CN103293304B - A kind of kit and method detecting sulfa drugs - Google Patents
A kind of kit and method detecting sulfa drugs Download PDFInfo
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Abstract
The invention discloses a kind of kit and the method that detect sulfa drugs.Kit comprises micropore reagent and test paper, and in described micropore reagent, freeze-drying has the sulfa drugs specific antibody of colloid gold label, and described sulfa drugs specific antibody can be sulfa drugs monoclonal antibody or sulfa drugs polyclonal antibody; Described test paper is connected to form successively by absorption of sample pad, reaction film, adsorptive pads, diaphragm, base plate, described reaction film comprises detection zone and quality control region, and detection zone is coated with sulfonamide hapten-carrier protein couplet thing, and quality control region is coated with antiantibody.The method of sulfa drugs is detected with kit of the present invention, simple, quick, directly perceived, accurate, applied widely, cost is low, easily promote the use of.
Description
Technical field
The present invention relates to a kind of kit and the method that detect sulfa drugs, be specifically related to a kind of colloidal gold strip for detecting sulfa drugs, it is particularly suitable for the detection of sulfa drug residue in milk sample.
Technical background
Sulfa drugs (Sulfonamides, SAs) be sulphanilamide derivant by Prof. Du Yucang, gram-positive bacteria and some negative bacterium can be suppressed, various bacteria can be treated infect, veterinary clinic is widely used in treat the various livestock and poultries infected by sensitive bacterial, can also improve the conversion ratio of feed as feed addictive and promote growth of animal simultaneously, SAs conventional at present has tens kinds.But such medicine exists serious side effects, the medium-term and long-term existence of human body can cause many bacteriums to produce drug resistance to sulfa drugs, and has potential carcinogenicity, has therefore caused great attention both domestic and external.The Ministry of Agriculture of China No. 235 file specifies that its residue limits is 100 μ g/kg.
At present, the detection method of sulfa drug residue mainly contains microbial method, physico-chemical analysis method and immunization etc.Microbial method is easy, expense is low, but during operating cost and susceptibility, specificity are all poor, qualitative, quantitative all has difficulties, can not meet the needs of modern residue detection, even some testing result does not also reach the requirement of maximum residue limit(MRL); Physics and chemistry method often as confirmation method for measuring the medicament residue in animal tissue, as gas chromatography (GC), high performance liquid chromatography (HPLC), compounds GC-MS (GC-MS), liquid-mass chromatography (HPLC-MS) etc., these methods are sensitive, accurate, high specificity, degree of separation are good, can Simultaneously test multi-medicament, but need the complex pretreatment of expensive instrument, sample, loaded down with trivial details cost that is time-consuming, that detect higher, can not execute-in-place, and need professional to operate, so limit its application; By comparison, immunological assay method has that specificity is good, highly sensitive, easy and simple to handle, testing cost is low, be suitable for the advantages such as the selective mechanisms of batch sample, can meet China's livestock and poultry cultivation family, food enterprise, government function supervision department etc. better and carry out testing.
Summary of the invention
An object of the present invention is to provide a kind of kit detecting sulfa drugs.
Kit provided by the present invention comprises micropore reagent and test paper, and micropore reagent has micropore plug, and test paper comprises base plate, absorption of sample pad, reaction film, adsorptive pads, diaphragm, and it connects successively; In described micropore reagent, freeze-drying has sulfa drugs specific antibody-colloid gold label thing; Sulfa drugs specific antibody can be sulfa drugs monoclonal antibody or sulfa drugs polyclonal antibody; Reaction film comprises detection zone and quality control region; When sulfa drugs specific antibody is sulfa drugs monoclonal antibody, detection zone bag is by sulfonamide hapten-carrier protein couplet thing, and quality control region bag is by sheep anti mouse antiantibody; When sulfa drugs specific antibody is sulfa drugs polyclonal antibody, detection zone bag is by sulfonamide hapten-carrier protein couplet thing, and quality control region bag is by goat-anti rabbit antiantibody.
Described sulfonamide hapten-carrier protein couplet thing is obtained by sulfonamide hapten and carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
Sulfa drugs specific antibody in described sulfa drugs specific antibody-colloid gold label thing prepares using sulfonamide hapten-carrier protein as immunogene.
Described sulfa drugs is sulphadiazine, madribon, 5-methoxysulfadiazine, sulfamethazine, daimeton, NU-445, sulfaquinoxaline, Sulfamethoxazole, sulfamethoxypyridazine, cistosulfa, sulfabenzamide, sulfadoxine.
Described absorption of sample pad is pasted with diaphragm, is test side, has MAX mark line above.
Described detection zone is positioned at the one end of the diaphragm being bordering on MAX mark, and described quality control region is positioned at the one end away from the diaphragm having MAX to mark.
Described base plate can be the material that PVC base plate or other hard do not absorb water; Described absorption of sample pad can be suction strainer paper or filter paper for oil; Described adsorptive pads is thieving paper; Described reaction film can be nitrocellulose filter or cellulose acetate membrane; Described diaphragm is PE material diaphragm.
Another object of the present invention is to provide a kind of method preparing mentioned reagent box, and it comprises step:
1) the micropore reagent that freeze-drying has sulfa drugs specific antibody-colloid gold label thing is prepared;
2) preparation has bag by the detection zone of sulfonamide hapten-carrier protein couplet thing and bag by the reaction film of the quality control region of antiantibody;
3) by 2) reaction film for preparing and absorption of sample pad, adsorptive pads, diaphragm, base plate be assembled into test paper;
4) by 1) and 3) freeze-drying for preparing has the micropore reagent of sulfa drugs specific antibody-colloid gold label thing and test paper to be assembled into kit.
Specifically, step comprises:
1) 4-ASC and 6-ACA 6-aminocaproic acid are reacted, preparation sulfonamide hapten;
2) by sulfonamide hapten and carrier protein couplet, preparation sulfonamide hapten-carrier protein couplet thing;
3) with sulfonamide hapten-carrier protein couplet thing immune mouse, mouse boosting cell and murine myeloma cell are passed through to merge, screen, obtain secreting sulfa drugs monoclonal antibody hybridoma cell strain or with sulfonamide hapten-carrier protein immunize rabbit, obtain sulfa drugs polyclonal antibody;
4) extract mouse IgG or rabbit igg immune health goat, obtain sheep anti-mouse igg antibody or goat-anti rabbit antiantibody;
5) react with trisodium citrate and gold chloride and prepare collaurum;
6) the sulfa drugs specific antibody of preparation is joined in the collaurum of preparation, obtain sulfa drugs specific antibody-colloid gold label thing;
7) by sulfa drugs specific antibody-colloid gold label thing freeze-drying in micropore reagent, micropore reagent is added micropore plug;
8) by absorption of sample pad be 7.2 containing 0.5% bovine serum albumin(BSA) (volumn concentration), pH, 0.1mol/L phosphate buffer soaks 2h, dries 2h at 37 DEG C;
9) on base plate, absorption of sample pad, reaction film, adsorptive pads and diaphragm is pasted in order;
10) the micropore reagent prepared and test paper are assembled into kit, preserve 12 months under 2 ~ 8 DEG C of conditions.
Another object of the present invention is to provide a kind of method applying sulfa drug residue in mentioned reagent box detection milk sample, and it comprises step:
(1) detect with kit;
(2) testing result is analyzed.
The Cleaning Principle of kit of the present invention: milk sample drop to be checked is added in micropore reagent, after mixing, hatch 5min, MAX mark line end will be indicated downward, insert the micropore reagent after hatching, spread to reaction film together with after measuring samples liquid combines with the golden labeling antibody in micropore; If the content of sulfa drugs is high in measuring samples liquid, sulfa drugs then in diffusion process in measuring samples liquid can combine with golden labeling antibody, and then close the antigen-combining site of sulfa drugs on golden labeling antibody completely, golden labeling antibody sulfonamide hapten-carrier protein couplet thing on reaction film is stoped to be combined, do not develop the color in detection zone, antiantibody then can be combined with golden labeling antibody, and quality control region develops the color; If the low or nothing of the content of sulfa drugs in measuring samples liquid, antigen binding site then on golden labeling antibody can not be closed, and then golden labeling antibody can be combined by sulfonamide hapten-carrier protein couplet antigen on reaction film, develop the color in detection zone, antiantibody also can be combined with golden labeling antibody simultaneously, and quality control region develops the color.If quality control region does not develop the color, then test paper lost efficacy.As shown in Figure 4.
Positive: when quality control region (C) demonstrates red stripes, and when detection zone (T) does not develop the color, to be judged to the positive.
Negative: when quality control region (C) demonstrates red stripes, detection zone (T) also demonstrates red stripes simultaneously, is judged to feminine gender.
Invalid: when quality control region (C) does not demonstrate red stripes, then no matter whether test section (T) demonstrates red stripes, and it is invalid that this test paper is judged to.
Kit of the present invention have highly sensitive, high specificity, cost are low, simple to operate, detection time is short, be applicable to various units uses, stores advantage that is simple, long shelf-life.Wherein, adopt the sulfa drugs monoclonal antibody of high specific, ensure that the reliability of testing result; By golden labeling antibody freeze-drying in micropore reagent, in testing process, golden labeling antibody can be made fully to contact with measuring samples liquid, fully react, thus reduce error, increase the reaction sensitivity of whole system.Detect that the antibiotic method of sulfa drugs is easy, quick, directly perceived, accurate, applied widely, cost is low with kit of the present invention, easily promote the use of.
Accompanying drawing explanation
Fig. 1 is test paper cross-sectional view.
Fig. 2 is test paper vertical view.
Fig. 3 is micropore reagent figure.
Fig. 4 is detection paper result process decision chart.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Embodiment 1 detects the formation of the kit of sulfa drugs
One, test paper (being called test strips) (Fig. 1)
Described test paper is made up of base plate, absorption of sample pad, reaction film, adsorptive pads, diaphragm;
Described absorption of sample pad 1, reaction film 2, adsorptive pads 3 and diaphragm 7 are pasted onto on base plate 6 successively in order, the end of absorption of sample pad is connected with reaction film, the end of reaction film is connected with adsorptive pads, the top of absorption of sample pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate;
Described test paper is pasted with diaphragm, and diaphragm 7 covers on absorption of sample pad, is test side, is printed on MAX printed words (Fig. 2) above;
Described reaction film there are detection zone 4 and quality control region 5, all in the ribbon vertical with the appearance of described test strips, detection zone is positioned at the one end of the diaphragm being bordering on MAX mark, quality control region is positioned at the one end away from the diaphragm having MAX to mark, detection zone is coated with sulfonamide hapten-carrier protein couplet thing (conjugate of sulfonamide hapten-ovalbumin), and quality control region is coated with sheep anti mouse antiantibody;
Described base plate is PVC base plate; Described absorption of sample pad is suction strainer paper; Described adsorptive pads is thieving paper; Described reaction film is nitrocellulose filter; Described diaphragm is PE material diaphragm.
Two, micropore reagent (Fig. 3)
On described micropore reagent 8, freeze-drying has sulfa drugs monoclonal antibody-colloid gold label thing, package amount 0.20 ~ 0.25 μ g/ml; Described micropore reagent has micropore plug 9.
Above-mentioned micropore reagent and test paper are assembled into kit, preserve in 2 ~ 8 DEG C of environment, the term of validity 12 months.
The preparation method of kit described in embodiment 2 embodiment 1
One, the preparation of kit
The preparation method of this kit mainly comprises the following steps:
1) the micropore reagent that freeze-drying has sulfa drugs monoclonal antibody-colloid gold label thing is prepared;
2) preparation has bag by the detection zone of sulfonamide hapten-carrier protein couplet thing and bag by the reaction film of the quality control region of sheep anti mouse antiantibody;
3) by 2) reaction film for preparing and absorption of sample pad, adsorptive pads, diaphragm, base plate be assembled into test paper;
4) by 1) and 3) freeze-drying for preparing has the micropore reagent of sulfa drugs monoclonal antibody-colloid gold label thing and test paper to be assembled into kit.
Substep describes in detail below:
(1) preparation of each parts
1. the synthesis of sulfonamide hapten-carrier protein couplet thing and qualification
Sulfa drugs is small-molecule substance, only has immunoreactivity, does not have immunogenicity, can not bring out body produce immune response, must with macromolecular carrier albumen coupling after just there is immunogenicity.
(1) preparation of sulfonamide hapten
A) in 25ml single port bottle, add 6-amino-nicotinic acid 0.87g, ethanol 20ml, 12mol/L hydrochloric acid 3.7ml, adds hot reflux, and reaction 8h, TLC monitoring, reacts complete, removal of solvent under reduced pressure, be dissolved in saturated NaHCO
3aqueous solution, extraction into ethyl acetate, anhydrous Na
2sO
4drying, column chromatography (ethyl acetate/petroleum ether, 2/1, v/v) purifying;
B) in 25ml single port bottle, a) products therefrom 0.89g is added, triethylamine (Et
3n) 1.6ml, CH
2cl
210ml, after stirring and dissolving, adds catalyzer DMAP (DMAP).The 2ml dichloromethane solution of slow instillation 4-ASC, TLC monitors, and 5h, processes after completion of the reaction, dry, column chromatography purification (ethyl acetate/petroleum ether, 1/10, v/v);
C) in 25ml single port bottle, add b) products therefrom 1g, 2mol/LNaOH aqueous solution 120ml, add hot reflux, TLC monitors, reaction 10h, complete, regulates pH4 ~ 5, has solid to separate out, be sulfonamide hapten.
(2) immunogenic preparation---sulfonamide hapten and bovine serum albumin(BSA) conjugate synthesize
Get 12mg sulfonamide hapten, be dissolved in 1mlN, in dinethylformamide (DMF), obtain solution I; Get 15mg carbodiimides (EDC) 0.2ml water fully dissolve after in adding in I, stirred at ambient temperature 24h, obtains solution II; Take bovine serum albumin(BSA) (BSA) 40mg, make it fully to be dissolved in 3mlPBS (pH7.2), solution II is dropwise slowly added drop-wise in protein solution, and in stirred at ambient temperature 24h; With 0.01mol/LPBS4 DEG C of dialysis 3 days, change 3 dislysates every day, to remove unreacted small-molecule substance, namely obtain sulfa drugs immunogene; Packing, saves backup in-20 DEG C.
(3) preparation of coating antigen---sulfonamide hapten and ovalbumin conjugate synthesize
Get 15mg sulfonamide hapten 0.8mlDMF to dissolve, be cooled to 10 DEG C, obtain solution I; Getting isobutyl chlorocarbonate 10 μ l adds in I, 10 DEG C of stirring reaction 30min; Get 30mg ovalbumin 2.2ml50mmol/LNa
2cO
3dissolve, 10 DEG C of reaction 4h, then 4 DEG C are spent the night; With 0.01mol/LPBS4 DEG C of dialysis 3 days, change 3 dislysates every day, to remove unreacted small-molecule substance, namely obtain sulfa drugs coating antigen; Packing, saves backup in-20 DEG C.
(4) qualification of sulfonamide hapten-carrier protein couplet thing
The PBS of carrier protein, sulfonamide hapten, sulfonamide hapten-carrier protein couplet thing pH7.4 is made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PBS, with ultraviolet spectrophotometer in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of carrier protein, sulfonamide hapten, sulfonamide hapten-carrier protein couplet thing.There is different absorption curves in three, shows sulfonamide hapten and carrier protein couplet success.
2. the preparation of sulfa drugs monoclonal antibody
(1) animal immune
Immunogene step 1 obtained is injected in Balb/c Mice Body, and immunizing dose is 150 μ g/, makes it produce antiserum.
(2) Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, in 8:1 (quantitative proportion) ratio and SP2/0 myeloma cell fusion, screening obtains the sulfa drugs monoclonal hybridoma strain of stably excreting sulfa drugs monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma cryopreserving liquid is made 1 × 10
9the cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
(4) preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, cultivates under 37 DEG C of conditions, by sad-saturated ammonium sulfate method, the nutrient solution obtained is carried out purifying, obtains monoclonal antibody ,-20 DEG C of preservations.
Described cell culture medium for add calf serum and sodium bicarbonate in RPMI1640 nutrient culture media, make the final concentration of calf serum in cell culture medium be 20% (mass percentage), make the final concentration of sodium bicarbonate in cell culture medium be 0.2% (mass percentage); The pH of described cell culture medium is 7.4.
3. the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, for immunogene, immunity is carried out to pathogen-free domestic sheep with mouse source antibody, obtain sheep anti mouse antiantibody.
4. the preparation of sulfa drugs monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
With two ionized water that boils off, 1% gold chloride (is purchased from sigma company, catalog number T09041) be diluted to 0.01% (mass percentage), get 100ml and be placed in conical flask, boiling is heated to thermostatic electromagnetic stirrer, under continuous high temperature, Keep agitation, add 2.5ml1% trisodium citrate (be purchased from Guangzhou Chemical Reagent Factory, catalog number BG11-AR-01KG), continue at the uniform velocity to be heated with stirring to solution be bright red time stopping, original volume is returned to deionized water, 4 DEG C of preservations after being cooled to room temperature.The collaurum outward appearance prepared is pure, bright, nothing precipitates and floating thing.
(2) preparation of sulfa drugs monoclonal antibody-colloid gold label thing
Under magnetic stirring, the pH value to 7.0 of collaurum is adjusted with 0.2mol/L solution of potassium carbonate, in colloidal gold solution, above-mentioned sulfa drugs monoclonal antibody is added by the standard adding 50 ~ 100 μ g in every milliliter of colloidal gold solution, continue to stir and evenly mix 10min, adding the final concentration that 10% bovine serum albumin(BSA) (BSA) makes it in colloidal gold solution is 1% (volumn concentration), leaves standstill 10min.12000rpm, 4 DEG C of centrifugal 40min, abandon supernatant, precipitation redissolution buffer solution twice, with volume be initial colloid gold volume 1/10 redissolution damping fluid will precipitate resuspended, the concentration of the sulfa drugs monoclonal antibody obtained-colloid gold label thing solution is 50 μ g/ml, put 4 DEG C for subsequent use.
Redissolution damping fluid: containing the 0.02mol/L phosphate buffer of bovine serum albumin(BSA) 0.1% ~ 0.3% (volumn concentration), Tween-80 0.05% ~ 0.2% (mass percentage), pH7.2.
5. by sulfa drugs monoclonal antibody-colloid gold label thing freeze-drying on micropore reagent
100 μ l sulfa drugs monoclonal antibody-colloid gold label things are added in micropore reagent microwell plate, put into freeze drier, under condenser temperature is-70 DEG C of conditions, after pre-freeze 4h, freeze-drying 14h again, can take out, obtain the micropore reagent that freeze-drying has sulfa drugs monoclonal antibody-colloid gold label thing.Sulfa drugs monoclonal antibody-colloid gold label thing freeze-drying amount is 0.20 ~ 0.25 μ g/ml.
6. the preparation of absorption of sample pad
Be placed in by absorption of sample pad and soak 2h containing 0.5% bovine serum albumin(BSA) (volumn concentration), pH7.2,0.1mol/L phosphate buffer, 37 DEG C of baking 2h are for subsequent use.
7. the preparation of reaction film
Bag is by process: with phosphate buffer, sulfonamide hapten-ovalbumin conjugate is diluted to 10mg/mL, be coated in the detection zone (T) on nitrocellulose filter with Biodot point film instrument, package amount is 1.0 μ g/cm
2; With the phosphate buffer of 0.01mol/L, pH7.4, sheep anti-mouse igg antibody is diluted to 200 μ g/ml, be coated in the quality control region (C) on nitrocellulose filter with Biodot point film instrument, package amount is 1.0 μ g/cm
2.Bag is placed in dry 2h under 37 DEG C of conditions by good reaction film, for subsequent use.
(2) assembling of each parts
1. the assembling of test paper
Described absorption of sample pad, reaction film, adsorptive pads, diaphragm are pasted onto on described base plate successively in order; The end of absorption of sample pad is connected with the top of reaction film, and the end of reaction film is connected with the top of adsorptive pads, and the top of absorption of sample pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate; Bonding protective film on the test paper assembled, one end diaphragm being printed on MAX mark line is pasted onto on absorption of sample pad.
2. the assembling of kit
The test paper obtain above-mentioned steps 1 and micropore reagent set dress up kit, store, the term of validity 12 months in the environment of 2 ~ 8 DEG C.
The detection of sulfa drugs in embodiment 3 milk
1. detect with kit
From original packing, take out reagent barrel (if low-temp storage needs pre-balance to room temperature), open rear taking-up requisite number object micropore reagent and test strips, and carry out mark; Draw milk soln to be checked, pipette 200 μ l in micropore with micropipettor, slowly suction and fully and in micropore reagent mix; The test strips marked is inserted in micropore---be printed on " MAX " line end down, make it fully to immerse in solution; After room temperature (20 DEG C-25 DEG C) hatches 5min, take out test strips, result of determination.
2. Analysis of test results
Positive: when quality control region (C) demonstrates red stripes, and when detection zone (T) does not develop the color, to be judged to the positive, as shown in fig. 4 a;
Negative: when quality control region (C) demonstrates red stripes, detection zone (T) also demonstrates red stripes simultaneously, is judged to feminine gender, as shown in Figure 4 b;
Invalid: when quality control region (C) does not demonstrate red stripes, then no matter whether detection zone (T) demonstrates red stripes, and it is invalid that this test paper is judged to, as shown in Fig. 4 c, 4d.
The determination of embodiment 4 kit technical parameter
1. sensitivity test
Sulphadiazine, madribon, 5-methoxysulfadiazine, sulfamethazine, daimeton, NU-445, sulfaquinoxaline, Sulfamethoxazole, sulfamethoxypyridazine, cistosulfa, sulfabenzamide, sulfadoxine standard items (available from Sigma) are diluted to following variable concentrations: sulphadiazine 0,30,60,120 μ g/L; Madribon, daimeton, sulfabenzamide 0,25,50,100 μ g/L; 5-methoxysulfadiazine, cistosulfa 0,15,30,60 μ g/L; Sulfamethazine 0,10,20,40 μ g/L; NU-445 0,5,10,20 μ g/L; Sulfaquinoxaline, Sulfamethoxazole 0,40,80,160 μ g/L; Sulfamethoxypyridazine, sulfadoxine 0,35,70,140 μ g/L; Used diluent is the phosphate buffer of pH7.2,0.2mol/L.
Detect with kit, result is: when sulphadiazine standard concentration is 0, 30 μ g/L, madribon, daimeton, sulfabenzamide standard concentration is 0, 25 μ g/L, 5-methoxysulfadiazine, cistosulfa standard concentration is 0, 15 μ g/L, sulfamethazine standard concentration is 0, 10 μ g/L, NU-445 standard concentration is 0, 5 μ g/L, sulfaquinoxaline, Sulfamethoxazole standard concentration is 0, 40 μ g/L, sulfamethoxypyridazine, sulfadoxine standard concentration is 0, during 35 μ g/L, test strips demonstrates macroscopic two red bar lines, be negative, when sulphadiazine standard concentration is 60, 120 μ g/L, madribon, daimeton, sulfabenzamide standard concentration is 50, 100 μ g/L, 5-methoxysulfadiazine, cistosulfa standard concentration is 30, 60 μ g/L, sulfamethazine standard concentration is 20, 40 μ g/L, NU-445 standard concentration is 10, 20 μ g/L, sulfaquinoxaline, Sulfamethoxazole standard concentration is 80, 160 μ g/L, sulfamethoxypyridazine, sulfadoxine standard concentration is 70, during 140 μ g/L, test strips quality control region develops the color, but not developing the color in detection zone, is positive, and shows that this kit is to sulphadiazine, madribon, 5-methoxysulfadiazine, sulfamethazine, daimeton, NU-445, sulfaquinoxaline, Sulfamethoxazole, sulfamethoxypyridazine, cistosulfa, sulfabenzamide, the detection sensitivity of sulfadoxine is respectively 60 μ g/L, 50 μ g/L, 30 μ g/L, 20 μ g/L, 50 μ g/L, 10 μ g/L, 80 μ g/L, 80 μ g/L, 70 μ g/L, 30 μ g/L, 50 μ g/L, 70 μ g/L.
2. false positive rate, false negative rate test
Get known Determination of sulfadiazine and be greater than the milk negative sample 20 parts that the milk positive sample 20 parts of 60 μ g/L and Determination of sulfadiazine be less than 60 μ g/L, detect respectively with the kit of 3 batches of productions, calculate its yin and yang attribute rate.The results are shown in Table 1, table 2.
Table 1 detects positive sample result
Table 2 detects negative sample result
Result shows: when the kit produced with 3 batches detects positive milk sample, and result is positive entirely, and known positive coincidence rate is 100%, and false negative rate is 0; When detecting 20 parts of negative milk samples, result is negative entirely, and known negative match-rate is 100%, and false positive rate is 0.Illustrate that detection sulfa drugs kit of the present invention can detect fast to sulfa drug residue in milk sample.
3. specific test
Specificity is commonly used cross reacting rate and is represented, refers to the ability of the antigenic determinant generation combination that antibody is different from structure.This kit is to sulphadiazine, madribon, 5-methoxysulfadiazine, sulfamethazine, daimeton, NU-445, sulfaquinoxaline, Sulfamethoxazole, sulfamethoxypyridazine, cistosulfa, sulfabenzamide, the detection sensitivity of sulfadoxine is respectively 60 μ g/L, 50 μ g/L, 30 μ g/L, 20 μ g/L, 50 μ g/L, 10 μ g/L, 80 μ g/L, 80 μ g/L, 70 μ g/L, 30 μ g/L, 50 μ g/L, 70 μ g/L, by the other drug (fluoroquinolones of inspection normal in milk, chloromycetin, aminoglycoside, Tetracyclines) use pH7.2, the phosphate buffer of 0.2mol/L dilutes, detect with the kit described in embodiment 1.Result shows, and when detecting the medicines such as fluoroquinolones, chloromycetin, aminoglycoside, Tetracyclines with this kit, all developing the color in test strips quality control region and detection zone, illustrates that this kit is to these medicine no cross reactions.
Claims (7)
1. detect a preparation method for sulfa drugs kit, it is characterized in that comprising step:
1) the micropore reagent that freeze-drying has sulfa drugs specific antibody-colloid gold label thing is prepared;
2) preparation has bag by the detection zone of sulfonamide hapten-carrier protein couplet thing and bag by the reaction film of the quality control region of antiantibody;
3) by 2) reaction film for preparing and absorption of sample pad, adsorptive pads, diaphragm, base plate be assembled into test paper;
4) by 1) and 3) freeze-drying for preparing has the micropore reagent of sulfa drugs specific antibody-colloid gold label thing and test paper to be assembled into kit;
Wherein, the synthetic method of described sulfonamide hapten is: a) in 25ml single port bottle, add 6-amino-nicotinic acid 0.87g, ethanol 20ml, 12mol/L hydrochloric acid 3.7ml, add hot reflux, reaction 8h, TLC monitoring, react complete, removal of solvent under reduced pressure, be dissolved in saturated NaHCO
3aqueous solution, extraction into ethyl acetate, anhydrous Na
2sO
4drying, ethyl acetate: sherwood oil=2:1 (v:v) column chromatography purification; B) in 25ml single port bottle, a) products therefrom 0.89g is added, triethylamine 1.6ml, CH
2cl
210ml, after stirring and dissolving, adds catalyzer DMAP, the 2ml dichloromethane solution of slow instillation 4-ASC, TLC monitors, 5h, process after completion of the reaction, dry, ethyl acetate: sherwood oil=1:10 (v:v) column chromatography purification; C) in 25ml single port bottle, add b) products therefrom 1g, 2mol/LNaOH aqueous solution 120ml, add hot reflux, TLC monitors, reaction 10h, complete, regulates pH4 ~ 5, has solid to separate out, be sulfonamide hapten; The molecular structural formula of described sulfonamide hapten is:
2. the preparation method of detection sulfa drugs kit according to claim 1, is characterized in that described test paper is pasted onto on base plate successively by absorption of sample pad, reaction film, adsorptive pads, diaphragm and forms, described micropore reagent has micropore plug.
3. the preparation method of the detection sulfa drugs kit according to any one of claim 1 or 2, is characterized in that described absorption of sample pad is pasted with diaphragm, as test side, has MAX mark line above.
4. the preparation method of detection sulfa drugs kit according to claim 3, it is characterized in that described detection zone is positioned at the one end of the diaphragm being bordering on MAX mark, described quality control region is positioned at the one end away from the diaphragm having MAX to mark.
5. the preparation method of detection sulfa drugs kit according to claim 1, it is characterized in that described sulfonamide hapten-carrier protein couplet thing is obtained by sulfonamide hapten and carrier protein couplet, described carrier protein is bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein or human serum albumins.
6. the preparation method of detection sulfa drugs kit according to claim 1, it is characterized in that the sulfa drugs specific antibody in described sulfa drugs specific antibody-colloid gold label thing prepares using sulfonamide hapten-carrier protein couplet thing as immunogene, described sulfa drugs specific antibody is sulfa drugs monoclonal antibody or sulfa drugs polyclonal antibody.
7. detect a method for sulfa drug residue in milk sample, it comprises step:
1) detect with kit prepared by the preparation method of the detection sulfa drugs kit described in any one of claim 1-6;
2) testing result is analyzed.
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| CN201210054226.0A CN103293304B (en) | 2012-03-02 | 2012-03-02 | A kind of kit and method detecting sulfa drugs |
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| CN103293304B true CN103293304B (en) | 2016-02-24 |
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| CN105137086A (en) * | 2015-07-29 | 2015-12-09 | 中国农业大学 | Kit and test strip for detecting sulfonamides and use thereof |
| CN112858683A (en) * | 2019-11-28 | 2021-05-28 | 清华大学 | Method for simultaneously and rapidly detecting multiple antibiotics by using immunobiosensor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101261274A (en) * | 2008-02-02 | 2008-09-10 | 王学生 | Method for combining colloidal gold immune leaching and immunity-chromatography for rapidly detecting medicament residue |
| CN201876457U (en) * | 2010-10-21 | 2011-06-22 | 北京望尔生物技术有限公司 | Colloidal gold test paper card for testing of sulfanilamide drug residue |
| CN201935920U (en) * | 2010-10-21 | 2011-08-17 | 北京勤邦生物技术有限公司 | Reagent kit for detecting beta-lactam antibiotics |
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| WO2010151485A1 (en) * | 2009-06-22 | 2010-12-29 | Charm Sciences, Inc. | Method and test strip for detection of residues |
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| CN101261274A (en) * | 2008-02-02 | 2008-09-10 | 王学生 | Method for combining colloidal gold immune leaching and immunity-chromatography for rapidly detecting medicament residue |
| CN201876457U (en) * | 2010-10-21 | 2011-06-22 | 北京望尔生物技术有限公司 | Colloidal gold test paper card for testing of sulfanilamide drug residue |
| CN201935920U (en) * | 2010-10-21 | 2011-08-17 | 北京勤邦生物技术有限公司 | Reagent kit for detecting beta-lactam antibiotics |
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| 磺胺母核结构半抗原的合成与表征;段振娟 等;《天津科技大学学报》;20070630;第22卷(第2期);第24-28页 * |
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