CN103713131B - Detection spectinomycin, streptomycin, gentamycin and the test strips of neomycin and method - Google Patents
Detection spectinomycin, streptomycin, gentamycin and the test strips of neomycin and method Download PDFInfo
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Abstract
The invention discloses a kind of test strips detecting spectinomycin, streptomycin, gentamycin and neomycin and method.Test strips includes capillary strip, micropore reagent, reagent paper and micropore plug, the spectinomycin of the described colloid gold label that micropore reagent is lyophilizing, streptomycin, gentamycin and neomycin specific antibody, described reagent paper is by sample absorption pad, reaction film, adsorptive pads, protecting film, base plate, it is sequentially connected with composition, four detection lines and a nature controlling line is included on described reaction film, article four, detection line is coated with spectinomycin-carrier protein couplet thing respectively, streptomycin-carrier protein couplet thing, gentamycin-carrier protein couplet thing and neomycin-carrier protein couplet thing, nature controlling line is coated with anti antibody.By ELISA test strip spectinomycin of the present invention, streptomycin, gentamycin and the method for neomycin, easy, quickly, directly perceived, accurately, be easy to carry, applied widely, low cost, easily promote the use of.<!--1-->
Description
Technical field
The present invention relates to a kind of test strips detecting spectinomycin, streptomycin, gentamycin and neomycin and method.
Background technology
Spectinomycin is also known as spectinomycin, spextinomyxin, grand rhzomorph etc., a kind of aminocyclitol antibiotic produced by grand sight streptomycete, gram negative bacteria (such as brucella, Klebsiella, Bacillus proteus, Salmonella, pasteurellosis bacillus etc.) is had and relatively pretends use, more weak to gram positive bacteria (streptococcus, staphylococcus etc.) effect, mycoplasma is also had certain effect, it is the novel specially good effect antibiotic of specially controlling gonorrhea, Diplococcus gonorrhoeae is had stronger antibacterial action.Growth of animal can be promoted, be good animal feed additive, be widely used in animal husbandry as veterinary drug.Some illegal retailers are ordered about abuse of antibiotics by economic interests, make a large amount of antibiotic remains in livestock products, if being eaten for a long time the animal derived food containing antibiotic remains, also it is equivalent to long-term low dose and absorbs antibiotic, in making human body, bacterial strain produces antibiotic resistance, harmful effect is brought to using antibiotic therapy time ill, organismic internal environment balance can be destroyed, cause dysbacteriosis, the people of antibiotic allergic constitution there will be anaphylaxis, cause immune hemolysis, cause the series of problems that food and environment drug residue pollute simultaneously.
Streptomycin is aminoglycoside antibiotics, and in milch cow and milk sheep cultivate, application is universal, but owing to using management lack of standardization, often results in fresh milk and milk powder streptomycin residual.Due to streptomycin, human body is had bigger toxic and side effects, anaphylactic shock and deafness, the particularly harm to child can be caused time serious relatively big, can pass through Placenta Hominis and enter amniotic fluid and fetal circulation, endanger foetus health.Streptomycin in milk product, dihydrostreptomycin have all been formulated strict residue limits by various countries, if total limitation of Japan's regulation milk streptomycin with dihydrostreptomycin is 0.2mg/kg, the U.S. specifies that in breast and milk, the limitation of dihydrostreptomycin is 0.125mg/kg, China's regulation milk streptomycin is 0.2mg/kg with the limitation of dihydrostreptomycin, so detecting the streptomycin in milk powder, dihydrostreptomycin residual quantity for controlling quality of milk powder, safeguard that the healthy diet of the mankind is significant.
Gentamycin is a kind of aminoglycoside antibiotics produced by small single-cell bacteria, and multiple Grain-positive and negative bacterium are all had significant antibacterial effect, and the antibiotic as feed additive and treatment disease is widely used in animal husbandry.But, gentamycin has obvious toxic action to the mankind, and the infringement to cranial nerve, audition and kidney is serious.The MRL of this drug residue in many countries and international organization's equal clear stipulaties food.European Union specifies, the maximum residue limit in milk is 100 μ g/kg, and the maximum residue limit in muscle, fat is 50 μ g/kg.
Neomycin belongs to aminoglycoside antibiotics, has a broad antifungal spectrum, is widely used on veterinary clinic, is important anti-infectives, be also widely used in treatment mastitis, keratitis, conjunctivitis, otitis, dermatitis, trauma infection contamination, foot rot etc. disease.Neomycin is also used by China and some other country as fodder antibiotics additive, for the wall losses and enteritis morning preventing poultry to infect to cause, improves efficiency of feed utilization, promotes to grow.Although neomycin uses on veterinary and herding and achieves huge success, but there is potential ototoxicity and nephrotoxicity in it to humans and animals, to this end, the country such as China and European Union has all formulated neomycin MRL in animal derived food.
Immunoassay field shortage can detect spectinomycin, streptomycin, gentamycin and the technology of neomycin these four medicine and application thereof simultaneously at present.Therefore, it is necessary in practice to set up the detection method that a Species sensitivity is high, simple to operate, low cost, detection medicament categories are many, be suitable for large-scale promotion.
Summary of the invention
It is an object of the present invention to provide a kind of test strips detecting spectinomycin, streptomycin, gentamycin and neomycin.
Test strips provided by the present invention includes capillary strip, micropore reagent, micropore plug and reagent paper, and reagent paper includes base plate, sample absorption pad, reaction film, adsorptive pads, protecting film, and it is sequentially connected with；Described micropore reagent is the spectinomycin specific antibody-colloid gold label thing of lyophilizing, streptomycin specific antibody-colloid gold label thing, gentamycin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing；Spectinomycin specific antibody is spectinomycin monoclonal antibody, and streptomycin specific antibody is streptomycin monoclonal antibody, and gentamycin specific antibody is gentamycin monoclonal antibody, and neomycin specific antibody is neomycin monoclonal antibody；Four detection lines and a nature controlling line it is coated with on reaction film；Micropore reagent comprises spectinomycin monoclonal antibody-colloid gold label thing, streptomycin monoclonal antibody-colloid gold label thing, gentamycin monoclonal antibody-colloid gold label thing, neomycin monoclonal antibody-colloid gold label thing, spectinomycin detection line is coated spectinomycin-carrier protein couplet thing, streptomycin detection line is coated streptomycin-carrier protein couplet thing, gentamycin detection line is coated gentamycin-carrier protein couplet thing, neomycin detection line is coated neomycin-carrier protein couplet thing, and nature controlling line is coated sheep anti mouse anti antibody.
Described protecting film is pasted onto the test side on sample absorption pad, has MAX mark line above.
Described spectinomycin detection line is near sample absorption pad one end, and nature controlling line is near adsorptive pads one end, and streptomycin detection line, gentamycin detection line, neomycin detection line are between spectinomycin detection line and nature controlling line.Wherein detection line (T1) of spectinomycin detection line represents, streptomycin detection line detection line (T2) represents, gentamycin detection line detection line (T3) represents, neomycin detection line detection line (T4) represents, nature controlling line nature controlling line (C) represents.With sample absorption pad one end of reagent paper as top, with adsorptive pads one end of reagent paper as end, one end that nature controlling line (C) distance reaction film is connected with adsorptive pads top is 5-8mm, detection line (T4) distance nature controlling line (C) 4-6mm, detection line (T3) distance detection line (T4) 4-6mm, detection line (T2) distance detection line (T3) 4-6mm, detection line (T1) distance detection line (T2) 4-6mm.
Described spectinomycin-carrier protein couplet thing is to be obtained with carrier protein couplet by spectinomycin, and described carrier protein can be bovine serum albumin, ovalbumin, hemocyanin, thyroprotein, human serum albumin.
Described streptomycin-carrier protein couplet thing is to be obtained with carrier protein couplet by streptomycin, and described carrier protein can be bovine serum albumin, ovalbumin, hemocyanin, thyroprotein, human serum albumin.
Described gentamycin-carrier protein couplet thing is to be obtained with carrier protein couplet by gentamycin, and described carrier protein can be bovine serum albumin, ovalbumin, hemocyanin, thyroprotein, human serum albumin.
Described neomycin-carrier protein couplet thing is to be obtained with carrier protein couplet by neomycin, and described carrier protein can be bovine serum albumin, ovalbumin, hemocyanin, thyroprotein, human serum albumin.
Spectinomycin specific antibody in described spectinomycin specific antibody-colloid gold label thing is to prepare using spectinomycin-carrier protein couplet thing as immunogen.
Streptomycin specific antibody in described streptomycin specific antibody-colloid gold label thing is to prepare using streptomycin-carrier protein couplet thing as immunogen.
Gentamycin specific antibody in described gentamycin specific antibody-colloid gold label thing is to prepare using gentamycin-carrier protein couplet thing as immunogen.
Neomycin specific antibody in described neomycin specific antibody-colloid gold label thing is to prepare using neomycin-carrier protein couplet thing as immunogen.
The material that described base plate can be PVC base plate or other hard do not absorb water；Sample absorption pad can be suction strainer paper or filter paper for oil；Adsorptive pads is absorbent paper；Reaction film can be nitrocellulose filter or cellulose acetate membrane；Adsorptive pads is absorbent paper；Protecting film is PE material protecting film.
Another object of the present invention is to provide a kind of method preparing above-mentioned test strips, its step includes:
1) on capillary strip, prepare the spectinomycin specific antibody-colloid gold label thing of lyophilizing, streptomycin specific antibody-colloid gold label thing, gentamycin specific antibody-colloid gold label thing and the micropore reagent of neomycin specific antibody-colloid gold label thing, and with micropore gag on capillary strip；
2) prepare four to be respectively provided with and be coated, spectinomycin-carrier protein couplet thing, streptomycin-carrier protein couplet thing, gentamycin-carrier protein couplet thing and the detection line of neomycin-carrier protein couplet thing and the reaction film of a nature controlling line being coated anti antibody；
3) by 2) reaction film for preparing is assembled into reagent paper with sample absorption pad, adsorptive pads, protecting film, base plate；
4) by 1) and 3) lyophilizing for preparing has spectinomycin specific antibody-colloid gold label thing, streptomycin specific antibody-colloid gold label thing, gentamycin specific antibody-colloid gold label thing and the micropore reagent of neomycin specific antibody-colloid gold label thing, reagent paper, capillary strip and micropore plug to be assembled into test strips.
Specifically, step includes:
1, the synthesis of spectinomycin antigen and the preparation of antibody thereof
1) by spectinomycin and carrier protein couplet, spectinomycin-carrier protein couplet thing is formed；
2) with spectinomycin-carrier protein couplet thing immune mouse, pass through to merge, screen by mouse boosting cell and murine myeloma cell, obtain secreting the hybridoma cell strain of spectinomycin monoclonal antibody.
2, the synthesis of Streptomycin antigen and the preparation of antibody thereof
1) by streptomycin and carrier protein couplet, streptomycin-carrier protein couplet thing is formed；
2) with streptomycin-carrier protein couplet thing immune mouse, pass through to merge, screen by mouse boosting cell and murine myeloma cell, obtain secreting the hybridoma cell strain of streptomycin monoclonal antibody.
3, the synthesis of gentamycin antigen and the preparation of antibody thereof
1) by gentamycin and carrier protein couplet, gentamycin-carrier protein couplet thing is formed；
2) with gentamycin-carrier protein couplet thing immune mouse, pass through to merge, screen by mouse boosting cell and murine myeloma cell, obtain secreting the hybridoma cell strain of gentamycin monoclonal antibody.
4, the synthesis of neomycin antigen and the preparation of antibody thereof
1) by neomycin and carrier protein couplet, neomycin-carrier protein couplet thing is formed；
2) with neomycin-carrier protein couplet thing immune mouse, pass through to merge, screen by mouse boosting cell and murine myeloma cell, obtain secreting the hybridoma cell strain of neomycin monoclonal antibody.
5, extract mouse IgG immune health goat, obtain sheep anti-mouse igg antibody.
6, gold colloidal is prepared with trisodium citrate and gold chloride reaction.
7, the spectinomycin specific antibody of preparation, streptomycin specific antibody, gentamycin specific antibody and neomycin specific antibody are added separately in the gold colloidal of preparation, obtain spectinomycin specific antibody-colloid gold label thing, streptomycin specific antibody-colloid gold label thing, gentamycin specific antibody-colloid gold label thing and the micropore reagent of neomycin specific antibody-colloid gold label thing.
8, by the micropore reagent lyophilizing of spectinomycin specific antibody-colloid gold label thing, streptomycin specific antibody-colloid gold label thing, gentamycin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing in capillary strip after, micropore plug will be covered on capillary strip.
9, sample absorption pad is soaked 2h with containing bovine serum albumin (bovine serum albumin final concentration of 0.5% (volumn concentration) in buffer), pH7.2,0.1mol/L phosphate buffer, at 37 DEG C, dry 2h.
10, on base plate, sample absorption pad, reaction film, adsorptive pads and protecting film are sticked in order.
11, the micropore reagent prepared, reagent paper, capillary strip and micropore plug are assembled into test strips, preserve 12 months under the conditions of 2-8 DEG C.
Another object of the present invention provides spectinomycin, streptomycin, gentamycin and the detection method of Detection of neomycin residues in a kind of milk sample.
Detect by any of the above-described described test strips of the present invention.
The Cleaning Principle of test strips of the present invention: milk sample drop to be checked is added in the capillary strip including micropore reagent, after mixing, hatch 5min, MAX labelling line end will be indicated downward, insert include in the micropore reagent capillary strip after hatching, measuring samples liquid is combined with the golden labeling antibody in micropore after together with to reaction film diffusion；nullIf the content of spectinomycin or streptomycin or gentamycin or neomycin is high in measuring samples liquid，Then in diffusion process, the spectinomycin in analyte sample fluid or streptomycin or gentamycin or neomycin can combine with gold labeling antibody，And then spectinomycin or streptomycin or gentamycin or the antigen-combining site of neomycin on completely enclosed gold labeling antibody，Stop gold labeling antibody spectinomycin-carrier protein couplet thing on reaction film to be combined or streptomycin-carrier protein couplet thing combines or gentamycin-carrier protein couplet thing combines or the combination of neomycin-carrier protein couplet thing，Corresponding spectinomycin detection line or streptomycin detection line or gentamycin detect line or neomycin detection line can not develop the color，Anti antibody then can be combined with spectinomycin gold labeling antibody or streptomycin gold labeling antibody or gentamycin gold labeling antibody or neomycin gold labeling antibody，Nature controlling line develops the color；nullIf in measuring samples liquid, the content of spectinomycin or streptomycin or gentamycin or neomycin is low or nothing，Antigen binding site on then corresponding spectinomycin gold labeling antibody or streptomycin gold labeling antibody or gentamycin gold labeling antibody or neomycin gold labeling antibody can not be closed，And then spectinomycin gold labeling antibody can spectinomycin-carrier protein couplet antigen be combined or streptomycin gold labeling antibody can streptomycin-carrier protein couplet antigen be combined or gentamycin gold labeling antibody can gentamycin-carrier protein couplet antigen be combined or neomycin gold labeling antibody can be combined by neomycin-carrier protein couplet antigen on reaction film on reaction film on reaction film on reaction film，Corresponding spectinomycin detection line colour developing or streptomycin detect line colour developing or gentamycin detection line colour developing or neomycin detection line develops the color，Anti antibody also can be combined with spectinomycin gold labeling antibody or streptomycin gold labeling antibody or gentamycin gold labeling antibody or neomycin gold labeling antibody simultaneously，Nature controlling line develops the color；If nature controlling line does not develops the color, then test strips lost efficacy.
Negative (-): C line develops the color, and four T lines all develop the color, no matter shade, all represents that spectinomycin in milk sample, streptomycin, gentamycin, neomycin concentration are below detection limit, as shown in Fig. 8 .a.
Positive (+): C line develops the color, T1Line does not develops the color, and represents that in milk sample, spectinomycin concentration is equal to or higher than detection limit；C line develops the color, T2Line does not develops the color, and represents that milk sample streptomycin concentration is equal to or higher than detection limit；C line develops the color, T3Line does not develops the color, and represents that in milk sample, gentamicin concentration is equal to or higher than detection limit；C line develops the color, T4Line does not develops the color, and represents that in milk sample, neomycin concentration is equal to or higher than detection limit, as shown in Fig. 8 .a-8.f.
Invalid: C line does not occurs, show that incorrect operating process or test strips lost efficacy, as shown in Fig. 8 .g-8.k.
Test strips of the present invention can detect medicine and include: spectinomycin, streptomycin, dihydrostreptomycin, gentamycin, neomycin.
The test strips of the present invention has the advantages that
1, highly sensitive: traditional test card is respectively provided with the fixing device that gets stuck of fixing reagent paper, reagent paper is fixed wherein by the fixing device that gets stuck tightly, when to sample detection, owing to getting stuck, the comparison being combined with reagent paper is tight, detection sample solution is caused can not fully to penetrate into sample absorption pad one section, and the speed that carries out chromatographing on reagent paper is very slow, causes the medicine detecting in sample can not be fully contacted with coated coating antigen on detection line, cause the sensitivity of test card to decline.Test strips of the present invention is higher than the detection sensitivity of traditional test card, owing to the reagent paper of test strips of the present invention is naked strip, without housing clamp device, detection sample can directly contact sample absorption pad one end of reagent paper, and the Tomography Velocity that sample is on reagent paper is than very fast, enable the medicine to be checked in sample sufficiently with detect coated coating antigen on line and be combined, thus improve the detection sensitivity of test strips.Spectinomycin, streptomycin, gentamycin and neomycin test strip are prepared from based on the monoclonal antibody of colloid gold label high-affinity, priceless strong formation between gold grain and antibody molecule in gold labeling antibody, the two is combined by the Van der Waals force between the charges of different polarity, monoclonal antibody specificity and affinity are affected the least by gold colloidal, and have higher mark rate.Spectinomycin monoclonal antibody-colloid gold label thing therein, streptomycin monoclonal antibody-colloid gold label thing, gentamycin monoclonal antibody-colloid gold label thing and neomycin monoclonal antibody-colloid gold label thing lyophilizing are in micropore reagent strip, during detection, gold labeling antibody can be made to be fully contacted with measuring samples liquid, fully reaction, thus reduce error, increase the reaction sensitivity of whole system.Therefore, test strips of the present invention has higher specificity and sensitivity.This test strips is respectively as follows: spectinomycin 20 μ g/L, streptomycin 80 μ g/L, dihydrostreptomycin 80 μ g/L, gentamycin 20 μ g/L, neomycin 500 μ g/L to various drug detection sensitivity.
The most easy and simple to handle quickly: for conventional test card detection raw material milk raw material milk have to add diluent is diluted can point sample detection, reason is owing to the plastic casing of fixing reagent paper is fixed on reagent paper in housing tightly, and raw material milk compares thickness, so directly by raw material milk if the well of test card carries out point sample detection, owing to housing and reagent paper are tightly combined and caused by milk thickness, raw material milk sample cannot completely penetrate in sample absorption pad and conjugate release pad, and the Tomography Velocity on reagent paper is slower, to such an extent as to cannot detect, therefore raw material milk added and can detect after diluent is diluted.Without other reagent any during use ELISA test strip of the present invention, during detection raw material milk, it is diluted also without to raw material milk, reason is naked strip due to the reagent paper in test strips of the present invention, i.e. without it being fixed with housing, as long as measuring samples solution directly being dripped and having spectinomycin monoclonal antibody-colloid gold label thing in lyophilizing, streptomycin monoclonal antibody-colloid gold label thing, in the micropore of gentamycin monoclonal antibody-colloid gold label thing and neomycin monoclonal antibody-colloid gold label thing, after mixing, hatch 5min, reagent paper is indicated MAX wire tag end again downward, insert in the micropore reagent after hatching, reagent paper can be fully contacted with sample solution, observed result after 5min.For the detection of milk sample, test strips of the present invention is shorter than traditional test card detection time, owing to test strips of the present invention is without being diluted sample, directly can detect sample.
Test strips sensitivity of the present invention is high, detection medicament categories is many, low cost, simple to operate, be easy to carry, the detection time is short, be suitable for various units uses, store simple, the advantage of long shelf-life.Wherein, the spectinomycin monoclonal antibody of high specific, streptomycin monoclonal antibody, gentamycin monoclonal antibody and neomycin monoclonal antibody are used, it is ensured that the reliability of testing result；By gold labeling antibody lyophilizing in capillary strip, during detection, it is possible to make gold labeling antibody be fully contacted with measuring samples liquid, fully react, thus reduce error, increase the reaction sensitivity of whole system.Test strips of the present invention can detect spectinomycin, streptomycin, gentamycin and four kinds of medicines of neomycin simultaneously, achieve the detection to multi-medicament of the test strips, meet the demand on market, spectinomycin, streptomycin, gentamycin and neomycin detected simultaneously.By ELISA test strip spectinomycin of the present invention, streptomycin, gentamycin and the method for neomycin, easy, quick, directly perceived, accurate, applied widely, low cost, easily promote the use of.
Accompanying drawing explanation
Fig. 1 lyophilizing has the capillary strip figure of golden labeling antibody.
Fig. 2 micropore plug top view.
Fig. 3 micropore plug cross-sectional view.
Fig. 4 reagent paper cross-sectional view.
The top view of Fig. 5 reagent paper.
Fig. 6 tetracycline medication hapten synthesis route map.
Fig. 7 ELISA test strip method schematic diagram.
Fig. 8 ELISA test strip result process decision chart.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
The composition of the test strips of embodiment 1, detection spectinomycin, streptomycin, gentamycin and neomycin
One, lyophilizing has the capillary strip of golden labeling antibody
Equipped with micropore reagent in described capillary strip, wherein 1 is capillary strip, and 2 is the spectinomycin specific antibody-colloid gold label thing of lyophilizing, streptomycin specific antibody-colloid gold label thing, gentamycin specific antibody-colloid gold label thing and the micropore reagent of neomycin specific antibody-colloid gold label thing；
There is on the described capillary strip including micropore reagent micropore plug 3 (Fig. 2 and Fig. 3)；
Spectinomycin monoclonal antibody-colloid gold label the thing of lyophilizing in described capillary strip, streptomycin monoclonal antibody-colloid gold label thing, gentamycin monoclonal antibody-colloid gold label thing and the micropore reagent of neomycin monoclonal antibody-colloid gold label thing, the lyophilizing amount of spectinomycin specific antibody-colloid gold label thing is 0.20-0.25 μ g/ml；The lyophilizing amount of streptomycin specific antibody-colloid gold label thing is 0.30-0.40 μ g/ml；The lyophilizing amount of gentamycin specific antibody-colloid gold label thing is 0.20-0.25 μ g/ml；The lyophilizing amount of neomycin specific antibody-colloid gold label thing is 0.20-0.25 μ g/ml.
Two, reagent paper (Fig. 4 and Fig. 5)
Described reagent paper is made up of base plate, sample absorption pad, reaction film, adsorptive pads, protecting film；
Described sample absorption pad 4, reaction film 5, adsorptive pads 6 and protecting film 7 are pasted the most in order on described base plate 8; the end of sample absorption pad is connected with reaction film; the end of reaction film is connected with adsorptive pads; the top of sample absorption pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate；
Protecting film 7 covers the test side on sample absorption pad, should have MAX printed words on the protecting film of test side.
Four detection lines are had respectively to detect line (T1) 9-1, detection line (T2) 9-2, detection line (T3) 9-3 and detection line (T4) 9-4 and a nature controlling line (C) 10 on described reaction film; article four, detection line and a nature controlling line are the ribbon perpendicular with the length of described reagent paper; article four, detection line is positioned at the side of the protecting film being bordering on MAX mark line, and nature controlling line (C) is located remotely from the side of the protecting film of MAX labelling.One end that the end of nature controlling line (C) distance reaction film is connected with adsorptive pads is 5-8mm, detection line (T4) distance nature controlling line (C) 4-6mm, detection line (T3) distance detection line (T4) 4-6mm, detection line (T2) distance detection line (T3) 4-6mm, detection line (T1) distance detection line (T2) 4-6mm.Detection line (T1) is coated with spectinomycin-carrier protein couplet thing (spectinomycin-oralbumin), detection line (T2) is coated with streptomycin-carrier protein couplet thing (streptomycin-oralbumin), detection line (T3) is coated with gentamycin-carrier protein couplet thing (gentamycin-oralbumin), detection line (T4) is coated with neomycin-carrier protein couplet thing (neomycin-oralbumin), and nature controlling line (C) is coated with sheep anti mouse anti antibody.
Base plate is PVC base plate；Sample absorption pad is suction strainer paper；Adsorptive pads is absorbent paper；Reaction film is nitrocellulose filter；Described protecting film is PE material protecting film.
Above-mentioned reagent paper is assembled into test strips with micropore reagent, capillary strip and micropore plug, stores in the environment of 2～8 DEG C, 12 months effect duration.
The preparation method of test strips described in embodiment 2, embodiment 1
One, the preparation of test strips
The preparation method of this test strips mainly comprises the steps:
1) in capillary strip, prepare the spectinomycin monoclonal antibody-colloid gold label thing of lyophilizing, streptomycin monoclonal antibody-colloid gold label thing, gentamycin monoclonal antibody-colloid gold label thing and the micropore reagent of neomycin monoclonal antibody-colloid gold label thing, and with micropore gag on capillary strip.
2) preparation has the detection line (T1) being coated spectinomycin-carrier protein couplet thing, the detection line (T2) being coated streptomycin-carrier protein couplet thing, the detection line (T3) being coated gentamycin-carrier protein couplet thing, is coated the detection line (T4) of neomycin-carrier protein couplet thing and is coated the reaction film of nature controlling line (C) of sheep anti mouse anti antibody；
3) by 2) reaction film for preparing is assembled into reagent paper with sample absorption pad, adsorptive pads, protecting film, base plate；
4) by 1) and 3) lyophilizing for preparing has spectinomycin monoclonal antibody-colloid gold label thing, streptomycin monoclonal antibody-colloid gold label thing, gentamycin monoclonal antibody-colloid gold label thing and the micropore reagent of neomycin monoclonal antibody-colloid gold label thing, reagent paper, capillary strip and micropore plug to be assembled into test strips.
Substep narration in detail below:
(1) preparation of each parts
1, the preparation of antigen
1), the synthesis of spectinomycin-carrier protein couplet thing and qualification
A. spectinomycin hapten synthesis: take spectinomycin 0.66g, carboxymethyl azanol 0.27g and pyridine 1ml mixed liquor in 20mlDMSO, it is stirred at room temperature reaction 20 hours, solvent is evaporated off, column chromatography is recrystallized to give the condensation substance of spectinomycin and carboxymethyl azanol after purification in ethanol-water system, and molecular structure is as follows:
The most immunogenic preparation-spectinomycin hapten synthesizes with BSA conjugate
Take spectinomycin hapten 38mg 5ml water dissolution；Take bovine serum albumin (BSA) 100mg 5ml water dissolution；Spectinomycin hapten aqueous solution is added in BSA aqueous solution, reacts 24h by magnetic stirrer；Dialyse 3 days with 0.01mol/LPBS, change liquid every day 3 times, to remove unreacted small-molecule substance.It is centrifuged 30min with 12000r/min, collects supernatant, subpackage, save backup in-20 DEG C.
C. the preparation of coating antigen-spectinomycin hapten synthesizes with OVA conjugate
By spectinomycin hapten 22mg, 15mgN, N'-carbonyl dimidazoles (CDI) 1.5mlN, dinethylformamide (DMF) dissolves, and reaction 1h is stirred at room temperature, i.e. can get reactant liquor A；Weigh ovalbumin (OVA) 36mg, be allowed to be substantially dissolved in 3.5ml50mmol/L sodium carbonate liquor, reactant liquor A is dropwise slowly dropped in this solution；Room temperature reaction 24h, dialyses 3 days with 0.01mol/LPBS, changes liquid every day 3 times, to remove unreacted small-molecule substance.It is centrifuged 30min with 12000r/min, collects supernatant, subpackage, save backup in-20 DEG C.
D. the qualification of spectinomycin hapten-carrier conjugates
Spectinomycin, bovine serum albumin, spectinomycin-bovine serum albumin conjugate and spectinomycin, ovalbumin, the PBS of spectinomycin-ovalbumin conjugate pH7.4 are made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PBS, scan in the range of wavelength 200～800nm with ultraviolet spectrophotometer, obtain spectinomycin, bovine serum albumin, the absorption curve of spectinomycin-bovine serum albumin conjugate and spectinomycin, ovalbumin, the absorption curve of spectinomycin-ovalbumin conjugate.There is different absorption curves in three, shows spectinomycin and spectinomycin and carrier protein couplet success.
2), the synthesis of streptomycin-carrier protein couplet thing and qualification
A. streptomycin hapten synthesis: 1.0g streptomycin, 0.30g phthalic anhydride and 1ml pyridine mixed liquor in 20mlDMSO, stirring reaction 24 hours at 80 DEG C, solvent being evaporated off, is repeatedly recrystallized to give phthalic acid strand mycin ester in ethanol-water system, molecular structure is as follows:
The most immunogenic preparation-streptomycin hapten synthesizes with BSA conjugate
Take hapten 40mg 2mlDMF and dissolve completely, make I liquid, take BSA80mg 7ml0.1MPBS (PH7.5) and dissolve completely, make II liquid, I liquid is added in II liquid, making III liquid, after taking EDC100mg 1ml water dissolution, buffering adds in III liquid, is stirred at room temperature 2 hours, dialyse three days with 0.01MPBS, change liquid every day three times, obtain immunogen, save backup in-20 DEG C.
C. the preparation of coating antigen-streptomycin hapten synthesizes with OVA conjugate
Take hapten 40mg 2mlDMF and dissolve completely, make I liquid, take OVA80mg 7ml0.1MPBS (PH7.5) and dissolve completely, make II liquid, I liquid is added in II liquid, making III liquid, after taking EDC100mg 1ml water dissolution, buffering adds in III liquid, is stirred at room temperature 2 hours, dialyse three days with 0.01MPBS, change liquid every day three times, obtain immunogen, save backup in-20 DEG C.
D. the qualification of streptomycin hapten-carrier conjugates
Streptomycin, bovine serum albumin, streptomycin-protein conjugate and streptomycin, ovalbumin, the PBS of streptomycin-ovalbumin conjugate pH7.4 are made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PBS, scan in the range of wavelength 200～800nm with ultraviolet spectrophotometer, obtain streptomycin, bovine serum albumin, the absorption curve of streptomycin-protein conjugate and streptomycin, ovalbumin, the absorption curve of streptomycin-ovalbumin conjugate.There is different absorption curves in three, shows streptomycin and streptomycin and carrier protein couplet success.
3), the synthesis of gentamycin-carrier protein couplet thing and qualification
A. gentamycin hapten synthesis: 0.39g bromo-acetic acid tert-butyl solution in 5mlDMSO, is slowly added dropwise at 40 DEG C in 0.90g gentamycin and 1ml pyridine mixture in 10mlDMSO.After dropping, after continuing reaction 6 hours, solvent is evaporated off.After simple column chromatography for separation, removing solvent, add 20mlDMSO and 5ml formic acid, room temperature reaction 20 hours, solvent is evaporated off, is recrystallized to give carboxymethyl gentamycin in ethanol-water system, molecular structure is as follows:
The most immunogenic preparation-gentamycin hapten synthesizes with BSA conjugate
Take hapten 35mg 2mlDMF and dissolve completely, make I liquid, take BSA100mg 7ml0.1MPBS (PH7.5) and dissolve completely, make II liquid, I liquid is added in II liquid, making III liquid, after taking EDC100mg 1ml water dissolution, buffering adds in III liquid, is stirred at room temperature 2 hours, dialyse three days with 0.01MPBS, change liquid every day three times, obtain immunogen, save backup in-20 DEG C.
C. the preparation of coating antigen-gentamycin hapten synthesizes with OVA conjugate
Take hapten 35mg 2mlDMF and dissolve completely, make I liquid, take OVA100mg 7ml0.1MPBS (PH7.5) and dissolve completely, make II liquid, I liquid is added in II liquid, making III liquid, after taking EDC100mg 1ml water dissolution, buffering adds in III liquid, is stirred at room temperature 2 hours, dialyse three days with 0.01MPBS, change liquid every day three times, obtain immunogen, save backup in-20 DEG C.
D. the qualification of gentamycin hapten-carrier conjugates
Gentamycin, bovine serum albumin, gentamycin-bovine serum albumin conjugate and gentamycin, ovalbumin, the PBS of gentamycin-ovalbumin conjugate pH7.4 are made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PBS, scan in the range of wavelength 200～800nm with ultraviolet spectrophotometer, obtain gentamycin, bovine serum albumin, the absorption curve of gentamycin-bovine serum albumin conjugate and gentamycin, ovalbumin, the absorption curve of gentamycin-ovalbumin conjugate.There is different absorption curves in three, shows gentamycin and gentamycin and carrier protein couplet success.
4), the synthesis of neomycin-carrier protein couplet thing and qualification
A. neomycin hapten synthesis: 0.61g neomycin and the mixed liquor of 10mlDMSO, it is slowly added dropwise under room temperature in the 10mlDMSO solution of 0.14g terephthalaldehyde, drip complete rear chamber temperature to react 2-4 hour to 60 DEG C, remove solvent, being recrystallized to give neomycin terephthalaldehyde list condensation substance in ethanol-water system, molecular structure is as follows:
The most immunogenic preparation-neomycin hapten synthesizes with BSA conjugate
Take hapten 30mg 3mlDMF and dissolve completely, make I liquid, take BSA100mg 7ml0.1MPBS (PH7.0) to dissolve completely, make II liquid, I liquid is added in II liquid, make III liquid, room temperature reaction 24 hours, dialyses three days with 0.01MPBS, changes liquid every day three times, obtain immunogen, save backup in-20 DEG C.
C. the preparation of coating antigen-neomycin hapten synthesizes with OVA conjugate
Take hapten 30mg 3mlDMF and dissolve completely, make I liquid, take OVA100mg 7ml0.1MPBS (PH7.0) to dissolve completely, make II liquid, I liquid is added in II liquid, make III liquid, room temperature reaction 24 hours, dialyses three days with 0.01MPBS, changes liquid every day three times, obtain immunogen, save backup in-20 DEG C.
D. the qualification of neomycin hapten-carrier conjugates
Neomycin, bovine serum albumin, neomycin-bovine serum albumin conjugate and neomycin, ovalbumin, the PBS of neomycin-ovalbumin conjugate pH7.4 are made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PBS, scan in the range of wavelength 200～800nm with ultraviolet spectrophotometer, obtain neomycin, bovine serum albumin, the absorption curve of neomycin-bovine serum albumin conjugate and neomycin, ovalbumin, the absorption curve of neomycin-ovalbumin conjugate.There is different absorption curves in three, shows neomycin and neomycin and carrier protein couplet success.
2, the preparation of the monoclonal antibody of spectinomycin, streptomycin, gentamycin and neomycin
(1) monoclonal antibody is prepared
A. animal immune
Four kinds of immunogens step 1 obtained are injected separately in Balb/c Mice Body, and immunizing dose is 150 μ g/ so that it is produce antiserum.
B. cell merges and cloning
Take immunity Balb/c mouse boosting cell, in 9:1 (quantitative proportion) ratio and SP2/0 myeloma cell fusion, screening obtains the spectinomycin monoclonal hybridoma strain of stably excreting spectinomycin monoclonal antibody, the streptomycin monoclonal hybridoma strain of stably excreting streptomycin monoclonal antibody, the gentamycin monoclonal hybridoma strain of stably excreting gentamycin monoclonal antibody and the neomycin monoclonal hybridoma strain of stably excreting neomycin monoclonal antibody respectively.
C. cell cryopreservation and recovery
Hybridoma frozen stock solution is made 1 × 109The cell suspension of individual/ml, preserves in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
D. the preparation of monoclonal antibody and purification
Increment culture method: be placed in cell culture medium by hybridoma, cultivates under the conditions of 37 DEG C, is purified by the culture fluid obtained by octanoic acid-saturated ammonium sulfate method, obtains monoclonal antibody ,-20 DEG C of preservations.
Described cell culture medium is to add calf serum and sodium bicarbonate in RPMI-1640 culture medium, make the calf serum final concentration of 20% (weight/mass percentage composition) in cell culture medium, make the sodium bicarbonate final concentration of 0.2% (weight/mass percentage composition) in cell culture medium；The pH of described cell culture medium is 7.4.
3, the preparation of sheep anti mouse anti antibody
Using sheep as immune animal, for immunogen, pathogen-free domestic sheep is carried out immunity with Mus source antibody, obtain sheep anti mouse anti antibody.
4, the preparation of monoclonal antibody-colloid gold label thing
(1) preparation of gold colloidal
By double distilled deionized water, 1% gold chloride is diluted to 0.01% (weight/mass percentage composition), put to stir on magnetic force heating rod agitator and boil, every 100ml0.01% gold chloride adds 2.5ml1% trisodium citrate, continue to stop heating when agitating heating reaction takes on a red color to liquid, after being cooled to room temperature, supply dehydration.The gold colloidal outward appearance prepared is pure, bright, without precipitation and floating thing.
(2) preparation of monoclonal antibody-colloid gold label thing
Under magnetic stirring, adjust the pH value of gold colloidal to 7.0 with 0.2mol/L potassium carbonate, in colloidal gold solution, above-mentioned spectinomycin monoclonal antibody, streptomycin monoclonal antibody, gentamycin monoclonal antibody and neomycin monoclonal antibody it is separately added into by the standard of 100-150 μ g antibody/ml gold colloidal, continue stirring and evenly mixing 30min, add the 10%BSA to BSA final concentration of 1% (volumn concentration) in colloidal gold solution, stand 30min.12000rpm, 4 DEG C of centrifugal 30min, abandon supernatant, precipitation uses redissolution buffer solution twice, to precipitate resuspended with the redissolution buffer that volume is initial colloid gold volume 1/20, the concentration of the spectinomycin monoclonal antibody respectively obtained-colloid gold label thing solution, streptomycin monoclonal antibody-colloid gold label thing solution, gentamycin monoclonal antibody-colloid gold label thing solution and neomycin monoclonal antibody-colloid gold label thing solution is 50 μ g/ml solution, put 4 DEG C standby.
Redissolve buffer: casein containing protein, the phosphate solution of 0.02mol/L, pH7.2 of tween 80, the wherein casein final concentration of 0.05%-0.1% (volumn concentration) in redissolution buffer, the tween 80 final concentration of 0.05%-0.15% (weight/mass percentage composition) in redissolution buffer.
5, in monoclonal antibody-colloid gold label thing micropore reagent lyophilizing to capillary strip
50 μ l spectinomycin monoclonal antibodies-colloid gold label thing is added in the micropore of capillary strip, 50 μ l streptomycin monoclonal antibodies-colloid gold label thing, 50 μ l gentamycin monoclonal antibodies-colloid gold label thing and 50 μ l neomycin monoclonal antibodies-colloid gold label thing, put in freezer dryer, under the conditions of condenser temperature is-70 DEG C, after pre-freeze 4h, lyophilizing 14h again, i.e. can be taken off, in the capillary strip obtained, lyophilizing has spectinomycin monoclonal antibody-colloid gold label thing, streptomycin monoclonal antibody-colloid gold label thing, gentamycin monoclonal antibody-colloid gold label thing and the micropore reagent of neomycin monoclonal antibody-colloid gold label thing.
6, the preparation of sample absorption pad
Sample absorption pad is placed in containing BSA (BSA final concentration of 0.5% (volumn concentration) in buffer), pH be 7.2,0.1mol/L phosphate buffer soak 2h, 37 DEG C dry 2h standby.
7, the preparation of reaction film
Being coated process: with phosphate buffer, spectinomycin-ovalbumin conjugate is diluted to 10mg/mL, detection line (T1) package amount being coated on nitrocellulose filter with Biodot point film instrument is 1.0 μ g/cm2；With phosphate buffer, streptomycin hapten-ovalbumin conjugate being diluted to 10mg/mL, the detection line (T2) being coated on nitrocellulose filter with Biodot point film instrument, package amount is 1.5 μ g/cm2；With phosphate buffer, gentamycin hapten-ovalbumin conjugate being diluted to 10mg/mL, the detection line (T3) being coated on nitrocellulose filter with Biodot point film instrument, package amount is 1.5 μ g/cm2；With phosphate buffer, neomycin hapten-ovalbumin conjugate being diluted to 10mg/mL, the detection line (T4) being coated on nitrocellulose filter with Biodot point film instrument, package amount is 1.5 μ g/cm2；With 0.01mol/L, pH7.4PBS buffer, sheep anti-mouse igg antibody being diluted to 200 μ g/ml, the nature controlling line (C) being coated on nitrocellulose filter with Biodot point film instrument, package amount is 1.0 μ g/cm2.2h it is dried under the conditions of the reaction film being coated is placed in 37 DEG C, standby.
(2) assembling of each parts
1, the assembling of reagent paper
Described sample absorption pad, reaction film, adsorptive pads, protecting film are pasted the most in order on described base plate；The end of sample absorption 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 sample absorption pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate；Protecting film is pasted in the absorption pad one end assembling reagent paper.
2, test strips assembles
Above-mentioned steps 1 is obtained reagent paper, micropore reagent, capillary strip and micropore plug and is assembled into test strips, store in the environment of 2～8 DEG C, 12 months effect duration.
Two, the sensitivity of test strips and specific assay
Test strips of the present invention can detect medicine and include: spectinomycin, streptomycin, dihydrostreptomycin, gentamycin, neomycin.
(1) sensitivity test
Spectinomycin, streptomycin, dihydrostreptomycin, gentamycin, neomycin standard substance (purchased from Germany Dr and China Veterinery Drug Inspection Office) are diluted to following variable concentrations: 10,20,40 μ g/L spectinomycin；40,80,160 μ g/L streptomycin；40,80,160 μ g/L dihydrostreptomycin；10,20,40 μ g/L gentamycin；250,500,1000 μ g/L neomycin.Diluent used be pH be 7.2, the phosphate buffer of 0.2mol/L.
Detect by test strips.In the capillary strip including gold labeling antibody micropore reagent, drip 200 μ l samples every time, mixing, after hatching 5min, test strips is inserted detection, result is: drip examination 10 μ g/L spectinomycin and gentamycins；40 μ g/L streptomycin and dihydrostreptomycins；During 250 μ g/L neomycin, reagent paper demonstrates macroscopic five red bar lines, is negative；When dripping examination 20,40 μ g/L spectinomycin and gentamycin；80,160 μ g/L streptomycin and dihydrostreptomycin；500, during 1000 μ g/L neomycin, reagent paper nature controlling line develops the color, and detection line (T1) does not develops the color, and detection line (T2) does not develops the color, and detection line (T3) does not develops the color, and detection line (T4) does not develops the color, and is positive.Showing, the sensitivity of this ELISA test strip is respectively as follows: spectinomycin 20 μ g/L, streptomycin 80 μ g/L, dihydrostreptomycin 80 μ g/L, gentamycin 20 μ g/L, neomycin 500 μ g/L.
(2) specific test
Specificity is commonly used cross reacting rate and is represented, refers to that the antibody antigenic determinant different from structure occurs the ability combined.The sensitivity of this ELISA test strip is respectively as follows: spectinomycin 20 μ g/L, streptomycin 80 μ g/L, dihydrostreptomycin 80 μ g/L, gentamycin 20 μ g/L, neomycin 500 μ g/L.By the other drug (lincomycin, norfloxacin, chloromycetin, tetracycline) of often inspection in milk with pH be 7.2, the phosphate buffer of 0.2mol/L is diluted.Detect by the test strips described in embodiment 1, result display lincomycin, norfloxacin, chloromycetin, tetracycline are when 500 μ g/L concentration, one nature controlling line of reagent paper and four detection lines all develop the color, it can be deduced that this test strips is not to these medicine generation cross reactions.
Embodiment 3, the application of test strips
One, with spectinomycin, streptomycin, gentamycin and neomycin in ELISA test strip milk described in embodiment 1
The test strips of the present invention can detect milk sample.General detection method is as follows:
1, detection method
The micropore plug covered on capillary strip is taken off, the sample solution that need to detect is dripped in the capillary strip including micropore reagent, obtain the mixed solution 11 of micropore reagent and sample solution, after mixing, hatch 5min, reagent paper 12 has MAX wire tag end insert down in capillary strip, in 5min, watches result, as shown in Figure 7.
2, testing result judges
nullIf the content of spectinomycin or streptomycin or gentamycin or neomycin is high in measuring samples liquid，Then in diffusion process, the spectinomycin in analyte sample fluid or streptomycin or gentamycin or neomycin can combine with gold labeling antibody，And then spectinomycin or streptomycin or gentamycin or the antigen-combining site of neomycin on completely enclosed gold labeling antibody，Stop gold labeling antibody spectinomycin-carrier protein couplet thing on reaction film to be combined or streptomycin-carrier protein couplet thing combines or gentamycin-carrier protein couplet thing combines or the combination of neomycin-carrier protein couplet thing，Corresponding spectinomycin detection line or streptomycin detection line or gentamycin detect line or neomycin detection line can not develop the color，Anti antibody then can be combined with spectinomycin gold labeling antibody or streptomycin gold labeling antibody or gentamycin gold labeling antibody or neomycin gold labeling antibody，Nature controlling line develops the color；nullIf in measuring samples liquid, the content of spectinomycin or streptomycin or gentamycin or neomycin is low or nothing，Antigen binding site on then corresponding spectinomycin gold labeling antibody or streptomycin gold labeling antibody or gentamycin gold labeling antibody or neomycin gold labeling antibody can not be closed，And then spectinomycin gold labeling antibody can spectinomycin-carrier protein couplet antigen be combined or streptomycin gold labeling antibody can streptomycin-carrier protein couplet antigen be combined or gentamycin gold labeling antibody can gentamycin-carrier protein couplet antigen be combined or neomycin gold labeling antibody can be combined by neomycin-carrier protein couplet antigen on reaction film on reaction film on reaction film on reaction film，Corresponding spectinomycin detection line colour developing or streptomycin detect line colour developing or gentamycin detection line colour developing or neomycin detection line develops the color，Anti antibody also can be combined with spectinomycin gold labeling antibody or streptomycin gold labeling antibody or gentamycin gold labeling antibody or neomycin gold labeling antibody simultaneously，Nature controlling line develops the color；If nature controlling line does not develops the color, then test strips lost efficacy.
Negative (-): C line develops the color, and four T lines all develop the color, no matter shade, all represents that spectinomycin in milk sample, streptomycin, gentamycin, neomycin concentration are below detection limit, as shown in Fig. 8 .a.
Positive (+): C line develops the color, T1Line does not develops the color, and represents that in milk sample, spectinomycin concentration is equal to or higher than detection limit；C line develops the color, T2Line does not develops the color, and represents that milk sample streptomycin concentration is equal to or higher than detection limit；C line develops the color, T3Line does not develops the color, and represents that in milk sample, gentamicin concentration is equal to or higher than detection limit；C line develops the color, T4Line does not develops the color, and represents that in milk sample, neomycin concentration is equal to or higher than detection limit, as shown in Fig. 8 .a-8.f.
Invalid: C line does not occurs, show that incorrect operating process or test strips lost efficacy, as shown in Fig. 8 .g-8.k.
Citing in detail below:
1, false positive rate measures
The negative milk sample 50 parts of instrumental method of learning from else's experience confirmation, numbered 1#-50#, detects test strips of the present invention for sample, calculates its false positive rate.
Result: in 50 parts of negative milk samples measure, ELISA test strip result is total negative, and test strips false positive rate of the present invention is 0.
2, false negative rate measures
The negative milk sample 50 parts of instrumental method of learning from else's experience confirmation, adds spectinomycin, streptomycin, gentamycin, neomycin by the detection limit concentration of regulation respectively, test strips of the present invention for sample is detected, calculate its false negative rate.
Result: in the mensuration of 50 parts of positive milk samples, the negative sample that ELISA test strip goes out is 0 part, and false negative rate is 0.
Spectinomycin, streptomycin, gentamycin and Detection of neomycin residues can be used for quickly detecting by the test strips of detection spectinomycin, streptomycin, gentamycin and the neomycin of the present invention simultaneously.
Claims (13)
- null1. a detection spectinomycin、Streptomycin、Gentamycin and the test strips of neomycin，It is characterized in that including reagent paper、Capillary strip、Micropore reagent and micropore plug，Described reagent paper includes reaction film、Sample absorption pad、Adsorptive pads、Protecting film、Base plate，Four detection lines and a nature controlling line is had on described reaction film，Article four, detection line is coated with spectinomycin-carrier protein couplet thing respectively、Streptomycin-carrier protein couplet thing、Gentamycin-carrier protein couplet thing and neomycin-carrier protein couplet thing，Described spectinomycin-carrier protein couplet thing is obtained with carrier protein couplet by spectinomycin hapten，Described streptomycin-carrier protein couplet thing is obtained with carrier protein couplet by streptomycin hapten，Described gentamycin-carrier protein couplet thing is obtained with carrier protein couplet by gentamycin hapten，Described neomycin-carrier protein couplet thing is obtained with carrier protein couplet by neomycin hapten，Nature controlling line is coated with anti antibody，Described micropore reagent is the spectinomycin specific antibody-colloid gold label thing of lyophilizing、Streptomycin specific antibody-colloid gold label thing、Gentamycin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing，The haptenic preparation method of described spectinomycin is: take spectinomycin 0.66g、Carboxymethyl azanol 0.27g and pyridine 1ml mixed liquor in 20mlDMSO，It is stirred at room temperature reaction 20 hours，Solvent is evaporated off，Column chromatography is recrystallized to give the condensation substance of spectinomycin and carboxymethyl azanol after purification in ethanol-water system；The haptenic preparation method of described streptomycin is: 1.0g streptomycin, 0.30g phthalic anhydride and 1ml pyridine mixed liquor in 20mlDMSO, stirring reaction 24 hours at 80 DEG C, solvent is evaporated off, ethanol-water system is repeatedly recrystallized to give phthalic acid strand mycin ester；The haptenic preparation method of described gentamycin is: 0.39g bromo-acetic acid tert-butyl solution in 5mlDMSO, it is slowly added dropwise at 40 DEG C in 0.90g gentamycin and 1ml pyridine mixture in 10mlDMSO, after dropping, after continuing reaction 6 hours, solvent is evaporated off, after simple column chromatography for separation, remove solvent, add 20mlDMSO and 5ml formic acid, room temperature reaction 20 hours, solvent is evaporated off, ethanol-water system is recrystallized to give carboxymethyl gentamycin；The haptenic preparation method of described neomycin is: 0.61g neomycin and the mixed liquor of 10mlDMSO, it is slowly added dropwise under room temperature in the 10mlDMSO solution of 0.14g terephthalaldehyde, drip complete rear chamber temperature to react 2-4 hour to 60 DEG C, remove solvent, ethanol-water system is recrystallized to give neomycin terephthalaldehyde list condensation substance.
- Test strips the most according to claim 1, it is characterised in that described reagent paper is pasted successively by sample absorption pad, reaction film, adsorptive pads, protecting film and formed on base plate, and described capillary strip has micropore plug.
- Test strips the most according to claim 1, it is characterised in that: described protecting film is pasted onto on the sample absorption pad of reagent paper, for test side, has MAX mark line above.
- Test strips the most according to claim 1, the detection line of spectinomycin-carrier protein couplet thing it is coated with near sample absorption pad one end described in it is characterized in that, nature controlling line near adsorptive pads one end, be coated with streptomycin-carrier protein couplet thing detection line, be coated with gentamycin-carrier protein couplet thing detection line, be coated with neomycin-carrier protein couplet thing detection line between detection line and the nature controlling line being coated with spectinomycin-carrier protein couplet thing.
- Test strips the most according to claim 1, it is characterized in that: spectinomycin-carrier protein couplet thing is by taking spectinomycin 0.66g, carboxymethyl azanol 0.27g and pyridine 1ml mixed liquor in 20mlDMSO, it is stirred at room temperature reaction 20 hours, solvent is evaporated off, column chromatography is recrystallized to give the condensation substance of spectinomycin and carboxymethyl azanol after purification in ethanol-water system, obtaining with carrier protein couplet, described carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroprotein or human serum albumin again.
- Test strips the most according to claim 1, it is characterized in that: streptomycin-carrier protein couplet thing is by taking 1.0g streptomycin, 0.30g phthalic anhydride and 1ml pyridine mixed liquor in 20mlDMSO, stirring reaction 24 hours at 80 DEG C, solvent is evaporated off, ethanol-water system is repeatedly recrystallized to give phthalic acid strand mycin ester, obtaining with carrier protein couplet, described carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroprotein or human serum albumin again.
- Test strips the most according to claim 1, it is characterized in that: gentamycin-carrier protein couplet thing is by taking 0.39g bromo-acetic acid tert-butyl solution in 5mlDMSO, it is slowly added dropwise at 40 DEG C in 0.90g gentamycin and 1ml pyridine mixture in 10mlDMSO, after dropping, after continuing reaction 6 hours, solvent is evaporated off, after simple column chromatography for separation, remove solvent, add 20mlDMSO and 5ml formic acid, room temperature reaction 20 hours, solvent is evaporated off, ethanol-water system is recrystallized to give carboxymethyl gentamycin, obtain with carrier protein couplet again, described carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroprotein or human serum albumin.
- Test strips the most according to claim 1, it is characterized in that: neomycin-carrier protein couplet thing is the mixed liquor by taking 0.61g neomycin and 10mlDMSO, it is slowly added dropwise under room temperature in the 10mlDMSO solution of 0.14g terephthalaldehyde, drip complete rear chamber temperature to react 2-4 hour to 60 DEG C, remove solvent, ethanol-water system is recrystallized to give neomycin terephthalaldehyde list condensation substance, obtaining with carrier protein couplet, described carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroprotein or human serum albumin again.
- Test strips the most according to claim 1, it is characterized in that: the spectinomycin specific antibody in spectinomycin specific antibody-colloid gold label thing is to prepare using spectinomycin-carrier protein couplet thing as immunogen, and described spectinomycin specific antibody is spectinomycin monoclonal antibody；Streptomycin specific antibody in streptomycin specific antibody-colloid gold label thing is to prepare using streptomycin-carrier protein couplet thing as immunogen, and described streptomycin specific antibody is streptomycin monoclonal antibody；Gentamycin specific antibody in gentamycin specific antibody-colloid gold label thing is to prepare using gentamycin-carrier protein couplet thing as immunogen, and described gentamycin specific antibody is gentamycin monoclonal antibody；Neomycin specific antibody in neomycin specific antibody-colloid gold label thing is to prepare using neomycin-carrier protein couplet thing as immunogen, and described neomycin specific antibody is neomycin monoclonal antibody.
- Test strips the most according to claim 1, it is characterised in that: containing the spectinomycin specific antibody-colloid gold label thing of lyophilizing, streptomycin specific antibody-colloid gold label thing, gentamycin specific antibody-colloid gold label thing and the micropore reagent of neomycin specific antibody-colloid gold label thing bottom described capillary strip.
- 11. test strips according to claim 1, it is characterised in that: described micropore plug can closely be filled in capillary strip.
- The method of 12. 1 kinds of test strips prepared described in any one of claim 1-9, its step includes:1) capillary strip that lyophilizing has the micropore reagent of spectinomycin specific antibody-colloid gold label thing, streptomycin specific antibody-colloid gold label thing, gentamycin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing is prepared respectively；2) prepare respectively to have and be coated four of spectinomycin-carrier protein couplet thing, streptomycin-carrier protein couplet thing, gentamycin-carrier protein couplet thing and neomycin-carrier protein couplet thing detection lines and be coated the reaction film of nature controlling line of anti antibody；3) by 2) reaction film for preparing is assembled into reagent paper with sample absorption pad, protecting film, adsorptive pads, base plate；4) by 1) and 3) lyophilizing for preparing has spectinomycin specific antibody-colloid gold label thing, streptomycin specific antibody-colloid gold label thing, gentamycin specific antibody-colloid gold label thing and the capillary strip of micropore reagent of neomycin specific antibody-colloid gold label thing, micropore plug and reagent paper to be assembled into test strips.
- 13. 1 kinds are detected spectinomycin, streptomycin, gentamycin and the detection method of Detection of neomycin residues in milk sample simultaneously, it is characterized in that directly being inserted by reagent paper in the micropore of the mixed solution including detection sample solution and micropore reagent and detect, it includes step:1) detect by the test strips described in any one of claim 1-9；2) testing result is analyzed.
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| CN106769915A (en) * | 2016-11-22 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of detection method of spectinomycin |
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