CN103995107B - A kind of method detecting lincomycin and special test paper thereof - Google Patents
A kind of method detecting lincomycin and special test paper thereof Download PDFInfo
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- CN103995107B CN103995107B CN201310053821.7A CN201310053821A CN103995107B CN 103995107 B CN103995107 B CN 103995107B CN 201310053821 A CN201310053821 A CN 201310053821A CN 103995107 B CN103995107 B CN 103995107B
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- 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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Abstract
The invention discloses a kind of method detecting lincomycin and special test paper thereof.This reagent paper includes sample absorption pad, conjugate release pad, reaction film and adsorptive pads, and it is sequentially connected with；The lincomycin specific antibody of colloid gold label it is coated with in described conjugate release pad；Described lincomycin specific antibody is lincomycin polyclonal antibody or lincomycin monoclonal antibody；Including detection zone and quality control region on described reaction film, detection zone is coated with the conjugate of hapten and carrier protein, and quality control region is coated with anti antibody.By the method for detection paper lincomycin of the present invention, easy, quick, directly perceived, accurate, applied widely, low cost, easy popularization and application.
Description
Technical field
The present invention relates to a kind of method detecting lincomycin and special test paper thereof.
Background technology
Lincomycin has antibacterial and antiinflammation, lawless person often adds in acne-eliminating cosmetic without authorization, thus reach quickly effectively to eliminate the purpose of small pox, acne, acne, but life-time service Diazolidinyl Urea, the drug resistance that also can cause renal dysfunction and gram positive bacteria increases, and causes serious harm to health.
At present, the illegal method of inspection adding lincomycin in acne-eliminating cosmetic, similar research mostly is instrument analytical method, and testing cost is high, the time is long, and needs multiple chemical reagent and the testing staff of specialty, the quick detection being not suitable under simple condition.Therefore make and sell, for high-tech, high intelligence, the new challenge that fake and inferior cosmetics case is brought to cosmetics supervision, it is necessary to set up the detection method of simple and easy to do lincomycin.
Summary of the invention
It is an object of the present invention to provide a kind of reagent paper detecting lincomycin.
The reagent paper of detection lincomycin provided by the present invention, including sample absorption pad, conjugate release pad, reaction film and adsorptive pads, it is sequentially connected with；The lincomycin specific antibody of colloid gold label it is coated with in described conjugate release pad；Lincomycin specific antibody can be lincomycin polyclonal antibody or lincomycin monoclonal antibody；Detection zone and quality control region it is coated with on reaction film；When lincomycin specific antibody is lincomycin polyclonal antibody, detection zone is coated lincomycin hapten-carrier protein conjugate, and quality control region is coated goat-anti rabbit anti antibody；When lincomycin specific antibody is lincomycin monoclonal antibody, detection zone is coated lincomycin hapten-carrier protein conjugate, and quality control region is coated sheep anti mouse anti antibody；Described hapten is following (Fig. 5):
Wherein, described conjugate release pad there is 1/3-1/2 region covered by described sample absorption pad.
Described reaction film is coated with detection zone and quality control region, and wherein said detection zone is positioned at the side of the end being bordering on described conjugate release pad, and described quality control region is located remotely from the side of the end of described conjugate release pad；The described detection zone end 5-8mm away from described conjugate release pad, preferably 6mm；Described quality control region is away from described detection zone 4-7mm, preferably 5mm.
Described detection zone and described quality control region are the ribbon perpendicular with the length of described test strips.
Described conjugate release pad is coated the lincomycin monoclonal antibody of colloid gold label, and it is coated density is 0.1-0.2 μ g/cm2, preferably 0.1 μ g/cm2。
Described lincomycin monoclonal antibody is the antibody produced by the lincomycin monoclonal antibody hybridoma cell strain F-1-4 secretion that preserving number is CGMCCNo.6508；Described anti antibody can be sheep anti-mouse igg；Described carrier protein can be ovoserum albumen, human serum albumin, bovine serum albumin, hemocyanin or thyroprotein etc..
Described reagent paper includes that base plate, described sample absorption pad, conjugate release pad, reaction film and adsorptive pads are pasted on described base plate.
Described reagent paper concretely strip；For the ease of using, the reagent paper of described strip can be coated with protecting film or be contained in box.
Described protecting film can cover the two ends (sample absorption pad, conjugate release pad and adsorptive pads) of described reagent paper
Described box is made up of base and lid, and the end is sat and is connected with hole clipping by latch with lid；The latch being connected with described lid and the groove matched with described reagent paper is had on described base；Described covering well, observation window and the hole clipping matched with base latch, the position of described well is corresponding with the position of described sample absorption pad, and described observation window is corresponding with the detection zone on described reaction film and quality control region；Described reagent paper is positioned at described groove；Being printed on T and C, T on described observation window and represent detection line, near described well side, C represents nature controlling line, away from described well side.
Base plate can be made up of the toughness material not absorbing water, and such as rigid plastics, do not absorb water cardboard, concretely PVC board；Sample absorption pad can be made up of glass fibre cotton, polyester material (polyester capillaries or polyester cotton), nylon membrane, PVDF membrane or polyester film；Conjugate release pad can be made up of glass fibre；Adsorptive pads can be suction strainer paper or consider oilpaper；Reaction film can be nitrocellulose filter or be pure cellulose film；Described protecting film can be the protecting film of PE material.
It is a further object to provide a kind of method preparing above-mentioned reagent paper.
The method preparing above-mentioned reagent paper provided by the present invention, is sample absorption pad, conjugate release pad, reaction film and adsorptive pads to be sequentially connected with；The lincomycin specific antibody of colloid gold label it is coated with in described conjugate release pad；Described lincomycin specific antibody is lincomycin polyclonal antibody or lincomycin monoclonal antibody；Including detection zone and quality control region on described reaction film, detection zone is coated with the conjugate of hapten and carrier protein, and quality control region is coated with anti antibody；
Described sample absorption pad, before carrying out described connection, first soaks 1-3h in following solution, then is dried: containing 0.3-0.5%(volumn concentration) bovine serum albumin, pH value be the carbonate buffer solution of 9.6-10.0,0.1-0.2mol/L；
The conjugate release pad of the described lincomycin specific antibody being coated with colloid gold label is prepared as follows: the conjugate release pad before being coated first is soaked the 20-40 second in following solution, it being dried, then the lincomycin specific antibody carrying out described colloid gold label is coated: containing 0.8-1.2%(volumn concentration) bovine serum albumin, pH value be 7.0-7.4,0.1-0.2mol/L phosphate buffer.
It is a further object to provide a kind of method detecting lincomycin.
The method of detection lincomycin provided by the present invention, is that detected sample carries out pre-treatment, then detects with any of the above-described described reagent paper；The method of described pre-treatment is as follows:
When described sample is Solic cosmetic, weigh Solic cosmetic sample in polystyrene centrifuge tube, add extracting solution vibration mixing, sampling detection.
When described sample is liquid make-up, absorption liquid cosmetic samples, in polystyrene centrifuge tube, adds extracting solution vibration mixing, and sampling detects.
The Cleaning Principle of reagent paper of the present invention: after the sample absorption pad end at reagent paper drips testing sample solution, solution to be checked is spread to reaction film together by the golden labeling antibody in conjugate release pad, and eventually penetrates in filter paper；If the content of lincomycin is high in detected sample solution, then in diffusion process, the lincomycin in detected sample solution can combine with gold labeling antibody, and then completely enclose the antigen-combining site of lincomycin on gold antibody, gold labeling antibody detection trace of coupling lincomycin carrier protein on reaction film is stoped to be combined, detection trace can not be shown, detection zone does not develops the color, sheep anti-mouse igg antibody then can be combined with gold labeling antibody, quality control region develops the color, form red comparison trace " ", be positive in a trace；If in detected sample solution, the content of lincomycin is low or nothing, then the antigen binding site on gold labeling antibody can not be closed, and then gold labeling antibody can be combined by the carrier protein detection trace of coupling lincomycin on cellulose membrane, the red trace " " of detection zone display, same sheep anti-mouse igg antibody is also combined with gold labeling antibody, quality control region displays that red comparison trace " ", forms two red lines " | | " negative marker；If not having red-label to show on cellulose membrane, then reagent paper is invalid.
The colloid gold test paper of the present invention has sensitivity height, specificity height, precision height, accuracy height, low cost, simple to operate, the detection time is short, be suitable for the use of various units, stores simple, the advantage of long shelf-life.Wherein, the lincomycin monoclonal antibody of high specific is used, it is ensured that the reliability of testing result；Conjugate release pad subregion sample absorption pad is covered, it is possible to extend testing result observing time, also make detected sample solution be fully absorbed simultaneously and then can completely attach to gold labeling antibody, fully react, thus reducing error；The design of distance between the design of monoclonal antibody package amount on reagent paper, detection zone and conjugate release pad and quality control region and the distance of detection zone all further increases the degree of accuracy of detection paper.By the method for detection paper lincomycin of the present invention, easy, quick, directly perceived, accurate, applied widely, low cost, easy popularization and application.
Accompanying drawing explanation
Fig. 1 is reagent paper cross-sectional view.
Fig. 2 is the top view of the reagent paper with protecting film.
Fig. 3 is the cross-sectional view of the reagent paper with paper box.
Fig. 4 is the judgement of the testing result of the reagent paper with paper box.
Fig. 5 is lincomycin hapten.
Fig. 6 is lincomycin hapten synthesis layout.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Embodiment 1, the composition of reagent paper of detection lincomycin
One, without the reagent paper (Fig. 1) of protecting film or test card:
Described reagent paper is made up of sample absorption pad 1, conjugate release pad 2, reaction film 3, adsorptive pads 4 and base plate 7；
Described sample absorption pad, conjugate release pad, reaction film and adsorptive pads are pasted the most in order on described base plate, conjugate release pad is absorbed by the sample pad and covers from Qi You1/3 region, its top, the end of conjugate release pad is connected with the top of reaction film, the end of reaction film is connected with the top of adsorptive pads, the top of sample absorption pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate；
Being coated with the antibody secreted by the lincomycin monoclonal antibody hybridoma cell strain F-1-4 that preserving number is CGMCCNo.6508 of colloid gold label in described conjugate release pad, package amount is 0.1 μ g/cm2。
Detection zone 5 and quality control region 6, detection zone and quality control region is had to be the ribbon perpendicular with the length of described test strips on described reaction film；Detection zone is positioned at the side being bordering on conjugate release pad end, away from conjugate release pad end 6mm；Quality control region is located remotely from the side of conjugate release pad end, away from described detection zone 5mm；Detection zone is coated with lincomycin hapten-carrier protein conjugate (lincomycin hapten and the conjugate of ovalbumin), and quality control region is coated with sheep anti-mouse igg；Described hapten is as follows:
Base plate is made up of PVC board；Sample absorption pad can be made up of glass fibre cotton；Conjugate release pad is made up of glass fibre；Adsorptive pads is suction strainer paper；Reaction film is nitrocellulose filter.
Described reagent paper is the rectangular strip that 3-5mm is wide.
Two, with the reagent paper (referred to as test strips) (Fig. 2) of protecting film:
Described reagent paper is made up of sample absorption pad 1, conjugate release pad 2, reaction film 3, adsorptive pads 4 and base plate 7；
Described sample absorption pad, conjugate release pad, reaction film and adsorptive pads are pasted the most in order on described base plate, conjugate release pad is absorbed by the sample pad and covers from Qi You1/3 region, its top, the end of conjugate release pad is connected with the top of reaction film, the end of reaction film is connected with the top of adsorptive pads, the top of sample absorption pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate；
Being coated with the antibody secreted by the lincomycin monoclonal antibody hybridoma cell strain F-1-4 that preserving number is CGMCCNo.6508 of colloid gold label in described conjugate release pad, package amount is 0.1 μ g/cm2。
Detection zone 5 and quality control region 6, detection zone and quality control region is had to be the ribbon perpendicular with the length of described test strips on described reaction film；Detection zone is positioned at the side being bordering on conjugate release pad end, away from conjugate release pad end 5mm；Quality control region is located remotely from the side of conjugate release pad end, away from described detection zone 4mm；Detection zone is coated with lincomycin hapten-carrier protein conjugate (lincomycin hapten and the conjugate of ovalbumin), and quality control region is coated with sheep anti-mouse igg；Described in described hapten is same identical.
Described reagent paper two ends are pasted protecting film, then is cut into the wide little bar of 3-5mm, preferably 3mm with machine.
Protecting film 8-1 covers at adsorptive pads left-hand seat pommel, and protecting film 8-2 covers the test side on sample absorption pad and conjugate release pad, and the intersection at sample absorption pad and conjugate release pad has shielding wire and is printed on max printed words.
Base plate is made up of PVC board；Sample absorption pad is made up of nylon membrane；Conjugate release pad is made up of glass fibre；Adsorptive pads is for considering oilpaper；Reaction film is pure cellulose film；Described protecting film is the protecting film of PE material.
Three, with the reagent paper (referred to as test card) (Fig. 3) of paper box
Described reagent paper is made up of sample absorption pad 1, conjugate release pad 2, reaction film 3, adsorptive pads 4 and base plate 7；
Described sample absorption pad, conjugate release pad, reaction film and adsorptive pads are pasted the most in order on described base plate, conjugate release pad is absorbed by the sample pad and covers from Qi You1/3 region, its top, the end of conjugate release pad is connected with the top of reaction film, the end of reaction film is connected with the top of adsorptive pads, the top of sample absorption pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate；
Being coated with the antibody secreted by the lincomycin monoclonal antibody hybridoma cell strain F-1-4 that preserving number is CGMCCNo.6508 of colloid gold label in described conjugate release pad, package amount is 0.1 μ g/cm2。
Detection zone 5 and quality control region 6, detection zone and quality control region is had to be the ribbon perpendicular with the length of described test strips on described reaction film；Detection zone is positioned at the side being bordering on conjugate release pad end, away from conjugate release pad end 5mm；Quality control region is located remotely from the side of conjugate release pad end, away from described detection zone 4mm；Detection zone is coated with lincomycin hapten-carrier protein conjugate (lincomycin hapten and the conjugate of ovalbumin), and quality control region is coated with sheep anti-mouse igg；Described in described hapten is same identical.
Described reagent paper is contained in box；Described box is made up of base 9 and lid 10, and the end is sat and lid is fitting to connection by latch 12 and hole clipping 13；
The latch 12 being connected with described lid and the groove matched with described reagent paper is had on described base；
Described well 11, observation window 14 and hole clipping 13(that is connected with base of covering matches with latch)；The position of described well is corresponding with the position of described sample absorption pad, and described observation window is corresponding with the detection zone on described reaction film and quality control region.Being printed on T and C, T on described observation window and represent detection line, near described well side, C represents nature controlling line, away from described well side.
Reagent paper after cutting is positioned at the groove of box base, and the lid and bottom seat of box connects into closed system by latch and hole clipping.
Base plate is made up of PVC board；Sample absorption pad is made up of nylon membrane；Conjugate release pad is made up of glass fibre；Adsorptive pads is for considering oilpaper；Reaction film is pure cellulose film；Described protecting film is the protecting film of PE material.Lid in test card is made of plastics, and base is made of plastics.
Above-mentioned test strips test card is all vacuum-packed with aluminium foil bag, stores in the environment of 4～30 DEG C, be valid for one year.
The preparation method of test kit described in embodiment 2, embodiment 1
One, the preparation of test kit
The preparation method of this test kit mainly comprises the steps:
1) preparation is coated the conjugate release pad of lincomycin monoclonal antibody-colloid gold label thing；
2) preparation reaction film containing the detection zone with the quality control region being coated sheep anti-mouse igg being coated lincomycin hapten-carrier protein conjugate；
3) sample absorption pad, above-mentioned conjugate release pad, reaction film, adsorptive pads and base plate are assembled into reagent paper.
4) protecting film is pasted at the reagent paper two ends assembled；It is cut into the wide little bar of 3mm with machine；Or the reagent paper machine assembled is cut into the wide little bar of 3mm, is contained in special plastic casing.
Substep narration in detail below:
(1) preparation of each parts
1, the synthesis of lincomycin hapten-carrier protein conjugate and qualification
Lincomycin is small-molecule substance, only immunoreactivity, does not has immunogenicity, it is impossible to induction body produce immunne response, it is necessary to just there is after macromolecular carrier albumen coupling immunogenicity.
1) haptenic synthesis
0.39g bromo-acetic acid tert-butyl solution in 5ml dimethyl sulfoxide, is slowly added dropwise at 40 DEG C in 0.81g lincomycin and 1ml pyridine mixture in 10ml dimethyl sulfoxide.After dropping, after continuing reaction 6 hours,
Solvent is evaporated off.After column chromatography for separation, remove solvent, add 20ml dimethyl sulfoxide and 5ml formic acid, room temperature reaction 24 hours, solvent is evaporated off, ethanol-water system is recrystallized to give carboxymethyl lincomycin, i.e. lincomycin hapten (as shown in Figure 6).
2) preparation of coating antigen-lincomycin hapten and the synthesis of ovalbumin conjugate
Lincomycin hapten 1.0ml N,N-Dimethylformamide step 1) prepared dissolves, and adds in hapten, reaction 1 hour is stirred at room temperature, obtains reactant liquor A after taking carbodiimides 20mg 0.5ml water dissolution；Weigh ovalbumin 36mg, be allowed to be substantially dissolved in 3.5ml50mmol/L sodium carbonate liquor, reactant liquor A is dropwise slowly dropped in this solution；Room temperature reaction 24 hours, dialyses 3 days with 0.01mol/LPBS, changes three dialysis solution every day, remove unreacted small-molecule substance, and 12000rpm is centrifuged 30min, collects supernatant, subpackage, saves backup in-20 DEG C.
3) immunogenic preparation-lincomycin hapten and the synthesis of bovine serum albumin
Lincomycin hapten 1.0ml N,N-Dimethylformamide step 1) prepared dissolves, and adds in hapten, reaction 1 hour is stirred at room temperature, i.e. can get reactant liquor B after taking carbodiimides 30mg 0.5ml water dissolution；Weigh bovine serum albumin 48mg, be allowed to be substantially dissolved in 3.5ml50mmol/L sodium carbonate liquor, reactant liquor B is dropwise slowly dropped in this solution；Room temperature reaction 24 hours, dialyses 3 days with 0.01mol/LPBS, changes three dialysis solution every day, remove unreacted small-molecule substance, and 12000rpm is centrifuged 30min, collects supernatant, subpackage, saves backup in-20 DEG C.
4) qualification of lincomycin hapten-carrier conjugates
Carrier protein, lincomycin hapten, the PBS of lincomycin hapten-carrier protein conjugate pH7.4 are made into the solution of 0.5mg/ml, return to zero with 0.01mol/LpH7.4PBS, scan in the range of wavelength 200-800nm with ultraviolet spectrophotometer, obtain carrier protein, lincomycin hapten, the absorption curve of lincomycin hapten-carrier protein conjugate.There is different absorption curves in three, shows lincomycin hapten and carrier protein couplet success.
2, the preparation of lincomycin monoclonal antibody
(1) monoclonal antibody is prepared
A. animal immune
The immunogen that step (1) obtains being injected in Balb/c Mice Body, immunizing dose is 150 μ g/ so that it is produce antiserum.
B. cell merges and cloning
Take immunity Balb/c mouse boosting cell, in 9:1(quantitative proportion) ratio and SP2/0 myeloma cell fusion, screening obtains the lincomycin monoclonal antibody hybridoma cell strain of stably excreting lincomycin monoclonal antibody, by named for this cell strain F-1-4, this cell strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 08 28th, 2012, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.6508.
C. cell cryopreservation and recovery
Hybridoma F-1-4 frozen stock solution is made 1 × 109The cell suspension of individual/ml, preserves in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
D. the preparation of monoclonal antibody and purification
Increment culture method: be placed in cell culture medium by hybridoma CGMCCNo.6508, cultivates under the conditions of 37 DEG C, is purified by the culture fluid obtained by octanoic acid-saturated ammonium sulfate method, obtains monoclonal antibody ,-20 DEG C of preservations.
Described cell culture medium is to add calf serum and sodium bicarbonate in RPMI-1640 culture medium, make the calf serum final concentration of 20%(weight/mass percentage composition in cell culture medium), make the sodium bicarbonate final concentration of 0.2%(weight/mass percentage composition in cell culture medium)；The pH of described cell culture medium is 7.4.
3, the preparation of sheep anti mouse anti antibody: sheep anti-mouse igg is purchased from creation biological place of production Genestsbio article No. 6102030.
4, the preparation of lincomycin monoclonal antibody-colloid gold label thing
(1) preparation of gold colloidal
By double distilled deionized water, 1% gold chloride (is purchased from sigma company, catalog number T09041) it is diluted to 0.01%(weight/mass percentage composition), put to stir on magnetic force heating rod agitator and boil, every 100ml0.01% gold chloride adds 2ml1% trisodium citrate and (is purchased from Guangzhou Chemical Reagent Factory, catalog number BG11-AR-01KG), continue to boil, stop heating when taking on a red color to liquid, after being cooled to room temperature, supply dehydration.The gold colloidal outward appearance prepared is pure, bright, nothing precipitates and floating thing, and effect duration is one month.
(2) preparation of lincomycin monoclonal antibody-colloid gold label thing
Under magnetic stirring, adjust the pH value of gold colloidal to 8.2 with 0.1mol/L potassium carbonate, in colloidal gold solution, above-mentioned lincomycin monoclonal antibody is added by the standard of 50-100 μ g antibody/ml gold colloidal, continue stirring and evenly mixing 30min, add the 10%BSA to BSA final concentration of 1%(volumn concentration in colloidal gold solution), stand 30min.12000rpm, 4 DEG C of centrifugal 30min, abandon supernatant, precipitation uses redissolution buffer solution twice, to precipitate resuspended with the redissolution buffer that volume is initial colloid gold volume 1/20, the concentration of the lincomycin monoclonal antibody obtained-colloid gold label thing solution is 50 μ g monoclonal antibodies/ml solution, put 4 DEG C standby, preserve 60 days.
Redissolution buffer: containing the boric acid solution of 0.02mol/L, pH9.0 of bovine serum albumin (BSA), tween 80 and PVP-300 ketopyrrolidine, the wherein bovine serum albumin (BSA) final concentration of 0.02-0.1%(volumn concentration in redissolution buffer), the tween 80 final concentration of 0.05-0.2%(weight/mass percentage composition in redissolution buffer), the PVP-300 ketopyrrolidine final concentration of 0.3%(weight/mass percentage composition in redissolution buffer).
5, lincomycin monoclonal antibody-colloid gold label thing is coated conjugate release pad
Conjugate release pad is soaked in containing bovine serum albumin (the bovine serum albumin final concentration of 1.0%(of having volumn concentration in buffer)) and pH value be 7.2,0.2mol/L phosphate buffer soak 30 seconds, 37 DEG C to dry 2h standby；With Biodot point film instrument, the lincomycin prepared monoclonal antibody-colloid gold label thing is uniformly coated in conjugate release pad, every 5cm2Conjugate release pad is coated 10 μ l lincomycin monoclonal antibodies-colloid gold label thing (i.e. every square centimeter conjugate release pad is coated 0.1 μ g antibody), vacuum drying, Vacuum Package, put 4 DEG C standby.
6, the preparation of sample absorption pad: sample absorption pad is placed in containing bovine serum albumin (bovine serum albumin final concentration of 0.4%(volumn concentration in buffer)), pH be 9.6,0.1mol/L carbonate buffer solution soak 2h, 37 DEG C dry 2h standby；
7, the preparation of reaction film
Being coated process: with phosphate buffer, lincomycin hapten-ovalbumin conjugate is diluted to 10mg/mL, the detection zone being coated on nitrocellulose filter with Biodot point film instrument, package amount is 0.8 μ g/cm2；With 0.01mol/L, pH7.4PBS buffer, sheep anti-mouse igg antibody being diluted to 200 μ g/ml, the quality control region being coated on nitrocellulose filter with Biodot point film instrument, package amount is 0.8 μ g/cm2。
Close: by the reaction film that is coated as 37 DEG C, seal after 2 hours and preserve.
Confining liquid: containing human serum albumin (human serum albumin's final concentration of 0.8%(weight/mass percentage composition in confining liquid)), sucrose (sucrose final concentration of 0.5%(weight/mass percentage composition in confining liquid)), pH value be 7.2,0.05mol/L phosphate buffer.
(2) assembling of each parts
1, the assembling of the reagent paper containing protecting film:
Described sample absorption pad, conjugate release pad, reaction film, adsorptive pads are pasted the most in order on described base plate；Conjugate release pad is absorbed by the sample pad and covers from Qi You1/3 region, its top, the end of conjugate release pad is connected with the top of reaction film, the end of reaction film is connected with the top of adsorptive pads, and the top of sample absorption pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate；Paste protecting film assembling reagent paper two ends, then be cut into the wide little bar of 3mm with machine, be contained in paper box, reagent paper aluminium foil bag is vacuum-packed, stores in the environment of 4～30 DEG C, be valid for one year.
2, the assembling of the reagent paper containing paper box:
Described sample absorption pad, conjugate release pad, reaction film, adsorptive pads are pasted the most in order on described base plate；Conjugate release pad is absorbed by the sample pad and covers from Qi You1/2 region, its top, the end of conjugate release pad is connected with the top of reaction film, the end of reaction film is connected with the top of adsorptive pads, and the top of sample absorption pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate；Reagent paper machine is cut into the wide little bar of 3mm, is contained in paper box.Reagent paper aluminium foil bag is vacuum-packed, stores in the environment of 4～30 DEG C, be valid for one year.
Two, the Sensitivity and Specificity inspection of test kit
(1) sensitivity tests
Lincomycin standard substance (purchased from Dr company of Germany, article No. C14635000) are diluted to following variable concentrations: 0,5,10,20,40mg/kg；Consisting of of diluent used: pH is 6.6, the phosphate buffer of 0.2mol/L.
Detect with test strips and test card respectively.Dripping standard substance to sample absorption pad, result is every time: when dripping examination 0mg/kg, 5mg/kg, 10mg/kg concentration, detection zone and quality control region demonstrate macroscopic two red bar lines, is negative；When dripping the titer of examination 20mg/kg, 40mg/kg concentration, detection zone does not develops the color, and is positive.Showing, the sensitivity of this test card detection lincomycin is 20mg/kg.
(2) specific test
Specificity is commonly used cross reacting rate and is represented, refers to that the antibody antigenic determinant different from structure occurs the ability combined.This test strips is 20mg/kg to lincomycin detection sensitivity, by clindamycin, chloromycetin, tetracycline, sulfamethazine, enrofloxacin is respectively by 0, 5, 10, 20, 40, 80, 100, 120mg/kg is diluted, detect respectively with two kinds of reagent paper described in embodiment 1, result display chloromycetin, tetracycline, sulfamethazine, when enrofloxacin adds concentrations above, test card detection line all develops the color, can show that the nothing of above medicine is intersected by lincomycin, clindamycin is when concentration is 100mg/kg, test card detection line does not develops the color, it is positive.
Embodiment 3, the application of reagent paper
One, with detection paper lincomycin described in embodiment 1
The reagent paper of the present invention can detect Solic cosmetic sample, liquid make-up sample.
1, the process of sample:
Solic cosmetic sample, liquid make-up sample: taking the extracting solution vibration mixing of 0.2g or 0.2ml cosmetic samples 4ml, then take mixing in 200 μ l mixing liquids to 600 μ l extracting solution, sampling detects；Extracting solution is the phosphate buffer containing 0.5% tween 20 activating agent (percentage ratio is percent by volume).
2, detection method and judgement
After the sample solution that in sample absorption pad in reagent paper in this test kit, dropping need to detect, in 5-8 minute, watch testing result.
Lincomycin medicine concentration in the sample is prescribed a time limit higher than the sample lowest detection of reagent paper, and colloidal gold antibody is combined with lincomycin, thus because competitive reaction will not be combined with lincomycin conjugate and occur without red stripes in detection zone, is positive.Negative sample during detection owing to lacking antibody antigen competitive reaction, it will in detection zone and quality control region, red stripes occurs.As shown in Figure 4.
Positive: when quality control region demonstrates red stripes, and when detection zone does not develops the color, to be judged to the positive.(Fig. 4 A)
Negative: when quality control region demonstrates red stripes, and detection zone also shows that red stripes, is judged to the positive simultaneously.(Fig. 4 B)
Invalid: when quality control region does not demonstrate red stripes, the most no matter whether test section demonstrates red stripes, and it is invalid that this test card is judged to.(Fig. 4 C)
Citing in detail below:
Take the known cosmetic samples 20 parts containing lincomycin concentration more than 2mg/kg and lincomycin concentration less than 2mg/kg cosmetic samples 20 parts, the reagent paper produced by 3 batches carries out detecting (every batch of test strips, each 10 of test card) respectively, calculates its yin and yang attribute rate.
Table 1, detection positive sample result
Table 2, detection negative sample result
Result shows: when the test strips produced by 3 batches and the positive cosmetic samples of test card detection, positive coincidence rate is all 100%；1 part of positive is occurred in that the when of the 2nd batch of Product checking liquid make-up of result during 20 parts of negative cosmetic samples of detection.Illustrate that lincomycin in cosmetics can be used for quickly detecting by the detection lincomycin reagent paper of the present invention.
Claims (9)
1. the reagent paper detecting lincomycin, including sample absorption pad, conjugate release pad, reaction film and adsorptive pads, it is sequentially connected with, it is characterized in that: in described conjugate release pad, be coated with the lincomycin monoclonal antibody of colloid gold label, described lincomycin monoclonal antibody is to be obtained by lincomycin monoclonal antibody hybridoma cell strain F-1-4 secretion, and the preserving number of described cell strain is CGMCCNo.6508；Including detection zone and quality control region on described reaction film, detection zone is coated with the conjugate of lincomycin hapten and carrier protein, and quality control region is coated with anti antibody, and described anti antibody is sheep anti-mouse igg.
Reagent paper the most according to claim 1, it is characterised in that: described sample absorption pad and the connected mode of described conjugate release pad are to have 1/3-1/2 region to be covered by described sample absorption pad in described conjugate release pad.
Reagent paper the most according to claim 1 and 2, it is characterised in that: described detection zone is positioned at the side of the end being bordering on described conjugate release pad, and described quality control region is located remotely from the side of the end of described conjugate release pad；The described detection zone end 5-8mm away from described conjugate release pad；Described quality control region is away from described detection zone 4-7mm.
Reagent paper the most according to claim 3, it is characterised in that: the described detection zone end 6mm away from described conjugate release pad；Described quality control region is away from described detection zone 5mm.
Reagent paper the most according to claim 1 and 2, it is characterised in that: in described conjugate release pad, the density that is coated of coated lincomycin monoclonal antibody is 0.1-0.2 μ g/cm2。
Reagent paper the most according to claim 1 and 2, it is characterised in that: described reagent paper includes that base plate, described sample absorption pad, conjugate release pad, reaction film and adsorptive pads are pasted on described base plate.
Reagent paper the most according to claim 1 and 2, it is characterised in that: described reagent paper is strip；The reagent paper of described strip is coated with protecting film or is contained in box.
8. prepare a method for arbitrary described reagent paper in claim 1-7, be that sample absorption pad, conjugate release pad, reaction film and adsorptive pads are sequentially connected with；The lincomycin monoclonal antibody of colloid gold label it is coated with in described conjugate release pad；Including detection zone and quality control region on described reaction film, detection zone is coated with the conjugate of lincomycin hapten and carrier protein, and quality control region is coated with anti antibody；
Described sample absorption pad, before carrying out described connection, is being first to soak 1-3h in the carbonate buffer solution of 9.6-10.0,0.1-0.2mol/L containing 0.3%-0.5% (volumn concentration) bovine serum albumin, pH value, then is being dried；
The conjugate release pad of the described lincomycin monoclonal antibody being coated with colloid gold label is prepared as follows: the conjugate release pad before being coated is being first the immersion 20-40 second in 7.0-7.4,0.1-0.2mol/L phosphate buffer containing 0.8%-1.2% (volumn concentration) bovine serum albumin, pH value, it is dried, then the lincomycin monoclonal antibody carrying out described colloid gold label is coated.
9. the method detecting lincomycin, is that detected sample carries out pre-treatment, then detects with described reagent paper arbitrary in claim 1-7.
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| CN105301252B (en) * | 2015-09-07 | 2017-07-21 | 北京勤邦生物技术有限公司 | A kind of immunomagnetic beads that purification is enriched with for ochratoxin A and its preparation method and application |
| CN106526189A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Lincomycin detection kit |
| 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 |
| CN109061171B (en) * | 2018-09-21 | 2021-04-30 | 中国烟草总公司郑州烟草研究院 | ELISA kit for detecting flumetralin and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102818895A (en) * | 2012-08-09 | 2012-12-12 | 河南省农业科学院 | Test strip for rapidly detecting lincomycin residues and preparation method of test strip |
| CN202710567U (en) * | 2012-06-15 | 2013-01-30 | 北京中检葆泰生物技术有限公司 | Colloidal gold test paper strip and kit both capable of rapidly detecting lincomycin |
| CN102928409A (en) * | 2011-08-09 | 2013-02-13 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting lincomycin, and applications thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102928409A (en) * | 2011-08-09 | 2013-02-13 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting lincomycin, and applications thereof |
| CN202710567U (en) * | 2012-06-15 | 2013-01-30 | 北京中检葆泰生物技术有限公司 | Colloidal gold test paper strip and kit both capable of rapidly detecting lincomycin |
| CN102818895A (en) * | 2012-08-09 | 2012-12-12 | 河南省农业科学院 | Test strip for rapidly detecting lincomycin residues and preparation method of test strip |
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