CN1944461A - Technology for preparing colorless malachite green complete antigen and monoclonal antibody - Google Patents
Technology for preparing colorless malachite green complete antigen and monoclonal antibody Download PDFInfo
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Abstract
The present invention discloses technology of preparing complete antigen of colorless malachite green and monoclonal antibody, and relates to fast detection of colorless malachite green in organism. Nitro colorless malachite green is first synthesized, reduced and purified to obtain amino colorless malachite green; the amino colorless malachite green is then cross-linked with rabbit albumin to form complete antigen of colorless malachite green; the complete antigen of colorless malachite green is used in immunizing the spleen cell of BALB/c mouse and fusing SP2/0 cell and through two-step process of positive cloning screen and several times of sub-cloning, monoclonal antibody of colorless malachite green with stable secretion is obtained. The complete antigen of colorless malachite green and the monoclonal antibody of colorless malachite green are used in fast detection of malachite green in aquatic product.
Description
Technical field
The invention belongs to biological technical field, relate to the fishery products detection technique, be specifically related to leucomalachite green rapid detection in vivo.
Background technology
(Leucomalachite green is that (malachite green, MG) main metabolic residue in vivo has carcinogenic danger to the mankind to malachite green LMG) to leucomalachite green.
It is green that malachite green is called aniline green, victoria green or China again, it is a kind of green crystal with metalluster, water-soluble, ethanol and methyl alcohol, the color of the aqueous solution is blue-greenish colour (maximum absorption wavelength is 618nm), there had the people to find that malachite green and other dyes can make some organize as far back as 1913 to be painted, can destroy pathogenic bacteria and not cause that the host damages, thereby make these dyestuffs be used as sanitas, trypanosomicide and other medical functions.From the appearance of sulfa drugs and other antibiotic, malachite green day by day fails as the application of antibiotic in livestock industry.Yet in aquaculture because cheap and antibiotic, anti-mycotic efficiency is good, be widely used in to carry out water body disinfection and prevent the fish and water mildew.
Discovered in recent years malachite green particularly its metabolite has in the aquatic products body and significantly accumulates residual phenomena, residence time is also longer, because its chemical functional group's tritane is a kind of carcinogenic substance, so abroad some developed countries have announced to forbid that as European Union, the U.S. it uses in economic fish (except the aquarium fish) breeding process." the pollution-free food China suede huge legendary turtle crab " of China Ministry of Agriculture issue (NY5064-2001) and in " veterinary drug and other compound inventory of food animal forbidding " file (agriculture and animal husbandry is sent out [2002] No. 1) of Ministry of Agriculture's issue clearly stipulates to forbid malachite green in all edible tissues.
" the residual Supervisory Surveillance Program of hazardous and noxious substances in the 2000 annual Chinese exports animal derived foods " lists the monitoring of malachite green project in the eel in annual plan first, and continues into the present.Test confirms that malachite green 50% changes leucomalachite green into after 8 hours in animal body, and 84% is transformed into leucomalachite green after 24 hours, and is trapped in the tissue for a long time.Detect MG both at home and abroad and mainly adopt with chromatographic technique (HPLC) detection technique, though sensitive, quantitatively accurate, speed is slow during mass detection, and cost is higher relatively.With competitiveness enzyme linked immunosorbent assay (ELISA) is that the rapid screening method of representative is the immunology mainstream technology, fast, cost is low, can mass detection to detect thing with protein be the product of target, and leucomalachite green does not have the group with albumen coupling, can not form complete antigen and be used to prepare specific antibody, can't use competitiveness enzyme linked immunosorbent assay (ELISA) batch detection, not have this technology report both at home and abroad.In order to set up rapid detection malachite green residual technology in the fishery products body, colorless malachite green complete antigen and monoclonal antibody successfully prepare becomes key.
Summary of the invention
Because leucomalachite green does not have the group with albumen coupling, thereby the present invention elder generation synthesizing nitryl leucomalachite green also reduces, purifying, obtains amino leucomalachite green.And become complete antigen with albumen coupling, immune balb/c mice, preparation monoclonal antibody.
1, the preparation process of colorless malachite green complete antigen comprises the steps:
A, be raw material with N, N xylidine, by 1: 3～4 molar weights than adding to nitro formaldehyde the synthesizing nitryl leucomalachite green;
B, nitro malachite green than the rare HCl that adds 10 times of amounts, 5～8 times metal-powder, are reduced to amino leucomalachite green by molar weight;
C, amino leucomalachite green drips of solution is added in the alkaline solution, separates out precipitation, throw out is the amino leucomalachite green of purifying;
D, be dissolved in the ratio of 6ml 0.3mol/L HCl,, under the ice bath agitation condition, slowly add 1%NaNO then the amino malachite green stirring and dissolving of purifying in the amino leucomalachite green of every 100mg purifying
2, make amino malachite green diazotization, get starch/liquor kalii iodide and drip in the white dried wave plate, drip an above-mentioned diazotization solution, mix and present black-and-bluely in back 30 seconds, be the diazotization of finishing amino malachite green;
E, above-mentioned diazotizing amino leucomalachite green continues reaction 20 minutes, take by weighing 4 times of rabbit albumin (RSA) to amino leucomalachite green weight, be dissolved in carbonate buffer solution and become 2% protein solution, under the ice bath agitation condition, slowly add diazotizing amino leucomalachite green solution, the limit edged is regulated pH to 7.5～8.0 with 0.01mol/L NaOH, obtain the albuminous conjugate of amino leucomalachite green and rabbit (NLMG-RSA), after putting 4 ℃ of refrigerator overnight, PBS with 0.1mol/L pH7.2 under 4 ℃ of conditions dialysed three days, change dialyzate every day three times, packing, after the freeze-drying ,-20 ℃ of preservations.
2, leucomalachite green MONOCLONAL ANTIBODIES SPECIFIC FOR process comprises the steps:
A, NLMG-RSA immune mouse;
B, immune mouse spleen cell and SP20 myeloma cell are with 2～5: 1 mixes, adding 50%PEG in 1 minute merges, the RPMI-1640 that adds 30～40ml within 90 seconds, left standstill 10 minutes, centrifugal, cell adds 96 well culture plate holes with the resuspended back of serum RPMI-1640 that contains that contains HAT and Turnover of Mouse Peritoneal Macrophages, puts CO
237 ℃ of cultivations in the incubator are changed liquid with 1640 complete culture solutions that contain HAT after 5 days and are cultivated, and change liquid with 1640 complete culture solutions that contain HT after 10 days and cultivate, and change 1640 complete culture solutions later on into and cultivate.
C, NLMG-OVA coated elisa plate, the hybridoma of screening positive clone;
The BALB/C mouse that the hybridoma of D, positive colony is transplanted the pristane processing prepares monoclonal antibody.
After described amino malachite green and the albuminous conjugate of rabbit (NLMG-RSA) are sampled, with its purity of UV spectrophotometer measuring, conjugate to amino leucomalachite green, carrier proteins and process dialysis scans respectively under the wavelength of 190～600nm, to formulate the coupling ratio of amino malachite green and carrier.
Above-mentioned concrete operations with the NLMG-RSA immune mouse are: selected for 3 monthly ages, BALB/c mouse about 30g, head exempts to be dissolved in 0.1mol/L PH7.4 PBS with NLMG-RSA, with isopyknic complete freund adjuvant mixing, and 150 μ gNLMG-RSA/ mouse, injection 0.1mL in the foot lift, after 2 weeks, add isopyknic incomplete freund adjuvant mixing, 250 μ gNLMG-RSA/ mouse subcutaneous injections with NLMG-RSA, after the amount intramuscular injection together of 2 weeks, the 4th tail vein injection.
What the hybridoma of above-mentioned steps C screening positive clone adopted is two step screening methods: the single clone of mark or two clones' (cell clone is separated from each other) cell cultures hole at first, wrap by NLMG-OVA to screen all index aperture; After 2 days, with doubtful positive colony (P/N＞2.1) with NLMG-OVA, OVA, carry out programmed screening, to detecting the cell in the positive hole of specific antibody, in time transferred species is also carried out cloning.
The concrete operations that above-mentioned steps D prepares monoclonal antibody are: the BALB/c mouse of getting for 7 weeks, with pristane 0.5ml/ only, the hybridoma that is in logarithmic phase that pneumoretroperitoneum inoculation in 7 days is diluted with serum free medium or physiological saline, every injected in mice cell count is 5 * 10
6Individual, after 4 days, observe the mouse ascites production every day at interval, treat that mouse web portion expands, spiritual variation, dying when motionless, gather ascites, centrifugal, get supernatant, mensuration is tired, packing, and-70 ℃ are frozen standby, add DEAE cellulose column desalting and purifying with ammonium sulfate precipitation method.
Description of drawings
Fig. 1 is nitro concealed malachite green thin layer and reduzate thin-layer chromatogram:
A is a nitro concealed malachite green thin-layer chromatogram among the figure: 1, paranitrobenzaldehyde; 2, synthetic product; 3, N, accelerine (synthesizing).B is the reduction thin-layer chromatogram of nitro concealed malachite green: 4, nitro concealed malachite green; 5, amino concealed malachite green (reduction).
Fig. 2 is a product infrared colour spectrogram:
A is a nitro concealed malachite green infrared colour spectrogram among the figure, nitro characteristic peak 1342 occurred; B is amino recessive malachite infrared colour spectrogram, amino hydrogen characteristic peak 3353.3 and 3433.1 occurred.
Fig. 3 is amino concealed malachite green, rabbit albumin and conjugate UV scanning figure:
A is amino concealed malachite green UV scanning figure among the figure; B is rabbit albumin UV scanning figure among the figure; C is amino concealed malachite green-rabbit albumin conjugate UV scanning figure among the figure.
Fig. 4 is that the standard of leucomalachite green suppresses curve, and the vertical seat table among the figure is inhibiting rate=OD logarithm that (adding LMG)/the horizontal seat table of OD (not adding LMG) is the LMG strength of solution.
Embodiment
Comparatively detailed technical operating procedure below is provided:
(1) the nitro leucomalachite green is synthetic: to nitro formaldehyde 10 grams, N, accelerine 30ml stirring heating reflux, and obtain nitro leucomalachite green 24.0 grams.
(2) nitro leucomalachite green reduction: nitro leucomalachite green 1 gram adding 20ml 2M HCl adds zinc powder 1 gram again and obtains amino leucomalachite green 0.80 gram.
(3) amino leucomalachite green purifying: amino leucomalachite green drips of solution is added on alkaline solution separates out amino malachite green precipitation.
(4) amino leucomalachite green diazotization: take by weighing the amino leucomalachite green of 50mg and be dissolved in 3ml 0.3mol/L HCl, constantly stirring and dissolving under the condition, slowly adds 1% NaNO in ice bath
2, the limit edged stirs.Detect NaNO simultaneously
2Whether react excessive.Detection method is: it is on glass in white dried to get a starch/liquor kalii iodide, gets a diazotization solution then, adds and mixes with it, and mixed solution becomes the black-and-blue diazotization that shows in 30 seconds.
(5) amino leucomalachite green and albumen coupling: above-mentioned solution continues reaction below 4 ℃ 20 minutes, take by weighing rabbit albumin 200mg and be dissolved in the 0.2M PH9.5 carbonate buffer solution of 10ml, in ice bath, under the condition, slowly add diazotization solution while stir.With 0.01mol/L NaOH adjust pH to 7.5～8.0, put then in 4 ℃ of refrigerators and react, spend the night.The coupling mixed solution is packed in the dialysis tubing, under 4 ℃, with 0.1mol/L PBS (pH7.2) dialysis 3 days, changes dialyzate every day three times.Sampling is with UV spectrophotometer measuring its purity, and with the packing of conjugate bottle, lyophilize, be stored in-20 ℃ standby.Conjugate to amino leucomalachite green, carrier proteins and process dialysis scans respectively under 190 to 600nm wavelength, calculates amino leucomalachite green and carrier protein couplet ratio.
(6) animal immune: selected for 3 monthly ages, 4 of the BALB/c mouse about 30g.Head exempts to be dissolved in 0.1mol/LpH7.4 PBS with NLMG-RSA, with complete freund adjuvant mixing, and 150 μ g NLMG-RSA/ mouse, injection 0.1mL in the sufficient lift.After 2 weeks, add incomplete freund adjuvant mixing with NLMG-RSA, 250 μ g NLMG-RSA/ mouse subcutaneous injections, after 2 weeks, the same intramuscular injection, the 4th time 300 μ g NLMG-RSA/ caudal vein injection.
The monoclonal antibody preparation process:
Asepticly get BALB/c mice spleen cell and SP20 myeloma cell, with 2～5: 1 mixes, adding 50% PEG in 1 minute merges, adding the 30ml RPMI-1640 in 90 seconds left standstill 10 minutes, centrifugal, centrifugal sediment add contain HAT and Turnover of Mouse Peritoneal Macrophages the serum RPMI-1640 arranged, cultivate in 96 holes, NLMG-OVA coated elisa plate screening positive clone, the BALB/c mouse of transplanting the pristane processing prepares antibody, evaluation.
Adopt two step screening methods: the single clone of mark or two clones' (cell clone is separated from each other) cell cultures hole at first, earlier with the NLMG-OVA bag by 96 hole enzyme plates to screen all index aperture; After 2 days, with doubtful positive colony (P/N＞2.1) with NLMG-OVA, OVA, carry out programmed screening.To detecting the cell in the positive hole of specific antibody, timely transferred species and carry out subclone.
(7) preparation of ascites and purifying
Get the BALB/c mouse in 7 weeks, only with pristane 0.5ml/.The hybridoma that is in logarithmic phase that pneumoretroperitoneum inoculation in 7 days is diluted with serum free medium or physiological saline.Every injected in mice cell count is 5 * 10
6Individual.After 4 days, observe the mouse ascites production every day at interval.Treat that mouse web portion expands, spiritual variation, dying when motionless, with No. 16 syringe needle aseptic collection ascites.Ascites is centrifugal, and supernatant liquor is the leucomalachite green monoclonal antibody, and ELISA measures and tires is 1: 20000, packing, and-70 ℃ are frozen standby.
Ammonium sulfate precipitation method adds the DEAE cellulose column with above-mentioned leucomalachite green monoclonal antibody desalting and purifying.
Be the reliability of checking effect of the present invention, carry out following evaluation:
1, chemical product is seen Fig. 2 through the infrared scan analysis:
In infrared scan figure, compare synthetics nitro leucomalachite green (Fig. 2 .A) and amino leucomalachite green (NLMG) (Fig. 2 .B) and leucomalachite green (LMG), find that nitro characteristic peak 1342 has appearred in the nitro leucomalachite green and amino hydrogen characteristic peak 3353.3 and 3433.1 appears in amino leucomalachite green (NLMG), other peak of three is consistent, illustrates that the synthetic material conforms to the target haptens.
2, conjugate is identified:
The conjugate of gained carries out the UV scanning analysis and sees Fig. 2, and A and B are respectively haptens and the carriers before the coupling, and C is a purified conjugate after the coupling, and the coupling characteristic peak drifts about, and the coupling success is described.
3, monoclonal antibody is at the evaluation of medicine determinant:
Part strong positive hole supernatant 1: 6 dilution with NLMG-OVA, OVA respectively coated elisa plate carry out ELISA and screen again, the OD value that one hole NLMG-OVA conjugate is wherein arranged is about 20 times of carrier, the antibody of generation that this clone is described is at the medicine determinant, and specificity is stronger.This clone's called after 5E9.
Table 1: with NLMG-OVA, OVA respectively coated elisa plate part strong positive clone secretory antibody is carried out the ELISA results of screening
4, the monoclonal antibody specificity is identified:
With the malachite green of serial dilution, colourless Victoria Green WPB, nitro malachite green, Viola crystallina, pararosaniline chloride, methyl orchid, paraxin, do indirect competitive ELISA: envelope antigen (NLMG-OVA), 100 μ l/ holes, 4 ℃ of effects are spent the night; Wash plate 3 times, add 200 μ l and contain the sealing of 1% gelatin, 37 ℃ of 2h wash plate 3 times; Add 50 μ l drug dilution liquid and 50 μ l monoclonal antibody diluents (1: 16000), 37 ℃ of 2h wash plate 3 times, add 1: the HRP mark goat anti-rabbit antibody of I000 dilution, and 37 ℃ of 2h add substrate reactions, and termination reaction behind the 15min detects OD490.Do to suppress curve respectively, calculate the half inhibiting rate, calculate the cross reacting rate (concentration of the other medicines the during cross reactivity=concentration of LMG when generations 50% suppresses/generation 50% inhibition) of medicine then, see Table 2 according to matched curve.The antibody that the 5E9 cell strain produces is except malachite green and its major metabolite leucomalachite green, to the basic no cross reaction of several structures medicine similar with purposes.It is the most obvious that synthetic former medicine NLMG suppresses effect, its IC
50Reach 6.4ng/ml; The IC of LMG and MG
50Be respectively 25.7 and 70.3ng/ml.As seen this antibody can carry out the detection of MG and metabolite LMG thereof to a great extent simultaneously.
Table 2: with 8 kinds of structures or act on the intercrossing of similar medicine
| Medicine | Cross reaction (%) |
| The blue chloramphenicol diethylstilbestrol of leucobase of malachite green malachite green paramagenta crystal | 100 36.56 ＜0.02 ＜0.06 ＜0.02 ＜0.02 ＜0.02 ＜0.02 |
5. the evaluation of 5E9 monoclonal antibody practicality is promptly used this antibody to explore and is set up detection LMG competitive ELISA, the line correlation calculation of parameter of going forward side by side
With NLMG-OVA 1ug/ml 100 μ l/ hole coated elisa plates, LMG is done 1 * 10
3To 1 * 10
-4As multiple proportions is 10 dilution, and concrete grammar is the same, calculates inhibiting rate and standard deviation, quantitative limit according to following formula.Result and correlation parameter see Table 3,
Table 3: competitive ELISA result and correlation parameter
| LMG concentration of standard solution (ug/ml) | 10 | 1 | 10 -1 | 10 -2 | 10 -3 | 10 -4 | 10 -5 | 0 |
| LMG concentration logarithm OD average (B) inhibiting rate (B/B 0) standard deviation (SD) variation coefficient (CV%) | 1 0.0825 0.0641 0.006 7.7139 | 0 0.216 0.1677 0 0 | -1 0.49 0.3804 0.0099 2.0203 | -2 0.7595 0.5897 0.0007 0.0931 | -3 0.917 0.712 0.0113 1.2338 | -4 0.9965 0.7737 0.0007 0.071 | -5 1.068 0.8292 0.0453 4.2373 | 1.288 1 0.0523 4.0626 |
Inhibiting rate=OD (adding LMG)/OD (not adding LMG)
Standard deviation (SD)=[∑ (X-X
i)
2/ (n-1)]
1/2
Detecting lower limit (LOD) is the value of (B-3SD) corresponding leucomalachite green concentration;
Quantitative limit (LOQ) is the value of (B-10SD) corresponding leucomalachite green concentration.
Can see that from Fig. 4 the logarithmic value of (B/B0) and LMG concentration is the One-place 2-th Order curved line relation in the 0.0016-1 μ g/ml scope, the regression curve equation is Y=-0.0139X
2-0.1928X+0.2282, relation conefficient is r=0.9848; Half-inhibition concentration is 25.7ng/ml; With the B0-3SD extrapotation as calculated sensitivity be 0.0023 μ g/ml.The scope of detection by quantitative LMG is: 0.955-10000ng/ml.Test-results shows that this 5E9 monoclonal anti physical efficiency is used for setting up detection LMG competitive ELISA.
Claims (6)
1, colorless malachite green complete antigen and Monoclonal Antibody technology is characterized in that the preparation process of described colorless malachite green complete antigen comprises the steps:
A, be raw material with N, N xylidine, by 1: 3～4 molar weights than adding to nitro formaldehyde the synthesizing nitryl leucomalachite green;
B, nitro malachite green than the rare HCl that adds about 10 times amounts, 5～8 times metal-powder, are reduced to amino leucomalachite green by molar weight;
C, amino leucomalachite green drips of solution is added in the alkaline solution, separates out precipitation, throw out is the amino leucomalachite green of purifying;
D, be dissolved in the ratio of 6ml 0.3mol/L HCl,, under the ice bath agitation condition, slowly add 1%NaNO then the amino malachite green stirring and dissolving of purifying in the amino malachite green of every 100mL purifying
2, make amino malachite green diazotization, get starch/liquor kalii iodide and drip in the white dried wave plate, drip an above-mentioned diazotization solution, mix and present black-and-bluely in back 30 seconds, be the diazotization of finishing amino malachite green;
E, above-mentioned diazotizing amino malachite green continues reaction 20 minutes, take by weighing 4 times of rabbit albumin to amino leucomalachite green weight, be dissolved in carbonate buffer solution and become 2% protein solution, under the ice bath agitation condition, slowly add diazotizing amino leucomalachite green solution, the limit edged is regulated pH to 7.5～8.0 with 0.01mol/L NaOH, obtain the albuminous conjugate of amino leucomalachite green and rabbit (NLMG-RSA), after putting 4 ℃ of refrigerator overnight, PBS with 0.1mol/L pH7.2 under 4 ℃ of conditions dialysed three days, change dialyzate every day three times, packing, after the freeze-drying ,-20 ℃ of preservations.
2, by described colorless malachite green complete antigen of claim 1 and Monoclonal Antibody technology, it is characterized in that described leucomalachite green MONOCLONAL ANTIBODIES SPECIFIC FOR process comprises the steps:
A, NLMG-RSA immune mouse;
B, immune mouse spleen cell and SP20 myeloma cell are with 2～5: 1 mixes, adding 50%PEG in 1 minute merges, the RPMI-1640 that adds 30～40ml within 90 seconds, left standstill 10 minutes, centrifugal, cell adds in the 96 well culture plate holes with the resuspended back of serum RPMI-1640 that contains that contains HAT and Turnover of Mouse Peritoneal Macrophages, puts CO
237 ℃ of cultivations in the incubator are changed liquid with 1640 complete culture solutions that contain HAT after 5 days and are cultivated, and change liquid with 1640 complete culture solutions that contain HT after 10 days and cultivate, and change 1640 complete culture solutions later on into and cultivate.
C, NLMG-OVA coated elisa plate, the hybridoma of screening positive clone;
The BALB/C mouse that the hybridoma of D, positive colony is transplanted the pristane processing prepares monoclonal antibody.
3, by described colorless malachite green complete antigen of claim 1 and Monoclonal Antibody technology, after it is characterized in that described amino leucomalachite green and the albuminous conjugate of rabbit (NLMG-RSA) are sampled, with its purity of UV spectrophotometer measuring, conjugate to amino leucomalachite green, carrier proteins and process dialysis scans respectively under the wavelength of 190～600nm, to formulate the coupling ratio of amino malachite green and carrier.
4, by described colorless malachite green complete antigen of claim 2 and Monoclonal Antibody technology, it is characterized in that described concrete operations with the NLMG-RSA immune mouse are: selected for 3 monthly ages, BALB/c mouse about 30g, head exempts to be dissolved in 0.1mol/L pH7.4PBS with NLMG-RSA, with isopyknic complete freund adjuvant mixing, 150 μ gNLMG-RSA/ mouse, injection 0.1mL in the foot lift, after 2 weeks, add isopyknic incomplete freund adjuvant mixing with NLMG-RSA, 250 μ gNLMG-RSA/ mouse subcutaneous injections are after the amount intramuscular injection together of 2 weeks, the 4th tail vein injection.
5, by described colorless malachite green complete antigen of claim 2 and Monoclonal Antibody technology, what it is characterized in that the hybridoma of described step C screening positive clone adopts is two step screening methods: the single clone of mark or two clones' (cell clone is separated from each other) cell cultures hole at first, bag by NLMG-OVA to screen all index aperture, after 2 days, with the doubtful positive colony of P/N＞2.1 with NLMG-OVA, OVA, carry out programmed screening, to detecting the cell in the positive hole of specific antibody, timely transferred species is also carried out cloning.
6, by described colorless malachite green complete antigen of claim 2 and Monoclonal Antibody technology, it is characterized in that the concrete operations that described step D prepares monoclonal antibody are: the BALB/c mouse of getting for 7 weeks, with pristane 0.5ml/ only, the hybridoma that is in logarithmic phase that pneumoretroperitoneum inoculation in 7 days is diluted with serum free medium or physiological saline, every injected in mice cell count is 5 * 10
6Individual, after 4 days, observe the mouse ascites production every day at interval, treat that mouse web portion expands, spiritual variation, dying when motionless, gather ascites, ascites is centrifugal, get supernatant, mensuration is tired, packing, and-70 ℃ are frozen standby, add DEAE cellulose column desalting and purifying with ammonium sulfate precipitation method.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101308140B (en) * | 2008-06-30 | 2012-08-08 | 江苏省苏微微生物研究有限公司 | Concealed malachite green gold mark detection test paper box and method for making same |
| CN103304662A (en) * | 2013-04-27 | 2013-09-18 | 上海海洋大学 | Monoclonal antibody of leucomalachite green and preparation method and usage thereof |
| CN104569372A (en) * | 2014-12-31 | 2015-04-29 | 中国农业科学院农业质量标准与检测技术研究所 | Preparation method of recessive malachite green hapten |
| CN105277692A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay method for determination of malachite green in aquatic products |
| CN111662879A (en) * | 2017-01-22 | 2020-09-15 | 杭州市农业科学研究院 | Preparation method of hybridoma cell strain secreting anti-malachite green monoclonal antibody |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101948531A (en) * | 2010-08-23 | 2011-01-19 | 上海海洋大学 | Complete antigen for detecting malachite green and preparation method thereof |
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- 2005-10-08 CN CNB2005100609947A patent/CN100488983C/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101308140B (en) * | 2008-06-30 | 2012-08-08 | 江苏省苏微微生物研究有限公司 | Concealed malachite green gold mark detection test paper box and method for making same |
| CN103304662A (en) * | 2013-04-27 | 2013-09-18 | 上海海洋大学 | Monoclonal antibody of leucomalachite green and preparation method and usage thereof |
| CN105277692A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay method for determination of malachite green in aquatic products |
| CN104569372A (en) * | 2014-12-31 | 2015-04-29 | 中国农业科学院农业质量标准与检测技术研究所 | Preparation method of recessive malachite green hapten |
| CN111662879A (en) * | 2017-01-22 | 2020-09-15 | 杭州市农业科学研究院 | Preparation method of hybridoma cell strain secreting anti-malachite green monoclonal antibody |
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