CN103439505A - Quick marbofloxacin detection test paper card and preparation method - Google Patents
Quick marbofloxacin detection test paper card and preparation method Download PDFInfo
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Abstract
The invention relates to a quick marbofloxacin detection test paper card and a preparation method. The test paper card comprises a housing and a test paper core, wherein the test paper core comprises a bottom plate, as well as a sample pad, a colloidal gold labeled pad, a detection reaction area and a water absorption pad which are arranged on the bottom plate; the colloidal gold labeled pad is absorbed with a specific marbofloxacin antibody; the detection reaction area is provided with a detection blot printed by a marbofloxacin coupled carrier protein solution, and a control blot printed by a goat (rabbit) anti-mouse IgG or goat anti-rabbit IgG solution; and the specific marbofloxacin antibody is a colloidal gold labeled marbofloxacin monoclonal antibody or polyclonal antibody. During preparation, a base is embedded with a panel, a detection window on the panel corresponds to the detection reaction area of the test paper core, and a sample adding hole corresponds to the sample pad of the test paper core. The test paper card is strong in specificity, high in sensitivity and timeliness, wide in application scope and convenient to popularize, detects simply, conveniently and quickly, and displays results vividly, visually and accurately, and the detection expense is low.
Description
technical field
The present invention relates to a kind of utensil of fast detecting marbofloxacin, particularly relate to a kind of test card and preparation method of fast detecting marbofloxacin.
Technical background
Marbofloxacin is fluoroquinolone antibiotics, have antibiotic general wide, bactericidal activity is strong, characteristics such as widely distributed in body, and with other antibacterials without cross resistance.At present, this microbiotic is widely used in livestock and poultry and aquaculture.Marbofloxacin is as the medicine for the foodstuff animal, and its residual carcinogenic toxicity in edible animal tissue and product receives much concern.Strict regulations have been done to its high residue amount in different meats by country.Because of marbofloxacin cheap, antibiotic general wide, bactericidal activity is strong, the raiser blindly pursues prevention and result for the treatment of, artificial excess is added, and causes the residual phenomenon ubiquity exceeded standard in meat.Therefore, accurately detect in meat the marbofloxacin residual quantity extremely important.
At present, the method for mensuration marbofloxacin mostly is the methods such as high performance liquid chromatography, liquid chromatograph mass spectrography, capillary zone electrophoresis and mass spectrometry.These methods are generally longer detection time, and testing cost is high, high to testing staff's competency profiling, seriously restricted the application in reality detects, and are difficult to promote.Due to limitation and the serious hysteresis quality of these methods, make the illegal use of monitoring marbofloxacin become difficult, can't guarantee the validity of food security supervision.
Summary of the invention
The technical problem to be solved in the present invention: provide a kind of special strong, highly sensitive, can detect fast, easily test card and the preparation method of marbofloxacin.
Technical scheme of the present invention is:
A kind of marbofloxacin quick detection test paper card, contain shell and the test paper core that is positioned at shell, the test paper core comprises base plate, fixedly be adhesive with successively sample pad, colloid gold label pad, detection reaction district and adsorptive pads on base plate, described colloid gold label pad absorption has the specific antibody of marbofloxacin, described detection reaction district is provided with the stealth detection trace that marbofloxacin coupling carrier protein solution is printed, and the stealth of printing with goat anti-mouse igg, the anti-mouse IgG of rabbit or goat anti-rabbit igg solution contrast trace; Marbofloxacin monoclonal antibody or polyclonal antibody that described marbofloxacin specific antibody is colloid gold label.
By following methods, prepared by described marbofloxacin coupling carrier protein solution:
Take in the DMF solvent that 2 mg marbofloxacins are dissolved in 500 μ L, add 2 mg NHS and 2 mg EDC, room temperature lucifuge reaction 24h, obtain the marbofloxacin liquid of activation; Take carrier protein 2 mg, fully be dissolved in the PBS solution of mol/L of 2 ml 0.13, the marbofloxacin liquid of activation is dropwise added in carrier protein solution, the sealing lucifuge stirs 4h under room temperature; Reaction product is placed in to the PBS dialysis of 0.01mol/L 3 days for 4 ℃ of chromatography cabinets, and the centrifugal precipitation of removing, obtain the conjugate of marbofloxacin carrier protein, packing, and-20 ℃ of preservations, standby.
Described marbofloxacin coupling carrier albumen is thyroprotein, mouse haemocyanin, bovine serum albumin(BSA), human serum albumins, albumin rabbit serum, ovoserum albumin or hemocyanin.
Described base plate is made by hard plastic bar or the cardboard bar that do not absorb water; Described for sample pad glass fibre cotton, nylon membrane, PVDF membrane or polyester film make; Described adsorptive pads is made with absorbent filter or filter paper for oil; Described detection reaction for district nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane make; Described colloid gold label for pad glass fibre, polyester film, acetate fiber or nylon membrane make.
Described shell consists of base and panel, base and panel connect together by chimeric, and base is provided with the groove of placing the test paper core, and panel is provided with detection window, well, detection window is corresponding with the detection reaction district of test paper core, and well is corresponding with the sample pad of test paper core.
The described stealthy arrangement mode that detects trace and stealthy contrast trace be "
︱ ︱", "
++" or " ".
The preparation method of described marbofloxacin quick detection test paper card comprises the following steps:
(1) coupling of marbofloxacin and carrier protein:
Take in the DMF solvent that 2 mg marbofloxacins are dissolved in 500 μ L, add 2 mg NHS and 2 mg EDC, room temperature lucifuge reaction 24h, obtain the marbofloxacin liquid of activation; Take carrier protein 2 mg, fully be dissolved in the PBS solution of mol/L of 2 ml 0.13, the marbofloxacin liquid of activation is dropwise added in carrier protein solution, the sealing lucifuge stirs 4h under room temperature; Reaction product is placed in to the PBS dialysis of 0.01mol/L 3 days for 4 ℃ of chromatography cabinets, and the centrifugal precipitation of removing, obtain the conjugate of marbofloxacin carrier protein, packing, and-20 ℃ of preservations, standby;
(2) preparation of anti-marbofloxacin specific antibody;
(3) preparation of marbofloxacin specific antibody;
Add freshly prepared 1% sodium citrate 8mL in 200mL 0.01～0.02% chlorauric acid solution of boiling, the colloidal gold solution that the acquisition diameter is 10-20nm, with the K of 0.1mol/L
2cO
3adjust pH to 8.5～9.5, put 2～8 ℃ of preservations, standby; Mark with 1:2000 adds in the aurosol of pH8.5～9.5 than by marbofloxacin monoclonal antibody to be marked or polyclonal antibody, after mark 10min, add 20% PEG10000 to final concentration be 0.05%, 4 ℃, the centrifugal 20min of 1500～3000rpm, remove unconjugated colloid gold particle, 4 ℃, the centrifugal 1h of 15000rpm, abandon supernatant, obtain the colloid gold label thing protein mixture of preliminary purification, carry out separation and purification with propylene glucosan S-400 column chromatography again, obtain the marbofloxacin colloidal gold labeled monoclonal antibody;
(4) preparation of goat-anti or the anti-mouse IgG antibody of rabbit;
Prepare the colloid gold label pad with the marbofloxacin specific antibody; Print the stealthy trace that detects with marbofloxacin coupling carrier protein solution, with goat-anti or the anti-mouse IgG antibody solution of rabbit, print stealthy contrast trace; Then fixedly paste successively sample pad, colloid gold label pad, detection reaction district and adsorptive pads on base plate, be assembled into the test paper core, be loaded in shell by the test paper core and then be assembled into test card.
Test paper jig of the present invention has following advantages:
high specificity, susceptibility is high.This colloidal gold chromatographic Test paper card be take monoclonal antibody or the polyclonal antibody of colloid gold label high-affinity and is prepared from as basis, in the colloid gold label thing, between gold grain and antibody molecule, without covalent bond, form, the two combines by the Van der Waals force between the charges of different polarity, the specificity of colloid gold label antagonist and affinity impact are very little, and have higher mark rate.Therefore, test card has stronger specificity and higher susceptibility, detects minimum the limiting the quantity of of marbofloxacin and can reach 10ppb.
easy, quick, ageing strong.Use this Test paper card, without any other reagent and instrument, but execute-in-place, after test card adds test sample liquid, can be judged testing result in 5 minutes.
5. the present invention adopts the sodium citrate reducing process to prepare colloidal gold solution, and the method course of reaction is gentleer, can under normal temperature and condition of neutral pH, carry out, and is easy to operate and control.Adopt the standby marbofloxacin carrier protein couplet thing of EDC legal system, the method operation steps is fewer, can save time, and to reduce the processing to sample, improves the efficiency of coupling.
The accompanying drawing explanation
Fig. 1, marbofloxacin quick detection test paper card plan structure schematic diagram;
The test card cross-sectional view of Fig. 2, Fig. 1;
The reaction principle figure of Fig. 3, marbofloxacin and carrier protein couplet.
In figure, 1: base plate, 2: sample pad, 3: colloid gold label pad, 4: detection reaction district, 5: the stealthy trace that detects, 6: stealthy contrast trace, 7: adsorptive pads, 8: well, 9: detection window, 10: panel, 11: pickup groove, 12: base, 13: groove.
Embodiment
In the present invention as not, specify, percentage composition wherein is weight content.
Embodiment mono-: marbofloxacin quick detection test paper card, referring to Fig. 1, Fig. 2, comprise shell and the test paper core that is positioned at shell, in figure, base plate 1, sample pad 2, colloid gold label pad 3, detection reaction district 4, stealthy trace 5, stealthy contrast trace 6 and the adsorptive pads 7 of detecting form the test paper core jointly; The test card sheathing material is engineering plastics, and base 12 is provided with the pickup groove 11 of test paper core; Well 8 on panel 10 is corresponding with the sample pad 2 of test paper core; Panel 10 is provided with the groove 13 combined with base, detection window 9 on panel 10 is corresponding with the detection reaction district 4 of test paper core, be the window of observing result of determination, its side is printed on T and C, corresponding with stealth detection trace 5 and stealthy contrast trace 6 in detection reaction district 4 respectively.The base of test card and panel connect together by chimeric, and the test card shell is strip flat plastic housing, long 7cm, wide 0.6cm, thick 0.3cm, the thick 0.1cm of shell wall.
Colloid gold label pad 3 adopts the anti-marbofloxacin polyclonal antibody that colloid gold label is arranged; Detection reaction district 4 adopts nitrocellulose filter; The stealthy trace 5 use marbofloxacins-carrier protein couplet thing that detects is printed in detection reaction district 4, and carrier protein is bovine serum albumin(BSA) BSA; The anti-mouse IgG antibody solution of stealthy contrast trace 6 use rabbit is printed in detection reaction district 4, two line parallel permutation and combination be "
︱ ︱"; Adsorptive pads 7 use absorbent filters are made.Sample pad 2, colloid gold label pad 3, detection reaction district 4, adsorptive pads 7 are pasted and fixed on base plate 1 from right to left successively, and the intersection fiber overlaps mutually each other.
Make marbofloxacin Test paper card, at first will prepare coupling marbofloxacin carrier protein and marbofloxacin colloid gold label thing, thus stealthy trace and the colloid gold label thing of detecting of preparation; Secondly need prepare goat-anti or the anti-mouse IgG of rabbit (or goat anti-rabbit igg) antibody, for the preparation of stealth contrast trace.
(1) marbofloxacin and carrier protein couplet
Adopt the synthetic marbofloxacin comlete antigen of EDC method and bovine serum albumin, reaction principle is referring to Fig. 3.
Take the N that 2 mg marbofloxacins are dissolved in 500 μ L, in dinethylformamide (DMF), add 2 mg NHS(N-N-Hydroxysuccinimide) and 2 mg EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), room temperature lucifuge reaction 24h, obtain the marbofloxacin liquid activated; Take bovine serum albumin 2 mg, fully be dissolved in the PBS solution of mol/L of 2 ml 0.13, the marbofloxacin liquid of activation is dropwise added in BSA solution, the sealing lucifuge stirs 4h under room temperature; Reaction product is placed in to the PBS dialysis of 0.01mol/L 3 days for 4 ℃ of chromatography cabinets, and the centrifugal precipitation of removing, obtain the conjugate of marbofloxacin carrier protein, packing, and-20 ℃ of preservations, standby.
(2) preparation of anti-marbofloxacin polyclonal antibody
By marbofloxacin carrier protein couplet thing immunity New Zealand white rabbit, immunizing dose is 200 μ g～500 μ g/ time, the injection of subcutaneous minute 4～6, back.Head exempts from, and with aseptic PBS, dissolves marbofloxacin carrier protein couplet thing, and with equivalent, FCA mixes, fully emulsified; Booster immunization, dissolve marbofloxacin carrier protein couplet thing with aseptic PBS, and with equivalent, FIA mixes, fully emulsified, after exempting from, head carries out continuous immunity 4～5 times in 2～3 weeks, every minor tick 2～3 weeks, after last immunity 10～15 days, measure it with the ELISA method and tire and reach 10
5when above, blood sampling separated and collected hyper-immune serum.Extract IgG antibody with the saturated ammonium sulfate salting out method, get 1 portion of hyper-immune serum and add 2 parts of PBS(pH7.2) mix, adding equal-volume saturated ammonium sulfate solution mixes, put 4 ℃ of refrigerator 12h, 4 ℃, the centrifugal 15min of 2500rpm, abandon supernatant, again with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration 33%, put 4 ℃ of refrigerator 2h, 4 ℃, the centrifugal 15min of 2500rpm, abandon supernatant, with appropriate PBS(pH7.2) dissolution precipitation, putting in 4 ℃ of refrigerators and use PBS(pH7.2) 48～72h dialyses, liquid is changed for several times in centre, 4 ℃, the centrifugal 15min of 12000rpm, collect supernatant, obtain the anti-marbofloxacin polyclonal antibody of purifying,-20 ℃ frozen, for the preparation of the colloid gold label pad.
(3) preparation of the glass fibre cotton of marbofloxacin colloid gold label thing and colloid gold label
Adopt the sodium citrate reducing process to prepare colloidal gold solution, in 200mL 0.01～0.02% chlorauric acid solution of boiling, add freshly prepared 1% sodium citrate 8mL, obtain the colloidal gold solution of diameter 15nm, use 0.1mol/L K
2cO
3adjust pH to 8.5～9.5, put 2～8 ℃ and save backup.Mark with 1:2000 adds in the aurosol of pH8.5～9.5 than by marbofloxacin monoclonal antibody to be marked or polyclonal antibody, after mark 10min, add 20% PEG10000 to final concentration be 0.05%, 4 ℃, the centrifugal 20min of 1500～3000rpm, remove unconjugated colloid gold particle, 4 ℃, the centrifugal 1h of 15000rpm, abandon supernatant, obtain the colloid gold label thing protein mixture of preliminary purification, carry out separation and purification with propylene glucosan S-400 column chromatography again, obtain the marbofloxacin colloidal gold labeled monoclonal antibody.
The colloidal gold labeled monoclonal antibody of 1:100～500 dilutions is adsorbed in the processed glass cellucotton, and 4 ℃ of low-temperature vacuum dryings, prepare marbofloxacin colloid gold label thing glass fibre cotton.
(4) preparation of goat-anti or the anti-mouse IgG antibody of rabbit
Extract the negative mice serum IgG of marbofloxacin with saturated ammonium sulfate, get 1 part of Mouse Blood and reset and add 2 parts of PBS(pH7.2) mix, adding equal-volume saturated ammonium sulfate solution mixes, put 4 ℃ of refrigerator 12h, 4 ℃, the centrifugal 15min of 2500rpm, abandon supernatant, again with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration 33%, put 4 ℃ of refrigerator 2h, 4 ℃, the centrifugal 15min of 2500rpm, abandon supernatant, with appropriate PBS(pH7.2) dissolution precipitation, put in 4 ℃ of refrigerators and use PBS(pH7.2) dialysis 48h, liquid is changed 3 times in centre, 4 ℃, the centrifugal 15min of 12000rpm, collect supernatant, measure its protein concentration with ultraviolet spectrophotometer, with the mice serum IgG of 50 μ g～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, measure its serum titer with ELISA and reach 1:2000 when above, heart or arterial blood drawing, the separated and collected hyper-immune serum, (method is identical with extraction mice serum IgG to extract goat-anti or the anti-mouse IgG of rabbit (or goat anti-rabbit igg) antibody with saturated ammonium sulfate, no longer repeat), preparation for the stealthy contrast of test card trace.
(5) detection reaction principle
After the well of test card adds testing sample solution, the colloid gold label thing that solution to be measured drives in marbofloxacin to be measured and colloid gold label thing by syphonic effect spreads to the detection reaction district together, and finally infiltrates adsorptive pads.In diffusion process, marbofloxacin to be measured can be combined with the colloid gold label phase, and then the antigen-combining site of marbofloxacin on sealing colloid gold label thing, stop the stealth detection trace of colloid gold label thing coupling marbofloxacin carrier protein on cellulose membrane to be combined, do not show the stealthy trace that detects, the anti-mouse IgG of sheep or rabbit (or goat anti-rabbit igg) antibody can be combined with the colloid gold label thing, the red stealthy contrast trace of formation "
︱", show a red line "
︱" positive expression.Otherwise can not stop the stealth of colloid gold label thing coupling marbofloxacin carrier protein on cellulose membrane to detect trace without marbofloxacin in sample solution, be combined, the red stealthy detection trace of demonstration "
︱", same goat-anti or the anti-mouse IgG of rabbit (or goat anti-rabbit igg) antibody also are combined with the colloid gold label thing, the red stealthy contrast trace of demonstration "
︱", form two red line "
︱ ︱" negative expression.If do not have red line to show on cellulose membrane, show that test card lost efficacy.
(6) sample preparation and detection method
Detecting sample is the meat sample: get meat 2g, add the extract (KH of extract employing 6.8g of 10mL
2pO
4be dissolved in the distilled water of 500 mL, its pH be adjusted to 7.0), homogeneous 1 minute, then with the rotating speed of 4000 rev/mins centrifugal 10 minutes; Collect supernatant, extracting is once again for sediment; The supernatant of twice is mixed, with the rotating speed of 10000 rev/mins centrifugal 10 minutes, get supernatant for detection of.
During detection, test card is kept flat, from well, drip analyte sample fluid, in 5 minutes, from detection window, judge testing result.
Result is judged: as the position display of the corresponding T in the detection reaction district have a red line "
︱" time, mean that testing result is positive, illustrate and contain marbofloxacin in testing sample; As the position display of corresponding T, C in the detection reaction district have two red line "
︱ ︱" time, mean that testing result is negative, illustrate that testing sample is not containing marbofloxacin; As do not had red line to show in the detection reaction district, show that test card lost efficacy.
Embodiment bis-: Test paper card structure and embodiment mono-are basic identical, and difference is: substitute anti-marbofloxacin polyclonal antibody with anti-marbofloxacin monoclonal antibody, prepare marbofloxacin colloid gold label thing and colloid gold label pad; The carrier protein of coupling marbofloxacin is chicken ovalbumin OVA, substitutes the anti-mouse IgG antibody solution of rabbit with goat anti-rabbit igg and prepare the contrast trace in the detection reaction district.Stealthy detect trace and stealthy contrast trace be arranged as "
+" or " ".
Other comprises that detection sample, method of operating and judgement as a result etc., all with embodiment mono-, no longer repeat.
Embodiment tri-: Test paper card structure and embodiment mono-are basic identical, and difference is:
Base plate is made with the hard plastic bar; Sample pad is made with nylon membrane; The detection reaction district adopts pure nitrocellulose filter; The marbofloxacin carrier protein is chicken ovalbumin; Stealthy contrast trace is printed with goat anti-mouse igg antibody solution.Other comprises that detection sample, method of operating and judgement as a result etc., all with embodiment mono-, no longer repeat.
Embodiment tetra-: Test paper card structure and embodiment mono-are basic identical, and difference is:
Base plate is made with the cardboard bar do not absorbed water; Sample pad is made with PVDF membrane; The detection reaction district adopts pure nitrocellulose filter; The marbofloxacin carrier protein is human serum albumins; Stealthy contrast trace is printed with goat anti-mouse igg antibody solution.Other comprises that detection sample, method of operating and judgement as a result etc., all with embodiment mono-, no longer repeat.
Embodiment five: Test paper card structure and embodiment mono-are basic identical, and difference is:
Base plate is made with the cardboard bar do not absorbed water; Sample pad is made with polyester film; Prepare marbofloxacin colloid gold label thing with anti-marbofloxacin monoclonal antibody; The detection reaction district adopts the carboxylation cellulose membrane; The marbofloxacin carrier protein is human serum albumins; Stealthy contrast trace is printed with goat anti-rabbit igg antibody solution.Other comprises that detection sample, method of operating and judgement as a result etc., all with embodiment mono-, no longer repeat.
Claims (10)
1. a marbofloxacin quick detection test paper card, contain shell and the test paper core that is positioned at shell, the test paper core comprises base plate, fixedly be adhesive with successively sample pad, colloid gold label pad, detection reaction district and adsorptive pads on base plate, it is characterized in that: described colloid gold label pad absorption has the specific antibody of marbofloxacin, described detection reaction district is provided with the stealth detection trace that marbofloxacin coupling carrier protein solution is printed, and the stealth of printing with goat anti-mouse igg, the anti-mouse IgG of rabbit or goat anti-rabbit igg solution contrast trace; Marbofloxacin monoclonal antibody or polyclonal antibody that described marbofloxacin specific antibody is colloid gold label.
2. test card according to claim 1, it is characterized in that: prepared by following methods by described marbofloxacin coupling carrier protein solution:
Take in the DMF solvent that 2 mg marbofloxacins are dissolved in 500 μ L, add 2 mg NHS and 2 mg EDC, room temperature lucifuge reaction 24h, obtain the marbofloxacin liquid of activation; Take carrier protein 2 mg, fully be dissolved in the PBS solution of mol/L of 2 ml 0.13, the marbofloxacin liquid of activation is dropwise added in carrier protein solution, the sealing lucifuge stirs 4h under room temperature; Reaction product is placed in to the PBS dialysis of 0.01mol/L 3 days for 4 ℃ of chromatography cabinets, and the centrifugal precipitation of removing, obtain the conjugate of marbofloxacin carrier protein, packing, and-20 ℃ of preservations, standby.
3. test card according to claim 1, is characterized in that: described marbofloxacin coupling carrier albumen
For thyroprotein, mouse haemocyanin, bovine serum albumin(BSA), human serum albumins, albumin rabbit serum, ovoserum albumin or hemocyanin.
4. according to the described test card of claim 1-3 any one, it is characterized in that: described base plate is made by hard plastic bar or the cardboard bar that do not absorb water; Described for sample pad glass fibre cotton, nylon membrane, PVDF membrane or polyester film make; Described adsorptive pads is made with absorbent filter or filter paper for oil; Described detection reaction for district nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane make; Described colloid gold label for pad glass fibre, polyester film, acetate fiber or nylon membrane make.
5. according to the described test card of claim 1-3 any one, it is characterized in that: described shell consists of base and panel, base and panel connect together by chimeric, base is provided with the groove of placing the test paper core, panel is provided with detection window, well, detection window is corresponding with the detection reaction district of test paper core, and well is corresponding with the sample pad of test paper core.
6. according to the described test card of claim 1-5 any one, it is characterized in that: the described stealthy arrangement mode that detects trace and stealthy contrast trace for "
︱ ︱", "
++" or " ".
7. the preparation method of the described marbofloxacin quick detection test paper of claim 1 card, it is characterized in that: described preparation method comprises:
(1) coupling of marbofloxacin and carrier protein:
Take in the DMF solvent that 2 mg marbofloxacins are dissolved in 500 μ L, add 2 mg NHS and 2 mg EDC, room temperature lucifuge reaction 24h, obtain the marbofloxacin liquid of activation; Take carrier protein 2 mg, fully be dissolved in the PBS solution of mol/L of 2 ml 0.13, the marbofloxacin liquid of activation is dropwise added in carrier protein solution, the sealing lucifuge stirs 4h under room temperature; Reaction product is placed in to the PBS dialysis of 0.01mol/L 3 days for 4 ℃ of chromatography cabinets, and the centrifugal precipitation of removing, obtain the conjugate of marbofloxacin carrier protein, packing, and-20 ℃ of preservations, standby;
(2) preparation of anti-marbofloxacin specific antibody;
(3) preparation of marbofloxacin specific antibody;
Add freshly prepared 1% sodium citrate 8mL in 200mL 0.01～0.02% chlorauric acid solution of boiling, the colloidal gold solution that the acquisition diameter is 10-20nm, with the K of 0.1mol/L
2cO
3adjust pH to 8.5～9.5, put 2～8 ℃ of preservations, standby; Mark with 1:2000 adds in the aurosol of pH8.5～9.5 than by marbofloxacin monoclonal antibody to be marked or polyclonal antibody, after mark 10min, add 20% PEG10000 to final concentration be 0.05%, 4 ℃, the centrifugal 20min of 1500～3000rpm, remove unconjugated colloid gold particle, 4 ℃, the centrifugal 1h of 15000rpm, abandon supernatant, obtain the colloid gold label thing protein mixture of preliminary purification, carry out separation and purification with propylene glucosan S-400 column chromatography again, obtain the marbofloxacin colloidal gold labeled monoclonal antibody;
(4) preparation of goat-anti or the anti-mouse IgG antibody of rabbit;
Prepare the colloid gold label pad with the marbofloxacin specific antibody; Print the stealthy trace that detects with marbofloxacin coupling carrier protein solution, with goat-anti or the anti-mouse IgG antibody solution of rabbit, print stealthy contrast trace; Then fixedly paste successively sample pad, colloid gold label pad, detection reaction district and adsorptive pads on base plate, be assembled into the test paper core, be loaded in shell by the test paper core and then be assembled into test card.
8. preparation method according to claim 7, it is characterized in that: the preparation method of described marbofloxacin specific antibody is as follows:
Add freshly prepared 1% sodium citrate 8mL in 200mL 0.01～0.02% chlorauric acid solution of boiling, the colloidal gold solution that the acquisition diameter is 10-20nm, with the K of 0.1mol/L
2cO
3adjust pH to 8.5～9.5, put 2～8 ℃ of preservations, standby; Mark with 1:2000 adds in the aurosol of pH8.5～9.5 than by marbofloxacin monoclonal antibody to be marked or polyclonal antibody, after mark 10min, add 20% PEG10000 to final concentration be 0.05%, 4 ℃, the centrifugal 20min of 1500～3000rpm, remove unconjugated colloid gold particle, 4 ℃, the centrifugal 1h of 15000rpm, abandon supernatant, obtain the colloid gold label thing protein mixture of preliminary purification, carry out separation and purification with propylene glucosan S-400 column chromatography again, obtain the marbofloxacin colloidal gold labeled monoclonal antibody.
9. preparation method according to claim 7, it is characterized in that: described marbofloxacin coupling carrier albumen is thyroprotein, mouse haemocyanin, bovine serum albumin(BSA), human serum albumins, albumin rabbit serum, ovoserum albumin or hemocyanin.
10. preparation method according to claim 7 is characterized in that: described base plate is made by hard plastic bar or the cardboard bar that do not absorb water; Described for sample pad glass fibre cotton, nylon membrane, PVDF membrane or polyester film make; Described adsorptive pads is made with absorbent filter or filter paper for oil; Described detection reaction for district nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane make, described colloid gold label for pad glass fibre, polyester film, acetate fiber or nylon membrane make;
Described shell consists of base and panel, base and panel connect together by chimeric, and base is provided with the groove of placing the test paper core, and panel is provided with detection window, well, detection window is corresponding with the detection reaction district of test paper core, and well is corresponding with the sample pad of test paper core.
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| CN103163296A (en) * | 2011-12-09 | 2013-06-19 | 大连普瑞康生物技术有限公司 | Immune colloidal gold test strip for detection of ractopamine residue in swine urine |
| CN102618502A (en) * | 2012-01-09 | 2012-08-01 | 浙江大学 | Hybridoma cell strain capable of secreting monoclonal antibodies to quinolones and application of monoclonal antibodies thereof |
| 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 |
| CN103091495A (en) * | 2013-01-16 | 2013-05-08 | 河南知微生物工程有限公司 | Test paper card for quickly detecting residues of fluoroquinolones and preparation method of test paper card |
| CN103175961A (en) * | 2013-03-12 | 2013-06-26 | 中国农业大学 | Enzyme linked immunosorbent assay kit for detecting 21 sulfonamides and 11 quinolones and application of enzyme linked immunosorbent assay kit |
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