CN103424545A - Malachite green oxalate ELISA (enzyme-linked immunosorbent assay) detection kit and method - Google Patents
Malachite green oxalate ELISA (enzyme-linked immunosorbent assay) detection kit and method Download PDFInfo
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- CN103424545A CN103424545A CN2013103502363A CN201310350236A CN103424545A CN 103424545 A CN103424545 A CN 103424545A CN 2013103502363 A CN2013103502363 A CN 2013103502363A CN 201310350236 A CN201310350236 A CN 201310350236A CN 103424545 A CN103424545 A CN 103424545A
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Abstract
The invention belongs to the technical filed of biology and provides a malachite green oxalate ELISA (enzyme-linked immunosorbent assay) detection kit and method. The detection kit comprises SPG protein, coating buffer solution, confining liquid, malachite green oxalate antibody, malachite green oxalate coupled with HRP (horse reddish peroxidase), color developing agent and terminator. The detection method includes: mixing the SPG protein and the coating buffer solution, and incubating the mixture on a solid-phase carrier; adding the confining liquid to the solid-phase carrier, and incubating; adding the confining liquid to the malachite green oxalate antibody, and incubating; adding a to-be-detected sample to the solid-phase carrier and adding the malachite green oxalate coupled with HRP simultaneously; adding the color developing agent and the terminator to the solid-phase carrier. The detection kit in high in detection flexibility.
Description
Technical field
The invention belongs to biological technical field, relate in particular to the detection method of a kind of malachite green ELISA detection kit and malachite green.
Background technology
Malachite green (Malachite Green oxalate, MG) being a kind of crystalline solid with the metallic luster green, is dirty-green crystalline solid, water-soluble, ethanol and methyl alcohol, aqueous solution is blue-green, and malachite green chemistry tetramethyl by name is for the diamido triphenylmethane.Because malachite green has the high-efficiency broad spectrum bactericidal action, therefore once be widely used in the aquaculture industry.Yet malachite green can be metabolised to concealed malachite green (LMG) in the aquatic animal body, MG and LMG all have the carcinogenic mutagenic toxicity of teratogenesis, in many countries, have all forbidden to use malachite green.But, due to the malachite green low price, the event of violated use occurs repeatedly, bring huge hidden danger to people's health.Therefore the detection for malachite green has larger demand, instrument detection method commonly used, and instrument as needed as detection methods such as high performance liquid chromatography, Liquid Chromatography/Mass Spectrometries is expensive, it is complicated to operate, sample pretreatment is complicated.
Immunological detection method is also the method for detectable antigens commonly used, wherein ELISA(Enzyme-linked Immunosorbent Assay) technology is the most frequently used a kind of immune analysis method during current medicament residue is analyzed.ELISA is widely used in the qualitative, quantitative of antigen-antibody and measures, and scope can relate to the quantitative detection of the hapten molecules such as some medicines, hormone, toxin.ELISA of the prior art detects that to take competitiveness enzyme-linked immuning adsorpting analysis (Competitive Inhibition Enzyme-linked Immunosorbent Assay ciELISA) be Typical Representative, this competitiveness enzyme immune absorption conalysis technology is to utilize immunology competition ratio juris, antigen/antibody is fixed on the solid phase carrier microwell plate, by the competition combination degree between the antibody/antigen in thing to be detected and pre-coated competition thing, obtains testing result.Due to the restriction that is limited by the antigen/antibody self character, characterization processes pattern of the prior art is single, and insufficient sensitivity, and antibody large usage quantity exist significantly not enough on technical matters.Therefore need to provide the detection method that a kind of cost is lower, easy and simple to handle, sensitivity is higher.
Summary of the invention
The purpose of the embodiment of the present invention is to provide a kind of malachite green ELISA detection kit and malachite green detection method, be intended to solve in prior art high for the malachite green testing cost, the problem that program is loaded down with trivial details.
The embodiment of the present invention is achieved in that a kind of malachite green ELISA detection kit, comprising: SPG albumen, coated damping fluid, confining liquid, malachite green antibody, coupling have malachite green, developer and the terminator of horseradish peroxidase (HRP).
Another purpose of the embodiment of the present invention is to provide a kind of malachite green detection method, and the method utilizes ELISA detection kit of the present invention to detect, and comprises the steps:
S01, SPG albumen is mixed with coated damping fluid, must be coated with mixed liquor and hatch on solid phase carrier, hatch rear removal liquid;
Add confining liquid and hatch on S02, the solid phase carrier that obtains to step S01, hatching rear removal liquid;
Add malachite green antibody and hatch on S03, the solid phase carrier that obtains to step S02, hatching rear removal liquid;
Add testing sample on S04, the solid phase carrier that obtains to step S03, add coupling that the malachite green of HRP is arranged simultaneously, hatch rear removal liquid;
Add developer on S05, the solid phase carrier that obtains to step S04, add terminator after colour developing.
The malachite green detection kit that the embodiment of the present invention provides, by being fixed in SPG very orderly being arranged on solid phase carrier by target IgG antibody on solid phase carrier, has increased the effective binding site of antibody to antigen greatly, significantly improves detection sensitivity.Recombinate to IgG, combination does not have the kind restriction to SPG simultaneously, can meet dissimilar antibody, highly versatile.And utilize detection kit of the present invention can lower the antibody use amount.That malachite green detection method of the present invention has advantages of is highly sensitive, stability is strong.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
The embodiment of the present invention provides a kind of malachite green ELISA detection kit, comprising: SPG albumen (streptococcal protein G), coated damping fluid, confining liquid, malachite green antibody, coupling have malachite green, developer and the terminator of horseradish peroxidase (HRP).
The malachite green that ELISA detection kit provided by the invention is treated in the test sample product by the competitiveness enzyme ELISA adsorption analysis method of improvement is detected, and ELISA detection kit of the present invention and ELISA Plate (96 orifice plate) are used in conjunction with.
Molecule of streptococcal protein G that the embodiment of the present invention is used can specific binding 2-5 IgG molecule, and the adhesion of SPG is strong, is equivalent to the adhesion of free antibodies and antigen, in conjunction with the rear activity on IgG without impact.The SPG albumen that the embodiment of the present invention is used can be restructuring SPG or natural SPG, and restructuring SPG has the intrinsic property of natural SPG, higher than natural SPG affinity.Consider that restructuring SPG preparation is simple, the height commercialization, the little stability of difference between batch is high, the uniform particles of restructuring SPG, can be absorbed and fixed on solid phase carrier, SPG is pre-coated on solid phase carrier, after washing the plate sealing, with target IgG antibody response, be combined, can allow very orderly being arranged on solid phase carrier of target IgG antibody, greatly increase the effective binding site of antibody to antigen, significantly improved detection sensitivity, therefore preferably used restructuring SPG.
Particularly, the ELISA detection kit of the embodiment of the present invention also comprises the malachite green standard items, for the preparation of typical curve.
Particularly, the coupling comprised in the ELISA detection kit of the embodiment of the present invention has the malachite green preparation method of HRP to be:
Carboxyl concealed malachite green (CLMG) is converted into to malachite green with the oxygenant chloranil, add the ascorbic acid neutralization, by products therefrom, with NHS, EDC, according to CLMG, with the mol ratio of NHS, EDC, be that 0.8-1:0.8-1:0.8-1 is mixed, stir to obtain mixed solution, described mixed solution is dropped in the HRP aqueous solution.
Particularly, above-mentioned ascorbic acid concentrations is 0.5-1.5%; Above-mentioned HRP concentration of aqueous solution is 15-25mg/ml.
ELISA detection kit of the present invention for the detection thing be malachite green (dominant), malachite green (dominant) is for concealed malachite green.
The malachite green antibody used in the embodiment of the present invention can be polyclonal antibody and also can be monoclonal antibody.
Developer in the embodiment of the present invention is hydrogen peroxide or urea peroxide and tetramethyl benzidine (TMB) mixed liquor.
The characteristics that the detection kit that the embodiment of the present invention provides utilizes SPG can the Fc fragment on people and some mammiferous IgG molecule to be combined, can allow very orderly being arranged on solid phase carrier of target IgG antibody, greatly increase the effective binding site of antibody to antigen, significantly improved detection sensitivity.Recombinate to IgG, combination does not have the kind restriction to SPG simultaneously, can meet dissimilar antibody, highly versatile.
The embodiment of the present invention also provides a kind of malachite green detection method, and the method utilizes ELISA detection kit of the present invention to detect, and comprises the following steps:
S01, the coated damping fluid that will contain SPG albumen are hatched on solid phase carrier, hatch rear removal liquid;
Add confining liquid and hatch on S02, the solid phase carrier that obtains to step S01, hatching rear removal liquid;
Add malachite green antibody and hatch on S03, the solid phase carrier that obtains to step S02, hatching rear removal liquid;
Add testing sample on S04, the solid phase carrier that obtains to step S03, add coupling that the malachite green of HRP is arranged simultaneously, hatch rear removal liquid;
Add developer on S05, the solid phase carrier that obtains to step S04, add terminator after colour developing.
Particularly, the confining liquid used in the detection method of the embodiment of the present invention is for containing 0.8-1.2%(w/v) the 0.01-0.02mol/L phosphate buffer of skimmed milk power and 0.4-0.6% (w/v) tryptone.
The malachite green antibody used in step S03 in the detection method of the embodiment of the present invention is polyclonal antibody or monoclonal antibody, preferably uses monoclonal antibody.
The material of the solid phase carrier used in the detection method of the embodiment of the present invention can be polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.; The form of solid phase carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.Preferred solid phase carrier is ELISA Plate.
The malachite green detection method that the embodiment of the present invention provides, utilize SPG albumen that antibody is fixed on solid phase carrier, with respect to conventional ELISA detection technique have advantages of highly sensitive, stability is strong.
Below by specific embodiment, describe the present invention.
Embodiment 1
1. prepare following reagent:
Protein protective agent: take KH
2PO
40.54g; Anhydrous Na
2HPO
42.28g; NaCl8.0g; KCl0.2g; 0.5g merthiolate; Add tri-distilled water 800ml, adjusting pH is 7.4; Add 0.5ml Tween-20(0.05%), mix, add 100g skimmed milk power and 50g bovine serum albumin(BSA), after dissolving, with tri-distilled water, be dissolved to 1000mL, 4 ℃ are in store for;
Coated damping fluid: first prepare 0.02M phosphate buffer (PBS, pH7.4): take KH
2PO
40.54g; Anhydrous Na
2HPO
42.28g; NaCl8.0g; KCl0.2g; 0.5g merthiolate; Be dissolved to 1000mL with tri-distilled water, adjusting pH is 7.4; Mix with 9:1 with above-mentioned protein protective agent, cross 0.25 μ m filter membrane, standby.
Confining liquid: take KH
2PO
40.27g; Anhydrous Na
2HPO
41.14g; NaCl8.0g; KCl0.2g; 0.5g merthiolate; Add tri-distilled water 800ml, adjusting pH is 7.4; Add 0.5mlTween-20(0.05%), mix, add 10g skimmed milk power and 5g tryptone, after dissolving, with tri-distilled water, be dissolved to 1000mL, standby.
Cleansing solution: take KH
2PO
40.27g; Anhydrous Na
2HPO
41.14g; NaCl8.0g; KCl0.2g; Add tri-distilled water 800ml, adjusting pH is 7.4; Add 0.5ml Tween-20(0.05%), mix, be dissolved to 1000mL with tri-distilled water.
Standard items dilution (sample dilution): take KH
2PO
40.27g; Anhydrous Na
2HPO
41.14g; NaCl8.0g; KCl0.2g; Add tri-distilled water 800ml, adjusting pH is 7.4, with tri-distilled water, is dissolved to 1000mL.
The malachite green standard solution: malachite green is dissolved in to above-mentioned standard items dilution, and concentration is respectively the malachite green solution of 0ng/ml, 0.001ng/ml, 0.003ng/ml, 0.009ng/ml, 0.027ng/ml, 0.081ng/ml.
Malachite green with the HRP coupling: accurately take 16.5mg carboxyl concealed malachite green (CLMG), be dissolved in 1mlDMF(N, dinethylformamide) in-water (DMF10%), add 0.01% oxygenant chloranil, stir 1min, splash into two 1% ascorbic acid solution neutralization reactions, add respectively 7mgNHS (N-hydroxy-succinamide) in this mixed solution, ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) 10.5mgEDC(1-(CLMG:NHS:EDC mol ratio=1:1:1), 4 ℃ of stirrings are spent the night, and obtain the carboxyl malachite green of activation.Take the HRP(horseradish peroxidase) 9mg, being dissolved to concentration with distilled water is 20mg/ml, under low temperature, the carboxyl malachite green of above-mentioned activation is slowly dropped to this HRP solution, 4 ℃ of stirrings are spent the night, with the PBS of 0.01mol/LpH7.4, dialyse 3 days, concentrated with PEG20000,4 ℃ of preservations.
Developer: hydrogen peroxide and tetramethyl benzidine (TMB) mixed liquor (mixing of 1:1 volume ratio).
Terminator: 2mol/L sulfuric acid solution.
2. the preparation of monoclonal antibody
1) preparation of malachite green immunizing antigen
Take 16.5mg carboxyl concealed malachite green (CLMG), 7mgNHS, 10.5mgEDC(CLMG:NHS:DCC=1:1:1) be dissolved in 5mlDMF(N, dinethylformamide) in, 4 ℃ of stirrings are spent the night, and obtain the carboxyl malachite green of activation.Taking 50mg oralbumin (OVA) is dissolved in the sodium borate buffer liquid that 5ml0.1mol/LpH is 8.5, precooling 30min on ice, the carboxyl malachite green of above-mentioned activation is slowly dropped to (white) in this damping fluid, 4 ℃ of stirrings are spent the night, the PBS that is 7.4 with the 0.01mol/LpH 3d that dialyses, concentrated with PEG20000, then use 0.22 μ m membrane filtration.
2) preparation of monoclonal antibody
A. animal immune: adopt BALB/c mouse as immune animal, immunizing antigen prepared by step 1) (conjugate of malachite green haptens and oralbumin) immunizing dose is 100 μ g/, first immunisation is mixed and made into emulsifying agent by the Freund's complete adjuvant of immunizing antigen and equivalent, the subcutaneous multi-point injection of nape section, interval is got the same dose immunizing antigen in 2 weeks and is added equivalent incomplete Freunds adjuvant mixing and emulsifying, peritoneal immunity is strengthened four times, the 5th immunity is that tail vein injection is strengthened, extracting spleen cell after 3 days;
B. Fusion of Cells and cloning: get the BALB/c mouse splenocyte after step a immunity, in 10:1 ratio and SP2/0 myeloma cell, merge, adopt indirect competitive ELISA to measure cell conditioned medium liquid specificity, screen positive hole, utilize limiting dilution assay to carry out cloning to positive hole, until obtain the hybridoma cell strain of stably excreting monoclonal antibody;
C. the preparation and purification of monoclonal antibody: adopt in body and induce method, by BALB/c mouse (8 weeks age) Intraperitoneal injection sterilizing paraffin oil, 0.5ml/ only, the hybridoma of 10 days pneumoretroperitoneums injection above-mentioned steps b acquisition, 5 * 10
6Individual/as only, after 8 days, to gather ascites, carry out ascites by sad-saturated ammonium sulfate method and purify, bottle packing ,-20 ℃ of preservations.
3. the acquisition of typical curve
Obtain SPG albumen, the 0.05M phosphate buffer (PBS, pH8.0) made by step 1 is diluted to 1 μ g/ml, obtains SPG albumen coating buffer.Get 96 hole ELISA Plate, in every hole, add the above-mentioned SPG albumen of 100 μ l coating buffer, 37 ℃ of incubation 30min, the coating buffer that inclines, wash respectively 3 times with cleansing solution, and each 20s, pat dry after washing;
To the confining liquid that adds 200 μ l steps 1 to make in every hole of above-mentioned ELISA Plate, 37 ℃ of incubation 1h, liquid in the hole of inclining, pat dry;
0.02MpH7.4 phosphate buffer dilution (0.1-0.3 μ g/ml) for monoclonal antibody by step 2 preparation, then be added in every hole of above-mentioned ELISA Plate, 37 ℃ of incubation 10min, and the liquid that inclines, with cleansing solution washing 3 times, each 20s, pat dry after washing;
To application of sample in above-mentioned ELISA Plate, the malachite green standard solution of 6 kinds of concentration that add respectively step 1 to make, 6 Duplicate Samples of every kind of concentration, every hole 50 μ l, then to that respectively add step 1 to make and the malachite green HRP coupling in the hole of above-mentioned application of sample, every hole 50 μ l, 37 ℃ of incubation 30min, the liquid that inclines, with cleansing solution washing 3 times, each 20s, pat dry after washing;
Add 100 μ l developers to every hole in above-mentioned ELISA Plate, 37 ℃ of incubation 15min, add the terminator cessation reaction, and the 405nm wavelength is measured the OD value.
According to above OD value drawing standard curve, obtain the IC50 value, as shown in table 1.Wherein IC50 be in curve between absorbance and 0ng/ml absorbance when being 50%, corresponding standard items concentration, IC50 value is lower, illustrates that the sensitivity of antibody is higher, and sensing range and the stability of IC50 fluctuation range demonstration system.
The IC50 value that the coated SPG method of table 1 obtains
| ? | Curve 1 | Curve 2 | Curve 3 | Curve 4 | Curve 5 | Curve 6 |
| IC50(ng/ml) | 0.0341 | 0.0318 | 0.0328 | 0.0317 | 0.0367 | 0.0335 |
| R 2 | 0.9994 | 0.9962 | 0.9917 | 0.9984 | 0.9985 | 0.9950 |
4. the sample Malachite Green is measured
A. solution preparation:
1) extract A: accurately take 3.854g ammonium acetate and 0.9511g p-toluenesulfonic acid, add the 800ml ultrapure water, stirring and dissolving, adjust pH to 5.0 with acetic acid, and constant volume is to 1000ml.
2) extract B: accurately take the 10.599g natrium carbonicum calcinatum, add ultrapure water, constant volume, to 1000ml, obtains 0.1mol/L sodium carbonate.
3) 1:1 acetonitrile: methylene chloride mixed liquor: get the 1ml acetonitrile and mix with the 1ml methylene chloride.
4) 2:1 acetonitrile: methylene chloride mixed liquor: get the 2ml acetonitrile and mix with the 1ml methylene chloride.
B. the pre-treatment of fish and shrimp sample:
A) meat is got in fish, shrimp peeling, with homogenizer, sample is ground evenly;
B) take the ground sample of 1g in the 15ml centrifuge tube, add the extract A of the above-mentioned preparation of 4ml, jolting 10 seconds, be uniformly dispersed fish, shrimp;
C) add the 6ml acetonitrile in this centrifuge tube, repeatedly vibrate 2 minutes;
D) 4000 rev/mins centrifugal 5 minutes, supernatant is poured in another 15ml centrifuge tube;
E) add 0.4ml said extracted liquid B, jolting 10 seconds in this centrifuge tube;
F) add the 2ml methylene chloride in this centrifuge tube, repeatedly vibrate 20 seconds, then 4000 rev/mins centrifugal 5 minutes, layering, draw upper organic phase in another 15ml centrifuge tube;
G) add the 0.5g anhydrous sodium sulfate in above-mentioned organic phase, vibration mixes, standing 3 minutes.4000 rev/mins centrifugal 5 minutes, the upper strata organic solvent is moved in another 15ml centrifuge tube to (whether water layer is arranged below attention, if having, liquid-transfering gun or dropper are drawn for the organic solvent on upper strata), at 50 ℃, with air/nitrogen, dry up;
H) add acetonitrile, the methylene chloride mixed liquor that the 2ml volume ratio is 1:1 in this centrifuge tube, 100 μ l oxidation solutions (chloranil), vibrate and within 2 minutes, make residue dissolve.The aluminium oxide solid phase column is put in a new 15ml centrifuge tube, pours aforesaid liquid into, after solution all enters post, the acetonitrile that is 2:1 by the 3ml volume ratio, methylene chloride mixed liquor are washed post (adding the aluminium oxide solid phase column naturally to flow through this mixed liquor).After this mixed liquor all enters post, by centrifuge tube with 700 rev/mins centrifugal 2 minutes, remove solid phase column, in 50 ℃, with air/nitrogen, dry up;
I) add 300 μ l acetonitriles in this centrifuge tube, abundant vortex 2 minutes (for avoiding the acetonitrile layer volatilization, cover tightly the centrifuge tube lid, also acetonitrile layer can be taken out to 4 ℃ of sealings and preserve) on the vortex instrument;
Get acetonitrile solution 25 μ l while j) measuring, add 175 μ l distilled water, (with embodiment mono-step 1), mix, get the above-mentioned solution of 50 μ l and carry out elisa assay, concrete operation step is with the acquisition of above-mentioned step 3 Plays curve for 50 μ l sample dilutions.
Measure respectively each 20 dummies of fish and shrimp sample, according to typical curve, obtained measured value, calculated its mean value, added 3 times of standard deviations, be the lowest detectable limit of malachite green vestigial in this sample.Testing result is in Table 2.
The detectability that the coated SPG method of table 2 records
The comparative example 1
In the embodiment of the present invention, utilize traditional antigen coated method ELISA to detect in contrast.
The processing of standard items and testing sample is with embodiment 1.The detecting step of standard items and testing sample is identical, and difference only is the application of sample difference, and operation steps is summarized as follows:
1) coupling of malachite green envelope antigen
Accurately take 16.5mg carboxyl concealed malachite green (CLMG), 7mgNHS, 10.5mgEDC(CLMG:NHS:DCC=1:1:1) be dissolved in 5mlDMF, 4 ℃ of stirrings are spent the night, and obtain the carboxyl malachite green solution of activation; The BSA that takes 50mg is dissolved in 5ml0.1MpH8.5 sodium borate buffer liquid, precooling 30min, the carboxyl malachite green activated is slowly dropped in above-mentioned carboxyl malachite green solution (white), 4 ℃ of stirrings are spent the night, use 0.01MPBS(pH7.4) dialyse 3 days, concentrated with PEG20000, then use 0.22 μ m membrane filtration, standby, obtain the malachite green with the BSA coupling;
2) by above-mentioned, with the malachite green BSA coupling, be coated with to 96 hole ELISA Plate, add 50 μ l malachite green standard items (adding testing sample during detection) in the every hole of this ELISA Plate, then add the malachite green monoclonal antibody (concentration is with embodiment 1) of 50 μ l with the dilution of 0.02mol/LpH7.4 phosphate buffer, 37 ℃ of incubation 30min, liquid inclines, with cleansing solution washing 3 times, each 20s, pat dry after washing;
2) to the HRP mark sheep anti-mouse igg (market is buied) that adds 100 μ l in the every hole of above-mentioned ELISA Plate, 37 ℃ of incubation 30min, the liquid that inclines, with cleansing solution washing 3 times, each 20s, pat dry after washing;
3) add 100 μ l developers in the every hole of above-mentioned ELISA Plate, 37 ℃ of incubation 15min, add the terminator cessation reaction, and the 405nm wavelength is measured OD value, acquisition standard curve I C50(table 3) and the detected value (table 4) of testing sample.
The curve IC50 of table 3 direct coated antigen measuring
The detectability that the coated malachite green antigen of table 4 records
Known with the IC50 value of table 3 by comparison sheet 1, the coated prepared detection kit sensitivity of SPG method is better than the standby detection kit of envelope antigen legal system greatly.R by comparison sheet 1 with table 3
2Be worth known, the coated prepared detection kit stability detection kit standby higher than the envelope antigen legal system of SPG method.
By comparison sheet 2 with table 4(because envelope antigen method detection sensitivity is told somebody what one's real intentions are, therefore in comparative example 1, the application of sample amount increases) known, the coated standby malachite green kit detectability of SPG legal system can reach 0.0195ng/ml, and sensitivity is much higher than the 0.17ng/ml of envelope antigen method.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (8)
1. a malachite green ELISA detection kit comprises: SPG albumen, coated damping fluid, confining liquid, malachite green antibody, coupling have malachite green, developer and the terminator of HRP.
2. detection kit as claimed in claim 1, is characterized in that, described coupling has the preparation method of the malachite green of HRP to be:
The carboxyl concealed malachite green is converted into to malachite green with the chloranil, add ascorbic acid, products therefrom mixes with NHS, EDC, the mol ratio of wherein said carboxyl concealed malachite green and NHS, EDC is 0.8-1:0.8-1:0.8-1, stir to obtain mixed solution, described mixed solution is added in the HRP aqueous solution.
3. detection kit as claimed in claim 1, is characterized in that, described malachite green antibody is polyclonal antibody or monoclonal antibody.
4. detection kit as claimed in claim 1, is characterized in that, also comprises the malachite green standard items.
5. detection kit as claimed in claim 1, is characterized in that, described developer is hydrogen peroxide and tetramethyl benzidine mixed liquor or urea peroxide and tetramethyl benzidine mixed liquor.
6. a malachite green detection method, utilize ELISA detection kit as described as any one in claim 1 to 5 to detect, and comprises the following steps:
S01, SPG albumen is mixed with coated damping fluid, must be coated with mixed liquor and hatch on solid phase carrier, hatch rear removal liquid;
Add confining liquid and hatch on S02, the solid phase carrier that obtains to step S01, hatching rear removal liquid;
Add malachite green antibody and hatch on S03, the solid phase carrier that obtains to step S02, hatching rear removal liquid;
Add testing sample on S04, the solid phase carrier that obtains to step S03, add coupling that the malachite green of HRP is arranged simultaneously, hatch rear removal liquid;
Add developer on S05, the solid phase carrier that obtains to step S04, add terminator after colour developing.
7. malachite green detection method as claimed in claim 6, is characterized in that, described solid phase carrier is made by polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber or Ago-Gel.
8. malachite green detection method as claimed in claim 6, is characterized in that, described solid phase carrier is ELISA Plate.
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| CN108152493A (en) * | 2017-12-19 | 2018-06-12 | 广州安诺科技股份有限公司 | The method for detecting aquatic products Malachite Green |
| CN108822005A (en) * | 2018-06-13 | 2018-11-16 | 郑州大学 | A kind of reversible colorimetric probe and its preparation, application based on malachite green and bisulfite addition product |
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| MEI-CHUN YANG ET AL.: "Production of Antibodies for Selective Detection of Malachite Green and the Related Triphenylmethane Dyes in Fish and Fishpond Water", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》, vol. 55, no. 22, 9 October 2007 (2007-10-09), XP009102201, DOI: doi:10.1021/jf071195y * |
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| CN107421938A (en) * | 2017-09-09 | 2017-12-01 | 合肥学院 | A kind of SiO for detecting malachite green2The preparation method of@ROX nanoparticle fluorescence probe arrays |
| CN108152493A (en) * | 2017-12-19 | 2018-06-12 | 广州安诺科技股份有限公司 | The method for detecting aquatic products Malachite Green |
| CN108822005A (en) * | 2018-06-13 | 2018-11-16 | 郑州大学 | A kind of reversible colorimetric probe and its preparation, application based on malachite green and bisulfite addition product |
| CN108822005B (en) * | 2018-06-13 | 2021-04-09 | 郑州大学 | Reversible colorimetric probe based on malachite green and bisulfite addition product, and preparation and application thereof |
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