CN101915831A - Enzyme-linked immunoassay kit for acid orange II - Google Patents
Enzyme-linked immunoassay kit for acid orange II Download PDFInfo
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- CN101915831A CN101915831A CN2010102620123A CN201010262012A CN101915831A CN 101915831 A CN101915831 A CN 101915831A CN 2010102620123 A CN2010102620123 A CN 2010102620123A CN 201010262012 A CN201010262012 A CN 201010262012A CN 101915831 A CN101915831 A CN 101915831A
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Abstract
The invention discloses an enzyme-linked immunoassay kit for acid orange II, which belongs to the technical field of enzyme-linked immunosorbent analysis. Reagents in the kit provided by the invention comprise an enzyme label plate coated by a coupled substance of the acid orange II serving as a coating antigen and a carrier protein, standard solution of the acid orange II, solution of enzyme labeled goat-anti-rabbit antigen, antibody solution of the acid orange II, luminous solution, washing solution, coating solution and closed solution. The carrier protein of the coupled substance is bovine serum albumin or egg albumin with a molecular weight of 6.7 to 6.8 KDa. The coating antigen of the coated enzyme label plate is prepared by coupling the acid orange II and egg albumin, and an artificial immunogen for preparing the angiten is prepared by coupling the acid orange II and the bovine serum albumin. The maximum acid orange II detection range of the kit is 0.1 to 10 ng/mL. The enzyme-linked immunoassay kit of the invention has the characteristics of simplicity, quickness, accuracy and high sensitivity and can play an important role in the acid orange II detection of synthetic non-edible pigment in foods (such as marinated products and cayenne pepper) and drinks.
Description
[technical field]
The invention belongs to the enzyme-linked immunosorbent analytical technique field, relate to a kind of enzyme-linked immunologic detecting kit, relate in particular to a kind of enzyme-linked immunologic detecting kit of Acid Orange II.
[background technology]
Azo dyes (azo dye) is a kind of important synthetic dyestuffs, is fabric clothing most widely used class synthetic dyestuffs in dyeing and printing process, is used for the dyeing and the stamp of multiple natural and synthon, also be used to paint, plastics, rubber etc. painted.The no any direct carcinogenesis of azo dyes itself, but reduction decomposition in vivo, can form aromatic amine compound, aromatic amine is in vivo through making DNA change with the target cell effect behind the metabolic activity, become the risk factor of human lesion, human body or animal are had potential carcinogenicity and mutagenicity.Because its carcinogenicity and mutagenicity, a lot of in the world countries have all implemented strict regulation to the use of azo dyes.The secondary colour that Japan's approval is once used have 27 kinds, has now banned use of wherein 16 kinds.The secondary colour that the U.S. allows to use nineteen sixty have 35 kinds, existing only remaining 7 kinds.Sweden, Finland, Norway, India, Denmark, France etc. ban use of azo class pigment already, wherein some countries such as Norway also total bans use any chemosynthesis pigment.Also have some countries to forbid in food such as meat, fish and processed goods thereof, fruit and goods thereof, flavouring, baby food, cake, adding synthetic dyestuff in addition.Also there is strict restriction in China to add synthetic dyestuff in food: flavouring, fruit and goods thereof, newborn class and dairy products, baby food, biscuit, cakes such as every meat and processed goods thereof, fish and processed goods thereof, vinegar, soy sauce, fermented bean curd all can not use synthetic food color.Having only carbonated drink, cold drink food, candy, assembled alcoholic drinks and fruit juice to reveal can use on a small quantity, generally must not surpass 1/10000.The edible synthesized coloring matter that China's approval is at present used has 9 kinds, i.e. red, carmine, newly red, acid red, lemon yellow, sunset yellow, indigo and light blue of amaranth, temptation.Acid Orange II is a class synthesis type azo dyes, is widely used in textile printing and dyeing, leather, papermaking, rubber, plastics, coating, cosmetics, wood working etc., forbids being used as food additives in China.But owing to Acid Orange II lovely luster, coloring stabilized, lower-price characteristic, some illegal retailers are used for food production and processing with it as food coloring in order to seek exorbitant profit.The preserved fruit that China is commercially available and outlet is overseas, bean product, spiced and stewed food, paprika, red shell melon seeds etc. all were detected similar synthetic non-food colorings such as containing Acid Orange II, serious harm consumer healthy, and usually cause export food to be forced to destroy on the spot.So the development research of Acid Orange II detection method is very urgent.
In the detection of azo class pigment, instrumental method such as high performance liquid chromatography and liquid phase-mass spectrometry are most widely used methods.These methods are accurate, and are stable, reliable, can be used as standard method.But instrumental method costs an arm and a leg, and is time-consuming longer, needs large-scale instrument and equipment, needs special technician, so be difficult to use in execute-in-place.
Enzyme linked immunosorbent detection method (ELISA) provides a kind of fabulous scanning means.This method has fast, and is accurately simple and easy, do not need advantages such as the professional operates, and it is a kind of desirable that this makes that the ELISA method becomes, and can be used for the detection method of conventional sweep, and the method for therefore setting up a kind of effective ELISA method detection of acidic orange II is significant.
[summary of the invention]
The present invention seeks to overcome the prior art above shortcomings, a kind of enzyme-linked immunologic detecting kit of Acid Orange II is provided.
The composition of reagent comprises in the Acid Orange II enzyme-linked immunologic detecting kit provided by the invention:
A, each hole are coated with 1 of the ELISA Plate that envelope antigen is the conjugate of Acid Orange II and carrier protein;
Respectively 1 bottle of B, Acid Orange II standard solution: 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL;
C, enzyme mark goat anti-rabbit antibody solution: horseradish peroxidase-goat anti-rabbit igg stoste is diluted to 1: 2000 working concentration with wash solution during use;
D, Acid Orange II antibody-solutions: the artificial immunity antigen-immunized animal prepares the polyclonal antibody stoste of gained, is diluted to 1: 40000 working concentration with wash solution during use;
E, luminous solution: use 400ul TMB solution, the citrate buffer solution of 10ml pH=5.0 and the H of 2ul 30%
2O
2Solution mixes, and wherein TMB solution is mixed by 10mgTMB+2ml DMSO;
F, wash solution:, wherein contain Tween-20 volume fraction 0.05% for pH=7.5, concentration are the phosphate buffer of 0.1mol/L; Described phosphate is prepared with sodium hydrogen phosphate and potassium dihydrogen phosphate.
G, bag are by solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in the solution of making in the 1L water, regulate pH=9.5;
H, lock solution: 10g OVA is dissolved in the 1L wash solution, and adding massfraction again is 0.05%NaN
3Make.
The carrier protein of the above conjugate is bovine serum albumin or the ovalbumin of molecular weight ranges at 6.7KDa～6.8KDa.The envelope antigen of coated elisa plate is made by Acid Orange II and ovalbumin coupling, and the artificial immunity reason Acid Orange II and the bovine serum albumin coupling of preparation antibody are made.
Described envelope antigen concentration is 1 μ g/mL.
Described kit is 0.1ng/mL～10ng/mL to the maximum detection range of Acid Orange II.
The preparation of the related solution that relates in the kit of the present invention
1, the preparation of ELISA Plate among the present invention
The ELISA Plate of bag quilt among the present invention, be adopt Acid Orange II-OVA the bag that is fit to by solution in, with suitable concentration, reaction overnight bag quilt in 4 ℃.
What the present invention adopted is that pH is sodium carbonate-sodium bicarbonate buffer solution of 9.5.Among the present invention in the ELISA Plate Acid Orange II-OVA of the quilt that wraps under alkaline environment, can well be combined on the ELISA Plate frosting, can stand repeatedly to wash plate, the coating protein concentration of employing is 1 μ g/mL.
Bag can be with containing the lock solution sealing by good ELISA Plate, and inert protein can be OVA in the confining liquid, needs to add NaN
3Prevent to go bad.
2, the preparation of Acid Orange II antibody-solutions and enzyme mark goat anti-rabbit antibody solution
Acid Orange II antibody-solutions among the present invention, enzyme mark goat anti-rabbit antibody solution concentration are the key factors of Acid Orange II enzyme linked immunological test kit measurement range and sensitivity among decision the present invention.
The Acid Orange II standard solution that relates among the present invention can be 0.1～10ng/mL with the wash solution compound concentration.
The enzyme mark goat anti-rabbit antibody solution that relates among the present invention can be 1: 2000 with the concentration of wash solution preparation.
The kit that can prepare according to above-mentioned Acid Orange II antibody-solutions concentration and enzyme mark goat anti-rabbit antibody solution concentration can reach the good range of linearity, and (the standard lines scope can reach 0.1ng/mL～10ng/mL) and the sensitivity of becoming reconciled (2.0ng/mL).
3, the preparation of luminous solution
The present invention adopts horseradish peroxidase labeled substrate luminescent system, mainly is tetramethyl benzidine (TMB)-hydrogen peroxide system.
Described luminescent solution is 400ul TMB solution (10mgTMB+2ml DMSO) and 10ml citrate buffer solution (pH=5.0) and 2ul 30% H
2O
2Solution mixes.
Advantage of the present invention and good effect
Principle of the present invention is that antibody-antigen reactive high degree of specificity and enzymatic high sensitivity are combined, and utilizes the luminescence-producing reaction of substrate for enzymatic activity to detect production concentration.
Enzyme-linked immunologic detecting kit of the present invention has highly sensitive, easy characteristics fast and accurately, can play a significant role in detecting by non-edible synthesized coloring matter Acid Orange II in food (as spiced and stewed food, paprika) and beverage.
[description of drawings]
Fig. 1 is the inhibiting rate curve of Acid Orange II antibody of the present invention.
Fig. 2 is a working curve of the present invention.
[embodiment]
The enzyme linked immunological kit component and the preparation process of embodiment 1, detection of acidic orange II
1, the composition of the enzyme linked immunological kit of detection of acidic orange II
A, be coated with the solid phase carrier (ELISA Plate) of envelope antigen (conjugate of Acid Orange II and carrier protein);
B, Acid Orange II standard solution: 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL.
C, enzyme mark goat anti-rabbit antibody solution: enzyme mark goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit igg stoste, packs into, is mixed with 1: 2000 working concentration during use with wash solution.
D, Acid Orange II antibody-solutions: prepare the polyclonal antibody of gained with artificial immunizing antigen immune animal, gained Acid Orange II antibody is diluted to 1: 40000 working concentration with wash solution.
E, luminous solution: use 400ul TMB solution (10mgTMB+2ml DMSO) and 10ml citrate buffer solution (pH5.0) and 2ul 30%H
2O
2Solution mixes.
F, wash solution: the pH7.5, the 0.1mol/L phosphate buffer that contain volume fraction 0.05% Tween-20.
G, bag are by solution: in 1.59g sodium carbonate and the 2.53g sodium bicarbonate solution 1L water, regulate pH9.5.
H, lock solution preparation: 10g OVA is dissolved in the 1L wash solution, adds 0.05%NaN again
3
2, the preparation of ELISA Plate
With coating buffer envelope antigen is diluted to 1 μ g/mL, every hole adds 100 μ L, hatches 2h for 36 ℃, and coating buffer inclines, every hole adds 300 μ L cleansing solutions washing 3 times, pat dry, every then hole adds confining liquid 250 μ L, and 4 ℃ are spent the night, liquid in the hole inclines, cleansing solution washing 3 times pats dry, and preserves with masking foil vacuum seal.
3, immunogenic synthetic
A, Acid Orange II and the thionyl chloride mol ratio with 1: 3 is dissolved in the toluene, stops behind 60 ℃ of reaction 2h.Revolve to steam and remove remaining thionyl chloride and toluene, suction filtration, infrared lamp oven dry down obtains Acid Orange II-sulfonic acid chloride.
B, Acid Orange II-sulfonic acid chloride and the phenyalanine methyl ester mol ratio with 1: 1 is dissolved in the acetone, is stopping behind the room temperature reaction 8h under the effect of 3 times of amount triethylamines, revolve to steam and remove acetone, suction filtration, infrared lamp is oven dry down, obtains Acid Orange II-phenyalanine methyl ester.
C, Acid Orange II-phenyalanine methyl ester is dissolved in methyl alcohol, in NaOH solution, reacts 0.5h under 30 ℃.It is acid regulating PH, suction filtration, and infrared lamp oven dry down obtains Acid Orange II-phenylalanine.
D, NHS and DCC are dissolved in the methylene chloride with 1: 1 mol ratio, Acid Orange II-phenylalanine are joined in the mixed solution of NHS and DCC again, 4 ℃ of reaction 24h revolve to steam and remove methylene chloride, obtain the Acid Orange II Acibenzolar.
E, Acid Orange II Acibenzolar and bovine serum albumin BSA are dissolved in the PBS buffer solution 4 ℃ of following stirring reaction 6-7h with 30: 1 mol ratio.Dialysis, high speed centrifugation, the freeze-drying supernatant obtains the immunogenic orange solids powder of Acid Orange II.
4, envelope antigen is synthetic
The Acid Orange II Acibenzolar that obtains in 3 and ovalbumin OVA are dissolved in the PBS buffer solution 4 ℃ of following stirring reaction 6～7h with 30: 1 mol ratio.Dialysis, high speed centrifugation, the freeze-drying supernatant obtains the orange solids powder of Acid Orange II envelope antigen.
5, Acid Orange II Polyclonal Antibody Preparation
Adopt new zealand white rabbit as immune animal, conjugate with Acid Orange II and bovine serum albumin is an immunogene, first immunisation dosage is 500 μ g/mL, when head exempts from immunogene is dissolved in into isopyknic physiological saline and Freund's complete adjuvant and makes emulsifying agent, nape portion multi-point injection, immunity is later on got the immunogene that dosage reduces by half and is dissolved in isopyknic physiological saline and incomplete Freund mixing and emulsifying, head exempts from and two exempted from 20 days at interval, once be total to immunity five times every two all immunity later on, do not add adjuvant for the last time.Last immunity is the heart blood sampling after 7 days, and the centrifugal antiserum that gets obtains the Acid Orange II polyclonal antibody.
The foundation of embodiment 2, ELISA detection method
(1) preferred (square formation) of antibody and envelope antigen concentration
Vertically with the serial dilution degree coated elisa plate of every kind of envelope antigen by 10.0 μ g/mL, 5.0 μ g/mL, 1.0 μ g/mL, 0.1 μ g/mL, 0.01 μ g/mL, 0.001 μ g/mL, 100 μ L/ holes, placed 2 hours for 36 ℃, wash plate three times, pat dry at every turn with cleansing solution; 250 μ L/ hole lock solution sealings, 0～4 ℃ of placement is spent the night, and washes plate three times, pats dry at every turn; The antibody (1: 5000 to 1: 640000) that adds the 100 a series of dilutions in μ L/ hole was placed 0.5 hour for 36 ℃, washed plate three times, patted dry at every turn; 1: 2000 the horseradish peroxidase-goat anti-rabbit igg antibody that adds 100 μ L/ holes was placed 0.5 hour for 36 ℃, washed plate three times, patted dry at every turn; The luminescent solution that adds 100 μ L/ holes is measured luminous value.With the concentration of envelope antigen the envelope antigen concentration of obvious graded and antibody dilution being arranged with luminous value is that optium concentration is carried out specific assay.
(2) mensuration of antibody sensitivity
According to the optimization experiment of above-mentioned antagonist and envelope antigen concentration, the applicant selects and determines that antibody concentration is 1: 40000, and envelope antigen concentration is the mensuration that 1 μ g/mL carries out the sensitivity of antibody:
A, bag quilt: the solution that by solution the envelope antigen of Acid Orange II is made into 1 μ g/mL with the carbonate bag of 0.05M pH9.6, add 100 μ L in the reacting hole of each polystyrene board, placed 2 hours for 36 ℃, discard solution in the hole, wash 3 times with lavation buffer solution, 300 μ L/ holes each 5 minutes, pat dry.(this step is called for short washing, down together).
B, sealing: with the above-mentioned ELISA Plate of having wrapped quilt of lock solution sealing, 250 μ L/ holes, 0～4 ℃ of placement is spent the night, then washing.
C, application of sample: placed 0.5 hour for 36 ℃, then washing in the above-mentioned reacting hole that has sealed in the Acid Orange II solution 50 μ L/ holes that add dilution Acid Orange II antibody (1: 40000) 50 μ L/ holes and variable concentrations.
D, add enzyme labelled antibody: in each reacting hole, add antibody (1: 2000) the 100 μ L/ holes of fresh dilution horseradish peroxidase-goat anti-rabbit igg, 0.5 hour, washing.
E, luminous: in each reacting hole, add the luminous solution 100 μ L/ holes of interim preparation, detect with the microplate reader instrument immediately.
F, testing result are calculated with inhibiting rate: % inhibiting rate=%B/B
0, B is the luminous value of the medicine of variable concentrations as the rival, B
0It is the luminous value of not dosing.Drug concentrations is the sensitivity of this antibody when calculating 50% inhibiting rate.
The application of the enzyme linked immunological kit of embodiment 3, detection of acidic orange II
(1) preparation of reagent
A, sample diluting liquid: the concentrated phosphoric acid salt buffer solution that provides in the kit is used with behind 10 times of the distilled water dilutings.
B, wash solution: the concentrated cleaning solution that provides in the kit is used with behind 10 times of the distilled water dilutings.
C, luminous solution: 400ul TMB solution (10mgTMB+2ml DMSO)+10ml citrate buffer solution (pH5.0)+2ul 30%H
2O
2Solution
(2) sample pre-treatments
A, orange juice at first with the orange juice sample bought under 4 ℃, 10000 rev/mins conditions, centrifugal 15 minutes, the orange juice supernatant is diluted to 1: 10 with cleansing solution, obtain testing sample.
B, spiced and stewed food, paprika take by weighing the 5g sample, grind, and add absolute ethyl alcohol-ammoniacal liquor-water (volume ratio 70: 1: 29) solution 20ml, and jolting 0.5h filters, filter wash slag 2 times, and merging filtrate, water (pH=6) constant volume is crossed 0.45 μ m filter membrane and is measured.
(3) detect step
A, application of sample: add Acid Orange II series standard concentration solution or the molten 50 μ L of sample in the ELISA Plate micropore, add Acid Orange II antibody-solutions 50 μ L then, room temperature (25 ℃) constant temperature is hatched 1h;
B, washing: the middle liquid that portals that inclines, every hole adds wash solution 300 μ L, washs 3 times, pats dry;
C, enzyme-added mark goat anti-rabbit antibody solution: every hole adds enzyme mark goat anti-rabbit antibody solution 100 μ L, and room temperature constant temperature is hatched 1h;
D, washing: the middle liquid that portals that inclines, every hole adds wash solution 300 μ L, washs 3 times, pats dry;
E, add luminous solution: every hole adds luminous solution 100 μ L;
F, detection: the luminous intensity of measuring every hole with microplate reader.
(4) result judges
The mean value of standard items that obtained and sample luminous value multiply by 100 again divided by the luminous value of first standard (0 standard), with the inhibiting rate is ordinate, the logarithm of Acid Orange II concentration is that horizontal ordinate is made typical curve, and the concentration of each sample can be read from typical curve.
% inhibiting rate=% standard items luminous value (or sample)/0 standard items luminous value.
Get 0.1,0.5,1,5, the Acid Orange II standard specimen of 10ppb adds in the orange juice sample, comes the detection of acidic orange II recovery.The interassay coefficient of variation of each concentration all calculates with 5 repeating datas of different 5 days, and the variation within batch coefficient calculates with 5 repeating datas on the same day.Carry out the quantitative Analysis of the recovery according to the linear equation of the typical curve of formulating.The results are shown in following table.
From the said determination result, the coefficient of variation is lower than 19.2%, and the recovery is between 80～118%.Show that this kit has good repeatability and accuracy.
Claims (5)
1. the enzyme-linked immunologic detecting kit of an Acid Orange II is characterized in that the composition of reagent in this kit comprises:
A, each hole are coated with 1 of the ELISA Plate that envelope antigen is the conjugate of Acid Orange II and carrier protein;
Respectively 1 bottle of B, Acid Orange II standard solution: 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL;
C, enzyme mark goat anti-rabbit antibody solution: horseradish peroxidase-goat anti-rabbit igg stoste is diluted to 1: 2000 working concentration with wash solution during use;
D, Acid Orange II antibody-solutions: the artificial immunity antigen-immunized animal prepares the polyclonal antibody stoste of gained, is diluted to 1: 40000 working concentration with wash solution during use;
E, luminous solution: use 400ul TMB solution, the citrate buffer solution of 10ml pH=5.0 and the H of 2ul 30%
2O
2Solution mixes, and wherein TMB solution is mixed by 10mgTMB+2ml DMSO;
F, wash solution:, wherein contain Tween-20 volume fraction 0.05% for pH=7.5, concentration are the phosphate buffer of 0.1mol/L;
G, bag are by solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in the solution of making in the 1L water, regulate pH=9.5;
H, lock solution: 10g OVA is dissolved in the 1L wash solution, adds massfraction 0.05%NaN again
3Make.
2. kit according to claim 1 is characterized in that: the carrier protein of described conjugate is that molecular weight ranges is bovine serum albumin or the ovalbumin of 6.7KDa～6.8KDa.
3. kit according to claim 1 is characterized in that: the envelope antigen of coated elisa plate is made by Acid Orange II and ovalbumin coupling, and the artificial immunity reason Acid Orange II and the bovine serum albumin coupling of preparation antibody are made.
4. kit according to claim 1 is characterized in that: described envelope antigen concentration is 1 μ g/mL.
5. want 1 described kit according to right, it is characterized in that: the maximum detection range of Acid Orange II is 0.1ng/mL～10ng/mL.
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Inventor after: Chi Rimo Inventor after: Wang Yabin Inventor after: Xue Huyin Inventor after: Wang Wenhu Inventor after: Meng Meng Inventor after: Zhang Taichang Inventor after: Zhao Xiujie Inventor after: Liu Tingting Inventor after: Zhang Yuanyang Inventor after: Xu Jing Inventor before: Chi Rimo Inventor before: Xue Huyin Inventor before: Meng Meng Inventor before: Zhang Taichang Inventor before: Zhang Yuanyang Inventor before: Xu Jing Inventor before: Wang Yabin |
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