CN 111718321 A CN111718321 A CN 111718321A CN 202010558461 A CN202010558461 A CN 202010558461A CN 111718321 A CN111718321 A CN 111718321A
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Abstract
The invention discloses a dioxin antibody and a preparation method and application thereof. Based on the immunological principle, the invention designs and synthesizes micromolecular hapten, couples with carrier protein to prepare effective artificial antigen, and prepares high specificity and high affinity antibody aiming at dioxin micromolecular compounds through immune animals. The specific immunoreaction of the antigen antibody and the amplification of the easily detected and identified marker are utilized to quantitatively detect trace dioxin small molecular target analytes in the sample, and the method has the characteristics of good specificity, high sensitivity, accuracy, rapidness, convenience, low price and the like.
Description
Technical Field
The invention belongs to the field of rapid detection, and relates to a dioxin antibody and a preparation method and application thereof.
Background
In recent years, environmental pollution incidents have become one of the social hotspots of public and government concern. Among the numerous environmental pollutants, persistent organic pollutants are of particular interest, wherein dioxin is of interest because of its very large potential toxicity. Experiments have shown that they can damage a variety of organs and systems. Dioxin, once it enters the human body, stays for a long time because it is chemically stable itself and easily absorbed by adipose tissues, and is accumulated in the body for a long time. Their half-life in vivo is estimated to be 7 to 11 years. In the environment, dioxins tend to accumulate in the food chain. And the higher the animal is in the food chain, the higher the degree of dioxin accumulation.
The chemical name of dioxin is: 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The name "dioxins" is commonly used to refer to polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) related in structure and chemical properties. Certain dioxin-like polychlorinated biphenyls (PCBs) have similar toxicity and are classified under the name of "dioxins". Approximately 419 dioxin-like compounds were identified, but only approximately 30 of them were considered to be quite toxic, with the greatest toxicity being TCDD, a typical persistent organic pollutant that is ubiquitous in soil, water, the atmosphere, and in food and feed.
Numerous epidemiological studies have shown that low concentrations of dioxins are present in human tissue, breast milk and serum. Today, high-resolution gas chromatography and high-resolution mass spectrometry (HRGC-HRMS) are "gold standards" for the determination of dioxins in media, but such analytical methods are time-consuming, expensive, require professional technicians to perform in specialized laboratories, and are not conducive to the screening analysis of large-scale samples. The immunological detection method is simple, rapid and sensitive in operation, can detect a plurality of samples simultaneously, and is an ideal rapid screening means.
In order to satisfy the analysis of samples in the fields of large-scale environment, food and sanitation, some simple and rapid methods need to be developed, particularly, rapid biological detection research and development are active.
Disclosure of Invention
The invention aims to provide a dioxin antibody and a preparation method and application thereof. In view of the defects of the method for testing the content of dioxin in soil, water, atmosphere, food and feed, the invention designs and synthesizes micromolecule hapten by relying on the basic immunological principle and the detection and analysis technical means, and the micromolecule hapten is coupled with carrier protein to prepare effective artificial antigen, immune animals are used for preparing specific antibody aiming at micromolecule analyte, thereby solving the problems of high cost, low sensitivity, low efficiency and the like of the traditional method for testing dioxin substances in media by using HRGC-HRMS. The method can be used for rapidly detecting dioxin substances.
The present invention claims hapten compounds of formula I,
the invention provides a method for preparing a hapten compound shown as a formula I, which comprises the following steps:
1) uniformly mixing 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and an organic solvent, adding ethyl 4-bromobutyrate, and reacting at the temperature of 50-70 ℃ for 6-12 hours to obtain a reaction mixture;
2) uniformly mixing the reaction mixture obtained in the step 1) with methanol, adding solid sodium hydroxide, and reacting at the temperature of 55-65 ℃ for 4-8 hours to obtain the catalyst.
In step 1) of the above method, the amount ratio of the feed materials of 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and ethyl 4-bromobutyrate is 1: 1-4; specifically 1: 2;
the organic solvent is DMF;
the dosage ratio of the 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin to the organic solvent is 20mg:1 mL;
in the reaction step, the temperature is 60 ℃; the time is 8 hours;
in the step 2), the volume ratio of the reaction mixture to the methanol is 15-16: 1; specifically, 15.5: 1;
the mass ratio of the sodium hydroxide to the feeding substance of the ethyl 4-bromobutyrate is 1: 5-8; specifically 1: 6.25;
in the reaction step, the temperature is 60 ℃; the time period required was 6 hours.
The method further comprises the following steps: after the reaction in the step 2) is finished, adding a saturated sodium bicarbonate aqueous solution at 25 ℃ into the obtained reaction solution for dissolving, extracting, removing an organic phase, adjusting the pH value of the obtained aqueous phase by using 0.5M hydrochloric acid until the precipitate is completely precipitated, washing with water, and evaporating to dryness;
specifically, in the extraction step, the extractant is ethyl acetate.
In addition, the application of the hapten compound shown in the formula I in preparing the dioxin antigen and the dioxin antigen containing the hapten compound shown in the formula I also belong to the protection scope of the invention.
Specifically, the hapten compound shown as the formula I is coupled with carrier protein;
the dosage ratio of the hapten compound shown in the formula I to the carrier protein is 1: 30-90; specifically 1: 60;
the carrier protein is bovine serum albumin, human serum albumin, keyhole limpet hemocyanin or ovalbumin.
In addition, the hapten compound shown in the formula I or the dioxin antigen provided by the invention is applied to preparing antibodies; and an antibody prepared by using the hapten compound shown in the formula I or the dioxin antigen as an immunogen, and an application of the hapten compound shown in the formula I or the dioxin antigen or the antibody in detecting dioxin also belong to the protection scope of the invention.
The invention also claims a kit or a test strip for detecting dioxin and dioxin-like pollutants, which comprises the dioxin antigen and the antibody.
The invention also claims a method for detecting dioxin and dioxin-like pollutants, which comprises the following steps: detecting with the dioxin antigen or the antibody; or the kit or the test strip for detecting the dioxin and dioxin pollutants is used for detection.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method quantitatively detects trace micromolecule target analytes in a sample by utilizing the specific immunological reaction of the antigen and the antibody and the amplification effect of the easily-detected and identified marker, and has the characteristics of high specificity, high sensitivity, accuracy, rapidness, convenience, low price and the like.
(2) The experimental result shows that the titer of antiserum obtained by immunizing animals by the antigen of the invention can reach 12.8 × 104The minimum detection limit is 3.1ng/L, the half inhibition concentration is 13.6ng/L, and the generated antibody has high specificity, high sensitivity and high accuracy.
(3) The antigen and the antibody of the invention can be used for establishing a competitive enzyme-linked immunosorbent assay technology, thereby being used for rapidly detecting the content of dioxin in a sample, and compared with the existing chromatography, the antigen and the antibody have the characteristics of low cost, simple operation, simple and convenient sample pretreatment, no pollution, convenience and quickness, are suitable for large-scale detection, have high yield of a preparation method, and have wide application prospects in the antigen, the antibody and the method.
(4) The dioxin detection kit or the test strip has high sensitivity, is simple and convenient to operate compared with an instrument method, has low personnel requirement, and can detect the magnitude order of magnitude of Pg.
(5) The dioxin detection kit or test strip has low detection cost, and compared with the HRGC-HRMS instrument detection method, the experimental cost is greatly reduced.
(6) The dioxin detection kit or test strip has short analysis period, and the advanced ASE sample pretreatment technology at home and abroad is introduced, so that the reaction time is greatly shortened.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
The invention discloses a preparation method of a dioxin antibody, which comprises the following steps:
1. preparation of hapten compound I of formula:
(1) uniformly mixing 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and DMF (dimethyl formamide) according to a (mg/ml)) ratio of 20:1, dropwise adding ethyl 4-bromobutyrate under the magnetic stirring condition of 400-500rpm, and reacting for 6-12 hours at a temperature of 50-70 ℃; the step is to dissolve the compound 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and make it fully react with ethyl 4-bromobutyrate; wherein, the temperature is preferably 60 ℃ and the time is preferably 8 hours; the mass ratio of the 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin to the ethyl 4-bromobutyrate is 1: 1-4, preferably the ratio is 1: 2.
(2) Mixing the reaction mixture in the step (1) and methanol according to a volume ratio of 15.5: 1, uniformly mixing, adding solid sodium hydroxide, and reacting for 4-8 hours at the temperature of 55-65 ℃; the mass ratio of the sodium hydroxide to the feeding substance of the ethyl 4-bromobutyrate is 1: 5-8, preferably 1: 6.25.
(3) adding 5ml of saturated sodium bicarbonate at 25 ℃ into the mixture obtained in the step (2) for dissolving, extracting by using ethyl acetate, discarding an organic phase, adjusting the pH value of a water phase by using 0.5M hydrochloric acid until the precipitate is complete, washing the precipitate by using distilled water, evaporating the precipitate by distillation to obtain a hapten compound I, and weighing the hapten compound I;
2. preparation of dioxin antigen compound (immunogen):
(4) the method comprises the following steps of mixing a compound shown as a formula I, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1: 1-2: 1-2, and reacting for 12-18 hours in a dark place at the temperature of 20-30 ℃ to obtain a uniformly dissolved solution I; this step is carried out in order to completely dissolve compound I, wherein the temperature is preferably 26 ℃ and the time is preferably 16 hours; the mass ratio of the compound shown in the step formula I, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1: 1-2: 1-2, preferably 1: 1.5;
(5) dissolving 10mg/ml carrier protein in 0.13M sodium bicarbonate water solution; wherein, the carrier protein can be bovine serum albumin, human serum albumin, keyhole limpet hemocyanin or ovalbumin, and most preferably bovine serum albumin;
(6) dropwise adding the solution I into a carrier protein solution, and standing and reacting for 16 hours at 4 ℃; then putting into a dialysis bag, dialyzing for 3 days in PBS buffer solution with the pH value of 7.4 and the concentration of 0.01M, and changing the PBS buffer solution for 3 times every day to obtain solution, namely immunogen, wherein the mass ratio of the carrier protein to the feeding substance of the compound I is 30-90: 1, preferably 60: 1;
3. preparation of dioxin antibodies:
(7) the preparation process of the antibody comprises the following steps: selecting female BALB/c healthy mice with 20 +/-2 g in 6-8 weeks as immune objects, injecting immunogen into the female BALB/c mice with 6-8 weeks every 2-3 weeks, emulsifying the immunogen by adopting a rapid emulsification method, completing the emulsification on ice for about 20min to ensure the stability and activity of the immunogen, generating antiserum in an immune period of 2-3 months, taking out spleens after the immunization is finished, grinding to obtain B cells, fusing the B cells with SP2/0 myeloma cells to prepare hybridoma cells, screening positive holes to obtain monoclonal cell strains which can stably secrete monoclonal cell strains capable of being specifically combined with dioxin, preparing the hybridoma cells into cell suspensions by using frozen stock solutions, preparing the hybridoma cells for later use, placing the hybridoma cells into cell culture media, culturing at 37 ℃, purifying the obtained culture solution by using an octanoic acid-saturated ammonium sulfate method to obtain an IgG antibody, storing at-20 deg.C.
Example 1
A preparation method of a dioxin antibody comprises the following steps:
1. preparation of hapten compound I of formula:
(1) uniformly mixing 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and DMF (dimethyl formamide) according to the (mg/ml)) ratio of 20:1, dropwise adding ethyl 4-bromobutyrate under the magnetic stirring condition of 400-500rpm, and reacting for 8 hours at the temperature of 60 ℃; the mass ratio of the 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin to the ethyl 4-bromobutyrate is 1: 2.
(2) Mixing the reaction mixture in the step (1) and methanol according to a volume ratio of 15.5: 1, uniformly mixing, adding solid sodium hydroxide, and reacting for 6 hours at the temperature of 60 ℃; the mass ratio of the sodium hydroxide to the ethyl 4-bromobutyrate is 1: 6.25.
(3) Adding 5ml of saturated sodium bicarbonate at 25 ℃ into the mixture obtained in the step (2) for dissolving, extracting by using ethyl acetate, discarding an organic phase, adjusting the pH value of a water phase by using 0.5M hydrochloric acid until the precipitate is complete, washing the precipitate by using distilled water, evaporating the precipitate by distillation to obtain a hapten compound I, and weighing the hapten compound I;
2. preparation of dioxin antigen compounds:
(4) mixing a compound shown in a formula I, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide according to the mass ratio of 1: 1.5 of the materials, and reacting for 16 hours in a dark place at the temperature of 26 ℃ to obtain a uniformly dissolved solution I; this step is to completely dissolve compound I;
(5) dissolving 10mg/ml bovine serum albumin in 0.13M sodium bicarbonate water solution;
(6) dropwise adding the solution I into a carrier protein solution, and standing and reacting for 16 hours at 4 ℃; then putting into a dialysis bag, dialyzing in PBS buffer solution with pH of 7.4 and 0.01M for 3 days, and changing the PBS buffer solution for 3 times every day to obtain solution which is immunogen, wherein the amount ratio of the bovine serum albumin to the feed material of the compound I is 60: 1;
3. preparation of dioxin antibodies:
(7) the preparation process of the antibody comprises the following steps: selecting a female BALB/c mouse with 6-8 weeks as an immune object, injecting immunogen into the female BALB/c mouse with 6-8 weeks every 2-3 weeks, generating antiserum after 2-3 months of the immune cycle, taking out spleen after the immune is finished, grinding to obtain B cells, fusing the B cells with SP2/0 myeloma cells to prepare hybridoma cells, screening positive holes to obtain a monoclonal cell strain which can be stably secreted and specifically combined with dioxin, preparing the hybridoma cells into cell suspension with cryopreservation liquid for later use, placing the hybridoma cells into a cell culture medium, culturing at 37 ℃, purifying the obtained culture liquid by an octanoic acid-saturated ammonium sulfate method to obtain a dioxin antibody, and storing at-20 ℃.
Example 2
A preparation method of a dioxin antibody comprises the following steps:
1. preparation of hapten compound I of formula:
(1) uniformly mixing 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and DMF (dimethyl formamide) according to the (mg/ml)) ratio of 20:1, dropwise adding ethyl 4-bromobutyrate under the magnetic stirring condition of 400-500rpm, and reacting for 12 hours at the temperature of 50 ℃; the mass ratio of the 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin to the ethyl 4-bromobutyrate is 1: 1;
(2) mixing the reaction mixture in the step (1) and methanol according to a volume ratio of 15.5: 1, uniformly mixing, adding solid sodium hydroxide, and reacting for 4 hours at the temperature of 65 ℃; the mass ratio of the sodium hydroxide to the feeding substance of the ethyl 4-bromobutyrate is 1: 5;
(3) adding 5ml of saturated sodium bicarbonate at 25 ℃ into the mixture obtained in the step (2) for dissolving, extracting by using ethyl acetate, discarding an organic phase, adjusting the pH value of a water phase by using 0.5M hydrochloric acid until the precipitate is complete, washing the precipitate by using distilled water, evaporating the precipitate by distillation to obtain a hapten compound I, and weighing the hapten compound I;
2. preparation of dioxin antigen compounds:
(4) the method comprises the following steps of mixing a compound shown as a formula I, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1: 1:2, mixing, and reacting for 18 hours in a dark place at the temperature of 20 ℃ to obtain a uniformly dissolved solution I;
(5) dissolving 10mg/ml bovine serum albumin in 0.13M sodium bicarbonate water solution;
(6) dropwise adding the solution I into a carrier protein solution, and standing and reacting for 16 hours at 4 ℃; then putting into a dialysis bag, dialyzing in 0.01M PBS buffer solution with pH7.4 for 3 days, changing the PBS buffer solution 3 times per day to obtain a solution which is the immunogen, wherein the dosage ratio of the carrier protein to the compound I is 30: 1;
3. preparation of dioxin antibodies:
(7) the preparation process of the antibody comprises the following steps: selecting a female BALB/c mouse with 6-8 weeks as an immune object, injecting immunogen into the female BALB/c mouse with 6-8 weeks every 2-3 weeks, generating antiserum after 2-3 months of the immune cycle, taking out spleen after the immune is finished, grinding to obtain B cells, fusing the B cells with SP2/0 myeloma cells to prepare hybridoma cells, screening positive holes to obtain a monoclonal cell strain which can be stably secreted and specifically combined with dioxin, preparing the hybridoma cells into cell suspension with cryopreservation liquid for later use, placing the hybridoma cells into a cell culture medium, culturing at 37 ℃, purifying the obtained culture liquid by an octanoic acid-saturated ammonium sulfate method to obtain a dioxin antibody, and storing at-20 ℃.
Example 3
A preparation method of a dioxin antibody comprises the following steps:
1. preparation of hapten compound I of formula:
(1) uniformly mixing 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and DMF (dimethyl formamide) according to the (mg/ml)) ratio of 20:1, dropwise adding ethyl 4-bromobutyrate under the magnetic stirring condition of 400-500rpm, and reacting for 6 hours at the temperature of 70 ℃; the step is to dissolve the compound 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and make it fully react with ethyl 4-bromobutyrate; the mass ratio of the 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin to the ethyl 4-bromobutyrate is 1: 4;
(2) mixing the reaction mixture in the step (1) and methanol according to a volume ratio of 15.5: 1, uniformly mixing, adding solid sodium hydroxide, and reacting for 8 hours at the temperature of 55 ℃; the mass ratio of the sodium hydroxide to the feeding substance of the ethyl 4-bromobutyrate is 1: 8;
(3) adding 5ml of saturated sodium bicarbonate at 25 ℃ into the mixture obtained in the step (2) for dissolving, extracting by using ethyl acetate, discarding an organic phase, adjusting the pH value of a water phase by using 0.5M hydrochloric acid until the precipitate is complete, washing the precipitate by using distilled water, evaporating the precipitate by distillation to obtain a hapten compound I, and weighing the hapten compound I;
2. preparation of dioxin antigen compounds:
(4) the method comprises the following steps of mixing a compound shown as a formula I, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1: 1-2: 1-2, and reacting for 12 hours in a dark place at the temperature of 30 ℃ to obtain a uniformly dissolved solution I;
(5) dissolving 10mg/ml egg white albumin in 0.13M sodium bicarbonate water solution;
(6) dropwise adding the solution I into a carrier protein solution, and standing and reacting for 16 hours at 4 ℃; then putting into a dialysis bag, dialyzing in PBS buffer solution with pH of 7.4 and 0.01M for 3 days, and changing the PBS buffer solution for 3 times every day to obtain solution which is immunogen, wherein the quantity ratio of the carrier protein to the feeding substance of the compound I is 90: 1;
3. preparation of dioxin antibodies:
(7) the preparation process of the antibody comprises the following steps: selecting a female BALB/c mouse with 6-8 weeks as an immune object, injecting immunogen into the female BALB/c mouse with 6-8 weeks every 2-3 weeks, generating antiserum after 2-3 months of the immune cycle, taking out spleen after the immune is finished, grinding to obtain B cells, fusing the B cells with SP2/0 myeloma cells to prepare hybridoma cells, screening positive holes to obtain a monoclonal cell strain which can be stably secreted and specifically combined with dioxin, preparing the hybridoma cells into cell suspension with cryopreservation liquid for later use, placing the hybridoma cells into a cell culture medium, culturing at 37 ℃, purifying the obtained culture liquid by an octanoic acid-saturated ammonium sulfate method to obtain a dioxin antibody, and storing at-20 ℃.
Example 4
A preparation method of a dioxin antibody comprises the following steps:
1. preparation of hapten compound I of formula:
(1) uniformly mixing 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and DMF (dimethyl formamide) according to the (mg/ml)) ratio of 20:1, dropwise adding ethyl 4-bromobutyrate under the magnetic stirring condition of 400-500rpm, and reacting for 10 hours at the temperature of 70 ℃; the step is to dissolve the compound 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and make it fully react with ethyl 4-bromobutyrate; the mass ratio of the 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin to the ethyl 4-bromobutyrate is 1: 2.5;
(2) mixing the reaction mixture in the step (1) and methanol according to a volume ratio of 15.5: 1, uniformly mixing, adding solid sodium hydroxide, and reacting for 6 hours at the temperature of 65 ℃;
(3) adding 5ml of saturated sodium bicarbonate at 25 ℃ into the mixture obtained in the step (2) for dissolving, extracting by using ethyl acetate, discarding an organic phase, adjusting the pH value of a water phase by using 0.5M hydrochloric acid until the precipitate is complete, washing the precipitate by using distilled water, evaporating the precipitate by distillation to obtain a hapten compound I, and weighing the hapten compound I;
2. preparation of dioxin antigen compounds:
(4) the method comprises the following steps of mixing a compound shown as a formula I, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1: 2: 2, mixing, and reacting for 14 hours in a dark place at the temperature of 24 ℃ to obtain a uniformly dissolved solution I;
(5) dissolving 10mg/ml human serum albumin in 0.13M sodium bicarbonate water solution;
(6) dropwise adding the solution I into a carrier protein solution, and standing and reacting for 16 hours at 4 ℃; then putting into a dialysis bag, dialyzing in 0.01M PBS buffer solution with pH7.4 for 3 days, and changing the PBS buffer solution 3 times per day to obtain a solution which is the immunogen, wherein the amount ratio of the carrier protein to the feed material of the compound I is 45:1, preferably 60: 1;
3. preparation of dioxin antibodies:
(7) the preparation process of the antibody comprises the following steps: selecting a female BALB/c mouse with 6-8 weeks as an immune object, injecting immunogen into the female BALB/c mouse with 6-8 weeks every 2-3 weeks, generating antiserum after 2-3 months of the immune cycle, taking out spleen after the immune is finished, grinding to obtain B cells, fusing the B cells with SP2/0 myeloma cells to prepare hybridoma cells, screening positive holes to obtain a monoclonal cell strain which can be stably secreted and specifically combined with dioxin, preparing the hybridoma cells into cell suspension with cryopreservation liquid for later use, placing the hybridoma cells into a cell culture medium, culturing at 37 ℃, purifying the obtained culture liquid by an octanoic acid-saturated ammonium sulfate method to obtain a dioxin antibody, and storing at-20 ℃.
Example 5
A preparation method of a dioxin antibody comprises the following steps:
1. preparation of hapten compound I of formula:
(1) uniformly mixing 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and DMF (dimethyl formamide) according to the (mg/ml)) ratio of 20:1, dropwise adding ethyl 4-bromobutyrate under the magnetic stirring condition of 400-500rpm, and reacting for 9 hours at the temperature of 65 ℃; the mass ratio of the 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin to the ethyl 4-bromobutyrate is 1: 3.3;
(2) mixing the reaction mixture in the step (1) and methanol according to a volume ratio of 15.5: 1, uniformly mixing, adding solid sodium hydroxide, and reacting for 6.5 hours at the temperature of 58 ℃; the mass ratio of the sodium hydroxide to the feeding substance of the ethyl 4-bromobutyrate is 1: 7;
(3) adding 5ml of saturated sodium bicarbonate at 25 ℃ into the mixture obtained in the step (2) for dissolving, extracting by using ethyl acetate, discarding an organic phase, adjusting the pH value of a water phase by using 0.5M hydrochloric acid until the precipitate is complete, washing the precipitate by using distilled water, evaporating the precipitate by distillation to obtain a hapten compound I, and weighing the hapten compound I;
2. preparation of dioxin antigen compounds:
(4) the method comprises the following steps of mixing a compound shown as a formula I, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1: 1.5: 2, mixing, and reacting for 16 hours in a dark place at the temperature of 30 ℃ to obtain a uniformly dissolved solution I;
(5) dissolving 10mg/ml egg white albumin in 0.13M sodium bicarbonate water solution;
(6) dropwise adding the solution I into a carrier protein solution, and standing and reacting for 16 hours at 4 ℃; then putting into a dialysis bag, dialyzing in PBS buffer solution with pH of 7.4 and 0.01M for 3 days, and changing the PBS buffer solution for 3 times every day to obtain solution which is immunogen, wherein the amount ratio of the carrier protein to the feed material of the compound I is 75: 1;
3. preparation of dioxin antibodies:
(7) the preparation process of the antibody comprises the following steps: selecting a female BALB/c mouse with 6-8 weeks as an immune object, injecting immunogen into the female BALB/c mouse with 6-8 weeks every 2-3 weeks, generating antiserum after 2-3 months of the immune cycle, taking out spleen after the immune is finished, grinding to obtain B cells, fusing the B cells with SP2/0 myeloma cells to prepare hybridoma cells, screening positive holes to obtain a monoclonal cell strain which can be stably secreted and specifically combined with dioxin, preparing the hybridoma cells into cell suspension with cryopreservation liquid for later use, placing the hybridoma cells into a cell culture medium, culturing at 37 ℃, purifying the obtained culture liquid by an octanoic acid-saturated ammonium sulfate method to obtain a dioxin antibody, and storing at-20 ℃.
Detailed implementation procedure
Preparing immunogen and coating antigen:
(1) hapten preparation
The first step is as follows: weighing 304mg of 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin, dissolving in 15.2ml of DMF, dropwise adding 280ul of 4-ethyl bromobutyrate under the magnetic stirring condition of 400-500rpm, and reacting for 8 hours at the controlled temperature of 60 ℃;
the second step is that: adding 1ml of methanol into the reaction mixture, adding 500mg of solid sodium hydroxide, and reacting for 6 hours under the condition of controlling the temperature to be 60 ℃;
the third step: evaporating the solvent of the reaction mixture, adding 5ml of saturated sodium bicarbonate at 25 ℃ for dissolving, extracting with ethyl acetate, and removing an organic phase; adjusting the pH value of the water phase with 0.5M hydrochloric acid until the precipitate is complete, washing the precipitate with water, evaporating the precipitate to dryness to obtain hapten, weighing 290mg, and converting the hapten into 74.3%.
(2) Preparation of immunogens
The first step is as follows: 3.9mg of the prepared hapten is weighed and dissolved in 0.2ml of DMF, 2.9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.7mg of N-hydroxysuccinimide are added, mixed evenly and reacted for 16 hours under the condition of magnetic stirring at 26 ℃ in a dark place.
The second step is that: taking 10mg of Bovine Serum Albumin (BSA), and dissolving in 1ml of 0.13M sodium bicarbonate water solution;
the third step: dropwise adding the hapten solution which is completely dissolved into the BSA solution, and standing for reaction for 16 hours at the temperature of 4 ℃; then filling into a dialysis bag, dialyzing in 0.01M PBS buffer solution with pH of 7.4 for 3 days, and changing the solution 3 times per day to obtain the solution which is the immunogen.
(3) Preparation of coating antigen
And (3) replacing BSA with egg albumin (OVA), and performing the same step (2) to obtain the coating antigen.
The first step is as follows: 3.9mg of the prepared hapten is weighed and dissolved in 0.2ml of DMF, 2.9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1.7mg of N-hydroxysuccinimide are added, mixed evenly and reacted for 16 hours under the condition of magnetic stirring at 26 ℃ in a dark place.
The second step is that: dissolving 10mg of egg albumin (OVA) in 1ml of 0.13M sodium bicarbonate water solution;
the third step: dropwise adding the hapten solution which is completely dissolved into the BSA solution, and standing for reaction for 16 hours at the temperature of 4 ℃; then filling into a dialysis bag, dialyzing in 0.01M PBS buffer solution with pH of 7.4 for 3 days, and changing the solution 3 times per day to obtain the solution which is the immunogen.
(II) animal immunization process
Taking the immunogen prepared in the step (I), and diluting the immunogen with PBS (phosphate buffer solution) with the pH value of 7.4 and the concentration of 0.01M to obtain immunogen diluent for preparing the monoclonal antibody. BALB/c was used as the immunized animal.
The immunization procedure was as follows (immunization dose is based on protein):
4 Balb/c female mice with 6-8 weeks of age are immunized by dioxin immunizing antigens respectively, and the numbers are 1#, 2#, 3#, and 4 #. The first immunization is carried out according to 100 mu g/mouse, the immunogen and Freund's complete adjuvant are dissolved by normal saline and uniformly mixed in equal volume, subcutaneous multipoint injection immunization is carried out on the neck and the back, after the first immunization, the immunogen and Freund's incomplete adjuvant are dissolved by normal saline and uniformly mixed in equal volume according to the immunization dose on 14, 28 and 42 days, and each additional immunization is carried out once. Blood is taken after tail breaking after one week of four-immunization, indirect ELISA and indirect competitive ELISA are adopted for detection, and the test method and the result are as follows:
preparing serum: after blood is taken, standing for 2h at room temperature, standing overnight at 4 ℃, freezing a centrifuge for 9000r/min, centrifuging for 10min, and taking supernatant, namely serum. Serum was diluted with PBS buffer to obtain serum dilutions.
The test procedure (dilution procedure, reaction steps, time) was as follows:
and (3) diluting: the dilution factor is larger than that of the dilution in steps (the added diluted body in the dilution, such as more than 5ul, is subtracted in the dilution and is not negligible).
Measuring the titer:
(1) diluting the coating source to 1ug/mL by PBS, and performing plate wrapping with each well at 100 ul;
(2) incubating overnight at 2-8 deg.C, sealing with sealing liquid, cleaning,
(3) adding 100ul serum diluent into the ELISA plate, incubating at 25 deg.C for 25min,
(4) pouring out the liquid in the hole, and washing the plate for 3 times by using the cleaning liquid;
(5) adding 100ul goat anti-mouse enzyme-labeled secondary antibody solution, incubating at 25 deg.C for 25min,
(6) pouring out the liquid in the hole, and washing the plate for 3 times by using the cleaning liquid;
(7) adding 100ul of color development solution, incubating at 25 deg.C for 15min,
(8) taking out and adding 100ul of stop solution, measuring OD value at 450nm, and the experimental data are shown in Table 1:
TABLE 1 OD values at different dilution times
Wherein OUT represents that the OD value is tested to be OUT of the testing range.
And (3) measuring inhibition:
(1) taking 50ul TCDD standard solution 200ppt, diluting gradually to 100ppt, 50ppt, 25ppt, 12.5ppt, 6.25ppt and 3.2 ppt. Adding into enzyme-linked immunosorbent assay plate, adding diluted serum antibody 50ul into the enzyme-linked immunosorbent assay plate, incubating for 25min at 25 ℃,
(2) pouring out the liquid in the hole, and washing the plate for 3 times by using the cleaning liquid;
(3) adding 100ul goat anti-mouse enzyme-labeled secondary antibody solution, incubating at 25 deg.C for 25min,
(4) pouring out the liquid in the hole, and washing the plate for 3 times by using the cleaning liquid;
(5) adding 100ul of color development solution, incubating at 25 deg.C for 15min,
(6) the solution was taken out and added with 100ul of stop solution, and OD was measured at 450 nm. The results obtained are shown in Table 2.
TABLE 2 OD values at different standard points
3. Calculation of monoclonal antibody sensitivity
3# Balb/c mice titer 12.8 × 104IC50 was 13.6ppt (ng/L) lowest, with a minimum detection limit of 3.1ppt (ng/L), boost immunity: 100 mu g of immunogen diluent is directly taken to carry out additional immunization once for the subcutaneous multi-point injection on the neck and the back of the 3# Balb/c mouse. Reference market kit, cell fusion conditions: the titer was 6 ten thousand or more, and the IC50 was 900 ppt.
4. Compared with the antibody (CAPE dioxin ELISA kit, cat # DF1-60) in the existing dioxin ELISA kit
TABLE 3 comparison with commercial antibodies
| Antibodies of the invention | OD value | Commercial antibodies | OD value |
| 200pg | 0.365 | —— | —— |
| 100pg | 0.437 | 100pg | 0.200 |
| 50pg | 0.547 | —— | —— |
| 25pg | 0.687 | —— | —— |
| 12.5pg | 0.850 | 32pg | 0.467 |
| 6.25pg | 0.968 | 10pg | 0.769 |
| 3.1pg | 1.173 | 3.2pg | 0.990 |
| 1.55pg | 1.305 | —— | —— |
| 0 | 1.668 | 0 | 1.159 |
| IC50 | 14.4 | IC50 | 20.3 |
Compared with the commercial antibody, the sensitivity of the commercial antibody is 3.2pg at most (the percentage is about 85 percent), and the sensitivity of the commercial antibody is at most 1.55pg (the percentage is about 78.8 percent).
(III) cell fusion
The mice are killed by pulling the neck, the body surface is disinfected by 75% alcohol, the spleen is taken out aseptically, the mice are put into RPMI-1640 basic culture solution, fascia and fat are carefully removed, the mice are cut into pieces, the pieces are placed into a stainless steel sieve (100 meshes), the cells are grinded aseptically to release single splenocytes, liquid containing the splenocytes is sucked and placed into a 50mL aseptic centrifuge tube, and the centrifugation is carried out.
Adding myeloma cells and the prepared spleen cells into the same 50mL centrifuge tube at a ratio of 5:1, adding 20mL of RPMI-1640 incomplete culture solution which is warm-bathed at 37 ℃, mixing uniformly, centrifuging (1500r/min, 6min), removing supernatant, tapping the bottom of the centrifuge tube with fingers, and mixing the precipitate uniformly to obtain paste. Taking 1mL of PEG preheated at 37 ℃ by a pipette, dripping the PEG into a centrifuge tube, standing for 1min, and dripping 10mL of RPMI-1640 complete culture solution into a water bath at 37 ℃ within 2 min. Centrifuging at 1000r/min for 6min, discarding supernatant, adding 75mL HAT culture solution, mixing, subpackaging the mixed suspension into 24-well plate with feeder cells, each well is 0.5mL, and culturing at 37 deg.C and 5% CO2Incubate in incubator saturated with humidity.
(IV) culture and selection of hybridoma cells
And (5) replacing the liquid with half amount of HT culture solution for 1 time 6-9 days after fusion, and changing to be the complete culture solution according to proliferation conditions after 12-14 days. The number of wells and total number of cells in which the hybridoma cells grew were counted when the cells adhered to the wells of the wells 1/3. Taking supernatant, and selecting positive hybridoma cells with high titer and strong inhibition by indirect competitive ELISA by indirect ELISA.
And (3) screening positive hybridoma cells by adopting an indirect ELISA method and an indirect competition ELISA method, wherein the hole which shows positive and has competition inhibition reaction is a hole for producing the dioxin antibody, and can be used for further subcloning.
Under the aseptic condition, eluting cells in positive holes, transferring the cells to a 96-hole culture plate paved with feeder cells in advance by using an elbow pipette, cloning each original hole into 8 holes, taking supernatant after the cells grow to the bottom of 1/2-1/3 holes fully adherent to the wall, carrying out indirect ELISA detection, taking subclones with strong positive, repeating the operation for 2-5 times, when the antibody positive rate in the supernatant of the 8 cloned holes is 100%, picking single-cell clones, transferring the detected cells to a 24-hole cell culture plate or a 25mL cell culture bottle for expanding culture, establishing strains, subpackaging, freezing at-20 ℃, injecting 0.5mL of pristane to the abdominal cavity of a Balb/c mouse one week ahead, taking the frozen cell strains, recovering, carrying out mass culture and propagation, collecting the cells, washing the cells twice by using incomplete culture medium, suspending by using 10mL of incomplete culture medium, counting, and transferring the cells (1 mL of each mouse, 3.1 × 10 contained in each mouse)7Individual cell) abdominal cavity injection mouse abdomen, and after 5-7 days, aseptically collecting ascites by using a No. 16 syringe when the mouse abdomen is obviously enlarged. Centrifuging at 2000r/min for 10min, removing upper layer fat, lower layer fibrin and cells, collecting middle layer, measuring titer by ELISA method, and subpackaging the rest at-70 deg.C for freezing.
(V) preparation and purification of monoclonal antibody
Taking about 3mL of ascites, adding 2 times of the volume of 0.06M, pH buffer solution of 4.5 sodium acetate, dropwise and slowly adding caprylic acid (33 mu g/mL) into the ascites sample while stirring, continuously stirring for 30min after the addition, centrifuging at 10000r/min at 4 ℃ for 30min, removing precipitates (albumin and other non-IgG proteins), filtering the supernatant through a 0.45 mu m microporous membrane, and mixing with 1/10 volume of 10 × PBS (10 × PBS: 80g NaCl, 2g KCl, 11.5g Na)2HPO4、2gKH2PO40.59g EDTA, 1L distilled water, pH7.4), adjusted to pH7.4 with 1M NaOH solution. The supernatant was cooled to 4 ℃ and ammonium sulfate (0.277g/mL, to give a final saturation of 45%) was added. Stirring for 30min at 4 deg.CCentrifuging at 10000r/min for 30min, and discarding the supernatant. The precipitate was dissolved in a small amount of PBS (pH7.4), dialyzed overnight against 50-100 volumes of PBS (pH7.4), and the solution was changed 3 times. The dialyzed solution was appropriately concentrated with PEG-6000 and stored at 4 ℃ for further use.
The technical solutions described above only represent the preferred technical solutions of the present invention, and some possible modifications to some parts of the technical solutions by those skilled in the art all represent the principles of the present invention, and fall within the protection scope of the present invention.
Claims (10)
1. Hapten compounds of the formula I,
2. a method of preparing a hapten compound of formula I comprising:
1) uniformly mixing 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin and an organic solvent, adding ethyl 4-bromobutyrate, and reacting at the temperature of 50-70 ℃ for 6-12 hours to obtain a reaction mixture;
2) uniformly mixing the reaction mixture obtained in the step 1) with methanol, adding solid sodium hydroxide, and reacting at the temperature of 55-65 ℃ for 4-8 hours to obtain the catalyst.
3. The method of claim 2, wherein: in the step 1), the mass ratio of the 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin to the ethyl 4-bromobutyrate is 1: 1-4; specifically 1: 2;
the organic solvent is DMF;
the dosage ratio of the 2-hydroxy-3, 7, 8-trichlorodibenzo-4-dioxin to the organic solvent is 20mg:1 mL;
in the reaction step, the temperature is 60 ℃; the time is 8 hours;
in the step 2), the volume ratio of the reaction mixture to the methanol is 15-16: 1; specifically, 15.5: 1;
the mass ratio of the sodium hydroxide to the feeding substance of the ethyl 4-bromobutyrate is 1: 5-8; specifically 1: 6.25;
in the reaction step, the temperature is 60 ℃; the time period required was 6 hours.
4. A method according to claim 2 or 3, characterized in that: the method further comprises the following steps: after the reaction in the step 2) is finished, adding a saturated sodium bicarbonate aqueous solution at 25 ℃ into the obtained reaction solution for dissolving, extracting, removing an organic phase, adjusting the pH value of the obtained aqueous phase by using 0.5M hydrochloric acid until the precipitate is completely precipitated, washing with water, and evaporating to dryness;
specifically, in the extraction step, the extractant is ethyl acetate.
5. Use of the hapten compound of the formula I according to claim 1 for the preparation of dioxin antigens;
a dioxin antigen comprising a hapten compound of the formula I according to claim 1.
6. The use or dioxin antigen according to claim 5, characterized in that: coupling the hapten compound shown in the formula I with carrier protein;
the dosage ratio of the hapten compound shown in the formula I to the carrier protein is 1: 30-90; specifically 1: 60;
the carrier protein is bovine serum albumin, human serum albumin, keyhole limpet hemocyanin or ovalbumin.
7. Use of the haptenic compound of formula I according to claim 1 or the dioxin antigen according to claim 5 or 6 for the preparation of antibodies;
an antibody prepared from the hapten compound of the formula I according to claim 1 or the dioxin antigen according to claim 5 or 6 as an immunogen.
8. Use of the haptenic compound of formula I according to claim 1 or the dioxin antigen according to claim 5 or 6 or the antibody according to claim 7 for the detection of dioxins.
9. A kit or a test strip for detecting dioxin and dioxin-like pollutants comprising the dioxin antigen according to claim 5 or 6 and the antibody according to claim 7.
10. A method of detecting dioxin and dioxin-like pollutants comprising: detecting with the dioxin antigen of claim 5 or 6 or the antibody of claim 7;
alternatively, the detection is carried out using the kit or the test strip for detecting dioxin and dioxin-like pollutants according to claim 10.
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