CN102798712B - Fluorescent brightening agent CBS-X detection kit and preparation method thereof - Google Patents
Fluorescent brightening agent CBS-X detection kit and preparation method thereof Download PDFInfo
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- CN102798712B CN102798712B CN201210281736.1A CN201210281736A CN102798712B CN 102798712 B CN102798712 B CN 102798712B CN 201210281736 A CN201210281736 A CN 201210281736A CN 102798712 B CN102798712 B CN 102798712B
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Abstract
The invention discloses a fluorescent brightening agent CBS-X detection kit, belonging to the technical field of additive detection. The kit comprises an enzyme label plate coated by a fluorescent brightening agent CBS-X specific antigen and a fluorescent brightening agent CBS-X specific antibody solution, wherein each micropore of the enzyme label plate is coated by the fluorescent brightening agent CBS-X specific antigen having a concentration of 1-3 mug/mL, the concentration of the fluorescent brightening agent CBS-X specific antibody is 1-3 mug/mL, and the dosage ratio of the fluorescent brightening agent CBS-X specific antigen to the fluorescent brightening agent CBS-X specific antibody solution is 1:1. The invention further discloses a preparation method of the detection kit. The detection kit disclosed herein can continuously detect a plurality of samples at a time when the fluorescent brightening agent CBS-X is detected, has the characteristics of convenience, rapidness, sensitivity, and low cost, and is suitable for rapidly and accurately detecting fluorescent brightening agent CBS-X in mass samples.
Description
Technical field
The present invention relates to a kind of adjuvant detection technique, specifically, particularly relate to a kind of enzyme-linked immunologic detecting kit for fluorescent brightener CBS-X content and preparation method thereof.
Background technology
Fluorescent brightener CBS-X, chemical name is 4,4 '-bis-(2-sodium sulfonate styryl) biphenyl, belongs to two talan-biphenyl type optical whitening agents, and feature is that to brighten intensity high, resistance to chlorine floats, oxygen floats function admirable, has excellent fastness to washing and dry, wet Exposure to Sunlight degree, and level dyeing is functional, be suitable for articles for washing, also can be directly used in the optical whitening of textile, paper.
For a long time, the security of fluorescer is quite disputed on, and has data to show that fluorescer is low toxicity, also there is data to show the molecular structure stabilized of fluorescer, be difficult for being decomposed, in vivo long-term accumulated, can make cell morph, produce potential carcinogenic factor.Therefore, in the case of fully not assessing the security of fluorescer, not all fluorescer is all allowed to use, European Union, the U.S., Japan, there is certain supervision in many countries such as China to fluorescer, relevant laws and regulations are as the 2002/72/EC of European Union instruction, CFR 21CFR178.3297 and GB 9685-2008 " food containers, wrappage use hygienic standard with adjuvant " all formulate the lists of additives that is allowed for producing plastic-food contact material and goods, the maximum addition that allows the fluorescer using is limited.Given this, set up the method for quick of the fluorescer of Related product, carry out periodic monitoring and safety evaluation, for standard industry development, guarantee product quality, Protection of consumer is healthy has profound significance with protection of the environment etc.And fluorescent brightener CBS-X is as the one in the fluorescer that allows to add, also need a kind of suitable method for quick badly.
At present, both at home and abroad the detection method of fluorescer is comprised following several: uviol lamp direct irradiation observation method, chromatography, combined gas chromatography mass spectrometry.Wherein, uviol lamp direct irradiation observation method is a kind of simple judgement just, can not differentiate the kind of fluorescer; Chromatography and combined gas chromatography mass spectrometry have that specificity is high, result advantage accurately, loaded down with trivial details but the shortcoming of these two kinds of methods is sample pre-treatments, consume a large amount of solvents, and need expensive laboratory equipment, and testing cost is high.
Summary of the invention
Based on this, the invention reside in the defect that overcomes prior art, a kind of fluorescent brightener CBS-X detection kit and a kind of fluorescent brightener CBS-X kit preparation method are provided, this kit can one-time continuous detect multiple samples in the time detecting fluorescent brightener CBS-X, it is convenient to have, fast, the low feature of sensitive, cost.
First object of the present invention is to provide a kind of fluorescent brightener CBS-X detection kit, and its technical scheme is as follows: a kind of fluorescent brightener CBS-X detection kit, mainly comprises:
1) ELISA Plate of coated fluorescent brightener CBS-X specific antigen: in each micropore of described ELISA Plate, the concentration of fluorescent brightener CBS-X specific antigen is 1-3 μ g/mL;
2) fluorescent brightener CBS-X specific antibody solution: the concentration of this fluorescent brightener CBS-X specific antibody is 1-3 μ g/mL;
The amount ratio of described fluorescent brightener CBS-X specific antigen and fluorescent brightener CBS-X specific antibody solution is 1:1.
In an embodiment, described fluorescent brightener CBS-X detection kit also includes ELIAS secondary antibody therein, and described ELIAS secondary antibody is that concentration is anti-by the horseradish peroxidase-labeled sheep anti mouse of 1:10000 dilution proportion two.
In an embodiment, the concentration of described fluorescent brightener CBS-X specific antigen is 2 μ g/mL therein; The concentration of described fluorescent brightener CBS-X specific antibody is 2 μ g/mL.
In an embodiment, described fluorescent brightener CBS-X detection kit also comprises fluorescent brightener CBS-X standard solution, substrate developer, cleansing solution, stop buffer, confining liquid and concentrating sample dilution therein.
In an embodiment, described specificity fluorescent whitening agent CBS-X specific antibody is mouse resource monoclonal antibody therein; Described fluorescent brightener CBS-X specific antigen is the conjugate of fluorescent brightener CBS-X and carrier protein; Described carrier protein is the one in bovine serum albumin(BSA), ovalbumin, albumin rabbit serum, thyroglobulin; Described fluorescent brightener CBS-X standard solution concentration is respectively 1 × 10
5μ g/L, 1 × 10
4μ g/L, 1 × 10
3μ g/L, 1 × 10
2μ g/L, 1 × 10
1μ g/L, 1 × 10
0μ g/L; Described developer is made up of developer A and developer B, and described developer A is hydrogen peroxide or urea peroxide, and described developer B is o-phenylenediamine or tetramethyl benzidine; Described stop buffer is the sulfuric acid solution of 1-2mol/L; Described cleansing solution is the 0.01M that contains 0.05%-0.5% Tween-20, the phosphate buffer of pH7.4; Described confining liquid is the 0.01M containing 5% skimmed milk power, the phosphate buffer of pH7.4; Described concentrating sample dilution is 0.01M, the phosphate buffer of pH7.4; The material of described ELISA Plate is the one in polystyrene, tygon, polypropylene.
Second object of the present invention is to provide a kind of fluorescent brightener CBS-X detection kit preparation method, mainly comprises the following steps:
1) ELISA Plate of the coated fluorescent brightener CBS-X specific antigen of preparation: 2-sulfonic benzo formaldehyde, 2-sulfonic group-5 hydroxyl-benzaldehyde and fluorescent brightener CBS-X are scattered in dry DMSO, add piperidines, microwave heating, reaction finishes to be poured into water, suction filtration solid, obtains the fluorescent brightener CBS-X derivant with phenolic hydroxyl group; Fluorescent brightener CBS-X derivant with phenolic hydroxyl group and sal tartari are scattered in dry DMF, stirring at normal temperature, add N-(3-bromopropyl) t-butyl carbamate, add hot reflux, the phenolic hydroxyl group of fluorescent brightener CBS-X is replaced by tertiary fourth oxanamide propoxyl group; Subsequently intermediate product is added in trifluoroacetic acid, return stirring, the protection of sloughing tertiary fourth oxygen acyl group, exposes amido, make can with the haptens of the fluorescent brightener CBS-X of protein coupling; Reaction after again it being mixed with N-hydroxy-succinamide (NHS) and carbodiimide (EDC), prepare fluorescent brightener CBS-X specific antigen with carrier protein couplet, fluorescent brightener CBS-X specific antigen is coated in ELISA Plate, and in each micropore of described ELISA Plate, the concentration of fluorescent brightener CBS-X specific antigen is 1-3 μ g/mL;
2) prepare fluorescent brightener CBS-X specific antibody: using step 1) in synthetic fluorescent brightener CBS-X specific antigen as immunogene, mouse is carried out to immunity; After immunity, get the mouse that serum titer is high, get its splenocyte and myeloma cell SP2/0 carries out Fusion of Cells; Adopt limiting dilution assay screening hybridoma, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain; And get Freund's incomplete adjuvant lumbar injection and carry out the mouse after sensitization, hybridoma suspension is expelled in mouse peritoneal, collect ascites, carry out ascites purifying through sad-ammonium sulfate precipitation method, obtain fluorescent brightener CBS-X monoclonal antibody of purifying; The concentration of described fluorescent brightener CBS-X specific antibody is 1-3 μ g/mL.
Therein in an embodiment, above-mentioned steps 1) in, the method of preparing fluorescent brightener CBS-X specific antigen is preferably: by 0.186g, be 2-sulfonic benzo formaldehyde, the 0.20g of 1.0mmol, be 2-sulfonic group-5 hydroxyl-benzaldehyde and the 0.40g of 1.0mmol, fluorescent brightener CBS-the X that is 1.0mmol is scattered in the dry DMSO of 30mL, add piperidines 3mL, 500 watts of heating 5min of microwave, reaction finishes to be poured into water, suction filtration solid, obtains the fluorescent brightener CBS-X derivant with phenolic hydroxyl group; By 0.53g, be fluorescent brightener CBS-X derivant and the 1.01g with phenolic hydroxyl group of 1.0mmol, the sal tartari that is 10mmol is scattered in the dry DMF of 80mL, stirring at normal temperature 30min, add 1.2g, be N-(3-bromopropyl) t-butyl carbamate of 5mmol, add hot reflux 5h, the phenolic hydroxyl group of fluorescent brightener CBS-X is replaced by tertiary fourth oxanamide propoxyl group; Subsequently intermediate product is added in the trifluoroacetic acid (TFA) of 30 times, return stirring 1h, the protection of sloughing tertiary fourth oxygen acyl group, exposes amido, make can with the haptens of the fluorescent brightener CBS-X of protein coupling; After it being mixed with N-hydroxy-succinamide and carbodiimide, reaction, prepares fluorescent brightener CBS-X specific antigen with bovine serum albumin(BSA) coupling again.
Therein in an embodiment, above-mentioned steps 2) be: using step 1) in synthetic fluorescent brightener CBS-X specific antigen as immunogene, the Ba lb/c mouse in 10 week age is carried out to immunity, first immunisation is used complete Freund's adjuvant and fluorescent brightener CBS-X specific antigen emulsifying soln, antigen concentration is 0.5mg/mL, dosage is 100 μ g/, later each booster immunization uses incomplete Freund's adjuvant and fluorescent brightener CBS-X specific antigen emulsifying soln, the same initial immunity of dosage; After initial immunity two weeks, at interval of 10 days booster immunizations once, immune 8-10 time altogether, in the time that antibody titer no longer raises, carry out last immunity; Directly do not use the direct lumbar injection of fluorescent brightener CBS-X specific antigen aqueous solution, the same initial immunity of dosage with immunologic adjuvant for the last time; Afterbody is got blood examination and is surveyed serum titer; Get the mouse that serum titer is high, under aseptic condition, get its splenocyte and carry out Fusion of Cells in 6:1 ratio and myeloma cell SP2/0; Adopt limiting dilution assay screening hybridoma, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain; And Balb/C mouse, before Yu Yizhou, adopt Freund's incomplete adjuvant lumbar injection to carry out sensitization, dosage is 0.5mL/; The hybridoma suspension that is 1.5 ten thousand/mL by cell concentration is expelled in mouse peritoneal, and dosage is 0.5mL/; Inoculation hybridoma, after 6-10 days, is collected ascites, repeatedly collects for several times; Be stored in 4 ℃ of Refrigerator stores; Carry out ascites purifying through sad-ammonium sulfate precipitation method, obtain fluorescent brightener CBS-X monoclonal antibody of purifying.
Fluorescent brightener CBS of the present invention-X detects principle:
Fluorescent brightener CBS-X specific antigen is adsorbed on solid phase carrier, add standard solution and the fluorescent brightener CBS-X specific antibody working fluid of sample or fluorescent brightener CBS-X, in testing sample, on fluorescent brightener CBS-X and solid phase carrier, coated fluorescent brightener CBS-X specific antigen is competed combined with fluorescent whitening agent CBS-X specific antibody, after hatching, add ELIAS secondary antibody to carry out the amplification of enzymatic activity, hatch, after colour developing, stop, the absorbance of working sample, in this value and sample, the amount of fluorescent brightener CBS-X is negative correlation, relatively can draw fluorescent brightener CBS-X concentration range with typical curve.
Below the advantage of aforementioned techniques scheme is described:
Fluorescent brightener CBS-X detection kit of the present invention, utilizes competitive enzyme-linked immune determination method to detect fluorescent brightener CBS-X, has specificity high, and result is accurate and pre-treatment is simple, and instrument and equipment is required to the feature low, testing cost is low.Meanwhile, the reagent in this kit provides with working fluid form, simple to operate, quick, for user has saved the time and reduced the error causing because of step complexity.Be suitable for the quick and accurate fluorescent brightener CBS-X that must detect in batch samples.
Embodiment
Below embodiments of the invention are elaborated, but content of the present invention are not caused to any restriction.
Embodiment 1
The chief component composition of the fluorescent brightener CBS-X detection kit described in the present embodiment is as follows:
1) fluorescent brightener CBS-X specific antigen working fluid;
Make by the following method: by 0.186g, be 2-sulfonic benzo formaldehyde, the 0.20g of 1.0mmol, be 2-sulfonic group-5 hydroxyl-benzaldehyde and the 0.40g of 1.0mmo l, fluorescent brightener CBS-the X that is 1.0mmol is scattered in the dry DMSO of 30mL, add piperidines 3mL, 500 watts of heating 5min of microwave, reaction finishes to be poured into water, suction filtration solid, obtains the fluorescent brightener CBS-X derivant with phenolic hydroxyl group; By 0.53g, be fluorescent brightener CBS-X derivant and the 1.01g with phenolic hydroxyl group of 1.0mmo l, the sal tartari that is 10mmol is scattered in the dry DMF of 80mL, stirring at normal temperature 30min, add 1.2g, be N-(3-bromopropyl) t-butyl carbamate of 5mmol, add hot reflux 5h, the phenolic hydroxyl group of fluorescent brightener CBS-X is replaced by tertiary fourth oxanamide propoxyl group; Subsequently intermediate product is added in the trifluoroacetic acid (TFA) of 30 times, return stirring 1h, the protection of sloughing tertiary fourth oxygen acyl group, exposes amido, make can with the haptens of the fluorescent brightener CBS-X of protein coupling; Again it is mixed with N-hydroxy-succinamide (NHS) and carbodiimide (EDC), mixes with bovine serum albumin(BSA), stirring at room temperature 1h, fluorescent brightener CBS-X specific antigen is prepared in coupling, and with concentrating sample diluted to concentration be 2 μ g/mL.
2) fluorescent brightener CBS-X specific antibody working fluid;
Make by the following method: the synthetic fluorescent brightener CBS-X specific antigen obtaining is carried out to immunity as immunogene to the Balb/c mouse in 10 week age.First immunisation is used the 0.01M of complete Freund's adjuvant and fluorescent brightener CBS-X specific antigen, the phosphate buffer emulsification of pH7.4, antigen concentration is 0.5mg/mL, dosage is 100 μ g/, later each booster immunization uses the 0.01M of incomplete Freund's adjuvant and fluorescent brightener CBS-X specific antigen, the phosphate buffer emulsification of pH7.4, the same initial immunity of dosage.After initial immunity two weeks, at interval of 10 days booster immunizations once, immune 8-10 time altogether, in the time that antibody titer no longer raises, carry out last immunity.The last 0.01M that does not directly use fluorescent brightener CBS-X specific antigen with immunologic adjuvant, the direct lumbar injection of phosphate buffer of pH7.4, the same initial immunity of dosage.Afterbody is got blood examination and is surveyed serum titer.Get the mouse that serum titer is high, under aseptic condition, get its splenocyte and carry out Fusion of Cells in 6:1 ratio and myeloma cell SP2/0.Adopt limiting dilution assay screening hybridoma, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain.
The preparation and purification of monoclonal antibody: Balb/C mouse, before Yu Yizhou, adopt Freund's incomplete adjuvant lumbar injection to carry out sensitization, dosage is 0.5mL/.The hybridoma suspension that is 1.5 ten thousand/mL by cell concentration is expelled in mouse peritoneal, and dosage is 0.5mL/.Inoculation hybridoma, after 6-10 days, is collected ascites, repeatedly collects for several times.Be stored in 4 ℃ of Refrigerator stores.Carry out ascites purifying through sad-ammonium sulfate precipitation method.Concrete grammar: add 3 parts of sodium acetate buffer (concentration 0.05mol/L in every 1 part of ascites, pH4.0),, with concentration 0.1mmol/L NaOH adjustment pH to 4.5, at 4 ℃, stir 30min, it is sad slowly to add during this time, calculates 40 μ L/mL by ascites volume before dilution; At 4 ℃ of static 3h, centrifugal (10140r/min, 30min), gets supernatant, remains in 4 ℃ of environment, adds (NH in 30min
4)
2sO
4making final concentration is 0.277g/mL, static 1h, 4 ℃ centrifugal (10140r/min, 30min), abandons supernatant, obtains monoclonal antibody precipitation, and with concentrating sample diluted to concentration be 2 μ g/mL.
3) horseradish peroxidase-labeled sheep anti mouse two is anti-;
The horseradish peroxidase-labeled sheep anti mouse two being provided by commercial company is anti-;
4) 6 bottles of fluorescent brightener CBS-X standard solutions;
Concentration is respectively: 1 × 10
5μ g/L, 1 × 10
4μ g/L, 1 × 10
3μ g/L, 1 × 10
2μ g/L, 1 × 10
1μ g/L, 1 × 10
0μ g/L;
5) ELISA Plate;
96 hole polystyrene ELISA Plate, are provided by commercial company;
6) substrate developer;
Be made up of developer A and developer B, developer A is that concentration is 30% aqueous hydrogen peroxide solution, and developer B is that concentration is the tetramethyl benzidine DMSO solution of 10mg/mL;
7) cleansing solution;
For containing the 0.01M that weight ratio is 0.05%-0.5% Tween-20, the phosphate buffer of pH7.4;
8) stop buffer;
For the sulfuric acid solution of 2mol/L;
9) confining liquid;
For the 0.01M containing 5% skimmed milk power, the phosphate buffer of pH7.4;
10) concentrating sample dilution;
For 0.01M, the phosphate buffer of pH7.4 (being that phosphate concentration is 0.01M/L, the phosphate buffer of pH 7.4);
11) valve bag;
Provided by commercial company.
Embodiment 2
The foundation of indirect competitive ELISA method.
Adopt indirect competitive ELISA method to detect the competition inhibiting rate of fluorescent brightener CBS-X monoclonal antibody, method is as follows:
1), by the coated 96 hole ELISA Plate of fluorescent brightener CBS-X specific antigen of 2 μ g/mL, 100 μ L/ holes, are positioned over that 4 ℃ of refrigerators are coated to spend the night, and wash, and pat dry with 250 μ L/ hole confining liquids (i.e. 5% skimmed milk power) after sealing with cleansing solution;
2) (concentration is respectively: 1 × 10 to add fluorescent brightener CBS-X standard solution
5μ g/L, 1 × 10
4μ g/L, 1 × 10
3μ g/L, 1 × 10
2μ g/L, 1 × 10
1μ g/L, 1 × 10
0μ g/L) or sample solution 50 μ L, then to add concentration be fluorescent brightener CBS-X specific antibody working fluid 50 μ L of 2 μ g/mL, mixes 37 ℃ of incubation 1h;
3) after washing pats dry, add with dilution by the anti-working fluid of horseradish peroxidase-labeled sheep anti mouse two of 1:10000 dilution proportion 100 μ L/ holes, 37 ℃ of incubation 1h;
4) washing pats dry, every hole adds 50 μ L nitrite ion B liquid, 10 μ L nitrite ion A liquid colour developing 15mi n, adding concentration is the sulfuric acid solution of 2mol/L, 50 μ L/ holes, cessation reaction, set microplate reader and (preferably detect with dual wavelength 450/630nm, in 5min, run through data) in 450nm place, measure OD value.
Take inhibiting rate I% as ordinate, take the logarithm 1g[fluorescent brightener CBS-X (ng/mL) of fluorescent brightener CBS-X concentration] as horizontal ordinate, draw fluorescent brightener CBS-X competition and suppress curve.By the inhibiting rate substitution typical curve regression equation of sample, read the corresponding concentration of sample from typical curve, be multiplied by its corresponding extension rate and be the actual content of fluorescent brightener CBS-X in sample.
Inhibiting rate computing formula is as follows:
Wherein: I---inhibiting rate
B---(the mean light absorbency value of sample solution)
B
0---(the mean light absorbency value of 0 μ g/L standard solution)
B
n---(reference blank mean light absorbency value)
Embodiment 3
Kit sensitivity, specificity, accuracy.
1. sensitivity determination.
Utilize the indirect competitive ELISA method measurement result of embodiment 2 to set up the standard working curve that fluorescent brightener CBS-X detects.Fluorescent brightener CBS-X monoclonal antibody is in 1 μ g/L～1 × 10
5within the scope of μ g/L, there are good linearity, IC
50=905.1ng/mL, lowest detection is limited to 0.553ng/mL, and sensing range (suppressing between 20%～80%) is 4.714ng/mL～1520.113 μ g/mL.The detection of paper and plastic products is limited to 0.277ng/cm
2, the detection of food samples is limited to 0.277ng/g, and the detection of daily chemical products is limited to 50ng/g.
2. specific assay.
Adopt the indirect competitive ELISA method of embodiment 2 to measure the cross reaction of fluorescent brightener CBS-X analogue (phenylbenzene, 2-vinylbenzenesulfonic acid sodium salt, 2-styrene benzene sulfonic acid sodium salt, 2-phenylbenzene vinylbenzenesulfonic acid sodium salt) and monoclonal antibody potpourri.By series concentration (1 × 10
7μ g/L, 1 × 10
6μ g/L, 1 × 10
5μ g/L, 1 × 10
4μ g/L, 1 × 10
3μ g/L, 1 × 10
2μ g/L, 1 × 10
1μ g/L) above-mentioned substance join in coated ELISA Plate of having sealed with antibody respectively simultaneously, concrete steps, with embodiment 2, are calculated respectively the inhibiting rate of each analog.Utilize the IC of monoclonal antibody to fluorescent brightener CBS-X
50value and the IC of monoclonal antibody to each analog
50the ratio of value obtains cross reacting rate (CR%), and formula is as follows:
Result shows that the cross reacting rate of fluorescent brightener CBS-X monoclonal antibody and phenylbenzene, 2-vinylbenzenesulfonic acid sodium salt, 2-styrene benzene sulfonic acid sodium salt, 2-phenylbenzene vinylbenzenesulfonic acid sodium salt all can't detect concrete numerical value, for being less than 0.05%.
3. accuracy determination.
To the fluorescent brightener CBS-X that adds 0.1 μ g/mL and 10 μ g/mL in plain pape goods, plastic products and food samples, in daily product sample, add fluorescent brightener CBS-X of 100 μ g/mL and 1000 μ g/mL, in triplicate, do at every turn three parallel, adopt the indirect competitive ELISA method of embodiment 2 to measure inhibiting rate, then by inhibiting rate (three parallel mean value) substitution typical curve regression equation, calculate content, and calculate recovery rate.
Recovery formula is as follows:
Result shows that the recovery of paper products is 87.5%～103.2%, and the recovery of plastic products is 90.5%～99.7%, and the recovery of food is 87.5%～98.2%, and the recovery of daily product is 86.5%～95.8%.
Embodiment 4
The migration of fluorescent brightener CBS-X in test paper sample.
1. the pre-treatment of sample.
Paper products are recorded after area, inject distilled water by the amount of every square centimeter of 2mL, or adopt the method for all soaking, its area, with two calculating of paper products, soaks after 2h in room temperature (>20 ℃), gets 50 μ L and analyzes.
2. indirect competitive ELISA method detects the residual of fluorescent brightener CBS-X in sample.
Adopt the method for embodiment 2 to detect, according to measurement result, calculate inhibiting rate, substitution typical curve equation is obtained the content of fluorescent brightener CBS-X, sees the following form 1.
The migration amount of fluorescent brightener CBS-X in table 1 paper products.
Embodiment 5
Detect the migration of fluorescent brightener CBS-X in plastic products.
1. the pre-treatment of sample.
1.1 hollw article.
Accurately measure distilled water by the sample volume recording and add in hollw article, soak 2h in room temperature (>20 ℃).The plastic containers that are greater than 1.1L also can be cut into test piece and measure.The plastic film bag that can hold solvent should soak the inner wall section without character pattern, sack can be opened to the beaker that is placed in suitable size, adds appropriate distilled water to soak in accordance with the law.Composite laminated food packaging bag, by every square centimeter of 2mL, injects distilled water and soaks according to upper method.Soak 2h in room temperature (>20 ℃), get 50 μ L and analyze.
1.2 flat articles.
Flat articles records after its area, injects distilled water soak in accordance with the law by the amount of every square centimeter of 2mL.Maybe can adopt the method for whole immersions, its area should be with two calculating.Soak 2h in room temperature (>20 ℃), get 50 μ L and analyze.
1.3 sheet materials, film and test piece.
With the pre-treating method of above-mentioned flat articles.
1.4 rubber.
Rubber adds 2mL distilled water by every square centimeter of contact area, cannot calculating contact area, add 20mL distilled water by every gram of sample.Soak 2h in room temperature (>20 ℃), get 50 μ L and analyze.
Mould pad for 1.5.
Can full wafer peel off by every square centimeter of 2mL adding distil water.The part that edge is thicker of getting of can not full wafer peeling off is cut into wide 0.3～0.5cm, and the strip of long 1.5～2.5cm, weighs.Add 60mL distilled water by every gram of sample.Soak 2h in room temperature (>20 ℃), get 50 μ L and analyze.
2. indirect competitive ELISA method detects the residual of fluorescent brightener CBS-X in sample.
Adopt the method for embodiment 2 to detect, according to measurement result, calculate inhibiting rate, substitution typical curve equation is obtained the content of fluorescent brightener CBS-X, sees the following form 2.
The migration amount of fluorescent brightener CBS-X in table 2 plastic products.
Embodiment 6
Detect fluorescent brightener CBS-X in food.
1. the pre-treatment of sample.
With homogenizer by sample homogeneous, taking 10g(and be accurate to 0.01g) sample adds in appropriate ultrapure water, the ultrapure water yield is as the criterion to soak sample completely, with after ultrasonic extraction 15min, with the centrifugal 10min of 10000r/min, with Filter paper filtering, sample liquid after filtering is crossed to the strata-X-AW(3mL through 10mL methyl alcohol and the activation of 10mL deionized water, 60mg) solid-phase extraction column, coutroi velocity is not more than 1mL/min, then use respectively 3mL deionized water and the drip washing of 3mL methyl alcohol, finally carry out wash-out with 6mL 2% ammoniacal liquor methanol solution, collecting eluent nitrogen in 50 ℃ of water-baths blows near doing, add 2mL ultrapure water to dissolve, getting 50 μ L analyzes.
2. indirect competitive ELISA method detects the residual of fluorescent brightener CBS-X in sample.
Adopt the method for embodiment 2 to detect, according to measurement result, calculate inhibiting rate, substitution typical curve equation is obtained the content of fluorescent brightener CBS-X, sees the following form 3.
The content of fluorescent brightener CBS-X in table 3 food.
Embodiment 7
Detect fluorescent brightener CBS-X in daily chemical products.
1. the pre-treatment of sample.
Sample (washing agent or the cosmetics) 2g(taking after mixing is accurate to 0.01g) sample is in 500mL volumetric flask, add after about 100mL ultrapure water, ultrasound wave dissolves, constant volume (can add a small amount of methyl alcohol to eliminate the foam producing before constant volume), the sample liquid 5mL getting after filtration crosses the strata-X-AW(3mL through 10mL methyl alcohol and the activation of 10mL deionized water, 60mg) solid-phase extraction column, coutroi velocity is not more than 1mL/min, then use respectively 3mL deionized water and the drip washing of 3mL methyl alcohol, finally carry out wash-out with 6mL 2% ammoniacal liquor methanol solution, collecting eluent nitrogen in 50 ℃ of water-baths blows near doing, add 2mL ultrapure water to dissolve, getting 50 μ L analyzes.
2. indirect competitive ELISA method detects the residual of fluorescent brightener CBS-X in sample.
Adopt the method for embodiment 2 to detect, according to measurement result, calculate inhibiting rate, substitution typical curve equation is obtained the content of fluorescent brightener CBS-X, sees the following form 4.
The content of fluorescent brightener CBS-X in table 4 daily chemical products.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (8)
1. fluorescent brightener CBS-X detection kit, is characterized in that, mainly comprises:
1) ELISA Plate of coated fluorescent brightener CBS-X specific antigen: in each micropore of described ELISA Plate, the concentration of fluorescent brightener CBS-X specific antigen is 1-3 μ g/mL;
2) fluorescent brightener CBS-X specific antibody solution: the concentration of this fluorescent brightener CBS-X specific antibody is 1-3 μ g/mL;
The amount ratio of described fluorescent brightener CBS-X specific antigen and fluorescent brightener CBS-X specific antibody solution is 1:1.
2. fluorescent brightener CBS-X detection kit according to claim 1, is characterized in that, also includes ELIAS secondary antibody, and described ELIAS secondary antibody is that concentration is anti-by the horseradish peroxidase-labeled sheep anti mouse of 1:10000 dilution proportion two.
3. fluorescent brightener CBS-X detection kit according to claim 1 and 2, is characterized in that, the concentration of described fluorescent brightener CBS-X specific antigen is 2 μ g/mL; The concentration of described fluorescent brightener CBS-X specific antibody is 2 μ g/mL.
4. fluorescent brightener CBS-X detection kit according to claim 1, is characterized in that, also comprises fluorescent brightener CBS-X standard solution, substrate developer, cleansing solution, stop buffer, confining liquid and concentrating sample dilution.
5. fluorescent brightener CBS-X detection kit according to claim 4, is characterized in that, described fluorescent brightener CBS-X specific antibody is mouse resource monoclonal antibody; Described fluorescent brightener CBS-X specific antigen is the conjugate of fluorescent brightener CBS-X and carrier protein; Described carrier protein is the one in bovine serum albumin(BSA), ovalbumin, albumin rabbit serum, thyroglobulin; Described fluorescent brightener CBS-X standard solution concentration is respectively 1 × 10
5μ g/L, 1 × 10
4μ g/L, 1 × 10
3μ g/L, 1 × 10
2μ g/L, 1 × 10
1μ g/L, 1 × 10
0μ g/L; Described developer is made up of developer A and developer B, and described developer A is hydrogen peroxide or urea peroxide, and described developer B is o-phenylenediamine or tetramethyl benzidine; Described stop buffer is the sulfuric acid solution of 1-2mol/L; Described cleansing solution is the 0.01M that contains 0.05%-0.5% Tween-20, the phosphate buffer of pH7.4; Described confining liquid is the 0.01M containing 5% skimmed milk power, the phosphate buffer of pH7.4; Described concentrating sample dilution is 0.01M, the phosphate buffer of pH7.4; The material of described ELISA Plate is the one in polystyrene, tygon, polypropylene.
6. fluorescent brightener CBS-X detection kit preparation method, is characterized in that, mainly comprises the following steps:
1) ELISA Plate of the coated fluorescent brightener CBS-X specific antigen of preparation: 2-sulfonic benzo formaldehyde, 2-sulfonic group-5 hydroxyl-benzaldehyde and fluorescent brightener CBS-X are scattered in dry DMSO, add piperidines, microwave heating, reaction finishes to be poured into water, suction filtration solid, obtains the fluorescent brightener CBS-X derivant with phenolic hydroxyl group; Fluorescent brightener CBS-X derivant with phenolic hydroxyl group and sal tartari are scattered in dry DMF, stirring at normal temperature, add N-(3-bromopropyl) t-butyl carbamate, add hot reflux, the phenolic hydroxyl group of fluorescent brightener CBS-X is replaced by tertiary fourth oxanamide propoxyl group; Subsequently intermediate product is added in trifluoroacetic acid, return stirring, the protection of sloughing tertiary fourth oxygen acyl group, exposes amido, make can with the haptens of the fluorescent brightener CBS-X of protein coupling; Reaction after again it being mixed with N-hydroxy-succinamide and carbodiimide, prepare fluorescent brightener CBS-X specific antigen with carrier protein couplet, fluorescent brightener CBS-X specific antigen is coated in ELISA Plate, and in each micropore of described ELISA Plate, the concentration of fluorescent brightener CBS-X specific antigen is 1-3 μ g/mL;
2) prepare fluorescent brightener CBS-X specific antibody: using step 1) in synthetic fluorescent brightener CBS-X specific antigen as immunogene, mouse is carried out to immunity; After immunity, get the mouse that serum titer is high, get its splenocyte and myeloma cell SP2/0 carries out Fusion of Cells; Adopt limiting dilution assay screening hybridoma, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain; And get Freund's incomplete adjuvant lumbar injection and carry out the mouse after sensitization, hybridoma suspension is expelled in mouse peritoneal, collect ascites, carry out ascites purifying through sad-ammonium sulfate precipitation method, obtain fluorescent brightener CBS-X monoclonal antibody of purifying; The concentration of described fluorescent brightener CBS-X specific antibody is 1-3 μ g/mL.
7. fluorescent brightener CBS-X detection kit preparation method according to claim 6, it is characterized in that, in step 1), the concrete grammar of preparing fluorescent brightener CBS-X specific antigen is: the 2-sulfonic benzo formaldehyde of 0.186g, 2-sulfonic group-5 hydroxyl-benzaldehyde of 0.20g and fluorescent brightener CBS-X of 0.40g are scattered in the dry DMSO of 30mL, add piperidines 3mL, 500 watts of heating 5min of microwave, reaction finishes to be poured into water, suction filtration solid, obtains the fluorescent brightener CBS-X derivant with phenolic hydroxyl group; The sal tartari of the fluorescent brightener CBS-X derivant with phenolic hydroxyl group of 0.53g and 1.01g is scattered in the dry DMF of 80mL, stirring at normal temperature 30min, add N-(3-bromopropyl) t-butyl carbamate of 1.2g, add hot reflux 5h, the phenolic hydroxyl group of fluorescent brightener CBS-X is replaced by tertiary fourth oxanamide propoxyl group; Subsequently intermediate product is added in the trifluoroacetic acid of 30 times, return stirring 1h, the protection of sloughing tertiary fourth oxygen acyl group, exposes amido, make can with the haptens of the fluorescent brightener CBS-X of protein coupling; After it being mixed with N-hydroxy-succinamide and carbodiimide, reaction, prepares fluorescent brightener CBS-X specific antigen with bovine serum albumin(BSA) coupling again.
8. fluorescent brightener CBS-X detection kit preparation method according to claim 6, is characterized in that step 2) be:
Using step 1) in synthetic fluorescent brightener CBS-X specific antigen as immunogene, the Balb/c mouse in 10 week age is carried out to immunity, first immunisation is used complete Freund's adjuvant and fluorescent brightener CBS-X specific antigen emulsifying soln, antigen concentration is 0.5mg/mL, dosage is 100 μ g/, later each booster immunization uses incomplete Freund's adjuvant and fluorescent brightener CBS-X specific antigen emulsifying soln, the same initial immunity of dosage; After initial immunity two weeks, at interval of 10 days booster immunizations once, immune 8-10 time altogether, in the time that antibody titer no longer raises, carry out last immunity; Directly do not use the direct lumbar injection of fluorescent brightener CBS-X specific antigen aqueous solution, the same initial immunity of dosage with immunologic adjuvant for the last time; Afterbody is got blood examination and is surveyed serum titer; Get the mouse that serum titer is high, under aseptic condition, get its splenocyte and carry out Fusion of Cells in 6:1 ratio and myeloma cell SP2/0; Adopt limiting dilution assay screening hybridoma, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain; And get Balb/C mouse, and before Yu Yizhou, adopt Freund's incomplete adjuvant lumbar injection to carry out sensitization, dosage is 0.5mL/; The hybridoma suspension that is 1.5 ten thousand/mL by cell concentration is expelled in mouse peritoneal, and dosage is 0.5mL/; Inoculation hybridoma, after 6-10 days, is collected ascites, repeatedly collects for several times; Be stored in 4 ℃ of Refrigerator stores; Carry out ascites purifying through sad-ammonium sulfate precipitation method, obtain fluorescent brightener CBS-X monoclonal antibody of purifying.
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| CN105476979B (en) * | 2015-12-12 | 2018-10-16 | 重庆理工大学 | A kind of pharmaceutical composition containing fluorescent brightener CBS and its application |
| CN108776223B (en) * | 2018-04-19 | 2021-04-06 | 广州质量监督检测研究院 | Ultraviolet absorbent UV-326 detection kit and preparation method and application thereof |
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