CN102768279B - Colloidal gold test strip for detecting residue of sulfonamides, usage method and application thereof - Google Patents
Colloidal gold test strip for detecting residue of sulfonamides, usage method and application thereof Download PDFInfo
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Abstract
The invention discloses a colloidal gold test strip, which comprises an absorption pad, a nitrocellulose membrane, a gold-labeled pad and a sample pad that are sequentially laminated and pasted on a plastic lining board. The cellulose nitrate membrane is provided with a detection line and a quality control line; the gold-labeled pad is coated with colloidal gold-labeled monoclonal antibodies capable of identifying sulfonamides; the detection line is coated with a sulfonamide coating antigen; and the quality control line is coated with a goat or rabbit anti-rat IgG. The invention also discloses a usage method of the colloidal gold test strip and application of the colloidal gold test strip to detection of residues of sulfonamides. The colloidal gold test strip provided by the invention can be used for detecting residues of sulfonamides in tissue samples, such as milk, pork, chicken, beef, lamb, fish, shrimp, pork liver, chicken liver, egg and honey, etc.; and the colloidal gold test strip provided by the invention can simultaneously detect 16 sulfonamides, and has characteristics of high sensitivity, strong specificity, simple operation, fastness, accuracy and low cost.
Description
Technical field
The invention belongs to residue of veterinary drug analysis and immunological technique field, be specifically related to a kind of colloidal gold strip and using method and application that detects sulfa drug residue.
Background technology
Sulfa drugs Chang Zuowei feed addictive or animal epidemic medicine are widely used, irrational medication meeting causes residual in animal food of sulfa drugs, affect food security, and then the health of harm humans, various countries have made more and more stricter regulation to the sulfa drug residue limitation in animal derived food.
Comprise high performance liquid chromatography, gas chromatography, High Performance Liquid Chromatography/Mass Spectrometry, gas phase-mass spectrum, thin-layered chromatography, capillary electrophoresis for the detection method of sulfa drug residue in food and feed.The advantages such as that colloidal gold immunity chromatography has is quick, highly sensitive, simple to operate, strong adaptability, are applicable to a large amount of sample field monitorings.Therefore be more suitable for residual in animal tissue of fast detecting sulfa drugs.
Application number is that the Chinese invention patent of CN200610019642.1 discloses a kind of immunity colloidal gold test paper strip that detects sulfa drug residue; Application number is that the Chinese invention patent of CN200810089431.4 discloses a kind of immunity colloidal gold test paper strip that detects sulfalene oxazole relict and preparation method thereof; Application number is that the Chinese invention patent of CN200910189654.2 discloses a kind of immunity colloidal gold test paper strip that detects sulfadiazine residue and preparation method thereof; Application number is that the Chinese invention patent of CN200610019642.1 discloses a kind of sulfadimidine colloidal-gold detecting-card and production and using method.These disclosed patented methods all can only detect one or more sulfa drugss.Application number is that the Chinese utility model patent of CN201020579015.5 discloses a kind of colloidal gold test paper card that detects sulfa drug residue, although this test card can detect the residual of 13 kinds of sulfa drugss, detectability is between 5-20ng/ml, but for the sulfa drugs that reaches more than 20 kind, or far from being enough.
Summary of the invention
The object of the invention is to, for the current insufficient problem of the disposable detection of drugs of sulfa drug residue test strip, provides colloidal gold strip and using method and the application of 16 kinds of sulfa drug residues of the disposable detection of a kind of energy.
The present invention is that (this monoclonal antibody is secreted by hybridoma 4E5 based on a kind of high sensitivity monoclonal antibody that can identify 16 kinds of sulfa drugss simultaneously, hybridoma 4E5 is by the center preservation of Chinese Typical Representative culture collection, preserving number is CCTCC NO:C201148), by preparing collaurum, antibody after mark purifying, optimizes that every chromatography condition etc. completes.
Above-mentioned purpose is achieved through the following technical solutions:
A kind of colloidal gold strip, comprise absorption pad, nitrocellulose filter, gold mark pad, sample pad, described nitrocellulose filter is provided with detection line and nature controlling line, described absorption pad, nitrocellulose filter, gold mark pad, sample pad stick on plastics lining board successively, described gold mark pad is coated with the monoclonal antibody that can identify sulfa drugs of colloid gold label, described detection line is coated with sulfanilamide (SN) coating antigen, and described nature controlling line is coated with sheep or rabbit anti-mouse igg.
The described monoclonal antibody that can identify sulfa drugs is secreted by hybridoma 4E5, described hybridoma 4E5 delivers Chinese Typical Representative culture collection center (CCTCC) preservation that is positioned at Wuhan City, Hubei Province Wuhan University on June 29th, 2011, preserving number is CCTCC NO:C201148.
Described sulfanilamide (SN) coating antigen is the conjugate (SM2-OVA) being formed by sulfadimidine (SM2) and ovalbumin (OVA).
The using method of described colloidal gold strip, comprises that with sample extracting solution, to tissue sample pre-treatment and the step that detects with colloidal gold strip, the pH value of described sample extracting solution is 7.6-8, and its compound method is: accurately take KH
2pO
40.41g, K
2hPO
45.59g, ultrapure water dissolves, and is settled to 1000mL, and the method for described pre-treatment is: take the tissue sample 1g of homogenate, add sample extracting solution 10ml, vibrate 5 minutes to mixing, draw supernatant.
Described tissue sample is one or more in pork, chicken, beef, mutton, the flesh of fish, shrimp, pork liver, chicken gizzard, egg, honey.
The application of described colloidal gold strip in sulfa drug residue detects.
Concrete steps are as follows:
(1) recovery hybridoma 4E5, immune mouse, preparation monoclonal antibody, and utilize sad ammonium sulfate to carry out purifying.
(2) utilize trisodium citrate reduction method to prepare collaurum, optimize flag condition, prepare the monoclonal antibody that can identify sulfa drugs of colloid gold label;
(3) coating antigen SM2-OVA and sheep or rabbit anti-mouse igg are coated on nitrocellulose filter;
(4) testing sample is extracted with sample extracting solution, utilize ELISA test strip.
When detection, if there is sulfa drugs in sample, the monoclonal antibody combination of medicine elder generation and colloid gold label, while arriving detection line, just can not there is not combination with the coating antigen (SM2-OVA) being coated on nitrocellulose filter in the monoclonal antibody that has combined the colloid gold label of sulfa drugs, just can not form at detection line place macroscopic red precipitate line, the monoclonal antibody of colloid gold label continues swimming forward, be enriched on nature controlling line, form red precipitate line, be judged to positive findings.If do not contain sulfa drugs in sample, when detection, will there is combination with the coating antigen (SM2-OVA) being coated on nitrocellulose filter in the monoclonal antibody of colloid gold label, form macroscopic red precipitate line at detection line place, the antibody of the complete colloid gold label of unreacted continues swimming forward, be enriched on nature controlling line, form red precipitate line, be judged to negative findings.
The invention has the beneficial effects as follows highly sensitive, sensing range is wide, can detect nearly 16 kinds of sulfa drugss simultaneously.Simple to operate is observable colour developing result in application of sample 5min-10min rapidly, is easy to widespread adoption.
Brief description of the drawings
Fig. 1 is Technology Roadmap of the present invention.
Fig. 2 is the structural representation of colloidal gold strip of the present invention, in figure: 1-absorption pad; 2-nitrocellulose filter; 3-gold mark pad; 4-sample pad; 5-detection line; 6-nature controlling line; 7-plastics lining board.
Fig. 3 is the colour developing schematic diagram of colloidal gold strip of the present invention, in figure: a-feminine gender, the b-positive, c-are invalid, and wherein T is detection line, and C is nature controlling line.
Embodiment
Below by embodiment, the invention will be further described, but do not limit the present invention.
The preparation of embodiment 1 colloidal gold strip
The preparation of 1.1 monoclonal antibodies and purifying
1.1.1 the preparation of ascites: only get Balb/c number of mice for first 7 days in inoculation, every mouse peritoneal injection 0.5ml incomplete Freund's adjuvant carries out pre-service.Suspending by preserving number with RPMI 1640 basal mediums is the hybridoma 4E5 expansion cultured cells of CCTCC NO:C201148, and cell number is adjusted to 1 × 10
6individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spiritual variation, the dying ascites that gathers when motionless.
1.1.2 the purifying of monoclonal antibody: the method with reference in Zhu Li equality " immunology common experimental method " People's Medical Officer Press 2000 editions: the mouse ascites 5ml that gets gained mixes with appropriate silicon dioxide, add isopyknic barbitol buffer solution, after room temperature vibration 1h, room temperature leaves standstill 30min, get supernatant in clean centrifuge tube, 4 DEG C, the centrifugal 10min of 3000 r/m, get supernatant 8ml, add 16ml 0.06mol sodium-acetate buffer, with HCL adjust pH to 4.5, under fully stirring, slowly add after sad 132 μ l, stirring at room temperature 30min, then proceed to 4 DEG C of refrigerators and fully precipitate 2h, 4 DEG C, the centrifugal 30min of 15000 r/m, obtain supernatant 22ml, add the phosphate buffer of 2.2ml 0.1M, with NaOH adjust pH to 7.6, under stirring, slowly adding ammonium sulfate to final concentration is 0.277g/ml, 4 DEG C of refrigerators fully precipitate after 2h, 4 DEG C, the centrifugal 30min of 12000r/m, abandon supernatant, precipitation is resuspended with the phosphate buffer of 5ml 0.1M, pack bag filter into, after 5000ml 0.01M pH7.2 phosphate buffer (PBS) is fully dialysed, again to 2000ml distill water dialysis, finally 3000ml tri-is boiled off to ionized water dialysis, the PEG-400 for protein solution (PEG-20000) of fully dialysing is concentrated into 3ml, then 4 DEG C, the centrifugal 30min of 12000r/m, abandon precipitation, collect supernatant, recording protein concentration is 1.6mg/ml.Be accredited as the monoclonal antibody of purifying through SDS-PAGE electrophoresis, purity is 98%.This monoclonal antibody can be used for the reagent of preparation detection sulfa drug residue.
The preparation of the monoclonal antibody of 1.2 collaurums, colloid gold label
1.2.1 the preparation of solution
(1) the preparation of gold chloride: boil off ionized water dissolved chlorine auric acid with two, be made into 1% solution, put 4 DEG C for subsequent use, the term of validity four months.1000ml1% chlorauric acid solution formula: 10g gold chloride; Two ionized waters that boil off are settled to 1000ml.
(2) the preparation of trisodium citrate: with two deionized water dissolving trisodium citrates that steam, be made into 1% solution, 0.22 μ m membrane filtration mistake, put 4 DEG C for subsequent use, the term of validity four months, two ionized waters that boil off are settled to 1000ml.
(3) the preparation of 0.1M sal tartari: boil off ionized waters preparation with two, 0.22 μ m membrane filtration mistake, put 4 DEG C for subsequent use, the term of validity four months.1000ml 0.1M solution of potassium carbonate formula: 13.8g sal tartari; Two ionized waters that boil off are settled to 1000ml.
(4) the preparation of 2% PEG-20000: boil off ionized waters preparation with two, 0.22 μ m membrane filtration mistake, put 4 DEG C for subsequent use, the term of validity four months.1000ml2% PEG-20000 solution formula: 20g PEG-20000; Two ionized waters that boil off are settled to 1000ml.
(5) the preparation that liquid is preserved in mark washing: 2% bovine serum albumin(BSA) (BSA), 0.05% Sodium azide (NaN
3), 0.01M pH 7.2 PBS solution, 0.22 μ m membrane filtration mistake, put 4 DEG C for subsequent use, the term of validity four months.Formula of liquid is preserved in the washing of 1000ml mark: 20g BSA, 0.5g NaN
3,0.01M pH7.2 PBS solution is settled to 1000ml.
1.2.2 the preparation of collaurum:
1% gold chloride is diluted to 0.01% with two ionized waters that boil off, puts electric furnace and boil, add 2ml 1% trisodium citrate by every 100ml 0.01% gold chloride, continue to boil, stop heating until liquid is shiny red, supply dehydration after being cooled to room temperature.The collaurum outward appearance preparing should be pure, bright, without precipitation and floating thing, the term of validity one week.
1.2.3 the preparation of the monoclonal antibody of colloid gold label:
20ml collaurum is added in 50ml small beaker, with magnetic stirrer 250rpm stirring, add the K of 0.1M
2cO
3be adjusted to best pH place.Slowly dropwise add the secreted monoclonal antibody of hybridoma 4E5, stirring reaction 30min.Add 10% BSA solution, make BSA final concentration reach 1%, continue to stir 10min.Under 4 DEG C of conditions, leave standstill 1h.By monoclonal anti nanocrystal composition good mark with 10000r/min, 4 DEG C of centrifugal 30min, supernatant discarded; Precipitation is resuspended in original volume, twice of repeated centrifugation with the 10mM phosphate buffer (PB, pH value is about 7.0) containing 1%BSA; Precipitation contains 0.02%NaN with the PB(of the 10mM containing 1%BSA preparing
3) damping fluid is resuspended in original volume 1/10,4 DEG C save backup.
The preparation of 1.3 gold medal mark pads 3
The preparation of confining liquid: 2% BSA, 0.1% Triton X-100 (Triton X-100), 0.05% NaN
3, 0.01M pH 7.2 PBS solution, 0.22 μ m miillpore filter filter, put 4 DEG C for subsequent use, the term of validity four months.1000 ml confining liquid formulas: 20g BSA, 0.5g NaN
3,1ml Triton X-100,0.01M pH7.2PBS solution are settled to 1000ml.
The preparation of gold mark pad 3: gold mark pad 3 is soaked in confining liquid after 30min, in 37 DEG C of oven dry.Then by the colloid gold label monoclonal antibody uniform spreading preparing on gold mark pad 3,20 square centimeters of every ml soln pavings, freeze drying, encapsulation, put 4 DEG C for subsequent use.
The preparation of 1.4 test strips sample pad 4
The preparation of confining liquid: 2% BSA, 0.1% Triton X-100,0.05% NaN
3, 0.01M pH 7.2 PBS solution, 0.22 μ m miillpore filter filter, put 4 DEG C for subsequent use, the term of validity four months.1000 ml confining liquid formulas: 20g BSA, 0.5g NaN
3,1ml Triton X-100,0.01M pH7.2PBS solution are settled to 1000ml.
The preparation of sample pad 4: sample pad 4 is soaked in confining liquid after 30 min, in 37 DEG C of oven dry, encapsulation, put 4 DEG C for subsequent use.
The assembling of 1.5 test strips
Using method and the application of embodiment 2 colloidal gold strips
The optimization of 2.1 colloidal gold strip detection methods
Coating antigen is dissolved, be mixed with the mother liquor of 1mg/ml, be diluted to 1mg/ml, 0.8mg/ml, a series of concentration of 0.6mg/ml, 0.4mg/ml, utilize the kynix standard items of 10 μ g/L concentration to detect, utilize PBS to detect as negative control simultaneously, when drug test line does not develop the color completely, negative control detection line develops the color when obvious, selects the coated concentration now coated concentration as colloidal gold strip coating antigen.
Sheep or rabbit anti-mouse igg are diluted to 1mg/ml, 0.8mg/ml, 0.6mg/ml, 0.4mg/ml, 0.2mg/ml concentration, are coated on nitrocellulose filter, as nature controlling line.Nature controlling line and detection line colour developing situation are consistent as far as possible, require negative blank two detection line bands colour developing clear, according to colour developing situation, and the finally coated concentration of sheep or rabbit anti-mouse igg in definite three kinds of different test strips.
Coating antigen dilutes a series of concentration on nitrocellulose filter, assembling test strips after dry, the methoxy pyridazine that utilization is diluted to 10 μ g/L detects, and in the time that the coated concentration of detection line is greater than 0.8mg/ml, detects sulfamethoxypyridazine 10 μ g/L standard items, detection line colour developing, testing result is negative, and when coated concentration is less than or equal to 0.8mg/ml, testing result is positive, blank detection line colour developing simultaneously also can shoal accordingly, and the coated concentration of finally selecting sulfanilamide (SN) coating antigen is 0.8mg/ml.
By sheep or the anti-mouse of rabbit two is anti-is diluted to a series of concentration, colour developing result develops the color and compares with detection line, finally selects the coated concentration of 0.2mg/ml as unmarked sheep or rabbit anti-mouse igg.
2.2 sample-pretreating method
The preparation (pH7.6-8.0) of sample extracting solution: accurately take KH
2pO
40.41g, K
2hPO
45.59g, ultrapure water dissolves, and is settled to 1000mL.
Milk sample disposal route: fresh milk sample directly dilutes after 10 times, drips 4 in sample well; Judged result in 5～10min.
Tissue sample disposal route: take in tissue sample (pork, chicken, beef, mutton, the flesh of fish, shrimp, pork liver, chicken gizzard or egg) 1.0 ± 0.01g to the 10ml centrifuge tube of homogenate, add Extraction buffer 10ml, 5min is to mixing in vibration, draw supernatant with dropper, drip 4 in sample well; Judged result in 5～10min.
Honey sample disposal route: get in honey 1.0 ± 0.01g to 50ml centrifuge tube, add Extraction buffer 9ml, 5min is to mixing in vibration, draws supernatant with dropper, drips 4 in sample well; Judged result in 5～10min.
Attention: this method is 10 to the extension rate of sample.
2.3 colloidal gold strip detecting steps
When each use ELISA test strip, all need to arrange contrast: the sample that drips 100 μ L water or feminine gender with pipettor or dropper is in sample port.
Sample detection method: after testing sample is processed, get 100 μ L and add in well, relatively judge the yin and yang attribute of testing sample by the colour developing situation of sample detection test strips and control stripes bar detection line.
Yin and yang attribute criterion: as consistent in the test strip shade of sample detection test strips and contrast or slightly shallow, result is judged as feminine gender; If sample detection ELISA test strip line does not occur or detection line is obviously shallower than control stripes bar detection line, result is judged as the positive.
Determining of 2.4 colloidal gold strip performance parameters
2.4.1 detectability
The instrument of learning from else's experience detects each 20 parts of (instrumental method carries out according to " the mensuration Liquid Chromatography-Tandem Mass Spectrometry of 16 kinds of residual quantity of sulfonamides in GB/T 20759-2006 livestock and poultry meat ") negative tissue to be measured (pork, chicken, beef, mutton, the flesh of fish, shrimp, pork liver, chicken gizzard, egg, honey or milk) sample, adds respectively each sulfa drug to different concentration.Get the colloidal gold strip of 3 batches and measure, under room temperature condition, operate, visual inspection judges, taking the least concentration that occurs positive findings as detectability.
Experimental result shows, adds concentration while being 20 μ g/kg, and it is clear that the sample nature controlling line that adds fanasil detects, and detection line is without colour developing; When interpolation concentration is 30 μ g/kg, the sample nature controlling line detection of adding 5-methoxysulfadiazine, sulphadiazine, daimeton is clear, and detection line is without colour developing; When interpolation concentration is 40 μ g/kg, the sample nature controlling line detection of adding sulfamethyldiazine is clear, and detection line is without colour developing; When interpolation concentration is 50 μ g/kg, the sample nature controlling line detection of adding sulfaclozine, cistosulfa, sulfaisodimidine is clear, and detection line is without colour developing; When interpolation concentration is 70 μ g/kg, the sample nature controlling line detection of adding sulphathiazole is clear, and detection line is without colour developing; When interpolation concentration is 80 μ g/kg, the sample nature controlling line detection of adding sulfadimethoxine, sulfaquinoxaline is clear, and detection line is without colour developing; When interpolation concentration is 90 μ g/kg, the sample nature controlling line detection of adding sulfadimidine is clear, and detection line is without colour developing; When interpolation concentration is 100 μ g/kg, the sample nature controlling line detection of adding sulfamethoxypyridazine is clear, and detection line is without colour developing; When interpolation concentration is greater than 250 μ g/kg, the sample nature controlling line detection of adding Sulfamethoxazole, ayerlucil, NU-445 is clear, and detection line is without colour developing.Therefore, can judge that this test card is as shown in table 1 at the detectability of each tissue.
The detectability of this test strip of table 1 to each medicine in different tissues
| Medicine | Detectability (μ g/kg) | Medicine | Detectability (μ g/kg) |
| Fanasil | 20 | Sulphathiazole | 70 |
| 5-methoxysulfadiazine | 30 | Sulfaquinoxaline | 80 |
| Daimeton | 30 | Sulfadimethoxine | 80 |
| Sulphadiazine | 30 | Sulfadimidine | 90 |
| Sulfamethyldiazine | 40 | Sulfamethoxypyridazine | 100 |
| Sulfaisodimidine | 50 | Sulfamethoxazole | 250 |
| Sulfaclozine | 50 | NU-445 | 300 |
| Cistosulfa | 50 | Ayerlucil | 300 |
2.4.2 specificity
By the test strip of 3 batches to 5-methoxysulfadiazine, sulphadiazine, daimeton, fanasil, sulfadimidine, sulfaclozine, sulfadimethoxine, cistosulfa, sulfamethoxypyridazine, Sulfamethoxazole, sulfamethyldiazine, ayerlucil, sulfaquinoxaline, NU-445, sulfaisodimidine, 16 kinds of sulfa drugss such as sulphathiazole (adding concentration is detectability concentration) and salbutamol, olaquindox, Enrofloxacin, Ciprofloxacin, terramycin, tetracycline, chloromycetin, tylosin, streptomysin, the positive of 10 kinds of other drug standard items such as gentamicin is added tissue sample (concentration is 100 μ g/kg) and is detected, every kind of medicine has 10 duplicate samples.Result is as shown in table 2.
Table 2 different pharmaceutical cross reaction experimental result
Visible, test strips specificity of the present invention is good.
2.4.3 false negative rate is measured
Each 50 parts of the negative tissue sample of the LC-MS/MS that learns from else's experience confirmation, adds respectively the sulfa drugs of detectability concentration, will after sample preparation, detect respectively by three batches of test strip, calculates false negative rate.The results are shown in Table 3, the false negative rate of visible test strips of the present invention is 0.
Table 3 test strip false negative rate measurement result
2.4.4 false positive rate is measured
Each 50 parts of the negative tissue sample of the LC-MS/MS that learns from else's experience confirmation.To after sample preparation, detect by three batches of test card respectively, calculate false positive rate.The results are shown in Table 4, the false positive rate of this test card is 0 as seen.
Table 4 test card false positive rate measurement result
Claims (3)
1. a colloidal gold strip, by absorption pad (1), nitrocellulose filter (2), gold mark pad (3), sample pad (4) composition, described nitrocellulose filter (2) is provided with detection line (5) and nature controlling line (6), described absorption pad (1), nitrocellulose filter (2), gold mark pad (3), sample pad (4) sticks on plastics lining board (7) successively, it is characterized in that described gold mark pad (3) is coated with the monoclonal antibody that can identify sulfa drugs of colloid gold label, described detection line (5) is coated with sulfanilamide (SN) coating antigen, described nature controlling line (6) is coated with sheep or rabbit anti-mouse igg, the described monoclonal antibody that can identify sulfa drugs is to be that the hybridoma 4E5 of CCTCC NO:C201148 is secreted by preserving number.
2. colloidal gold strip as claimed in claim 1, is characterized in that described sulfanilamide (SN) coating antigen is the conjugate being formed by sulfadimidine and ovalbumin.
3. the application of the colloidal gold strip described in claim 1 or 2 in sulfa drug residue detects.
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| CN104597243B (en) * | 2014-12-26 | 2016-11-23 | 华中农业大学 | For detecting antibody chip test kit and the method for sulfa drug residue in food |
| CN104977411A (en) * | 2015-05-20 | 2015-10-14 | 集美大学 | Colloidal gold immunochromatography test paper strip for detecting sulfanilamide drugs, and preparation method thereof |
| CN105116094A (en) * | 2015-08-14 | 2015-12-02 | 舟山出入境检验检疫局综合技术服务中心 | Fast detecting method for sulfonamide residues in shrimps |
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