CN102230937A - Brown meat essence multi-residue combined detection test paper card and preparation method thereof - Google Patents
Brown meat essence multi-residue combined detection test paper card and preparation method thereof Download PDFInfo
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- CN102230937A CN102230937A CN2011101639038A CN201110163903A CN102230937A CN 102230937 A CN102230937 A CN 102230937A CN 2011101639038 A CN2011101639038 A CN 2011101639038A CN 201110163903 A CN201110163903 A CN 201110163903A CN 102230937 A CN102230937 A CN 102230937A
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Abstract
The invention discloses a gold-labeled chromatographic detection test paper card for brown meat essence multi-residue analysis, and the test paper card comprises a plastic support and a test paper core, wherein the test paper core is formed by sequentially bonding a sample pad, a gold-labeled material combination pad, a chromatographic film and a water absorption pad onto a lining board; the gold-labeled material combination pad is glass fiber cotton adsorbed with 2-5 mixed gold-labeled antibodies; the gold-labeled antibodies are colloidal-gold-labeled monoclonal antibodies or polyclonal antibodies for resisting brown meat essence; detection lines are 2-5 corresponding brown meat essence-carrier protein conjugated antigen strips; control lines are formed by printing of goat or rabbit anti-mouse IgG, goat anti-rabbit IgG or rabbit anti-carrier protein IgG antibodies; and the detection lines and the control lines are arranged in parallel in the transverse direction along the chromatographic film. The test paper card has high specificity and sensitivity, and the minimum detection limit can be 1ppb, thereby realizing fast detection of brown meat essence multi-residue gold-labeled chromatography. By using the test paper card, multiple brown meat essence residues can be simultaneously detected after one-step sample application, thereby greatly saving the detection time, improving the working efficiency and saving the detection cost.
Description
Technical field
The present invention relates to the utensil of a kind of fast detecting trace " clenbuterol hydrochloride ", particularly relate to gold mark chromatography detecting test paper card of a kind of " clenbuterol hydrochloride " multi-residue analysis and preparation method thereof.
Background technology
" clenbuterol hydrochloride " is meant a class medicine, any material of lean meat growth, minimizing fat deposition that can promote can be called " clenbuterol hydrochloride ", comprise " Clenbuterol " (Clenbuterol, CL), clenobuterol hydrochloride is its hydrochloride, " Ractopamine " (Ractopamine, RAC also is translated into Ractopamine), " salbutamol " (Salbutamol, SAL), " Terbutaline " (Terbutaline, TBL) and " Cimaterol " (Cimaterol, CIM) etc.When " clenbuterol hydrochloride " dosage in meat animals is produced is 5-10 times of therapeutic dose, have the muscle development of promotion and lipolysis effect, can improve the meat animals carcass lean meat percentage.Because the consumer is to the special preferences of lean meat, the pig that the lean meat output capacity is high can be fetched a good price, and adds " clenbuterol hydrochloride " and has brought extra returns to the raiser.Therefore, be subjected to ordering about of undue profits, the raiser is everlasting cultivating link interpolation " clenbuterol hydrochloride " in animal feed.
" but clenbuterol hydrochloride " is a kind of selectivity β
2-adrenoceptor agonists, spinoff is very big, can cause the infringement of cardiovascular system and serious nervous symptoms occur when intake is big.When people eaten contain " clenbuterol hydrochloride " residual pluck and meat after, can cause acute or chronic kreotoxism, harm the consumer healthy and safe.Neither one state approval " clenobuterol hydrochloride " can be used in the animal production so far, but exists illegal use for a long time always, causes the incident of serious food poisoning not rarely seen all over the world.The poisoning of " clenbuterol hydrochloride " has also appearred on China Beijing, Hong Kong, Shanghai, Zhejiang, Guangdong, Shandong, Hebei, Guangxi, Heilungkiang and Anhui and other places, the physical and mental health that is greatly threatening the consumer.Because of the strict management and control of country to clenobuterol hydrochloride, so-called " novel clenbuterol hydrochloride " products such as " Ractopamine ", " salbutamol " have appearred now.
The method that is used for " clenbuterol hydrochloride " residue detection is more, (1) physico-chemical analysis method, as high pressure liquid chromatograph (High performance liquid chromatography, HPLC), gas chromatography (Gas chromatography, GC), thin-layer chromatography (Thin-layer chromatography, TLC), the capillary zone electrophoresis method (Capillary zone electrophoresis, CE) etc.; (2) immunoassay, as radioimmunology (Radio-immunoassay, RIA), enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) etc.; (3) bioassay method, as microbiological assay (microbiological assays, MA), radioreceptor assay (redioreceptor assays, RA) etc.But the instrument and equipment of the needs costliness that these methods have needs skilled professional's operation, and complicated operating process needs the time longer, has limited its range of application, is difficult to apply in production reality.The immune test paper method has sxemiquantitative and certain quantitation capabilities, the preliminary information of determinand can be provided, this method is highly sensitive, analytic process is simple, examination as " clenbuterol hydrochloride " has special advantages, be the detection technique that to first develop, but " clenbuterol hydrochloride " how residual detection paper technology still belong to blank at home at present, suddenly waits to research and solve.
Summary of the invention
The technical problem to be solved in the present invention: a kind of on-the-spot how residual joint inspection test card of special, responsive, quick and easy clenbuterol hydrochloride that detects and preparation method thereof that is applicable to is provided.
Technical scheme of the present invention:
The how residual joint inspection test card of a kind of clenbuterol hydrochloride, comprise plastic support thing and test paper core, the plastic support thing comprises base and panel, wherein base is provided with the pickup groove that the test paper core is installed, panel is provided with well and display window as a result, the test paper core is by sample pad, gold mark thing pad, chromatographic film and adsorptive pads paste on the liner plate successively and make, chromatographic film is provided with detection line and control line, described gold mark thing pad is to be adsorbed with two～five kinds of glass fibre cottons that mix golden labeling antibody, the gold labeling antibody is the monoclonal antibody or the polyclonal antibody of the anti-clenbuterol hydrochloride of colloid gold label, detection line is the simulation conjugated antigen of two～five corresponding clenbuterol hydrochloride-carrier protein conjugated antigens or synthetic, control line is the anti-mouse IgG of goat-anti or rabbit, the anti-carrier protein IgG of goat anti-rabbit igg or rabbit antibody is printed and is formed, and detection line and control line laterally are arranged in parallel along chromatographic film.
The simulation conjugated antigen of described golden labeling antibody and corresponding clenbuterol hydrochloride-carrier protein conjugated antigen or synthetic is: first group: the anti-clenobuterol hydrochloride CL antibody of gold mark, and detection line is a CL-carrier protein conjugated antigen; Second group: the anti-Ractopamine RAC antibody of gold mark, detection line is RAC-carrier protein conjugated antigen; The 3rd group: gold mark desertification butylamine alcohol SAL antibody, detection line is a SAL-carrier protein conjugated antigen; The 4th group: the special special woods TBL antibody of step of gold mark, detection line is a TBL-carrier protein conjugated antigen; The 5th group: the anti-Cimaterol CIM antibody of gold mark, detection line is a CIM-carrier protein conjugated antigen; Adopt wherein two groups, three groups, four groups or five groups, form respectively that bigeminy is inspected paper card, three joint inspection test cards, tetrad inspects paper card or 5-linked is inspected paper card.
Described adsorptive pads adopts filter paper, sponge or nonwoven fabrics to make, and described liner plate is made of the hard plastic bar that not absorbing water, and described sample pad adopts glass fibre cotton, nylon membrane, PVDF membrane or polyester film to make; Chromatographic film adopts nylon membrane, PVDF membrane, nitrocellulose filter or cellulose acetate membrane to make, and control line adopts nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane to make; Described carrier protein is bovine serum albumin(BSA), the pure albumen of ovum gallinaceum, human serum albumins or keyhole limpet hemocyanin.
The preparation method of the how residual joint inspection test card of described clenbuterol hydrochloride, comprise the preparation of clenbuterol hydrochloride-carrier protein conjugated antigen, the preparation of lean meat fine gold mark thing, the preparation of IgG antibody, the preparation of test paper core, it is characterized in that: described clenbuterol hydrochloride-carrier protein conjugated antigen comprises following lean meat fine gold labeling antibody and corresponding conjugated antigen, first group: the anti-antibody of clenbuteral hydrochloride of gold mark, detection line is a CL-carrier protein conjugated antigen; Second group: the anti-Ractopamine antibody of gold mark, detection line is RAC-carrier protein conjugated antigen; The 3rd group: gold mark desertification butylamine alcohol antibody, detection line is a SAL-carrier protein conjugated antigen; The 4th group: the special special woods antibody of step of gold mark, detection line is a TBL-carrier protein conjugated antigen; The 5th group: the anti-Cimaterol antibody of gold mark, detection line is a CIM-carrier protein conjugated antigen.
Described CL-carrier protein conjugated antigen may further comprise the steps when preparing: under 0～4 ℃ of condition of ice bath, take by weighing 5～15mg CL and be dissolved in the watery hydrochloric acid of 1～3mL, 0.1mol/L, dropping concentration is 10% NaNO under stirring
2Solution, reaction 2～4h obtains reactant liquor; Take by weighing the 5-10mg carrier protein and be dissolved in the 1-3mLPBS liquid, stirring slowly adds in the described reactant liquor down, regulates pH to 8.5～9.5,0～4 ℃ stirring, spends the night; Collect reactant liquor in bag filter, under 4 ℃,, change dislysate every day 2～3 times, obtain described CL-carrier protein conjugated antigen at last with PBS dialysis 3～5 days.
Described RAC-carrier protein conjugated antigen may further comprise the steps when preparing:
The carrier protein that takes by weighing 5～20mg places the screw socket bottle, and with the distilled water dissolving, the NaOH adjusting pH to 10.8 with 1 mol/L adds 1, the 4-butanediol diglycidyl ether, and the room temperature lucifuge stirs 24 h under the nitrogen protection, the carrier protein solution that must activate; Taking by weighing 15～20mg TBL is dissolved among the NaOH of 2-5ml, 1 mol/L; slowly add in the described carrier protein solution under stirring, 20 h are stirred in nitrogen protection down, and reaction product was dialysed 3 days with PBS; change liquid every day 3～6 times, obtain the RAC-carrier protein conjugated antigen of purifying after the dialysis.
Described SAL-carrier protein conjugated antigen may further comprise the steps when preparing:
The 80-100mg salbutamol sulfate is added in the methyl alcohol, stir under the room temperature and make it dissolving, add the 40-60mg succinic anhydride, stir reaction 72h down; Reacted solution is placed on 12h in the fuming cupboard, makes the methyl alcohol volatilization fully, put into vacuum drying chamber then and dry, obtain the salbutamol succinic acid derivative; Taking by weighing this derivant 20-30 mg, to be dissolved in pH be in 4.8 the tris-HCL damping fluid, adds carbodiimide 30～40mg, stirring reaction 12h, reactant liquor; Take by weighing carrier protein 20-30mg and be dissolved in the tris-HCL damping fluid of 2-5mL, obtain carrier protein solution; Described carrier protein solution is dropwise added in the described reactant liquor stirring reaction 24h under the room temperature; The 4 ℃ of PBS dialysis 72h in the bag filter that pack into after reaction finishes change liquid 6～9 times, promptly obtain SAL-carrier protein conjugated antigen.
Described TBL-carrier protein conjugated antigen may further comprise the steps when preparing:
The carrier protein that takes by weighing 20～30mg places the screw socket bottle, with the distilled water dissolving, regulates pH value to 10.8 with NaOH solution, adds 1,4-BDDE 12 μ L, and the room temperature lucifuge stirs 20 h under the nitrogen protection, the carrier protein solution that obtains activating; Taking by weighing 10～20mg TBL is dissolved in the NaOH solution of 1-5ml, 1 mol/L; stir down and slowly add in the described carrier protein solution; the room temperature lucifuge stirs 20h under the nitrogen protection; product stirs down with PBS dialysis 3 days at 4 ℃; change liquid every day 3～6 times, obtain the TBL-carrier protein conjugated antigen of purifying after the dialysis.
Described CIM-carrier protein conjugated antigen may further comprise the steps when preparing:
The CIM that takes by weighing 3.5～10mg places the screw socket bottle, with the HCL dissolving of 0.1 mol/L, after the ice bath cooling, dropwise adds NaNO under stirring
2Solution is the black-and-blue NaNO that stops to drip with the starch potassium iodide paper check
2Solution, reaction 6 h get diazotizing CIM under 4 ℃ of conditions; Take by weighing 10～30mg carrier protein and be dissolved in 1-5mL pH and in 7.4 the PBS solution, dropwise add diazotizing CIM after the precooling while stirring, regulate PH to 8.5～9.5,4 ℃ reaction overnight with NaOH; Product with PBS dialysis 3 days, changes liquid every day 3 times under 4 ℃ of stirrings, obtain the CIM-carrier protein conjugated antigen of purifying after the dialysis.
Positive beneficial effect of the present invention:
The multi-joint gold mark of clenbuterol hydrochloride of the present invention chromatography detecting test paper jig has following outstanding advantage:
5. test card of the present invention can be saved testing cost, and is applied widely.Use this test card to decline to a great extent than expense with instrumental analysis and conventional individual event test strip; This test card is applied widely simultaneously, can satisfy different levels personnel's needs, comprise professional inspection, customs quarantine control, health quarantine, quality monitoring, processing enterprise and plant family etc., can keep deterrence illegal interpolation clenbuterol hydrochloride, ensure food safety, easy to utilize.
Description of drawings
Fig. 1, test paper core side-looking structural representation.
Fig. 2, test paper core plan structure synoptic diagram.
Fig. 3, binomial joint inspection test card panel structure vertical view.
Fig. 4, test card base organigram.
Fig. 5, three joint inspection test paper core plan structure synoptic diagram.
Fig. 6, three joint inspection test card panel structure vertical views.
Fig. 7, five joint inspection test paper core plan structure synoptic diagram.
Fig. 8, five joint inspection test card panel structure vertical views.
Among the figure, 1: liner plate; 2: sample pad; 3: gold mark thing pad; 4: chromatographic film; 5: adsorptive pads; 6: control line; 7: detection line; 8: well; 9: display window; 10: panel; 11: base; 12: pickup groove.
Embodiment
When preparation " clenbuterol hydrochloride " how residual joint inspection chromatographic test paper card, need certain " clenbuterol hydrochloride "-carrier protein conjugated antigen of preparation, " clenbuterol hydrochloride " gold mark thing earlier, preparation goat-anti or anti-mouse IgG antibody of rabbit or goat anti-rabbit igg antibody, or the anti-carrier protein IgG of rabbit antibody, and then be prepared into the test paper core, and be installed in the pickup groove of plastic housing base, last and panel is entrenched togather.
1. the preparation of " clenbuterol hydrochloride "-carrier protein conjugated antigen
1.1 adopt the diazotising legal system to be equipped with CL-carrier protein conjugated antigen
With BSA is carrier protein, the summary preparation process.The diazotising legal system is equipped with CL-BSA, and (0～4 ℃) takes by weighing CL 5mg under the condition of ice bath, and it is dissolved in the 0.1mol/L watery hydrochloric acid of 1mL pH value ≈ 1, and dropping 0.3～0.5mL concentration is 10% NaNO under stirring
2Solution, reaction 2～4h; Take by weighing BSA 5mg and be dissolved in the 1mL PBS liquid, under the stirring condition it is slowly added NaNO
2In the solution, about the NaOH adjusting pH to 8.5 with 1mol/L, 0～4 ℃ of stirring reaction spends the night.Collect reactant liquor and pack in the bag filter, 4 ℃ down with 0.1mol/L(pH 7.4) PBS dialysis 3～5 days, change dislysate every day 2～3 times; Collect the good artificial conjugated antigen CL – BSA of dialysis at last, be stored in 20 ℃ of refrigerators of ﹣, standby.
Same method substitutes BSA with OVA, HAS or KLH, makes artificial conjugated antigen CL-OVA, CL-HAS or CL-KLH.
Course of reaction during preparation CL – BSA is as follows:
1.2 adopt the BDDE legal system to be equipped with RAC-carrier protein conjugated antigen.
Adopt the synthetic BSA-RAC conjugated antigen of BDDE, concrete grammar is as follows: take by weighing 20 mg BSA and place 10 mL screw socket bottles, dissolve with 2 ml distilled waters (DDW); NaOH with 1 mol/L regulates pH to 10.8; add BDDE, the room temperature lucifuge stirs 24 h under the nitrogen protection, the BSA that must activate.
The TBL that takes by weighing 15 mg is dissolved among the NaOH of 2 ml, 1 mol/L (suitably DMF hydrotropy); slowly add in the BSA solution of activation under the stirring condition; the room temperature lucifuge stirs 20 h under the nitrogen protection; reaction product was dialysed 3 days with PBS under 4 ℃ of stirrings; change liquid every day 3 ~ 6 times, obtain the RAC-BSA of purifying after the dialysis.
Same method substitutes BSA with OVA, HAS or KLH, makes artificial conjugated antigen RAC-OVA, RAC-HAS or RAC-KLH.
Course of reaction during preparation RAC-BSA is as follows:
1.3 the EDC legal system is equipped with SAL-carrier protein conjugated antigen
The 80mg salbutamol sulfate is added in the 10mL methyl alcohol, and the room temperature lower magnetic force stirs and makes it dissolving, adds the 45mg succinic anhydride, and magnetic agitation makes it to react 72h; Reacted solution is placed in the fuming cupboard, makes methyl alcohol volatilization 12h, put into vacuum drying chamber again and dry, promptly get salbutamol succinic acid derivative (SAL-HS).
Taking by weighing SAL-HS20 mg, to be dissolved in 2mL PH be in 4.8 the tris-HCL damping fluid, adds EDC 40mg, stirring reaction 12h; Take by weighing BSA 20mg and be dissolved in the 2mL tris-HCL damping fluid, BSA solution is dropwise added in the above-mentioned reactant liquor, room temperature lower magnetic force stirring reaction 24h.After reaction finishes, 4 ℃ of dialysis of bag filter 72h that pack into, during be that 7.4 PBS solution changes liquid 6～9 times with 0.01mol/L, pH, promptly get immunizing antigen SAL-BSA.Same method substitutes BSA with OVA, HAS or KLH, makes artificial conjugated antigen SAL-OVA, SAL-HAS or SAL-KLH.Course of reaction during preparation SAL-BSA is as follows:
1.4 adopt the synthetic CIM-carrier protein conjugated antigen of diazotising method
The CIM that takes by weighing 3.5 mg places 10 mL screw socket bottles, and with the HCl dissolving of 1 mL, 0.1 mol/L, after the ice bath cooling, lucifuge dropwise adds the sterilization distilled water while stirring and dissolves 1 mol/L NaNO of precooling
2Solution an amount of (being black-and-blue with starch potassium iodide paper is advisable), reaction 6 h get diazotizing CIM under 4 ℃ of conditions.Take by weighing 10 mg BSA and be dissolved among the 1 mL PBS (pH=7.4), dropwise add diazotizing CIM after the precooling while stirring, about NaOH adjusting PH to 8.5 with 1 mol/L, 4 ℃ of reaction overnight.Reaction product with the PBS 3d that dialyses, is changed liquid every day 3 times under 4 ℃ of stirrings, obtain the CIM-BSA of purifying after the dialysis.
Same method substitutes BSA with OVA, HAS or KLH, makes artificial conjugated antigen CIM-OVA, CIM-HAS or CIM-KLH.
Course of reaction during preparation CIM-BSA is as follows:
1.5 adopt the BDDE legal system to be equipped with TBL-carrier protein conjugated antigen
Adopt the synthetic BSA-TBL comlete antigen of BDDE method, concrete grammar is as follows: the BSA that takes by weighing 30 mg places 10 ml screw socket bottles, dissolve with 1 ml distilled water (DDW), 1 mol/L NaOH regulates pH to 10.8, add 1,4 fourth diether, the room temperature lucifuge stirs 20 h under the nitrogen protection, the BSA that must activate; Taking by weighing 12 mg TBL is dissolved in 1 ml, the 1 mol/L NaOH solution; stir the BSA solution that slowly adds activation down, the room temperature lucifuge stirs 20 h under the nitrogen protection, and reaction product was dialysed 3 days with PBS under 4 ℃ of stirrings; change liquid every day 3 ~ 6 times, obtain the TBL-BSA of purifying after the dialysis.Same method substitutes BSA with OVA, HAS or KLH, makes artificial conjugated antigen TBL-OVA, TBL-HAS or TBL-KLH.
Course of reaction during preparation TBL-BSA is as follows:
2, anti-certain " clenbuterol hydrochloride " polyclonal antibody or MONOCLONAL ANTIBODIES SPECIFIC FOR
Many anti-preparations (is example with the anti-mouse of rabbit): with CL is example, with CL-carrier protein conjugated antigen immunity New Zealand white rabbit, immunizing dose be 200～500 μ g/time, the subcutaneous branch 4 in back～6 injections.Head exempts from, and with sterile phosphate damping fluid (PBS) dissolving CL-carrier protein conjugated antigen, (FCA) mixes with the equivalent Freund's complete adjuvant, and be fully emulsified; Booster immunization, with aseptic PBS dissolving CL-carrier protein conjugated antigen, (FIA) mixes with the equivalent incomplete Freund, fully emulsified, head exempts from the back and carried out in 2～3 weeks, continuous immunity 4～5 times, each 2～3 weeks at interval, last immunity back 10～15 days is measured it with blocking-up ELISA method and is tired and reach 10
5When above, blood sampling and separated and collected hyper-immune serum; The saturated ammonium sulfate salting out method extracts IgG antibody, place-20 ℃ frozen standby.
The monoclonal antibody preparation: with CL is example, press 50～100 μ g/ dosage immunity BALB/c mouse in 8 age in week only 3～4 times with CL-carrier protein conjugated antigen, each immunity 3～5 weeks of interval time, last superpower immunity back 3～4 days, asepticly get its splenocyte and hybridize fusion, put 37 ℃, 5%CO with NS0 myeloma cell
2In the incubator, cultivated 10～14 days, and selected strong positive, inhibiting rate height, the eugonic hole of cell to carry out limited dilution cloningization, then enlarged culture 3 times with indirect ELISA method, set up hybridoma cell strain, the monoclonal antibody of its secretion can be reacted with CL specifically.Carry out monoclonal antibody preparation to induce method in the body, with sad-ammonium sulfate method purification monoclonal antibody, put-20 ℃ frozen standby.
Said method can be used for any " clenbuterol hydrochloride " monoclonal antibody or Polyclonal Antibody Preparation.
3, the preparation of gold mark thing pad
The sodium citrate reducing process is an example, at first prepares colloidal gold solution: add freshly prepared 1% sodium citrate 8ml in 200ml 0.01～0.02% chlorauric acid solution of boiling, obtain the colloidal gold solution of the about 15nm of diameter, with the K of 0.1mol/L
2CO
3Adjust pH to 8.5～9.5, it is standby to put 2～8 ℃ of preservations; Mark ratio with 1:2000 adds monoclonal antibody to be marked or polyclonal antibody in the aurosol of pH8.5～9.5, behind the mark 10min, add 20% PEG10000 to final concentration be 0.05%, 4 ℃, the centrifugal 20min of 1500～2000rpm remove unconjugated colloid gold particle, 4 ℃, the centrifugal 1h of 15000rpm, abandon supernatant, obtain the golden labeling antibody albumen of preliminary purification, carry out separation and purification with propylene glucosan S-400 column chromatography again, obtain colloid gold label antibody.The colloid gold label antibody (monoclonal antibody or polyclonal antibody) of 1:100～500 dilutions is adsorbed in the processed glass cellucotton, and 4 ℃ of low-temperature vacuum dryings prepare golden labeling antibody pad.
The preparation of said method can be used for any " clenbuterol hydrochloride " golden labeling antibody pad.
4, anti-mouse IgG(of goat-anti or rabbit or goat anti-rabbit igg) preparation of antibody
Extract " clenbuterol hydrochloride " negative mice serum IgG(or negative rabbit anteserum IgG with the saturated ammonium sulfate salting out method), with mice serum (or rabbit anteserum) IgG with the dosage of 50～100 μ g/kg body weight through the subcutaneous and healthy goat of intramuscular injection or rabbit 3～4 times, the last injection is after 10 days, measuring its serum fine jade expands to tire and reaches 1:40 when above, heart or arterial blood drawing, the separated and collected hyper-immune serum, extracting goat-anti or anti-mouse IgG(of rabbit or goat anti-rabbit igg with saturated ammonium sulfate salting out method or sodium sulphate method etc.) (method is identical with extraction mice serum IgG for antibody, no longer repeat), place-20 ℃ frozen standby.
5, the preparation of the anti-carrier protein IgG of rabbit antibody
With carrier protein BSA immunity New Zealand white rabbit, immunizing dose be 200～500 μ g/time, the subcutaneous branch 4 in back～6 injections.Head exempts from, and with aseptic PBS dissolving BSA, FCA mixes with equivalent, and is fully emulsified; Booster immunization, with aseptic PBS dissolving BSA, FIA mixes with equivalent, and is fully emulsified, and head exempts from the back and carried out in 2～3 weeks, and continuous immunity 3～4 times in each 2～3 weeks at interval, after the last immunity 10～15 days, is measured its serum fine jade expansion and is tired and reach 10
5When above, blood sampling and separated and collected hyper-immune serum.Extract anti-BSA antibody with the saturated ammonium sulfate salting out method, put-20 ℃ frozen standby.
Prepare anti-OVA antibody, anti-HSA antibody, anti-KLH antibody with method.
6, test card detection reaction principle
Because colloid gold label object difference, its detection reaction principle is variant slightly.
When the colloid gold label thing was antibody, behind the adding testing sample solution, solution to be measured spread to chromatographic film together by the golden labeling antibody that syphonic effect drives in determinand and the gold mark thing pad; Medicine to be measured can combine with golden labeling antibody in diffusion process, thereby sealed the binding site on the golden labeling antibody, stop golden labeling antibody to be tackled by the conjugated antigen detection line on the chromatographic film, detection line does not show, anti-mouse IgG(of goat-anti or rabbit or goat anti-rabbit igg) antibody then can tackle golden labeling antibody, show a brownish red control line "
︱", positive expression; Otherwise, during no medicine to be measured, then can not stop golden labeling antibody in the sample solution by the interception of the conjugated antigen detection line on the chromatographic film, show the brownish red detection line "
︱", but anti-mouse IgG(of goat-anti or rabbit or goat anti-rabbit igg) antibody also tackles golden labeling antibody, show the brownish red control line "
︱", negative expression; If do not have reddish brown colo(u)r streak to show on the chromatographic film, show that then test card lost efficacy.
When the colloid gold label thing is conjugated antigen, after adding testing sample solution, sample solution spreads to chromatographic film together by the gold mark conjugated antigen that syphonic effect drives in medicine to be measured and the gold mark thing pad, medicine free to be measured in the sample and antibody competition combination, stop gold mark conjugated antigen to combine with antibody detection line on the chromatographic film, detection line do not show, anti-carrier protein antibody then can be tackled gold mark conjugated antigen, show a brownish red control line "
︱", positive expression; Otherwise, in the sample solution during no medicine to be measured, when gold mark conjugated antigen arrives detection line on the chromatographic film by the interception of relevant detection line antibody, show the brownish red detection line "
︱", anti-simultaneously carrier protein antibody also can be tackled gold mark conjugated antigen, demonstration brownish red control line "
︱", negative expression; If do not have reddish brown colo(u)r streak to show on the chromatographic film, show that then test card lost efficacy.
Embodiment one:" clenbuterol hydrochloride " binomial joint inspection gold mark chromatographic test paper card.
On the basis that obtains clenbuterol hydrochloride-carrier protein conjugated antigen, lean meat fine gold mark thing and IgG antibody, assembling obtains test card of the present invention (is example with clenobuterol hydrochloride and Ractopamine two joint inspections).
The test card plastic housing comprises base 11 and panel 10, and base is provided with the pickup groove 12 that the test paper core is installed, and panel is provided with well 8 and display window 9, and the display window next door is provided with T
1, T
2With the C sign, referring to Fig. 1-4.
The structure of test paper core: liner plate 1 usefulness hard plastic bar is made; Sample pad 2 usefulness glass fibre cottons are made; Gold mark thing pad 3 is for being adsorbed with the anti-CL of colloid gold label and the glass fibre cotton of anti-RAC monoclonal antibody; Chromatographic film 4 is used nitrocellulose filter, uses two detection line 7(of conjugated antigen preparation stealth of coupling CL-carrier protein and RAC-carrier protein on it respectively), carrier protein is a bovine serum albumin(BSA).Article two, detection line is arranged in order by the sample end, prepares control line 6(stealth with goat anti-mouse igg antibody), control line 6 adopts nitrocellulose filter; Detection line be arranged in parallel with control line "
︱ ︱ ︱"; Adsorptive pads 5 usefulness absorbent filters are made.With sample pad, gold mark thing pad, chromatographic film, adsorptive pads overlaps successively mutually and be pasted and fixed on the liner plate, is assembled into CL and RAC joint inspection test paper core.
The test paper core is installed in the pickup groove 12 on the base 11, and sample pad is corresponding with well and display window on the panel respectively with chromatographic film, two stealthy detection lines and stealthy control line respectively with the T on display window next door
1, T
2Corresponding with C, promptly make test card behind the chimeric panel.
Test sample: test sample can be urine sample.
The test sample preparation: urine sample need not special processing, as too muddy, leave standstill back centrifuging and taking supernatant and gets final product.
Method of operating: during detection, test card is kept flat, drip testing sample, judge testing result from display window in 1～5min from well.
The result judges: as have only stealthy control line place, C position show a reddish brown colo(u)r streak "
︱", expression CL and RAC testing result are all positive, illustrate and contain this two kinds of medicines to be measured in testing sample; As at T
1With two reddish brown colo(u)r streaks of C position display "
︱ ︱" time, expression CL testing result is positive, and the RAC testing result is negative, illustrates to contain CL and do not contain RAC in testing sample; As at T
2With two reddish brown colo(u)r streaks of C position display "
︱ ︱", expression CL testing result is positive, and the RAC testing result is negative, illustrates to contain CL and do not contain RAC in testing sample; As at T
1, T
2With three reddish brown colo(u)r streaks of C position display "
︱ ︱ ︱", expression CL and RAC testing result are all negative, illustrate not contain CL and RAC in testing sample; As on chromatographic film, there not being reddish brown colo(u)r streak to show, show that then test card lost efficacy.
Embodiment two: "Clenbuterol hydrochloride
"Binomial joint inspection gold mark chromatographic test paper card (is example with clenobuterol hydrochloride and Ractopamine two joint inspections)
Test card structure and embodiment one are basic identical, and difference is: sample pad adopts nylon membrane to make; With the alternative anti-CL of polyclonal antibody of anti-CL and anti-RAC and the monoclonal antibody of anti-RAC, preparation gold-marking binding pad; Chromatographic film is made with nylon membrane, with goat anti-rabbit igg antibody substitute sheep anti-mouse igg antibody on chromatographic film, prepare stealthy control line "
︱", control line adopts the pure cellulose film to make; Other, repeats no more all with embodiment one as test sample preparation, method of operating and judgement as a result etc.
Embodiment three: "Clenbuterol hydrochloride " binomial joint inspection gold mark chromatographic test paper card (is example with clenobuterol hydrochloride and Ractopamine two joint inspections).
Test card structure and embodiment one are basic identical, and difference is: sample pad adopts PVDF membrane to make; With the monoclonal antibody of CL-carrier protein and alternative anti-CL of RAC-carrier protein conjugated antigen and anti-RAC, preparation gold-marking binding pad; The chromatographic film PVDF membrane, with the anti-carrier protein IgG of rabbit antibody surrogate sheep anti-mouse igg antibody on chromatographic film, prepare stealthy control line "
︱", control line adopts the carboxylation cellulose membrane to make; Other, repeats no more all with embodiment one as test sample preparation, method of operating and judgement as a result etc.
Embodiment four: "Clenbuterol hydrochloride " three joint inspection gold mark chromatographic test paper cards (is example with clenobuterol hydrochloride, Ractopamine and salbutamol three joint inspections).
The structure of test card plastic housing comprises base and panel, and base is provided with the pickup groove that the test paper core is installed, and panel is provided with well and display window, and the display window next door is provided with T
1, T
2, T
3Identify with C.Referring to Fig. 5-6.
The structure of test paper core: liner plate is made with the hard plastic bar; Sample pad adopts polyester film to make; Gold mark thing pad is the glass fibre cotton of the anti-CL that is adsorbed with colloid gold label, anti-RAC and anti-SAL monoclonal antibody; The chromatographic film cellulose acetate membrane, three stealthy detection lines that prepare with coupling CL-carrier protein, RAC-carrier protein and SAL-carrier protein conjugated antigen respectively on it "
︱", described carrier protein is the pure albumen of ovum gallinaceum.Article three, stealthy detection line is arranged in order by the sample end, with the anti-mouse IgG antibody of rabbit prepare stealthy control line "
︱", control line adopts the pure cellulose film, three detection lines and control line be arranged in parallel and be combined into "
︱ ︱ ︱ ︱"; Adsorptive pads is made with absorbent filter.Sample pad, gold mark thing pad, chromatographic film, adsorptive pads are overlapped mutually successively and be pasted and fixed on the liner plate, be assembled into CL, RAC and SAL joint inspection test paper core.The test paper core is installed in the pickup groove of base, the sample pad of test paper core is corresponding with well and display window on the panel respectively with chromatographic film, three detection lines on the chromatographic film and control line respectively with the T on display window next door
1, T
2, T
3Corresponding with C, make test card behind the chimeric panel.
Other comprises that test sample preparation and method of operating all with embodiment one, repeat no more.
The result judges: as have only 1 reddish brown colo(u)r streak of C position display "
︱" time, expression CL, RAC and SAL testing result are all positive, illustrate to contain this 3 kinds of medicines to be measured in testing sample; As show 1 brownish red T line and 1 brownish red C line "
︱ ︱" time, representing that 2 kinds of drug test results are positive, other a kind of drug test result is negative, illustrates to contain 2 kinds of corresponding medicines and do not contain other a kind of medicine in testing sample; As show 2 brownish red T lines and 1 brownish red C line "
︱ ︱ ︱" time, representing that a kind of drug test result is positive, other 2 kinds of drug test results are negative, illustrate to contain a kind of medicine and do not contain other 2 kinds of medicines in testing sample; As show 3 brownish red T lines and 1 brownish red C line "
︱ ︱ ︱ ︱" time, represent that 3 kinds of drug test results are all negative; As on chromatographic film, there not being reddish brown colo(u)r streak to show, show that then test card lost efficacy.
Embodiment five: "Clenbuterol hydrochloride " three joint inspection gold mark chromatographic test paper cards (is example with Clenbuterol, Ractopamine and salbutamol three joint inspection test paper)
Test card structure and embodiment four are basic identical, and difference is: sample pad adopts glass fibre cotton to make; Substitute the monoclonal antibody of anti-CL, anti-RAC and anti-SAL, preparation gold-marking binding pad with the polyclonal antibody of anti-CL, anti-RAC and anti-SAL; With goat anti-rabbit igg antibody substitute rabbit anti-mouse igg antibody on chromatographic film, prepare stealthy control line "
︱".
Other comprises that test sample preparation, method of operating and judgement as a result etc. all with embodiment four, no longer repeat.
Embodiment six: "Clenbuterol hydrochloride " three joint inspection gold mark chromatographic test paper cards (is example with Clenbuterol, Ractopamine and salbutamol three joint inspection test paper).
Test card structure and embodiment four are basic identical, and difference is: with the monoclonal antibody of alternative anti-CL, anti-RAC and anti-SAL of CL-carrier protein, RAC-carrier protein and SAL-carrier protein conjugated antigen, and the preparation gold-marking binding pad; With the anti-carrier protein IgG of rabbit antibody surrogate rabbit anti-mouse igg antibody on chromatographic film, prepare stealthy control line "
︱".
Other comprises that test sample preparation, method of operating and judgement as a result etc. all with embodiment four, no longer repeat.
Embodiment seven: "Clenbuterol hydrochloride " five joint inspection gold mark chromatographic test paper cards
The structure of test card plastic housing comprises base and panel, and base is provided with the pickup groove that the test paper core is installed, and panel is provided with well and display window, the other T that is provided with of display window
1, T
2, T
3, T
4, T
5Identify with C.Referring to Fig. 7-8.
The structure of test paper core: liner plate is made with the hard plastic bar; Sample pad is made with glass fibre cotton; Gold mark thing pad is the glass fibre cotton of the anti-CL that is adsorbed with colloid gold label, anti-RAC, anti-SAL, anti-CML and anti-TBL monoclonal antibody; The chromatographic film nitrocellulose filter, the stealthy detection line for preparing with coupling CL-carrier protein, RAC-carrier protein, SAL-carrier protein, CML-carrier protein and TBL-carrier protein conjugated antigen respectively on it "
︱", carrier protein is human serum albumins or keyhole limpet hemocyanin.
Article five, detection line is arranged in order by the sample end, with goat anti-rabbit igg antibody prepare stealthy control line "
︱", control line adopts the carboxylation cellulose membrane, six line parallel permutation and combination become "
︱ ︱ ︱ ︱ ︱ ︱"; Adsorptive pads is made with absorbent filter.Sample pad, gold mark thing pad, chromatographic film, adsorptive pads are overlapped mutually successively and be pasted and fixed on the liner plate, be assembled into CL, RAC, SAL, CML and TBL joint inspection test paper core.The test paper core is installed in the pickup groove of base, the sample pad of test paper core and chromatographic film are corresponding with well and display window on the panel respectively, five detection lines on the chromatographic film and control line respectively with the T on display window next door
1, T
2, T
3, T
4, T
5Corresponding with C, make test card behind the chimeric panel.
Other comprises that test sample preparation and method of operating all with embodiment one, repeat no more.
The result judges: if the C position do not show 1 reddish brown colo(u)r streak "
︱" time, represent that this detection is invalid; 1 reddish brown colo(u)r streak of C position display "
︱" time, represent that this detects effectively, and T
1, T
2, T
3, T
4, T
5The corresponding the sort of determinand of which i.e. expression of bar colour developing does not detect in this detects, and the corresponding the sort of determinand of the i.e. expression that does not develop the color is detected in this detects, and the result is positive.So can judge the yin and yang attribute of five kinds of checking matters in once measuring, promptly 5-linked is inspected paper card.
Embodiment eight:Five joint inspection gold of clenbuterol hydrochloride mark chromatographic test paper card
Test card structure and embodiment seven are basic identical, and difference is: the monoclonal antibody with polyclonal antibody alternative anti-CL, anti-RAC, anti-SAL, anti-CML and the anti-TBL of anti-CL, anti-RAC, anti-SAL, anti-CML and anti-TBL prepares gold-marking binding pad; With rabbit anti-mouse igg antibody surrogate goat anti-rabbit igg antibody on chromatographic film, prepare stealthy control line "
︱".
Other comprises that test sample preparation, method of operating and judgement as a result etc. all with embodiment seven, repeat no more.
Embodiment nine:Five joint inspection gold of clenbuterol hydrochloride mark chromatographic test paper card
Test card structure and embodiment seven are basic identical, difference is: with the monoclonal antibody of alternative anti-CL, anti-RAC, anti-SAL, anti-CML and anti-TBL of CL-carrier protein, RAC-carrier protein, SAL-carrier protein, CML-carrier protein and TBL-carrier protein conjugated antigen, and the preparation gold-marking binding pad; With the anti-carrier protein IgG of rabbit antibody surrogate goat anti-rabbit igg antibody on chromatographic film, prepare stealthy control line "
︱".
Other comprises that test sample preparation, method of operating and judgement as a result etc. all with embodiment seven, repeat no more.
Claims (10)
1. how residual joint inspection test card of clenbuterol hydrochloride, comprise plastic support thing and test paper core, the plastic support thing comprises base and panel, wherein base is provided with the pickup groove that the test paper core is installed, panel is provided with well and display window as a result, the test paper core is by sample pad, gold mark thing pad, chromatographic film and adsorptive pads paste on the liner plate successively and make, chromatographic film is provided with detection line and control line, it is characterized in that: described gold mark thing pad is to be adsorbed with two～five kinds of glass fibre cottons that mix golden labeling antibody, the gold labeling antibody is the monoclonal antibody or the polyclonal antibody of the anti-clenbuterol hydrochloride of colloid gold label, detection line is the simulation conjugated antigen of two～five corresponding clenbuterol hydrochloride-carrier protein conjugated antigens or synthetic, control line is the anti-mouse IgG of goat-anti or rabbit, the anti-carrier protein IgG of goat anti-rabbit igg or rabbit antibody is printed and is formed, and detection line and control line laterally are arranged in parallel along chromatographic film.
2. test card according to claim 1, it is characterized in that: the simulation conjugated antigen of described golden labeling antibody and corresponding clenbuterol hydrochloride-carrier protein conjugated antigen or synthetic is: first group: the anti-clenobuterol hydrochloride CL antibody of gold mark, and detection line is a CL-carrier protein conjugated antigen; Second group: the anti-Ractopamine RAC antibody of gold mark, detection line is RAC-carrier protein conjugated antigen; The 3rd group: gold mark desertification butylamine alcohol SAL antibody, detection line is a SAL-carrier protein conjugated antigen; The 4th group: the special special woods TBL antibody of step of gold mark, detection line is a TBL-carrier protein conjugated antigen; The 5th group: the anti-Cimaterol CIM antibody of gold mark, detection line is a CIM-carrier protein conjugated antigen; Adopt wherein two groups, three groups, four groups or five groups, form respectively that bigeminy is inspected paper card, three joint inspection test cards, tetrad inspects paper card or 5-linked is inspected paper card.
3. test card according to claim 1, it is characterized in that: described adsorptive pads adopts filter paper, sponge or nonwoven fabrics to make, described liner plate is made of the hard plastic bar that not absorbing water, and described sample pad adopts glass fibre cotton, nylon membrane, PVDF membrane or polyester film to make; Chromatographic film adopts nylon membrane, PVDF membrane, nitrocellulose filter or cellulose acetate membrane to make, and control line adopts nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane to make; Described carrier protein is bovine serum albumin(BSA), the pure albumen of ovum gallinaceum, human serum albumins or keyhole limpet hemocyanin.
4. the preparation method of the how residual joint inspection test card of the described clenbuterol hydrochloride of claim 1, comprise the preparation of clenbuterol hydrochloride-carrier protein conjugated antigen, the preparation of lean meat fine gold mark thing, the preparation of IgG antibody, the preparation of test paper core, it is characterized in that: described clenbuterol hydrochloride-carrier protein conjugated antigen comprises following lean meat fine gold labeling antibody and corresponding conjugated antigen, first group: the anti-antibody of clenbuteral hydrochloride of gold mark, detection line is a CL-carrier protein conjugated antigen; Second group: the anti-Ractopamine antibody of gold mark, detection line is RAC-carrier protein conjugated antigen; The 3rd group: gold mark desertification butylamine alcohol antibody, detection line is a SAL-carrier protein conjugated antigen; The 4th group: the special special woods antibody of step of gold mark, detection line is a TBL-carrier protein conjugated antigen; The 5th group: the anti-Cimaterol antibody of gold mark, detection line is a CIM-carrier protein conjugated antigen.
5. the described preparation method of claim 4, it is characterized in that: described CL-carrier protein conjugated antigen may further comprise the steps when preparing: under 0～4 ℃ of condition of ice bath, take by weighing 5～15mg CL and be dissolved in the watery hydrochloric acid of 1～3mL, 0.1mol/L, dropping concentration is 10% NaNO under stirring
2Solution, reaction 2～4h obtains reactant liquor; Take by weighing the 5-10mg carrier protein and be dissolved in the 1-3mLPBS liquid, stirring slowly adds in the described reactant liquor down, regulates pH to 8.5～9.5,0～4 ℃ stirring, spends the night; Collect reactant liquor in bag filter, under 4 ℃,, change dislysate every day 2～3 times, obtain described CL-carrier protein conjugated antigen at last with PBS dialysis 3～5 days.
6. test card according to claim 4, it is characterized in that: described RAC-carrier protein conjugated antigen may further comprise the steps when preparing: the carrier protein that takes by weighing 5～20mg places the screw socket bottle, dissolve with distilled water, NaOH with 1 mol/L regulates pH to 10.8, add 1, the 4-butanediol diglycidyl ether, the room temperature lucifuge stirs 24 h under the nitrogen protection, the carrier protein solution that must activate; Taking by weighing 15～20mg TBL is dissolved among the NaOH of 2-5ml, 1 mol/L; slowly add in the described carrier protein solution under stirring, 20 h are stirred in nitrogen protection down, and reaction product was dialysed 3 days with PBS; change liquid every day 3～6 times, obtain the RAC-carrier protein conjugated antigen of purifying after the dialysis.
7. test card according to claim 4, it is characterized in that: described SAL-carrier protein conjugated antigen may further comprise the steps when preparing: the 80-100mg salbutamol sulfate is added in the methyl alcohol, stir under the room temperature and make it dissolving, add the 40-60mg succinic anhydride, stir reaction 72h down; Reacted solution is placed on 12h in the fuming cupboard, makes the methyl alcohol volatilization fully, put into vacuum drying chamber then and dry, obtain the salbutamol succinic acid derivative; Taking by weighing this derivant 20-30 mg, to be dissolved in pH be in 4.8 the tris-HCL damping fluid, adds carbodiimide 30～40mg, stirring reaction 12h, reactant liquor; Take by weighing carrier protein 20-30mg and be dissolved in the tris-HCL damping fluid of 2-5mL, obtain carrier protein solution; Described carrier protein solution is dropwise added in the described reactant liquor stirring reaction 24h under the room temperature; The 4 ℃ of PBS dialysis 72h in the bag filter that pack into after reaction finishes change liquid 6～9 times, promptly obtain SAL-carrier protein conjugated antigen.
8. test card according to claim 4, it is characterized in that: described TBL-carrier protein conjugated antigen may further comprise the steps when preparing: the carrier protein that takes by weighing 20～30mg places the screw socket bottle, dissolve with distilled water, regulate pH value to 10.8 with NaOH solution, add 1,4-BDDE 12 μ L, the room temperature lucifuge stirs 20 h under the nitrogen protection, the carrier protein solution that obtains activating; Taking by weighing 10～20mg TBL is dissolved in the NaOH solution of 1-5ml, 1 mol/L; stir down and slowly add in the described carrier protein solution; the room temperature lucifuge stirs 20h under the nitrogen protection; product stirs down with PBS dialysis 3 days at 4 ℃; change liquid every day 3～6 times, obtain the TBL-carrier protein conjugated antigen of purifying after the dialysis.
9. test card according to claim 4, it is characterized in that: described CIM-carrier protein conjugated antigen may further comprise the steps when preparing: the CIM that takes by weighing 3.5～10mg places the screw socket bottle, with the HCL dissolving of 0.1 mol/L, after the ice bath cooling, dropwise add NaNO under stirring
2Solution is the black-and-blue NaNO that stops to drip with the starch potassium iodide paper check
2Solution, reaction 6 h get diazotizing CIM under 4 ℃ of conditions; Take by weighing 10～30mg carrier protein and be dissolved in 1-5mL pH and in 7.4 the PBS solution, dropwise add diazotizing CIM after the precooling while stirring, regulate PH to 8.5～9.5,4 ℃ reaction overnight with NaOH; Product with PBS dialysis 3 days, changes liquid every day 3 times under 4 ℃ of stirrings, obtain the CIM-carrier protein conjugated antigen of purifying after the dialysis.
10. according to each described preparation method of claim 4-9, it is characterized in that: described carrier protein conjugated antigen is bovine serum albumin(BSA), the pure albumen of ovum gallinaceum, human serum albumins or keyhole limpet hemocyanin.
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Application publication date: 20111102 |