CN103713131A - Spectinomycin, streptomycin, gentamycin and neomycin detection test strip and method thereof - Google Patents
Spectinomycin, streptomycin, gentamycin and neomycin detection test strip and method thereof Download PDFInfo
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- CN103713131A CN103713131A CN201210372008.1A CN201210372008A CN103713131A CN 103713131 A CN103713131 A CN 103713131A CN 201210372008 A CN201210372008 A CN 201210372008A CN 103713131 A CN103713131 A CN 103713131A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention discloses a spectinomycin, streptomycin, gentamycin and neomycin detection test strip and a method thereof. The test strip comprises micro-porous strips, micro-porous reagents, a test paper and micro-porous plugs, the micro-porous reagents are lyophilized colloidal gold labeled spectinomycin, streptomycin, gentamycin and neomycin specific antibodies respectively, the test paper is formed by sequentially connecting a sample absorption pad, a reaction film, a water absorption pad, a protection film and a substrate, the reaction film comprises four detection lines and a quality control line, the four detection lines are coated with a spectinomycin-carrier protein conjugate, a streptomycin-carrier protein conjugate, a gentamycin-carrier protein conjugate and a neomycin-carrier protein conjugate respectively, and the quality control line is coated with an antiantibody. The method for detecting the spectinomycin, streptomycin, gentamycin and neomycin by using the test strip has the advantages of simplicity, rapidness, perceptual intuition, accuracy, portability, wide application range, low cost, and easy popularized use.
Description
Technical field
The present invention relates to a kind of test strips and method that detects spectinomycin, streptomysin, gentamicin and neomycin.
Background technology
Spectinomycin claims again spectinomycin, spextinomyxin, grand rhzomorph etc., a kind of aminocyclitol antibiotic producing for grand sight streptomycete, Gram-negative bacteria (as brucella, Klebsiella, proteus, salmonella, Pasteurella etc.) is had and pretends use, a little less than gram-positive bacteria (streptococcus, staphylococcus etc.) effect, mycoplasma is also had to certain effect, be the microbiotic that a novel special efficacy is specially controlled gonorrhoea, NEISSERIA GONORRHOEAE is had to stronger antibacterial action.Can promote growth of animal, be good animal feed additive, as veterinary drug, is widely used in animal husbandry.Some illegal retailers are subject 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 be just equivalent to long-term low dose and absorb microbiotic, make bacterial strain in human body produce antibiotic resistance, giving when ill uses antibiotic therapy to bring harmful effect, can destroy organismic internal environment balance, cause flora imbalance, the people of microbiotic allergic constitution there will be allergic reaction, cause immune hemolysis, the series of problems that simultaneously causes food and environment drug residue to pollute.
Streptomysin is aminoglycoside antibiotics, applies generally, but owing to using management lack of standardization, often cause fresh milk and milk powder streptomycin residual in milk cow and milch goat cultivation.Due to streptomysin, human body is had to larger toxic and side effect, when serious, can cause anaphylactic shock and deafness, particularly larger to child's harm, can see through placenta and enter amniotic fluid and fetal circulation, harm foetus health.Various countries have all formulated strict residue limits to the streptomysin in dairy products, dihydrostreptomycin, if always limiting the quantity of of Japanese regulation milk streptomycin and dihydrostreptomycin is 0.2 mg/kg, in U.S.'s regulation breast and milk, limiting the quantity of of dihydrostreptomycin is 0.125 mg/kg, China's regulation milk streptomycin and limiting the quantity of of dihydrostreptomycin are 0.2 mg/kg, so the streptomysin in milk powder, dihydrostreptomycin residual quantity are detected for controlling quality of milk powder, safeguard that the mankind's healthy diet is significant.
Gentamicin is a kind of aminoglycoside antibiotics being produced by small single-cell bacteria, and multiple Grain-positive and negative bacterium are all had to significant antibacterial effect, as feed addictive and the microbiotic for the treatment of disease, is widely used in animal husbandry.Yet gentamicin has obvious toxic action to the mankind, 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 food.European Union's regulation, the maximum residue limit in milk is 100 μ g/kg, the maximum residue limit in muscle, fat is 50 μ g/kg.
Neomycin belongs to aminoglycoside antibiotics, and has a broad antifungal spectrum is widely used on veterinary clinic, is important anti-infectives, is also widely used in the diseases such as treatment mastitis, keratitis, conjunctivitis, otitis, dermatitis, trauma infection contamination, foot rot.China and some other country are also used neomycin as fodder antibiotics adjuvant, and the wall losses and enteritis morning for preventing livestock and poultry to infect to cause, improves efficiency of feed utilization, promotes growth.Although neomycin use in animal doctor and herding has obtained huge success, there is potential ototoxicity and renal toxicity to humans and animals in it, and for this reason, the countries such as China and European Union have all formulated neomycin maximum residue limit(MRL) in animal derived food.
Immunoassay field lacks technology and the application thereof that can simultaneously detect these four kinds of medicines of spectinomycin, streptomysin, gentamicin and neomycin at present.Therefore, be necessary in practice to set up that a Species sensitivity is high, simple to operate, cost is low, detection of drugs kind is many, be applicable to the detection method of large-scale promotion.
Summary of the invention
An object of the present invention is to provide a kind of test strips that detects spectinomycin, streptomysin, gentamicin and neomycin.
Test strips provided by the present invention 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; Spectinomycin specific antibody-colloid gold label thing that described micropore reagent is freeze-drying, streptomysin specific antibody-colloid gold label thing, gentamicin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing; Spectinomycin specific antibody is spectinomycin monoclonal antibody, and streptomysin specific antibody is streptomysin monoclonal antibody, and gentamicin specific antibody is gentamicin monoclonal antibody, and neomycin specific antibody is neomycin monoclonal antibody; On reaction film, be coated with four detection lines and a nature controlling line; In micropore reagent, comprise spectinomycin monoclonal antibody-colloid gold label thing, streptomysin monoclonal antibody-colloid gold label thing, gentamicin monoclonal antibody-colloid gold label thing, neomycin monoclonal antibody-colloid gold label thing, spectinomycin detection line is coated with spectinomycin-carrier protein couplet thing, streptomysin detection line is coated with streptomysin-carrier protein couplet thing, gentamicin detection line is coated with gentamicin-carrier protein couplet thing, neomycin detection line is coated with neomycin-carrier protein couplet thing, and nature controlling line is coated with sheep anti mouse antiantibody.
Described diaphragm sticks on the test side on absorption of sample pad, has MAX mark line above.
Described spectinomycin detection line is near absorption of sample pad one end, and nature controlling line is near adsorptive pads one end, and streptomysin detection line, gentamicin detection line, neomycin detection line are between spectinomycin detection line and nature controlling line.Wherein spectinomycin detection line (T1) expression for detection line, streptomysin is detection line (T2) expression for detection line, and gentamicin is detection line (T3) expression for detection line, and neomycin is detection line (T4) expression for detection line, and nature controlling line for nature controlling line (C) represents.Take absorption of sample pad one end of test paper is top, take adsorptive pads one end of test paper is end, one end that nature controlling line (C) is connected with adsorptive pads top apart from reaction film is 5-8mm, 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.
Described spectinomycin-carrier protein couplet thing is to be obtained by spectinomycin and carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
Described streptomysin-carrier protein couplet thing is to be obtained by streptomysin and carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
Described gentamicin-carrier protein couplet thing is to be obtained by gentamicin and carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
Described neomycin-carrier protein couplet thing is to be obtained by neomycin and carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
Spectinomycin specific antibody in described spectinomycin specific antibody-colloid gold label thing is to using spectinomycin-carrier protein couplet thing to prepare as immunogene.
Streptomysin specific antibody in described streptomysin specific antibody-colloid gold label thing is to using streptomysin-carrier protein couplet thing to prepare as immunogene.
Gentamicin specific antibody in described gentamicin specific antibody-colloid gold label thing is to using gentamicin-carrier protein couplet thing to prepare as immunogene.
Neomycin specific antibody in described neomycin specific antibody-colloid gold label thing is to using neomycin-carrier protein couplet thing to prepare as immunogene.
Described base plate can be the material that PVC base plate or other hard do not absorb water; 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; Adsorptive pads is thieving paper; Diaphragm is PE material diaphragm.
Another object of the present invention has been to provide a kind of method of preparing above-mentioned test strips, and its step comprises:
1) on capillary strip, prepare the micropore reagent 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 of freeze-drying, and with micropore gag on capillary strip;
2) prepare four have respectively coated, spectinomycin-carrier protein couplet thing, streptomysin-carrier protein couplet thing, the reaction film of gentamicin-carrier protein couplet thing and the detection line of neomycin-carrier protein couplet thing and the nature controlling line of a coated antiantibody;
3) by 2) reaction film and absorption of sample pad, adsorptive pads, diaphragm, the base plate that prepare be assembled into test paper;
4) by 1) and 3) freeze-drying for preparing has micropore reagent, test paper, capillary strip and the micropore plug 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 be assembled into test strips.
Specifically, step comprises:
1, the preparation of the synthetic and antibody of spectinomycin antigen
1), by spectinomycin and carrier protein couplet, form spectinomycin-carrier protein couplet thing;
2) with spectinomycin-carrier protein couplet thing immune mouse, mouse boosting cell and murine myeloma cell are passed through to merge, screen, obtain secreting the hybridoma cell strain of spectinomycin monoclonal antibody.
2, the preparation of the synthetic and antibody of Streptomycin antigen
1), by streptomysin and carrier protein couplet, form streptomysin-carrier protein couplet thing;
2) with streptomysin-carrier protein couplet thing immune mouse, mouse boosting cell and murine myeloma cell are passed through to merge, screen, obtain secreting the hybridoma cell strain of streptomysin monoclonal antibody.
3, the preparation of the synthetic and antibody of gentamicin antigen
1), by gentamicin and carrier protein couplet, form gentamicin-carrier protein couplet thing;
2) with gentamicin-carrier protein couplet thing immune mouse, mouse boosting cell and murine myeloma cell are passed through to merge, screen, obtain secreting the hybridoma cell strain of gentamicin monoclonal antibody.
4, the preparation of the synthetic and antibody of neomycin antigen
1), by neomycin and carrier protein couplet, form neomycin-carrier protein couplet thing;
2) with neomycin-carrier protein couplet thing immune mouse, mouse boosting cell and murine myeloma cell are passed through to merge, screen, obtain secreting the hybridoma cell strain of neomycin monoclonal antibody.
5, extract mouse IgG immune health goat, obtain sheep anti-mouse igg antibody.
6, with trisodium citrate, react and prepare collaurum with gold chloride.
7, the spectinomycin specific antibody of preparation, streptomysin specific antibody, gentamicin specific antibody and neomycin specific antibody are joined respectively in the collaurum of preparation, obtain the micropore reagent 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.
8, by after the micropore reagent freeze-drying 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 is in capillary strip, micropore plug will be covered on capillary strip.
9, by absorption of sample pad with containing bovine serum albumin(BSA) (final concentration of bovine serum albumin(BSA) in damping fluid is 0.5%(volumn concentration)), pH7.2,0.1mol/L phosphate buffer soak 2h, at 37 ℃, dries 2h.
10, on base plate, stick in order absorption of sample pad, reaction film, adsorptive pads and diaphragm.
11, the micropore reagent preparing, test paper, capillary strip and micropore plug are assembled into test strips, under 2-8 ℃ of condition, preserve 12 months.
Another object of the present invention provides the detection method of spectinomycin, streptomysin, gentamicin and Detection of neomycin residues in a kind of milk sample.
By the above-mentioned arbitrary described test strips of the present invention, detect.
The detection principle of test strips of the present invention: milk sample drop to be checked is added in the capillary strip that includes micropore reagent, after mixing, hatch 5min, to indicate MAX mark line end downward, insertion includes in the micropore reagent capillary strip after hatching, sample liquid to be checked and golden labeling antibody in micropore in conjunction with after together with to reaction film, spread, if the content of spectinomycin or streptomysin or gentamicin or neomycin is high in sample liquid to be checked, in diffusion process, the spectinomycin in analyte sample fluid or streptomysin or gentamicin or neomycin can combine with golden labeling antibody, and then seal the antigen-combining site of spectinomycin on golden labeling antibody or streptomysin or gentamicin or neomycin completely, stop golden labeling antibody spectinomycin-carrier protein couplet thing on reaction film be combined or streptomysin-carrier protein couplet thing in conjunction with gentamicin-carrier protein couplet thing in conjunction with or the combination of neomycin-carrier protein couplet thing, corresponding spectinomycin detection line or streptomysin detection line or gentamicin detection line or neomycin detection line can not develop the color, antiantibody can be combined with spectinomycin gold labeling antibody or streptomysin gold labeling antibody or gentamicin gold labeling antibody or neomycin gold labeling antibody, nature controlling line colour developing, if the low or nothing of the content of spectinomycin or streptomysin or gentamicin or neomycin in sample liquid to be checked, the antigen binding site on corresponding spectinomycin gold labeling antibody or streptomysin gold labeling antibody or gentamicin gold labeling antibody or neomycin gold labeling antibody can not be closed, and then spectinomycin gold labeling antibody can be on reaction film spectinomycin-carrier protein couplet antigen be combined or streptomysin gold labeling antibody can be on reaction film streptomysin-carrier protein couplet antigen be combined or the golden labeling antibody of gentamicin can be on reaction film gentamicin-carrier protein couplet antigen be combined or the golden labeling antibody of neomycin can be combined by neomycin-carrier protein couplet antigen on reaction film, corresponding spectinomycin detection line colour developing or the colour developing of streptomysin detection line or the colour developing of gentamicin detection line or the colour developing of neomycin detection line, simultaneously antiantibody also can be combined with spectinomycin gold labeling antibody or streptomysin gold labeling antibody or gentamicin gold labeling antibody or neomycin gold labeling antibody, nature controlling line colour developing, if nature controlling line does not develop the color, test strips lost efficacy.
Negative (-): the colour developing of C line, four T lines all develop the color, and no matter shade, all represents that spectinomycin in milk sample, streptomysin, gentamicin, neomycin concentration are all lower than detectability, as shown in Fig. 8 .a.
Positive (+): the colour developing of C line, T1 line does not develop the color, and represents that in milk sample, spectinomycin concentration is equal to or higher than detectability; The colour developing of C line, T2 line does not develop the color, and represents that milk sample streptomycin concentration is equal to or higher than detectability; The colour developing of C line, T3 line does not develop the color, and represents that in milk sample, gentamicin concentration is equal to or higher than detectability; The colour developing of C line, T4 line does not develop the color, and represents that in milk sample, neomycin concentration is equal to or higher than detectability, as shown in Fig. 8 .a-8.f.
Invalid: not occur C line, 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 detection of drugs comprise: spectinomycin, streptomysin, dihydrostreptomycin, gentamicin, neomycin.
Test strips of the present invention has following beneficial effect:
1, highly sensitive: traditional test card all has the fixedly stationary installation that gets stuck of test paper, the stationary installation that gets stuck is fixing wherein by test paper tightly, when to sample detection, tightr due to what get stuck and be combined with test paper, cause detection sample solution can not fully be penetrated into one section, absorption of sample pad, and the speed of carrying out chromatography on test paper is very slow, the medicine that causes detecting in sample can not fully contact with coating antigen coated on detection line, causes the sensitivity of test card to decline.Test strips of the present invention is higher than the detection sensitivity of traditional test card, because the test paper of test strips of the present invention is naked strip, without housing fastener, detect absorption of sample pad one end that sample can directly contact test paper, and the chromatography velocity ratio of sample on test paper is very fast, make medicine to be checked in sample can be fully on detection line coated coating antigen be combined, thereby improved the detection sensitivity of test strips.Spectinomycin, streptomysin, gentamicin and neomycin test strip be take the monoclonal antibody of colloid gold label high-affinity and are prepared from as basis, priceless strong formation between gold grain and antibody molecule in gold labeling antibody, the two combines by the Van der Waals force between the charges of different polarity, collaurum is very little on monoclonal antibody specificity and affinity impact, 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 the freeze-drying of neomycin monoclonal antibody-colloid gold label thing are in micropore reagent strip, in testing process, can make golden labeling antibody fully contact with sample liquid to be checked, fully reaction, thereby minimizing error, increases the reaction sensitivity of whole system.Therefore, test strips of the present invention has higher specificity and sensitivity.This test strips is respectively various drug test sensitivity: spectinomycin 20 μ g/L, streptomysin 80 μ g/L, dihydrostreptomycin 80 μ g/L, gentamicin 20 μ g/L, neomycin 500 μ g/L.
Easy and simple to handle fast: for test card in the past detect raw material milk raw material milk must be added dilution dilute can point sample detection, reason is tightly test paper to be fixed in housing due to the fixing plastic casing of test paper, and raw material milk compares thickness, so directly raw material milk is carried out to the words of point sample detection at the well of test card, due to housing be combined with test paper closely and milk thickness due to, raw material milk sample cannot be penetrated into absorption of sample pad completely and bond discharges in pad, and the chromatography velocity ratio on test paper is slower, to such an extent as to cannot detect, therefore can detect after will adding dilution to dilute raw material milk.While using ELISA test strip of the present invention without any other reagent, while detecting raw material milk, do not need raw material milk to dilute yet, reason is because the test paper in test strips of the present invention is naked strip, without with housing, it being fixed, as long as sample solution to be checked is directly dripped in freeze-drying and has spectinomycin monoclonal antibody-colloid gold label thing, 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, after mixing, hatch 5min, again test paper is indicated to MAX wire tag end downward, in micropore reagent after insertion is hatched, test paper can fully contact with sample solution, observations after 5min.For the detection of milk sample, test strips of the present invention is shorter detection time than traditional test card, because test strips of the present invention is without sample is diluted, can directly to sample, detect.
Test strips susceptibility of the present invention is high, detection of drugs kind is many, cost is low, simple to operate, be easy to carry, detection time is short, be applicable to various units uses, stores advantage simple, long shelf-life.Wherein, adopt spectinomycin monoclonal antibody, streptomysin monoclonal antibody, gentamicin monoclonal antibody and the neomycin monoclonal antibody of high specific, guaranteed the reliability of testing result; In capillary strip, in testing process, can make golden labeling antibody fully contact with sample liquid to be checked golden labeling antibody freeze-drying, fully reaction, thus reduce error, increase the reaction sensitivity of whole system.Test strips of the present invention can detect spectinomycin, streptomysin, gentamicin and four kinds of medicines of neomycin simultaneously, realize the detection of a test strips to multi-medicament, met the demand on market, spectinomycin, streptomysin, gentamicin and neomycin being detected simultaneously.By the method for ELISA test strip spectinomycin of the present invention, streptomysin, gentamicin and neomycin, easy, quick, directly perceived, accurate, applied widely, cost is low, easily promotes the use of.
Accompanying drawing explanation
Fig. 1 freeze-drying has the capillary strip figure of golden labeling antibody.
Fig. 2 micropore plug vertical view.
Fig. 3 micropore plug cross-sectional view.
Fig. 4 test paper cross-sectional view.
The vertical view of Fig. 5 test paper.
Fig. 6 tetracycline medication haptens synthetic route chart.
Fig. 7 ELISA test strip method schematic diagram.
Fig. 8 ELISA test strip result process decision chart.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
The formation of the test strips of embodiment 1, detection spectinomycin, streptomysin, gentamicin and neomycin
One, freeze-drying has the capillary strip of golden labeling antibody
Micropore reagent is housed in described capillary strip, wherein 1 is capillary strip, the 2 micropore reagent for spectinomycin specific antibody-colloid gold label thing of freeze-drying, streptomysin specific antibody-colloid gold label thing, gentamicin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing;
On the described capillary strip that includes micropore reagent, there is micropore plug 3(Fig. 2 and Fig. 3);
The micropore reagent of the spectinomycin of freeze-drying 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 in described capillary strip, the freeze-drying amount of spectinomycin specific antibody-colloid gold label thing is 0.20-0.25 μ g/ml; The freeze-drying amount of streptomysin specific antibody-colloid gold label thing is 0.30-0.40 μ g/ml; The freeze-drying amount of gentamicin specific antibody-colloid gold label thing is 0.20-0.25 μ g/ml; The freeze-drying amount of neomycin specific antibody-colloid gold label thing is 0.20-0.25 μ g/ml.
Two, test paper (Fig. 4 and Fig. 5)
Described test paper is comprised of base plate, absorption of sample pad, reaction film, adsorptive pads, diaphragm;
Described absorption of sample pad 4, reaction film 5, adsorptive pads 6 and diaphragm 7 are pasted successively in order on described base plate 8, the end of absorption of sample pad is connected with reaction film, the end of reaction film is connected with adsorptive pads, the top of absorption of sample pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate;
Diaphragm 7 covers the test side on absorption of sample pad, on the diaphragm of test side, should have MAX printed words.
On described reaction film, there are four detection lines to be respectively detection 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; article four, detection line is the ribbon vertical with the appearance of described test paper with a nature controlling line; article four, detection line is positioned at a side of the diaphragm that is bordering on MAX mark line, and nature controlling line (C) is positioned at away from a side that has the diaphragm of MAX mark.One end that nature controlling line (C) is connected with adsorptive pads apart from the end of reaction film is 5-8mm, 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 (spectinomycin-oralbumin), detection line (T2) is coated with streptomysin-carrier protein couplet thing (streptomysin-oralbumin), detection line (T3) is coated with gentamicin-carrier protein couplet thing (gentamicin-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 antiantibody.
Base plate is PVC base plate; Absorption of sample pad is suction strainer paper; Adsorptive pads is thieving paper; Reaction film is nitrocellulose filter; Described diaphragm is PE material diaphragm.
Above-mentioned test paper and micropore reagent, capillary strip and micropore plug are assembled into test strips, in the environment of 2～8 ℃, store the term of validity 12 months.
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 micropore reagent of 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 of freeze-drying, and with micropore gag on capillary strip.
2) preparation has the detection line (T1) of coated spectinomycin-carrier protein couplet thing, the reaction film of the detection line (T3) of the detection line (T2) of coated streptomysin-carrier protein couplet thing, coated gentamicin-carrier protein couplet thing, the coated detection line (T4) of neomycin-carrier protein couplet thing and the nature controlling line (C) of coated sheep anti mouse antiantibody;
3) by 2) reaction film and absorption of sample pad, adsorptive pads, diaphragm, the base plate that prepare be assembled into test paper;
4) by 1) and 3) freeze-drying for preparing has micropore reagent, test paper, capillary strip and the micropore plug of 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 to be assembled into test strips.
Substep describes in detail below:
(1) preparation of each parts
1, the preparation of antigen
1), the synthetic and evaluation of spectinomycin-carrier protein couplet thing
A. 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, after column chromatography purification, in ethanol-water system, recrystallization obtains the condensation product of spectinomycin and ethyloic azanol, and molecular structure is as follows:
B. immunogenic preparation-spectinomycin haptens and BSA conjugate are synthetic
Get the water-soluble solution of 5ml for spectinomycin haptens 38mg; Get the water-soluble solution of 5ml for bovine serum albumin(BSA) (BSA) 100mg; Spectinomycin haptens aqueous solution is added in BSA aqueous solution, by magnetic stirrer, react 24h; 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, saves backup in-20 ℃.
C. preparation-spectinomycin haptens of coating antigen and OVA conjugate are synthetic
By spectinomycin haptens 22mg, 15mg N, N'-carbonyl dimidazoles (CDI) dissolves with 1.5ml DMF (DMF), and stirring at room reaction 1h, can obtain reactant liquor A; Take ovalbumin (OVA) 36mg, make it to be fully dissolved in 3.5ml 50mmol/L sodium carbonate liquor, reactant liquor A is dropwise slowly added drop-wise 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, saves backup in-20 ℃.
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, with 0.01mol/L pH7.4 PBS, return to zero, with ultraviolet spectrophotometer, 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.There is different absorption curves in three, shows spectinomycin and spectinomycin and carrier protein couplet success.
2), the synthetic and evaluation of streptomysin-carrier protein couplet thing
A. 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, at 80 ℃, stirring reaction is 24 hours, steaming desolventizes, and in ethanol-water system, repeatedly recrystallization obtains phthalic acid strand mycin ester, and molecular structure is as follows:
(formula II)
B. immunogenic preparation-streptomysin haptens and BSA conjugate are synthetic
Getting haptens 40mg dissolves completely with 2mlDMF, make I liquid, get BSA80mg 7ml0.1MPBS(PH7.5) dissolve completely, make II liquid, I liquid is added in II liquid, make III liquid, get EDC100mg with cushioning and add in III liquid after the water-soluble solution of 1ml, stirring at room 2 hours, with 0.01MPBS dialysis three days, change liquid every day three times, obtain immunogene, in-20 ℃, save backup.
C. preparation-streptomysin haptens of coating antigen and OVA conjugate are synthetic
Getting haptens 40mg dissolves completely with 2mlDMF, make I liquid, get OVA80mg 7ml0.1M PBS(PH7.5) dissolve completely, make II liquid, I liquid is added in II liquid, make III liquid, get EDC100mg with cushioning and add in III liquid after the water-soluble solution of 1ml, stirring at room 2 hours, with 0.01M PBS dialysis three days, change liquid every day three times, obtain immunogene, in-20 ℃, save backup.
D. the evaluation of streptomysin hapten-carrier conjugates
Streptomysin, bovine serum albumin(BSA), streptomycin-protein conjugate and streptomysin, ovalbumin, streptomysin-ovalbumin conjugate are made into the solution of 0.5mg/ml with the PBS of pH7.4, with 0.01mol/L pH7.4 PBS, return to zero, with ultraviolet spectrophotometer, in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of the absorption curve of streptomysin, bovine serum albumin(BSA), streptomycin-protein conjugate and streptomysin, ovalbumin, streptomysin-ovalbumin conjugate.There is different absorption curves in three, shows streptomysin and streptomysin and carrier protein couplet success.
3), the synthetic and evaluation of gentamicin-carrier protein couplet thing
A. gentamicin haptens is synthetic: the solution of 0.39 g bromo-acetic acid tert-butyl in 5 ml DMSO, is slowly added dropwise in 0.90 g gentamicin and the potpourri of 1 ml pyridine in 10 ml DMSO at 40 ℃.After dropwising, continue reaction after 6 hours, steaming desolventizes.After simple column chromatography for separation, except desolventizing, add 20 ml DMSO and 5 ml formic acid, room temperature reaction 20 hours, steaming desolventizes, and in ethanol-water system, recrystallization obtains ethyloic gentamicin, and molecular structure is as follows:
B. immunogenic preparation-gentamicin haptens and BSA conjugate are synthetic
Getting haptens 35mg dissolves completely with 2mlDMF, make I liquid, get BSA100mg 7ml0.1MPBS(PH7.5) dissolve completely, make II liquid, I liquid is added in II liquid, make III liquid, get EDC100mg with cushioning and add in III liquid after the water-soluble solution of 1ml, stirring at room 2 hours, with 0.01MPBS dialysis three days, change liquid every day three times, obtain immunogene, in-20 ℃, save backup.
C. preparation-gentamicin haptens of coating antigen and OVA conjugate are synthetic
Getting haptens 35mg dissolves completely with 2mlDMF, make I liquid, get OVA100mg 7ml0.1MPBS(PH7.5) dissolve completely, make II liquid, I liquid is added in II liquid, make III liquid, get EDC100mg with cushioning and add in III liquid after the water-soluble solution of 1ml, stirring at room 2 hours, with 0.01MPBS dialysis three days, change liquid every day three times, obtain immunogene, in-20 ℃, save backup.
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, with 0.01mol/L pH7.4 PBS, return to zero, with ultraviolet spectrophotometer, 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.There is different absorption curves in three, shows gentamicin and gentamicin and carrier protein couplet success.
4), the synthetic and evaluation of neomycin-carrier protein couplet thing
A. neomycin haptens is synthetic: the mixed liquor of 0.61 g neomycin and 10ml DMSO, under room temperature, be slowly added dropwise in 10 ml DMSO solution of 0.14 g terephthalaldehyde, dropwise rear room temperature to 60 ℃ reaction 2-4 hour, except desolventizing, in ethanol-water system, recrystallization obtains neomycin terephthalaldehyde list condensation product, and molecular structure is as follows:
B. immunogenic preparation-neomycin haptens and BSA conjugate are synthetic
Getting haptens 30mg dissolves completely with 3mlDMF, make I liquid, get BSA100mg 7ml0.1MPBS(PH7.0) dissolve completely, make II liquid, I liquid is added in II liquid, make III liquid, room temperature reaction 24 hours, with 0.01MPBS dialysis three days, changes liquid every day three times, obtain immunogene, in-20 ℃, save backup.
C. preparation-neomycin haptens of coating antigen and OVA conjugate are synthetic
Getting haptens 30mg dissolves completely with 3mlDMF, make I liquid, get OVA100mg 7ml0.1MPBS(PH7.0) dissolve completely, make II liquid, I liquid is added in II liquid, make III liquid, room temperature reaction 24 hours, with 0.01MPBS dialysis three days, changes liquid every day three times, obtain immunogene, in-20 ℃, save backup.
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, with 0.01mol/L pH7.4 PBS, return to zero, with ultraviolet spectrophotometer, 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.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, streptomysin, gentamicin and neomycin
(1) prepare monoclonal antibody
A. animal immune
Four kinds of immunogenes that step 1 is obtained are injected into respectively in Balb/c Mice 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 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 gentamicin monoclonal hybridoma strain of the streptomysin monoclonal hybridoma strain of stably excreting streptomysin monoclonal antibody, 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 to the cell suspension of 1 * 109/ml with cryopreserving liquid, in liquid nitrogen, preserved for a long time.During recovery, take out cryopreservation tube, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
D. the preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in to cell culture medium, cultivates under 37 ℃ of conditions, by sad-saturated ammonium sulfate method, the nutrient solution obtaining is carried out to purifying, obtain monoclonal antibody ,-20 ℃ of preservations.
Described cell culture medium for to add calf serum and sodium bicarbonate in 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
Using sheep as immune animal, and the mouse source antibody of take carries out immunity as immunogene to pathogen-free domestic sheep, obtains 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 on magnetic force heating rod stirrer and stir and boil, every 100ml 0.01% gold chloride adds 2.5 ml 1% trisodium citrates, 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, without precipitation and floating thing.
(2) preparation of monoclonal antibody-colloid gold label thing
Under magnetic agitation, with 0.2mol/L sal tartari, adjust the pH value to 7.0 of collaurum, by the standard of 100-150mg antibody/ml collaurum, in colloidal gold solution, add respectively above-mentioned spectinomycin monoclonal antibody, streptomysin monoclonal antibody, gentamicin monoclonal antibody and neomycin monoclonal antibody, continue to stir and evenly mix 30min, adding the final concentration of 10%BSA to BSA in colloidal gold solution is 1%(volumn concentration), standing 30min.12000rpm, 4 ℃ of centrifugal 30min, abandon supernatant, precipitation is with redissolving damping fluid washed twice, with the redissolution damping fluid that volume is initial collaurum volume 1/20, will precipitate resuspended, the concentration of the spectinomycin monoclonal antibody-colloid gold label thing solution obtaining respectively, 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 redissolution damping fluid is 0.05%-0.1%(volumn concentration), the final concentration of Tween-80 in redissolution damping fluid is 0.05%-0.15%(quality percentage composition).
5, the freeze-drying of monoclonal antibody-colloid gold label thing micropore reagent 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, after pre-freeze 4h, freeze-drying 14h again, can take out, in the capillary strip obtaining, freeze-drying has spectinomycin monoclonal antibody-colloid gold label thing, 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
It is 0.5%(volumn concentration at the final concentration of damping fluid that absorption of sample pad is placed in containing BSA(BSA)), pH is 7.2,0.1mol/L phosphate buffer soaks 2h, 37 ℃ to dry 2h standby.
7, the preparation of reaction film
Coated process: with phosphate buffer, spectinomycin-ovalbumin conjugate being diluted to 10mg/mL, is 1.0mg/cm2 with detection line (T1) package amount that Biodot point film instrument is coated on nitrocellulose filter; With phosphate buffer, streptomysin haptens-ovalbumin conjugate is diluted to 10mg/mL, with Biodot point film instrument, is coated in the detection line (T2) on nitrocellulose filter, package amount is 1.5mg/cm2; With phosphate buffer, gentamicin haptens-ovalbumin conjugate is diluted to 10mg/mL, with Biodot point film instrument, is coated in the detection line (T3) on nitrocellulose filter, package amount is 1.5mg/cm2; With phosphate buffer, neomycin haptens-ovalbumin conjugate is diluted to 10mg/mL, with Biodot point film instrument, is coated in the detection line (T4) on nitrocellulose filter, package amount is 1.5mg/cm2; With 0.01mol/L, pH 7.4 PBS damping fluids, sheep anti-mouse igg antibody is diluted to 200mg/ml, with Biodot point film instrument, is coated in the nature controlling line (C) on nitrocellulose filter, package amount is 1.0mg/cm2.The reaction film being coated with is placed in to dry 2h under 37 ℃ of conditions, standby.
(2) assembling of each parts
1, the assembling of test paper
Described absorption of sample pad, reaction film, adsorptive pads, diaphragm are pasted successively in order on described base plate; The end of absorption of sample pad is connected with the top of reaction film, and the end of reaction film is connected with the top of adsorptive pads, and the top of absorption of sample pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate; In the absorption pad one end that assembles test paper, paste diaphragm.
2, test strips assembling
Above-mentioned steps 1 is obtained to test paper, micropore reagent, capillary strip and micropore plug and be assembled into test strips, in the environment of 2～8 ℃, store the term of validity 12 months.
Two, the sensitivity of test strips and specificity check
Test strips of the present invention can detection of drugs comprise: spectinomycin, streptomysin, dihydrostreptomycin, gentamicin, neomycin.
(1) sensitivity test
Spectinomycin, streptomysin, dihydrostreptomycin, gentamicin, neomycin standard items (purchased from German DrHe China Veterinery Drug Inspection Office) are diluted to following variable concentrations: 10,20,40 μ g/L spectinomycins; 40,80,160 μ g/L streptomysins; 40,80,160 μ g/L dihydrostreptomycins; 10,20,40 μ g/L gentamicins; 250,500,1000 μ g/L neomycins.Dilution used is that pH is 7.2, the phosphate buffer of 0.2mol/L.
By test strips, detect.At every turn to including in the capillary strip of golden labeling antibody micropore reagent, drip 200 μ l samples, mix, hatch after 5min, test strips is inserted and detected, result is: drip examination 10 μ g/L spectinomycin and gentamicins; 40 μ g/L streptomysin and dihydrostreptomycins; During 250 μ g/L neomycin, on test paper, demonstrate macroscopic five red bar lines, be negative; When dripping examination 20,40 μ g/L spectinomycin and gentamicins; 80,160 μ g/L streptomysin and dihydrostreptomycins; 500, during 1000 μ g/L neomycin, the colour developing of test paper nature controlling line, detection line (T1) does not develop the color, and detection line (T2) does not develop the color, and detection line (T3) does not develop the color, and detection line (T4) does not develop the color, and is positive.Show, the sensitivity of this ELISA test strip is respectively: spectinomycin 20 μ g/L, streptomysin 80 μ g/L, dihydrostreptomycin 80 μ g/L, gentamicin 20 μ g/L, neomycin 500 μ g/L.
(2) specific test
The conventional cross reacting rate of specificity represents, refers to the ability of the antigenic determinant generation combination that antibody is different from structure.The sensitivity of this ELISA test strip is respectively: spectinomycin 20 μ g/L, streptomysin 80 μ g/L, dihydrostreptomycin 80 μ g/L, gentamicin 20 μ g/L, neomycin 500 μ g/L.By the other drug (lincomycin, Norfloxacin, chloromycetin, tetracycline) of normal inspection in milk with pH be 7.2, the phosphate buffer of 0.2mol/L dilutes.By the test strips described in embodiment 1, detect, result shows that lincomycin, Norfloxacin, chloromycetin, tetracycline are when 500 μ g/L concentration, nature controlling line of test paper and four detection lines all develop the color, and can show that this test strips is not to these medicine generation cross reactions.
The application of embodiment 3, test strips
One, with spectinomycin, streptomysin, gentamicin and neomycin in ELISA test strip milk described in embodiment 1
Test strips of the present invention can detect milk sample.General detection method is as follows:
1, detection method
The micropore plug covering on capillary strip is taken off, to including to drip in the capillary strip of micropore reagent, need the sample solution that detects, obtain the mixed solution 11 of micropore reagent and sample solution, after mixing, hatch 5min, there is MAX wire tag end to insert in capillary strip down on test paper 12, in 5min, watch result, as shown in Figure 7.
2, testing result is judged
If the content of spectinomycin or streptomysin or gentamicin or neomycin is high in sample liquid to be checked, in diffusion process, the spectinomycin in analyte sample fluid or streptomysin or gentamicin or neomycin can combine with golden labeling antibody, and then seal the antigen-combining site of spectinomycin on golden labeling antibody or streptomysin or gentamicin or neomycin completely, stop golden labeling antibody spectinomycin-carrier protein couplet thing on reaction film be combined or streptomysin-carrier protein couplet thing in conjunction with gentamicin-carrier protein couplet thing in conjunction with or the combination of neomycin-carrier protein couplet thing, corresponding spectinomycin detection line or streptomysin detection line or gentamicin detection line or neomycin detection line can not develop the color, antiantibody can be combined with spectinomycin gold labeling antibody or streptomysin gold labeling antibody or gentamicin gold labeling antibody or neomycin gold labeling antibody, nature controlling line colour developing, if the low or nothing of the content of spectinomycin or streptomysin or gentamicin or neomycin in sample liquid to be checked, the antigen binding site on corresponding spectinomycin gold labeling antibody or streptomysin gold labeling antibody or gentamicin gold labeling antibody or neomycin gold labeling antibody can not be closed, and then spectinomycin gold labeling antibody can be on reaction film spectinomycin-carrier protein couplet antigen be combined or streptomysin gold labeling antibody can be on reaction film streptomysin-carrier protein couplet antigen be combined or the golden labeling antibody of gentamicin can be on reaction film gentamicin-carrier protein couplet antigen be combined or the golden labeling antibody of neomycin can be combined by neomycin-carrier protein couplet antigen on reaction film, corresponding spectinomycin detection line colour developing or the colour developing of streptomysin detection line or the colour developing of gentamicin detection line or the colour developing of neomycin detection line, simultaneously antiantibody also can be combined with spectinomycin gold labeling antibody or streptomysin gold labeling antibody or gentamicin gold labeling antibody or neomycin gold labeling antibody, nature controlling line colour developing, if nature controlling line does not develop the color, test strips lost efficacy.
Negative (-): the colour developing of C line, four T lines all develop the color, and no matter shade, all represents that spectinomycin in milk sample, streptomysin, gentamicin, neomycin concentration are all lower than detectability, as shown in Fig. 8 .a.
Positive (+): the colour developing of C line, T1 line does not develop the color, and represents that in milk sample, spectinomycin concentration is equal to or higher than detectability; The colour developing of C line, T2 line does not develop the color, and represents that milk sample streptomycin concentration is equal to or higher than detectability; The colour developing of C line, T3 line does not develop the color, and represents that in milk sample, gentamicin concentration is equal to or higher than detectability; The colour developing of C line, T4 line does not develop the color, and represents that in milk sample, neomycin concentration is equal to or higher than detectability, as shown in Fig. 8 .a-8.f.
Invalid: not occur C line, show that incorrect operating process or test strips lost efficacy, as shown in Fig. 8 .g-8.k.
Concrete example below:
1, false positive rate is measured
50 parts of the negative milk samples of the instrumental method of learning from else's experience confirmation, are numbered 1#-50#, and sample is detected by test strips of the present invention, calculate its false positive rate.
Result: in 50 parts of negative milk samples are measured, ELISA test strip result is total negative, and test strips false positive rate of the present invention is 0.
2, false negative rate is measured
50 parts of the negative milk samples of the instrumental method of learning from else's experience confirmation, difference detectability concentration is in accordance with regulations added spectinomycin, streptomysin, gentamicin, neomycin, and sample is detected by test strips of the present invention, calculates 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.
The test strips of detection spectinomycin of the present invention, streptomysin, gentamicin and neomycin can be carried out fast detecting to spectinomycin, streptomysin, gentamicin and Detection of neomycin residues simultaneously.
Claims (13)
1. one kind is detected spectinomycin, streptomysin, the test strips of gentamicin and neomycin, it is characterized in that comprising test strips, capillary strip, micropore reagent and micropore plug, described test paper comprises reaction film, absorption of sample pad, adsorptive pads, diaphragm, base plate, on described reaction film, there are four detection lines and a nature controlling line, article four, detection line is coated with respectively spectinomycin-carrier protein couplet thing, streptomysin-carrier protein couplet thing, gentamicin-carrier protein couplet thing and neomycin-carrier protein couplet thing, nature controlling line is coated with antiantibody, spectinomycin specific antibody-colloid gold label thing that described micropore reagent is freeze-drying, streptomysin specific antibody-colloid gold label thing, gentamicin specific antibody-colloid gold label thing and neomycin specific antibody-colloid gold label thing.
2. test strips according to claim 1, is characterized in that described test paper pasted on base plate and form successively by absorption of sample pad, reaction film, adsorptive pads, diaphragm, has micropore plug described in claim 1 on capillary strip.
3. test strips according to claim 2, is characterized in that: described diaphragm sticks on the absorption of sample pad of test paper, is test side, has MAX mark line above.
4. test strips according to claim 1, is characterized in that described spectinomycin detection line is near absorption of sample pad one end, and nature controlling line is near adsorptive pads one end, and streptomysin, gentamicin, neomycin detection line are between spectinomycin detection line and nature controlling line.
5. test strips according to claim 1, it is characterized in that: spectinomycin-carrier protein couplet thing is by getting 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, after column chromatography purification, in ethanol-water system, recrystallization obtains the condensation product of spectinomycin and ethyloic azanol, obtain with carrier protein couplet, described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins again.
6. test strips according to claim 1, it is characterized in that: streptomysin-carrier protein couplet thing is by getting 1.0 g streptomysins, 0.30 g phthalic anhydride and the mixed liquor of 1 ml pyridine in 20 ml DMSO, at 80 ℃, stirring reaction is 24 hours, steaming desolventizes, in ethanol-water system, repeatedly recrystallization obtains phthalic acid strand mycin ester, obtain with carrier protein couplet, described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins again.
7. test strips according to claim 1, it is characterized in that: gentamicin-carrier protein couplet thing is by getting the solution of 0.39 g bromo-acetic acid tert-butyl in 5 ml DMSO, be slowly added dropwise in 0.90 g gentamicin and the potpourri of 1 ml pyridine in 10 ml DMSO at 40 ℃.After dropwising, continue reaction after 6 hours, steaming desolventizes.After simple column chromatography for separation, except desolventizing, add 20 ml DMSO and 5 ml formic acid, room temperature reaction 20 hours, steaming desolventizes, in ethanol-water system, recrystallization obtains ethyloic gentamicin, then obtains with carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
8. test strips according to claim 1, it is characterized in that: neomycin-carrier protein couplet thing is by getting the mixed liquor of 0.61 g neomycin and 10ml DMSO, under room temperature, be slowly added dropwise in 10 ml DMSO solution of 0.14 g terephthalaldehyde, dropwise rear room temperature to 60 ℃ reaction 2-4 hour, except desolventizing, in ethanol-water system, recrystallization obtains neomycin terephthalaldehyde list condensation product, obtain with carrier protein couplet, described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins again.
9. test strips according to claim 1, it is characterized in that: the spectinomycin specific antibody in spectinomycin specific antibody-colloid gold label thing is to using spectinomycin-carrier protein couplet thing to prepare as immunogene, and described spectinomycin specific antibody is spectinomycin monoclonal antibody; Streptomysin specific antibody in streptomysin specific antibody-colloid gold label thing is to using streptomysin-carrier protein couplet thing to prepare as immunogene, and described streptomysin specific antibody is streptomysin monoclonal antibody; Gentamicin specific antibody in gentamicin specific antibody-colloid gold label thing is to using gentamicin-carrier protein couplet thing to prepare as immunogene, and described gentamicin specific antibody is gentamicin monoclonal antibody; Neomycin specific antibody in neomycin specific antibody-colloid gold label thing is to using neomycin-carrier protein couplet thing to prepare as immunogene, and described neomycin specific antibody is neomycin monoclonal antibody.
10. test strips according to claim 1, is characterized in that: the micropore reagent 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 that freeze-drying is contained in described capillary strip bottom.
11. test strips according to claim 1, is characterized in that: described micropore plug can closely be filled in capillary strip.
12. 1 kinds of methods of preparing the test strips described in claim 1-9 any one, its step comprises:
1) prepare respectively the micropore reagent that freeze-drying 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;
2) prepare respectively the reaction film with coated spectinomycin-carrier protein couplet thing, streptomysin-carrier protein couplet thing, gentamicin-carrier protein couplet thing and four detection lines of neomycin-carrier protein couplet thing and the nature controlling line of coated antiantibody;
3) by 2) reaction film and absorption of sample pad, diaphragm, adsorptive pads, the base plate that prepare be assembled into test paper;
4) by 1) and 3) freeze-drying for preparing has micropore reagent, capillary strip, micropore plug and the test paper 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 be assembled into test strips.
13. 1 kinds of detection methods that simultaneously detect spectinomycin in milk sample, streptomysin, gentamicin and Detection of neomycin residues, it is characterized in that directly test paper being inserted in the micropore that includes the mixed solution that detects sample solution and micropore reagent and being detected, it comprises step:
1) by the test strips described in claim 1-9 any one, detect;
2) analyzing and testing result.
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| CN106680485A (en) * | 2016-11-22 | 2017-05-17 | 云南辉瑞贝尔生物科技有限公司 | Enzyme-linked immune sorbent assay kit for spectinomycin medicine and application of enzyme-linked immune sorbent assay kit |
| 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 |
| CN109959795A (en) * | 2017-12-26 | 2019-07-02 | 北京勤邦生物技术有限公司 | A kind of development and application of matrix type test strips |
| CN110726836A (en) * | 2019-10-28 | 2020-01-24 | 南京海关动植物与食品检测中心 | Time-resolved fluorescence immunoassay test strip for quantitatively determining tetracycline and norfloxacin |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101281195A (en) * | 2008-05-19 | 2008-10-08 | 中国计量学院 | Colloidal gold immune chromatography test paper for detecting biotoxin and detecting method thereof |
| CN101435823A (en) * | 2007-11-12 | 2009-05-20 | 无锡中德伯尔生物技术有限公司 | Fluorescent micro-ball immune chromatography test paper strip for detecting residual animal medicine and preparing method thereof |
| CN201935920U (en) * | 2010-10-21 | 2011-08-17 | 北京勤邦生物技术有限公司 | Reagent kit for detecting beta-lactam antibiotics |
| CN102288753A (en) * | 2011-05-10 | 2011-12-21 | 重庆市科学技术研究院 | Quadruple colloidal gold immunochromatography testing strip for rapid detection of residual tetracycline, chlortetracycline, oxytetracycline and doxycycline and preparation method of same |
-
2012
- 2012-09-29 CN CN201210372008.1A patent/CN103713131B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101435823A (en) * | 2007-11-12 | 2009-05-20 | 无锡中德伯尔生物技术有限公司 | Fluorescent micro-ball immune chromatography test paper strip for detecting residual animal medicine and preparing method thereof |
| CN101281195A (en) * | 2008-05-19 | 2008-10-08 | 中国计量学院 | Colloidal gold immune chromatography test paper for detecting biotoxin and detecting method thereof |
| CN201935920U (en) * | 2010-10-21 | 2011-08-17 | 北京勤邦生物技术有限公司 | Reagent kit for detecting beta-lactam antibiotics |
| CN102288753A (en) * | 2011-05-10 | 2011-12-21 | 重庆市科学技术研究院 | Quadruple colloidal gold immunochromatography testing strip for rapid detection of residual tetracycline, chlortetracycline, oxytetracycline and doxycycline and preparation method of same |
Non-Patent Citations (3)
| Title |
|---|
| MARJORIE B. MEDINA: "Development of a fluorescent latex immunoassay for detection of a spectinomycin antibiotic", 《J. AGRIC. FOOD CHEM.》 * |
| PETRA SCHNAPPINGER，ET AL: "Enzyme immunoassay for the detection of streptomycin and dihydrostreptomycin in milk", 《FOOD AND AGRICULTURAL IMMUNOLOGY》 * |
| TOUICHI TANAKA,ET AL: "Easy enzyme-linked immunosorbent assay for spectinomycin in chicken plasma.", 《JOURNAL OF AOAC INTERNATIONAL》 * |
Cited By (8)
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| CN105004844A (en) * | 2014-05-30 | 2015-10-28 | 北京勤邦生物技术有限公司 | Gentamicin residue diagnosis strip and application thereof |
| CN105004844B (en) * | 2014-05-30 | 2016-06-22 | 北京勤邦生物技术有限公司 | A kind of gentamicin residue test strip and application thereof |
| CN106405103A (en) * | 2016-08-31 | 2017-02-15 | 杨世东 | Colloidal gold immunochromatography kit for detecting rheumatoid arthritis and preparation method thereof |
| CN106680485A (en) * | 2016-11-22 | 2017-05-17 | 云南辉瑞贝尔生物科技有限公司 | Enzyme-linked immune sorbent assay kit for spectinomycin medicine and application of enzyme-linked immune sorbent assay kit |
| 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 |
| CN109959795A (en) * | 2017-12-26 | 2019-07-02 | 北京勤邦生物技术有限公司 | A kind of development and application of matrix type test strips |
| CN110726836A (en) * | 2019-10-28 | 2020-01-24 | 南京海关动植物与食品检测中心 | Time-resolved fluorescence immunoassay test strip for quantitatively determining tetracycline and norfloxacin |
| CN110726836B (en) * | 2019-10-28 | 2023-06-09 | 南京海关动植物与食品检测中心 | Time-resolved fluorescence immunoassay test strip for quantitatively determining tetracycline and norfloxacin |
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