CN101655498A - Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method - Google Patents
Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method Download PDFInfo
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- CN101655498A CN101655498A CN200910093010A CN200910093010A CN101655498A CN 101655498 A CN101655498 A CN 101655498A CN 200910093010 A CN200910093010 A CN 200910093010A CN 200910093010 A CN200910093010 A CN 200910093010A CN 101655498 A CN101655498 A CN 101655498A
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- malachite green
- colloidal gold
- concealed malachite
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Abstract
The invention discloses an immune colloidal gold test stripe for detecting leucomalachite green in aquatic products and a preparation method; the colloidal gold test stripe provided by the invention detects leucomalachite green with a direct competition method, and comprises a sample pad, a colloidal gold combined pad, a nitrocellulose membrane (NC), a water adsorption pad and a PVC back lining, one end of the PVC back lining is sequentially adhered with the sample pad and the colloidal gold combined pad, and the middle thereof is adhered with the NC layer, and the other end thereof is sequentially adhered with water adsorption pad; the immune colloidal gold test stripe has the characteristics that colloidal gold combined pad is enveloped with colloidal gold marker which is monoclonal antibody of the specificity of the leucomalachite green, and the NC is adsorbed with leucomalachite green-OVA and sheep anti-mouse IgG. The invention has high detection sensitivity and low price and is simple and rapid, the whole reaction only needs 30 minutes; the immune colloidal gold test stripe can be used for large-batch screening of samples and can rapidly detect the residual leucomalachite green in the aquatic products at site.
Description
Technical field
The invention belongs to and relate to immune colloid gold and detection of veterinary drugs in food field in the food security field.Particularly, the present invention relates to a kind of being applicable to and detect the residual immunity colloidal gold test paper strip of concealed malachite green in the aquatic products.
Background technology
Malachite green (Malachite green) molecular formula is: C
23H
25ClN
2It is a kind of industrial triphenylmethane triphenylmehtane class dyestuff, owing to have the effect of antibiotic and resisting gram-positive bacteria, and low price, so be widely used in culturing saprolegniasis, branchiomycosis, ich of the prevention fish of fishery etc., and in order to make the impaired fish of squama prolong life, deposit in the pond in the transportation neutralization, also often use malachite green.When culturing water when containing malachite green, meeting fast Absorption malachite green such as fish body, shrimp and then metabolism become concealed malachite green (Le μ comalachite green, LMG), i.e. N, accelerine, molecular formula: C
23H
26N
2, the procrypsis malachite green is water insoluble, and residual toxicity is stronger than malachite green, can hoard to reach the several months in muscle.The toxicological study of relevant in recent years this type of dyestuff is found: malachite green and major metabolite concealed malachite green thereof have multiple toxicity, comprise carcinogenicity, cause gene mutation, cause chromosome breakage, teratogenesis tire and respiratory tract toxicity.Given this, many countries all classify malachite green as the aquaculture forbidden drugs.
Nutrition is complete, the characteristics of balance because aquatic products (flesh of fish, shrimp etc.) have, and more and more is subjected to people's favor.By 1996, China's output of aquatic products surpassed 2,000 ten thousand tons, accounts for 1/3 of national meat food production, leaps to the No. 1 in the world.Along with the raising of living standards of the people, the quality and quantity of aquatic products there has been the requirement of renewal.Yet China fishery development is faced with a series of new challenges, and wherein food-safety problem has seriously restricted the industrialized development of China's fishery to a certain extent, and illegal interpolation malachite green is at present distinct issues.China lists malachite green in May, 2002 veterinary drug of forbidding.
The Information Office of the Ministry of Agriculture has issued the monitoring result of the aquatic product quality safety first time in 2007.In last year in the concrete the first tenday period of a month in time, the Ministry of Agriculture organizes relevant quality inspection organization that malachite green pollution condition in 22 municipal water products of China has been carried out the routine monitor first time in the current year, and 10 cities all have sample to detect malachite green.Because the driving of huge interests is difficult to the use that malachite green is stopped in real control effectively, this just means that also to malachite green detection demand be long-term.
At present in the world the detection technique route of malachite green and concealed malachite green is mainly contained two: one is to be the chemical detection technique of core with look (matter) spectral technology, and another is the quick bio detection technique.High performance liquid chromatography cost height, the running time is long, and step is many and complicated, and the scene truly of therefore can't realizing is detected.And advantages such as the colloidal gold strip detection method is highly sensitive, high specificity, good stability, easy and simple to handle, on-the-spot detection soon, grasped by basic unit easily and the large tracts of land popularization, the field quick detection that is suitable for batch samples, do not need specific instrument equipment, do not need other specific detectable yet, reduced the burden that detects unit, can fast detecting obtain preliminary testing result, being fit to China's current social economic and technique level, therefore is the main developing direction that following malachite green detects.
Summary of the invention
The object of the present invention is to provide a kind of residual colloidal gold strip of golden mark technology field quick detection concealed malachite green that adopts.
The invention provides the residual colloidal gold strip of a kind of fast detecting concealed malachite green, the monoclonal antibody that is produced is the anti-concealed malachite green monoclonal antibody of specificity.Described concealed malachite green monoclonal antibody is that concealed malachite green haptens and carrier protein couplet thing make by immune animal (for example rabbit, mouse) as immunogene.Described concealed malachite green haptens is to adopt chemical method to make it contain reactive group, and described carrier protein is the blue albumen (KLH) of bovine serum albumin(BSA), human serum albumins or key hole copper.Described concealed malachite green-carrier protein couplet thing adopts couplings such as mixed anhydride method, diazonium method or glutaraldehyde method to obtain.Described colloid gold label can be used commercial gold chloride, makes by gold chloride and the coupling of concealed malachite green monoclonal antibody that produces.
Be convenient on-the-spot the detection and the great amount of samples examination, the immunity colloidal gold test paper strip of described concealed malachite green comprises: NC film, sample pad, glue gold pad, thieving paper, trisodium citrate, PVC backing and pad, PVC backing one end sticks sample pad, collaurum pad, centre successively and sticks the NC rete, the other end sticks adsorptive pads successively, it is characterized in that collaurum pad bag by anti-concealed malachite green monoclonal antibody specific-colloid gold label thing, has adsorbed concealed malachite green-OVA and sheep or rabbit anti-mouse igg on the NC film.
The immune colloid gold reagent method that preparation the present invention detects product concealed malachite green in the water is:
(1) concealed malachite green haptens and holoantigen are synthetic by at phenyl ring contraposition coupling one reactive group-COOH, synthesize the concealed malachite green haptens; Concealed malachite green haptens and carrier protein are pressed 1: 10～30 mol ratio mixings, and synthetic stealthy malachite green-carrier protein couplet things such as glutaraldehyde or diazonium method obtain concealed malachite green hapten-carrier protein conjugate after separation and purification;
(2) Monoclonal Antibody is the repeatedly immune Balb/c mouse inbred lines of immunogene with haptens-BSA, gets the external fusion of mouse boosting cell and myeloma cell and produces hybridoma, obtains the positive hybridoma cell strain through the selective medium screening.Hybridoma cell strain is injected mouse peritoneal obtain methods such as ascites or cell in vitro cultivation collection supernatant, prepare anti-concealed malachite green monoclonal antibody in a large number;
(3) the monoclonal antibody colloid gold label is reduced into gold chloride with reductive agents such as trisodium citrates the colloid gold particle of 20～40nm; Then collaurum and anti-concealed malachite green monoclonal antibody are pressed 1: 0.005～0.015 (volume mass ratio) mixing, make it in conjunction with forming stable colloid gold particle, purified and concentrated generation resists recessive peacock stone monoclonal antibody-colloid gold label thing;
(4) the anti-mouse IgG preparation of sheep (rabbit) is obtained antiserum with many immune mouses of haptens-OVA, and immune goat or rabbit obtain the anti-mouse IgG of sheep (rabbit) after separation and purification again;
(5) preparation of collaurum pad is coated on concealed malachite green specific monoclonal antibody-colloid gold label thing on the collaurum pad, concealed malachite green-OVA conjugate and the anti-mouse IgG of sheep (rabbit) are adsorbed on the detection zone and the control zone of NC film, fully dry at 37 ℃;
(6) colloidal gold strip assembling: NC film, collaurum pad, sample pad and adsorptive pads etc. are sticked on the PVC backing successively, the PVC material that glues is cut into the test strips of certain width, be assembled at last in a certain size the plastic casing and get final product.
Beneficial effect
Detection concealed malachite green immunity colloidal gold test paper strip of the present invention adopts the direct competitive immunization, with colloid gold label concealed malachite green specific monoclonal antibody, can detect by fast qualitative, the result is accurate, need not reagent wash and standard control, can be in batches or single sample in time detect.
Detection concealed malachite green immunity colloidal gold test paper strip of the present invention uses starting material to produce, and the production flow process is simple, and cost is low, and testing cost only is about 1/5 of a gas phase and liquid phase chromatogram, is 1/3 of enzyme marking reagent box.For the user saves time, reduce the error cause because of operation steps is miscellaneous, advantage such as the reagent holding time is long, execute-in-place is convenient can play a significant role in aquatic products detect.
Description of drawings
Fig. 1 is stealthy malachite green immunity colloidal gold test paper strip structural drawing
Fig. 2 is a colloidal gold solution visible light scanning spectra
Fig. 3 detects data and curves figure the stealthy malachite green immunity colloidal gold test paper strip shelf-life
Concrete to implement to provide following embodiment be in order further to understand the present invention better, and never content of the present invention and protection domain are constituted any restriction.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but not as a limitation of the invention.
Embodiment 1 haptens and comlete antigen (immunogene) are synthetic
1.1 preparation to the concealed malachite green ethyl ketone
In the 100mL three-necked bottle, add 20mL methylene chloride, 18g (0.135mol) aluminum trichloride (anhydrous).Cryosel is bathed and is cooled to-10 ℃, drips the mixed liquor of 7.1g (0.07mol) aceticanhydride and 21.61g (0.065mol) concealed malachite green, and temperature is no more than-5 ℃ in the control.After dropwising, continue to stir 15h, solution becomes kermesinus from yellow.In the potpourri with reactant liquor impouring 50mL concentrated hydrochloric acid and 50g trash ice, divide water-yielding stratum with separating funnel then, and with twice of dichloromethane extraction.Merge organic phase, wash to neutrality anhydrous sodium sulfate drying with water.Distillation remove desolvate yellow oil 9.8g.Crude product can be directly used in bromoform reaction.
1.2 preparation to concealed malachite green formic acid
NaOH 6.3g is dissolved in the 54mL water, is cooled to-5 ℃.Dripping bromine 6.4g (0.04mol), temperature is no more than 0 ℃ in the control.To drop in the reactant liquor concealed malachite green ethyl ketone crude product 5.04g then, temperature is no more than 10 ℃ in the control.Dropwise, insulation 1h is then in stirring at room 2h.Standing demix divides and to remove the bromofom that generates.Water layer transfers to pH1～2 with concentrated hydrochloric acid, separates out white solid.Suction filtration is washed to neutrality.Solid is dissolved in the 20mL sodium hydroxide solution (5%), filters, light yellow clarifying liquid body and function concentrated hydrochloric acid transfers to pH1～2, separates out white crystal.Suction filtration is washed to neutrality, and oven dry gets white crystal 1.7g, yield 81% (by concealed malachite green).m.p.117～120℃。HNMR(300MHz，CDCl
3)，δ：1.28(d，6H，J＝6.9Hz)；2.99(m，1H，J＝6.9Hz)；7.33(d，2H，J＝8.3Hz)；8.05(d，2H，J＝8.3Hz)。
1.3 it is immunogenic synthetic
Active ester method prepares immunogene and is achieved in that concealed malachite green haptens and the N-hydroxy-succinamide (NHS) of getting equimolar amounts, ring dihexyl carbodiimide (DCC), (DFM) dissolves potpourri with dimethyl formamide, the lucifuge reaction is spent the night under the room temperature, centrifugal place to go post precipitation, get the supernatant drying, getting its 225 μ L adding contains in the 8mL carbonate buffer solution (Ph9.6 contains 5% methyl alcohol) of 250mgBSA, potpourri stirred 2 hours at 4 ℃ of lower magnetic forces, dialysed overnight 4 times (phosphate buffer of pH7.4) under 4 ℃ of conditions is carried out full wavelength scanner through ultraviolet scanner and is identified the coupling result.
Fig. 2 is the UV scanning figure of the haptenic conjugate of BSA-(immunogene) of employing active ester method preparation, three kinds of materials are respectively carrier protein BSA, the haptenic conjugate of carrier protein BSA-, haptenic ultraviolet absorpting spectrum from top to bottom among the figure, carrier protein BSA characteristic absorption peak is at the 278nm place as can be seen from Figure, the haptens characteristic absorption peak is at the 260nm place, the haptenic conjugate characteristic absorption peak of carrier protein BSA-is at the 255nm place, and the conjugate characteristic peak drifts about.
The embodiment 2 holoantigen MONOCLONAL ANTIBODIES SPECIFIC FOR and the detection of tiring
2.1 MONOCLONAL ANTIBODIES SPECIFIC FOR
With the BSA-haptens is immunogene, 4 BALB/C mice of immunity, and every mouse is got 100 μ g immunogenes, and is even with equal-volume Freund's complete adjuvant mixing and emulsifying, injects in the abdominal cavity film along groin.After 4 week, booster immunization, dosage is constant, and adjuvant changes incomplete Freund into.Behind the booster immunization three times, blood sampling is surveyed and is tired, treat that serum titer no longer rises, antigen with two multiple doses does not add the adjuvant immunity mouse, get spleen cell and murine myeloma cell after three days in 5-10 under aseptic condition: 1 ratio is mixed in the 50mL centrifuge tube, adds 30mL serum-free IPMI1640 nutrient culture media, and 1200r/min is centrifugal, and 10min abandons supernatant, with the cell mass pine that shakes gently, place 37 ℃ of water-baths.1mL 50%PEG-4000 is slowly added in the cell, in 1min, drip off, stir bottom settlings gently simultaneously, leave standstill 1min.Slowly add serum free medium along tube wall and stop fusion process.Slowly at the uniform velocity added in preceding 30 seconds behind the 1mL and to add 2mL in 30 seconds, add the 27mL serum free medium then fast, the centrifugal 10min of 1200r/min abandons supernatant.Cell after the fusion screens in HAT selectivity nutrient solution earlier, changes the HT nutrient solution after 5 days into, treats that the hybrid cell quantity hole in reaches 300 when above, carries out the detection of multiple hole with ELISA pair cell culture supernatant, the definite result of duplicate detection next day.Cell in the strong positive hole is cloned cultivation with limiting dilution assay, and track record, to cultivate and detect through the clone more than 3 times, the hole inner cell that all is positive is the hybridoma of secrete monoclonal antibody.Hybridoma through enlarged culture, is selected 4 multiparity BALB/C mice, lumbar injection saxol 0.5mL/, 7 days pneumoretroperitoneum injection hybridomas 5 * 10
5-10
6/ only, after 10 days, treat to collect when mouse web portion obviously expands ascites.Come purifying ascites with caprylic acid-ammonium sulfate precipitation method, the albumen that obtains through the preliminary judgement of nucleic acid-protein ultraviolet scanner analysis of protein is IgG albumen.
2.2 detection antibody titer
By every hole 100 μ L coated elisa plates, 4 ℃ of bags are spent the night, and wash 5 times with 1 μ g/mL concentration, pat dry, and seal 12h down for 4 ℃ by every hole 200 μ L confining liquids, wash 3 times, pat dry.Adding the antiserum extension rate by every hole 100 μ L is 2000,10000,250000,50000,250000,1250000, and negative serum and blank (do not add antiserum, only add its dilution) room temperature effect 30min washs five times, pats dry.Add every hole 100 μ L enzymes mark goat-anti rabbit (mouse) antibody, room temperature effect 30min washs five times, pats dry.Add every hole 100 μ L colour developing liquid, 37 ℃ of lucifuge effect 15min.Add every hole 50 μ L stop buffer cessation reactions, microplate reader detects A value (450nm).The antiserum dilutability corresponding with the serum OD value that doubles negative serum OD value is antiserum titre.Testing result sees Table 1:
The table 1 monoclonal antibody testing result of tiring
| Extension rate | ??2000 | ??10000 | ??15000 | ??30000 | ??50000 | ??250000 | Negative serum | Blank |
| The OD1 value | ??0.988 | ??0.692 | ??0.415 | ??0.324 | ??0.317 | ??0.362 | ??0.172 | ??0.024 |
| The OD1 value | ??0.975 | ??0.673 | ??0.442 | ??0.398 | ??0.365 | ??0.354 | ??0.207 | ??0.02 |
| The OD1 value | ??1.136 | ??0.943 | ??0.754 | ??0.503 | ??0.387 | ??0.351 | ??0.193 | ??0.032 |
| The OD1 value | ??0.982 | ??0.669 | ??0.411 | ??0.376 | ??0.344 | ??0.351 | ??0.223 | ??0.03 |
Can infer that from the data of table 1 the tiring of monoclonal antibody that the present invention prepares reaches 15000.
The preparation of the anti-mouse IgG of sheep (rabbit) among embodiment 3 the present invention
Choosing healthy male new zealand white rabbit or goat, is that immunogene and equivalent Freund's complete adjuvant are mixed into water in oil emulsion by syringe to the method for taking out with the OVA-haptens, carries out first immunisation by the amount of 1mg/kg body weight, takes the subcutaneous multi-point injection in back.Every two all booster immunizations once, replace Freund's complete adjuvant, the same first immunisation of dosage and method with incomplete Freund.From immunity for the third time, back 10 days of each immunity, auricular vein is got blood 1mL, carrying out antibody titer detects, when antibody titer no longer raises, do not add adjuvant and carry out for the last time (the 7th time) immunity, the leg muscle injection, rear neck artery bloodletting in 7 days, room temperature is solidified behind the 2h 4 ℃ and is spent the night, and centrifugal 10 minutes of 8000r/min removes clot, serum partly precipitates with 50% saturated ammonium sulfate solution, the centrifugal supernatant that goes, precipitation is resuspended with phosphate buffer, precipitates twice with 33% saturated ammonium sulfate solution again, sediment is with the dissolving of the least possible phosphate buffer, through dialyse concealed malachite green-anti-mouse IgG of OVA sheep (rabbit).
The preparation of colloidal gold strip among embodiment 4 the present invention
4.1 the preparation of collaurum
The ultimate principle of immune colloidal gold technique is, gold chloride is under the effect of reductive agent, and polymerizable is a certain size gold grain, form electronegative, because electrostatic interaction and stable hydrophobic sol solution.Our company adopts this reductive agent reducing process of trisodium citrate to prepare collaurum, detailed process is as follows: get 0.01% gold chloride 100mL aqueous solution and be heated to and boil, stir and accurately add 1% citrate three sodium aqueous solution 0.7mL down, flavous gold chloride became aubergine in 2 minutes, continue to boil 15min, original volume is recovered with distilled water in the cooling back, is the colloidal gold solution of preparation.Whether this colloidal gold solution meets the production demand, except that the visual inspection color need be for the aubergine, also need to adopt the ultra-violet and visible spectrophotometer analysis, colloidal gold solution needs at visible range 535nm the highest absorption peak (see figure 2) is arranged, simultaneously, Electronic Speculum figure show preparation the colloid gold particle homogeneity better, grain size is about 40m.
4.2 the preparation of colloid gold label antibody
Regulate colloidal gold solution pH value to 8.2, evenly stir, dropwise add the monoclonal antibody of anti-concealed malachite green simultaneously, and add other protective agent, after all adding, continue to stir 15 minutes with constant speed stirrer.Centrifugal acquisition homogeneity gold labeling antibody precipitation by twice different rotating speeds.Add resuspended liquid again, move in the clean beaker standby behind the mixing.
4.3 the preparation of malachite green colloidal gold immune chromatography rapid detecting test paper strip
Successively thieving paper, the NC film that is sprayed with amino procrypsis malachite green-BSA (detection line) and the anti-mouse IgG of sheep (rabbit) (nature controlling line), the gold pad that is sprayed with anti-procrypsis malachite green antibody and sample pad are superimposed with each other and are fixed on the PVC base plate, be cut into test strips again, be contained in the plastic module, make the colloidal gold immunochromatographimethod rapid detection card.
The residual detection of concealed malachite green in embodiment 5 samples
5.1 kit of the present invention detects principle
Antibody labeling is on colloid gold particle; collaurum and sample move on the NC film simultaneously; in the process that moves; malachite green in the sample or procrypsis malachite green and the reaction that is marked at the antibody specificity on the colloid gold particle; when the malachite green in the sample or procrypsis malachite green content are lower than the detectability of test strips; then immune gold grain meeting and the antigen line that is sprayed on the NC film react the detection tape that forms a redness, otherwise, then can not form coloured detection tape.
5.2 sample preparation
Getting 5g sample (flesh of fish/shrimp) rubbing inserts in the 50mL centrifuge tube, add 10mL ethyl acetate homogeneous, with 4000g centrifugal 5 minutes, get the 5mL supernatant and insert in the glass tube, under 60 ℃, dry up with nitrogen, in this glass tube, add 1mL normal hexane and 1mL distilled water, vibration mixing 2 minutes, centrifugal 10 minutes of room temperature 4000g gets 100 μ L subnatants, through 5 times of sample diluting liquid dilutions, to be measured.
Embodiment 6 colloidal gold strip sensitivity tests
Get 3ng/ml and suppress product fully, handle the stealthy malachite green mark of adding product in the sample, make its final concentration be respectively 0.1,1,2,3,4,5,6,7,10,30ng/mL at buffer solution and 5 negative aquatic products.Every kind of sample repeats 5 times, judges the detection sensitivity of test strips, the results are shown in following table:
Table 2 the present invention adopts the colloidal gold strip sensitivity test of monoclonal antibody
As can be seen from the above results, the detection threshold of concealed malachite green colloid gold immune quick detection test paper bar is 3ng/mL.
The specificity test of embodiment 7 colloidal gold strips of the present invention
(ELISA is determined as feminine gender) adds malachite green, recessive crystal violet, crystal violet, Clenbuterol, sulfadimidine, chloromycetin, furazolidone respectively in the aquatic products of feminine gender, and making its final concentration is 1,5,10,50,100,500ng/mL aquatic products Treatment Solution.The standard method that detects with test strips detects, and judges the specificity that test strips detects, and the aquatic products of every kind of concentration are handled sample and done 5 repetitions.
Concealed malachite green immunity colloidal gold test paper strip specificity experimental result in table 3 aquatic products
Experimental result is as shown in table 3, and recessive crystal violet, crystal violet, Clenbuterol, sulfadimidine, chloromycetin, these analogues of furazolidone do not produce cross reaction in stealthy malachite green colloid gold immune quick detection test paper bar.
The accuracy test of embodiment 8 colloidal gold strips
Stealthy malachite green colloid gold immune quick detection test paper bar is qualitative, rather than quantitative card.This product all shows as the positive to the sample of every 3ng/mL of equaling or exceeding.
Add stealthy malachite green when negative aquatic products are handled in the sample,, only need directly aquatic products are handled sample and in well, get final product with the vertical 3 bubble-free samples to be checked (about 70ul) that drip of plastic suction pipe without any leaching process.Therefore when adding concealed malachite green 〉=3ng/mL, then detection line does not develop the color, and promptly is judged to the positive, thus the recovery test that is not similar to ELISA as a result the accuracy rate of this test card greater than 96%.
The test of embodiment 9 colloidal gold strip storage lives
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, concealed malachite green added the actual sample measured value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed 6 days under 37 ℃ of preservation conditions, carries out the accelerated stability experiment, and the result shows that the every index of this colloidal gold strip meets the requirements fully.
Claims (2)
1. detect concealed malachite green immunity colloidal gold test paper strip and preparation method thereof, comprising: comprise sample pad, collaurum pad, nitrocellulose filter (NC), adsorptive pads and PVC backing, PVC backing one end sticks sample pad, collaurum pad, NC rete successively, adsorptive pads, it is characterized in that collaurum pad bag by anti-concealed malachite green monoclonal antibody specific-colloid gold label thing, has adsorbed the anti-mouse IgG of concealed malachite green-OVA or concealed malachite green-BSA and sheep (rabbit) on the NC film.
2. concealed malachite green immune colloid gold reagent according to claim 1 and preparation method thereof, the preparation method who it is characterized in that this reagent may further comprise the steps: (1) concealed malachite green holoantigen synthetic: by at phenyl ring contraposition coupling one reactive group-COOH, and synthetic concealed malachite green haptens; Concealed malachite green haptens and carrier protein are pressed 1: 10-30 mol ratio mixing, synthetic stealthy malachite green-carrier protein couplet things such as glutaraldehyde or diazonium method obtain concealed malachite green hapten-carrier protein conjugate after separation and purification; (2) preparation of concealed malachite green monoclonal antibody specific: with haptens-BSA is the repeatedly immune Balb/c mouse inbred lines of immunogene, get the external fusion of mouse boosting cell and myeloma cell and produce hybridoma, obtain the positive hybridoma cell strain through the selective medium screening.Hybridoma cell strain is injected mouse peritoneal obtain methods such as ascites or cell in vitro cultivation collection supernatant, prepare anti-concealed malachite green monoclonal antibody in a large number; (3) acquisition of the anti-mouse IgG of sheep (rabbit): with many immune mouses of haptens-OVA, obtain antiserum, immune goat or rabbit obtain the anti-mouse IgG of sheep (rabbit) after separation and purification again; (4) monoclonal antibody specific Preparation of Colloidal Gold: the colloid gold particle that gold chloride is reduced into 20～40nm with reductive agents such as trisodium citrates; Then collaurum and anti-concealed malachite green monoclonal antibody are pressed 1: 0.005～0.015 (volume mass ratio) mixing, make it in conjunction with forming stable colloid gold particle, purified and concentrated generation resists recessive peacock stone monoclonal antibody-colloid gold label thing; (5) preparation of collaurum pad: concealed malachite green specific monoclonal antibody-colloid gold label thing is coated on the collaurum pad, concealed malachite green-OVA conjugate and the anti-mouse IgG of sheep (rabbit) are adsorbed on the detection zone and the control zone of NC film, fully dry at 37 ℃; (6) colloid gold reagent bar assembling: NC film, collaurum pad, sample pad and adsorptive pads etc. are sticked on the PVC backing successively, the PVC material that glues is cut into the reagent strip of certain width, be assembled at last in a certain size the plastic casing and get final product.
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Denomination of invention: Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method Effective date of registration: 20160615 Granted publication date: 20130703 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Zhongde Bio-Engrg Co., Ltd., Jiangxi Registration number: 2016360000008 |
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Address after: 330046 Jiangxi city in Nanchang Province, the provincial government compound Dongsilu dragon building C, Room 308 Patentee after: Jiangxi Sino German bioengineering Limited by Share Ltd Address before: High tech Development Zone, Jiangxi, Nanchang high and new way (Chang Jian Industrial Park) Patentee before: Jiangxi Zodogenes Biotechnology Co., Ltd. |
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Denomination of invention: Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method Effective date of registration: 20170811 Granted publication date: 20130703 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Jiangxi Sino German bioengineering Limited by Share Ltd Registration number: 2017990000738 |
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Denomination of invention: Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method Effective date of registration: 20180730 Granted publication date: 20130703 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Jiangxi Sino German bioengineering Limited by Share Ltd Registration number: 2018990000621 |
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