CN102183634A - Immune nano gold test strip for quickly detecting four nitrofuran medicaments - Google Patents
Immune nano gold test strip for quickly detecting four nitrofuran medicaments Download PDFInfo
- Publication number
- CN102183634A CN102183634A CN2011100513935A CN201110051393A CN102183634A CN 102183634 A CN102183634 A CN 102183634A CN 2011100513935 A CN2011100513935 A CN 2011100513935A CN 201110051393 A CN201110051393 A CN 201110051393A CN 102183634 A CN102183634 A CN 102183634A
- Authority
- CN
- China
- Prior art keywords
- solution
- concentration
- phosphate buffer
- antibody
- minutes
- 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.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to an immune nano gold test strip for quickly detecting four nitrofuran medicaments. The immune nano gold test strip is prepared by the following steps of: synthesizing general hapten, immunogen and captured antigen aiming at the four nitrofuran medicaments comprising nitrofurazone, furaltadone, nitrofurantoin and furazolidone by using 5-nitrofurfural as a raw material, immunizing a domestic rabbit serving as an experimental animal to acquire polyclonal IgG antibody, preparing gold labeled antibody by using nano gold and the polyclonal IgG antibody, coating the gold labeled antibody solution on a Glass33 glass cellulose membrane, namely a combined release pad, coating the captured antigen and goat anti-rabbit antibody on a detection line and a quality control line of an AE99 nitrocellulose membrane respectively, sequentially sticking the AE99 nitrocellulose membrane, the combined release pad, namely the Glass33 glass cellulose membrane, an absorption pad Cotton linters 2668 and a sample pad Glass33 on a polyvinyl chloride (PVC) back board, and finally, cutting the back board into 4*50mm test strips. The test strip can simultaneously and quickly detect the four medicaments comprising nitrofurazone, furaltadone, nitrofurantoin and furazolidone in feed; and the cost of the test strip is less than 5 yuan, so the test strip is suitable to be widely applied in feed detection or food animal detection.
Description
Technical field
The present invention relates to the immune nanometer gold test paper strip of four kinds of itrofurans medicines of a kind of fast detecting, totally belong to agriculture field (herding and aquatic products industry), specifically belong to animal derived product safety detection/detection of veterinary drugs in food field.
Background technology
Furazolidone, nitrofurazone, furantoin and furaltadone all belong to the itrofurans extensive pedigree antibiotic of chemosynthesis, but, classified as the medicine of forbidding being used for food animal by the Ministry of Agriculture because their original shape medicine and metabolin have carcinogenicity and mutagenesis.But these medicines often are added to illegal use in the animal feed, and therefore the residue of veterinary drug that causes can have a strong impact on animal product safety, threaten the consumer healthy.Therefore, detecting this type of medicine that illegally adds in the feed can be from the quality safety of source watching for animals property product.
At present, some researcher adopts immunological method that this type of medicine is detected.As patent 200510111229, its statement directly is connected furazolidone as immunogene with carrier protein, and acquisition Antibody Preparation immune nanometer gold test paper strip can detect the furazolidone in the feed; Patent 200610041481 for another example, and its statement has prepared the monoclonal antibody of nitrofurazone, and the ELISA method of foundation can detect nitrofurazone.But it does not provide immunogenic preparation method, presses immunology principle and infers that the applicant directly is connected nitrofurazone with carrier protein.These method and technologies all can only detect a kind of itrofurans medicine, and can not detect other several itrofurans medicines simultaneously.Therefore, the research preparation method and technology that can be simultaneously these four kinds of itrofurans medicines in the feed be carried out fast detecting is extremely important.At present the existent method technology can not detect this four kinds of itrofurans medicines simultaneously, be because their immunogene when synthetic at a kind of medicine, have very big deficiency.The molecular structure parent nucleus of itrofurans medicine is a 5-nitrofuran ring, and above-mentioned four kinds of medicines all contain this structure.And a kind of medicinal intermediates 5-nitryl furfural also contains this structure, just can discern these four kinds of medicines simultaneously at the antibody of 5-nitryl furfural.
Summary of the invention
The problem to be solved in the present invention provides the immune nanometer gold test paper strip of four kinds of itrofurans medicines of a kind of energy fast detecting.
The technical scheme that the present invention takes is as follows:
The immune nanometer gold test paper strip of four kinds of itrofurans medicines of a kind of fast detecting, its preparation method is as follows:
(1) synthetic universal hapten: take by weighing 5-nitryl furfural 50-80mg be dissolved in the 3-5mL ethanol A liquid; Take by weighing sulfuric acid hydrazine 70-100mg be dissolved in the 5-10mL distilled water B liquid; Under the room temperature A liquid dropwise is added drop-wise in the B liquid, stirring reaction 30-60 minute, gets yellow mercury oxide; With in the saturated sodium carbonate solution and above-mentioned reactant liquor to pH6.0-8.0, with this mixed liquid suction filtration to doing; Last solid washs with 10-20mL distilled water, and suction filtration is to doing again, and air dry promptly gets universal hapten;
(2) synthetic immunogen: take by weighing described haptens 10-20mg and be dissolved in the 5-10mL methyl alcohol, dropwise being added drop-wise to the concentration that contains bovine serum albumin(BSA) 30-50mg under the stirring condition is among 0.01mol/L, the pH7.0-7.4 phosphate buffer 5-10mL; Dropwise add concentration then and be 25% glutaraldehyde solution 60-100 μ L, 20-25 ℃ of reaction promptly get immunogen solution in 4 hours, dialysed 72 hours the freezing preservation of dislysate then in 4 ℃ of physiological saline;
(3) synthetic capture antigen: take by weighing described haptens 10-20mg and be dissolved in the 5-10mL methyl alcohol, be cooled to 4 ℃; Then, regulating pH with hydrochloric acid under 4 ℃ of conditions is 1.0-3.0, dropwise adds the sodium nitrite solution 1-4mL of 0.2-0.5mol/L again, and stirring reaction is till the dusty blue to the starch potassium iodide paper check; Transfer pH to 7.5-9.0 with sodium hydroxide solution then; Gained solution is dropwise joined among the phosphate buffer 5-10mL that the concentration that contains ovalbumin 10-20mg is 0.01mol/L, pH7.0-7.4,4 ℃ of stirring reaction 8-16h promptly get capture antigen solution, in physiological saline, dialysed 72 hours the freezing preservation of dislysate then;
(4) preparation polyclone IgG antibody: is 1mg/mL with described immunogene with the physiological saline dilution, add with the Fu Shi Freund's complete adjuvant of dilution back immunogen solution equivalent and make emulsifying agent, carry out head at the back of animal used as test rabbit intracutaneous multi-point injection and exempt from, immunizing dose 0.2-0.8mg/ only; 3-4 is after week, the immunogen solution of getting 1mg/mL carries out booster immunization at the subcutaneous multi-point injection of animal used as test man's rabbit back after adding Fu Shi Freund emulsification with immunogen solution equivalent, immunizing dose 0.2-0.8mg/, per 4 all booster immunizations once are total to immune 6-8 time; Last immunity is the direct auricular vein injection of immunogene, and immunizing dose 0.2-0.8mg/ only; Then, get the rabbit whole blood separation of serum, adopt the saturated ammonium sulfate method that the IgG in the serum is slightly carried; Crude extract is dialysed 2-3 days with desalination in the phosphate buffer of concentration 0.01mol/L, pH7.0-7.4; Dislysate carries out purifying with the DEAE-52 chromatographic column again; IgG solution behind the purifying is again at concentration 0.01mol/L, and dialysis promptly got polyclone IgG antibody-solutions, last freezing preservation with desalination in 2-3 days in the phosphate buffer of pH7.0-7.4;
(5) preparation nano-Au solution: the chlorauric acid solution 100mL constant temperature of getting 0.01%-0.05% is heated with stirring to boiling, at the citric acid three sodium solution 1-4mL that continues to add under the condition of stirring 1%-4%, continued agitating heating 20-30 minute, be bright redness to solution, the room temperature cooling, return to 100mL with deionized water, promptly get nano-Au solution, 4 ℃ of preservations are standby;
(6) prepare golden labeling antibody: the nano-Au solution of step (5) gained is regulated pH to 6.5-8.5 with the solution of potassium carbonate of concentration 0.01mol/L; To wherein adding step (4) gained polyclone IgG solution, making the IgG antibody concentration is 20-25 μ g/mL, shakes up back 4 ℃ and leaves standstill 30-60 minute; Adding bovine serum albumin(BSA) then, to make its concentration be 1%-2%, and stirring reaction 1-2 hour, 4 ℃ left standstill 2-3 hour; The centrifugal 20-30 of 1500r/min minute then, discard precipitation, again with supernatant with 4 ℃ of centrifugal 30-60 of 20000r/min minutes, abandoning supernatant; Precipitation is suspended in the phosphate buffer of 10-20mL concentration 0.01mol/L, pH7.0-7.4, with 4 ℃ of centrifugal 30-60 of 20000r/min minutes, precipitation is suspended in the phosphate buffer of 5-10mL concentration 0.01mol/L, pH7.0-7.4, is golden labeling antibody solution, 4 ℃ of preservations;
(7) preparation immune nanometer gold test paper strip: (a): the phosphate buffer of step (6) gained gold labeling antibody solution concentration 0.01mol/L, pH7.0-7.4 is diluted 4-6 doubly, contain 3% sucrose, 0.1% bovine serum albumin(BSA) and 0.05% Tween-20 in the described phosphate buffer, by 20 μ l/cm
2Dosage be sprayed on the plain film of Glass 33 type glass fibre promptly in conjunction with discharging on the pad, 37 ℃ of bakings are 10-20 minute then; (b): is 8 μ g/ml with described capture antigen with the carbonic acid buffer dilution that contains 3% methyl alcohol of concentration 0.01mol/L, pH9.0-9.5, at the detection line position of AE99 type nitrocellulose filter spraying 1-2 μ l; At the goat anti-rabbit igg antibody 1-2 μ l that spraying 1000-1500 in the nature controlling line position of this nitrocellulose filter doubly dilutes, detection line and nature controlling line spacing 5-8mm; Nitrocellulose filter was dried by the fire 10-20 minute at 37 ℃; Again nitrocellulose filter was immersed in the phosphate buffer of concentration 0.01mol/L, pH7.0-7.4 sealing 10 minutes under 20～25 ℃, and contained 0.5%BSA and 0.05% Sodium azide in the described phosphate buffer, then with nitrocellulose filter 37 ℃ of bakings 10-20 minute; (c): successively AE99 nitrocellulose filter, combination are discharged the plain film of pad Glass 33 glass fibre, absorption pad Cotton linters 2668 and sample pad Glass 33 and stick on the PVC backboard; Be cut to the test strips of 4 * 50mm at last.
When prepared immune nanometer gold test paper strip is used for on-the-spot the detection, handle sample to be checked earlier, the sample to be checked 100 μ L that will handle well then drip to sample pad, read the result in 5 minutes, if it is that nature controlling line and detection line all develop the color that two red colour developing bands are arranged, then be judged to feminine gender; If it is nature controlling line colour developing and detection line does not develop the color that a red colour developing band is arranged, then be judged to the positive.
Prepared immune nanometer gold test paper strip is respectively 2 μ g/g, 2 μ g/g, 3 μ g/g and 5 μ g/g to the detectability of nitrofurazone, furaltadone, furantoin and furazolidone.
Compare with existing method and technology, the good effect that the present invention obtains is:
The molecular structure parent nucleus of itrofurans medicine is a 5-nitrofuran ring, nitrofurazone, furaltadone, furantoin and furazolidone all contain this structure, the 5-nitryl furfural also contains this structure, just can discern these four kinds of medicines simultaneously at the antibody of 5-nitryl furfural.The present invention is a raw material with the 5-nitryl furfural, the universal hapten that has synthesized the itrofurans medicine, the immune nanometer gold test paper strip that has prepared four kinds of medicines such as nitrofurazone, furaltadone, furantoin and furazolidone in the energy while fast detecting feed, prepared immune nanometer gold test paper strip can detect whether contain these four kinds of medicines in the sample in 10 minutes, and the cost price of this test strips is lower than 5 yuan, is adapted at widespread use in feed detection or the food animal detection.
Description of drawings
Fig. 1 is the synoptic diagram of synthetic haptens, immunogene and capture antigen among the present invention;
Fig. 2 is an immune nanometer gold test paper strip structural representation of the present invention.
Embodiment
Embodiment 1
(1) synthetic universal hapten: as shown in Figure 1, take by weighing 5-nitryl furfural 60mg and be dissolved in (A liquid) in the 3mL ethanol, take by weighing sulfuric acid hydrazine 70mg and be dissolved in (B liquid) in the 7mL distilled water.Under the room temperature A liquid dropwise is added drop-wise in the B liquid, stirring reaction 30 minutes gets yellow mercury oxide.With in the saturated sodium carbonate solution and above-mentioned reactant liquor to pH6.0-8.0, with this mixed liquid suction filtration to doing.Last solid washs with 15mL distilled water, and suction filtration is to doing again, and air dry promptly gets universal hapten, the productive rate 74% of synthetic reaction, fusing point 243-245 ℃ (39 ℃ of 5-nitryl furfural fusing points), ft-ir characteristic absorption peak: 3150,3129,3099,1641,1349,1251,1027,825,738 cm
-1Ultraviolet spectrophotometer carries out full wavelength scanner to 5-nitryl furfural and haptens, and both ultraviolet absorption curves are basic identical as a result, illustrates that the structure of 5-nitryl furfural itself is not destroyed, and the structure that contains the 5-nitryl furfural in the haptens shows that haptens synthesizes successfully;
(2) synthetic immunogen: take by weighing described haptens 10mg and be dissolved in the 7mL methyl alcohol, dropwise be added drop-wise to the phosphate buffer 8mL(0.01mol/L that contains bovine serum albumin(BSA) 40mg under the stirring condition, pH7.0-7.4) in, dropwise add 25% glutaraldehyde solution 80 μ L then, room temperature reaction 4 hours.In 4 ℃ of physiological saline, dialysed 72 hours the freezing preservation of dislysate then;
(3) synthetic capture antigen: take by weighing described haptens 20mg and be dissolved in the 8mL methyl alcohol, be cooled to 4 ℃.Then, regulating pH with hydrochloric acid under 4 ℃ of conditions is 1.0-3.0, dropwise adds the sodium nitrite solution 1mL of 0.5mol/L again, and stirring reaction is till the dusty blue to the starch potassium iodide paper check, transfers pH to 7.5-9.0 with sodium hydroxide solution then.Gained solution is dropwise joined (0.01mol/L among the phosphate buffer 8mL that contains ovalbumin 20mg, pH7.0-7.4), 4 ℃ of stirring reaction 8-16h, in physiological saline, dialysed 72 hours then, the ultraviolet full wavelength scanner curve of comlete antigen is equivalent to the stack of the scanning curve and the haptens ultraviolet curve of carrier, shows that immunogene and capture antigen synthesize successfully; The freezing preservation of dislysate;
(4) preparation polyclonal antibody: is 1mg/mL with described immunogene with the physiological saline dilution, add with the Fu Shi Freund's complete adjuvant of dilution back immunogen solution equivalent and make emulsifying agent, carry out head at the back of New Zealand white rabbit intracutaneous multi-point injection and exempt from, immunizing dose 0.5mg/ only; After 4 weeks, the immunogen solution of getting 1mg/mL carries out booster immunization at the subcutaneous multi-point injection in animal used as test New Zealand white rabbit back after adding the Fu Shi Freund emulsification of immunogen solution equivalent therewith, immunizing dose 0.5mg/, per 4 all booster immunizations once are total to immunity 8 times; Last immunity is the direct auricular vein injection of immunogene, and immunizing dose 0.5mg/ only.Then, get the rabbit whole blood separation of serum, adopt the saturated ammonium sulfate method that the IgG in the serum is slightly carried; Crude extract phosphate buffer (0.01mol/L, pH7.0-7.4) in the dialysis 3 days with desalination; Dislysate carries out purifying with the DEAE-52 chromatographic column again; IgG solution behind the purifying again phosphate buffer (0.01mol/L, pH7.0-7.4) in the dialysis 3 days with desalination, last freezing preservation;
(5) preparation nano-Au solution: get 0.05% chlorauric acid solution 100mL and be heated with stirring to boiling with the constant temperature magnetic stirrer, at the citric acid three sodium solution 2mL that continues adding 3% under the condition of stirring, continued agitating heating 30 minutes, be bright redness to solution, the room temperature cooling, return to 100mL with deionized water, promptly get nano-Au solution, 4 ℃ of preservations are standby;
(6) prepare golden labeling antibody: (0.01mol/L pH9.0-9.5) regulates pH to 6.5-8.5 with solution of potassium carbonate with step (5) gained nano-Au solution.To wherein adding IgG solution, making IgG concentration is 20 μ g/mL, shakes up back 4 ℃ and leaves standstill 50 minutes.Adding bovine serum albumin(BSA) then, to make its concentration be 2%, stirring reaction 2 hours, and 4 ℃ left standstill 3 hours.1500r/min is centrifugal 25 minutes then, discards precipitation, again with supernatant with centrifugal 60 minutes of 4 ℃ of 20000r/min, abandoning supernatant.With precipitation be suspended in the 10mL phosphate buffer (0.01mol/L, pH7.0-7.4) in, with 20000r/min 4-6 ℃ centrifugal 30 minutes, the precipitation be suspended in 8mL(0.01mol/L, pH7.0-7.4) in the phosphate buffer, 4 ℃ of preservations.Adopt the specificity of indirect competitive enzyme-linked immunosorbent absorption method antagonist to detect, gained antibody is respectively 95%, 68%, 61% and 59% to the cross reacting rate of nitrofurazone, furazolidone, furaltadone and furantoin as a result, and detectability is respectively 22.5,31.5,35.1,36.3ng/mL.Show that with the 5-nitryl furfural be the antibody that haptens has obtained to discern four kinds of itrofurans medicines;
(7) prepare immune nanometer gold test paper strip: (a): after step (6) gained gold labeling antibody is diluted 4 times with phosphate buffer (0.01mol/L, pH7.0-7.4 contain 3% sucrose, 0.1% bovine serum albumin(BSA), 0.05% Tween-20), by 20 μ l/cm
2Dosage be sprayed on (in conjunction with discharging pad) on the plain film of Glass 33 type glass fibre, 37 ℃ of bakings are 20 minutes then; (b): with described capture antigen carbonic acid buffer (0.01mol/L, pH9.0-9.5, contain 3% methyl alcohol) dilution be 8 μ g/ml, spray 1 μ l in the detection line position of AE99 type nitrocellulose filter, at the goat anti-rabbit igg antibody 1 μ l of 1000 times of dilutions of the nature controlling line position of this nitrocellulose filter spraying, detection line and nature controlling line spacing 8mm.Nitrocellulose filter was dried by the fire 10 minutes at 37 ℃.Again with nitrocellulose filter at ambient temperature (20～25 ℃) be immersed in the phosphate buffer (0.01mol/L, pH7.0-7.4 contain 0.5%BSA, 0.05% Sodium azide) sealing after 10 minutes, 37 ℃ of bakings 10 minutes; (c): as shown in Figure 2, successively AE99 nitrocellulose filter, combination are discharged the plain film of pad Glass 33 glass fibre, absorption pad Cotton linters 2668 and sample pad Glass 33 and stick on the PVC backboard, be cut to the test strips of 4 * 50mm at last.
(8) feed sample detection: the feed sample 5 that takes by weighing after grinding restrains in test tube, and (1:1 v/v), vibrated 5 minutes centrifugal 5 minutes of 4000r/min to the methanol/water solution of adding 10mL 50%.Draw 100 μ L sample extraction drops of 10 times of dilutions during detection to the test strips sample pad, read the result in 5 minutes, nature controlling line colour developing and detection line does not develop the color then is judged as the positive, promptly contains a kind of in nitrofurazone, furaltadone, furantoin and the furazolidone in the sample at least.
The present invention can detect nitrofurazone in the feed, furaltadone, furantoin and furazolidone simultaneously, and the detectability of four kinds of medicines is respectively 2,2,3,5 μ g/g.
Embodiment 2
(1) synthetic universal hapten: take by weighing 5-nitryl furfural 50mg and be dissolved in (A liquid) in the 5mL ethanol; Take by weighing sulfuric acid hydrazine 85mg and be dissolved in (B liquid) in the 5mL distilled water; Under the room temperature A liquid dropwise is added drop-wise in the B liquid, stirring reaction 60 minutes gets yellow mercury oxide; With in the saturated sodium carbonate solution and above-mentioned reactant liquor to pH6.0-8.0, with this mixed liquid suction filtration to doing; Last solid washs with 20mL distilled water, and suction filtration is to doing again, and air dry promptly gets universal hapten;
(2) synthetic immunogen: take by weighing described haptens 15mg and be dissolved in the 5mL methyl alcohol, dropwise be added drop-wise under the stirring condition 5mL phosphate buffer that contains bovine serum albumin(BSA) 30mg (0.01mol/L, pH7.0-7.4) in; Dropwise add 25% glutaraldehyde solution 60 μ L, room temperature reaction 4 hours then; In 4 ℃ of physiological saline, dialysed 72 hours the freezing preservation of dislysate then;
(3) synthetic capture antigen: take by weighing described haptens 10mg and be dissolved in the 10mL methyl alcohol, be cooled to 4 ℃; Then, regulating pH with hydrochloric acid under 4 ℃ of conditions is 1.0-3.0, dropwise adds the sodium nitrite solution 4mL of 0.3mol/L again, and stirring reaction is till the dusty blue to the starch potassium iodide paper check; Transfer pH to 7.5-9.0 with sodium hydroxide solution then; Gained solution slowly joined in the 5mL phosphate buffer that contains ovalbumin 10mg (0.01mol/L, pH7.0-7.4), 4 ℃ of stirring reaction 8-16h; In physiological saline, dialysed 72 hours the freezing preservation of dislysate then;
(4) preparation polyclonal antibody: is 1mg/mL with described immunogene with the physiological saline dilution, add with the Fu Shi Freund's complete adjuvant of dilution back immunogen solution equivalent and make emulsifying agent, carry out head at the back of New Zealand white rabbit intracutaneous multi-point injection and exempt from, immunizing dose 0.2mg/ only; After 4 weeks, the immunogen solution of getting 1mg/mL carries out booster immunization at the subcutaneous multi-point injection in New Zealand white rabbit back after adding the Fu Shi Freund emulsification of immunogen solution equivalent therewith, immunizing dose 0.2mg/ only, per 4 all booster immunizations once, immunity is 7 times altogether; Last immunity is the direct auricular vein injection of immunogene, and immunizing dose 0.2mg/ only; Then, get the rabbit whole blood separation of serum, adopt the saturated ammonium sulfate method that the IgG in the serum is slightly carried; Crude extract phosphate buffer (0.01mol/L, pH7.0-7.4) in the dialysis 2 days with desalination; Dislysate carries out purifying with the DEAE-52 chromatographic column again; IgG solution behind the purifying again phosphate buffer (0.01mol/L, pH7.0-7.4) in the dialysis 3 days with desalination, last freezing preservation;
(5) preparation nano-Au solution: get 0.01% chlorauric acid solution 100mL and be heated with stirring to boiling with the constant temperature magnetic stirrer, at the citric acid three sodium solution 1mL that continues adding 4% under the condition of stirring, continued agitating heating 20 minutes, be bright redness to solution, the room temperature cooling, return to 100mL with deionized water, promptly get nano-Au solution, 4 ℃ of preservations are standby;
(6) the golden labeling antibody solution of preparation: (0.01mol/L pH9.0-9.5) regulates pH to 6.5-8.5 with solution of potassium carbonate with step (5) gained nano-Au solution; To wherein adding IgG solution, making antibody concentration is 25 μ g/mL, shakes up back 4 ℃ and leaves standstill 30 minutes; Adding bovine serum albumin(BSA) then, to make its concentration be 1%, stirring reaction 1 hour, and 4 ℃ left standstill 2 hours; 1500r/min is centrifugal 20 minutes then, discards precipitation, again with supernatant with centrifugal 30 minutes of 4 ℃ of 20000r/min, abandoning supernatant; With precipitation be suspended in the 15mL phosphate buffer (0.01mol/L, pH7.0-7.4) in, with centrifugal 60 minutes of 4 ℃ of 20000r/min, precipitation was suspended in 5mL(0.01mol/L, pH7.0-7.4) in the phosphate buffer, promptly gets golden labeling antibody solution, 4 ℃ of preservations;
(7) prepare immune nanometer gold test paper strip: (a): after step (6) gained gold labeling antibody solution is diluted 6 times with phosphate buffer (0.01mol/L, pH7.0-7.4 contain 3% sucrose, 0.1% bovine serum albumin(BSA), 0.05% Tween-20), by 20 μ l/cm
2Dosage be sprayed on (in conjunction with discharging pad) on the plain film of Glass 33 type glass fibre, 37 ℃ of bakings are 10 minutes then; (b): with described capture antigen carbonic acid buffer (0.01mol/L, pH9.0-9.5,3% methyl alcohol) dilution is 8 μ g/ml, spray 2 μ l in the detection line position of AE99 type nitrocellulose filter, at the goat anti-rabbit igg antibody 2 μ l of 1500 times of dilutions of the nature controlling line position of this nitrocellulose filter spraying, detection line and nature controlling line spacing 5mm; Nitrocellulose filter was dried by the fire 15 minutes at 37 ℃; Again with nitrocellulose filter at ambient temperature (20～25 ℃) be immersed in the phosphate buffer (0.01mol/L, pH7.0-7.4,0.5%BSA, 0.05% Sodium azide) sealing after 10 minutes, 37 ℃ of bakings 15 minutes; (c): successively AE99 nitrocellulose filter, combination are discharged the plain film of pad Glass 33 glass fibre, absorption pad Cotton linters 2668 and sample pad Glass 33 and stick on the PVC backboard; Be cut to the test strips of 4 * 50mm at last.
The feed sample 5 that takes by weighing after grinding restrains in test tube, and (1:1 v/v), vibrated 5 minutes centrifugal 5 minutes of 4000r/min to the methanol/water solution of adding 10mL 50%.Draw 100 μ L sample extraction drops of 10 times of dilutions during detection to the test strips sample pad, read the result in 5 minutes, nature controlling line and detection line all develop the color (two red line), then be judged to feminine gender, do not contain any in nitrofurazone, furaltadone, furantoin and the furazolidone in the sample.
Embodiment 3
(1) synthetic universal hapten: take by weighing 5-nitryl furfural 80mg and be dissolved in (A liquid) in the 4mL ethanol; Take by weighing sulfuric acid hydrazine 100mg and be dissolved in (B liquid) in the 10mL distilled water; Under the room temperature A liquid dropwise is added drop-wise in the B liquid, stirring reaction 40 minutes gets yellow mercury oxide; With in the saturated sodium carbonate solution and above-mentioned reactant liquor to pH6.0-8.0, with this mixed liquid suction filtration to doing; Last solid washs with 10mL distilled water, and suction filtration is to doing again, and air dry promptly gets universal hapten;
(2) synthetic immunogen: take by weighing described haptens 20mg and be dissolved in the 10mL methyl alcohol, under the stirring condition dropwise be added drop-wise to the phosphate buffer that contains bovine serum albumin(BSA) 50mg (0.01mol/L, pH7.0-7.4) in; Dropwise add 25% glutaraldehyde solution 100 μ L, room temperature reaction 4 hours then; In 4 ℃ of physiological saline, dialysed 72 hours the freezing preservation of dislysate then;
(3) synthetic capture antigen: take by weighing described haptens 15mg and be dissolved in the 5mL methyl alcohol, be cooled to 4 ℃; Then, regulating pH with hydrochloric acid under 4 ℃ of conditions is 1.0-3.0, dropwise adds the sodium nitrite solution 3mL of 0.2mol/L again, and stirring reaction is till the dusty blue to the starch potassium iodide paper check; Transfer pH to 7.5-9.0 with sodium hydroxide solution then; Gained solution dropwise joined in the 10mL phosphate buffer that contains ovalbumin 15mg (0.01mol/L, pH7.0-7.4), 4 ℃ of stirring reaction 8-16h; In physiological saline, dialysed 72 hours the freezing preservation of dislysate then;
(4) preparation polyclonal antibody: is 1mg/mL with described immunogene with the physiological saline dilution, add with the Fu Shi Freund's complete adjuvant of dilution back immunogen solution equivalent and make emulsifying agent, carry out head at the back of New Zealand white rabbit intracutaneous multi-point injection and exempt from, immunizing dose 0.8mg/ only; After 3 weeks, the immunogen solution of getting 1mg/mL carries out booster immunization at the subcutaneous multi-point injection in New Zealand white rabbit back after adding the Fu Shi Freund emulsification of immunogen solution equivalent therewith, immunizing dose 0.8mg/ only, per 4 all booster immunizations once, immunity is 6 times altogether; Last immunity is the direct auricular vein injection of immunogene, and immunizing dose 0.8mg/ only; Then, get the rabbit whole blood separation of serum, adopt the saturated ammonium sulfate method that the IgG in the serum is slightly carried; Crude extract phosphate buffer (0.01mol/L, pH7.0-7.4) in the dialysis 2 days with desalination; Dislysate carries out purifying with the DEAE-52 chromatographic column again; IgG solution behind the purifying again phosphate buffer (0.01mol/L, pH7.0-7.4) in the dialysis 2 days with desalination, last freezing preservation;
(5) preparation nano-Au solution: get 0.03% chlorauric acid solution 100mL and be heated with stirring to boiling with the constant temperature magnetic stirrer, at the citric acid three sodium solution 4mL that continues adding 1% under the stirring, continued agitating heating 25 minutes, be bright redness to solution, the room temperature cooling, return to 100mL with deionized water, promptly get nano-Au solution, 4 ℃ of preservations are standby;
(6) the golden labeling antibody solution of preparation: (0.01mol/L pH9.0-9.5) regulates pH to 6.5-8.5 to the nano-Au solution of step (5) gained with solution of potassium carbonate; To wherein adding IgG solution, making antibody concentration is 23 μ g/mL, shakes up back 4 ℃ and leaves standstill 60 minutes; Adding bovine serum albumin(BSA) then, to make its concentration be 1%, stirring reaction 2 hours, and 4 ℃ left standstill 3 hours; 1500r/min is centrifugal 25 minutes then, discards precipitation, again with supernatant with centrifugal 50 minutes of 4 ℃ of 20000r/min, abandoning supernatant; With precipitation be suspended in the 20mL phosphate buffer (0.01mol/L, pH7.0-7.4) in, with centrifugal 40 minutes of 4 ℃ of 20000r/min, precipitation was suspended in 10mL(0.01mol/L, pH7.0-7.4) in the phosphate buffer, promptly gets golden labeling antibody solution, 4 ℃ of preservations;
(7) prepare immune nanometer gold test paper strip: (a): after step (6) gained gold labeling antibody solution is diluted 5 times with phosphate buffer (0.01mol/L, pH7.0-7.4 contain 3% sucrose, 0.1% bovine serum albumin(BSA), 0.05% Tween-20), by 20 μ l/cm
2Dosage be sprayed on (in conjunction with discharging pad) on the plain film of Glass 33 type glass fibre, 37 ℃ of bakings are 15 minutes then; (b): with described capture antigen carbonic acid buffer (0.01mol/L, pH9.0-9.5,3% methyl alcohol) dilution is 8 μ g/ml, spray 2 μ l in the detection line position of AE99 type nitrocellulose filter, at the goat anti-rabbit igg antibody 2 μ l of 1000 times of dilutions of the nature controlling line position of this nitrocellulose filter spraying, detection line and nature controlling line spacing 6mm; Nitrocellulose filter was dried by the fire 20 minutes at 37 ℃; Again nitrocellulose filter is immersed in the phosphate buffer (0.01mol/L, pH7.0-7.4,0.5%BSA, 0.05% Sodium azide) sealing after 10 minutes under 20～25 ℃, 37 ℃ of bakings 20 minutes; (c): successively AE99 nitrocellulose filter, combination are discharged the plain film of pad Glass 33 glass fibre, absorption pad Cotton linters 2668 and sample pad Glass 33 and stick on the PVC backboard; Be cut to the test paper of 4 * 50mm at last.
The feed sample 5 that takes by weighing after grinding restrains in test tube, and (1:1 v/v), vibrated 5 minutes centrifugal 5 minutes of 4000r/min to the methanol/water solution of adding 10mL 50%.Draw 100 μ L sample extraction drops of 10 times of dilutions during detection to the test strips sample pad, read the result in 5 minutes, nature controlling line colour developing and detection line does not develop the color then is judged as the positive, promptly contains a kind of in nitrofurazone, furaltadone, furantoin and the furazolidone in the sample at least.
Claims (3)
1. the immune nanometer gold test paper strip of four kinds of itrofurans medicines of a fast detecting is characterized in that its preparation method is as follows:
(1) synthetic universal hapten: take by weighing 5-nitryl furfural 50-80mg be dissolved in the 3-5mL ethanol A liquid; Take by weighing sulfuric acid hydrazine 70-100mg be dissolved in the 5-10mL distilled water B liquid; Under the room temperature A liquid dropwise is added drop-wise in the B liquid, stirring reaction 30-60 minute, gets yellow mercury oxide; With in the saturated sodium carbonate solution and above-mentioned reactant liquor to pH6.0-8.0, with this mixed liquid suction filtration to doing; Last solid washs with 10-20mL distilled water, and suction filtration is to doing again, and air dry promptly gets universal hapten;
(2) synthetic immunogen: take by weighing described haptens 10-20mg and be dissolved in the 5-10mL methyl alcohol, dropwise being added drop-wise to the concentration that contains bovine serum albumin(BSA) 30-50mg under the stirring condition is among 0.01mol/L, the pH7.0-7.4 phosphate buffer 5-10mL; Dropwise add concentration then and be 25% glutaraldehyde solution 60-100 μ L, 20-25 ℃ of reaction promptly get immunogen solution in 4 hours, dialysed 72 hours the freezing preservation of dislysate then in 4 ℃ of physiological saline;
(3) synthetic capture antigen: take by weighing described haptens 10-20mg and be dissolved in the 5-10mL methyl alcohol, be cooled to 4 ℃; Then, regulating pH with hydrochloric acid under 4 ℃ of conditions is 1.0-3.0, dropwise adds the sodium nitrite solution 1-4mL of 0.2-0.5mol/L again, and stirring reaction is till the dusty blue to the starch potassium iodide paper check; Transfer pH to 7.5-9.0 with sodium hydroxide solution then; Gained solution is dropwise joined among the phosphate buffer 5-10mL that the concentration that contains ovalbumin 10-20mg is 0.01mol/L, pH7.0-7.4,4 ℃ of stirring reaction 8-16h promptly get capture antigen solution, in physiological saline, dialysed 72 hours the freezing preservation of dislysate then;
(4) preparation polyclone IgG antibody: is 1mg/mL with described immunogene with the physiological saline dilution, add with the Fu Shi Freund's complete adjuvant of dilution back immunogen solution equivalent and make emulsifying agent, carry out head at the back of animal used as test rabbit intracutaneous multi-point injection and exempt from, immunizing dose 0.2-0.8mg/ only; 3-4 is after week, the immunogen solution of getting 1mg/mL carries out booster immunization at the subcutaneous multi-point injection of animal used as test man's rabbit back after adding Fu Shi Freund emulsification with immunogen solution equivalent, immunizing dose 0.2-0.8mg/, per 4 all booster immunizations once are total to immune 6-8 time; Last immunity is the direct auricular vein injection of immunogene, and immunizing dose 0.2-0.8mg/ only; Then, get the rabbit whole blood separation of serum, adopt the saturated ammonium sulfate method that the IgG in the serum is slightly carried; Crude extract is dialysed 2-3 days with desalination in the phosphate buffer of concentration 0.01mol/L, pH7.0-7.4; Dislysate carries out purifying with the DEAE-52 chromatographic column again; IgG solution behind the purifying is again at concentration 0.01mol/L, and dialysis promptly got polyclone IgG antibody-solutions, last freezing preservation with desalination in 2-3 days in the phosphate buffer of pH7.0-7.4;
(5) preparation nano-Au solution: the chlorauric acid solution 100mL constant temperature of getting 0.01%-0.05% is heated with stirring to boiling, at the citric acid three sodium solution 1-4mL that continues to add under the condition of stirring 1%-4%, continued agitating heating 20-30 minute, be bright redness to solution, the room temperature cooling, return to 100mL with deionized water, promptly get nano-Au solution, 4 ℃ of preservations are standby;
(6) prepare golden labeling antibody: the nano-Au solution of step (5) gained is regulated pH to 6.5-8.5 with the solution of potassium carbonate of concentration 0.01mol/L; To wherein adding step (4) gained polyclone IgG solution, making the IgG antibody concentration is 20-25 μ g/mL, shakes up back 4 ℃ and leaves standstill 30-60 minute; Adding bovine serum albumin(BSA) then, to make its concentration be 1%-2%, and stirring reaction 1-2 hour, 4 ℃ left standstill 2-3 hour; The centrifugal 20-30 of 1500r/min minute then, discard precipitation, again with supernatant with 4 ℃ of centrifugal 30-60 of 20000r/min minutes, abandoning supernatant; Precipitation is suspended in the phosphate buffer of 10-20mL concentration 0.01mol/L, pH7.0-7.4, with 4 ℃ of centrifugal 30-60 of 20000r/min minutes, precipitation is suspended in the phosphate buffer of 5-10mL concentration 0.01mol/L, pH7.0-7.4, is golden labeling antibody solution, 4 ℃ of preservations;
(7) preparation immune nanometer gold test paper strip: (a): the phosphate buffer of step (6) gained gold labeling antibody solution concentration 0.01mol/L, pH7.0-7.4 is diluted 4-6 doubly, contain 3% sucrose, 0.1% bovine serum albumin(BSA) and 0.05% Tween-20 in the described phosphate buffer, by 20 μ l/cm
2Dosage be sprayed on the plain film of Glass 33 type glass fibre promptly in conjunction with discharging on the pad, 37 ℃ of bakings are 10-20 minute then; (b): is 8 μ g/ml with described capture antigen with the carbonic acid buffer dilution that contains 3% methyl alcohol of concentration 0.01mol/L, pH9.0-9.5, at the detection line position of AE99 type nitrocellulose filter spraying 1-2 μ l; At the goat anti-rabbit igg antibody 1-2 μ l that spraying 1000-1500 in the nature controlling line position of this nitrocellulose filter doubly dilutes, detection line and nature controlling line spacing 5-8mm; Nitrocellulose filter was dried by the fire 10-20 minute at 37 ℃; Again nitrocellulose filter was immersed in the phosphate buffer of concentration 0.01mol/L, pH7.0-7.4 sealing 10 minutes under 20～25 ℃, and contained 0.5%BSA and 0.05% Sodium azide in the described phosphate buffer, then with nitrocellulose filter 37 ℃ of bakings 10-20 minute; (c): successively AE99 nitrocellulose filter, combination are discharged the plain film of pad Glass 33 glass fibre, absorption pad Cotton linters 2668 and sample pad Glass 33 and stick on the PVC backboard; Be cut to the test strips of 4 * 50mm at last.
2. the immune nanometer gold test paper strip of four kinds of itrofurans medicines of a kind of fast detecting according to claim 1, when it is characterized in that prepared immune nanometer gold test paper strip is used for on-the-spot the detection, handle sample to be checked earlier, the sample to be checked 100 μ L that will handle well then drip to sample pad, read the result in 5 minutes, if it is that nature controlling line and detection line all develop the color that two red colour developing bands are arranged, then be judged to feminine gender; If it is nature controlling line colour developing and detection line does not develop the color that a red colour developing band is arranged, then be judged to the positive.
3. the immune nanometer gold test paper strip of four kinds of itrofurans medicines of a kind of fast detecting according to claim 1 is characterized in that prepared immune nanometer gold test paper strip is respectively 2 μ g/g, 2 μ g/g, 3 μ g/g and 5 μ g/g to the detectability of nitrofurazone, furaltadone, furantoin and furazolidone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100513935A CN102183634A (en) | 2011-03-04 | 2011-03-04 | Immune nano gold test strip for quickly detecting four nitrofuran medicaments |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100513935A CN102183634A (en) | 2011-03-04 | 2011-03-04 | Immune nano gold test strip for quickly detecting four nitrofuran medicaments |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102183634A true CN102183634A (en) | 2011-09-14 |
Family
ID=44569845
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011100513935A Pending CN102183634A (en) | 2011-03-04 | 2011-03-04 | Immune nano gold test strip for quickly detecting four nitrofuran medicaments |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102183634A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102890155A (en) * | 2012-09-12 | 2013-01-23 | 暨南大学 | Fluorescent test strip based on resonance energy transfer, and preparation method and application for fluorescent test strip |
| CN103364554A (en) * | 2012-04-01 | 2013-10-23 | 北京勤邦生物技术有限公司 | Test paper and method for detecting nitrofuran-like medicines |
| CN103364555A (en) * | 2012-04-07 | 2013-10-23 | 北京勤邦生物技术有限公司 | Kit and method for detecting furacilin metabolin |
| CN105283761A (en) * | 2013-06-10 | 2016-01-27 | 旭化成纤维株式会社 | Immunochromatographic diagnosis kit |
| CN107515299A (en) * | 2017-07-07 | 2017-12-26 | 西北农林科技大学 | A kind of bioprobe, the test strips for detecting furazolidone and its application |
| CN109239328A (en) * | 2018-09-26 | 2019-01-18 | 南京财经大学 | A kind of immunochromatographic method of four kinds of metabolites of nitrofuran of quick detection |
| CN112578110A (en) * | 2020-12-16 | 2021-03-30 | 西北农林科技大学 | Biological probe and kit for detecting furacilin and furazolidone and application |
| CN114137206A (en) * | 2021-12-02 | 2022-03-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colloidal gold test strip, kit and detection method for detecting fumonisins |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793926A (en) * | 2005-12-08 | 2006-06-28 | 上海交通大学 | Preparation method of immune nanometer gold test paper strip for fast detecting furazolidone |
| WO2008115580A2 (en) * | 2007-03-21 | 2008-09-25 | Charm Sciences, Inc. | Method for detecting nitrofuran |
-
2011
- 2011-03-04 CN CN2011100513935A patent/CN102183634A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793926A (en) * | 2005-12-08 | 2006-06-28 | 上海交通大学 | Preparation method of immune nanometer gold test paper strip for fast detecting furazolidone |
| WO2008115580A2 (en) * | 2007-03-21 | 2008-09-25 | Charm Sciences, Inc. | Method for detecting nitrofuran |
Non-Patent Citations (2)
| Title |
|---|
| 徐一平: "动物源性食品中硝基呋喃类药物残留分析研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 李军等: "硝基呋喃类药物广谱特异性多克隆抗体的制备与鉴定", 《畜牧与兽医》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103364554A (en) * | 2012-04-01 | 2013-10-23 | 北京勤邦生物技术有限公司 | Test paper and method for detecting nitrofuran-like medicines |
| CN103364554B (en) * | 2012-04-01 | 2016-02-24 | 北京勤邦生物技术有限公司 | A kind of test paper and method detecting Nitrofuran metabolites |
| CN103364555A (en) * | 2012-04-07 | 2013-10-23 | 北京勤邦生物技术有限公司 | Kit and method for detecting furacilin metabolin |
| CN103364555B (en) * | 2012-04-07 | 2016-02-24 | 北京勤邦生物技术有限公司 | A kind of kit and method detecting Furacilin metabolite |
| CN102890155A (en) * | 2012-09-12 | 2013-01-23 | 暨南大学 | Fluorescent test strip based on resonance energy transfer, and preparation method and application for fluorescent test strip |
| CN102890155B (en) * | 2012-09-12 | 2015-04-29 | 暨南大学 | Fluorescent test strip based on resonance energy transfer, and preparation method and application for fluorescent test strip |
| CN105283761A (en) * | 2013-06-10 | 2016-01-27 | 旭化成纤维株式会社 | Immunochromatographic diagnosis kit |
| CN105283761B (en) * | 2013-06-10 | 2018-02-09 | 旭化成株式会社 | Immunochromatography diagnostic kit |
| CN107515299A (en) * | 2017-07-07 | 2017-12-26 | 西北农林科技大学 | A kind of bioprobe, the test strips for detecting furazolidone and its application |
| CN109239328A (en) * | 2018-09-26 | 2019-01-18 | 南京财经大学 | A kind of immunochromatographic method of four kinds of metabolites of nitrofuran of quick detection |
| CN112578110A (en) * | 2020-12-16 | 2021-03-30 | 西北农林科技大学 | Biological probe and kit for detecting furacilin and furazolidone and application |
| CN114137206A (en) * | 2021-12-02 | 2022-03-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colloidal gold test strip, kit and detection method for detecting fumonisins |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102183634A (en) | Immune nano gold test strip for quickly detecting four nitrofuran medicaments | |
| CN106940373B (en) | The immunochromatography time-resolved fluorescence kit of four kinds of mycotoxin composite pollutions such as synchronous detection fumonisin B1 and its application | |
| CN104017070B (en) | The synthetic method of the derosal complete antigen of a kind of highly sensitive | |
| CN103575889B (en) | A kind of test strips and method detecting vancomycin | |
| CN102746403A (en) | Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin | |
| CN108409749B (en) | Hexa-gelsemicine haptens and holoantigen and the preparation method and application thereof | |
| CN109239336A (en) | A kind of test strips and its application detecting Mobucin | |
| CN103849604B (en) | Hybridoma cell strain ST03, anti-aflatoxin biosynthesis precursor ST monoclonal antibody and application thereof | |
| CN104502554A (en) | Immunoassay method for nano-colloidal gold marker of tadalafil and analogs thereof | |
| CN109265404A (en) | A kind of preparation method and application of carbendazim haptens and antigen | |
| CN109061146A (en) | A kind of test strips and its preparation method and application detecting Acetamiprid | |
| CN109265401A (en) | A kind of preparation method and application of iprodione haptens and antigen | |
| CN103193883B (en) | Method for synthesizing artificial antigen of specific ractopamine | |
| CN103255109B (en) | Hybridoma cell strain AFG1G6, its monoclonal antibody and aflatoxin G1 immunochromatographic test strip | |
| CN104892445A (en) | Preparation and application of highly specific amantadine artificial antigen | |
| CN103713133A (en) | Test strip for detecting spiramycin, streptomycin, gentamycin and neomycin, and method | |
| CN110108875A (en) | The synchronous immuno-chromatographic test paper strip for detecting aflatoxin, cyclopiazonic acid, ochratoxin A and zearalenone | |
| CN108931640A (en) | A kind of test strips and its application detecting organophosphorus pesticide | |
| CN102702345B (en) | Melamine antigen, related antibody and preparation method thereof | |
| CN104479007A (en) | Carbodiimide synthesis method for citrinin-protein carrier artificial coupled antigen | |
| CN109187948B (en) | Paroxarsone and nitrophenylarsonic acid duplex detection test paper | |
| CN109324183A (en) | A kind of glucocorticoid immunochromatography wide spectrum detection card and the preparation method and application thereof | |
| CN102363634A (en) | Method for preparing pyrethroid pesticide polyclonal antibody | |
| CN106978402B (en) | Hybridoma cell strain Fm7A11 and fumonisin B produced by same1Monoclonal antibodies | |
| CN114262376A (en) | Oyster four-insulin-like peptide polyclonal antibody and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110914 |