CN101206223A - Direct racing method ELISA reagent box for detecting melamine - Google Patents
Direct racing method ELISA reagent box for detecting melamine Download PDFInfo
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- CN101206223A CN101206223A CNA2007101794751A CN200710179475A CN101206223A CN 101206223 A CN101206223 A CN 101206223A CN A2007101794751 A CNA2007101794751 A CN A2007101794751A CN 200710179475 A CN200710179475 A CN 200710179475A CN 101206223 A CN101206223 A CN 101206223A
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Abstract
The invention discloses a direct competition enzyme linked immunity detection reagent kit for detection of melamines. The enzyme linked immunity detection reagent kit for the melamines provided by the invention comprises an enzyme target coated by specific antibodies of the melamines and enzyme labeling melamines. The enzyme linked immunity detection reagent kit for the melamines of the invention has the advantages of sensitivity, quickness and accuracy and is mainly used for screening of a large portion of samples; main reagents in the kit are provided in the form of operating fluid, have the characteristics of convenient use, high specificity, high flexibility, high precision, high accuracy and so on, and can quickly detect residual melamines in feedingstuff and animal products.
Description
Technical field
The present invention relates to enzyme linked immunological and detection of veterinary drugs in food field.Particularly, the present invention relates to a kind of direct competition method enzyme linked immunological kit that is used to detect melamine.
Technical background
Melamine (Melamine) is called for short triamine, formal name used at school three ammonia triazines, and another name melamine, cyanuramide, three polyamide are a kind of important azacyclo-Organic Chemicals, white crystalline powder is tasteless.This chemicals often is used to produce plastics, glue and fire retardant, and in part Asian countries, it also is used to make chemical fertilizer.It is a kind of chemical substance of forbidding being used for pet food and animal feed, can make animal generation kidney failure behind the animal edible and cause death.
Since in mid-March, 2007, a lot of pet cats, the dog incident of being poisoned to death takes place in the U.S..Through investigation, be because some feed for pet suppliers in order to increase Protein content in the feed, have added melamine, thereby caused cat, dog to be poisoned to death in gluten powder and rice protein powder.
Therefore need badly and set up convenient and swift and highly sensitive detection method, forbid the generation of this type of incident.
Existing melamine detection method mostly is chromatography, but these methods need perfect laboratory, experienced technician, and complicated instrument and equipment, and testing process is loaded down with trivial details, the examination of incompatible on-the-spot great amount of samples.Provide a new approach based on the immunoreactive immunology detection technology of antigen-antibody for the analyzing and testing of micromolecule residue.The direct competition method enzyme linked immunosorbent detection technology of setting up based on this principle has further shortened detection time than indirect method.The key of this technology is the preparation of the synthetic and antibody of comlete antigen and enzyme-labelled antigen.
At present, the direct competitive enzyme-linked immune detection method of melamine does not appear in the newspapers as yet.
Summary of the invention
The object of the present invention is to provide a kind of enzyme linked immunological kit of fast detecting melamine.
The invention provides a kind of enzyme linked immunological kit of fast detecting melamine, this kit comprises: melamine specific antibody ELISA Plate, enzyme mark melamine.Wherein the melamine specific antibody is the polyclonal antibody of rabbit anti-melamine, makes by immune animal (for example rabbit) as immunogene with melamine and carrier protein couplet thing.Described enzyme labeling melamine adopts the chemical method coupling to obtain, and marker enzyme is a horseradish peroxidase, and described coupling method is the succinic anhydride method.
Be used to prepare the solid phase material of described ELISA Plate, include but not limited to, for example, polystyrene, tygon, polypropylene.The form of carrier is the micro-reaction plate shrinkage pool.
Be convenient on-the-spot the detection and the great amount of samples examination, described kit can further include ELISA Plate, melamine standard solution, developer, cleansing solution, stop buffer, enzyme mark thing dilution and the concentrating sample dilution that is coated with the melamine specific antibody.
Described cleansing solution is a 0.05%-0.5% Tween-20 phosphate buffer.Described developer is formed (for example, volume ratio is 1: 1) by developer A liquid and developer B liquid, and developer A liquid is hydrogen peroxide or urea peroxide, and developer B liquid is o-phenylenediamine or tetramethyl benzidine.Described concentrating sample dilution is the phosphate buffer that contains 0.1% Tween-20, and described enzyme mark thing dilution is the phosphate buffer that contains 0.01% Tween-20.
On the other hand, the present invention also provides a kind of method that detects content of melamine in the feed sample, comprises step:
(1) sample pre-treatments;
(2) detect with the arbitrary described kit of claim 1-9, in the ELISA Plate hole that is coated with melamine antibody, add standard items or sample solution, add the enzyme labeling melamine again, hatch the back washing and pat dry, add chromogenic reagent, termination, measure absorbance with microplate reader;
(3) analyzing and testing result.
The detection principle of kit of the present invention is:
With the melamine antibody bag by on solid phase carrier, add sample or melamine standard solution and add enzyme labeling melamine working fluid, melamine in the testing sample and enzyme labeling melamine are competed immobilised melamine antibody, go out unconjugated enzyme labeling thing by washing, the colour developing back stops, the absorbance of working sample, the amount of melamine is negative correlation in this value and the sample, relatively can draw the melamine concentration scope with typical curve.
Beneficial effect:
The kit that the present invention detects melamine mainly adopts content of melamine in the qualitative or detection by quantitative sample of direct competitive enzymoimmunoassay; Further shortened detection time, had 2 hours of the indirect competition method to shorten to 1 hour, low to the pre-treatment requirement of sample, sample pretreatment process is simple, simultaneously the fast detecting gross sample.
This kit of the present invention adopts the melamine polyclonal antibody of high specific, main agents provides with the working fluid form, can reduce the operation steps of kit, for the user saves time and reduces the error that causes because of operation steps is miscellaneous, that the present invention has is highly sensitive, high specificity, pinpoint accuracy, pin-point accuracy, low to the instrument and equipment requirement, the reagent holding time is long, automaticity is high, "dead" isotopic contamination etc. advantage, can in feed and animal derived product detect, play a significant role.
Description of drawings
Fig. 1 suppresses curve for melamine.
It is in order further to understand the present invention better that following embodiment is provided, and never content of the present invention and protection domain is constituted any restriction.
The immunogenic synthetic preparation that reaches immune serum of embodiment 1 melamine
1.1 reagent and instrument
Melamine (Beijing lark prestige reagent company limited), succinic anhydride (available from Beijing northern Bioisystech Co., Ltd that shines), pyridine (Pyridine, AR.WM=79.10, content＞99.5%, Tianjin section close europeanized reagent development centre), N, dinethylformamide (Dimethylformamide, DMF, N.J. ACROSORGANICS company produces, available from lark prestige chemical reagents corporation), positive tri-n-butylamine (Tributylamine, N.J. ACROSORGANICS company produces, available from lark prestige chemical reagents corporation), isobutyl chlorocarbonate is purchased (from lark prestige chemical reagents corporation), bovine serum albumin(BSA) (BSA), horseradish peroxidase (HRP) etc. (available from Beijing northern Bioisystech Co., Ltd that shines), other reagent be analyze pure.
Twin-beam UV, visible light spectrophotometer (TU-1909, the general all purpose instrument company limited of analysing in Beijing), chromatographic apparatus (3057 type potable recording instrument, river, Chongqing instrument four factories; SBS series numerical control drop recorder, constant flow pump, fraction collector automatically; chromatographic column, Shanghai Hu Xi analytical instrument factory), magnetic stirring apparatus (east, Shanghai Rong Feng scientific instrument company limited); desk centrifuge (Minispin maximum (top) speed 13400rpm maximum centrifugal force 12100rcf, 2ml * 12).
1.2 artificial antigen of melamine is synthetic
Take by weighing melamine 63.06mg (0.5mmol) and be dissolved in the 5ml pyridine solution, add the reaction of succinic anhydride 50mg stirring at room and spend the night.
Dry up pyridine, with 10ml solvent (DMF and 1, the 4-dioxan mixes at 1: 1) dissolving Rac-glutaric anhydride semialdehyde, add 131 μ l (about 0.5mmol) tri-n-butylamine, stir 10min in the ice, add isobutyl chlorocarbonate 72 μ l (about 0.5mmol), change stirring at room reaction 1 hour over to.The melamine solution of activation dropwise joins in 0.1M pH 8.5 ice-cold carrier proteins (BSA or the OVA) dobell's solution, adds in 1 hour, and the stirring at room reaction is spent the night.
Then, conjugate is crossed the SephadexG-25M gel chromatography,, regulate flow velocity to 3ml/min with three times of bed volume balances of 0.01M pH 7.4PBS, sample concentration joins chromatography purification conjugate in the good chromatographic column of balance to 5ml, eluent pH 7.40.01M PBS.
1.3 the preparation of immunity and specific antibody
Above-mentioned melamine-BSA conjugate is diluted to the 1mg/ml solution for standby with physiological saline.
Choose the healthy male new zealand white rabbit of 6 body weight 2～2.5Kg.Conjugate and equivalent Freund's complete adjuvant are mixed into water in oil emulsion by syringe to the method for taking out, carry out first immunisation, take the subcutaneous multi-point injection in back by the amount of 1mg/Kg body weight.Every two all booster immunizations once, replace Freund's complete adjuvant, the same first immunisation of dosage and method with incomplete Freund.From immunity for the third time, back 10 days of each immunity, auricular vein is got blood 1ml, carry out antibody titer and detect, when antibody titer no longer raises, do not add adjuvant and carry out for the last time (the 7th time) immunity, the leg muscle injection, rear neck artery bloodletting in 7 days, room temperature are solidified behind the 2h 4 ℃ and are spent the night centrifugal 10 minutes of 8000r/min, remove clot, partly with 50% saturated ammonium sulfate solution precipitation, the centrifugal supernatant that goes precipitates with phosphate buffer resuspended serum, precipitate twice with 33% saturated ammonium sulfate solution again, sediment is used the SephadexG-25M chromatography with the least possible phosphate buffer dissolving after dialysing, promptly get polyclonal antibody.
The foundation of embodiment 2 immunologic detection methods
2.1ELISA the square formation method is determined optimum response concentration
The melamine antibody of 1000 μ g/ml, 100 μ g/ml, 10 μ g/ml, 5 μ g/ml, 1 μ g/ml, 0.25 μ g/ml series concentration is pressed every hole 100 μ l coated elisa plates, and 4 ℃ of bags are spent the night, and wash 3 times, pat dry, spend the night by 4 ℃ of following sealings of every hole 200 μ l confining liquids, wash 3 times, pat dry.Adding is since the enzyme mark melamine of 1: 200 doubling dilution, and room temperature effect 30 minutes is washed three times, adds 50 μ l substrate A liquid and 50 μ, 1 substrate B liquid, room temperature lucifuge effect 15min, and 50 μ l stop buffer cessation reactions, microplate reader detects A value (450nm).Be provided with simultaneously two parallel, getting the bag of OD value when being 1.0 left and right sides is optium concentration by concentration.Test figure is listed in table 1.
Table 1 square formation method OD value
By determining in the data of table 1 that the optimum antibody bag is 5 μ g/ml by concentration.The enzyme-labelled antigen extension rate reaches optimum response concentration at 1: 800.
2.2ELISA detect antibody antibody I C50 value
The ELISA method of operating is with 2.1, and the optimum antibody bag is 5 μ g/ml by concentration, and enzyme-labelled antigen extension rate 1: 800 is prepared the free melamine standard items of 0 μ g/L, 1 μ g/L, 5 μ g/L, 10 μ g/L, 50 μ g/L, 100 μ g/L series concentration.Every hole adds 50 μ, 1 standard items, adds 50 μ l enzyme-labelled antigen dilutions subsequently, and room temperature reaction 30 minutes is washed plate 3 times, adds the developer incubation 15 minutes, cessation reaction, microplate reader reading.The data of testing result see the following form 2
Table 2 antiserum IC
50Value
| The inhibition concentration of melamine (ng/ml) | 0 | 1 | 5 | 10 | 50 | 100 |
| The OD value | 1.541 | 1.353 | 1.026 | 0.736 | 0.423 | 0.154 |
By data in the table 2 as can be known, melamine antiserum IC
50Value shows that the antiserum that the present invention prepares is to have specific preferably anti-melamine polyclonal antibody about 10ng/ml, can satisfy melamine and detect requirement.
Embodiment 3 detects the establishment of the enzyme linked immunological kit of melamine
Set up the enzyme linked immunological kit that detects melamine, make it comprise following component:
(1) bag is by the ELISA Plate of melamine antibody;
(2) concentration is the enzyme labeling melamine of 1mg/L;
(3) the melamine standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 1 μ g/L, 5 μ g/L, 10 μ g/L, 50 μ g/L, 100 μ g/L;
(4) substrate colour developing liquid A liquid is urea peroxide, and substrate colour developing liquid B liquid is tetramethyl benzidine;
(6) cleansing solution is the phosphate buffer that contains 0.05% polysorbas20;
(7) the concentrating sample dilution is the phosphate buffer of 0.1% Tween-20;
(8) enzyme labeling melamine dilution is the phosphate buffer that contains 0.01% Tween-20;
(9) stop buffer is the hydrochloric acid solution of 2mol/L.
The detection of melamine residual in embodiment 4 samples
4.1 sample pre-treatments
Accurately take by weighing the 5g feed in the 40ml centrifuge tube.Add 20mlDMSO vibration 1 hour, the centrifuging and taking supernatant, with the dilution of sample sample diluting liquid, the preparation sample solution.
4.2 detect with kit
In the ELISA Plate micropore of melamine antibody bag quilt, add series standard product or sample solution 50 μ 1, add enzyme mark melamine working fluid 50 μ l again, room temperature reaction 30 minutes.Pour out liquid in the hole, every hole adds 250 μ l through 10 times of cleansing solutions that diluted, and pours out liquid in the hole after 30 seconds, and so repetitive operation is washed plate 3 times altogether, pats dry with thieving paper.Every hole adds substrate colour developing liquid A liquid (urea peroxide) 50 μ l, adds B liquid (tetramethyl benzidine) 50 μ l again, and mixing gently vibrates, room temperature lucifuge colour developing 15min, every hole adds stop buffer (2mol/L hydrochloric acid) 50 μ l, and the mixing that vibrates is gently measured every hole absorbance (OD value) with microplate reader.
4.3 interpretation of result:
Calculate percentage absorbance and drawing standard curve, the concentration of melamine can be read from typical curve in corresponding each sample, also can calculate the content of melamine in sample with regression equation method.Utilize the be more convenient for express-analysis of a large amount of samples of professional computer software.According to the comparison of the depth and the series concentration standard solution color of the sample of color on the ELISA Plate, can judge the concentration range of melamine in the sample.
The test of experimental example 1 kit precision
This experimental example is the repeatable test of standard.Concrete operations are:
In the ELISA Plate of the method preparation from every crowd of embodiment 2 or 3, each extracts 10 holes, measures the absorbance (OD) of the standard solution of 5 μ g/L, repeats 3 times, calculates coefficient of variation CV%.
The result shows coefficient of variation scope between 4.8-7.6%, has met the coefficient of variation less than 10% regulation, illustrates that this kit standard items precision has reached requirement.
The recovery test of experimental example 2 kits
Get the melamine standard specimen of two concentration, sample added recovery test, each concentration do 4 parallel, calculate recovery rate respectively.The result shows that the interpolation recovery in the feed is 75%-105%.
The test of experimental example 3 kit storage lives
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value of kit (zero adds), 50% inhibition concentration, melamine added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed for two weeks under 37 ℃ of preservation conditions, carries out the accelerated deterioration experiment, and the result shows that the every index of this kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 12 months at 2-8 ℃.
Claims (9)
1. direct competition method enzyme linked immunological kit that detects melamine, comprising: melamine specific antibody, melamine standard items and enzyme labeling melamine.
2. enzyme linked immunological kit according to claim 1 is characterized in that: described kit also comprises by enzyme labeling melamine, the ELISA Plate that is coated with the melamine specific antibody, melamine standard solution, substrate developer, cleansing solution, stop buffer, concentrating sample dilution and enzyme mark thing dilution.
3. enzyme linked immunological kit according to claim 1 and 2 is characterized in that: described melamine specific antibody is a polyclonal antibody.
4. enzyme linked immunological kit according to claim 1 and 2 is characterized in that: the used enzyme of described enzyme labeling melamine is a horseradish peroxidase.
5. enzyme linked immunological kit according to claim 2 is characterized in that: described cleansing solution is for containing 0.05%-0.5% Tween-20 phosphate buffer.
6. enzyme linked immunological kit according to claim 2 is characterized in that: described developer is made up of developer A and developer B, and developer A is hydrogen peroxide or urea peroxide, and developer B is o-phenylenediamine or tetramethyl benzidine.
7. enzyme linked immunological kit according to claim 2 is characterized in that: described concentrating sample dilution is the phosphate buffer that contains 0.1% Tween-20.
8. enzyme linked immunological kit according to claim 2 is characterized in that: described enzyme mark thing dilution is the phosphate buffer that contains 0.01% Tween-20.
9. the method for content of melamine in the test sample comprises step:
(1) sample pre-treatments;
(2) detect with the arbitrary described kit of claim 1-9, in the ELISA Plate hole that is coated with melamine antibody, add standard items or sample solution, add the enzyme labeling melamine again, hatch the back washing and pat dry, add developer, end on end, measure absorbance with microplate reader;
(3) analyzing and testing result.
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| CN101413947A (en) * | 2008-11-08 | 2009-04-22 | 江苏省苏微微生物研究有限公司 | Enzyme-linked immunologic test box for detecting melamine and preparation and detection method thereof |
| WO2010101777A1 (en) * | 2009-03-06 | 2010-09-10 | Idexx Laboratories, Inc. | Detection of melamine |
| CN101477051B (en) * | 2008-10-28 | 2010-12-22 | 中国检验检疫科学研究院 | Raman spectrum method and reagent kit used for on-site fast detection of melamine in liquid milk |
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| CN101407580B (en) * | 2008-11-28 | 2011-06-08 | 吉林大学 | Method for synthesizing melamine complete antigen and ELISA reagent kit for detecting melamine |
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| CN102435729A (en) * | 2011-12-02 | 2012-05-02 | 杭州迪恩科技有限公司 | One-step enzyme-linked immunoassay method for quickly detecting melamine and kit thereof |
| CN102439445A (en) * | 2009-01-16 | 2012-05-02 | 理德免疫诊断有限责任公司 | Detection devices and methods |
| CN101413943B (en) * | 2008-12-02 | 2012-09-05 | 北京望尔生物技术有限公司 | Method for detecting melamine and specific enzyme-linked immunologic reagent kit |
| CN101839864B (en) * | 2009-03-19 | 2012-10-03 | 兰州大学 | Method for rapidly detecting melamine in liquid milk |
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| CN103102319A (en) * | 2011-11-11 | 2013-05-15 | 北京勤邦生物技术有限公司 | Melamine semiantigen, preparation method and application thereof |
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| CN101477051B (en) * | 2008-10-28 | 2010-12-22 | 中国检验检疫科学研究院 | Raman spectrum method and reagent kit used for on-site fast detection of melamine in liquid milk |
| CN101413947A (en) * | 2008-11-08 | 2009-04-22 | 江苏省苏微微生物研究有限公司 | Enzyme-linked immunologic test box for detecting melamine and preparation and detection method thereof |
| CN101413956B (en) * | 2008-11-08 | 2012-10-17 | 江苏省苏微微生物研究有限公司 | Colloidal gold detection card for rapidly detecting melamine and preparing method thereof |
| CN101407580B (en) * | 2008-11-28 | 2011-06-08 | 吉林大学 | Method for synthesizing melamine complete antigen and ELISA reagent kit for detecting melamine |
| CN101413943B (en) * | 2008-12-02 | 2012-09-05 | 北京望尔生物技术有限公司 | Method for detecting melamine and specific enzyme-linked immunologic reagent kit |
| CN101706469B (en) * | 2009-01-09 | 2012-11-14 | 中国科学院烟台海岸带研究所 | Method and device for detecting tripolycyanamide |
| CN102439445A (en) * | 2009-01-16 | 2012-05-02 | 理德免疫诊断有限责任公司 | Detection devices and methods |
| WO2010101777A1 (en) * | 2009-03-06 | 2010-09-10 | Idexx Laboratories, Inc. | Detection of melamine |
| CN101839864B (en) * | 2009-03-19 | 2012-10-03 | 兰州大学 | Method for rapidly detecting melamine in liquid milk |
| CN102207505A (en) * | 2010-03-29 | 2011-10-05 | 上海友科生物科技有限公司 | Method for in vitro detection of zinc-alpha2-glycoprotein, and kit thereof |
| CN102087217A (en) * | 2011-01-14 | 2011-06-08 | 河北省食品质量监督检验研究院 | Laser Raman spectroscopy method for quickly analyzing content of melamine in milk powder |
| CN103102319A (en) * | 2011-11-11 | 2013-05-15 | 北京勤邦生物技术有限公司 | Melamine semiantigen, preparation method and application thereof |
| CN103102319B (en) * | 2011-11-11 | 2016-02-24 | 北京勤邦生物技术有限公司 | Melamine hapten and its preparation method and application |
| CN102435729A (en) * | 2011-12-02 | 2012-05-02 | 杭州迪恩科技有限公司 | One-step enzyme-linked immunoassay method for quickly detecting melamine and kit thereof |
| CN108152284A (en) * | 2018-02-07 | 2018-06-12 | 衡阳市食品药品检验检测中心 | A kind of detection method of melamine in fluid milk |
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