CN102313806A - Method and enzyme-linked immunosorbent assay (ELISA) kit for detecting sunset yellow - Google Patents
Method and enzyme-linked immunosorbent assay (ELISA) kit for detecting sunset yellow Download PDFInfo
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Abstract
The invention discloses a method and enzyme-linked immunosorbent assay (ELISA) kit for detecting sunset yellow. The ELISA kit for detecting sunset yellow comprises sunset yellow hapten and a specific antibody of sunset yellow, wherein the specific antibody is a polyclonal antibody or monoclonal antibody of sunset yellow. A high-specificity monoclonal antibody of sunset yellow is adopted in the kit, thus ensuring the reliability of the detection result. The experimental results show that the kit has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like. Main reagents of the kit adopt working solutions, thus being convenient to use and low in cost. The method for detecting sunset yellow by using the kit is simple and convenient to operate and has low requirements for pre-processing of samples; and by adopting the method, multitudinous samples can be simultaneously detected quickly. Therefore, the method for detecting sunset yellow by using the ELISA kit can be used for on-site supervision and is suitable for qualitative and quantitative screening of multitudinous samples.
Description
Technical field
The present invention relates to a kind of method and enzyme linked immunological kit that detects sunset yellow.
Background technology
Sunset yellow is one of six kinds of edible synthesized coloring matters of present China approval use, is mainly used in the painted of food and medicine, also can be used for making aluminium salt mordant pigment.China's " food additives use hygienic standard " (GB2760-2007) stipulates: sunset yellow can be used in fruit drink, soda, inspissated juice, candy, cake, watermelon jam can, prawn slice class swelling product, vegetable protein beverage and the sour milk beverage, and maximum use amount is 0.10g/kg; Being used for the maximum use amount of frozen is 0.09g/kg; Be used for candy dressing and red and green pickled fruit, maximum use amount is 0.20g/kg.Its molecular structure is an azo-compound, and the product of metabolism is for having the aromatic compounds of potential hazard to human body in vivo.Therefore, there is strict restriction in China to the sunset yellow in the food.If people are long-term or the food of disposable a large amount of edible sunset yellow content overproofs, may cause symptoms such as allergy, diarrhoea, excessive when intake, when surpassing the liver load, can accumulate in vivo, kidney, liver are produced certain injury.
At present, the detection method taked of sunset yellow national standard is TLC, spectrophotometer method, fluorescent spectrometry and high performance liquid chromatography.In recent years; China's researcher has been done a large amount of work in the sunset yellow context of detection; But all mainly concentrate on the physics and chemistry context of detection; The report about the EL ISA detection method of sunset yellow is not found in literature search as yet, and Given this, the present invention studies and set up the competition EL ISA detection method of sunset yellow.
Summary of the invention
An object of the present invention is to provide a kind of enzyme linked immunological kit that detects sunset yellow.The enzyme linked immunological kit of detection sunset yellow provided by the present invention comprises the specific antibody of sunset yellow haptens and sunset yellow; Said specific antibody is the polyclonal antibody or the monoclonal antibody of sunset yellow; Wherein, the specific antibody of said haptens and sunset yellow can exist with following any form:
1) sunset yellow haptens and carrier protein are carried out coupling, obtain the conjugate (below be called the hapten-carrier protein conjugate) of sunset yellow haptens and carrier protein, as coating antigen, said specific antibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) said specific antibody is a coating antigen, and said haptens carries out after the enzyme labeling as the enzyme labeling thing.
Said kit can also both comprise the specific antibody of haptens and sunset yellow, comprised antiantibody again; Said antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody; Wherein, said haptens and antiantibody exist with following arbitrary form:
1) said haptens and carrier protein are carried out coupling, obtain the hapten-carrier protein conjugate, as coating antigen, said antiantibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) said antiantibody is a coating antigen, and said haptens carries out after the enzyme labeling as the enzyme labeling thing.
Said sunset yellow polyclonal antibody or sunset yellow monoclonal antibody all obtain as immunogene with said sunset yellow hapten-carrier protein conjugate; Said carrier protein can be thyroprotein, mouse haemocyanin, human albumin, bovine serum albumin, rabbit anteserum albumen, hemocyanin or ovalbumin etc.
Said monoclonal antibody obtains through hybridoma cell technology.Said sunset yellow polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody.
Detect for ease, said kit also comprises ELISA Plate, when said sunset yellow specific antibody or antiantibody carry out mark with enzyme, is coated with said sunset yellow haptens or sunset yellow hapten-carrier protein conjugate on the said ELISA Plate; When said haptens carries out mark with enzyme, be coated with said sunset yellow specific antibody or said antiantibody on the said ELISA Plate.
For more convenient on-site supervision and great amount of samples examination, said kit also can comprise sunset yellow standard solution, colour developing liquid, stop buffer, concentrated cleaning solution, concentrate and redissolve at least a in the liquid;
Wherein, said concentrated cleaning solution is that the pH value is 6-9, the final concentration that contains in said concentrated cleaning solution is that 0.02-0.05% (quality percentage composition) antiseptic, the final concentration in said concentrated cleaning solution are the phosphate buffer of 1.0-2.0% (quality percentage composition) Tween-20,0.1-0.2mol/L; Said concentrated redissolution liquid is that to contain final concentration in said concentrated redissolution liquid be that DMSO, the PH of 5-10% (quality percentage composition) is the phosphate buffer of 6-8,0.1-0.2mol/L.
Used marker enzyme is horseradish peroxidase or alkaline phosphatase in the said enzyme labeling; When marker enzyme was horseradish peroxidase, said substrate colour developing liquid was hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, and said stop buffer is 1-2mol/L sulfuric acid solution or hydrochloric acid solution.When being labeled as alkaline phosphatase, developer is the nitro phosphate buffer, and stop buffer is the 1-2mol/L sodium hydroxide solution.
Said concentrated cleaning solution can be 7.4 for the pH value specifically, contain final concentration 0.03% antiseptic, the final concentration in said concentrated cleaning solution in said concentrated cleaning solution is 1.50% Tween-20,0.2mol/L phosphate buffer; Said concentrated redissolution liquid can be that 8% DMSO, pH are 7.4, the 0.2mol/L phosphate buffer for containing final concentration in said concentrated redissolution liquid specifically; Said substrate colour developing liquid is hydrogen peroxide or urea peroxide, and tetramethyl benzidine sulfate mixed solution, said stop buffer are 2mol/L sulfuric acid solution or hydrochloric acid solution.
Sunset yellow is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.Adopt carbodiimide method to carry out coupling sunset yellow haptens and bovine serum albumin and obtain immunogene; The ratio of sunset yellow haptens and carrier protein is crossed low or too high all unfavorable to immunity, and haptens is 23-28 with the mol ratio that combines of bovine serum albumin: 1 is more suitable.In preparation during coating antigen, the mole proportioning of sunset yellow haptens and said carrier protein be 26: 1 proper.The sunset yellow haptens is through obtaining the sunset yellow and the oxalic acid tert-butyl ester through chemical reaction.
When making was coated with the ELISA Plate of coating antigen, the said damping fluid that encapsulates can be 9.0-9.6,0.05mol/L carbonate buffer solution for the pH value, and confining liquid is pH6-8, contains 5%-8% lowlenthal serum, the caseic 0.02mol/L phosphate buffer of 10g/L.
Another object of the present invention provides a kind of method that detects sunset yellow.
The method of the sunset yellow content in a kind of test sample provided by the present invention may further comprise the steps:
1) with sunset yellow haptens or sunset yellow hapten-carrier protein conjugate coated elisa plate;
2) sunset yellow standard items or testing sample are added in the said ELISA Plate hole that encapsulates, every then hole adds the sunset yellow specific antibody of enzyme labeling, hatches washing;
3) add the colour developing of substrate colour developing liquid;
4) add the reaction terminating liquid cessation reaction;
5) through comparing the color of sunset yellow standard items and testing sample, infer the sunset yellow content that in the testing sample; Perhaps, measure the absorbance in each hole, set up the typical curve of sunset yellow concentration, and extrapolate the sunset yellow content in the testing sample by the absorbance of testing sample according to this typical curve with respect to absorbance.
The method of the sunset yellow content in the another kind of test sample provided by the present invention may further comprise the steps:
1) with sunset yellow haptens or sunset yellow hapten-carrier protein conjugate coated elisa plate;
2) add sunset yellow standard items or testing sample;
3) add the sunset yellow specific antibody, hatch, washing;
4) antiantibody of adding enzyme labeling is hatched, washing;
5) add the colour developing of substrate colour developing liquid;
6) add the reaction terminating liquid cessation reaction;
7) through comparing the color of sunset yellow standard items and testing sample, infer the sunset yellow content that in the testing sample; Perhaps, measure the absorbance in each hole, set up the typical curve of sunset yellow concentration, and extrapolate the sunset yellow content in the testing sample by the absorbance of testing sample according to this typical curve with respect to absorbance.
The method of the sunset yellow content in test sample provided by the present invention can also comprise the sample pre-treatments step:
When said sample was liquid food, said sample-pretreating method was: liquid food is removed gas, with the sample dilution with volume ratio 1: 10-15 mixes, and gets to mix the back sample and be used for analyzing;
When said sample is candy, preserved plum based food; Said sample-pretreating method is: thing to be checked is pulverized, with the sample dilution be 1 with mass volume ratio: the 5-10 mixing adds the normal hexane degreasing of certain volume again; Centrifugal, get the dilution of supernatant and sample and detect with certain proportion dilution back.
When said sample was ice cream, sample placed water-bath dissolving, adds a certain amount of normal hexane degreasing, with the sample dilution be 1 with mass volume ratio: the 10-15 mixing, centrifugal, take off layer and detect.
When said sample is cake, homogenize, add the normal hexane of certain volume, mixing, with the sample dilution with mass volume ratio 1: the 10-15 mixing, centrifugal, take off layer and detect.
The enzyme linked immunological kit of detection sunset yellow of the present invention adopts direct competitive and the indirect competitive ELISA method is qualitative or the detection by quantitative sample in the content of sunset yellow.Adopt the sunset yellow monoclonal antibody of high specific in this kit, guaranteed the reliability of testing result.Experimental result shows that this kit has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height; The main agents of this kit all adopts the working fluid form, and is easy to use, with low cost.Detect the method for sunset yellow with kit of the present invention, easy and simple to handle, simplified the step of traditional detection method, shortened the time of detecting, the pre-treatment of sample is required low, fast detecting gross sample simultaneously.Therefore, the method for utilizing enzyme linked immunological kit of the present invention to detect can be carried out the qualitative and quantitative examination of on-site supervision and suitable a large amount of samples, will in the detection of sunset yellow, play a significant role.
Description of drawings
Fig. 1 is a sunset yellow ELISA examination criteria curve.
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Embodiment 1, be that coating antigen, enzyme labelled antibody are the kit and the ELISA detection method of enzyme labeling thing with the hapten-carrier protein conjugate
One, be that coating antigen, enzyme labelled antibody are the ELISA detection principle of enzyme labeling thing with sunset yellow haptens and carrier protein couplet thing:
When the coating antigen on the ELISA Plate capillary strip is sunset yellow hapten-carrier protein conjugate; After in enzyme plate micropore, adding standard solution or sample solution; Sunset yellow coupled antigen competition enzyme mark sunset yellow specific antibody is washed plate, colour developing on sunset yellow in the sample and the ELISA Plate; The content of sample light absorption value and sunset yellow is negative correlation, relatively can draw the content of sunset yellow in the sample with typical curve.Simultaneously also can be according to the depth of color on the ELISA Plate, through with the more rough judgement sample of series concentration sunset yellow standard solution color in the content of sunset yellow.
Two, be that coating antigen, enzyme labelled antibody are that the kit of enzyme labeling thing generally can comprise with sunset yellow hapten-carrier protein conjugate:
1, be coated with the ELISA Plate of sunset yellow hapten-carrier protein conjugate, the concentration of coating antigen can be 0.1-0.5 μ g/ml.
2, enzyme labelled antibody working fluid: enzyme labelled antibody is the sunset yellow specific antibody with horseradish peroxidase-labeled; The dilution of enzyme labelled antibody is that to contain final concentration in dilution be that lowlenthal serum, the pH of 6-8% (quality percentage composition) is 6-8,0.2-0.3mol/L phosphate buffer, and enzyme mark antiantibody working dilution is 1: 1500.
3, the sunset yellow standard solution is 6 bottles, and concentration is respectively 0 μ g/ml, 1 μ g/ml; 3 μ g/ml; 9 μ g/ml, 27 μ g/ml, 81 μ g/ml; 100 μ g/ml, the preparation standard items solution be contain the preparation standard items solution in final concentration be that pH is the phosphate buffer of 7.0-7.5,0.1-0.2mol/L.
4, substrate colour developing liquid: substrate colour developing liquid is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, 7ml/ bottle, 1 bottle.
5, stop buffer: 1-2mol/L hydrochloric acid or sulfuric acid.
6, concentrated cleaning solution: the final concentration that the pH value is 6-9, contain in cleansing solution is that 0.02-0.05% (quality percentage composition) antiseptic and the final concentration in cleansing solution are the Tween-20 of 1.0-2.0% (quality percentage composition), the phosphate buffer of 0.1-0.2mol/L; The 40ml/ bottle, 1 bottle.
7, concentrate to redissolve liquid: containing redissolving final concentration in the liquid is that DMSO, the pH of 5-10% (quality percentage composition) is the phosphate buffer of 6-8,0.1-0.2mol/L; The 200ml/ bottle, 1 bottle.
Three, be that coating antigen, enzyme labelled antibody are the concrete composition and the preparation thereof of the kit of enzyme labeling thing with sunset yellow hapten-carrier protein conjugate:
(1) forms
1, be coated with the ELISA Plate of sunset yellow haptens-bovine serum albumin(BSA) conjugate, the concentration of coating antigen can be 0.20 μ g/ml.
2, enzyme labelled antibody working fluid: enzyme labelled antibody is the sunset yellow specific antibody with horseradish peroxidase-labeled; The dilution of enzyme labelled antibody is that to contain final concentration in dilution be that lowlenthal serum, the pH of 6% (quality percentage composition) is 7.4, the 0.2mol/L phosphate buffer, and enzyme mark antiantibody working dilution is 1: 1500.
3, the sunset yellow standard solution is 6 bottles, and concentration is respectively 0 μ g/ml, 1 μ g/ml, 3 μ g/ml, 9 μ g/ml, 27 μ g/ml, 81 μ g/ml.The solution of preparation standard items is that pH is 7.2, the phosphate buffer of 0.1mol/L for final concentration in the preparation standard solution.
4, substrate colour developing liquid: substrate colour developing liquid is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, 7ml/ bottle, 1 bottle.
5, stop buffer: 2mol/L hydrochloric acid.
6, concentrated cleaning solution: the pH value is 7.4, and the final concentration that contains in cleansing solution is that 0.03% (quality percentage composition) antiseptic, the final concentration in cleansing solution are 1.5% (quality percentage composition) Tween-20,0.2mol/L phosphate buffer; The 40ml/ bottle, 1 bottle.
7, concentrate to redissolve liquid: containing redissolving final concentration in the liquid is that 8% (quality percentage composition) DMSO, PH are 7.4, the 0.2mol/L phosphate buffer; The 200ml/ bottle, 1 bottle.
(2) preparation
1, is coated with the preparation of the ELISA Plate of haptens-bovine serum albumin(BSA) conjugate
1) preparation of sunset yellow coating antigen:
A.200mg the amino sunset yellow of purifying adds 0.3mol/L HCL 12mL, with the amino sunset yellow stirring and dissolving of purifying;
B. under the ice bath stirring condition, slowly add 10g/L NaNO
2, make amino sunset yellow diazotising, monitor NaNO simultaneously
2Whether excessive, detection method: get starch/liquor kalii iodide and drip on the white dried glass sheet, drip 1 above-mentioned diazotising solution, mix and present the black-and-blue diazotising of promptly accomplishing amino sunset yellow in back 30 seconds;
C. above-mentioned diazotizing amino sunset yellow continues reaction 20 minutes, takes by weighing the BSA of 800mg, is dissolved in carbonate buffer solution, processes the 20g/L protein solution, under the ice bath stirring condition, slowly adds the amino sunset yellow solution of diazotising; The limit edged is regulated pH to 7.5～8.0 with 0.01mol/L NaOH, obtains the conjugate of sunset yellow and bovine serum albumin(BSA), places 4 ℃ of refrigerator overnight;
D. under 4 ℃ of conditions, with lower rare HCL (0.01mol/L) solution dialysis of pH value, the PBS with 0.1mol/L pH7.2 dialysed 3 days then, changed dislysate every day 3 times earlier, after the packing freeze-drying, and-20 ℃ of preservations.
2) be coated with the preparation of the ELISA Plate of coating antigen
With encapsulating damping fluid coating antigen is diluted to 0.2 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubations 2 hours; 4 ℃ are spent the night again, and the coating buffer that inclines is with cleansing solution washing 5 times; Each 30 seconds, clap and do, in every hole, go into 200-300 μ l confining liquid then; 37 ℃ incubation 1-2 hour, liquid clap to be done in the hole of inclining, deposit with the password protection of aluminium film vacuum dry back.
Wherein, the used damping fluid that encapsulates is that the pH value is 9.5, the 0.05mol/L carbonate buffer solution; Used confining liquid is pH7.4, contains 5% lowlenthal serum, the caseic 0.02mol/L phosphate buffer of 10g/L.
2, sunset yellow MONOCLONAL ANTIBODIES SPECIFIC FOR
1) immunogenic preparation
Sunset yellow is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.
Adopt carbodiimide method to carry out coupling the above-mentioned haptens that obtains and thyroprotein and obtain immunogene, concrete steps are following:
Haptens 10mg, 40mg carbodiimides (DEC) and the 15mg N-hydroxy-succinamide (NHS) of above-mentioned preparation fully are dissolved among the 1mL DMF, under heating condition, stirred 24 hours, obtain reactant liquor a liquid; Take by weighing thyroprotein 25mg, make it fully to be dissolved among the 3.0mLPBS (PH 8.2), obtain anti-liquid b liquid; A liquid is slowly joined in the b liquid, and under room temperature, stirred 5 hours, the mol ratio of haptens and thyroprotein is 26: 1; With 0.01mol/L PBS damping fluid dialysis 3 days, change dislysate every day 2 times, to remove the small-molecule substance of not answering; With the speed of 12000rpm centrifugal 30 minutes, collect supernatant, obtain the conjugate of haptens and thyroprotein; Packing, subsequent use in-20 ℃ of preservations.
2) sunset yellow MONOCLONAL ANTIBODIES SPECIFIC FOR
Animal immune: with the hapten-carrier protein conjugate is that immunogene is carried out the interval immunity to the Balb/c mouse, and indirect ELISA detects and obtain containing in the blood mouse spleen of sunset yellow specific antibody.
Fusion of Cells and clone: get the Balb/c mouse boosting cell and the myeloma cell SP2/0 that produce specific antibody and merge, adopt the indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay or microclone method that cloning is carried out in positive hole, obtain and set up the hybridoma cell strain that produces monoclonal antibody.
Cell cryopreservation and recovery: get the hybridoma that is in exponential phase and process cell suspension, be sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen with cryopreserving liquid.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is injected the sterilization paraffinum liquidum, 7～14 days pneumoretroperitoneum injection hybridomas were gathered ascites after 7～10 days.Carry out the ascites purifying through sad-saturated ammonium sulfate method or affinity chromatography. purity is identified through the SDS-PAGE electrophoresis, bottle packing ,-20 ℃ of preservations.
3, sunset yellow Polyclonal Antibody Preparation
Adopting new zealand white rabbit as immune animal, is immunogene with haptens-thyroprotein conjugate, and immunizing dose is 1.5mg/kg; Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent; The subcutaneous multi-point injection of femoribus internus, getting the same dose immunogene 3-4 week adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once; Immunity is 5 times altogether, does not add adjuvant for the last time.Serum antibody titer is measured in last immunity blood sampling after 10 days, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
4, horseradish peroxidase-labeled antibody:
Use the sunset yellow monoclonal antibody or the polyclonal antibody of the above-mentioned preparation of horseradish peroxidase-labeled.
Four, with the hapten-carrier protein conjugate be the application of the enzyme linked immunological kit of coating antigen
1, kit of the present invention can be used for tracer liquid food, cake, the sunset yellow in the samples such as ice cream.
(1) beverage, fruit juice (extension rate 10):
Containing the vapour beverage needs after the water-bath jolting is fully removed vapour, gets 1ml and adds the 9ml sample diluting liquid, gets 50 μ l as liquid to be checked.
(2) candy, preserved plum, preserved fruit class (extension rate 20):
Get the sample (having nuclear preserved fruit or preserved plum to need to remove earlier stoning) that 2g pulverizes, add the 10ml sample diluting liquid, the 5ml normal hexane fully shook even 10 minutes; Centrifugal 10 minutes of 4000g gets the 1ml water, the 3ml sample diluting liquid, and fully mixing is got 50 μ l as liquid to be checked.
(3) ice cream (extension rate 10):
Place 60 ℃ of water-baths of beaker to melt on ice cream; Get the sample that 1g melts, add the 5ml normal hexane, it is even to shake, and adds the 10ml sample diluting liquid, fully shakes even 3 minutes; Centrifugal 10 minutes of 4000g gets 50 μ l waters, as liquid to be checked.
(4) cake (extension rate 10):
Get the sample that 1g pulverizes, add the 5ml normal hexane, mixing adds the 10ml sample diluting liquid, and fully mixing is 3 minutes; Centrifugal 5 minutes of 4000g gets 50 μ l waters, as liquid to be checked.
2, detect
In the ELISA Plate micropore that is coated with the sunset yellow coupled antigen, add sunset yellow standard solution or sample solution 50 μ l, add enzyme labeling sunset yellow specific antibody working fluid 50 μ l again, with cover plate mould shrouding; Reaction is 30 minutes in 25 ℃ of constant temperature; Pour out liquid in the hole, every hole adds 300 μ l cleansing solutions, pours out liquid in the hole after 30 seconds; Repetitive operation 5 times is clapped dried with thieving paper; Add chromogenic substrate liquid 100 μ l, the mixing that vibrates gently, reaction is 30 minutes in 25 ℃ of constant temperature, and every hole adds 50 μ l2mol/L stop buffer sulfuric acid, and mixing at the 450nm place, is measured every hole absorbance (OD value) with the ELIASA wavelength set.
3, testing result analysis
The absorbance mean value (B) of standard solution of using each concentration that is obtained multiply by 100% again divided by the absorbance (B0) of first standard items liquid (0 standard), obtains the percentage absorbance.
B is the mean light absorbency value of standard solution or sample solution in the formula, and B0 is the mean light absorbency value of 0 μ g/L standard solution.
With sunset yellow standard items concentration (μ g/mL) value is the X axle, and the percentage absorbance is the Y axle, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample then can be read the content of sunset yellow the sample from typical curve.The analysis of testing result also can be adopted regression equation method among the present invention, calculates sample solution concentration.The be more convenient for express-analysis of a large amount of samples of the analysis of testing result computer professional software also capable of using among the present invention, this method, whole testing process only needed just can accomplish in 60 minutes.
Embodiment 2, be that coating antigen, ELIAS secondary antibody are the kit and the ELISA detection method of enzyme labeling thing with the hapten-carrier protein conjugate
One, be that coating antigen, ELIAS secondary antibody are the ELISA detection principle of enzyme labeling thing with sunset yellow hapten-carrier protein conjugate:
When the coating antigen on the ELISA Plate capillary strip is sunset yellow hapten-carrier protein conjugate; After in the ELISA Plate micropore, adding standard solution or sample solution; Add the sunset yellow specific antibody, sunset yellow coupled antigen competition sunset yellow specific antibody is washed plate on sunset yellow in the sample and the ELISA Plate; Add enzyme mark antiantibody again and carry out amplification; With colour developing liquid colour developing, the content of sample absorbance and sunset yellow is negative correlation, relatively can draw the content of sunset yellow in the sample with typical curve.Simultaneously also can be according to the depth of color on the ELISA Plate, through with the more rough judgement sample of series concentration sunset yellow standard solution color in the concentration range of sunset yellow.
Two, be that coating antigen, ELIAS secondary antibody are that the kit of enzyme labeling thing generally can comprise with sunset yellow hapten-carrier protein conjugate:
1, be coated with the ELISA Plate of sunset yellow hapten-carrier protein conjugate, the concentration of coating antigen can be 0.2 μ g/ml.
2, enzyme mark antiantibody working fluid: ELIAS secondary antibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody with horseradish peroxidase-labeled; The dilution of ELIAS secondary antibody is that the final concentration that contains in dilution is lowlenthal serum, pH7.4, the 0.2mol/L phosphate buffer of 8% (quality percentage composition), and enzyme mark antiantibody working fluid dilutability is 1: 5000.
3, sunset yellow specific antibody working fluid: can be sunset yellow polyclonal antibody or sunset yellow monoclonal antibody working fluid; With 3000 times of sunset yellow monoclonal antibody dilutions, obtain the specific antibody working fluid with dilution, said dilution is that the pH value is 7.4, the phosphate buffer of 0.2mol/L.
4, the sunset yellow standard solution is 6 bottles, and concentration is respectively 0 μ g/ml, 1 μ g/ml, 3 μ g/ml, 9 μ g/ml, 27 μ g/ml, 81 μ g/ml.The solution of preparation standard items is that pH is 7.4, the phosphate buffer of 0.1mo/L for final concentration in the preparation standard solution.
5, substrate colour developing liquid: substrate colour developing liquid is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, 7ml/ bottle, 1 bottle.
6, stop buffer: 2mol/L hydrochloric acid or sulfuric acid.
7, concentrated cleaning solution: the pH value is 7.4, and the final concentration that contains in cleansing solution is that 0.03% (quality percentage composition) sodium azide, the final concentration in cleansing solution are 0.05% (quality percentage composition) polysorbas20,0.02mol/L phosphate buffer; The 40ml/ bottle, 1 bottle.
8, concentrate to redissolve liquid: containing redissolving final concentration in the liquid is that 8% (quality percentage composition) DMSO, pH are 7.4, the 0.2mol/L phosphate buffer; The 200ml/ bottle, 1 bottle.
Embodiment 3, kit precision, accuracy, keeping quality test
1, the precision of kit experiment
(1) standard solution replica test
From 3 batches of ELISA Plates according to the preparation of the method the embodiment 1, each extracts 10 micropores out, measures the absorbance (OD value) of 9 μ g/mL standard solutions, repeats 10 times, calculates coefficient of variation CV, and the result sees table 1.
Table 1 standard solution replica test
| cv％ | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 01 batch | 8 | 6.5 | 7.3 | 6.6 | 6.9 | 7.1 | 8.2 | 6.8 | 7.4 | 8.1 |
| 02 batch | 8.2 | 6.8 | 6.7 | 6.9 | 7.2 | 7.4 | 8.4 | 6.3 | 6.6 | 8.3 |
| 03 batch | 7.2 | 6.8 | 6.3 | 6.1 | 8 | 7.5 | 6.3 | 7.6 | 8.1 | 7.7 |
The result shows variation within batch coefficient scope that the kit standard items detect between 6.1～8.4%, and interassay coefficient of variation is 9.6%, meet the coefficient of variation criticize in less than 10%, between batch less than 20% regulation.
(2) repeatability of sample test
In fruit juice, ice cream and cake, add standard items with 10 μ g/ml concentration, get each five of the kits of three different batches respectively, each concentration repeats five times, calculates the coefficient of variation respectively, and the result sees table 2-4.
The repeatable test of table 2 fruit juice
The repeatable test of table 3 ice cream
The repeatable test of table 4 cake
The result shows; The fruit juice sample coefficient of variation is lower than 7%, and the ice cream sample coefficient of variation is lower than 10%, and the cake sample coefficient of variation is lower than 6%; Meet the coefficient of variation less than 20% regulation, explain that the precision of this kit standard items meets the requirement of the Ministry of Agriculture about kit precision standard.
2, the accuracy test of kit
Get standard items 5 μ g/ml, the 15 μ g/ml of two concentration, sample is added recovery test, it is parallel that each concentration is done 3 holes, and calculate recovery rate is seen table 5. respectively
The result shows that the recovery of fruit juice is between 75～78%, and the recovery of ice cream is about 76%, and the recovery of cake is 78～80%, and the recovery of steamed bun stuffed with sugar fruit is 70.5～72.8%.
3, kit storage life test
(1) kit is positioned over 2～8 ℃; Get 0,2,4,6,8,9,10,11 and 12 months kit respectively, to absorbance, 50% inhibition concentration of sunset yellow standard model (1 μ g/ml), add the recovery, each parameter of variation within batch coefficient is measured.
(2) with kit the condition held of 37 ℃ of preservations 12 days, every day to absorbance, 50% inhibition concentration of sunset yellow standard model (1 μ g/ml), add the recovery, each parameter of variation within batch coefficient is measured.
(3) kit was preserved 12 days at-20 ℃ of refrigerators, measure absorbance, 50% inhibition concentration, the interpolation recovery, each parameter of variation within batch coefficient of sunset yellow standard model (1 μ g/ml) every day.
Can find out from the result, preserve test, sunset yellow standard model 1 μ g/ml through three kinds of conditions) absorbance descend less than 5%, and OD is not less than 1.5; 50% inhibiting rate is between 20～40 μ g/ml; Add the recovery between 70～105%; Variation within batch coefficient coefficient is less than 10%; Each item index all conforms to quality requirements, and therefore, kit can be preserved 12 months at 2～8 ℃.
Claims (10)
1. enzyme linked immunological kit that detects sunset yellow comprises the specific antibody of sunset yellow haptens and sunset yellow; Said specific antibody is the polyclonal antibody or the monoclonal antibody of sunset yellow.
2. kit according to claim 1 is characterized in that:
1) said sunset yellow haptens and carrier protein couplet, the hapten-carrier protein conjugate that obtains be as coating antigen, and said specific antibody is an enzyme labeling; Perhaps
2) said specific antibody is as coating antigen, and said haptens is an enzyme labeling.
3. kit according to claim 1 is characterized in that: said kit also comprises antiantibody, like sheep anti mouse antiantibody or goat-anti rabbit antiantibody; And:
1) said sunset yellow haptens and carrier protein couplet, the hapten-carrier protein conjugate that obtains be as coating antigen, and said antiantibody is an enzyme labeling;
2) said antiantibody is as coating antigen, and said haptens is an enzyme labeling.
4. according to each described kit among the claim 1-3, it is characterized in that: said kit also comprises ELISA Plate, sunset yellow standard solution, substrate colour developing liquid, stop buffer, concentrated cleaning solution, concentrate and redissolve at least a in the liquid.
5. according to each described kit among the claim 1-3, it is characterized in that: any in said sunset yellow haptens or sunset yellow hapten-carrier protein conjugate, said sunset yellow specific antibody, the said antiantibody is coated on the ELISA Plate.
6. according to arbitrary described kit among the claim 1-3, it is characterized in that: used marker enzyme is horseradish peroxidase or alkaline phosphatase in the said enzyme labeling; When marker enzyme was horseradish peroxidase, the substrate that uses colour developing liquid was hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution, and stop buffer is 1-2mol/L sulfuric acid solution or hydrochloric acid solution; When marker enzyme was alkaline phosphatase, the substrate colour developing liquid that uses was the nitro phosphate buffer, and stop buffer is the 1-2mol/L sodium hydroxide solution.
7. kit according to claim 4; It is characterized in that: said concentrated cleaning solution is the pH value for 6-9, to contain antiseptic, the final concentration that final concentration is 0.02-0.05% (quality) be the Tween-20 of 1.0-2.0% (quality), the phosphate buffer of 0.1-0.2mol/L; Preferably, said concentrated cleaning solution is that the pH value is 7.4, contains the 0.2mol/L phosphate buffer of antiseptic that final concentration is 0.03% (quality), Tween-20 that final concentration is 1.5% (quality); Said concentrated redissolution liquid is for containing the DMSO that final concentration is 5-10% (quality), the 0.1-0.2mol/L phosphate buffer that pH is 6-8; Preferably, said concentrated redissolution liquid is 7.4 0.2mol/L phosphate buffer for containing DMSO, pH that final concentration is 8% (quality); Said substrate colour developing liquid is hydrogen peroxide or urea peroxide, tetramethyl benzidine sulfate mixed solution; Said stop buffer is 2mol/L sulfuric acid solution or hydrochloric acid solution.
8. the method for the sunset yellow in the test sample may further comprise the steps:
1) with sunset yellow haptens or sunset yellow hapten-carrier protein conjugate coated elisa plate;
2) sunset yellow standard items or testing sample are added in the said ELISA Plate hole that encapsulates, every then hole adds the sunset yellow specific antibody of enzyme labeling, hatches washing;
3) add the colour developing of substrate colour developing liquid;
4) add the reaction terminating liquid cessation reaction;
5) through comparing the color of sunset yellow standard items and testing sample, infer the sunset yellow content that in the testing sample; Perhaps, measure the absorbance in each hole, set up the typical curve of sunset yellow concentration, and extrapolate the sunset yellow content in the testing sample by the absorbance of testing sample according to this typical curve with respect to absorbance.
9. the method for the sunset yellow content in the test sample may further comprise the steps:
1) with sunset yellow haptens or sunset yellow hapten-carrier protein conjugate coated elisa plate;
2) add sunset yellow standard items or testing sample;
3) add the sunset yellow specific antibody, hatch, washing;
4) antiantibody of adding enzyme labeling is hatched, washing;
5) add the colour developing of substrate colour developing liquid;
6) add the reaction terminating liquid cessation reaction;
7) through comparing the color of sunset yellow standard items and testing sample, infer the sunset yellow content that in the testing sample; Perhaps, measure the absorbance in each hole, set up the typical curve of sunset yellow concentration, and extrapolate the sunset yellow content in the testing sample by the absorbance of testing sample according to this typical curve with respect to absorbance.
10. the method for sunset yellow content according to Claim 8 or in the 9 described test sample also comprises the sample pre-treatments step, wherein:
When said sample was liquid food, said sample-pretreating method was: liquid food is removed gas, with the sample dilution with volume ratio 1: 10-15 mixes, and gets to mix the back sample and be used for analyzing;
When said sample is candy, preserved plum based food; Said sample-pretreating method is: thing to be checked is pulverized, with the sample dilution be 1 with mass volume ratio: the 5-10 mixing adds the normal hexane degreasing of certain volume again; Centrifugal, get supernatant and sample and detect with certain proportion dilution back;
When said sample is ice cream, sample is placed water-bath dissolving, add a certain amount of normal hexane degreasing, with the sample dilution be 1 with mass volume ratio: the 10-15 mixing, centrifugal, take off layer and detect;
When said sample is cake, homogenize, add the normal hexane of certain volume, mixing, with the sample dilution with mass volume ratio 1: the 10-15 mixing, centrifugal, take off layer and detect.
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| CN113754568A (en) * | 2021-09-22 | 2021-12-07 | 江南大学 | Acid orange I hapten, artificial antigen, synthesis and application thereof |
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