CN101413956A - Colloidal gold detection card for rapidly detecting melamine and preparing method thereof - Google Patents
Colloidal gold detection card for rapidly detecting melamine and preparing method thereof Download PDFInfo
- Publication number
- CN101413956A CN101413956A CNA2008102363748A CN200810236374A CN101413956A CN 101413956 A CN101413956 A CN 101413956A CN A2008102363748 A CNA2008102363748 A CN A2008102363748A CN 200810236374 A CN200810236374 A CN 200810236374A CN 101413956 A CN101413956 A CN 101413956A
- Authority
- CN
- China
- Prior art keywords
- melamine
- gold
- solution
- preparation
- pad
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a colloidal gold cassette for rapid detection of melamine, the colloidal gold cassette has rapid detection, convenient carry and simple operation, the invention also provides a preparation method of the cassette, the preparation process thereof is simple, and the cost is low. The colloidal gold cassette comprises a test strip, the test strip is packaged in a shell body of the cassette, a sample adding hole and an observation hole are arranged on the shell body of the cassette, the test strip comprises a bottom plate, the colloidal gold cassette is characterized in that water absorbent paper, a nitrocellulose membrane, a gold-labeled pad and a sample pad are sequentially arranged on the bottom plate of the test strip, the water absorbent paper and the gold-labeled pad are respectively arranged at the two ends of the nitrocellulose membrane, small sections of the water absorbent paper and the gold-labeled pad are respectively overlapped on the nitrocellulose membrane at the junctions at the two ends of the nitrocellulose membrane, the sample pad is arranged at the other end of the gold-labeled pad, a small section of the sample pad is overlapped on the gold-labeled pad; a gold-labeled anti-melamine monoclonal antibody is sprayed on the gold-labeled pad; a detection line with the material of a melamine-ovalbumin coupled conjugate and a quality control line with the material of goat anti-mouse IgG are sequentially sprayed on the nitrocellulose membrane.
Description
(1) technical field
The present invention relates to the rapid detection technical field of the content of melamine in feed and the food, be specially collaurum test card of a kind of fast detecting melamine and preparation method thereof.
(2) background technology
Melamine is a kind of triazines nitrogen heterocyclic ring organic compound, and important azacyclo-Organic Chemicals is called for short triamine, is melamine, melamine, cyanogen urea triamide again.Melamine is a kind of broad-spectrum basic organic chemical industry's intermediate product, and topmost purposes is as the raw material of producing melamine formaldehyde resin (MF).Owing to contain a large amount of nitrogen elements in the melamine molecule, often artificially added in precession thing feed and the vegetable protein to improve protein content in the product, and for a long time or repeatedly a large amount of edible feed or foods that contain melamine, can cause the infringement of the reproduction of animal or human's class, urinary system, bladder, kidney portion calculus, and can further bring out carcinoma of urinary bladder.Therefore, be necessary the content of the melamine in feed and the food is detected, adopt the more high performance liquid chromatography that has at present, this method has good sensitivity and specificity, but complex operation, length consuming time, need not be suitable for detection, and need expensive instrument and equipment, detect the cost height batch samples; Also can adopt enzyme linked immunosorbent assay to detect,, not need expensive instrument and equipment though the method operation is simple relatively, the detection cost is low, but in testing process, need to repeat repeatedly to wash, and will measure, can not realize real single stage method fast detecting by means of the microplate reader reading.
(3) summary of the invention
At the problems referred to above, the invention provides a kind of collaurum test card of fast detecting melamine, can satisfy the needs of field quick detection, detect fast, easy to carry, simple to operate, need not specific installation and instrument, the present inventor also provides the preparation method of the collaurum test card of fast detecting melamine for this reason, and its preparation technology is simple, and cost is low.
A kind of collaurum test card of fast detecting melamine, it comprises test strips, described test strips is packaged in the test card housing, have well and observation port on the described test card housing, described test strips comprises base plate, it is characterized in that: be pasted with thieving paper on the base plate of described test strips successively, nitrocellulose filter, gold mark pad, sample pad, described thieving paper and described gold mark pad stick on described nitrocellulose filter two ends respectively, junction, two ends at described nitrocellulose filter, the a bit of pressed and overlapped of described thieving paper and gold mark pad is on described nitrocellulose filter, described sample pad sticks on the other end of described gold mark pad, and a bit of of described sample pad overlaps on the described gold mark pad; Be coated with gold mark anti-melamine monoclonal antibody on the described gold mark pad; Being coated with material on the described nitrocellulose filter successively is that the melamine-detection line of ovalbumin coupling bond, material are the nature controlling line of sheep anti-mouse igg.
It is further characterized in that: described sample pad position is over against described well, and described cellulose nitrate film location is over against described observation port.
A kind of preparation method of colloidal gold strip of fast detecting melamine is characterized in that: it may further comprise the steps:
(1), forms conjugate melamine-ovalbumin as artificial antigen with carbodlimide method coupling melamine hapten and ovalbumin;
(2) merge with the mouse boosting cell of immunogen immune and murine myeloma cell hybridization, the preparation hybridoma, the cell line that screening can stably excreting anti-melamine monoclonal antibody prepares the anti-melamine monoclonal antibody;
(3) preparation colloidal gold solution;
(4) with the colloidal gold solution mark anti-melamine monoclonal antibody for preparing;
(5) with the envelope antigen and the sheep anti-mouse igg treatment of nitric acid cellulose membrane that prepare;
(6) handle gold mark pad with the gold mark anti-melamine monoclonal antibody for preparing;
(7) assembling gold test strip bar.
It is further characterized in that:
The preparation of described melamine-ovalbumin: take by weighing melamine, be dissolved in the pyridine solution, add 10%～20% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction is 1 hour～3 hours under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction dropped to this reactant liquor in ovalbumin (OVA) solution after 20 minutes, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 24 hours～72 hours, after the freeze drying in-20 ℃～-40 ℃ preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: mg μ l:(6-600 sodium cyanoborohydride=(10～1000) mg:(50～8000)), haptens: EDC:OVA:NHS=(10～2000) mg:(5～10000) mg:(10-2000) mg:(20～5000) mg;
The preparation of described melamine-bovine serum albumin(BSA) coupled antigen: take by weighing melamine, be dissolved in the pyridine solution, add 10%～20% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction is 2 hours under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction is after 20 minutes, this reactant liquor is dropped to bovine serum albumin(BSA) (BSA, be dissolved in the ultrapure water) in the solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 24 hours～72 hours, after the freeze drying in-20 ℃～-40 ℃ preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: μ l:(6～600 sodium cyanoborohydride=(10～1000) mg:(50～8000)) mg, haptens: EDC:BSA:NHS=(10～2000) mg:(5～10000) mg:(10～2000) mg:(20～5000) mg;
The preparation of described anti-melamine monoclonal antibody and solution thereof: get melamine-bovine serum albumin(BSA) (Melamine-BSA) conjugate and be made into 0.1 μ g/ μ l～10 μ g/ μ l antigenic solutions with physiological saline, with the complete freund adjuvant mixing of equal-volume, fully emulsified, use for first immunisation, replace complete freund adjuvant with incomplete freund adjuvant during booster immunization with amount, first immunisation adopts directly injection in the mouse peritoneal, immunizing dose is 10 μ g/～100 a μ g/ mouse, later on once every all booster immunizations in 2 week～4, booster immunization adopts tail vein injection, immunizing dose 10 μ g/～100 μ g/ only, intrasplenic injection is adopted in last immunity, getting spleen after 4 days merges, the immune mouse spleen cell that separates is mixed with the 10:1 ratio with the myeloma cell SP-2/o that is in exponential phase, centrifugal, remove supernatant, in 50S～90S, 0.1ml～10ml 50% polyglycol (molecular weight is 1500) is added in the cell, abundant mixing, make its fusion, add 10ml～50ml DMEM nutrient solution after 1 minute～3 minutes, stop merging, water-bath is centrifugal after static 10 minutes～30 minutes, removes supernatant; After fused cell suspended with the HAT selective medium that contains 5%～30% calf serum, be that 1 * 104～1 * 106 feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 5%～10%CO with final concentration
2Cultivate under 35 ℃～45 ℃ conditions, after 5 days～10 days, every culture hole is changed the 2/3HT nutrient solution, after 10 days～20 days, begin microscopy is had the culture hole of hybridoma clonal growth, getting supernatant screens, the cell that testing result is positive is cloned with limiting dilution assay immediately, the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, with melamine-ovalbumin (Melamine-OVA) is envelope antigen, with the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A test-A blank)/(A contrast-A blank)〉2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope, dilution are 70/ml, get 20 times of 1ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.3ml/ of 10～13 all age～0.5ml/, after 8 days～10 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 105 cells/only, note after 5 days observing are collected ascites, centrifugal removal precipitation, glycerol adding is in-20 ℃～-40 ℃ preservations; The ratio of mentioned component is: Melamine-BSA: ml physiological saline=(10～1000) μ g:(0.1～10).
The preparation of described colloidal gold solution: with new system distilled water preparation 0.01%～0.1% chlorauric acid solution, place conical flask, be heated to 100 ℃～120 ℃ with the constant temperature magnetic stirrer, under 100r/min～200r/min magnetic agitation, add rapidly in advance 1%～3% citric acid three sodium solution 35 ℃～45 ℃ of insulations, keep temperature and rotating speed constant, continue agitating heating about 15 minutes～30 minutes, present limpid claret until solution; The room temperature cooling, 2 ℃～8 ℃ refrigerators are preserved standby; The ratio of mentioned component is: gold chloride: ml trisodium citrate=(10～1000) ml:(1～20).
Described colloid gold label anti-melamine MONOCLONAL ANTIBODIES SPECIFIC FOR: get colloidal gold solution in centrifuge tube, with 0.1M～1.0M K
2CO
3Or about 0.1M～1.0M HCl adjusting pH to 7～8, encase centrifuge tube with aluminium-foil paper, gently vibration; Dropwise add antibody protein solution while vibrating, continue vibration about 10 minutes～30 minutes; Dropwise add 1%BSA solution with saturated free collaurum, then in 2 ℃～8 ℃, 6000rpm～12000rpm, centrifugal 30 minutes～60 minutes; Centrifuge tube is taken out in centrifugal back, supernatant is sopped up a part gently after, will manage the end with liquid-transfering gun and precipitate and carefully be drawn in the centrifuge tube, 2 ℃～8 ℃ preservations are standby; The ratio of mentioned component is: colloidal gold solution: antibody protein: BSA=(10～1000) ml:(50～5000) μ g:(1～100) ml.
The processing of described nitrocellulose filter: spray two parallel lines on nitrocellulose filter with gold mark point sample instrument, melamine coupling ovalbumin bond melamine-ovalbumin is as detection line, sheep anti-mouse igg is as nature controlling line, puts after 35 ℃～45 ℃ vacuum drying standby;
The processing of described gold mark pad: a certain amount of gold mark anti-melamine monoclonal antibody evenly is sprayed on the gold mark pad, puts after 35 ℃～45 ℃ vacuum drying standby;
The assembling of described gold test strip bar: get the base plate of making the test strips special use, the nitrocellulose filter that drying is good sticks on the relevant position earlier, again thieving paper and dry good gold mark pad are sticked on the relevant position and pushes down nitrocellulose filter a little, at last sample pad is glued and pushes down gold mark pad, cutting into the wide test strips of 3mm with cutting cutter packs in the test card, at this moment, the observation port of the position of sample pad over against the well of test card, cellulose nitrate film location over against test card, pack in the aluminium foil bag with drying agent, pack.
The colloidal gold strip of fast detecting melamine of the present invention, it detects fast, only need 5min detection time, can satisfy the needs of field quick detection, and detect accuracy rate height, high specificity, it has changed the limitation that must just can be detected by the professional of professional institution melamine detection, and is easy to carry, easy and simple to handle, each milk cattle cultivating field, milk purchasing station, inspection and quarantine department at different levels and consumer all can test immediately; The preparation technology of test strip is simple, and cost is low, and can preserve at normal temperatures, need not specific installation and instrument, detects good reproducibility.
(4) description of drawings
Fig. 1 is the collaurum test card synoptic diagram of fast detecting melamine of the present invention;
Fig. 2 is the structural representation of test strips described in the collaurum test card of fast detecting melamine of the present invention;
Fig. 3 is the left view of Fig. 2.
(5) embodiment
See Fig. 1, Fig. 2, a kind of collaurum test card of fast detecting melamine, it comprises test card housing 1 and test strips 2, test strips 2 is packaged in the test card housing 1, have well 3 and observation port 4 on the test card housing 1, test strips 2 comprises base plate 5, be pasted with thieving paper 6 successively on the base plate 5 of test strips 2, nitrocellulose filter 7, gold mark pad 8, sample pad 9, thieving paper 6 and gold mark pad 8 stick on nitrocellulose filter 7 both sides respectively, junction, two ends at nitrocellulose filter 7, the a bit of pressed and overlapped of thieving paper 6 and gold mark pad 8 is on nitrocellulose filter 7, and sample pad 9 sticks on the opposite side of gold mark pad 8, and a bit of of sample pad 9 overlaps on the gold mark pad 8; Be coated with gold mark anti-melamine monoclonal antibody on the gold mark pad 8; Being coated with material on the nitrocellulose filter 7 successively is the detection line 10 of melamine coupling ovalbumin bond, the nature controlling line 11 that material is sheep anti-mouse igg.
A kind of preparation method of collaurum test card of fast detecting melamine, it may further comprise the steps:
(1), forms conjugate melamine-ovalbumin as artificial antigen with carbodlimide method coupling melamine hapten and ovalbumin;
(2) merge with the mouse boosting cell of immunogen immune and murine myeloma cell hybridization, the preparation hybridoma, the cell line that screening can stably excreting anti-melamine monoclonal antibody prepares the anti-melamine monoclonal antibody;
(3) preparation colloidal gold solution;
(4) with the colloidal gold solution mark anti-melamine monoclonal antibody for preparing;
(5) with the envelope antigen and the sheep anti-mouse igg treatment of nitric acid cellulose membrane that prepare;
(6) handle gold mark pad with the gold mark anti-melamine monoclonal antibody for preparing;
(7) assembling gold test strip bar.
The preparation method of test card is described below in conjunction with embodiment
The preparation of melamine-ovalbumin: take by weighing melamine, be dissolved in the pyridine solution, add 10% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction is 1 hour under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction dropped to this reactant liquor in ovalbumin (OVA) solution after 20 minutes, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 72 hours, after the freeze drying in-40 ℃ of preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: sodium cyanoborohydride=1000mg:4000 μ l:6mg, haptens: EDC:OVA:NHS=2000mg:5000mg:10mg:2500mg.
The preparation of melamine-bovine serum albumin(BSA) coupled antigen: take by weighing melamine, be dissolved in the pyridine solution, add 15% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction 2h under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction is after 20 minutes, this reactant liquor is dropped to bovine serum albumin(BSA) (BSA, be dissolved in the ultrapure water) in the solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 48 hours, after the freeze drying in-40 ℃ of preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: sodium cyanoborohydride=1000mg:8000 μ l:600mg, haptens: EDC:BSA:NHS=1000mg:5mg:10mg:2500mg.
The preparation of anti-melamine monoclonal antibody and solution thereof: get melamine-bovine serum albumin(BSA) (Melamine-BSA) conjugate and be made into 5.05 μ g/ μ l antigenic solutions with physiological saline, with the complete freund adjuvant mixing of equal-volume, fully emulsified, use for first immunisation, replace complete freund adjuvant with incomplete freund adjuvant during booster immunization with amount, first immunisation adopts directly injection in the mouse peritoneal, immunizing dose is 55 a μ g/ mouse, later on every 4 all booster immunizations once, booster immunization adopts tail vein injection, and immunizing dose 100 μ g/ only, intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, the immune mouse spleen cell that separates is mixed with the 10:1 ratio with the myeloma cell SP-2/o that is in exponential phase, centrifugal, remove supernatant, in 50S 5.05ml50% polyglycol (molecular weight is 1500) is added in the cell, fully mixing makes its fusion, add 10ml DMEM nutrient solution after 3 minutes, stop merging, water-bath is centrifugal after static 20 minutes, removes supernatant; After fused cell suspended with the HAT selective medium that contains 30% calf serum, be that 1 * 104 feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 10%CO with final concentration
2Cultivate under 40 ℃ of conditions, after 5 days, every culture hole is changed the 2/3HT nutrient solution, after 20 days, begin microscopy is had the culture hole of hybridoma clonal growth, getting supernatant screens, the cell that testing result is positive is cloned with limiting dilution assay immediately, the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, with melamine-ovalbumin (Melamine-OVA) is envelope antigen, with the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A test-A blank)/(A contrast-A blank)〉2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope, dilution are 70/ml, get 20 times of 1ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.4ml/ of 10 all age, after 8 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only, note after 5 days observing is collected ascites, centrifugal removal precipitation, and glycerol adding is in-40 ℃ of preservations; The ratio of mentioned component is: Melamine-BSA: physiological saline=1000 μ g:5.05ml.
The preparation of colloidal gold solution: prepare 0.1% chlorauric acid solution with the new system distilled water, place conical flask, be heated to the constant temperature magnetic stirrer and boil 120 ℃, under the 150r/min magnetic agitation, add rapidly in advance 3% citric acid three sodium solution 35 ℃ of insulations, keep temperature and rotating speed constant, continued agitating heating 22.5 minutes, present limpid claret until solution; The room temperature cooling, 8 ℃ of refrigerators are preserved standby; The ratio of mentioned component is: gold chloride: trisodium citrate=1000ml:10.5ml.
Colloid gold label anti-melamine MONOCLONAL ANTIBODIES SPECIFIC FOR: get colloidal gold solution in centrifuge tube, use 0.1M K
2CO
3Or 1.0M HCl adjusting pH to 7, encase centrifuge tube with aluminium-foil paper, gently vibration; Dropwise add antibody protein solution while vibrating, continue vibration about 20 minutes; Dropwise add 1%BSA solution with saturated free collaurum, then in 5 ℃, 12000rpm, centrifugal 45 minutes; Centrifuge tube is taken out in centrifugal back, supernatant is sopped up a part gently after, will manage the end with liquid-transfering gun and precipitate and carefully be drawn in the centrifuge tube, 8 ℃ of preservations are standby; The ratio of mentioned component is: colloidal gold solution: antibody protein: BSA=1000ml:2500 μ g:50.5ml;
The processing of described nitrocellulose filter: spray two parallel lines on nitrocellulose filter with gold mark point sample instrument, melamine coupling ovalbumin bond melamine-ovalbumin is as detection line, sheep anti-mouse igg is as nature controlling line, puts after 45 ℃ of vacuum drying standby;
The processing of described gold mark pad: a certain amount of gold mark anti-melamine monoclonal antibody evenly is sprayed on the gold mark pad, puts after 35 ℃ of vacuum drying standby.
Embodiment 2
The preparation of described melamine-ovalbumin: take by weighing melamine, be dissolved in the pyridine solution, add 20% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction 3h under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction dropped to this reactant liquor in ovalbumin (OVA) solution after 20 minutes, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 48 hours, after the freeze drying in-30 ℃ of preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: sodium cyanoborohydride=10mg:8000 μ l:300mg, haptens: EDC:OVA:NHS=10mg:10000mg:1000mg:5000mg.
The preparation of described melamine-bovine serum albumin(BSA) coupled antigen: take by weighing melamine, be dissolved in the pyridine solution, add 10% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction 2h under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction is after 20 minutes, this reactant liquor is dropped to bovine serum albumin(BSA) (BSA, be dissolved in the ultrapure water) in the solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 24 hours, after the freeze drying in-20 ℃ of preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: sodium cyanoborohydride=10mg:4000 μ l:6mg, haptens: EDC:BSA:NHS=2000mg:10000mg:1000mg:5000mg.
The preparation of described anti-melamine monoclonal antibody and solution thereof: get melamine-bovine serum albumin(BSA) (Melamine-BSA) conjugate and be made into 0.1 μ g/ μ l antigenic solution with physiological saline, with the complete freund adjuvant mixing of equal-volume, fully emulsified, use for first immunisation, replace complete freund adjuvant with incomplete freund adjuvant during booster immunization with amount, first immunisation adopts directly injection in the mouse peritoneal, immunizing dose is 10 a μ g/ mouse, later on every 2 all booster immunizations once, booster immunization adopts tail vein injection, and immunizing dose 10 μ g/ only, intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, the immune mouse spleen cell that separates is mixed with the 10:1 ratio with the myeloma cell SP-2/o that is in exponential phase, centrifugal, remove supernatant, in 90S 0.1ml50% polyglycol (molecular weight is 1500) is added in the cell, fully mixing makes its fusion, add 50ml DMEM nutrient solution after 1 minute, stop merging, water-bath is centrifugal after static 10 minutes, removes supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 5% calf serum, be 1 * 10 with final concentration
6Feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 5%CO
2Cultivate under 35 ℃ of conditions, after 7 days, every culture hole is changed the 2/3HT nutrient solution, after 10 days, begin microscopy is had the culture hole of hybridoma clonal growth, getting supernatant screens, the cell that testing result is positive is cloned with limiting dilution assay immediately, the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, with melamine-ovalbumin (Melamine-OVA) is envelope antigen, with the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A test-A blank)/(A contrast-A blank)〉2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope, dilution are 70/ml, get 20 times of 1ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.3ml/ of 13 all age, after 10 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only, note after 5 days observing is collected ascites, centrifugal removal precipitation, and glycerol adding is in-20 ℃ of preservations; The ratio of mentioned component is: Melamine-BSA: physiological saline=10 μ g:5.05ml.
The preparation of described colloidal gold solution: prepare 0.01% chlorauric acid solution with the new system distilled water, place conical flask, be heated to the constant temperature magnetic stirrer and boil 100 ℃, under the 100r/min magnetic agitation, add rapidly in advance 1% citric acid three sodium solution 45 ℃ of insulations, keep temperature and rotating speed constant, continue agitating heating about 15 minutes, present limpid claret until solution; The room temperature cooling, 5 ℃ of refrigerators are preserved standby; The ratio of mentioned component is: gold chloride: trisodium citrate=10ml:20ml.
Described colloid gold label anti-melamine MONOCLONAL ANTIBODIES SPECIFIC FOR: get colloidal gold solution in centrifuge tube, use 1.0M K
2CO
3Or about 0.1M HCl adjusting pH to 7.5, encase centrifuge tube with aluminium-foil paper, gently vibration; Dropwise add antibody protein solution while vibrating, continue vibration about 10 minutes; Dropwise add 1%BSA solution with saturated free collaurum, then in 8 ℃, 6000rpm, centrifugal 60 minutes; Centrifuge tube is taken out in centrifugal back, supernatant is sopped up a part gently after, will manage the end with liquid-transfering gun and precipitate and carefully be drawn in the centrifuge tube, 5 ℃ of preservations are standby; The ratio of mentioned component is: colloidal gold solution: antibody protein: BSA=10ml:5000 μ g:100ml.
The processing of described nitrocellulose filter: spray two parallel lines on nitrocellulose filter with gold mark point sample instrument, melamine coupling ovalbumin bond melamine-ovalbumin is as detection line, sheep anti-mouse igg is as nature controlling line, puts after 40 ℃ of vacuum drying standby;
The processing of described gold mark pad: a certain amount of gold mark anti-melamine monoclonal antibody evenly is sprayed on the gold mark pad, puts after 45 ℃ of vacuum drying standby.
The preparation of melamine-ovalbumin: take by weighing melamine, be dissolved in the pyridine solution, add 15% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction 2h under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction dropped to this reactant liquor in ovalbumin (OVA) solution after 20 minutes, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 24 hours, after the freeze drying in-20 ℃ of preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: sodium cyanoborohydride=505mg:50 μ l:600mg, haptens: EDC:OVA:NHS=1005mg:5mg:2000mg:20mg.
The preparation of melamine-bovine serum albumin(BSA) coupled antigen: take by weighing melamine, be dissolved in the pyridine solution, add 20% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction 2h under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction is after 20 minutes, this reactant liquor is dropped to bovine serum albumin(BSA) (BSA, be dissolved in the ultrapure water) in the solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 72 hours, after the freeze drying in-30 ℃ of preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: sodium cyanoborohydride=505mg:50 μ l:303mg, haptens: EDC:BSA:NHS=10mg:5000mg:2000mg:20mg.
The preparation of anti-melamine monoclonal antibody and solution thereof: get melamine-bovine serum albumin(BSA) (Melamine-BSA) conjugate and be made into 10 μ g/ μ l antigenic solutions with physiological saline, with the complete freund adjuvant mixing of equal-volume, fully emulsified, use for first immunisation, replace complete freund adjuvant with incomplete freund adjuvant during booster immunization with amount, first immunisation adopts directly injection in the mouse peritoneal, immunizing dose is 100 a μ g/ mouse, later on every 3 all booster immunizations once, booster immunization adopts tail vein injection, and immunizing dose 55 μ g/ only, intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, the immune mouse spleen cell that separates is mixed with the 10:1 ratio with the myeloma cell SP-2/o that is in exponential phase, centrifugal, remove supernatant, in 70S 10ml 50% polyglycol (molecular weight is 1500) is added in the cell, fully mixing makes its fusion, add 30ml DMEM nutrient solution after 2 minutes, stop merging, water-bath is centrifugal after static 30 minutes, removes supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 17.5% calf serum, be 1 * 10 with final concentration
5Feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 7.5%CO
2Cultivate under 45 ℃ of conditions, after 10 days, every culture hole is changed the 2/3HT nutrient solution, after 15 days, begin microscopy is had the culture hole of hybridoma clonal growth, getting supernatant screens, the cell that testing result is positive is cloned with limiting dilution assay immediately, the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, with melamine-ovalbumin (Melamine-OVA) is envelope antigen, with the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A test-A blank)/(A contrast-A blank)〉2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope, dilution are 70/ml, get 20 times of 1ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.5ml/ of 11 all age, after 9 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only, note after 5 days observing is collected ascites, centrifugal removal precipitation, and glycerol adding is in-30 ℃ of preservations; The ratio of mentioned component is: Melamine-BSA: physiological saline=505 μ g:10ml.
The preparation of colloidal gold solution: prepare 0.055% chlorauric acid solution with the new system distilled water, place conical flask, be heated to 110 ℃ with the constant temperature magnetic stirrer, under the 200r/min magnetic agitation, add rapidly in advance 2% citric acid three sodium solution 40 ℃ of insulations, keep temperature and rotating speed constant, continue agitating heating about 30 minutes, present limpid claret until solution; The room temperature cooling, 2 ℃ of refrigerators are preserved standby; The ratio of mentioned component is: gold chloride: trisodium citrate=505ml:1ml.
Colloid gold label anti-melamine MONOCLONAL ANTIBODIES SPECIFIC FOR: get colloidal gold solution in centrifuge tube, use 0.55M K
2CO
3Or 0.55M HCl adjusting pH to 8, encase centrifuge tube with aluminium-foil paper, gently vibration; Dropwise add antibody protein solution while vibrating, continue vibration about 30 minutes; Dropwise add 1%BSA solution with saturated free collaurum, then in 2 ℃, 9000rpm, centrifugal 30 minutes; Centrifuge tube is taken out in centrifugal back, supernatant is sopped up a part gently after, will manage the end with liquid-transfering gun and precipitate and carefully be drawn in the centrifuge tube, 2 ℃ of preservations are standby; The ratio of mentioned component is: colloidal gold solution: antibody protein: BSA=505ml:50 μ g:1ml.
The processing of nitrocellulose filter: on nitrocellulose filter, melamine coupling ovalbumin bond melamine-ovalbumin is as detection line with two parallel lines of gold mark point sample instrument spraying, and sheep anti-mouse igg is as nature controlling line, puts after 35 ℃ of vacuum drying standby;
The processing of gold mark pad: a certain amount of gold mark anti-melamine monoclonal antibody evenly is sprayed on the gold mark pad, puts after 40 ℃ of vacuum drying standby;
The assembling of described gold test strip bar 2: get the base plate of making the test strips special use, the nitrocellulose filter 7 that drying is good sticks on the relevant position earlier, again thieving paper 6 and dry good gold mark pad 8 are sticked on the relevant position and pushes down nitrocellulose filter 7 a little, at last sample pad is glued and pushes down gold mark pad 8, cutting into the wide test strips of 3mm with cutting cutter packs in the test card, at this moment, the observation port 4 of the position of sample pad 9 over against the well 3 of test card, nitrocellulose filter 7 positions over against test card, pack in the aluminium foil bag with drying agent, pack.
When sample is detected, test card is kept flat, get 100 μ L samples and splash into well 3, observations in 30 minutes night; Sheep anti-mouse igg and melamine-ovalbumin conjugate is sprayed on the nature controlling line 11 (C) and the detection line 10 (T) of nitrocellulose filter respectively, the side sample for the treatment of that contains melamine moves to the other end according to chromatographic theory through sample pad 9, and successively through nature controlling line 11 and detection line 10, be solidificated in golden mark anti-melamine monoclonal antibody specific on the gold mark pad 8 and the melamine in the sample and play specific reaction, and competition suppresses it and combines with melamine on the detection line, therefore, when the content of melamine in the sample is lower than a certain amount, gold mark anti-melamine monoclonal antibody specific combines back gold grain aggegation with melamine on the detection line 10 and develops the color, when the content of melamine in the sample surpasses when a certain amount of, the gold labelled antibody is suppressed fully and does not develop the color, wherein, nature controlling line 11 (C) is set for judging that whether effective the method for inspection itself is, colour developing is effective, and the illustration method itself that do not develop the color is invalid.
Claims (10)
1, a kind of collaurum test card of fast detecting melamine, it comprises test strips, described test strips is packaged in the test card housing, have well and observation port on the described test card housing, described test strips comprises base plate, it is characterized in that: be disposed with thieving paper on the base plate of described test strips, nitrocellulose filter, gold mark pad, sample pad, described thieving paper and described gold mark pad are separately positioned on described nitrocellulose filter two ends, junction, two ends at described nitrocellulose filter, the a bit of pressed and overlapped of described thieving paper and gold mark pad is on described nitrocellulose filter, described sample pad is arranged on the other end of described gold mark pad, and a bit of of described sample pad overlaps on the described gold mark pad; Be coated with gold mark anti-melamine monoclonal antibody on the described gold mark pad; Being coated with material on the described nitrocellulose filter successively is that the melamine-detection line of ovalbumin coupling bond, material are the nature controlling line of sheep anti-mouse igg.
2, according to the collaurum test card of the described a kind of fast detecting melamine of claim 1, it is characterized in that: described sample pad position is over against described well; Described cellulose nitrate film location is over against described observation port.
3, a kind of preparation method of collaurum test card of fast detecting melamine, it is characterized in that: it may further comprise the steps:
(1), forms conjugate melamine-ovalbumin as artificial antigen with carbodlimide method coupling melamine hapten and ovalbumin;
(2) merge with the mouse boosting cell of immunogen immune and murine myeloma cell hybridization, the preparation hybridoma, the cell line that screening can stably excreting anti-melamine monoclonal antibody prepares the anti-melamine monoclonal antibody;
(3) preparation colloidal gold solution;
(4) with the colloidal gold solution mark anti-melamine monoclonal antibody for preparing;
(5) with the envelope antigen and the sheep anti-mouse igg treatment of nitric acid cellulose membrane that prepare;
(6) handle gold mark pad with the gold mark anti-melamine monoclonal antibody for preparing;
(7) assembling gold test strip bar.
4, preparation method according to the collaurum test card of the described a kind of fast detecting melamine of claim 3, it is characterized in that: the preparation of described melamine-ovalbumin, take by weighing melamine, be dissolved in the pyridine solution, add 10%～20% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction is 1 hour～3 hours under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction dropped to this reactant liquor in ovalbumin (OVA) solution after 20 minutes, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 24 hours～72 hours, after the freeze drying in-20 ℃～-40 ℃ preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: μ l:(6～600 sodium cyanoborohydride=(10～1000) mg:(50～8000)) mg, haptens: EDC:OVA:NHS=(10～2000) mg:(5～10000) mg:(10～2000) mg:(20～5000) mg.
5, preparation method according to the collaurum test card of the described a kind of fast detecting melamine of claim 3, it is characterized in that: the preparation of described melamine-bovine serum albumin(BSA) coupled antigen, take by weighing melamine, be dissolved in the pyridine solution, add 10%～20% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri of stirring reaction is 2 hours under the room temperature, cryoconcentration is to doing then, get haptens, get this haptens, be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction is after 20 minutes, this reactant liquor is dropped to bovine serum albumin(BSA) (BSA, be dissolved in the ultrapure water) in the solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stir under the room temperature and spend the night, phosphate buffer (PBS) dialysis 24 hours～72 hours, after the freeze drying in-20 ℃～-40 ℃ preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: μ l:(6～600 sodium cyanoborohydride=(10～1000) mg:(50～8000)) mg, haptens: EDC:BSA:NHS=(10～2000) mg:(5～10000) mg:(10～2000) mg:(20～5000) mg.
6, preparation method according to the collaurum test card of the described a kind of fast detecting melamine of claim 3, it is characterized in that: the preparation of described anti-melamine monoclonal antibody and solution thereof, get melamine-bovine serum albumin(BSA) (Melamine-BSA) conjugate and be made into 0.1 μ g/ μ l～10 μ g/ μ l antigenic solutions with physiological saline, with the complete freund adjuvant mixing of equal-volume, fully emulsified, use for first immunisation, replace complete freund adjuvant with incomplete freund adjuvant during booster immunization with amount, first immunisation adopts directly injection in the mouse peritoneal, immunizing dose is 10 μ g/～100 a μ g/ mouse, later on once every 2～4 all booster immunizations, booster immunization adopts tail vein injection, immunizing dose 10 μ g/～100 μ g/ only, intrasplenic injection is adopted in last immunity, getting spleen after 4 days merges, the immune mouse spleen cell that separates is mixed with 10: 1 ratios with the myeloma cell SP-2/o that is in exponential phase, centrifugal, remove supernatant, in 50S～90S, 0.1ml～10ml50% polyglycol (molecular weight is 1500) is added in the cell, abundant mixing, make its fusion, add 10ml～50ml DMEM nutrient solution after 1 minute～3 minutes, stop merging, water-bath is centrifugal after static 10 minutes～30 minutes, removes supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 5%～30% calf serum, be 1 * 10 with final concentration
4～1 * 10
6Feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 5%～10%CO
2Cultivate under 35 ℃～45 ℃ conditions, after 5 days～10 days, every culture hole is changed the 2/3HT nutrient solution, after 10 days～20 days, begin microscopy is had the culture hole of hybridoma clonal growth, getting supernatant screens, the cell that testing result is positive is cloned with limiting dilution assay immediately, the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, with melamine-ovalbumin (Melamine-OVA) is envelope antigen, with the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A test-A blank)/(A contrast-A blank)〉2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope, dilution are 70/ml, get 20 times of 1ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.3ml/ of 10～13 all age～0.5ml/, after 8 days～10 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only, note after 5 days observing is collected ascites, centrifugal removal precipitation, and glycerol adding is in-20 ℃～-40 ℃ preservations; The ratio of mentioned component is: Melamine-BSA: ml physiological saline=(10～1000) μ g:(0.1～10).
7, preparation method according to the collaurum test card of the described a kind of fast detecting melamine of claim 3, it is characterized in that: the preparation of described colloidal gold solution, with new system distilled water preparation 0.01%～0.1% chlorauric acid solution, place conical flask, be heated to 100 ℃～120 ℃ with the constant temperature magnetic stirrer, under 100r/min～200r/min magnetic agitation, add rapidly in advance 1%～3% citric acid three sodium solution 35 ℃～45 ℃ of insulations, keep temperature and rotating speed constant, continue agitating heating about 15 minutes～30 minutes, present limpid claret until solution; The room temperature cooling, 2 ℃～8 ℃ refrigerators are preserved standby; The ratio of mentioned component is: gold chloride: ml trisodium citrate=(10～1000) ml:(1～20).
8, according to the preparation method of the collaurum test card of the described a kind of fast detecting melamine of claim 3, it is characterized in that: described colloid gold label anti-melamine MONOCLONAL ANTIBODIES SPECIFIC FOR, get colloidal gold solution in centrifuge tube, with 0.1M～1.0M K
2CO
3Or about 0.1M～1.0M HCl adjusting pH to 7～8, encase centrifuge tube with aluminium-foil paper, gently vibration; Dropwise add antibody protein solution while vibrating, continue vibration 10 minutes～30 minutes; Dropwise add 1%BSA solution with saturated free collaurum, then in 2 ℃～8 ℃, 6000rpm～12000rpm, centrifugal 30 minutes～60 minutes; Centrifuge tube is taken out in centrifugal back, supernatant is sopped up a part gently after, will manage the end with liquid-transfering gun and precipitate and carefully be drawn in the centrifuge tube, 2 ℃～8 ℃ preservations are standby; The ratio of mentioned component is: colloidal gold solution: antibody protein: BSA=(10～1000) ml:(50～5000) μ g:(1～100) ml.
9, according to the preparation method of the collaurum test card of the described a kind of fast detecting melamine of claim 3, it is characterized in that: the processing of described nitrocellulose filter, spray two parallel lines on nitrocellulose filter with gold mark point sample instrument, melamine coupling ovalbumin bond melamine-ovalbumin is as detection line, sheep anti-mouse igg is as nature controlling line, puts after 35 ℃～45 ℃ vacuum drying standby; The processing of described gold mark pad: a certain amount of gold mark anti-melamine monoclonal antibody evenly is sprayed on the gold mark pad, puts after 35 ℃～45 ℃ vacuum drying standby.
10, preparation method according to the collaurum test card of claim 3 or 9 described a kind of fast detecting melamines, it is characterized in that: the assembling of described gold test strip bar, get the base plate of making the test strips special use, the nitrocellulose filter that drying is good sticks on the relevant position earlier, again thieving paper and dry good gold mark pad are sticked on the relevant position and pushes down nitrocellulose filter a little, at last sample pad is glued and pushes down gold mark pad, cutting into the wide test strips of 3mm with cutting cutter packs in the test card, at this moment, the position of sample pad is over against the well of test card, the cellulose nitrate film location is over against the observation port of test card, pack in the aluminium foil bag with drying agent, pack.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810236374A CN101413956B (en) | 2008-11-08 | 2008-11-08 | Colloidal gold detection card for rapidly detecting melamine and preparing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810236374A CN101413956B (en) | 2008-11-08 | 2008-11-08 | Colloidal gold detection card for rapidly detecting melamine and preparing method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101413956A true CN101413956A (en) | 2009-04-22 |
| CN101413956B CN101413956B (en) | 2012-10-17 |
Family
ID=40594589
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810236374A Expired - Fee Related CN101413956B (en) | 2008-11-08 | 2008-11-08 | Colloidal gold detection card for rapidly detecting melamine and preparing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101413956B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102297970A (en) * | 2011-05-27 | 2011-12-28 | 重庆市科学技术研究院 | Two-joint detection test strip for rapidly detecting residue of cyromazine (CA) and melamine (MA) and preparation method thereof |
| CN102297964A (en) * | 2011-05-20 | 2011-12-28 | 广州万孚生物技术有限公司 | Immunity chromatography detection test strip of melamine |
| CN102353773A (en) * | 2011-06-13 | 2012-02-15 | 清华大学深圳研究生院 | Immune chromatographic test paper for detecting melamine and preparation method thereof |
| CN101750410B (en) * | 2010-01-22 | 2012-05-30 | 大连理工大学 | Method for detecting melamine by electrochemical luminescence |
| CN102707047A (en) * | 2012-06-05 | 2012-10-03 | 南通市产品质量监督检验所 | Colloidal gold test card for quickly testing ergot alkaloid and preparation method thereof |
| CN106771170A (en) * | 2017-01-21 | 2017-05-31 | 上海为然环保科技有限公司 | A kind of colloidal gold fast detecting reagent kit of soybean medicinal extract Daidzein |
| CN108982862A (en) * | 2018-08-01 | 2018-12-11 | 山西公大智检科技有限公司 | It is a kind of for detecting the immuno-chromatographic test paper strip and preparation method thereof of capsaicine in gutter oil |
| CN111443205A (en) * | 2020-02-25 | 2020-07-24 | 古镜科技(深圳)有限公司 | Quintuplet test paper for detecting abuse of mental drugs and preparation thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101183105B (en) * | 2007-12-13 | 2011-06-29 | 中国农业科学院农业质量标准与检测技术研究所 | ELISA reagent kit for detecting melamine |
| CN101206223B (en) * | 2007-12-13 | 2011-06-29 | 中国农业科学院农业质量标准与检测技术研究所 | Direct competition method ELISA reagent box for detecting melamine |
-
2008
- 2008-11-08 CN CN200810236374A patent/CN101413956B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101750410B (en) * | 2010-01-22 | 2012-05-30 | 大连理工大学 | Method for detecting melamine by electrochemical luminescence |
| CN102297964A (en) * | 2011-05-20 | 2011-12-28 | 广州万孚生物技术有限公司 | Immunity chromatography detection test strip of melamine |
| CN102297970A (en) * | 2011-05-27 | 2011-12-28 | 重庆市科学技术研究院 | Two-joint detection test strip for rapidly detecting residue of cyromazine (CA) and melamine (MA) and preparation method thereof |
| CN102353773A (en) * | 2011-06-13 | 2012-02-15 | 清华大学深圳研究生院 | Immune chromatographic test paper for detecting melamine and preparation method thereof |
| CN102353773B (en) * | 2011-06-13 | 2014-01-01 | 清华大学深圳研究生院 | Immune chromatographic test paper for detecting melamine and preparation method thereof |
| CN102707047A (en) * | 2012-06-05 | 2012-10-03 | 南通市产品质量监督检验所 | Colloidal gold test card for quickly testing ergot alkaloid and preparation method thereof |
| CN106771170A (en) * | 2017-01-21 | 2017-05-31 | 上海为然环保科技有限公司 | A kind of colloidal gold fast detecting reagent kit of soybean medicinal extract Daidzein |
| CN108982862A (en) * | 2018-08-01 | 2018-12-11 | 山西公大智检科技有限公司 | It is a kind of for detecting the immuno-chromatographic test paper strip and preparation method thereof of capsaicine in gutter oil |
| CN111443205A (en) * | 2020-02-25 | 2020-07-24 | 古镜科技(深圳)有限公司 | Quintuplet test paper for detecting abuse of mental drugs and preparation thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101413956B (en) | 2012-10-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101413956B (en) | Colloidal gold detection card for rapidly detecting melamine and preparing method thereof | |
| CN101413955B (en) | ELISA test box for detecting zearalenone and preparing and detecting method thereof | |
| CN104569399B (en) | A kind of test strips detecting ochratoxin A and application thereof | |
| CN109324182B (en) | Fluorescent microsphere immunochromatography test strip for detecting pendimethalin and preparation method and application thereof | |
| CN109239336B (en) | Test strip for detecting isoprocarb and application thereof | |
| CN105807055B (en) | A kind of test strips for detecting dichloro quinolinic acid and its preparation method and application | |
| CN109061146B (en) | Test strip for detecting acetamiprid and preparation method and application thereof | |
| CN106918705B (en) | Test paper for detecting fenpropathrin and application thereof | |
| CN112067811B (en) | Test strip for detecting alternaria alternate and application thereof | |
| CN103777015B (en) | A kind of colloidal gold strip detecting erythromycin and method | |
| CN103376316A (en) | Test strip for detecting streptomycin and method | |
| CN111500546A (en) | Cell strain secreting four subtype antibodies against aflatoxin, antibody secreted by cell strain and immunochromatography detection card | |
| CN110551220A (en) | Preparation and application of DDT monoclonal antibody | |
| CN110927375A (en) | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof | |
| CN113030463B (en) | Test strip for detecting impurities such as protein A in vaccine and application thereof | |
| CN111896738B (en) | Test strip for detecting serpentine bacterial element and application thereof | |
| CN111751535B (en) | Test strip for detecting endosulfan and application thereof | |
| CN111454912B (en) | Cyperazine monoclonal antibody hybridoma cell strain and application thereof | |
| CN111735951B (en) | Test strip for detecting fenpropathrin and application thereof | |
| CN109061145B (en) | Test strip for detecting flumetralin, preparation method and application thereof | |
| CN109265364B (en) | Preparation and application of pendimethalin hapten and antigen | |
| CN107192826B (en) | The colloid gold test paper and its preparation and detection method of a kind of detection diethylene triamine pentacetic acid (DTPA) and its chelate | |
| CN101413947A (en) | Enzyme-linked immunologic test box for detecting melamine and preparation and detection method thereof | |
| CN111505293B (en) | Test strip for detecting variegated aspergillin and application thereof | |
| CN111646959A (en) | Dinotefuran hapten, colloidal gold labeled dinotefuran monoclonal antibody and dinotefuran colloidal gold detection device |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121017 Termination date: 20181108 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |