CN102818895A - Test strip for rapidly detecting lincomycin residues and preparation method of test strip - Google Patents
Test strip for rapidly detecting lincomycin residues and preparation method of test strip Download PDFInfo
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- CN102818895A CN102818895A CN2012102816439A CN201210281643A CN102818895A CN 102818895 A CN102818895 A CN 102818895A CN 2012102816439 A CN2012102816439 A CN 2012102816439A CN 201210281643 A CN201210281643 A CN 201210281643A CN 102818895 A CN102818895 A CN 102818895A
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Abstract
The invention relates to a test strip for rapidly detecting residues and a preparation method of the test strip. The bottom layer of the test strip is used as a supporting layer; an intermediate adsorption layer is fixed on the supporting layer; an external protection layer is fixed on the adsorption layer; the adsorption layer comprises an adsorption fiber layer, a gold-labeled antibody fiber layer for adsorbing and coupling lincomycin and a cellulose membrane layer sequentially arranged on the testing sample end and further comprises a water absorbing material layer arranged on the handle end; a detection blot printed by a lincomycin carried protein solution and a control blot printed by a goat anti- or rabbit anti-mouse IgG antibody solution are arranged on the cellulose membrane layer. The test strip is high in detection specificity, high in sensitivity and simple and fast in operation; detection results are displayed visually, directly and accurately; a special apparatus and a professional are not in need in the detection; and the test strip has the following characteristics of low cost, low investment as well as easiness for promotion and application.
Description
Technical field
The present invention relates to the residual appliance of a kind of lincomycin, particularly relate to test strips of the residual fast detecting of a kind of lincomycin and preparation method thereof.
Background technology
Lincomycin (lincomycin; LIN) claiming lincomycinum again, is the lincosamides that is produced by the streptomyces lincolnensis fermentation, molecular formula: C18H34N2O6S; Molecular weight: 406.53; Having stronger bacteriostatic activity, mainly act on gram-positive bacterium, is the acetylcholinesteraseinhibitors inhibitors that RNA relies on.LIN does not possess outstanding toxicity, but is prone to accumulation after its residual in food and medicine gets into human body through food chain, can cause drug-resistance of bacteria, causes disease is lacked therapeutic efficiency.Therefore, each state is all to the requirement of lincomycin finite quantity.The Ministry of Agriculture has stipulated the MRL of LIN in ox, sheep, pig, fowl, wherein muscle 100 μ g/kg, fatty 100 μ g/kg, liver 500 μ g/kg, kidney 1500 μ g/kg.
Both at home and abroad the residual detection method commonly used of limiting the quantity of of LIN is mainly contained at present: microbial method, high performance liquid chromatography (HPLC), vapor-phase chromatography, high performance liquid chromatography mass spectrum series process (UPLC-MS/MS), liquid chromatography/mass spectrometry coupling technique (LC-MS), thin-layered chromatography (TLC) etc.Said method not only needs expensive instrument and equipment and complicated loaded down with trivial details operating process, and reviewer's technical ability is had relatively high expectations, and is not suitable for the examination of on-site supervision and great amount of samples.Therefore, need a kind of fast and convenient LIN detection technique in a hurry.
Summary of the invention
The technical matters that the present invention will solve: the test strips of a kind of high specificity, susceptibility height, fast and convenient detection lincomycin is provided, the preparation method of this test strips also is provided.
Technical scheme of the present invention:
The residual quick test strip of a kind of lincomycin; Bottom is a supporting layer; The middle layer adsorbed layer is fixed on the supporting layer, and outer protective layer is fixed on the adsorbed layer, and adsorbed layer is followed successively by the absorbent material layer of adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete and handle end from test lead; On the cellulose rete, be provided with the detection trace of printing with coupling lincomycin carrier protein solution, with the contrast trace of goat-anti or the anti-mouse IgG antibody solution printing of rabbit; Said golden labeling antibody fibrage is processed with the golden labeling antibody glass fibre cotton of absorption lincomycin, and golden labeling antibody is the anti-lincomycin monoclonal antibody of colloid gold label.
Said supporting layer is processed with hard plastic slip or the cardboard bar that do not absorb water; Said adsorbing fiber layer is processed with glass fibre cotton, nylon membrane, PVDF membrane or polyester film; Said absorbent material layer is processed with thieving paper.
Said cellulose rete is processed with nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane.
Said carrier protein is bovine serum albumin(BSA), the pure albumen of ovum gallinaceum or hemocyanin.
Said outer protective layer is the diaphragm that covers on adsorbing fiber layer, golden labeling antibody fibrage and the absorbent material layer; On the adsorbing fiber layer diaphragm corresponding, be printed with the sample mark line, this mark line deflection adsorbing fiber layer one side 0.3～0.7cm with the fibrolaminar intersection of golden labeling antibody.
Said detection trace is " ‖ " orthoscopic trace that is arranged in parallel with the contrast trace; Or be that " 10 " font is arranged trace, or be that " ┬ ┬ " font is arranged trace, or be that " ┴ ┴ " font is arranged trace; Or be that " ├ ├ " font is arranged trace, or be that " ┤ ┤ " font is arranged trace.
The preparation method of said test strips may further comprise the steps:
(1) preparation of coupling lincomycin carrier protein solution
Take by weighing the 12.6mg lincomycin and place reactor, add the PBS damping fluid dissolving of 500～1000 μ L, obtain LIN solution; Take by weighing 6.7mg NaIO
4Place another reactor,, obtain NaIO4 solution with the dissolving of 500～1000 μ L distilled waters; With NaIO
4Solution under agitation dropwise joins in the LIN solution, stirring at room reaction 2h;
Take by weighing carrier protein 15mg,, obtain carrier protein solution, place 4 ℃ of refrigerator 2h with the dissolving of 5mL PBS damping fluid, subsequent use; With LIN and NaIO
4Reactant liquor under agitation dropwise join in the carrier protein solution 4 ℃ of stirring reaction 2h; The NaIO that adds 50 μ L, 2mg/mL then
4Solution, stirring at room reaction 2h; With the reactant liquor bag filter of packing into, with the PBS solution dialysis 3d of pH7.4, every 8h changes dislysate once, collects reactant liquor, promptly obtains the conjugate solution of lincomycin at last;
(2) anti-lincomycin MONOCLONAL ANTIBODIES SPECIFIC FOR;
(3) preparation of lincomycin gold labeling antibody;
(4) preparation of goat-anti or the anti-mouse IgG antibody of rabbit;
Conjugate solution with gained is processed the detection trace on the cellulose rete, on the cellulose rete, prepare the contrast trace with goat-anti or the anti-mouse IgG antibody of rabbit; Bravo gold labeling antibody is used to prepare golden labeling antibody fibrage, successively supporting layer, adsorbed layer and protective seam is assembled into test strips then.
Said lincomycin gold labeling antibody prepare by following method: 0.01～0.05% the chlorauric acid solution of measuring that 50～l00mL seethes with excitement; In chlorauric acid solution, add 2～4mL, 0.5～2% citric acid three sodium solution, the reaction back obtains the colloid gold particle of particle diameter 10～15nm; K with 0.1mol/L
2CO
3Regulate pH to 8.5～9.5 of collaurum colloidal sol, will resist the LIN monoclonal antibody to join in the collaurum colloidal sol mark l0min with the mark ratio of 1:1000～1300; The final concentration of the PEG 10000 to PEG 10000 of adding 20% is 0.05%, 4 ℃, the centrifugal 20min of 1500～3000rpm, removes unconjugated colloid gold particle, and 4 ℃, the centrifugal 1h of 15000rpm abandon supernatant; With the golden labeling antibody protein mixture of the preliminary purification that obtains with propylene glucosan S-400 column chromatography, separation and purification gold mark albumen, acquisition LIN colloid gold label antibody; The colloid gold label antibody of 1:100～500 dilutions is adsorbed in the glass fibre cotton, in 4 ℃ of low-temperature vacuum dryings, preparation LIN gold labeling antibody.
The positive effect of having a mind to of the present invention:
(1) high specificity, susceptibility is high.LIN quick detection test paper bar is that the basis is prepared from the monoclonal antibody of colloid gold label high-affinity; No covalent bond formation between gold grain and the antibody molecule in the gold labeling antibody; The two combines through the Van der Waals force between the charges of different polarity; The collaurum mark is less to monoclonal antibody specificity and affinity influence, and has higher mark rate.
(2) easy and simple to handle quick.Need not any other reagent when using test strips of the present invention, as long as be inserted in the sample to be checked about 10 seconds, is the decidable testing result in 2 minutes.
(3) result shows image, directly perceived, accurate.Test strips of the present invention is to show that rufous " ︱ " (or " ten ", " ┬ ", " ┴ ", " ├ ", " ┤ ") trace is as the positive and the negative marker that detect; Showing on cellulose membrane promptly that a brownish red " ︱ " trace is illustrated in the sample to be detected contains LIN, shows that two brownish reds " ︱ ︱ " trace is illustrated in not contain LIN in the test sample.The result judges image, directly perceived, accurate, simple and clear, is not prone to artificial erroneous judgement such as false negative and false positive.
(4) cost is low, small investment.Use this test strips not need to join in addition instrument and equipment and other reagent, can detect whenever and wherever possible, testing cost is cheap.Adopting common instrumental analysis expense is 300 yuan of every head/parts; Adopting the testing cost of import ELISA kit is 50 yuan of every head/parts; The testing cost of test strips of the present invention is 8～10 yuan of every head/parts, and testing cost declines to a great extent, and can save a large amount of instrument and equipment expenses.
(5) applied range is convenient to promote.Test strips of the present invention is simple to operate, can satisfy different levels personnel's needs, comprises aspects such as professional chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, livestock products processing, intensive culture and individual breed.The present invention is extremely important aspect the consumer health ensuring food safety and protect, and has favorable economic benefit and social benefit.
(6) the present invention uses sodium metaperiodate and prepares immunogene and detect trace; And data by MoM and MEI; Sodium periodate oxidation is that carbohydrate or the adjacent diol structure that contains in the glycosyl compound molecule are aldehyde radical by sodium periodate oxidation, then with protein molecular in amino form SchiffShi alkali, course of reaction is relatively gentleer; Can under normal temperature and condition of neutral pH, carry out, be easy to operate and control.
Description of drawings
Fig. 1 is the structural representation of test strips of the present invention;
Fig. 2 is the plan structure synoptic diagram of test strips of the present invention.
Embodiment
Following examples are in order to further specify content of the present invention, not represent limitation of the present invention.Wen Zhongru does not specify that percentage composition wherein is the quality percentage composition.
Embodiment one: referring to Fig. 1, Fig. 2.1 is supporting layer among the figure; Process with the plastic slice bar, 2 is adsorbing fiber layer (test lead), processes with glass fibre cotton; The golden labeling antibody glass fibre cotton of gold labeling antibody fibrage 3 usefulness absorption lincomycin is processed, and golden labeling antibody is the monoclonal antibody of the anti-lincomycin of colloid gold label.
8-1 covers adsorbing fiber layer 2 and the white of the sample end above the golden labeling antibody fibrage 3 diaphragm; On the diaphragm 8-1 of adsorbing fiber layer 2 and golden labeling antibody fibrage 3 intersections correspondence; Be partial to adsorbing fiber layer 2 one side 0.5cm place and be printed on mark line 9; The right-hand member of mark line 9 is printed on arrow and max printed words, is coated with yellow diaphragm 8-2 on the absorbent material layer 5 (handle end).
Be used to detect the coupling LIN carrier protein of trace and the preparation of golden labeling antibody, and it is following to be used to contrast the preparation method of goat-anti or the anti-mouse IgG antibody of rabbit of trace:
(1) coupling of LIN and carrier protein
Adopt sodium periodate method, LIN and carrier protein are carried out coupling, the preparation immunizing antigen.
Take by weighing 12.6mg LIN and place the 10mL reaction bulb, dissolve with 500 μ L PBS; Take by weighing 6.7mg NaIO
4Place the 10mL reaction bulb, dissolve with 500 μ L distilled waters; Then NaIO4 solution is dropwise joined in the LIN solution stirring at room reaction 2h under stirring condition; Take by weighing carrier protein BSA (or OVA, KLH) 15mg and place the 10mL reaction bulb,, place 4 ℃ of refrigerator 2h then with 5mL PBS dissolving, subsequent use; Afterwards the reaction mixture of LIN and NaIO4 is dropwise stirred and join in the BSA solution 4 ℃ of stirring reaction 2h; The NaIO that in mixed liquor, adds 2mg/mL once more
450 μ L, stirring at room reaction 2h; With the reaction mixture bag filter of packing into, with the PBS solution dialysis 3d of pH=7.4, every 8h changes dislysate once, collects the conjugate that obtains LIN and carrier protein.
(2) anti-LIN MONOCLONAL ANTIBODIES SPECIFIC FOR
LIN carrier protein couplet thing with preparation is a mouse with 20 μ g～50 μ g/ consumption immunity BALB/c only, adopts the subcutaneous multi-point injection in back, and immunity is four times altogether, each 15～30 days at interval.Behind the 4th booster immunization 3～4 days, with the bloodletting of immune mouse eyeball, draw neck to cause death, the alcohol-pickled 5～l0min with 75%, aseptic its splenocyte of getting shreds and through 100 order nylon net filters, the centrifugal l0min of l000r/min, collection splenocyte; With 1 * 10
8Individual splenocyte and 2 * 10
7～5 * 10
7Individual NS0 oncocyte mixes, and the centrifugal l0min of l000r/min abandons supernatant, and cell precipitation is slowly added PEG4000 (pH 8.5～9.0) 0.7～1.0mL of 40～50%, effect 1min in 37 ℃ of water-baths; Slowly add serum-free 1640 nutrient culture media 15mL then, with the effect of termination PEG, 37 ℃ of water-bath 5～l0min, the centrifugal l0min of 1000r/min abandons supernatant; The cell precipitation that obtains is resuspended in HAT selects to add in 4 96 well culture plates (100 μ L～200 μ L/ holes) in the nutrient culture media, place 37 ℃, 5% CO2 incubator to cultivate.Cultivate after 7～10 days,, detect the culture supernatant of hybridoma with EUSA (ELISA) with the carrier protein coated elisa plate (96 holes/piece) of 5～10 μ g/mL coupling LIN.
Obtain positive cell clone (more than the OD492=0.6) 325 holes after the fusion, positive rate is 83%; Picking strong positive cell clone (more than the OD492=1.8) carries out continuous three times limited dilution cloningization, and the hybridoma chromosome number of being produced is 92～98.Continuous once more three times limited dilution cloningization is chosen the strong positive cell clone, continues limited dilution cloningization.Through identifying; The monoclonal antibody of its secretion specifically with LIN reaction, and not with other albumen generation cross reaction, affinity constant reaches l09～10; Light chain subtype is κ or λ; The heavy chain hypotype is IgG1, IgG2a, IgG2b, IgG3, to the monoclonal antibody of LIN specific antigen determinant, is used to prepare golden labeling antibody.
(3) preparation of LIN gold labeling antibody and golden labeling antibody glass fibre cotton
Prepare collaurum colloidal sol with the sodium citrate reducing process.The citric acid three sodium solution that in 0.01～0.05% aqueous solution of chloraurate of 50～l00mL boiling, adds 2～4mL 0.5～2%, the reaction back obtains the collaurum about particle diameter 15nm.K2CO3 with 0.1mol/L regulates collaurum pH to 8.5～9.5; Mark with 1:1000～1300 joins in the above-mentioned aurosol than with LIN monoclonal antibody to be marked, behind the mark l0min, add 20% PEG 10000 to final concentration be 0.05%; 4 ℃, the centrifugal 20min of 1500～3000rpm; Remove unconjugated colloid gold particle, 4 ℃, the centrifugal 1h of 15000rpm abandon supernatant; After obtaining the golden labeling antibody protein mixture of preliminary purification, with propylene glucosan S-400 column chromatography, separation and purification gold mark albumen obtains LIN colloid gold label antibody.With 1: the colloid gold label antibody of (100～500) dilution is adsorbed in the processed glass cellucotton, 4 ℃ of low-temperature vacuum dryings, preparation LIN gold labeling antibody glass fibre cotton.
(4) preparation of goat-anti or the anti-mouse IgG antibody of rabbit
Extract mice serum IgG with saturated ammonium sulfate.Get 1 part of mice serum and add 2 parts of PBS (pH=7.4) mixing, add equal-volume saturated ammonium sulfate mixing, put 4 ℃ of refrigerator 2h, 4 ℃, the centrifugal 15min of 1200r/min abandon supernatant; With an amount of PBS (pH=7.4) dissolution precipitation, add saturated ammonium sulfate to final concentration 33%, put in 4 ℃ of refrigerators with PBS (pH=7.4) dialyzed overnight; Change liquid 2～3 times, 4 ℃, the centrifugal 15min of 12000r/min collect supernatant; Measure its protein concentration with ultraviolet spectrophotometer, through subcutaneous and intramuscular injection immune health sheep or rabbit 3～4 times, the last immunity is after 10 days with 50 μ g～100 μ g/kg body weight (mice serum IgG); Venous blood collection is measured its serum antibody titer more than 1:2000 with ELISA, heart blood sampling or arteria carotis bloodletting; Collect its serum, be used for the preparation of test strips contrast trace.Extract goat-anti or the anti-mouse IgG of rabbit with saturated ammonium sulfate, method is identical with extraction mice serum IgG, no longer repeats.
(5) the detection reaction principle of test strips
After LIN test strip sample end inserts detected sample solution; Solution to be measured spreads to the cellulose rete through the golden labeling antibody that syphonic effect drives in LIN to be checked and the golden labeling antibody cotton together, and finally infiltrates in the absorbent material layer of handle end, and LIN to be checked combines with golden labeling antibody in the diffusion process; And then seal the antigen-combining site of LIN on the golden labeling antibody; Stop the detection trace of coupling LIN carrier protein on golden labeling antibody and the cellulose membrane to combine, can not show the detection trace, the anti-mouse IgG of goat-anti or rabbit then can combine with golden labeling antibody; Form rufous contrast trace " ︱ ", the i.e. positive mark of a rufous " ︱ " trace; If do not have LIN in the sample solution; Then can not stop the carrier protein of coupling LIN on golden labeling antibody and the cellulose membrane to detect trace combines; Show that rufous detects trace " ︱ ", same goat-anti or the anti-mouse IgG of rabbit also combine with golden labeling antibody, show rufous contrast trace " ︱ "; Form two rufous " ︱ ︱ ", negative mark; If do not have the rufous mark to show on the cellulose membrane, show that then test strips lost efficacy.
(6) detection method
A. sample liquid preparation: when detecting chicken, chicken meat sample is rubbed the back add a little hydrochloric acid, reaction 5 ~ 10min adds damping fluid afterwards, and vibration 1～2min pours out extract and detects.The preparation of other meat sample liquid similarly.
B. method of operating: test strips sample end is inserted in the sample liquid, and insertion depth is no more than mark line, takes out test strips, about 2 minutes of horizontal positioned, observations in about 10 seconds.
C. the result judges: if on cellulose membrane, there is a rufous mark " ︱ " to show, surveys the inspection result and be positive, explain in sample liquid to be detected and contain LIN; If two rufous marks " ︱ ︱ " appear in the cellulose membrane on the test strip, testing result is negative, and does not contain LIN in the sample to be checked.
Embodiment two: test strips structure and embodiment one are basic identical, and difference is:
Supporting layer l processes with the cardboard bar that does not absorb water; Adsorbing fiber layer 2 is used nylon membrane; Gold labeling antibody fibrage 3 is adsorbed with the polyclonal antibody of the anti-lincomycin of colloid gold label; Cellulose rete 4 adopts the pure cellulose film, and the carrier protein that detects coupling LIN in the trace 6 is the pure albumen of ovum gallinaceum (OVA), and the anti-mouse IgG of contrast trace 7 usefulness rabbits solution is processed on cellulose membrane.
Embodiment three: test strips structure and embodiment one are basic identical, and difference is:
Adsorbing fiber layer 2 usefulness PVDF (PVDF) film, cellulose rete 4 adopts the carboxylation cellulose membrane, and the carrier protein that detects coupling LIN in the trace 6 is the pure albumen of ovum gallinaceum (OVA), and contrast trace 7 usefulness goat anti-mouse igg solution are processed on cellulose membrane.
Embodiment four: test strips structure and embodiment one are basic identical, and difference is:
Adsorbing fiber layer 2 is used polyester film, and the carrier protein that detects coupling LIN in the trace 6 is a hemocyanin.
Embodiment five: test strips structure and embodiment one are basic identical, and difference is:
Among the embodiment two ~ five, test sample preparation, method of operating and result judge, all with embodiment one.
Embodiment six: the susceptibility of test strips, specific detection
Test strips with embodiment one detects, and the test strips test findings in other examples is similar with this.
(1) use the test strips of embodiment one to detect respectively to add LIN concentration to be 20 parts of the chicken meat samples of 0ng/g, 100ng/g, 200ng/g, 400ng/g and 800ng/g; Room temperature condition is operation down, and observation is with the naked eye judged; The result shows that the sample of 0ng/mL is all negative; The interpolation sample of 100ng/mL and above concentration thereof is all positive.
Getting test strips detects respectively and adds 500 parts of the chicken meat samples that LIN concentration is 0ng/g and 100ng/g; Room temperature condition is operation down, and naked-eye observation is judged; Interpolation sample with 0ng/g concentration calculates false positive rate, false positive rate=false positive sample number/500.Interpolation sample with 100ng/g concentration calculates false negative rate, false negative rate=false negative sample number/500.The result shows that false positive is 3, and false positive rate is≤1%; False negative rate is 0%.
(2) with the standard sample of known negative chicken meat sample preparation lincomycin class medicine, make its final concentration be respectively 1000ng/g, detect with the test strips of embodiment one.The result shows: the sample that is positive, contains clindamycin except that the sample that contains LIN is the weak positive, with the equal no cross reaction of other multiple medicine, explains that the atopic of test strips is stronger.
Claims (10)
1. residual quick test strip of lincomycin; Bottom is a supporting layer; The middle layer adsorbed layer is fixed on the supporting layer, and outer protective layer is fixed on the adsorbed layer, it is characterized in that: adsorbed layer is followed successively by adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete and absorbent material layer from test lead; On the cellulose rete, be provided with the detection trace of printing with coupling lincomycin carrier protein solution, with the contrast trace of goat-anti or the anti-mouse IgG antibody solution printing of rabbit; Said golden labeling antibody fibrage is processed with the golden labeling antibody glass fibre cotton of absorption lincomycin, and golden labeling antibody is the anti-lincomycin monoclonal antibody of colloid gold label.
2. test strips according to claim 1 is characterized in that: said supporting layer is processed with hard plastic slip or the cardboard bar that do not absorb water; The adsorbing fiber layer is processed with glass fibre cotton, nylon membrane, PVDF membrane or polyester film; Absorbent material layer is processed with thieving paper.
3. test strips according to claim 1 is characterized in that: said cellulose rete is processed with nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane.
4. according to the described test strips of claim l, it is characterized in that: wherein coupling lincomycin carrier protein solution is prepared by following method:
Take by weighing the 12.6mg lincomycin and place reactor, add the PBS damping fluid dissolving of 500～1000 μ L, obtain LIN solution; Take by weighing 6.7mg NaIO4 and place another reactor,, obtain NaIO4 solution with the dissolving of 500～1000 μ L distilled waters; NaIO4 solution is under agitation dropwise joined in the LIN solution stirring at room reaction 2h;
Take by weighing carrier protein 15mg,, obtain carrier protein solution, place 4 ℃ of refrigerator 2h with the dissolving of 5mL PBS damping fluid, subsequent use; The reactant liquor of LIN and NaIO4 is under agitation dropwise joined in the carrier protein solution 4 ℃ of stirring reaction 2h; The NaIO4 solution that adds 50 μ L, 2mg/mL then, stirring at room reaction 2h; With the reactant liquor bag filter of packing into, with the PBS solution dialysis 3d of pH7.4, every 8h changes dislysate once, collects reactant liquor, promptly obtains the conjugate solution of lincomycin at last.
5. according to each described test strips of claim l～4, it is characterized in that: said carrier protein is bovine serum albumin(BSA), the pure albumen of ovum gallinaceum or hemocyanin.
6. according to each described test strips of claim l～4; It is characterized in that: said outer protective layer is the diaphragm that covers on adsorbing fiber layer, golden labeling antibody fibrage and the absorbent material layer; On the adsorbing fiber layer diaphragm corresponding, be printed with the sample mark line, this sample mark line deflection adsorbing fiber layer one side 0.3～0.7cm with the fibrolaminar intersection of golden labeling antibody.
7. according to each described test strips of claim 1～4; It is characterized in that: said detection trace is " ‖ " orthoscopic trace that is arranged in parallel with the contrast trace; Or be that " 10 " font is arranged trace, or be that " ┬ ┬ " font is arranged trace, or be that " ┴ ┴ " font is arranged trace; Or be that " ├ ├ " font is arranged trace, or be that " ┤ ┤ " font is arranged trace.
8. the preparation method of the said test strips of claim 1, it is characterized in that: this method may further comprise the steps:
(1) preparation of coupling lincomycin carrier protein solution
Take by weighing the 12.6mg lincomycin and place reactor, add the PBS damping fluid dissolving of 500～1000 μ L, obtain LIN solution; Take by weighing 6.7mg NaIO4 and place another reactor,, obtain NaIO4 solution with the dissolving of 500～1000 μ L distilled waters; NaIO4 solution is under agitation dropwise joined in the LIN solution stirring at room reaction 2h; Take by weighing carrier protein 15mg,, obtain carrier protein solution, place 4 ℃ of refrigerator 2h with the dissolving of 5mL PBS damping fluid, subsequent use; The reactant liquor of LIN and NaIO4 is under agitation dropwise joined in the carrier protein solution 4 ℃ of stirring reaction 2h; The NaIO4 solution that adds 50 μ L, 2mg/mL then, stirring at room reaction 2h; At last with the reactant liquor bag filter of packing into, with the PBS solution dialysis of pH7.4 3 days, every 8h changed dislysate once, collects reactant liquor, obtains the conjugate solution of lincomycin;
(2) anti-lincomycin MONOCLONAL ANTIBODIES SPECIFIC FOR;
(3) preparation of lincomycin gold labeling antibody;
(4) preparation of goat-anti or the anti-mouse IgG antibody of rabbit;
Conjugate solution with gained is processed the detection trace on the cellulose rete; On the cellulose rete, prepare the contrast trace with goat-anti or the anti-mouse IgG antibody of rabbit; Bravo gold labeling antibody is used to prepare golden labeling antibody fibrage, successively supporting layer, adsorbed layer and protective seam is assembled into test strips then.
9. the preparation method of said test strips according to Claim 8, it is characterized in that: said carrier protein is bovine serum albumin(BSA), the pure albumen of ovum gallinaceum or hemocyanin.
10. it is characterized in that according to Claim 8 or the preparation method of 9 said test strips: said lincomycin gold labeling antibody is prepared by following method:
Measure 0.01～0.05% chlorauric acid solution of 50～l00mL boiling, in chlorauric acid solution, add 2～4mL, 0.5～2% citric acid three sodium solution, the reaction back obtains the colloid gold particle of particle diameter 10～15nm; Regulate pH to 8.5～9.5 of collaurum colloidal sol with the K2CO3 of 0.1mol/L, will resist the LIN monoclonal antibody to join in the collaurum colloidal sol mark l0min with the mark ratio of 1:1000～1300; The final concentration of the PEG 10000 to PEG 10000 of adding 20% is 0.05%, 4 ℃, the centrifugal 20min of 1500～3000rpm, removes unconjugated colloid gold particle, and 4 ℃, the centrifugal 1h of 15000rpm abandon supernatant; With the golden labeling antibody protein mixture of the preliminary purification that obtains with propylene glucosan S-400 column chromatography, separation and purification gold mark albumen, acquisition LIN colloid gold label antibody; The colloid gold label antibody of 1:100～500 dilutions is adsorbed in the glass fibre cotton, in 4 ℃ of low-temperature vacuum dryings, preparation lincomycin gold labeling antibody.
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| CN103995107A (en) * | 2013-02-19 | 2014-08-20 | 北京勤邦生物技术有限公司 | Detection method for lincomycin and special test paper therefor |
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| CN109813890A (en) * | 2017-11-18 | 2019-05-28 | 镇江亿特生物科技发展有限公司 | Lincomycin rapid time resolved fluorometric immunochromatographiassay assay quantitative detection test paper |
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| CN103454420B (en) * | 2013-08-03 | 2015-07-01 | 河南百奥生物工程有限公司 | Test strip for rapidly detecting trace sulfachlorpyridazine and preparation method thereof |
| CN104678100A (en) * | 2013-11-27 | 2015-06-03 | 北京维德维康生物技术有限公司 | Gentamycin and quinolones two-in-one gold-labeled microporous test paper strip |
| CN104678095A (en) * | 2013-11-27 | 2015-06-03 | 北京维德维康生物技术有限公司 | Kanamycin and erythromycin integrated gold-labeled microporous test strip |
| CN104678069A (en) * | 2013-11-27 | 2015-06-03 | 中国农业大学 | Kanamycin, erythrocin and lincomycin three-in-one micro-hole gold-labeled test strip |
| CN104678101A (en) * | 2013-11-27 | 2015-06-03 | 中国农业大学 | Melamine and quinolone two-in-one gold-labeled microporous test paper strip |
| CN104678069B (en) * | 2013-11-27 | 2016-08-10 | 中国农业大学 | Kanamycin, erythromycin, lincomycin three-in-one gold mark micropore test strips |
| CN104678100B (en) * | 2013-11-27 | 2017-01-11 | 北京维德维康生物技术有限公司 | Gentamycin and quinolones two-in-one gold-labeled microporous test paper strip |
| CN104678101B (en) * | 2013-11-27 | 2017-01-11 | 中国农业大学 | Melamine and quinolone two-in-one gold-labeled microporous test paper strip |
| CN109813890A (en) * | 2017-11-18 | 2019-05-28 | 镇江亿特生物科技发展有限公司 | Lincomycin rapid time resolved fluorometric immunochromatographiassay assay quantitative detection test paper |
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