CN103102319A - Melamine semiantigen, preparation method and application thereof - Google Patents
Melamine semiantigen, preparation method and application thereof Download PDFInfo
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- CN103102319A CN103102319A CN2011103567710A CN201110356771A CN103102319A CN 103102319 A CN103102319 A CN 103102319A CN 2011103567710 A CN2011103567710 A CN 2011103567710A CN 201110356771 A CN201110356771 A CN 201110356771A CN 103102319 A CN103102319 A CN 103102319A
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Abstract
The invention provides a melamine semiantigen, a preparation method and an application thereof. According to the invention, a synthetic method of the melamine semiantigen comprises: mixing cyanuric acid diamide and succinic anhydride, adding pyridine, stirring for 12-24 hours under a room temperature, removing a solvent, and then re-crystallizing in ethanol-water to obtain hemisuccinate of cyanuric acid amine, that is, the melamine semiantigen. Experimental results show that the method for synthesizing the melamine semiantigen is simple; the obtained melamine semiantigen is relatively high in yield and purity; by using antigens prepared by the synthesized melamine semiantigen to immunize experimental animals, monoclonal antibodies with good specificity and high sensitivity can be obtained; and ELISA kits established with the antibodies are easy to use, cheap, convenient to carry, and high-efficient, accurate and simple in detection methods, can simultaneously detect large quantities of samples, and is suitable for on-site monitoring of residual melamine in animal source food and screening of large quantities of samples.
Description
Technical field
The present invention relates to a kind of melamine hapten and its preparation method and application.
Background technology
Trimeric cyanamide formal name used at school 1,3,5-triazines-2,4, the 6-triamine is a kind of important nitrogen heterocyclic Organic Chemicals, is the post-treatment product of urea.Main application is and the aldehyde condensation, generates melamine formaldehyde resin, produces plastics, and part Asian countries also is used for making chemical fertilizer.Because the trimeric cyanamide nitrogen content is 66.6%, be 4.16 times of protein average content, 151 times of milk, 23 times of milk powder.Add the 0.1g trimeric cyanamide in every 100g milk, just can improve 0.4% protein.Foodstuffs industry always general protein testing method is Kjeldahl determination, estimates protein content by measuring total nitrogen.This method can only be known the total amount of nitrogen, can not identify the source of nitrogen and the kind of nitrogenous source, there is defective just because of food and feed industrial protein content assaying method, stay the chance of making food of poor quality for illegal businessman, they with trimeric cyanamide as foodstuff additive, pretending to be the protein in food, so trimeric cyanamide also is called " extract of protein ".In September, 2008, the Sanlu infant problem milk powder case of China's outburst causes the infant of edible contaminated milk powder to suffer from the urinary stone disease illness, and its reason is to contain trimeric cyanamide in milk powder.State General Administration for Quality Supervision promptly carries out the special examination of baby milk powder content of melamine in the whole nation, all the other 109 Dairy Enterprises are investigated, and has checked altogether 491 batch products of these enterprises.Check result shows, has 69 batch products of 22 baby milk powder manufacturing enterprises to detect the different trimeric cyanamide of content.This time event involvement aspect of outburst is wide, endanger bigly, has caused huge negative impact to people's daily life.Five departments such as China Ministry of Health unite the issue bulletin, have stipulated that in China's infant formula, the trimeric cyanamide Limited Doses is 1mg/kg, and in other food, the Limited Doses of trimeric cyanamide is 2.5mg/kg.
The most popular method that is used at present detecting trimeric cyanamide is instrumental method, comprises gas chromatography-mass spectrography, Liquid Chromatography-Tandem Mass Spectrometry, superelevation phase High Performance Liquid Chromatography-Electrospray Ionization Tandem Mass method, reversed-phased high performace liquid chromatographic, high performance liquid chromatography-level Four bar MS etc.The instrumental method testing cost is high, and instrument is expensive, and pre-treating process is loaded down with trivial details, and detection time is long, but and need just operating instrument of professional and technical personnel, be not suitable for the examination of on-site supervision and a large amount of samples.By comparison, enzyme-linked immunoassay method has pinpoint accuracy and sensitivity, the operative technique requirement that testing cost is low, lower, of short duration detection time, the larger characteristics such as detection sample size, can satisfy better China's livestock and poultry cultivation family, slaughterhouse, food enterprise, government function supervision department etc. and carry out testing.
Summary of the invention
The purpose of this invention is to provide a kind of melamine hapten and preparation method thereof
The compound of melamine hapten provided by the invention, molecular structural formula are shown in formula I:
The preparation method of the compound of melamine hapten provided by the present invention comprises the steps: cyanurodiamide is mixed with succinyl oxide, add pyridine at room temperature to stir 12-24h, after desolventizing in alcohol-water recrystallization get the hemisuccinic acid ester of Cyanuramide, be the compound shown in formula I; The molar ratio of cyanurodiamide and succinyl oxide is 1: (1-3), be preferably 1: 2.
The compound of melamine antigen provided by the present invention, molecular structural formula are shown in formula II:
The compounds process for production thereof of melamine antigen provided by the present invention comprises the steps:
Get in the DMF (DMF) that melamine hapten is dissolved in, obtain solution I; After carbodiimide (EDC) is fully dissolved with water, add in solution I, stir 12-24h under room temperature, obtain solution II, the time is preferably 24h; The carrier proteins of getting coupling makes it fully to be dissolved in phosphate buffered saline buffer (PBS), and solution II is added drop-wise in protein solution, and with room temperature under stir 12-24h, obtain solution III, the time is preferably 24h; In 4 ℃ of dialysis, obtain melamine antigen with PBS solution, packing, and cryopreservation is standby.
Wherein, carrier proteins can be bovine serum albumin, mouse serum protein, rabbit anteserum albumen, thyroprotein, ovalbumin, hemocyanin or human serum albumin.
The application in the preparation melamine antibody of above-mentioned melamine hapten or melamine antigen compound also belongs to protection scope of the present invention.
The antibody that is obtained by above-claimed cpd formula II immune animal also belongs to protection scope of the present invention.Described antibody is monoclonal antibody.
Above-mentioned arbitrary described compound and (or) application of antibody in detecting trimeric cyanamide also belong to protection scope of the present invention.
The present invention is take cyanurodiamide as synthetic initial substance, react with succinyl oxide, thereby the active group-COOH of acquisition and the direct coupling of carrier proteins, the hemisuccinic acid ester of the melamine hapten cyanurodiamide of acquisition prepares artificial antigen with carrier protein couplet again.Can produce for the affinity of antibody of trimeric cyanamide high after the antigen-immunized animal that the melamine hapten that synthesizes with this method prepares, high specificity, adopted the former medicine of trimeric cyanamide as synthetic initial substance in the past, with succinyl oxide reaction, the antibody that can produce the haptens preparation that obtains for the avidity of the antibody of trimeric cyanamide and synthesis of semiantigen that specificity adopts than the present invention after the antigen-immunized animal that the melamine hapten that obtains prepares is low.Preparation method's yield of hapten of the present invention and purity are all higher, the method simple possible, and cost is lower, is suitable for the on-site supervision of melamine residual in animal derived food and the examination of great amount of samples.
Description of drawings
Fig. 1: melamine hapten composite diagram.
Fig. 2: trimeric cyanamide hydrogen nuclear magnetic resonance spectrogram.
Fig. 3: trimeric cyanamide ELISA typical curve.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are used for explanation the present invention, and be not used for limiting the scope of the invention.
Embodiment one: the synthetic and evaluation of melamine hapten
One, melamine hapten is synthetic
Get 0.64g cyanurodiamide and 1.0g succinyl oxide, add the 10ml pyridine, stir 12-24h under room temperature, after desolventizing in alcohol-water recrystallization obtain the white powder crystal, hemisuccinic acid ester for Cyanuramide, be melamine hapten, molecular structure is as follows, synthetic route as shown in Figure 1:
Two, the evaluation of melamine hapten
Get above-mentioned formula I compound and carry out structural analysis through proton nmr spectra, as shown in Figure 2, near in the discovery collection of illustrative plates 12ppm (mg/L) peak is the H on carboxylic acid, and the peak about 2.7ppm (mg/L) is two methylene radical on the hemisuccinic acid acid anhydride, illustrates that melamine hapten synthesizes successfully.
The preparation of embodiment two, melamine antigen
One, immunogenic synthetic
Get the 30mg melamine hapten, be dissolved in 1.5ml DMF and obtain solution I, then the EDC that gets 20mg joins in solution I after fully dissolving with 0.2m water, stirring at room 24h can obtain solution II; Take BSA50mg, make it fully to be dissolved in 8.3ml pH and be in 7.2 PBS, solution II dropwise slowly is added drop-wise in above-mentioned BSA solution, stir 24h and obtain solution III under room temperature; Dialysed 3 days under 4 ℃ with 0.02mol/LPBS, to remove unreacted small-molecule substance.Immunogen packing with obtaining saves backup in-20 ℃.
Two, coating antigen is synthetic
Get the 30mg melamine hapten, be dissolved in 1.5ml DMF and obtain solution I, then the EDC that gets 20mg joins in solution I after fully dissolving with 0.2ml water, stirring at room 24h namely gets solution II; Take OVA40mg, make it fully to be dissolved in 5.3ml pH and be in 7.2 PBS, solution II dropwise slowly is added drop-wise in above-mentioned OVA solution, and stir 24h obtain solution III under room temperature; PBS with 0.02mol/L dialysed 3 days under 4 ℃, to remove unreacted small-molecule substance; Coating antigen packing with obtaining saves backup in-20 ℃.
Three, the evaluation of melamine antigen
Carrier proteins, melamine hapten, melamine hapten-carrier protein couplet thing are made into the solution of 0.5mg/ml with the PBS of pH7.4, return to zero with 0.01mol/L pH7.4PBS, with ultraviolet spectrophotometer in the interscan of wavelength 200～800nm scope, obtain the absorption curve of carrier proteins, melamine hapten, melamine hapten-carrier protein couplet thing, and calculate its in conjunction with than.Found that, different absorption curves appears in the three, shows the success of melamine hapten and carrier protein couplet, and the combination of half melamine antigen and bovine serum albumin is than being 16-21: 1, and the combination of haptens and oralbumin is than being 18-22: 1.Molecular structural formula is shown in formula II:
Embodiment three, trimeric cyanamide monoclonal antibody
One, trimeric cyanamide monoclonal antibody preparation
Animal immune: melamine hapten and carrier protein couplet thing immunity 8-10 Bal b/c mouse in age in week.
Cytogamy and cloning: get the mice spleen cell after immunity, merge under the effect of fusogen polyoxyethylene glycol (PEG) 4000 with SP2/0 myeloma cell, screening obtain can the stably excreting monoclonal antibody hybridoma cell strain.
Obtain the monoclonal hybridoma strain of trimeric cyanamide through screening.The monoclonal hybridoma strain of trimeric cyanamide can be endless generation trimeric cyanamide specific antibody, this antibodies specific is for trimeric cyanamide, sensitivity reaches 0.05 μ g/L.
Cell cryopreservation and recovery: hybridoma is made 1 * 10 with frozen storing liquid
9The cell suspension of individual/ml is preserved in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal frozen storing liquid, move in culturing bottle and cultivate.
Two, the mensuration of antibody titer
Measuring tiring of antibody with the indirect competitive ELISA method is 1: 100000～170000.
Indirect competitive ELISA method: with melamine hapten-oralbumin conjugate coated elisa plate, add trimeric cyanamide standard substance working fluid solution, monoclonal antibody working fluid and ELIAS secondary antibody, 25 ℃ of reaction 30min, pour out liquid in the hole, with PBST washings washing 3～5 times, pat dry with thieving paper; Add the substrate nitrite ion, after 25 ℃ of reaction 15min, add the stop buffer termination reaction; Set microplate reader in the wavelength 450nm place every hole of mensuration absorbance.
Three, the specificity of monoclonal antibody
The ability that antibodies specific refers to its homospecificity antigen combination and comparison with such antigen-analogues ability, cross reacting rate commonly used is as judgement criteria.Cross reaction is less, and the specificity of antibody is higher.
This experiment is done serial dilution with trimeric cyanamide, tricyanic acid and 3 kinds of medicines of trimer acid diamide, carries out indirect competitive ELISA with monoclonal antibody respectively, and the production standard curve is analyzed and obtained IC
50, then be calculated as follows cross reacting rate:
Result shows that cross reacting rate is: trimeric cyanamide 100%, tricyanic acid＜1, cyanurodiamide＜1.Antibody of the present invention has stronger specificity and avidity to trimeric cyanamide.
Embodiment four, by the enzyme linked immunological kit of trimeric cyanamide monoclonal antibody preparation
One, trimeric cyanamide enzyme linked immunological kit comprises following each component:
(1) be coated with the enzyme plate of coating antigen.
(2) enzyme labelling anti-antibody: horseradish peroxidase-sheep anti mouse anti-antibody.
(3) trimeric cyanamide monoclonal antibody working fluid.
(4) standardized solution: adopt gradient dilution method preparation standard solution, obtain 6 bottles of series standard product, concentration is respectively 0.0 μ g/L, 1.0 μ g/L, 3.0 μ g/L, 9.0 μ g/L, 27.0 μ g/L, 81.0 μ g/L, and high density standard substance 10mg/L.
(5) substrate nitrite ion A liquid is urea peroxide solution, and substrate nitrite ion B liquid is the tetramethyl biphenyl amine aqueous solution.
(6) stop buffer is the sulphuric acid soln of 2mol/L.
(7) concentrated cleaning solution is the PBS of 0.3～0.6mol/LpH7.4 of the sodiumazide sanitas of 0.6%～1.0% tween 20 and 0.01%～0.02%.
(8) the concentrated liquid that redissolves is that the pH value is 7.4, contains the PBS of 9%～12% oralbumin, 0.1～0.3mol/L.
The main agents of this test kit provides with the form of working fluid, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, tolerance range is high, the accuracy high.
Two, enzyme linked immunological kit detects the application of actual sample
1. determination
(1) milk sample pre-treating process
Get 400 μ l fresh milk samples, add 800 μ l acetonitriles, whirling motion 1min gets 100 μ l supernatant liquors, then adds the redissolution working fluid (mixing by 1: 1 volume ratio with the concentrated liquid that redissolves with deionized water) of 500 μ l, whirling motion mixing.
(2) powdered milk sample pre-treating process
Take 1.0 ± 0.05g powdered milk sample to the 10ml centrifuge tube, add the 2.5ml deionized water, whirling motion 1min.Pipette 400 μ l in the 2ml centrifuge tube, add 400 μ l acetonitriles, then add 400 μ l methyl alcohol, whirling motion 1min, 4000r/min, the centrifugal 5min of room temperature.Get 100 μ l supernatant liquors and join in 500 μ l redissolution working fluids, the whirling motion mixing is got 50 μ l and is used for analyzing.
(3) tissue (pork, chicken, fish, shrimp) sample-pretreating method
Take 1.0 ± 0.02g tissue sample to the 50ml centrifuge tube, add 2ml acetone, then add the 2ml deionized water with the vibrator 5min that vibrates, 4000r/min, centrifugal 5min under room temperature.Get supernatant liquor 100 μ l, then add 900 μ l redissolution working fluids, the whirling motion mixing is got 50 μ l and is used for analyzing.
(4) Feed Sample pre-treating process
Take 1.0 ± 0.05 Feed Samples of pulverizing to the 50ml centrifuge tube, add respectively 5ml acetone, 4ml methyl alcohol, 0.5ml1M hydrochloric acid soln and 0.5ml deionized water with the vibrator 5min that vibrates.4000r/min, centrifugal 10min under room temperature pipettes 100 μ l supernatant liquors, adds 900 μ l redissolution working fluids, and the whirling motion mixing is got 50 μ l and is used for analyzing.
(5) egg sample pre-treating process
Take 1.0 ± 0.05g egg sample to centrifuge tube, add respectively 4.5ml deionized water and 0.5ml methyl alcohol, with the vibrator 5min that vibrates.4000r/min, centrifugal 10min under room temperature pipettes 100 μ l supernatant liquors, adds 300 μ l redissolution working fluids, and the whirling motion mixing is got 50 μ l and is used for analyzing.
2, detect with test kit
Required reagent is taken out from cold storage environment, and the direct application of sample of not rising again is noted must shaking up before every kind of liquid reagent uses.Taking-up needs the microwell plate of quantity, and no microwell plate is put into valve bag, is stored in 2-8 ℃.Numbering: the corresponding micropore of sample and standard substance is numbered according to the order of sequence, and it is parallel that each sample and standard substance are done 2 holes, and the position at record standard hole and sample aperture place.Add standard substance/sample 50 μ l in the micropore of correspondence.Add again ELIAS secondary antibody 50 μ l/ holes, add at last antibody working fluid 50 μ l/ holes to vibrate gently and shake up, with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.Wash plate: carefully open the cover plate film, liquid in the hole is dried, with wash operating solution (20 * concentrated cleaning solution being diluted by 1: 19 volume ratio with deionized water) 250 μ l/ holes, fully wash 4-5 time, pat dry with thieving paper.Colour developing: add substrate solution A liquid 50 μ l/ holes, then add substrate solution B liquid 50 μ l/ holes, the mixing that vibrates gently is with reacting 15min in 25 ℃ of lucifuge environment of cover plate membrane cover plate postposition.Measure: add stop buffer 50 μ l/ holes, the mixing that vibrates is gently set microplate reader in 450nm place (suggestion with dual wavelength 450/630nm detection), measures every hole OD value.
3, Analysis of test results
The percentage light absorption ratio of standard substance or sample equals the mean value (diplopore) of the absorbance of standard substance or sample divided by the absorbance of first standard (0 standard), then multiply by 100%, namely obtains the percentage light absorption ratio.Take standard substance percentage light absorption ratio as ordinate zou, take the logarithm of trimeric cyanamide standard substance concentration as X-coordinate, drawing standard graphic representation (as Fig. 3).In percentage light absorption ratio substitution typical curve with sample, read the corresponding concentration of sample from typical curve, multiply by its corresponding extension rate and be trimeric cyanamide actual concentrations in sample.
Three, the enzyme linked immunological kit technical parameter determines
1. test kit sensitivity determination
50% inhibition concentration (is IC
50, the 50% corresponding drug level in place of the absorbance of nulling standard solution) and Chang Zuowei estimates the sensitivity index of competitive enzyme-linked immune test kit.Utilize the trimeric cyanamide standard solution to react, according to experimental result drawing standard curve, test kit trimeric cyanamide typical curve is between 1.0～81.0 μ g/L as seen from the figure.In milk, milk powder, tissue (pork, chicken, fish, shrimp), egg sample, trimeric cyanamide sensitivity is 1 μ g/L.
2. test kit lowest detectable limit
20 parts of blank samples are detected, find concentration corresponding to each percentage light absorption value from typical curve, mean value with the trimeric cyanamide measured value of 20 duplicate samples adds that 3 times of standard deviations represent detectability, and result gets the method and detects to be limited to detect in 18 μ g/L, milk powder to be limited in milk and detect the detection that is limited in 50 μ g/kg, feed in 45 μ g/kg, tissue (pork, chicken, fish, shrimp) and be limited to detect in 100 μ g/kg, egg sample and be limited to 20 μ g/kg.
3. test kit accuracy and precision
Add respectively the trimeric cyanamide of 36 μ g/L and 72 μ g/L concentration in the blank milk sample; Add the trimeric cyanamide of 90 μ g/kg and 180 μ g/kg concentration in blank powdered milk sample; Add the trimeric cyanamide of 100 μ g/kg and 200 μ g/kg concentration in blank tissue sample; Add the trimeric cyanamide of 100 μ g/kg and 200 μ g/kg concentration in blank Feed Sample; Add the trimeric cyanamide of 40 μ g/kg and 80 μ g/kg concentration in blank egg sample; Measure, get the test kit of 3 different batches, and each batch extracts 2 test kits and detects, every kind of sample do 6 times parallel, each sample repeats 2 times, it is equal 90 ± 15% to the rate of recovery of milk, milk powder, tissue (pork, chicken, fish, shrimp), feed, egg sample that result gets the method, variation within batch coefficient＜15%, interassay coefficient of variation＜25%.
4. preservation period experiment
The test kit preservation condition is 2-8 ℃, through 12 months mensuration, the maximum absorbance value of test kit, IC
50Value, trimeric cyanamide add the practical measurement value all within normal range.Do simultaneously accelerated deterioration and freezing test, test kit is placed in 37 ℃ ,-20 ℃ 6 days, measurement result shows that also the indices of test kit is normal.Obtaining the trimeric cyanamide test kit from above result can preserve 12 months at 2-8 ℃.
Claims (8)
1. compound, its molecular structural formula is shown in formula I:
2. method for preparing the compound shown in claim 1 Chinese style I, comprise the steps: cyanurodiamide is mixed with succinyl oxide, add pyridine at room temperature to stir 12-24h, after desolventizing in alcohol-water recrystallization get the hemisuccinic acid ester of Cyanuramide, be the compound shown in formula I.
3. a compound, obtained by compound claimed in claim 1 and carrier protein couplet, it is characterized in that molecular structural formula is shown in formula II:
4. compound as claimed in claim 3, is characterized in that its preparation method comprises the steps:
Get in the DMF (DMF) that melamine hapten is dissolved in, obtain solution I; After carbodiimide (EDC) water is fully dissolved, add in solution I, stir 12-24h under room temperature, obtain solution II; The carrier proteins of getting coupling makes it fully to be dissolved in phosphate buffered saline buffer (PBS), and solution II is added drop-wise in protein solution, and with room temperature under stir 12-24h, obtain solution III; In 4 ℃ of dialysis, obtain melamine antigen with PBS solution, packing, and cryopreservation is standby.
5. compound as claimed in claim 3, is characterized in that described carrier proteins is bovine serum albumin, mouse serum protein, rabbit anteserum albumen, thyroprotein, ovalbumin, hemocyanin or human serum albumin.
6. a trimeric cyanamide monoclonal antibody, be to be prepared by the described compound immune animal of claim 3.
7. claim 1,3 or 5 described compounds prepare the trimeric cyanamide enzyme linked immunological kit.
8. a detection method that detects melamine residual in animal food, is characterized in that utilizing the described test kit of claim 7 to detect.
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| CN103105494A (en) * | 2011-11-11 | 2013-05-15 | 北京勤邦生物技术有限公司 | Kit and method for detecting beta-lactam antibiotic and melamine |
| CN105503757A (en) * | 2014-09-25 | 2016-04-20 | 北京维德维康生物技术有限公司 | Preparation methods of melamine hapten and melamine antigen, and application thereof in quantum dot fluorescence immunoassay kit |
| CN105503759A (en) * | 2014-09-25 | 2016-04-20 | 北京维德维康生物技术有限公司 | Preparation methods of melamine hapten and melamine antigen, and application thereof in chemiluminescent immunoassay kit |
| CN110938040A (en) * | 2019-11-25 | 2020-03-31 | 广东达元绿洲食品安全科技股份有限公司 | Melamine hapten and artificial antibody as well as preparation method and application thereof |
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| CN105503759A (en) * | 2014-09-25 | 2016-04-20 | 北京维德维康生物技术有限公司 | Preparation methods of melamine hapten and melamine antigen, and application thereof in chemiluminescent immunoassay kit |
| CN110938040A (en) * | 2019-11-25 | 2020-03-31 | 广东达元绿洲食品安全科技股份有限公司 | Melamine hapten and artificial antibody as well as preparation method and application thereof |
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| Publication number | Publication date |
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| CN103102319B (en) | 2016-02-24 |
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