CN102279262A - Enzyme-linked immuno-detection assay kit of sunset yellow - Google Patents
Enzyme-linked immuno-detection assay kit of sunset yellow Download PDFInfo
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- CN102279262A CN102279262A CN2011101825775A CN201110182577A CN102279262A CN 102279262 A CN102279262 A CN 102279262A CN 2011101825775 A CN2011101825775 A CN 2011101825775A CN 201110182577 A CN201110182577 A CN 201110182577A CN 102279262 A CN102279262 A CN 102279262A
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Abstract
The invention relates to an enzyme-linked immuno-detection assay kit of a sunset yellow, belonging to the technical field of enzyme-linked immunosorbent assay (ELISA). The reagents in the kit provided by the invention comprise: an ELISA plate which is enveloped by an envelope antigen, i.e. a conjugate of the sunset yellow and a carrier protein, a sunset yellow standard solution, an ELISA goat anti-rabbit antibody solution, a sunset yellow antibody solution, a light-emitting solution, a washing solution, an enveloping solution and a closing solution. The carrier protein of the conjugate is a bovine serum albumin or an ovalbumin with a molecular weight of 6.7-6.8 KDa. The envelope antigen which envelopes the ELISA plate is made by conjugating the sunset yellow with the ovalbumin, and the artificial immunogen for preparing an antibody is made from the sunset yellow and the bovine serum albumin. The kit has the maximal detecting range of 0.01-10 ng/mL to the sunset yellow. The enzyme-linked immuno-detection assay kit provided by the invention has the characteristics of simplicity, fastness, accuracy and high sensitivity and can exert an important function in detection of the sunset yellow in edible synthetic pigments of foodstuff (such as a pot stewed product) and beverages.
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 sunset yellow.
[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.Sunset yellow is a class synthesis type azo dyes, and synthetic dyestuff owing to low cost, bright in colour, strong coloring force, stablize, can allocate arbitrarily and find broad application, partly also has been used for food color since 19th century came out.But, because synthetic dyestuff belongs to the synthetic dyestuff of coal tar mostly, not only nonnutritive value own, and also most of harmful, so all there is the regulation of strictness in the World Health Organization (WHO) to use kind, the use amount of synthetic food colors.Think from the conclusion that big quantity research draws: the carcinogenicity of synthetic dyestuff generally may mostly to be azo-compound relevant with it.All contain azo bond in the azopigment, it is the colour carrier of azopigment, and under the strong reductant effect, azo bond fracture takes place generates amine compound, obtain the arylamine of 2 molecules at last, and arylamine class material often has carcinogenicity.The preserved fruit that China is commercially available and outlet is overseas, bean product, spiced and stewed food, beverage etc. all contain similar synthetic food colors such as sunset yellow, so the development research of sunset yellow 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 sunset yellow 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 sunset yellow is provided.
The composition of reagent comprises in the sunset yellow 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 sunset yellow and carrier protein;
Respectively 1 bottle of B, sunset yellow standard solution: 0.01ng/mL, 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, sunset yellow antibody-solutions: the artificial immunity antigen-immunized animal prepares the sunset yellow polyclonal antibody stoste of gained, is diluted to 1: 80000 working concentration with wash solution during use;
E, luminous solution: the citrate buffer solution of use 400ul TMB solution, 10ml pH=5.0 and 2ul percent by volume are 30% H
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 above F of 1L and goes on foot in the described 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 sunset yellow and ovalbumin coupling, and the artificial immunity reason sunset yellow and the bovine serum albumin coupling of preparation antibody are made.
Described envelope antigen concentration is 1 μ g/mL.
Described kit is 0.01ng/mL～10ng/mL to the maximum detection range of sunset yellow.
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 is to adopt sunset yellow-OVA to make certain density bag by solution, reaction overnight bag quilt in 4 ℃.Wherein wrap by the concentration of solution and determine through the square formation optimization experiment.
The bag that the present invention adopts is that pH is sodium carbonate-sodium bicarbonate buffer solution of 9.5 by solution.Among the present invention in the ELISA Plate sunset yellow-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 lock solution, needs to add NaN
3Prevent to go bad.
2, the preparation of sunset yellow antibody-solutions and enzyme mark goat anti-rabbit antibody solution
Sunset yellow antibody-solutions among the present invention, enzyme mark goat anti-rabbit antibody solution concentration are the key factors of sunset yellow enzyme linked immunological test kit measurement range and sensitivity among decision the present invention.
The sunset yellow antibody-solutions that relates among the present invention can go on foot described wash solution with above F and be mixed with the antibody-solutions that concentration is 0.01～10ng/mL.
The enzyme mark goat anti-rabbit antibody solution that relates among the present invention can go on foot described wash solution with above F and be formulated as 1: 2000 enzyme of concentration mark solution.
The kit that can prepare according to above-mentioned sunset yellow 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 0.01ng/mL～10ng/mL) and good sensitivity (0.024ng/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% (v/v) 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 the edible synthesized coloring matter sunset yellow in food (as spiced and stewed food) and beverage.
[description of drawings]
Fig. 1 is the inhibiting rate curve of sunset yellow antibody of the present invention, antigen coated concentration 1 μ g/ml, and antibody dilution multiple 1/40000, the IC50 minimum of this moment, the sensitivity of method is best.The log value that adds medicine (sunset yellow) concentration in addition is horizontal ordinate, and the absorbance ratio B/B0 of absorbance and blank is the ordinate mapping, and rate curve can be inhibited.
Fig. 2 is a working curve of the present invention, Fig. 1 inhibiting rate curve is added drug concentration at home and abroad to be intercepted from the straight line portion of 0.01ng/mL～10ng/mL, be working curve of the present invention, i.e. the present invention is 0.01ng/mL～10ng/mL to the maximum detection range of sunset yellow.
[embodiment]
The enzyme linked immunological kit component and the preparation process of embodiment 1, detection sunset yellow
1, detects the composition of the enzyme linked immunological kit of sunset yellow
A, be coated with the solid phase carrier (ELISA Plate) of envelope antigen (conjugate of sunset yellow and carrier protein);
B, sunset yellow standard solution: 0.01ng/mL, 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, sunset yellow antibody-solutions: prepare the sunset yellow polyclonal antibody of gained with artificial immunizing antigen immune animal, gained sunset yellow antibody is diluted to 1: 80000 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% (v/v) 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: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in the 1L water, regulate pH9.5.
H, lock solution preparation: 10g OVA is dissolved in the 1L wash solution, and adding massfraction again is 0.05%NaN
3Make.
2, the preparation of ELISA Plate
By solution envelope antigen is diluted to 1 μ g/mL with bag, every hole adds 100 μ L, hatches 2h for 36 ℃, and the bag that inclines is by solution, every hole adds 300 μ L wash solutions washing 3 times, pat dry, every then hole adds lock solution 250 μ L, and 4 ℃ are spent the night, liquid in the hole inclines, wash solution washing 3 times pats dry, and preserves with masking foil vacuum seal.
3, immunogenic synthetic
A, 0.001mol compd A (2-amino-5-sodium sulfonate benzoic acid) is dissolved in the 4mL water, adds massfraction and be 3% NaNO
2The solution mixing drops to mixed liquor in the ice-cold dense HCl solution (12mol/L), dropwises to be placed on 0-5 ℃ of reaction 1h, carries out diazo-reaction.
B, 0.001mol compd B (2-sodium sulfonate-6-naphthols) is dissolved in the solution that contains 2mL water and 200 μ LDMF, hot bath is dissolving down.Mixed liquor is dropped in the diazotising solution that obtains of step, regulate pH value to 10, react 5h down at 4 ℃ with concentrated NaOH solution.
C, reaction solution is carried out suction filtration, oven dry obtains sunset yellow modification (being Compound C, 2-(2 '-sodium sulfonate-6 '-hydroxyl naphthalene) diazo-5-sodium sulfonate Sodium Benzoate).
D, sunset yellow modification 0.06mmol is dissolved in the 3mL PBS solution, takes by weighing 0.6mmol EDC and 0.3mmolNHS again and be dissolved in the 3mLPBS solution, 4 ℃ of above-mentioned solution are reaction 2h down.
E, the above-mentioned D solution in the step mixed with bovine serum albumin BSA solution be dissolved in the PBS buffer solution, 4 ℃ times stirrings are spent the night.Dialysis, high speed centrifugation, the freeze-drying supernatant obtains the immunogenic pressed powder of sunset yellow.
4, envelope antigen is synthetic
A, sunset yellow modification (the being Compound C) 0.05mmol that obtains in 3 steps is dissolved in the solution that contains 3mL DMF and 1mLPBS and dissolves.
B, under 4 ℃ of ice baths, upwards go on foot in the solution and to add 13 μ L tri-n-butylamines, stirring reaction 0.5h.
C, in B step solution, adding 9.1 μ L chloromethanes alcohol isobutyl ester, stirring reaction 1h under 4 ℃ of ice baths.
D, above-mentioned C step solution mixed with oralbumin OVA solution be dissolved in the PBS buffer solution, 4 ℃ times stirrings are spent the night.Dialysis, high speed centrifugation, the freeze-drying supernatant obtains the pressed powder of sunset yellow envelope antigen.
5, sunset yellow Polyclonal Antibody Preparation
Adopt new zealand white rabbit as immune animal, conjugate with sunset yellow and bovine serum albumin is an immunogene, first immunisation dosage is 500 μ g/mL, when head exempts from immunogene is dissolved in 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 sunset yellow 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 wash 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 luminous 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 sunset yellow 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 washing 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 sunset yellow solution 50 μ L/ holes that add dilution sunset yellow 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 microplate reader 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 sunset yellow
(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, soda at first boil the soda of buying, and remove CO wherein
2, obtain sample solution.Solution dilution to 1 is obtained testing sample for ten thousand times.
B, spiced and stewed food 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 sunset yellow series standard concentration solution or sample solution 50 μ L in the ELISA Plate micropore, add sunset yellow 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) testing sample assay
The mean value of standard items that obtained and sample luminous value multiply by 100 rates that promptly are inhibited again divided by the luminous value of first standard (0 standard).With the inhibiting rate is ordinate, and the logarithm of sunset yellow concentration is that horizontal ordinate is made typical curve (Fig. 2), then in the testing sample each testing result can from typical curve read its correspondence concentration.
% inhibiting rate=% standard items luminous value (or sample)/0 standard items luminous value.
It is 1,/10,000,1,/20,000,1,/10 ten thousand three concentration that the soda that will contain sunset yellow allows the dilution of addition appropriateness according to maximum, obtains the sunset yellow addition of every kind of concentration beverage by detection, detects the sunset yellow recovery with this.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.
The % recovery=%[mark-on sample determination value (ppm)-contrast solution measured value (ppm)]/interpolation concentration (ppm)
From the said determination result, the coefficient of variation is lower than 15.86%, and the recovery is between 80～120%.Show that this kit has good repeatability and accuracy.
Claims (5)
1. the enzyme-linked immunologic detecting kit of a sunset yellow 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 sunset yellow and carrier protein;
B, sunset yellow standard solution: respectively 1 bottle of 0.01 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL;
C, enzyme mark goat anti-rabbit antibody solution: horseradish peroxidase-goat anti-rabbit igg stoste is diluted to the working concentration of 1:2000 with wash solution during use;
D, sunset yellow antibody-solutions: the artificial immunity antigen-immunized animal prepares the sunset yellow polyclonal antibody stoste of gained, is diluted to the 1:80000 working concentration with wash solution during use;
E, luminous solution: the citrate buffer solution of use 400ul TMB solution, 10ml pH=5.0 and 2ul percent by volume are 30% H
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 0.1 mol/L;
G, bag are by solution: 1.59 g sodium carbonate and 2.53 g sodium bicarbonates are dissolved in the solution of making in the 1 L water, regulate pH=9.5;
H, lock solution: 10 g OVA are dissolved in the described wash solution of 1 L, add 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 6.7 KDa～6.8 KDa.
3. kit according to claim 1 is characterized in that: the envelope antigen of coated elisa plate is made by sunset yellow and ovalbumin coupling, and the artificial immunity reason sunset yellow 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
μG/mL.
5. want 1 described kit according to right, it is characterized in that: the maximum detection range of sunset yellow is 0.01 ng/mL ~ 10 ng/mL.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1834654A (en) * | 2006-04-13 | 2006-09-20 | 廖伟 | Kit for diagnosing high triglyceride |
| CN101915861A (en) * | 2010-08-04 | 2010-12-15 | 威胜集团有限公司 | Relay and PCB assembly of electric energy meter |
| CN101949933A (en) * | 2010-08-25 | 2011-01-19 | 南开大学 | Indirect chemiluminescent enzyme linked immunosorbent assay (ELISA) kit for detecting residual ractopamine by using monoclonal antibody |
-
2011
- 2011-06-30 CN CN2011101825775A patent/CN102279262A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1834654A (en) * | 2006-04-13 | 2006-09-20 | 廖伟 | Kit for diagnosing high triglyceride |
| CN101915861A (en) * | 2010-08-04 | 2010-12-15 | 威胜集团有限公司 | Relay and PCB assembly of electric energy meter |
| CN101949933A (en) * | 2010-08-25 | 2011-01-19 | 南开大学 | Indirect chemiluminescent enzyme linked immunosorbent assay (ELISA) kit for detecting residual ractopamine by using monoclonal antibody |
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Application publication date: 20111214 |