CN203069593U - Test strip for detecting spectinomycin, streptomycin, gentamicin and neomycin - Google Patents
Test strip for detecting spectinomycin, streptomycin, gentamicin and neomycin Download PDFInfo
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- CN203069593U CN203069593U CN201220510206.5U CN201220510206U CN203069593U CN 203069593 U CN203069593 U CN 203069593U CN 201220510206 U CN201220510206 U CN 201220510206U CN 203069593 U CN203069593 U CN 203069593U
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Abstract
The utility model discloses a test strip for detecting spectinomycin, streptomycin, gentamicin and neomycin. The test strip comprises a microporous strip, a microporous reagent, a test strip and a microporous plug, wherein a piece of test paper comprises a sample absorption pad, a reaction membrane, a water absorption pad, a protective film, and a bottom plate, which are sequentially connected, the reaction membrane comprises four detection lines and a quality control line, the four detection lines are enveloped by a spectinomycin-carrier protein conjugate, a streptomycin-carrier protein conjugate, a gentamicin-carrier protein conjugate, and a neomycin-carrier protein conjugate respectively, and the quality control line is enveloped by a goat anti mouse antibody. The application method of the test strip disclosed by the utility model to the detection of spectinomycin, streptomycin, gentamicin and neomycin is simple, convenient, fast, visual, accurate, wide in application range, low in cost and easy in promotion and application, and the test strip is convenient to carry about.
Description
Technical field
The utility model relates to a kind of test strips and method that detects spectinomycin, streptomysin, gentamicin and neomycin.
Background technology
Spectinomycin claims spectinomycin, spextinomyxin, grand rhzomorph etc. again, a kind of aminocyclitol antibiotic that produces for the grand sight streptomycete, Gram-negative bacteria (as brucella, Klebsiella, proteus, salmonella, Pasteurella etc.) is had and pretends usefulness, a little less than gram-positive bacteria (streptococcus, staphylococcus etc.) effect, mycoplasma also there is certain effect, be the microbiotic that a novel special efficacy is specially controlled gonorrhoea, NEISSERIA GONORRHOEAE is had stronger antibacterial action.Can promote growth of animal, be good animal feed additive, is widely used in the animal husbandry as veterinary drug.Some illegal retailers are subjected to the abuse of antibiotics of ordering about of economic interests, make a large amount of antibiotic residues in livestock products, if the long-term edible animal derived food that contains antibiotic residue, also just be equivalent to long-term low dose and absorb microbiotic, make bacterial strain generation antibiotic resistance in the human body, give and to use antibiotic therapy to bring harmful effect when ill, can destroy the organismic internal environment balance, cause flora imbalance, allergic reaction can appear in the people of microbiotic allergic constitution, cause immune hemolysis, cause a series of problems of food and environment Chinese traditional medicine residual contamination simultaneously.
Streptomysin is aminoglycoside antibiotics, uses generally in milk cow and milch goat are cultured, but because use and management is lack of standardization, often causes in fresh milk and the milk powder streptomysin residual.Owing to streptomysin, human body is had bigger toxic and side effect, can cause anaphylactic shock and deafness when serious, particularly the harm to child is bigger, can see through placenta and enter amniotic fluid and fetal circulation, the harm foetus health.Various countries have all formulated strict residual limiting the quantity of to the streptomysin in the dairy products, dihydrostreptomycin, be 0.2 mg/kg as streptomysin and always limiting the quantity of of dihydrostreptomycin in the Japanese regulation milk, limiting the quantity of of dihydrostreptomycin is 0.125 mg/kg in U.S.'s regulation breast and the milk, streptomysin and limiting the quantity of of dihydrostreptomycin are 0.2 mg/kg in China's regulation milk, so the streptomysin in the milk powder, dihydrostreptomycin residual quantity are detected for the control quality of milk powder, safeguard that human diet health is significant.
Gentamicin is a kind of aminoglycoside antibiotics that is produced by small single-cell bacteria, and multiple Grain-positive and negative bacterium are all had significant antibacterial effect, is widely used in animal husbandry as feed addictive and the microbiotic for the treatment of disease.Yet gentamicin has tangible toxic action to the mankind, and is serious to the infringement of cranial nerve, the sense of hearing and kidney.Many countries and international organization have all clearly stipulated the maximum residue limit of this medicament residue in the food.European Union's regulation, the maximum residue limit in the milk is 100 μ g/kg, the maximum residue limit in muscle, the fat is 50 μ g/kg.
Neomycin belongs to aminoglycoside antibiotics, and has a broad antifungal spectrum is widely used at veterinary clinic, is important anti-infectives, also is widely used in diseases such as treatment mastitis, keratitis, conjunctivitis, otitis, dermatitis, trauma infection contamination, foot rot.China and some other country also use neomycin as the fodder antibiotics adjuvant, be used for the prevention livestock and poultry and infect the wall losses and enteritis morning that causes, improve efficiency of feed utilization, promote growth.Though neomycin use in animal doctor and herding has obtained great success, it exists potential ototoxicity and renal toxicity to humans and animals, and for this reason, countries such as China and European Union have all formulated neomycin maximum residue limit(MRL) in the animal derived food.
The immunoassay field lacks technology and the application thereof that can detect these four kinds of medicines of spectinomycin, streptomysin, gentamicin and neomycin simultaneously at present.Therefore, be necessary in practice to set up a kind of susceptibility height, simple to operate, cost is low, the detection of drugs kind is many, be fit to the detection method of large-scale promotion.
The utility model content
The purpose of this utility model provides a kind of test strips that detects spectinomycin, streptomysin, gentamicin and neomycin simultaneously.
Test strips provided by the utility model comprises capillary strip, micropore reagent, micropore plug and test paper, and test paper comprises base plate, absorption of sample pad, reaction film, adsorptive pads, diaphragm, and it connects successively; The freeze-drying of described micropore reagent has spectinomycin specific antibody-colloid gold label thing, streptomysin specific antibody-colloid gold label thing, gentamicin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing; Have four detection lines being coated with spectinomycin-carrier protein couplet thing, streptomysin-carrier protein couplet thing, gentamicin-carrier protein couplet thing and neomycin-carrier protein couplet thing and bag on the reaction film by a nature controlling line of sheep anti mouse antiantibody.Article four, detection line and a nature controlling line are parallel to each other, and the spectinomycin detection line is near absorption of sample pad one end, and nature controlling line is near adsorptive pads one end.Streptomysin detection line, gentamicin detection line, neomycin detection line are between spectinomycin detection line and nature controlling line.Wherein the spectinomycin detection line is represented with detection line (T1), and the streptomysin detection line is represented with detection line (T2), detection line (T3) expression of gentamicin detection line, and detection line (T4) expression of neomycin detection line, nature controlling line uses nature controlling line (C) to represent.Nature controlling line (C) is 5-8mm apart from the end that the end of reaction film links to each other with adsorptive pads, detection line (T4) is apart from nature controlling line (C) 4-6mm, detection line (T3) is apart from detection line (T4) 4-6mm, detection line (T2) is apart from detection line (T3) 4-6mm, and detection line (T1) is apart from detection line (T2) 4-6mm.Diaphragm is pasted the test side on the absorption of sample pad, and the MAX mark line is arranged above.In that being housed, freeze-drying have the capillary strip of spectinomycin specific antibody-colloid gold label thing, streptomysin specific antibody-colloid gold label thing, gentamicin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing to have the micropore plug.
For the ease of transportation and use, test strips also has holding appliance and is fixed in wherein and is used for the reagent bucket that splendid attire test paper and freeze-drying have the capillary strip of spectinomycin specific antibody-colloid gold label thing, streptomysin specific antibody-colloid gold label thing, gentamicin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing, has gland bonnet on the described reagent bucket.
The utility model test strips pilot scale Cardboard Bottom Boards can be the material that PVC base plate or other hard do not absorb water; The absorption of sample pad can be suction strainer paper or filter paper for oil; Adsorptive pads is thieving paper; Reaction film can be nitrocellulose filter or cellulose acetate membrane; Diaphragm is PE material diaphragm; The test strips box body is carton box; The reagent bucket is plastics reagent bucket; Holding appliance is the rigid support material.
The utility model test strips has following beneficial effect:
1. highly sensitive: traditional test card all has the fixedly stationary installation that gets stuck of test strips, the stationary installation that gets stuck is fixing wherein with test paper tightly, to sample detection the time, because what get stuck and be combined with test paper is tightr, cause test sample solution can not fully be penetrated into one section on absorption of sample pad, and it is very slow to carry out the speed of chromatography at test paper, causes the medicine in the test sample fully to contact with the coating antigen of bag quilt on the detection line, causes the sensitivity of test card to descend.The utility model test strips is than the detection sensitivity height of traditional test card, because the test paper of the utility model test strips is naked strip, need not the housing fastener, test sample can directly contact absorption of sample pad one end of test paper, and the chromatography velocity ratio of sample on test paper is very fast, make medicine to be checked in the sample can be fully on detection line the coating antigen of bag quilt be combined, thereby improved the detection sensitivity of test strips.Spectinomycin, streptomysin, gentamicin and neomycin test strip are that the basis is prepared from the monoclonal antibody of colloid gold label high-affinity, priceless strong formation between gold grain and the antibody molecule in the gold labeling antibody, the two combines by the Van der Waals force between the charges of different polarity, collaurum is very little to monoclonal antibody specificity and affinity influence, and has higher mark rate.Spectinomycin monoclonal antibody-colloid gold label thing wherein, streptomysin monoclonal antibody-colloid gold label thing, gentamicin monoclonal antibody-colloid gold label thing and neomycin monoclonal antibody-colloid gold label thing freeze-drying is in the micropore reagent strip, in testing process, golden labeling antibody is fully contacted with sample liquid to be checked, fully reaction, thereby the minimizing error increases the reaction sensitivity of whole system.Therefore, the utility model test strips has higher specificity and sensitivity.This test strips to the spectinomycin detection sensitivity is: 20 μ g/L; To the streptomysin detection sensitivity be: 80 μ g/L; To the gentamicin detection sensitivity be: 20 μ g/L; To the neomycin detection sensitivity be: 500 μ g/L.
Easy and simple to handle fast: detect raw material milk in the past test card and raw material milk must be added dilution and dilute and can detect by point sample, reason be since fixedly the plastic casing of test paper tightly test paper is fixed in the housing, and raw material milk compares thickness, so directly raw material milk is carried out the words that point sample detects at the well of test card, since housing be combined tight with test strips and the liquid thickness of suckling due to, the raw material milk sample can't be penetrated into the absorption of sample pad fully and bond discharges in the pad, and the chromatography velocity ratio on test paper is slower, to such an extent as to can't detect, can detect after dilution dilutes so raw material milk will be added.Need not any other reagent when using the utility model test strips to detect, when detecting raw material milk, do not need raw material milk is diluted yet, reason is because the test paper in the utility model test strips is naked strip, namely need not with housing it to be fixed, as long as sample solution to be checked directly dripped in freeze-drying spectinomycin monoclonal antibody-colloid gold label thing is arranged, streptomysin monoclonal antibody-colloid gold label thing, in the micropore of gentamicin monoclonal antibody-colloid gold label thing and neomycin monoclonal antibody-colloid gold label thing, behind the mixing, hatch 5min, it is downward test paper to be indicated MAX wire tag end again, in the micropore reagent after insertion is hatched, test paper can fully contact with sample solution, observations behind the 5min.For the detection of milk sample, the utility model test strips is shorter detection time than traditional test card, because the utility model test strips need not sample is diluted, can directly detect sample.
3. show testing result image, accurately directly perceived.Whether the judgement of testing result is to develop the color as negative and positive criterion with four detection lines of test strips and a nature controlling line.If the spectinomycin detection line on the reaction film shows a red stripes, the streptomysin detection line shows a red stripes, the gentamicin detection line shows a red stripes, the neomycin detection line also shows a red stripes, nature controlling line also shows a red stripes simultaneously, and spectinomycin, streptomysin, gentamicin and neomycin are lower than test strips respectively to the lowest detectable limit of spectinomycin, streptomysin, gentamicin and neomycin in the expression sample; If the detection line of streptomysin, gentamicin, neomycin shows red stripes on the reaction film, nature controlling line also shows a red stripes simultaneously, and the spectinomycin detection line does not develop the color, and has only spectinomycin content to be equal to or higher than test strips to the low detectability of spectinomycin in the expression test sample; If the detection line of spectinomycin, gentamicin, neomycin shows red stripes on the reaction film, nature controlling line also shows a red stripes simultaneously, and the streptomysin detection line does not develop the color, and has only content of streptomycin to be equal to or higher than test strips to the low detectability of streptomysin in the expression test sample; If the detection line of spectinomycin, streptomysin, neomycin shows red stripes on the reaction film, nature controlling line also shows a red stripes simultaneously, and the gentamicin detection line does not develop the color, and has only gentamicin content to be equal to or higher than test strips to the low detectability of gentamicin in the expression test sample; If the detection line of spectinomycin, streptomysin, gentamicin shows red stripes on the reaction film, nature controlling line also shows a red stripes simultaneously, and the neomycin detection line does not develop the color, and has only neomycin content to be equal to or higher than test strips to the low detectability of neomycin in the expression test sample; If have only nature controlling line to show red stripes on the reaction film, and spectinomycin, streptomysin, gentamicin, neomycin detection line all do not show red stripes, and the content of the spectinomycin in the expression test sample, streptomysin, gentamicin, neomycin is equal to or higher than test strips simultaneously to the lowest detectable limit of spectinomycin, streptomysin, gentamicin, neomycin; If the nature controlling line on the reaction film does not show red stripes, no matter spectinomycin, streptomysin, gentamicin and (or) whether the neomycin detection line show red stripes, it is invalid that the test strips testing result all is considered as.The result judges image, directly perceived, accurate, simple and clear, is not easy to occur result's erroneous judgement.
4. cost is low, small investment: use the utility model test strips, do not need to join in addition instrument and equipment and other reagent, also do not need the fixedly stationary installation that gets stuck of test strips, and with low cost, small investment, instant effect settles on-the-spot the detection at one go.
5. be easy to apply on a large scale: test strips is simple to operate, can better satisfy different levels personnel's needs, as specialty chemical examination, health and epidemic prevention, quality monitoring, dairy industry enterprise etc., has vast market prospect and bigger economical, societal benefits.
Description of drawings
Fig. 1 is the utility model test paper section of structure;
Fig. 2 is the utility model test paper vertical view;
Fig. 3 has the capillary strip of golden labeling antibody for the utility model freeze-drying;
Fig. 4 is the utility model micropore plug vertical view;
Fig. 5 is the utility model micropore plug side view;
Fig. 6 is the utility model test strips testing result process decision chart;
Fig. 7 is the utility model reagent barrel structure synoptic diagram;
Fig. 8 is the vertical view of the utility model holding appliance;
Fig. 9 is the side view of the utility model box body and holding appliance.
Embodiment
One, the assembling of spectinomycin, streptomysin, gentamicin, neomycin test strip preparation
1) test paper
Absorption of sample pad 1, reaction film 2, adsorptive pads 3 and diaphragm 7 are pasted successively in order on described base plate 6; the end of absorption of sample pad links to each other with reaction film top; the end of reaction film links to each other with adsorptive pads; the top of absorption of sample pad aligns with the top of base plate; the end of adsorptive pads aligns with the end of base plate; diaphragm 7 covers the test side of absorption of sample pad, is printed on the MAX mark line at the test side diaphragm.Four detection lines and a nature controlling line on the reaction film are parallel to each other, and vertical with the appearance of test paper.Detection line 4-1 is detection line (T1), near absorption of sample pad one end, nature controlling line 5 is nature controlling line (C), near adsorptive pads one end, detection line 4-2 is detection line (T2), detection line 4-3 is detection line (T3), and detection line 4-4 is detection line (T4), between detection line (T) and nature controlling line (C).Nature controlling line (C) is 5-8mm apart from the end that the end of reaction film links to each other with adsorptive pads, detection line (T4) is apart from nature controlling line (C) 4-6mm, detection line (T3) is apart from detection line (T4) 4-6mm, detection line (T2) is apart from detection line (T3) 4-6mm, and detection line (T1) is apart from detection line (T2) 4-6mm.Detection line (T1) is coated with spectinomycin-carrier protein couplet thing, detection line (T2) is coated with streptomysin-carrier protein couplet thing, detection line (T3) is coated with gentamicin-carrier protein couplet thing, detection line (T4) is coated with neomycin-carrier protein couplet thing, and nature controlling line (C) is coated with the sheep anti mouse antiantibody.
Base plate is the PVC base plate, and the absorption of sample pad is suction strainer paper, and reaction film is nitrocellulose filter, and adsorptive pads is thieving paper, and diaphragm is PE material diaphragm.
2) freeze-drying has the capillary strip of golden labeling antibody
The bottom freeze-drying of described capillary strip 8 has spectinomycin monoclonal antibody-colloid gold label thing, streptomysin monoclonal antibody-colloid gold label thing, gentamicin monoclonal antibody-colloid gold label thing and neomycin monoclonal antibody-colloid gold label thing, i.e. golden labeling antibody 9.Capillary strip 8 has micropore plug 10.
3) with 1) test strips and 2) capillary strip packs in the reagent bucket 11 with sealing-plug, the reagent bucket is positioned in the holding appliance 12 in the test strips box body 13.
Two, the use of test strips
Get milk sample solution to be checked and drip 200 μ l in micropore reagent, behind the mixing, hatch 5min, it is downward that test paper is indicated MAX wire tag end, timing behind the micropore reagent after insertion is hatched, observations behind the 5min.
Three, testing result analysis
Negative (-): the colour developing of C line, four T lines all develop the color, and no matter shade represents that all spectinomycin in the milk sample, streptomysin, gentamicin, neomycin concentration all are lower than detectability, shown in Fig. 6 .a.
Positive (+): the colour developing of C line, T
1Line does not develop the color, and spectinomycin concentration is equal to or higher than detectability in the expression milk sample; The colour developing of C line, T
2Line does not develop the color, and streptomysin concentration is equal to or higher than detectability in the expression milk sample; The colour developing of C line, T
3Line does not develop the color, and gentamicin concentration is equal to or higher than detectability in the expression milk sample; The colour developing of C line, T
4Line does not develop the color, and neomycin concentration is equal to or higher than detectability in the expression milk sample, shown in Fig. 6 .a-6.f.
Invalid: as the C line not occur, show that incorrect operating process or test strips lost efficacy, shown in Fig. 6 .g-6.k.
Four, the material preparation method of using in the test strip is as follows:
1, the preparation of antigen
1), the synthetic and evaluation of spectinomycin-carrier protein couplet thing
A. the spectinomycin haptens is synthetic: get spectinomycin 0.66 g, ethyloic azanol 0.27 g and the mixed liquor of pyridine 1 ml in 20 ml DMSO, at room temperature stirring reaction is 20 hours, steaming desolventizes, behind the column chromatography purification in ethanol-water system recrystallization obtain the condensation product of spectinomycin and ethyloic azanol, molecular structure is as follows:
(formula I)
B. immunogenic preparation-spectinomycin haptens and BSA conjugate are synthetic
Get the spectinomycin haptens 38mg water-soluble solution of 5ml; Get bovine serum albumin(BSA) (BSA) the 100mg water-soluble solution of 5ml; Spectinomycin haptens aqueous solution is added in the BSA aqueous solution, react 24h with magnetic stirrer; With 0.01mol/L PBS dialysis 3 days, change liquid every day 3 times, to remove unreacted small-molecule substance.With the centrifugal 30min of 12000r/min, collect supernatant, packing, standby in-20 ℃ of preservations.
C. the preparation of coating antigen-spectinomycin haptens and OVA conjugate are synthetic
With spectinomycin haptens 22mg, 15mg N, N'-carbonyl dimidazoles (CDI) is used 1.5ml N, dinethylformamide (DMF) dissolving, and stirring at room reaction 1h can obtain reactant liquor A; Take by weighing ovalbumin (OVA) 36mg, make it fully to be dissolved in the 3.5ml 50mmol/L sodium carbonate liquor, dropwise slowly be added drop-wise to reactant liquor A in this solution; Room temperature reaction 24h with 0.01mol/L PBS dialysis 3 days, changes liquid every day 3 times, to remove unreacted small-molecule substance.With the centrifugal 30min of 12000r/min, collect supernatant, packing, standby in-20 ℃ of preservations.
D. the evaluation of spectinomycin hapten-carrier conjugates
Spectinomycin, bovine serum albumin(BSA), spectinomycin-bovine serum albumin(BSA) conjugate and spectinomycin, ovalbumin, spectinomycin-ovalbumin conjugate are made into the solution of 0.5mg/ml with the PBS of pH7.4, return to zero with 0.01mol/L pH7.4 PBS, in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of the absorption curve of spectinomycin, bovine serum albumin(BSA), spectinomycin-bovine serum albumin(BSA) conjugate and spectinomycin, ovalbumin, spectinomycin-ovalbumin conjugate with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of spectinomycin and spectinomycin and carrier protein couplet.
2), the synthetic and evaluation of streptomysin-carrier protein couplet thing
A. the streptomysin haptens is synthetic: 1.0 g streptomysins, 0.30 g phthalic anhydride and the mixed liquor of 1 ml pyridine in 20 ml DMSO, 80 ℃ of following stirring reactions 24 hours, steaming desolventizes, in the ethanol-water system repeatedly recrystallization obtain phthalic acid strand mycin ester, molecular structure is as follows:
B. immunogenic preparation-streptomysin haptens and BSA conjugate are synthetic
Get haptens 40mg with the 2mlDMF dissolving fully, make I liquid, get BSA80mg 7ml0.1MPBS(PH7.5) dissolve fully, make II liquid, I liquid is added in the II liquid, make III liquid, get EDC100mg and add in the III liquid stirring at room 2 hours with the water-soluble solution of 1ml back buffering, with 0.01MPBS dialysis three days, change liquid every day three times, get immunogene, standby in-20 ℃ of preservations.
C. the preparation of coating antigen-streptomysin haptens and OVA conjugate are synthetic
Get haptens 40mg with the 2mlDMF dissolving fully, make I liquid, get OVA80mg 7ml0.1M PBS(PH7.5) dissolve fully, make II liquid, I liquid is added in the II liquid, make III liquid, get EDC100mg and add in the III liquid stirring at room 2 hours with the water-soluble solution of 1ml back buffering, with 0.01M PBS dialysis three days, change liquid every day three times, get immunogene, standby in-20 ℃ of preservations.
D. the evaluation of streptomysin hapten-carrier conjugates
Streptomysin, bovine serum albumin(BSA), streptomysin-bovine serum albumin(BSA) conjugate and streptomysin, ovalbumin, streptomysin-ovalbumin conjugate are made into the solution of 0.5mg/ml with the PBS of pH7.4, return to zero with 0.01mol/L pH7.4 PBS, in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of the absorption curve of streptomysin, bovine serum albumin(BSA), streptomysin-bovine serum albumin(BSA) conjugate and streptomysin, ovalbumin, streptomysin-ovalbumin conjugate with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of streptomysin and streptomysin and carrier protein couplet.
3), the synthetic and evaluation of gentamicin-carrier protein couplet thing
A. the gentamicin haptens is synthetic: the solution of 0.39 g bromo-acetic acid tert-butyl in 5 ml DMSO slowly is added dropwise in 0.90 g gentamicin and the potpourri of 1 ml pyridine in 10 ml DMSO under 40 ℃.After dropwising, continue reaction after 6 hours, steaming desolventizes.After the simple column chromatography for separation, desolventizing adds 20 ml DMSO and 5 ml formic acid, room temperature reaction 20 hours, and steaming desolventizes, and recrystallization obtains the ethyloic gentamicin in the ethanol-water system, and molecular structure is as follows:
B. immunogenic preparation-gentamicin haptens and BSA conjugate are synthetic
Get haptens 35mg with the 2mlDMF dissolving fully, make I liquid, get BSA100mg 7ml0.1MPBS(PH7.5) dissolve fully, make II liquid, I liquid is added in the II liquid, make III liquid, get EDC100mg and add in the III liquid stirring at room 2 hours with the water-soluble solution of 1ml back buffering, with 0.01MPBS dialysis three days, change liquid every day three times, get immunogene, standby in-20 ℃ of preservations.
C. the preparation of coating antigen-gentamicin haptens and OVA conjugate are synthetic
Get haptens 35mg with the 2mlDMF dissolving fully, make I liquid, get OVA100mg 7ml0.1MPBS(PH7.5) dissolve fully, make II liquid, I liquid is added in the II liquid, make III liquid, get EDC100mg and add in the III liquid stirring at room 2 hours with the water-soluble solution of 1ml back buffering, with 0.01MPBS dialysis three days, change liquid every day three times, get immunogene, standby in-20 ℃ of preservations.
D. the evaluation of gentamicin hapten-carrier conjugates
Gentamicin, bovine serum albumin(BSA), gentamicin-bovine serum albumin(BSA) conjugate and gentamicin, ovalbumin, gentamicin-ovalbumin conjugate are made into the solution of 0.5mg/ml with the PBS of pH7.4, return to zero with 0.01mol/L pH7.4 PBS, in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of the absorption curve of gentamicin, bovine serum albumin(BSA), gentamicin-bovine serum albumin(BSA) conjugate and gentamicin, ovalbumin, gentamicin-ovalbumin conjugate with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of gentamicin and gentamicin and carrier protein couplet.
4), the synthetic and evaluation of neomycin-carrier protein couplet thing
A. the neomycin haptens is synthetic: the mixed liquor of 0.61 g neomycin and 10ml DMSO, slowly be added dropwise under the room temperature in the 10 ml DMSO solution of 0.14 g terephthalaldehyde, dropwise back room temperature to 60 ℃ reaction 2-4 hour, desolventizing, recrystallization obtains neomycin terephthalaldehyde list condensation product in the ethanol-water system, and molecular structure is as follows:
B. immunogenic preparation-neomycin haptens and BSA conjugate are synthetic
Get haptens 30mg with the 3mlDMF dissolving fully, make I liquid, get BSA100mg 7ml0.1MPBS(PH7.0) dissolve fully, make II liquid, I liquid is added in the II liquid, make III liquid, room temperature reaction 24 hours with 0.01MPBS dialysis three days, changes liquid every day three times, get immunogene, standby in-20 ℃ of preservations.
C. the preparation of coating antigen-neomycin haptens and OVA conjugate are synthetic
Get haptens 30mg with the 3mlDMF dissolving fully, make I liquid, get OVA100mg 7ml0.1MPBS(PH7.0) dissolve fully, make II liquid, I liquid is added in the II liquid, make III liquid, room temperature reaction 24 hours with 0.01MPBS dialysis three days, changes liquid every day three times, get immunogene, standby in-20 ℃ of preservations.
D. the evaluation of neomycin hapten-carrier conjugates
Neomycin, bovine serum albumin(BSA), neomycin-bovine serum albumin(BSA) conjugate and neomycin, ovalbumin, neomycin-ovalbumin conjugate are made into the solution of 0.5mg/ml with the PBS of pH7.4, return to zero with 0.01mol/L pH7.4 PBS, in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of the absorption curve of neomycin, bovine serum albumin(BSA), neomycin-bovine serum albumin(BSA) conjugate and neomycin, ovalbumin, neomycin-ovalbumin conjugate with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of neomycin and neomycin and carrier protein couplet.
2, the MONOCLONAL ANTIBODIES SPECIFIC FOR of spectinomycin, streptomysin, gentamicin and neomycin
(1) preparation monoclonal antibody
A. animal immune
Four kinds of immunogenes that step 1 is obtained are injected into respectively in the Balb/c mouse body, and immunizing dose is 150 μ g/, make it produce antiserum.
B. Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, in the 9:1(quantitative proportion) ratio and SP2/0 myeloma cell's fusion, screening obtains the spectinomycin monoclonal hybridoma strain of stably excreting spectinomycin monoclonal antibody, the streptomysin monoclonal hybridoma strain of stably excreting streptomysin monoclonal antibody, the gentamicin monoclonal hybridoma strain of stably excreting gentamicin monoclonal antibody and the neomycin monoclonal hybridoma strain of stably excreting neomycin monoclonal antibody respectively.
C. cell cryopreservation and recovery
Hybridoma is made 1 * 10 with cryopreserving liquid
9The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
D. MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Increment cultivation: hybridoma is placed cell culture medium, under 37 ℃ of conditions, cultivate, with sad-saturated ammonium sulfate method the nutrient solution that obtains is carried out purifying, obtain monoclonal antibody ,-20 ℃ of preservations.
Described cell culture medium is to add calf serum and sodium bicarbonate in the RPMI-1640 nutrient culture media, making the final concentration of calf serum in cell culture medium is 20%(quality percentage composition), making the final concentration of sodium bicarbonate in cell culture medium is 0.2%(quality percentage composition); The pH of described cell culture medium is 7.4.
3, the preparation of sheep anti mouse antiantibody
As immune animal, be that immunogene to pathogen-free domestic sheep carry out immunity with mouse source antibody with sheep, obtain the sheep anti mouse antiantibody.
4, the preparation of monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
With two ionized waters that boil off 1% gold chloride is diluted to 0.01%(quality percentage composition), put to stir on the magnetic force heating rod stirrer and boil, every 100ml 0.01% gold chloride adds 2.5 ml, 1% trisodium citrate, continue agitating heating and react and when liquid takes on a red color, stop heating, supply dehydration after being cooled to room temperature.The collaurum outward appearance for preparing is pure, bright, nothing precipitates and floating thing.
(2) preparation of monoclonal antibody-colloid gold label thing
Under magnetic agitation, transfer the pH value to 7.0 of collaurum with 0.2mol/L sal tartari, standard by 100-150 μ g antibody/ml collaurum adds above-mentioned spectinomycin monoclonal antibody, streptomysin monoclonal antibody, gentamicin monoclonal antibody and neomycin monoclonal antibody respectively in colloidal gold solution, continue to stir and evenly mix 30min, adding the final concentration of 10%BSA to BSA in colloidal gold solution is the 1%(volumn concentration), leave standstill 30min.12000rpm, 4 ℃ of centrifugal 30min, abandon supernatant, precipitation is with redissolving the damping fluid washed twice, be that the redissolution damping fluid of initial collaurum volume 1/20 will precipitate resuspended with volume, the concentration of the spectinomycin monoclonal antibody that obtains respectively-colloid gold label thing solution, streptomysin monoclonal antibody-colloid gold label thing solution, gentamicin monoclonal antibody-colloid gold label thing solution and neomycin monoclonal antibody-colloid gold label thing solution is 50 μ g/ml solution, put 4 ℃ standby.
Redissolution damping fluid: the 0.02mol/L of casein containing protein, Tween-80, the phosphate solution of pH7.2, wherein the final concentration of casein in the redissolution damping fluid is the 0.05%-0.1%(volumn concentration), the final concentration of Tween-80 in the redissolution damping fluid is 0.05%-0.15%(quality percentage composition).
5, monoclonal antibody-colloid gold label thing micropore reagent freeze-drying is in capillary strip
In the micropore of capillary strip, add 50 μ l spectinomycin monoclonal antibody-colloid gold label things, 50 μ l streptomysin monoclonal antibody-colloid gold label things, 50 μ l gentamicin monoclonal antibody-colloid gold label things and 50 μ l neomycin monoclonal antibody-colloid gold label things, put into freeze drier, condenser temperature is under-70 ℃ of conditions, behind the pre-freeze 4h, freeze-drying 14h again, can take out, freeze-drying has spectinomycin monoclonal antibody-colloid gold label thing in the capillary strip that obtains, streptomysin monoclonal antibody-colloid gold label thing, the micropore reagent of gentamicin monoclonal antibody-colloid gold label thing and neomycin monoclonal antibody-colloid gold label thing.
6, the preparation of absorption of sample pad
The absorption of sample pad placed to contain BSA(BSA be the 0.5%(volumn concentration at the final concentration of damping fluid)), pH is 7.2, the 0.1mol/L phosphate buffer soaks 2h, 37 ℃ of baking 2h are standby.
7, the preparation of reaction film
Bag is by process: with phosphate buffer spectinomycin-ovalbumin conjugate being diluted to 10mg/mL, is 1.0 μ g/cms with its bag by the detection line on nitrocellulose filter (T1) package amount with Biodot point film instrument
2With phosphate buffer streptomysin haptens-ovalbumin conjugate is diluted to 10mg/mL, with Biodot point film instrument it is wrapped by the detection line on nitrocellulose filter (T2), package amount is 1.5 μ g/cm
2With phosphate buffer gentamicin haptens-ovalbumin conjugate is diluted to 10mg/mL, with Biodot point film instrument it is wrapped by the detection line on nitrocellulose filter (T3), package amount is 1.5 μ g/cm
2With phosphate buffer neomycin haptens-ovalbumin conjugate is diluted to 10mg/mL, with Biodot point film instrument it is wrapped by the detection line on nitrocellulose filter (T4), package amount is 1.5 μ g/cm
2With 0.01mol/L, pH 7.4 PBS damping fluids sheep anti-mouse igg antibody is diluted to 200 μ g/ml, with Biodot point film instrument it is wrapped by the nature controlling line on nitrocellulose filter (C), package amount is 1.0 μ g/cm
2Bag is placed dry 2h under 37 ℃ of conditions by good reaction film, standby.
Claims (9)
1. one kind is detected spectinomycin; streptomysin; gentamicin; the test strips of neomycin; it is characterized in that comprising test paper; capillary strip; micropore reagent and micropore plug; described test paper comprises reaction film; the absorption of sample pad; adsorptive pads; diaphragm; base plate; four detection lines and bag are arranged by a nature controlling line of sheep anti mouse antiantibody on the described reaction film; article four, detection line is coated with spectinomycin-carrier protein couplet thing respectively; streptomysin-carrier protein couplet thing; gentamicin-carrier protein couplet thing and neomycin-carrier protein couplet thing, described micropore reagent are the spectinomycin-colloid gold label thing of freeze-drying; streptomysin-colloid gold label thing; gentamicin-colloid gold label thing and neomycin-colloid gold label thing.
2. test strips according to claim 1 is characterized in that four detection lines and the nature controlling line on the described reaction film is parallel to each other, and vertical with the appearance of test strips.
3. test strips according to claim 1, it is characterized in that described spectinomycin detection line is near absorption of sample pad one end, nature controlling line is near adsorptive pads one end, and streptomysin detection line, gentamicin detection line and neomycin detection line are between spectinomycin detection line and nature controlling line.
4. test strips according to claim 1 is characterized in that described test paper is pasted at base plate successively by absorption of sample pad, reaction film, adsorptive pads, diaphragm to form.
5. according to claim 1 or 4 described test strips, it is characterized in that: described diaphragm is pasted the test side on the absorption of sample pad, and the MAX mark line is arranged above.
6. according to the described test strips of claim 1, it is characterized in that having the micropore plug on the described capillary strip.
7. according to the described test strips of claim 1, it is characterized in that described test strips comprises also that for splendid attire test paper and freeze-drying the reagent bucket of capillary strip of spectinomycin specific antibody-colloid gold label thing, streptomysin specific antibody-colloid gold label thing, gentamicin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing and the holding appliance of fixating reagent bucket are arranged.
8. test strips according to claim 7 is characterized in that having gland bonnet on the described reagent bucket.
9. test strips according to claim 7 is characterized in that described test strips box body is carton box, and the reagent bucket is plastics reagent bucket, and holding appliance is the rigid support material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201220510206.5U CN203069593U (en) | 2012-09-29 | 2012-09-29 | Test strip for detecting spectinomycin, streptomycin, gentamicin and neomycin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201220510206.5U CN203069593U (en) | 2012-09-29 | 2012-09-29 | Test strip for detecting spectinomycin, streptomycin, gentamicin and neomycin |
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| Publication Number | Publication Date |
|---|---|
| CN203069593U true CN203069593U (en) | 2013-07-17 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201220510206.5U Expired - Fee Related CN203069593U (en) | 2012-09-29 | 2012-09-29 | Test strip for detecting spectinomycin, streptomycin, gentamicin and neomycin |
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| CN (1) | CN203069593U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104678102A (en) * | 2013-11-27 | 2015-06-03 | 北京维德维康生物技术有限公司 | Neomycin and tylosin two-in-one gold-labeled microporous test paper strip |
-
2012
- 2012-09-29 CN CN201220510206.5U patent/CN203069593U/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104678102A (en) * | 2013-11-27 | 2015-06-03 | 北京维德维康生物技术有限公司 | Neomycin and tylosin two-in-one gold-labeled microporous test paper strip |
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