CN101413956B - 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
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- CN101413956B CN101413956B CN200810236374A CN200810236374A CN101413956B CN 101413956 B CN101413956 B CN 101413956B CN 200810236374 A CN200810236374 A CN 200810236374A CN 200810236374 A CN200810236374 A CN 200810236374A CN 101413956 B CN101413956 B CN 101413956B
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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 the detection to batch samples, and need expensive instrument and equipment, it is high to detect cost; 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 repeat repeatedly to wash, and will measure, can not realize real single stage method fast detecting by means of the ELIASA reading.
(3) summary of the invention
To 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; Said test strips is packaged in the test card housing, has well and observation port on the said test card housing, and said test strips comprises base plate; It is characterized in that: be pasted with thieving paper, nitrocellulose filter, gold mark pad, sample pad on the base plate of said test strips successively; Said thieving paper and said gold mark pad stick on said nitrocellulose filter two ends respectively, and in the junction, two ends of said nitrocellulose filter, a bit of pressed and overlapped of said thieving paper and gold mark pad is on said nitrocellulose filter; Said sample pad sticks on the other end of said gold mark pad, and a bit of of said sample pad overlaps on the said gold mark pad; Be coated with gold mark anti-melamine monoclonal antibody on the said gold mark pad; Being coated with material on the said 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: said sample pad position is over against said well, and said cellulose nitrate film location is over against said 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 preparation hybridoma, the cell line that screening can stably excreting anti-melamine monoclonal antibody, preparation anti-melamine monoclonal antibody with the mouse boosting cell of immunogen immune and murine myeloma cell hybridization;
(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 said 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, and cryoconcentration gets haptens to doing then; 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; Stirred overnight under the room temperature, 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: sodium cyanoborohydride=(10～1000) mg: (50～8000) μ l: (6-600) mg, haptens: EDC:OVA:NHS=(10～2000) mg: (5～10000) mg: (10-2000) mg: (20～5000) mg;
The preparation of said 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, and cryoconcentration gets haptens to doing then; Get this haptens, be dissolved in N, in dinethylformamide (DMF) solution; Drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction dropped to bovine serum albumin(BSA) (BSA with this reactant liquor after 20 minutes; Be dissolved in the ultrapure water) in the solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stirred overnight under the room temperature; 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: sodium cyanoborohydride=(10～1000) mg: (50～8000) μ l: (6～600) mg, haptens: EDC:BSA:NHS=(10～2000) mg: (5～10000) mg: (10～2000) mg: (20～5000) mg;
The preparation of said 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; Confession first immunisation usefulness replaces complete freund adjuvant with the incomplete freund adjuvant with amount during booster immunization, and first immunisation adopts directly injection in the mouse peritoneal; Immunizing dose is 10 μ g/～100 a μ g/ mouse, and later on whenever at a distance from all booster immunizations in 2 week～4 once, booster immunization adopts tail vein injection; Immunizing dose 10 μ g/ only～100 μ g/ only, intrasplenic injection is adopted in last immunity, gets spleen after 4 days and merges; The immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase is with the 10:1 mixed, centrifugal, remove supernatant; In 50S～90S, 0.1ml～10ml50% polyglycol (molecular weight is 1500) is added in the cell, fully mixing makes 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
2, cultivate under 35 ℃～45 ℃ conditions, after 5 days～10 days; Every culture hole is changed the 2/3HT nutrient solution, after 10 days～20 days, begins microscopy is had the culture hole of hybridoma clonal growth; Getting supernatant screens; Cell to testing result is positive is cloned with limiting dilution assay immediately, and the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, and is envelope antigen with melamine-ovalbumin (Melamine-OVA); 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 is blank)/(blank) > of A contrast-A; 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 deposition, glycerol adding is in-20 ℃～-40 ℃ preservations; The ratio of mentioned component is: Melamine-BSA: physiological saline=(10～1000) μ g: (0.1～10) ml.
The preparation of said 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 subsequent use; The ratio of mentioned component is: gold chloride: trisodium citrate=(10～1000) ml: (1～20) ml.
Said 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 subsequent use; 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 said 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 subsequent use;
The processing of said 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 subsequent use;
The assembling of said gold test strip bar: get and make the special-purpose base plate of test strips; 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 push down nitrocellulose filter a little, at last sample pad is glued and pushes down gold mark pad, cut into the wide test strips of 3mm with cutting cutter and pack 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 packed in the aluminium foil bag with drying agent, packs.
The colloidal gold strip of fast detecting melamine of the present invention, it detects fast, only needs 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, has well 3 and observation port 4 on the test card housing 1, and test strips 2 comprises base plate 5; Be pasted with thieving paper 6, nitrocellulose filter 7, gold mark pad 8, sample pad 9 on the base plate 5 of test strips 2 successively; Thieving paper 6 sticks on nitrocellulose filter 7 both sides respectively with gold mark pad 8, and in the junction, two ends of nitrocellulose filter 7, a bit of pressed and overlapped of thieving paper 6 and gold mark pad 8 is on nitrocellulose filter 7; 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 preparation hybridoma, the cell line that screening can stably excreting anti-melamine monoclonal antibody, preparation anti-melamine monoclonal antibody with the mouse boosting cell of immunogen immune and murine myeloma cell hybridization;
(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
Embodiment 1
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 gets haptens to doing then, gets this haptens; Be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC); Behind the stirring reaction 20 minutes, this reactant liquor is dropped in ovalbumin (OVA) solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately; Stirred overnight under the room temperature, 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 gets haptens to doing then; Get this haptens, be dissolved in N, in dinethylformamide (DMF) solution; Drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction dropped to bovine serum albumin(BSA) (BSA with this reactant liquor after 20 minutes; Be dissolved in the ultrapure water) in the solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stirred overnight under the room temperature; 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, fully emulsified with the complete freund adjuvant mixing of equal-volume, supply first immunisation to use; Replace complete freund adjuvant with the incomplete freund adjuvant with amount during booster immunization, first immunisation adopts directly injection in the mouse peritoneal, and immunizing dose is 55 a μ g/ mouse; Every later at a distance from 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, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with the 10:1 mixed; Centrifugal, remove supernatant, in 50S, 5.05ml 50% polyglycol (molecular weight is 1500) is added in the cell; Fully mixing makes its fusion, adds 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
2, cultivate under 40 ℃ of conditions, after 5 days; Every culture hole is changed the 2/3HT nutrient solution, after 20 days, begins microscopy is had the culture hole of hybridoma clonal growth; Getting supernatant screens; Cell to testing result is positive is cloned with limiting dilution assay immediately, and the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, and is envelope antigen with melamine-ovalbumin (Melamine-OVA); 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 is blank)/(blank) > of A contrast-A; 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 deposition, 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 subsequent use; 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 subsequent use; The ratio of mentioned component is: colloidal gold solution: antibody protein: BSA=1000ml:2500 μ g:50.5ml;
The processing of said 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 subsequent use;
The processing of said 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 subsequent use.
The preparation of said 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 gets haptens to doing then, gets this haptens; Be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC); Behind the stirring reaction 20 minutes, this reactant liquor is dropped in ovalbumin (OVA) solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately; Stirred overnight under the room temperature, 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 said 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 gets haptens to doing then; Get this haptens, be dissolved in N, in dinethylformamide (DMF) solution; Drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction dropped to bovine serum albumin(BSA) (BSA with this reactant liquor after 20 minutes; Be dissolved in the ultrapure water) in the solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stirred overnight under the room temperature; 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 said 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, fully emulsified with the complete freund adjuvant mixing of equal-volume, supply first immunisation to use; Replace complete freund adjuvant with the incomplete freund adjuvant with amount during booster immunization, first immunisation adopts directly injection in the mouse peritoneal, and immunizing dose is 10 a μ g/ mouse; Every later at a distance from 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, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with the 10:1 mixed; Centrifugal, remove supernatant, in 90S, 0.1ml50% polyglycol (molecular weight is 1500) is added in the cell; Fully mixing makes its fusion, adds 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
2, cultivate under 35 ℃ of conditions, after 7 days; Every culture hole is changed the 2/3HT nutrient solution, after 10 days, begins microscopy is had the culture hole of hybridoma clonal growth; Getting supernatant screens; Cell to testing result is positive is cloned with limiting dilution assay immediately, and the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, and is envelope antigen with melamine-ovalbumin (Melamine-OVA); 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 is blank)/(blank) > of A contrast-A; 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 deposition, 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 said 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 subsequent use; The ratio of mentioned component is: gold chloride: trisodium citrate=10ml:20ml.
Said 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 subsequent use; The ratio of mentioned component is: colloidal gold solution: antibody protein: BSA=10ml:5000 μ g:100ml.
The processing of said 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 subsequent use;
The processing of said 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 subsequent use.
Embodiment 3
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 gets haptens to doing then, gets this haptens; Be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC); Behind the stirring reaction 20 minutes, this reactant liquor is dropped in ovalbumin (OVA) solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately; Stirred overnight under the room temperature, 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 gets haptens to doing then; Get this haptens, be dissolved in N, in dinethylformamide (DMF) solution; Drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), stirring reaction dropped to bovine serum albumin(BSA) (BSA with this reactant liquor after 20 minutes; Be dissolved in the ultrapure water) in the solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stirred overnight under the room temperature; 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, fully emulsified with the complete freund adjuvant mixing of equal-volume, supply first immunisation to use; Replace complete freund adjuvant with the incomplete freund adjuvant with amount during booster immunization, first immunisation adopts directly injection in the mouse peritoneal, and immunizing dose is 100 a μ g/ mouse; Every later at a distance from 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, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with the 10:1 mixed; Centrifugal, remove supernatant, in 70S, 10ml 50 ℅ polyglycol (molecular weight is 1500) are added in the cell; Fully mixing makes its fusion, adds 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
2, cultivate under 45 ℃ of conditions, after 10 days; Every culture hole is changed the 2/3HT nutrient solution, after 15 days, begins microscopy is had the culture hole of hybridoma clonal growth; Getting supernatant screens; Cell to testing result is positive is cloned with limiting dilution assay immediately, and the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, and is envelope antigen with melamine-ovalbumin (Melamine-OVA); 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 is blank)/(blank) > of A contrast-A; 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 deposition, 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 subsequent use; 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 subsequent use; 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 subsequent use;
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 subsequent use;
The assembling of said gold test strip bar 2: get and make the special-purpose base plate of test strips; 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 push down nitrocellulose filter 7 a little, at last sample pad is glued and pushes down gold mark pad 8, cut into the wide test strips of 3mm with cutting cutter and pack 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 packed in the aluminium foil bag with drying agent, packs.
When sample is detected, test card is kept flat, get 100 μ L appearance 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 of treating 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, and the golden mark anti-melamine monoclonal antibody specific and the melamine in the sample that are solidificated on the gold mark pad 8 play specific reaction; And competition suppresses it and combines with melamine on the detection line; Therefore, when the content of melamine in the sample was lower than a certain amount, golden mark anti-melamine monoclonal antibody specific combined back gold grain aggegation colour developing with melamine on the detection line 10; 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, and 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 (9)
1. the collaurum test card of a fast detecting melamine; It comprises test strips; Said test strips is packaged in the test card housing, has well and observation port on the said test card housing, and said test strips comprises base plate; It is characterized in that: be disposed with thieving paper, nitrocellulose filter, gold mark pad, sample pad on the base plate of said test strips; Said thieving paper and said gold mark pad are separately positioned on said nitrocellulose filter two ends, and in the junction, two ends of said nitrocellulose filter, a bit of pressed and overlapped of said thieving paper and gold mark pad is on said nitrocellulose filter; Said sample pad is arranged on the other end of said gold mark pad, and a bit of of said sample pad overlaps on the said gold mark pad; Be coated with gold mark anti-melamine monoclonal antibody on the said gold mark pad; Being coated with material on the said nitrocellulose filter successively is that the melamine-detection line of ovalbumin coupling bond, material are the nature controlling line of sheep anti-mouse igg;
The preparation of said collaurum test card may further comprise the steps:
⑴ form conjugate melamine-ovalbumin as envelope antigen with carbodlimide method coupling melamine hapten and ovalbumin;
⑵ merge with melamine-bovine serum albumin(BSA) coupled antigen mice immunized splenocyte and murine myeloma cell hybridization; The preparation hybridoma; Screening can stably excreting anti-melamine monoclonal antibody cell line, preparation anti-melamine monoclonal antibody;
⑶ prepare colloidal gold solution;
⑷ with the colloidal gold solution mark anti-melamine monoclonal antibody for preparing;
⑸ with the envelope antigen and the sheep anti-mouse igg treatment of nitric acid cellulose membrane that prepare;
⑹ handle gold mark pad with the gold mark anti-melamine monoclonal antibody for preparing;
⑺ assembling gold test strip bar;
The preparation of said melamine-ovalbumin takes by weighing melamine, is dissolved in the pyridine solution; Add 10% ~ 20% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri 1 h ~ of stirring reaction is 3 hours under the room temperature, and 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 in ovalbumin (OVA) solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stirred overnight under the room temperature; Phosphate buffer (PBS) dialysis 24 h ~ 72 hours, after the freeze drying in-20 ℃ ~-40 ℃ preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: sodium cyanoborohydride=(10 ~ 1000) mg: (50 ~ 8000) μ l: (6 ~ 600) mg, haptens: EDC:OVA:NHS=(10 ~ 2000) mg: (5 ~ 10000) mg: (10 ~ 2000) mg: (20 ~ 5000) mg.
2. according to the collaurum test card of the said a kind of fast detecting melamine of claim 1, it is characterized in that: said sample pad position is over against said well; Said cellulose nitrate film location is over against said observation port.
3. the preparation method of the collaurum test card of a fast detecting melamine, it is characterized in that: it may further comprise the steps:
⑴ form conjugate melamine-ovalbumin as envelope antigen with carbodlimide method coupling melamine hapten and ovalbumin;
⑵ merge with melamine-bovine serum albumin(BSA) coupled antigen mice immunized splenocyte and murine myeloma cell hybridization; The preparation hybridoma; Screening can stably excreting anti-melamine monoclonal antibody cell line, preparation anti-melamine monoclonal antibody;
⑶ prepare colloidal gold solution;
⑷ with the colloidal gold solution mark anti-melamine monoclonal antibody for preparing;
⑸ with the envelope antigen and the sheep anti-mouse igg treatment of nitric acid cellulose membrane that prepare;
⑹ handle gold mark pad with the gold mark anti-melamine monoclonal antibody for preparing;
⑺ assembling gold test strip bar;
The preparation of said melamine-ovalbumin takes by weighing melamine, is dissolved in the pyridine solution; Add 10% ~ 20% succinaldehyde acid solution and sodium cyanoborohydride respectively, this potpourri 1 h ~ of stirring reaction is 3 hours under the room temperature, and 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 in ovalbumin (OVA) solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stirred overnight under the room temperature; Phosphate buffer (PBS) dialysis 24 h ~ 72 hours, after the freeze drying in-20 ℃ ~-40 ℃ preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: sodium cyanoborohydride=(10 ~ 1000) mg: (50 ~ 8000) μ l: (6 ~ 600) mg, haptens: EDC:OVA:NHS=(10 ~ 2000) mg: (5 ~ 10000) mg: (10 ~ 2000) mg: (20 ~ 5000) mg.
4. according to the preparation method of the collaurum test card of the said a kind of fast detecting melamine of claim 3, it is characterized in that: the preparation of said melamine-bovine serum albumin(BSA) coupled antigen takes 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 gets haptens to doing then, gets this haptens; Be dissolved in N, in dinethylformamide (DMF) solution, drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC); Behind the stirring reaction 20 minutes; This reactant liquor is dropped in the bovine serum albumin solution, add N-hydroxy thiosuccinimide (Sulfo-NHS) immediately, stirred overnight under the room temperature; Phosphate buffer (PBS) dialysis 24 h ~ 72 hours, after the freeze drying in-20 ℃ ~-40 ℃ preservations; The ratio of above-mentioned reacted constituent is: melamine: amber aldehydic acid: sodium cyanoborohydride=(10 ~ 1000) mg: (50 ~ 8000) μ l: (6 ~ 600) mg, haptens: EDC:BSA:NHS=(10 ~ 2000) mg: (5 ~ 10000) mg: (10 ~ 2000) mg: (20 ~ 5000) mg.
5. according to the preparation method of the collaurum test card of the said a kind of fast detecting melamine of claim 3, it is characterized in that: the preparation of said 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, supply first immunisation to use; Replace complete freund adjuvant with the incomplete freund adjuvant of measuring together during booster immunization, first immunisation adopts directly injection in the mouse peritoneal, and immunizing dose is 10 μ g/ ~ 100 a μ g/ mouse; Every later at a distance from 2 ~ 4 all booster immunizations once booster immunization adopts tail vein injection, and immunizing dose 10 μ g/ ~ 100 μ g/ only; Intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with the 10:1 mixed; Centrifugal, remove supernatant, in 50 S～90 S, be that 1500 polyglycol adds in the cell with 0.1 ml ~ 10 ml, 50% molecular weight; Abundant mixing; Make its fusion, add 10 ml ~ 50 ml DMEM nutrient solutions 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
2, cultivate under 35 ℃ ~ 45 ℃ conditions, after 5 days ~ 10 days; Every culture hole is changed 2/3 HT nutrient solution, after 10 days～20 days, begins microscopy is had the culture hole of hybridoma clonal growth; Getting supernatant screens; Cell to testing result is positive is cloned with limiting dilution assay immediately, and the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, and is envelope antigen with melamine-ovalbumin (Melamine-OVA); 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 is blank)/(blank) > of A contrast-A; 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 is 70/m1; Get 20 times of 1 ml 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 0.3 ml/ of 10～13 all age～0.5 ml/, 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 deposition, and glycerol adding is in-20 ℃ ~-40 ℃ preservations.
6. according to the preparation method of the collaurum test card of the said a kind of fast detecting melamine of claim 3; It is characterized in that: the preparation of said colloidal gold solution with new system distilled water preparation 0.01% ~ 0.1% chlorauric acid solution, places conical flask; Be heated to 100 ℃ ~ 120 ℃ with the constant temperature magnetic stirrer; Under 100r/min ~ 200r/min magnetic agitation, adding keeps temperature and rotating speed constant in advance at 1% ~ 3% citric acid three sodium solution of 35 ℃ ~ 45 ℃ of insulations rapidly; Continued agitating heating 15 minutes ~ 30 minutes, and presented limpid claret until solution; The room temperature cooling, 2 ℃ ~ 8 ℃ refrigerators are preserved subsequent use; The ratio of mentioned component is: gold chloride: trisodium citrate=(10 ~ 1000) ml: (1 ~ 20) m1.
7. according to the preparation method of the collaurum test card of the said a kind of fast detecting melamine of claim 3, it is characterized in that: said colloid gold label anti-melamine MONOCLONAL ANTIBODIES SPECIFIC FOR, get colloidal gold solution in centrifuge tube, with 0.1 M ~ 1.0M K
2CO
3Or 0.1 M ~ 1.0M HCl regulates pH to 7 ~ 8, encases 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 ℃, 6000 rpm ~ 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 subsequent use; The ratio of mentioned component is: colloidal gold solution: antibody protein: BSA=(10 ~ 1000) ml: (50 ~ 5000) μ g: (1 ~ 100) ml.
8. according to the preparation method of the collaurum test card of the said a kind of fast detecting melamine of claim 3; It is characterized in that: the processing of said 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, and sheep anti-mouse igg is as nature controlling line, puts after 35 ℃ ~ 45 ℃ vacuum drying subsequent use; The processing of said 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 subsequent use.
9. according to the preparation method of the collaurum test card of claim 3 or 8 said a kind of fast detecting melamines; It is characterized in that: the assembling of said gold test strip bar; Get and make the special-purpose base plate of test strips, the nitrocellulose filter that drying is good sticks on the relevant position earlier, again thieving paper and dry good gold mark pad is sticked on the relevant position and pushes down nitrocellulose filter a little; At last sample pad is glued and pushes down gold mark pad; Cut into the wide test strips of 3mm with cutting cutter and pack 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.
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| 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 |
| 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 |
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| CN101183105A (en) * | 2007-12-13 | 2008-05-21 | 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 |
| CN101206223A (en) * | 2007-12-13 | 2008-06-25 | 中国农业科学院农业质量标准与检测技术研究所 | Direct racing method ELISA reagent box for detecting melamine |
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| CN101183105A (en) * | 2007-12-13 | 2008-05-21 | 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 |
| CN101206223A (en) * | 2007-12-13 | 2008-06-25 | 中国农业科学院农业质量标准与检测技术研究所 | Direct racing method ELISA reagent box for detecting melamine |
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