CN108776223B - Ultraviolet absorbent UV-326 detection kit and preparation method and application thereof - Google Patents
Ultraviolet absorbent UV-326 detection kit and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an ultraviolet absorbent UV-326 detection kit, a preparation method and application thereof, and belongs to the technical field of additive detection. The kit comprises: the ultraviolet absorbent UV-326 specific antigen working solution is a conjugate of an ultraviolet absorbent UV-326 and carrier protein; an ultraviolet absorbent UV-326 specific antibody working solution, wherein the ultraviolet absorbent UV-326 specific antibody is a murine monoclonal antibody. The kit detects the ultraviolet absorbent UV-326 by utilizing a competitive enzyme-linked immunoassay method, and has the characteristics of high specificity, accurate result, simple pretreatment, low requirements on instruments and equipment and low detection cost. Meanwhile, the reagent in the kit is provided in the form of working solution, the operation is simple and rapid, the time is saved for a user, and the error caused by complicated steps is reduced. Is suitable for quickly and accurately detecting the ultraviolet absorbent UV-326 in a large batch of samples.
Description
Technical Field
The invention relates to the technical field of additive detection, in particular to an ultraviolet absorbent UV-326 detection kit and a preparation method and application thereof.
Background
The ultraviolet absorbent UV-326 is 2'- (2' -hydroxy-3 '-tert-butyl-5' -methylphenyl) -5-chlorobenzotriazole which is a representative benzotriazole ultraviolet absorbent and can effectively absorb ultraviolet rays with the wavelength of 270nm to 380 nm.
In the field of food packaging, various packaging films, bags, drums, boxes, bottles, cans, composite packaging materials and the like made of plastics are widely used, and the usage amount is increasing year by year. However, under the irradiation of sunlight and some artificial light, these high molecular materials are liable to generate autoxidation reaction under the action of ultraviolet light, resulting in degradation of the polymer, deterioration of appearance and mechanical properties, and reduction of service life. To prevent the damaging effects of ultraviolet light, ultraviolet light stabilizers have been used to address the degradation of plastics under sun exposure.
UV absorbers are one of the main classes of light stabilizers, which effectively absorb UV light and release it as harmless heat energy, thereby acting to inhibit the degradation of plastics by UV light. Research shows that part of ultraviolet absorbers are toxic and harmful, and can affect reproduction, development and the like of organisms after being taken for a long time, and materials in contact with food can be transferred into the food through absorption, dissolution, diffusion and other ways if the ultraviolet absorbers are contained, so that the health of consumers is harmed. The European Union directive (EU) No.10/2011 specifies the maximum residual levels of UV-326, UV-327 and UV-234 in food-contact plastic materials and articles. GB 9685-2016 national Standard for food safety standards for the use of food contact materials and additives for products also specifies the types of UV absorbers (including UV-326, UV-327, UV-329, UV-531, UV-71, UV-24, UV-9, UV-0), the range of use and the maximum amount of use and the associated maximum residual amount or specific migration limit, such as a specific migration limit [ SML (T) ] of UV-326 of 30 mg/kg. In addition, in the field of daily chemical products such as cosmetics, some illegal trade people also add ultraviolet absorbers to the sunscreen cosmetics in an over-range manner. In view of this, a rapid detection method of the ultraviolet absorbent of the related product is established, regular monitoring and safety evaluation are carried out, and the method has profound significance for standardizing the industrial development, ensuring the product quality, protecting the physical health of consumers, protecting the environment and the like.
At present, the ultraviolet absorbent is mainly determined by liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry and the like; the pretreatment method mainly comprises Solid Phase Extraction (SPE), Soxhlet Extraction (SE), rapid solvent extraction (ASE), microwave-assisted extraction (MAE), Supercritical Fluid Extraction (SFE), Ultrasonic Extraction (UE) and the like; the method combining chromatography and chromatographic mass spectrometry has the advantages of high specificity and accurate result, but the two methods have the defects of complicated sample pretreatment, large solvent consumption, expensive laboratory equipment and high detection cost.
The ultraviolet absorbent UV-326 is one of the ultraviolet absorbents, and a suitable rapid detection method is also needed.
Disclosure of Invention
Therefore, it is necessary to provide an ultraviolet absorbent UV-326 detection kit and a preparation method thereof, which can continuously detect a plurality of samples at one time when detecting the ultraviolet absorbent UV-326 by using a cover kit, and have the characteristics of convenience, rapidness, sensitivity and low cost.
An ultraviolet absorber UV-326 detection kit comprising:
the ultraviolet absorbent UV-326 specific antigen working solution is a conjugate of an ultraviolet absorbent UV-326 and carrier protein;
an ultraviolet absorbent UV-326 specific antibody working solution, wherein the ultraviolet absorbent UV-326 specific antibody is a murine monoclonal antibody.
The kit detects the ultraviolet absorbent UV-326 by utilizing a competitive enzyme-linked immunoassay method, and has the characteristics of high specificity, accurate result, simple pretreatment, low requirements on instruments and equipment and low detection cost.
In one embodiment, the carrier protein is one of bovine serum albumin, ovalbumin, rabbit serum albumin, and thyroglobulin.
In one embodiment, the ultraviolet absorbent UV-326 specific antigen is coated on the ELISA plate to form a specific antigen ELISA plate; the kit also comprises an enzyme-labeled secondary antibody, a washing solution, a substrate color developing agent, a stop solution, a standard solution, a confining solution and a concentrated sample diluent.
In one embodiment, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat-anti-mouse secondary antibody diluted by a ratio of 1:10000 in concentration.
In one embodiment, the material of the ELISA plate is one of polystyrene, polyethylene and polypropylene;
the washing solution is 0.01M phosphate buffer solution with pH7.4 containing 0.05-0.5% Tween-20;
the substrate color developing agent consists of a color developing agent A and a color developing agent B, wherein the color developing agent A is hydrogen peroxide or carbamide peroxide, and the color developing agent B is o-phenylenediamine or tetramethyl benzidine;
the stop solution is 1-2mol/L sulfuric acid solution;
the concentration of UV-326 in the standard solution is 2 x 10 respectively5μg/L、2×104μg/L、2×103μg/L、2×102μg/L、2×101μg/L、2×100μg/L；
The confining liquid is 0.01M phosphate buffer solution with pH of 7.4 and containing 5% of skimmed milk powder;
the concentrated sample diluent was 0.01M phosphate buffer ph 7.4.
In one embodiment, the ultraviolet absorber UV-326 specific antigen is prepared by the following method: dispersing ultraviolet absorbers UV-326, 3-amino-1-propanol and triphenylphosphine in dry tetrahydrofuran, performing ultrasonic treatment, adding diisopropyl azodicarboxylate in the ultrasonic treatment process, continuing the ultrasonic reaction, and separating by column chromatography to obtain hapten of the ultraviolet absorber UV-326 capable of being coupled with protein; then mixing the antigen with N-hydroxysuccinimide and carbodiimide, reacting, and coupling with carrier protein to prepare an ultraviolet absorbent UV-326 specific antigen;
the ultraviolet absorbent UV-326 specific antibody is prepared by the following method: immunizing a mouse by taking the synthesized UV-326 specific antigen of the ultraviolet absorbent as an immunogen; after immunization, taking a mouse with high serum titer, and taking spleen cells of the mouse to perform cell fusion with myeloma cells SP 2/0; screening hybridoma cells by adopting a limiting dilution method to obtain a completely homogeneous monoclonal antibody and a stable monoclonal hybridoma cell strain; and injecting the mice sensitized by the incomplete adjuvant into the abdominal cavity of the mice, injecting the hybridoma cell suspension into the abdominal cavity of the mice, collecting ascites, and purifying the ascites by an octanoic acid-ammonium sulfate precipitation method to obtain the purified UV-326 monoclonal antibody as the ultraviolet absorbent.
In one embodiment, the ultraviolet absorber UV-326 specific antigen is prepared by the following method: dissolving 0.32g of ultraviolet absorbent UV-326, 0.075g of 3-amino-1-propanol and 0.29g of triphenylphosphine in 2.0mL of dry Tetrahydrofuran (THF), carrying out ultrasonic treatment at normal temperature for 5min, dropwise adding 0.22mL of diisopropyl azodicarboxylate in the ultrasonic treatment process, continuing the ultrasonic reaction for 20min, and separating by using column chromatography after the reaction is finished to prepare hapten of the ultraviolet absorbent UV-326 capable of being coupled with protein; mixing the antigen with N-hydroxysuccinimide and carbodiimide, reacting, and coupling with bovine serum albumin to obtain an ultraviolet absorbent UV-326 specific antigen;
the ultraviolet absorbent UV-326 specific antibody is prepared by the following method: immunizing a 10-week-old Balb/C mouse by taking the synthesized UV-326 specific antigen of the ultraviolet absorbent as an immunogen, emulsifying complete Freund's adjuvant and a UV-326 specific antigen solution of the ultraviolet absorbent for the first immunization, wherein the antigen concentration is 0.5mg/mL, the dosage is 100 mu g/mouse, emulsifying the incomplete Freund's adjuvant and the UV-326 specific antigen solution of the ultraviolet absorbent for each subsequent immunization enhancement, and the dosage is the same as the primary immunization; two weeks after the primary immunization, the boosting immunization is carried out once every 10 days, the total immunization is 8-10 times, and the last immunization is carried out when the titer of the antibody is not increased any more; directly injecting ultraviolet absorbent UV-326 specific antigen aqueous solution into abdominal cavity without adding immunological adjuvant in the last time, and the dosage is the same as that of the first immunization; tail blood is taken to detect the serum titer; taking a mouse with high serum titer, and carrying out cell fusion on spleen cells and myeloma cells SP2/0 according to a ratio of 6:1 under the aseptic condition; screening hybridoma cells by adopting a limiting dilution method to obtain a completely homogeneous monoclonal antibody and a stable monoclonal hybridoma cell strain; and the Balb/C mice are sensitized by adopting the intraperitoneal injection of an incomplete adjuvant before one week, and the dosage is 0.5 mL/mouse; injecting the hybridoma cell suspension with the cell concentration of 1.5 ten thousand/mL into the abdominal cavity of a mouse, wherein the dosage is 0.5 mL/mouse; inoculating hybridoma cells for 6-10 days, collecting ascites, and repeatedly collecting for several times; storing in a refrigerator at 4 deg.C; and carrying out ascites purification by an octanoic acid-ammonium sulfate precipitation method to obtain the purified UV-326 monoclonal antibody as the ultraviolet absorbent.
In one embodiment, the dosage mass ratio of the ultraviolet absorbent UV-326 specific antigen to the ultraviolet absorbent UV-326 specific antibody is 1: 2.
the invention also discloses a preparation method of the ultraviolet absorbent UV-326 detection kit, which mainly comprises the following steps:
1) preparing an ultraviolet absorbent UV-326 specific antigen working solution: preparing the concentration of the UV-326 specific antigen of the ultraviolet absorbent to be 0.5-2.5 mu g/mL;
2) preparing an ultraviolet absorbent UV-326 specific antibody working solution: the concentration of the UV-326 specific antibody of the UV absorber was formulated to be 1-5. mu.g/mL.
In one embodiment, in the step 1), the concentration of the ultraviolet absorbent UV-326 specific antigen working solution is 1.5 mu g/mL; in the step 2), the concentration of the ultraviolet absorbent UV-326 specific antibody working solution is 3 mug/mL.
The invention also discloses a using method of the ultraviolet absorbent UV-326 detection kit, namely
If the detected object is a plastic food packaging product, the method comprises the following pretreatment steps: weighing distilled water according to a predetermined amount, and soaking the object to be tested at 30-50 deg.C for 200-300hr to obtain solution to be tested;
if the detection object is food, the method comprises the following pretreatment steps: homogenizing the substance to be detected, adding ammonium formate solution and ammonia water for extraction, filtering, adding the filtrate into an activated HLB solid phase extraction column, leaching the small column by using 20-40% formic acid aqueous solution, discarding the leaching solution, and then performing mass flow rate control on the volume ratio of 0.5-1.5: 1, eluting the small column with the methanol-dichloromethane mixed solution, collecting eluent, recovering the solvent, dissolving a proper amount of acetone, and adding water to obtain a solution to be detected;
if the detection object is a daily chemical product, the method comprises the following pretreatment steps: uniformly mixing the substances to be detected, adding an ammonium formate solution and ammonia water for extraction, filtering, adding the filtrate into an activated HLB solid-phase extraction column, leaching the small column by using a 20-40% formic acid aqueous solution, discarding the leaching solution, and then performing mass flow rate control on the molecular weight ratio of 0.5-1.5: 1, eluting the small column with the methanol-dichloromethane mixed solution, collecting eluent, recovering the solvent, dissolving with a proper amount of acetone, and adding water to obtain the solution to be detected.
The detection principle of the ultraviolet absorbent UV-326 is as follows:
adsorbing an ultraviolet absorbent UV-326 antigen on a solid phase carrier, adding a sample or a standard solution of the ultraviolet absorbent UV-326 and an ultraviolet absorbent UV-326 antibody working solution, competitively binding the ultraviolet absorbent UV-326 antigen in the sample to be detected and the ultraviolet absorbent UV-326 antigen coated on the solid phase carrier with the ultraviolet absorbent UV-326 antibody, adding an enzyme-labeled secondary antibody for amplification of enzyme activity after incubation, incubating, terminating after color development, determining the absorbance of the sample, wherein the value is negatively related to the amount of the ultraviolet absorbent UV-326 in the sample, and comparing with a standard curve to obtain the concentration range of the ultraviolet absorbent UV-326.
Compared with the prior art, the invention has the following beneficial effects:
the ultraviolet absorbent UV-326 detection kit provided by the invention detects the ultraviolet absorbent UV-326 by utilizing a competitive enzyme-linked immunoassay method, and has the characteristics of high specificity, accurate result, simple pretreatment, low requirements on instruments and equipment and low detection cost. Meanwhile, the reagent in the kit is provided in the form of working solution, the operation is simple and rapid, the time is saved for a user, and the error caused by complicated steps is reduced. Is suitable for quickly and accurately detecting the ultraviolet absorbent UV-326 in a large batch of samples.
Detailed Description
In order that the invention may be more fully understood, the following description is included. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The starting materials used in the following examples are all commercially available.
Example 1
An ultraviolet absorbent UV-326 detection kit mainly comprises the following components:
1) ultraviolet absorbent UV-326 specific antigen working solution;
the preparation method comprises the following steps: 0.32g (i.e. 1.0mmol) of the ultraviolet absorber UV-326 having a phenolic hydroxyl group; dissolving 0.075g (namely 1.0mmol) of 3-amino-1-propanol and 0.29g (namely 1.1mmol) of triphenylphosphine in 2.0mL of dry Tetrahydrofuran (THF), carrying out ultrasonic treatment at normal temperature for 5min, dropwise adding 0.22mL (namely 1.1mmol) of diisopropyl azodicarboxylate in the ultrasonic treatment process, continuing the ultrasonic reaction for 20min, and separating by using a chromatographic column after the reaction is finished to prepare the hapten of the ultraviolet absorbent UV-326 capable of being coupled with protein; then mixing the antigen with N-hydroxysuccinimide (NHS) and carbodiimide (EDC), mixing the antigen with bovine serum albumin, stirring the mixture for 1h at room temperature, coupling to prepare an ultraviolet absorbent UV-326 specific antigen, and diluting the antigen with a concentrated sample diluent (0.01M, phosphate buffer solution with pH 7.4) to the concentration of 1.5 mu g/mL.
2) Ultraviolet absorbent UV-326 specific antibody working solution;
the preparation method comprises the following steps: the ultraviolet absorbent UV-326 specific antigen obtained by the synthesis is used as immunogen to immunize 10-week-old Balb/C mice. The first immunization was carried out by emulsifying complete Freund's adjuvant with 0.01M, pH7.4 phosphate buffer solution of UV-326 specific antigen as ultraviolet absorbent, the antigen concentration was 0.5mg/mL, the dose was 100. mu.g/mouse, and the subsequent booster immunization was carried out by emulsifying complete Freund's adjuvant with 0.01M, pH7.4 phosphate buffer solution of UV-326 specific antigen as ultraviolet absorbent, and the dose was the same as that of the first immunization. Two weeks after the initial immunization, booster immunizations were performed every 10 days for 8-10 total immunizations, and the last immunization was performed when the antibody titer did not increase any more. Directly performing intraperitoneal injection with 0.01M of ultraviolet absorbent UV-326 specific antigen and phosphate buffer solution with pH of 7.4 without adding immunologic adjuvant at the last time, wherein the dosage is the same as that of the initial immunization. And (3) taking blood from the tail part to detect the serum titer, taking a mouse with high serum titer, and taking spleen cells of the mouse to perform cell fusion with myeloma cells SP2/0 according to a ratio of 6:1 under the aseptic condition. And (3) screening the hybridoma cells by adopting a limiting dilution method to obtain a completely homogeneous monoclonal antibody and a stable monoclonal hybridoma cell strain.
Preparation and purification of monoclonal antibody: Balb/C mice were sensitized by intraperitoneal injection with incomplete adjuvant at a dose of 0.5 mL/mouse one week before. The hybridoma cell suspension with the cell concentration of 1.5 ten thousand/mL is injected into the abdominal cavity of the mouse, and the dosage is 0.5 mL/mouse. Ascites was collected 6-10 days after the hybridoma cells were inoculated, and the collection was repeated several times. Storing in a refrigerator at 4 deg.C. Purifying ascites by caprylic acid-ammonium sulfate precipitation. The specific method comprises the following steps: adding 3 parts of sodium acetate buffer solution (concentration 0.05mol/L, pH4.0) into 1 part of ascites, adjusting pH to 4.5 with concentration 0.1mmol/LNaOH, stirring at 4 deg.C for 30min, slowly adding octanoic acid, and calculating by ascites volume before dilution to 40 μ L/mL; standing at 4 deg.C for 3 hr, centrifuging (10140r/min,30min), collecting supernatant, maintaining at 4 deg.C, and adding (NH) within 30min4)2SO4The final concentration was made 0.277g/mL, allowed to stand for 1h, centrifuged at 4 deg.C (10140r/min,30min), the supernatant was discarded to give a monoclonal antibody precipitate, and diluted with a concentrated sample diluent to a concentration of 3. mu.g/mL.
3) An ELISA plate;
96-well polystyrene elisa plates, available from commercial companies;
4) horse radish peroxidase is used for marking a goat anti-mouse secondary antibody;
horseradish peroxidase-labeled goat anti-mouse secondary antibody supplied by commercial company;
5) washing liquid;
is 0.01M phosphate buffer solution with pH of 7.4 and containing 0.05-0.5% of Tween-20 by weight ratio;
6) a substrate color-developing agent;
the color developing agent A is a 30% hydrogen peroxide aqueous solution, and the color developing agent B is a 10mg/mL tetramethyl benzidine DMSO solution;
7) a stop solution;
is a 2mol/L sulfuric acid solution;
8) 6 bottles of ultraviolet absorbent UV-326 standard substance solution;
the concentrations are respectively: 2X 105μg/L、2×104μg/L、2×103μg/L、2×102μg/L、2×101μg/L、2×100μg/L；
9) Sealing liquid;
is 0.01M phosphate buffer solution with pH7.4 and containing 5% skimmed milk powder;
10) concentrating the sample diluent;
a phosphate buffer at pH7.4 at 0.01M (i.e., a phosphate buffer at a phosphate concentration of 0.01M/L at pH 7.4);
11) a valve bag;
provided by a commercial company.
Example 2
And (3) establishing an indirect competition ELISA method.
The competitive inhibition rate of the UV-326 monoclonal antibody of the ultraviolet absorbent is detected by adopting an indirect competitive ELISA method, which comprises the following steps:
1) coating a 96-well enzyme label plate with 1.5 mu g/mL of ultraviolet absorbent UV-326 specific antigen working solution, coating 100 mu L/well, placing in a refrigerator at 4 ℃ for overnight, sealing with 250 mu L/well sealing solution (namely 5% skimmed milk powder), washing with a washing solution, and patting dry;
2) adding an ultraviolet absorbent UV-326 standard solution (the concentration is respectively: 2X 105μg/L、2×104μg/L、2×103μg/L、2×102μg/L、2×101μg/L、2×100Mu g/L) or 50 mu L of sample solution to be detected, then adding 100 mu L of ultraviolet absorbent UV-326 specific antibody working solution with the concentration of 3 mu g/mL, uniformly mixing, and incubating for 1h at 37 ℃;
3) washing, beating to dry, adding a horseradish peroxidase-labeled goat anti-mouse secondary antibody working solution diluted by a diluent according to the proportion of 1:10000, incubating for 1h at 37 ℃ in a 100 mu L/hole manner;
4) washing and drying, adding 50 mu L of developing solution B and 10 mu L of developing solution A into each hole for developing for 15min, adding 2mol/L sulfuric acid solution, adding 50 mu L/hole, terminating the reaction, setting an enzyme-labeling instrument at 450nm (preferably using double-wavelength 450/630nm for detection, reading the data within 5 min), and determining the OD value.
The inhibition rate I% is plotted on the ordinate, and the logarithm lg of the concentration of the ultraviolet absorbent UV-326 [ ultraviolet absorbent UV-326(ng/mL) ] is plotted on the abscissa, thereby drawing an ultraviolet absorbent UV-326 competitive inhibition curve. Substituting the inhibition rate of the sample into a regression equation of a standard curve, reading out the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the corresponding dilution factor to obtain the actual content of the ultraviolet absorbent UV-326 in the sample.
The inhibition rate calculation formula is as follows:
wherein: i-inhibition rate
B- (average absorbance of sample solution)
B0- (average absorbance value of 0. mu.g/L standard solution)
BN- (reference blank control average absorbance value)
Example 3
Sensitivity, specificity and accuracy of the kit.
1. And (4) measuring the sensitivity.
The results of the indirect competitive ELISA assay of example 2 were used to establish a standard working curve for the detection of the UV absorber UV-326. The ultraviolet absorbent UV-326 monoclonal antibody is 2 mu g/L-2 multiplied by 105Good linearity in the mu g/L range, IC50958.4ng/mL, the lowest detection limit is 5.81ng/mL, and the detection range (between 20% and 80% inhibition) is 49.50ng/mL to 1650 mu g/mL. The detection limit of the plastic food packaging product is 2.91ng/cm2The detection limit of the food sample is 1.16ng/g, and the daily chemicalThe detection limit of the product is 5.81 ng/g. 2. Specific assay
The cross-reactivity of the UV absorber UV-326 structural analogs (UV absorbers UV-327, UV absorbers UV-234, UV absorbers UV-329, UV absorbers UV-71, UV absorbers UV-360) with the monoclonal antibody mixture was determined by the indirect competitive ELISA method of example 2. The series of concentrations (1X 10)7μg/L、1×106μg/L、1×105μg/L、1×104μg/L、1×103μg/L、1×102μg/L、1×101μ g/L) of the substances and the antibodies are added into the enzyme label plate which is coated and sealed at the same time, the specific steps are the same as the example 2, and the inhibition rates of various analogues are calculated respectively. IC Using monoclonal antibody to UV-326, an ultraviolet absorber50Value and IC of monoclonal antibody against various analogs50The ratio of values gives the cross-reactivity (CR%), the formula is as follows:
the result shows that the cross-reactivity of the ultraviolet absorbent UV-326 monoclonal antibody with the ultraviolet absorbents UV-327, UV-234, UV-329, UV-71 and UV-360 can not be detected to be a specific value, and is less than 0.05%.
3. Determination of accuracy
Adding 0.1 mu g/mL and 10 mu g/mL of ultraviolet absorbent UV-326 into a blank plastic food packaging product and a food sample, adding 1 mu g/mL and 10 mu g/mL of ultraviolet absorbent UV-326 into a cosmetic sample, repeating the steps three times, performing three parallels each time, measuring the inhibition rate by adopting the indirect competitive ELISA method of example 2, substituting the inhibition rate (the average value of the three parallels) into a regression equation of a standard curve, calculating the content, and calculating the recovery rate.
The recovery formula is as follows:
the result shows that the recovery rate of the plastic product is 95.6-102%, the recovery rate of the food is 92.5-99.4%, and the recovery rate of the daily chemical product is 89.5-97.2%.
Example 4
The migration of the ultraviolet absorber UV-326 in the plastic food packaging article was detected.
1. And (4) pretreating a sample.
Accurately weighing distilled water according to the measured volume of the sample, adding the distilled water into the hollow product, and soaking for 240h at 40 ℃. Plastic containers, plastic film bags, films, etc. of more than 1.1L can also be cut into test pieces for measurement. The plastic film bag capable of containing the solvent is soaked in the inner wall part without characters and patterns, the bag opening is opened and placed in a beaker with proper size, and a proper amount of distilled water is added for soaking according to the method. The composite food packaging bag is measured by 2mL per square centimeter and is soaked by injecting distilled water according to the method. Soaking at 40 deg.C for 240h, and collecting 50 μ L for analysis.
2. The indirect competitive ELISA method detects the residue of the UV-326 absorbent in the sample.
The method of example 2 was used to calculate the inhibition ratio according to the measurement results, and the inhibition ratio was substituted into the standard curve equation to determine the content of UV-326, as shown in Table 2 below.
Table 2 migration of UV-326, an ultraviolet absorber, in the plastic article.
Example 5
Detecting the ultraviolet absorbent UV-326 in the food.
1. And (4) pretreating a sample.
Homogenizing the sample by a homogenizer, weighing 10g (accurate to 0.01g) of the sample, adding 30mL of 0.5mol/L ammonium formate solution and 200 mu L of ammonia water, uniformly mixing, carrying out ultrasonic extraction for 15min, centrifuging for 10min at 10000r/min, filtering by using filter paper, adding the filtrate into an HLB (3mL, 60mg) solid phase extraction column which is activated by 6mL of dichloromethane, 6mL of methanol and 6mL of deionized water in advance, controlling the flow rate to be not more than 1mL/min, leaching the column by using 6mL of 30% formic acid aqueous solution after all the sample liquid passes through, discarding leaching liquid, leaching the column by using 6mL of methanol-dichloromethane mixed solution (1:1, V/V), collecting eluent, blowing nitrogen in a water bath at 40 ℃ until the sample liquid is nearly dried, adding 100 mu L of acetone, carrying out vortex dissolution on the residue, then adding 1.9mL of ultrapure water, uniformly mixing, and analyzing by using 50 mu L.
2. The indirect competitive ELISA method detects the residue of the UV-326 absorbent in the sample.
The method of example 2 was used to calculate the inhibition ratio according to the measurement results, and the inhibition ratio was substituted into the standard curve equation to determine the content of UV-326, as shown in Table 3 below.
Table 3 content of UV-326, an ultraviolet absorber, in the food product.
Example 6
Detecting the ultraviolet absorbent UV-326 in the daily chemical products.
1. And (4) pretreating a sample.
Weighing 2g (accurate to 0.01g) of the uniformly mixed sample (detergent or cosmetic) in a 50mL plastic centrifuge tube, adding 30mL of 0.5mol/L ammonium formate solution and 200 μ L ammonia water, uniformly mixing, carrying out ultrasonic extraction for 15min, centrifuging for 10min at 10000r/min, filtering with filter paper, adding the filtrate into an HLB (3mL, 60mg) solid phase extraction column activated by 6mL of dichloromethane, 6mL of methanol and 6mL of deionized water in advance, controlling the flow rate to be not more than 1mL/min, leaching the column with 6mL of 30% formic acid aqueous solution after all sample liquid passes through, discarding the leaching solution, leaching the column with 6mL of methanol-dichloromethane mixed solution (1:1, V/V), collecting the eluent, blowing nitrogen to be nearly dry in a 40 ℃ water bath, adding 100 μ L of acetone, carrying out vortex to dissolve residues, then adding 1.9mL of ultrapure water, uniformly mixing, 50 μ L was taken for analysis.
2. The indirect competitive ELISA method detects the residue of the UV-326 absorbent in the sample.
The method of example 2 was used to calculate the inhibition ratio according to the measurement results, and the inhibition ratio was substituted into the standard curve equation to determine the content of UV-326, as shown in Table 4 below.
Table 4 content of UV-326, an ultraviolet absorber, in the daily chemical products.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (5)
1. An ultraviolet absorber UV-326 detection kit, comprising:
the ultraviolet absorbent UV-326 specific antigen working solution is a conjugate of an ultraviolet absorbent UV-326 and carrier protein;
an ultraviolet absorbent UV-326 specific antibody working solution, wherein the ultraviolet absorbent UV-326 specific antibody is a murine monoclonal antibody;
the ultraviolet absorbent UV-326 specific antigen is prepared by the following method: 0.32g of an ultraviolet absorber UV-326 having a phenolic hydroxyl group; dissolving 0.075g of 3-amino-1-propanol and 0.29g of triphenylphosphine in 2.0mL of dry tetrahydrofuran, carrying out ultrasonic treatment at normal temperature for 5min, dropwise adding 0.22mL of diisopropyl azodicarboxylate in the ultrasonic treatment process, continuing the ultrasonic reaction for 20min, and separating by using a chromatographic column after the reaction is finished to prepare a hapten of an ultraviolet absorbent UV-326 capable of being coupled with protein; mixing the antigen with N-hydroxysuccinimide and carbodiimide, mixing the antigen with bovine serum albumin, stirring the mixture for 1 hour at room temperature, and coupling to prepare an ultraviolet absorbent UV-326 specific antigen;
the ultraviolet absorbent UV-326 specific antibody is prepared by the following method: immunizing a 10-week-old Balb/C mouse by taking the synthesized UV-326 specific antigen of the ultraviolet absorbent as an immunogen; the first immunization uses the complete Freund adjuvant and the ultraviolet absorbent UV-326 specific antigen 0.01M, pH7.4 phosphate buffer solution emulsification, the antigen concentration is 0.5mg/mL, the dose is 100 mug/piece, later each boosting immunization uses the incomplete Freund adjuvant and the ultraviolet absorbent UV-326 specific antigen 0.01M, pH7.4 phosphate buffer solution emulsification, the dose is the same as the first immunization; two weeks after the primary immunization, the boosting immunization is carried out once every 10 days, the total immunization is 8-10 times, and the last immunization is carried out when the titer of the antibody is not increased any more; directly performing intraperitoneal injection with 0.01M of ultraviolet absorbent UV-326 specific antigen and phosphate buffer solution with pH of 7.4 without adding immunologic adjuvant for the last time, wherein the dosage is the same as that of the initial immunization; taking blood from the tail part to detect the serum titer, taking a mouse with high serum titer, and taking spleen cells of the mouse to perform cell fusion with myeloma cells SP2/0 according to the ratio of 6:1 under the aseptic condition; screening hybridoma cells by adopting a limiting dilution method to obtain a completely homogeneous monoclonal antibody and a stable monoclonal hybridoma cell strain;
before one week, the Balb/C mice are sensitized by adopting the intraperitoneal injection of an incomplete adjuvant, and the dosage is 0.5 mL/mouse; injecting the hybridoma cell strain suspension with the cell concentration of 1.5 ten thousand/mL into the abdominal cavity of a mouse, wherein the dosage is 0.5 mL/mouse; inoculating hybridoma cells for 6-10 days, collecting ascites, and repeatedly collecting for several times; purifying ascites by an octanoic acid-ammonium sulfate precipitation method, which comprises the following steps: adding 3 parts of sodium acetate buffer solution into 1 part of ascites, adjusting the pH value to 4.5 by using NaOH with the concentration of 0.1mmol/L, stirring for 30min at 4 ℃, slowly adding octanoic acid during stirring, and calculating 40 mu L/mL according to the volume of the ascites before dilution; standing at 4 deg.C for 3h, centrifuging at 10140r/minCollecting supernatant for 30min, maintaining at 4 deg.C, and adding (NH) within 30min4)2SO4Standing for 1h until the final concentration is 0.277g/mL, centrifuging at 10140r/min at 4 ℃ for 30min, and removing supernatant to obtain monoclonal antibody precipitate;
the dosage mass ratio of the ultraviolet absorbent UV-326 specific antigen to the ultraviolet absorbent UV-326 specific antibody is 1: 2;
the ultraviolet absorbent UV-326 specific antigen is coated on the ELISA plate to form a specific antigen ELISA plate; the kit also comprises an enzyme-labeled secondary antibody, a washing solution, a substrate color developing agent, a stop solution, a standard solution, a confining solution and a concentrated sample diluent.
2. The ultraviolet absorbent UV-326 detection kit as claimed in claim 1, wherein the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-mouse secondary antibody diluted at a concentration of 1: 10000.
3. The ultraviolet absorbent UV-326 detection kit as claimed in claim 1, wherein the elisa plate is made of one of polystyrene, polyethylene and polypropylene;
the washing solution is 0.01M phosphate buffer solution with pH7.4 containing 0.05-0.5% Tween-20;
the substrate color developing agent consists of a color developing agent A and a color developing agent B, wherein the color developing agent A is hydrogen peroxide or carbamide peroxide, and the color developing agent B is o-phenylenediamine or tetramethyl benzidine;
the stop solution is 1-2mol/L sulfuric acid solution;
the concentration of UV-326 in the standard solution is 2 x 10 respectively5μg/L、2×104μg/L、2×103μg/L、2×102μg/L、2×101μg/L、2×100μg/L；
The confining liquid is 0.01M phosphate buffer solution with pH of 7.4 and containing 5% of skimmed milk powder;
the concentrated sample diluent was 0.01M phosphate buffer ph 7.4.
4. The method for preparing the ultraviolet absorbent UV-326 detection kit as claimed in any one of claims 1 to 3, which is characterized by essentially comprising the steps of:
1) preparing an ultraviolet absorbent UV-326 specific antigen working solution: preparing the concentration of the UV-326 specific antigen of the ultraviolet absorbent to be 0.5-2.5 mu g/mL;
2) preparing an ultraviolet absorbent UV-326 specific antibody working solution: the concentration of the UV-326 specific antibody of the UV absorber was formulated to be 1-5. mu.g/mL.
5. The method of using the UV-326 detection kit for ultraviolet absorber as claimed in any one of claims 1 to 3, wherein,
if the detected object is a plastic food packaging product, the method comprises the following pretreatment steps: weighing distilled water according to a predetermined amount, and soaking the object to be tested at 30-50 deg.C for 200-300hr to obtain solution to be tested;
if the detection object is food, the method comprises the following pretreatment steps: homogenizing the substance to be detected, adding ammonium formate solution and ammonia water for extraction, filtering, adding the filtrate into an activated HLB solid phase extraction column, leaching the small column by using 20-40% formic acid aqueous solution, discarding the leaching solution, and then performing mass flow rate control on the volume ratio of 0.5-1.5: 1, eluting the small column with the methanol-dichloromethane mixed solution, collecting eluent, recovering the solvent, dissolving a proper amount of acetone, and adding water to obtain a solution to be detected;
if the detection object is a daily chemical product, the method comprises the following pretreatment steps: uniformly mixing the substances to be detected, adding an ammonium formate solution and ammonia water for extraction, filtering, adding the filtrate into an activated HLB solid-phase extraction column, leaching the small column by using a 20-40% formic acid aqueous solution, discarding the leaching solution, and then performing mass flow rate control on the molecular weight ratio of 0.5-1.5: 1, eluting the small column with the methanol-dichloromethane mixed solution, collecting eluent, recovering the solvent, dissolving with a proper amount of acetone, and adding water to obtain the solution to be detected.
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