CN102680675A - Detection method for enzyme-linked immune adsorption of 2-naphthylamine in food - Google Patents
Detection method for enzyme-linked immune adsorption of 2-naphthylamine in food Download PDFInfo
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- CN102680675A CN102680675A CN2012101899701A CN201210189970A CN102680675A CN 102680675 A CN102680675 A CN 102680675A CN 2012101899701 A CN2012101899701 A CN 2012101899701A CN 201210189970 A CN201210189970 A CN 201210189970A CN 102680675 A CN102680675 A CN 102680675A
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Abstract
The invention relates to a detection method for enzyme-linked immune adsorption of 2-naphthylamine in food and belongs to the field of immunology detection analysis. The detection method comprises the following steps: enveloping the same amount of antigens in holes of an ELISA plate; adding to-be-tested 2-naphthylamine-containing food sample and 2-naphthylamine polyclonal antibody, and then causing the 2-naphthylamine in the to-be-tested sample and solid-phase envelope antigen to fight for limited first antibodies and combine with the antibody, wherein few antibodies are combined with the solid-phase envelope antigen when the concentration of the 2-naphthylamine in the to-be-tested sample is high; after achieving reaction balance, pouring out supernatant; washing with washing liquor; remaining the antibodies combined with the solid-phase envelope antigen in the holes of the ELISA plate and reacting with the added ELIAS secondary antibody; and if the combining volume of the ELIAS secondary antibody with the fixed antibodies is reduced, the color reaction is shallow and OD value of a 450nm part detected by an ELIASA is low, confirming that the inhibition ratio is high, vice versa. The concentration of the 2-naphthylamine in the to-be-tested sample is calculated according to a drawn standard curve. The detection method provided by the invention has the advantages of simple sample-pretreatment process, less time consumption, low cost, mild reaction condition, environment-friendly method and capability of realizing large-flux detection of food.
Description
Technical field
A kind of enzyme-linked immunosorbent assay method that detects azo dyes in the food belongs to the immunology detection analysis field.
Background technology
Azo is the material that forms base color in the dyestuff, and the azo dyes synthetic method is comparatively simple, structure is changeable, thereby of a great variety, and a lot of dyestuffs all belong to this.Azo dyes because of its be easy to take place the azo reductive cleavage can produce 20 surplus kind of a carcinogenic aromatic amine receive much concern; And become an extremely important content of chemicals constituent analysis in the products such as food, leather, toy; And biphenylamine and 2-naphthylamines are two kinds of the strongest aromatic amines of carcinogenicity wherein, and they are intermediates of synthetic many industrial dyes and food colour.Yet continuous development along with food industry; People also improve constantly for the requirement of food organoleptic quality; The incomparable superiority of other dyestuff such as azo dyes is bright-colored, painted simply by means of himself, stablize; And the illegal retailer who is reaped staggering profits adds in the food more and more, like tonyred.For the detection of azo dyes, mainly be to realize its monitoring through the aromatic amine that detects its generation.At present; The detection of aromatic amine mainly relies on instrumental analysis such as traditional gas chromatography, liquid chromatography, gas chromatography mass spectrometry, LC-MS to detect; Though these detection methods are sensitive, accurate, all rely on expensive and huge instrument and equipment, it is higher therefore to detect cost; And consuming time, complex pretreatment is loaded down with trivial details, be difficult to satisfy the on-the-spot fast and convenient and sensitive requirement efficiently that detects.So, work out a kind of quick, sensitive, easy, accurate, efficient and inexpensive immunological detection method, imperative.Yet enzyme linked immunological is learned to rarely have in this field so far and is set foot in, thereby the necessity and the importance of carrying out this research have some idea of.
Summary of the invention
The technical matters that (one) will solve
The present invention is intended to set up a kind of enzyme-linked immunosorbent assay method with characteristics such as high specific, high sensitivity, pin-point accuracy, pinpoint accuracy, method of operating are simple, quick, inexpensive, is used to contain batch, the fast detecting analysis of the food of azo dyes.
(2) technical scheme
For realizing above-mentioned purpose, the present invention has set up the enzyme-linked immunosorbent assay method of 2-naphthylamines, and detection method has been carried out condition optimizing.
Wherein, sample pre-treatments: according to GB GB/T 17592-2006: food samples is cut into the fragment of 5mm * 5mm, accurately takes by weighing the 3.00g sample after evenly mixing and place tool plug conical flask; The citrate buffer solution that adds 50mL pH 6.0,70 ℃ of stirring in water bath reaction 30min add 10mL hydrosulfurous acid sodium solution then; Continue above-mentioned stirring in water bath reaction 30min; Question response is cooled to room temperature with mobile tap water rapidly with test tube after finishing, and so can the azo dyes in the food be reduced to corresponding aroma amine; The elimination residue is subsequent use then.
Wherein, Encapsulate in the preparation process of ELISA Plate of 2-naphthylamines antigen; Used coating antigen is the coupled complex of haptens 2-naphthylamines and ovalbumin (OVA), and the used damping fluid that encapsulates is 0.05 M pH9.6 sodium carbonate buffer, and used confining liquid is the above-mentioned damping fluid that encapsulates that contains 2% gelatin.
Wherein, 2-naphthylamines polyclonal antibody is the diazotising method coupled complex with 2-naphthylamines and bovine serum albumin(BSA) (BSA), measures protein concentration with the Coomassie brilliant blue method, and with certain immunizing dose repeatedly booster immunization healthy new zealand white rabbit prepare.
Wherein, ELIAS secondary antibody is the goat anti-rabbit antibody (GAR-HRP) of horseradish peroxidase-labeled.
The wherein preparation of main solution:
0.01M phosphate (PBS) damping fluid: sodium chloride 8 g, potassium dihydrogen phosphate 0.24 g, sodium hydrogen phosphate 3.62 g, potassium chloride 0.2g are settled to 1L with ultrapure water.
Cleansing solution (PBST): be the 0.01M that contains 0.05% Tween-20, the phosphate buffer of pH7.4.
Antibody diluent: the PBST solution that contains 0.1% gelatin.
The standard items dilution is a methanol solution.
0.05M the carbonate of pH 9.6 (CBS) buffer solution: take by weighing 1.59g Na
2CO
3, 2.93g NaHCO
3, add ultrapure water and be diluted to 1L.
Colour developing liquid: be made up of A liquid and two kinds of solution of B liquid, the A formula of liquid is: 9.33 g citric acids, 36.8 g Na
2HPO
412H
2O, the H of 180 μ L 30%
2O
2, be settled to 1L with ultrapure water; The B formula of liquid is that 600 mg tetramethyl benzidines are dissolved in 1L monoethylene glycol, and sonic oscillation a few minutes.The two is all stored in 4 ℃ of refrigerators and preserves, and during use A liquid and the B liquid volume ratio mixing with A ︰ B=5 ︰ 1 is got final product.
Stop buffer: be the H of 2mol/L
2SO
4
The check and analysis principle of the inventive method is:
Antigen coated in each hole of ELISA Plate with same amount (100 μ L), add to be measured contain 2-naphthylamines food samples and 2-naphthylamines polyclonal antibody after, the vie each other antibody (one is anti-) of limited anti-determinand of 2-naphthylamines in the testing sample and solid-phase coating antigen; And combine with it, because the antibody content of solid phase antigen in each hole and adding is all identical, so when the contained 2-naphthylamines concentration of testing sample is high; The antibody that combines with solid phase antigen will reduce, and reacts when reaching balance, and supernatant is toppled over out; And wash with washing lotion; So have only the antibody that combines with solid phase antigen to stay in the ELISA Plate hole, and react with the ELIAS secondary antibody that adds, obviously this moment, ELIAS secondary antibody reduced with the antibodies amount that is fixed; Add substrate solution (liquid A liquid promptly develops the color) and colour developing liquid (being B liquid) with cleansing solution washing back; Chromogenic reaction is shallow, and the OD value that detects the 450nm place with ELIASA is low, shows that inhibiting rate is high; Otherwise when 2-naphthylamines concentration to be measured was low, the OD value of then being surveyed was high, and inhibiting rate is low.According to the typical curve of being done, can extrapolate the concentration height of contained 2-naphthylamines in the testing sample.
The enzyme-linked immunosorbent assay method of the reduzate 2-naphthylamines of azo dyes in a kind of food, step is:
(1) antigen encapsulates
With 2-naphthylamines and the coupling of ovalbumin OVA diazotising method; And with this coupled complex as envelope antigen; With the sodium carbonate buffer dilution envelope antigen of 0.05 M, pH 9.6, the antigen diluent multiple is respectively 2000,4000,8000,16000 times, in every hole of ELISA Plate, adds the coating buffer 100 μ L of above-mentioned different extension rates; 4 ℃ of incubated overnight are sealed as confining liquid with the above-mentioned damping fluid that encapsulates that is added with 2% gelatin;
(2) competitive reaction
After the 2-naphthylamines polyclonal antibody of immunity preparation diluted 1000,3000,9000 times respectively with antibody diluent, add in the ELISA Plate, every hole adds 50 μ L, and adding concentration simultaneously respectively is 0 ng/mL; 2 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL; 50 ng/mL, the 2-naphthylamines standard items of 100 ng/mL, every hole adds 50 μ L; Hatch 1h for 37 ℃, place on the shaking table with the PBST cleansing solution then and wash 3 times, each 3min;
Said cleansing solution PBST: be the 0.01M that contains 0.05% Tween-20, the phosphate buffer of pH7.4;
Antibody diluent: the PBST solution that contains 0.1% gelatin;
(3) add ELIAS secondary antibody
Add the goat anti-rabbit antibody GAR-HRP of horseradish peroxidase-labeled, carry out 5000 times of dilutions with antibody diluent, every hole adds 100 μ L, and 37 ℃ of incubation 1h place on the shaking table washing 3 times with PBST, each 3min;
(4) colour developing
In each hole of ELISA Plate, add 100 μ L colour developing liquid respectively, reaction 15 min take out ELISA Plate in 37 ℃ of constant temperature ovens, add the sulfuric acid of 100 μ L stop buffers, 2 mol/L to every hole, measure the light absorption value A at 450nnm place with ELIASA
450
(3) beneficial effect
Food azo dyes detection method provided by the invention has adopted 2-naphthylamines polyclonal antibody; Can accurately detect the part azo dyes in the food delicately; The pre-treatment process of sample is simple, consuming time less, detect that cost is low, reaction conditions is gentle, the method environmental protection, can realize that the big flux of food detects; And the inventive method good stability, the pollution of basic organic solvent-free.The present invention detects for the scene that contains azo dyes and has important practical significance, and has important directive significance for the foundation of the enzyme-linked immunosorbent assay method of other relevant azo dyes.
Description of drawings
The standard of Fig. 1 2-naphthylamines suppresses curve.
Embodiment
The operation of embodiment 1 detection method and result calculate:
The enzyme linked immunological adsorption method of 2-naphthylamines in a kind of detection by quantitative food may further comprise the steps:
(1) preparation 2-naphthylamines-BSA immunogene;
(2) polyclonal antibody of the anti-2-naphthylamines of preparation;
(3) preparation 2-naphthylamines-OVA coating antigen;
(4) preparation encapsulates plate;
(5) competitive reaction;
(6) add ELIAS secondary antibody;
(7) colour developing;
Wherein:
(1) preparation 2-naphthylamines-BSA immunogene
Take by weighing the 2-naphthylamines (about 0.05mmol) of 7.2 mg, with about its dissolving and transferring pH to 1,0 ℃ of precooling 30min obtains A liquid with 1mol/L HCl solution; Take by weighing 6g NaNO
2Be dissolved in the 20mL pure water, be made into 30% NaNO
2Solution, 0 ℃ of precooling 30min; 4 ℃, the lasting stirring down are 30% NaNO of configuration
2Drips of solution is added in the A liquid, NaNO
2The addition of solution is used the starch-kalium iodide detection paper, and when it becomes black-and-bluely, and nondiscolouring can stop to add in 2 seconds, this continued reflection 15min; Take by weighing 67mg BSA, be dissolved in the CB solution of 9mL, 0.1M pH9.6, and place 0 ℃ of precooling 30min; 4 ℃, continue to stir down diazotizing 2-naphthylamines slowly be added drop-wise in the BSA solution, and stirring reaction spends the night in 4 ℃ of refrigerators, in this process, wants continuous detection reaction system pH, and with 1M NaOH it is remained on about pH8～9.After reaction finishes, reactant liquor is transferred in the bag filter, then in the PBS of 0.01M, pH7.4,72h dialyses under 4 ℃ of temperature.Measure protein concentration with the Coomassie brilliant blue method then.4 ℃ of preservations are subsequent use.
(2) polyclonal antibody of the anti-2-naphthylamines of preparation
Used immune animal is 4 of new zealand white rabbits, is numbered respectively No. 1, No. 2, No. 3, No. 4.Adopt subcutaneous and intramuscular injection to carry out immunity.
Immunization method: initial immunity is with the immunogene and the isopyknic Freund's complete adjuvant mixing and emulsifying in having the syringe of connecting pipe that make, and dosage is 0.5mg/kg, adopts the subcutaneous multi-point injection in back; Carry out the booster immunization first time all around, carry out emulsification with incomplete Freund, dosage is 1 mg/kg, adopts intramuscular injection; Whenever at a distance from carrying out booster immunization two weeks one time, carry out emulsification with incomplete Freund later on, immunizing dose is 1 mg/kg, adopts intramuscular injection.Since the booster immunization second time, ear edge blood sampling after each immune 8-10 days, and with blood in 5000r/min, 4 ℃ centrifugal 10min down, collect serum and measure and tire and inhibiting rate.After all reaching maximum when tiring with inhibiting rate or meeting the demands; With the blood sampling of heart puncture blood collecting method, blood is collected serum after same centrifugal condition is centrifugal, adopt the immune-affinity chromatography antagonist to carry out purifying; In gained antibody, add equivalent glycerine and 0.1% Sodium azide ,-20 ℃ of preservations.
(3) preparation 2-naphthylamines-OVA coating antigen
The above-mentioned diazotising method of the synthetic employing of 2-naphthylamines-OVA coating antigen is synthetic, changes BSA into OVA and gets final product.
(4) encapsulate the preparation of plate
To encapsulate plate be 96 hole ELISA Plates and in micropore, encapsulate 2-naphthylamines-OVA conjugate; The carbonate buffer solution that adopts 0.05M pH 9.6 is as coating buffer dilution 2-naphthylamines-OVA conjugate, and the antigen diluent multiple is respectively 2000,4000,8000,16000 times, in each hole that encapsulates plate, adds 100 μ L coating buffers then; 37 ℃ of baking ovens are placed 2h or 4 ℃ and are spent the night; Wash 3min with cleansing solution, do at the thieving paper arsis at every turn, repeat 2 times.Add the above-mentioned damping fluid that encapsulates that contains 2% gelatin then and seal as confining liquid, 37 ℃ of baking oven incubation 2h take out with above-mentioned cleansing solution washing 3 times.
(5) competitive reaction
Respectively a series of concentration gradients are respectively 0 ng/mL, 2ng/mL, 5 ng/mL; 10 ng/mL, 20 ng/mL, 50 ng/mL; The 2-naphthylamines standard items of 100 ng/mL and join respectively through the sample extracting solution of pre-treatment encapsulate in the plate micropore separately, and every hole 50 μ L want the parallel application of sample of diplopore.Again the 2-naphthylamines polyclonal antibody of immunity preparation is diluted 9000 times respectively with antibody diluent (contain 0.1% gelatin, contain 0.05% Tween-20, the PBS PBST of pH 7.4); Add in the ELISA Plate; Every hole addition is 50 μ L; 37 ℃ of constant temperature ovens wash 3 times with PBST after hatching 1h, wash 3min at every turn;
(6) add ELIAS secondary antibody
The goat-anti rabbit (GAR-HRP) that adds horseradish peroxidase-labeled with 5000 times of antibody diluent dilutions, adds 100 μ L in every hole, wash 3 times each 3min behind 37 ℃ of constant temperature oven incubation 1h with PBST;
(7) colour developing
Every hole adds 100 μ L colour developing liquid, places 37 ℃ of light tight constant temperature oven reaction 15 min, takes out every hole, back and adds 100 μ L stop buffers, measures the 450nm light absorption value A of place with ELIASA
450
The method for drafting that standard suppresses curve is: deduct the OD value gained difference that adds Cmax standard items hole to the OD value that adds 0 ng/mL standard items hole and be decided to be B
0, the difference that deducts the OD value that adds 0 ng/mL standard items hole the OD value that adds other each hole of concentration is designated as B, calculates the corresponding B/B of each concentration
0Value; With standard items concentration is horizontal ordinate, with its corresponding B/B
0Value is ordinate, draws 2-naphthylamines standard and suppresses curve.Can obtain the concentration of counter sample according to the regression equation of curve, also can obtain the minimum detectable level IC of 2-naphthylamines
90(concentration of correspondence during B/Bo=90%) is 2.0ng/mL, IC
50(concentration of correspondence during B/Bo=50%) is 14.5 ng/mL.
Confirming of embodiment 2 recovery and sample extraction method
(1) adds sample with fresh color bean curd as experiment; According to the said pre-treating method of preamble it is shredded; Level with 10.0 ng/g, 20.0 ng/g, 50.0 ng/g adds the 2-naphthylamines to grinding in the broken fresh color bean curd; Each concentration prepares 4 parts in sample respectively, handles food samples according to the sample-pretreating method in the technique scheme, and wherein azo dyes is reduced to corresponding aroma amine.Set blank experiment simultaneously.
(2) according to preceding method, accurately pipette 50 μ L sample extracting solutions with liquid-transfering gun, directly application of sample carries out ELISA and measures in 96 hole micropores.
(3) calculating of the recovery:, find concentration separately according to corresponding inhibition ratio from typical curve again according to the sample OD value computing corresponding inhibition ratio of difference interpolation concentration.Detectable concentration is the recovery of corresponding concentration with the ratio of actual concentration.The result sees table 1.
Table 1 adds the mensuration of the recovery
Claims (3)
1. the enzyme-linked immunosorbent assay method of the reduzate 2-naphthylamines of azo dyes in the food is characterized in that:
(1) antigen encapsulates
With 2-naphthylamines and the coupling of ovalbumin OVA diazotising method; And with this coupled complex as envelope antigen; With the sodium carbonate buffer dilution envelope antigen of 0.05 M, pH 9.6, the antigen diluent multiple is respectively 2000,4000,8000,16000 times, in every hole of ELISA Plate, adds the coating buffer 100 μ L of above-mentioned different extension rates; 4 ℃ of incubated overnight are sealed as confining liquid with the above-mentioned damping fluid that encapsulates that is added with 2% gelatin;
(2) competitive reaction
After the 2-naphthylamines polyclonal antibody of immunity preparation diluted 1000,3000,9000 times respectively with antibody diluent, add in the ELISA Plate, every hole adds 50 μ L, and adding concentration simultaneously respectively is 0 ng/mL; 2 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL; 50 ng/mL, the 2-naphthylamines standard items of 100 ng/mL, every hole adds 50 μ L; Hatch 1h for 37 ℃, place on the shaking table with the PBST cleansing solution then and wash 3 times, each 3min;
Said cleansing solution PBST: be the 0.01M that contains 0.05% Tween-20, the phosphate buffer of pH7.4;
Antibody diluent: the PBST solution that contains 0.1% gelatin;
(3) add ELIAS secondary antibody
Add the goat anti-rabbit antibody GAR-HRP of horseradish peroxidase-labeled, carry out 5000 times of dilutions with antibody diluent, every hole adds 100 μ L, and 37 ℃ of incubation 1h place on the shaking table washing 3 times with PBST, each 3min;
(4) colour developing
In each hole of ELISA Plate, add 100 μ L colour developing liquid respectively, reaction 15 min take out ELISA Plate in 37 ℃ of constant temperature ovens, add the sulfuric acid of 100 μ L stop buffers, 2 mol/L to every hole, measure the light absorption value A at 450nnm place with ELIASA
450
2. 2-naphthylamines enzyme-linked immunosorbent assay method according to claim 1; It is characterized in that the prescription of PBST cleansing solution is to add sodium chloride 8 g, potassium dihydrogen phosphate 0.24 g, sodium hydrogen phosphate 3.62 g, potassium chloride 0.2g and Tween-20 0.5 mL in the 1000 mL distilled water.
3. 2-naphthylamines enzyme-linked immunosorbent assay method according to claim 1 is characterized in that, colour developing liquid is made up of A liquid and two kinds of solution of B liquid, and the A formula of liquid is 9.33 g citric acids, 36.8 g Na
2HPO
412H
2O, the H of 180 μ L 30%
2O
2, be settled to 1L with ultrapure water; The B formula of liquid is that 600 mg tetramethyl benzidines are dissolved in 1L monoethylene glycol, and sonic oscillation a few minutes, and packing is used then; The two is all stored in 4 ℃ of refrigerators and preserves, during use with A liquid and B liquid volume ratio mixing with A ︰ B=5 ︰ 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111781361A (en) * | 2020-05-20 | 2020-10-16 | 量准(武汉)生命科技有限公司 | Method for determining binding kinetic parameters of virus and antibody by utilizing enzyme label instrument |
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|---|---|---|---|---|
| US5738693A (en) * | 1993-09-04 | 1998-04-14 | Basf Aktiengesellschaft | Detection of naphthylamines in mineral oils |
| CN101871936A (en) * | 2010-06-03 | 2010-10-27 | 江南大学 | Rhodamine B ELISA detection method |
| CN102060713A (en) * | 2010-12-09 | 2011-05-18 | 中华人民共和国嘉兴出入境检验检疫局 | Aromatic amine hapten for detecting azo dyes and preparation method thereof |
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2012
- 2012-06-11 CN CN2012101899701A patent/CN102680675A/en active Pending
Patent Citations (3)
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|---|---|---|---|---|
| US5738693A (en) * | 1993-09-04 | 1998-04-14 | Basf Aktiengesellschaft | Detection of naphthylamines in mineral oils |
| CN101871936A (en) * | 2010-06-03 | 2010-10-27 | 江南大学 | Rhodamine B ELISA detection method |
| CN102060713A (en) * | 2010-12-09 | 2011-05-18 | 中华人民共和国嘉兴出入境检验检疫局 | Aromatic amine hapten for detecting azo dyes and preparation method thereof |
Non-Patent Citations (1)
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| CN111781361A (en) * | 2020-05-20 | 2020-10-16 | 量准(武汉)生命科技有限公司 | Method for determining binding kinetic parameters of virus and antibody by utilizing enzyme label instrument |
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Application publication date: 20120919 |