CN109813896A - Chloramphenicol medicament residue Rapid detection test strip - Google Patents
Chloramphenicol medicament residue Rapid detection test strip Download PDFInfo
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- CN109813896A CN109813896A CN201711151019.6A CN201711151019A CN109813896A CN 109813896 A CN109813896 A CN 109813896A CN 201711151019 A CN201711151019 A CN 201711151019A CN 109813896 A CN109813896 A CN 109813896A
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Abstract
Test strips of the present invention belong to food safety field of fast detection.Test strips include bottom plate, are attached to sample pad successively closely coupled on bottom plate, conjugate release pad, reaction film and water absorption pad, and the partial region of conjugate release pad is covered under sample absorption pad.The detection line and the nature controlling line for being coated with sheep anti-mouse igg composition that chloramphenicol-carrier protein couplet object is constituted are coated on reaction film；Conjugate release pad is coated with the colloid gold label object of chloramphenicol.The test strips can extend testing result observing time, and sample absorption pad, which can also will test liquid and fully absorb, to be completely attached to and sufficiently react with gold labeling antibody, then is chromatographed to being reacted on reaction film, can effectively reduce error.It can also prevent some interfering components in detection sample from the albumen in gold labeling antibody being made to lose activity, influence the combination of gold labeling antibody and coating antigen.
Description
Technical field
The present invention relates to a kind of detection chloramphenicol medicament residue Rapid detection test strips, belong to food safety and quickly detect neck
Domain.
Technical background
Chloramphenicol is a kind of broad-spectrum antibiotic, is usually used in the treatment of the various infectious diseases of animal, for a variety of pathogens have compared with
Strong inhibiting effect.There are serious side effects for chloramphenicol, can inhibit human bone marrow's hematopoiesis function, cause the aregeneratory of the mankind
Property the diseases such as anaemia, therefore the residual chloromycetin in animal foodstuff constitutes grave danger to the health of the mankind.
The current measuring method in relation to chloramphenicol in animal derived food and its metabolin is mainly Physico-chemical tests method, is such as used
Chemical analysis measures the chloramphenicol residue in shrimp, and detection is limited to 0.0125ug/kg, uses high effective liquid chromatography for measuring
Chloramphenicol residue in the flesh of fish, detection are limited to 0.0125ug/kg.But these method instrument and equipments are expensive, complicated for operation, sensitive
Spend detection that is low, and not being suitable for batch samples, be unable to satisfy domestic food safety detection market there is an urgent need to.
Immuno analytical method simple, quick, sensitivity with the high specificity of its antigen-antibody reaction and measuring method
The advantages that height, expense are low and are suitable for live batch samples screening, illustrates in agricultural and veterinary chemicals retention analysis before being widely applied
Scape.As pesticide residue immunology detection technology is constantly completed and is commercialized, immunology detection technology can become food agriculture beast
Effective quick detection means of medicine residual and the control of food safety quality.Research based on current residue of veterinary drug diagnostic reagent is existing
Shape, being developed in time better than current, rapid sensitive detection reagent is the task of top priority.Although agricultural and veterinary chemicals retention analysis
Although method has very much, but carrying out analysis using immunological technique is specific best, the shortest method of detection time.
The prior art is mainly the detections sides such as gas chromatography-mass spectrography, high performance liquid chromatography for the detection of chloramphenicol
Method, but the defect of these technologies is obvious: equipment is expensive, it is complicated for operation, be difficult to promote.So establishing a kind of simple, effective, suitable
It is extremely necessary for closing the measuring method that base uses.The purpose of the present invention is develop one kind to be more suitable for enterprise's progress on-site test
And fast and simple, low-cost qualitative checking method.
Summary of the invention
The purpose of the present invention: special, sensitive, quick, easy chloramphenicol medicament residue Rapid detection test strip is developed.
Technical solution of the present invention:
A kind of chloramphenicol medicament residue Rapid detection test strip, bottom are supporting layer, and middle layer adsorption layer is fixed on supporting layer,
Outer protective film is fixed on adsorption layer, and adsorption layer is followed successively by fibrous layer from test lead, adsorbs gold labeling antibody fibrous layer, cellulose
The absorbent material layer of film layer and handle end, wherein the carrier protein solution of useful coupling chloramphenicol drug prints in cellulose film layer
The linear detection trace of system, the linear control trace printed with goat-anti or rabbit anti-mouse IgG solution, detects trace and control
Trace combines be arranged as " II " in parallel.Supporting layer is made of the hard plastic slip or cardboard item not absorbing water.Test lead fiber
Layer mineral wool or nylon membrane or polyvinylidene fluoride (PVDF) film or poly- phenol film are made, preferably mineral wool.Absorbent material layer
It is made of blotting paper.Cellulose film layer nitrocellulose filter or pure cellulose film or shuttle cellulose film are made.Gold mark is anti-
The gold labeling antibody mineral wool of body fibrous layer absorption chloramphenicol drug, chloramphenicol drug gold labeling antibody are colloid gold label chloramphenicol
The monoclonal antibody or polyclonal antibody of drug, the carrier protein solution of coupling chloramphenicol drug are chloramphenicol drug and carrier egg
The composite solution of white coupling.It is covered with protective film on test lead adsorbing fiber layer, gold labeling antibody fibrous layer and absorbent material layer,
It is printed with sample mark line on test lead fibrous layer protective film corresponding with gold labeling antibody fibrous layer intersection, the mark line is inclined
To at the about 0.5cm of test lead fibrous layer side.The carrier protein for being coupled chloramphenicol drug is bovine serum albumin(BSA) (BSA), or is
Chicken ovalbumin (OVA), or be ferritin, or be hemocyanin.
Chloramphenicol medicament residue Rapid detection test strip of the invention has the advantage that
(1) high specificity, sensibility are high.The test strips prepared based on the monoclonal antibody of colloid gold label high-affinity and
At, formed between gold particle and antibody molecule without covalent bond in gold labeling antibody, the two pass through the charges of different polarity between Van der Waals force phase
In conjunction with colloidal gold mark influences very little, and mark rate with higher to monoclonal antibody specificity and affinity.Therefore, quickly
Test strip specificity with higher and sensibility.
(2) easy to operate, quick.Using any other reagent is not necessarily to when test strips of the present invention, it is left that 100 μ l need to only be added dropwise
Right measuring samples can determine that testing result in sample pad in 5 minutes.
(3) vivid, intuitive, accurate as the result is shown.This test paper slip is to show rufous " I " and " II " trace as detection
The positive and negative marker, i.e., one brownish red " l " trace of display indicates to contain in being detected sample liquid on cellulose membrane
Chloramphenicol, two brownish red " II " traces indicate in test sample without chloramphenicol, result judgement image, intuitive, accurate, letter
It is single to be illustrated, it is less prone to false negative and false positive erroneous judgement.
(4) at low cost, small investment.Using Rapid detection test strip, be not required to separately match instrument and equipment and other reagents, at any time with
Ground is detected, low-cost, can save a large amount of expensive instruments and cost of equipment.
(5) have a wide range of application, it is easy to promote and utilize.Quick detection test paper of the present invention is easy to operate, is able to satisfy different levels
The needs of personnel, including professional chemical examination, customs quarantine control, health and epidemic prevention, quality-monitoring, livestock products processing, intensive culture are to a
Body cultivation etc., has a vast market foreground and preferable economical, societal benefits.
Detailed description of the invention
Fig. 1 is chloramphenicol medicament residue Rapid detection test strip structural schematic diagram (wherein 1, sample absorption pad；2, conjugate
Release pad；3, reaction film；4, water absorption pad；5, detection line；6. nature controlling line；7, bottom plate).
Fig. 2 is that (wherein 8-1 and 8-2 is to be covered on to chloramphenicol medicament residue Rapid detection test strip overlooking structure diagram
The protective film at test card both ends, 9 be mark line).
Fig. 3 is chloramphenicol drug quick detection test paper card negative (Fig. 3 .a), positive (Fig. 3 .b), invalid (Fig. 3 .c) colour developing
Schematic diagram, wherein T quality control region, C are detection zone.
Specific embodiment
Prepare chloramphenicol medicament residue Rapid detection test strip, need first preparation coupling chloramphenicol drug carrier protein and
Gold labeling antibody, to prepare detection trace and gold labeling antibody fiber;Secondly goat-anti or rabbit anti-mouse IgG antibody need to be prepared, be used for
Preparation control trace.
1. the coupling of chloramphenicol drug and carrier protein
Mixed anhydride method and carbodiimide method is respectively adopted to be coupled to obtain by chloramphenicol drug and ovalbumin and hemocyanin
Coating antigen (for being fixed on reaction film) and immunogene (being used to prepare monoclonal antibody).
(1) preparation of coating antigen:
1. 0. 5mL formamide (DMF) of 5mg chloramphenicol drug is dissolved, 10 DEG C are cooled to, 5 μ 1 of isobutyl chlorocarbonate is added,
4 DEG C are stirred to react 30min, and I liquid of reaction solution can be obtained;
2. weighing ovalbumin (OVA) 30mg, it is allowed to be substantially dissolved in 2mL 50mM sodium carbonate liquor, reaction solution can be obtained
II liquid;Wherein the mol ratio of chloramphenicol drug haptens and the ovalbumin is (15-20): 1.
3. I liquid of reaction is slowly dropped to dropwise in II solution of reaction, 4 DEG C of 4 h of reaction, 4 DEG C overnight.It takes final anti-
It answers object 24 h of dialysis purification in pH7.4,0.02M phosphate buffer, 3000 g or more to be centrifuged 30 min, collects supernatant,
Obtain coating antigen.
(2) preparation of immunogene:
1. by 5mg chloramphenicol drug, 10mg n-hydroxysuccinimide (NHS) and 12.5 mg carbodiimides (EDC)
It is completely dissolved in 1 mL DMF, stirs 24 h at room temperature, I liquid of reaction solution can be obtained;
2. weighing 40 mg of hemocyanin, it is allowed to be substantially dissolved in 3 mL, 40 mM sodium carbonate liquor to get reaction solution II is arrived
Liquid, wherein the mol ratio of chloramphenicol drug haptens and the hemocyanin is (12-15): 1.
3. I liquid of reaction solution is slowly dropped to dropwise in II liquid of reaction solution, and 3 h are stirred at room temperature, then 4 DEG C of mistakes
Night crosses column, is balanced and is eluted with buffer, obtains immunogene after being further purified.
2. the preparation of chloramphenicol resistance anti-drug monoclonal antibody
(1) animal immune: immunogene is injected into Balb/c Mice Body, and immunizing dose is 100 μ g/, generates it anti-
Serum.
(2) cell fusion and cloning: after mice serum measurement result is higher, taking its splenocyte, in 7:1 ratio (quantity
Proportion) it is merged with SP2/0 myeloma cell, cell supernatant is measured using indirect competitive ELISA, screens positive hole.Using having
It limits dilution method and cloning is carried out to positive hole, until obtaining the hybridoma cell strain of secrete monoclonal antibody.
(3) 1X10 cell cryopreservation and recovery: is made with frozen stock solution in aforementioned monoclonal hybridoma strain6A/mL's is thin
Born of the same parents' suspension, saves for a long time in liquid nitrogen.Cryopreservation tube is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and centrifugation removal freezes
After liquid, culture culture in glassware is moved into.
(4) production and purifying of monoclonal antibody: injecting sterilizing 0. 5 mL/ of paraffin oil only for Balb/c mouse peritoneal, and 7
The monoclonal hybridoma strain 5X10 of chloramphenicol drug is injected intraperitoneally after it7A/only, ascites is acquired after 7 days.It is full with octanoic acid-
Ascites purifying is carried out with ammonium sulfate method, obtains monoclonal antibody, is saved in -20 DEG C.
3. the preparation of chloramphenicol drug gold labeling antibody and gold labeling antibody mineral wool
Aurosol is prepared with reduction of sodium citrate method, i.e., is added in 0.01 ~ 0.05% aqueous solution of chloraurate of 50 ~ 100 mL boiling
Enter 0.5 ~ 2% citric acid three sodium solution of 2 ~ 4 mL, obtains the colloidal gold of 15 nm of diameter or so.With the K of 0.1 mol/L2C03
Colloidal gold pH to 8.5 ~ 9.5 is adjusted, is placed in the label of 1:2000 ~ 3000 than adding chloramphenicol anti-drug monoclonal antibody to be marked
Enter in the aurosol of pH8.5 ~ 9.5, after 10 min of label, adds 20%PEG 10000 to final concentration 0.05%, 4 DEG C, 1500 ~ 3000
Rpm is centrifuged 20 min, removes unbonded colloid gold particle, and 4 DEG C, 15000 rpm are centrifuged 1 h, abandon supernatant, obtain preliminary purification
It after gold labeling antibody protein mixture, is chromatographed with propylene glucan S-400 column, isolates and purifies gold mark albumen, obtain chloramphenicol drug
Colloidal gold labeled monoclonal antibody.By 1:(100 ~ 500) diluted glue gold labelled antibody is adsorbed in processed glass cotton, and 4 DEG C of low temperature are true
Sky is dry, prepares chloramphenicol drug gold labeling antibody mineral wool.
4. the preparation of goat-anti or rabbit anti-mouse IgG
Mice serum IgG is extracted with saturated ammonium sulfate, takes 1 part of mice serum that 2 parts of PBS (pH 7.2) is added to mix, is added isometric full
It is mixed with ammonium sulfate, sets 4 DEG C of 2 h of refrigerator, 4 DEG C, 1200 r/min are centrifuged 15 min, abandon supernatant;With appropriate PBS (pH7.2)
Dissolution precipitating, adds saturated ammonium sulfate to final concentration 33%, sets 4 DEG C of refrigerator 2h, and 4 DEG C, 1200 r/min are centrifuged 15 min, in abandoning
Clearly, H7.2 is printed with a small amount of PBS) dissolution precipitating, it sets and is dialyzed overnight with PBS (pH7.2) in 4 DEG C of refrigerators, change liquid 2 ~ 3 times, 4 DEG C,
12000 r/min are centrifuged 15 min, collect supernatant, its protein concentration are measured with ultraviolet specrophotometer, with 50 ~ 100 μ g/kg
Weight (mice serum IgG) is subcutaneously with intramuscular injection immune health sheep or rabbit 3 ~ 4 times, and after final immunization 10 days, vein is adopted
Blood measures its serum antibody titer in 1:2000 or more, Culling heart blood or arteria carotis bloodletting with ELISA, collects its height and exempt from blood
Clearly, goat-anti or rabbit anti-mouse IgG (method is identical as mice serum IgG is extracted, and does not repeat) are extracted with saturated ammonium sulfate, is used for chlorine
The preparation of mycin drug Rapid detection test strip control display trace.
5. chloramphenicol drug Rapid detection test strip reaction principle
(1) after measuring samples solution instills test strips well, sample solution is made because of the capillary of nitrocellulose membrane carrier
It is spread with to the other end.In the process of moving, it may occur that corresponding antigen-antibody reaction, and the color for passing through immune colloid gold
It shows.If sample solution contains chloramphenicol medicament residue, chloramphenicol drug elder generation and the antibody response on colloid gold particle,
Therefore when colloid gold particle diffuses to detection line with sample solution, the active site of antibody is because molten by sample on colloid gold particle
Chloramphenicol drug in liquid occupies and can not cannot show detection trace in conjunction with chloramphenicol drug antigenic in detection line, and sheep
Dynamics then can be in conjunction with gold labeling antibody, quality control region colour developing, forms red control trace " I ", is in a trace
It is positive；It cannot then prevent chloramphenicol drug antigenic on gold labeling antibody and cellulose membrane even in opposite sample solution without chloramphenicol drug
Join carrier protein detection trace to combine, detection zone is displayed in red trace " I ", same sheep anti-mouse igg antibody also with gold labeling antibody knot
It closes, quality control region also shows red control trace " I ", two red line " II " negative markers is formed, if not red on cellulose membrane
Color marker shows that then test card is invalid.
(2) structure of chloramphenicol medicament residue Rapid detection test strip, referring to Fig. 1, Fig. 2.1 absorbs in figure for sample
Pad, is made of mineral wool, and 2 discharge bed course for conjugate, according to preparation method described in above-mentioned specific embodiment 4, preparation
It is adsorbed with the gold labeling antibody fibrous layer of chloramphenicol anti-drug monoclonal antibody, 3 be reaction film layer, and using nitrocellulose filter, 4 be suction
Water material layer, is made of absorbent filter, and by number 1,2,3,4 each layers are successively pasted and fixed on plastic strip 7 from left to right, respectively
Layer intersection fiber crosses one another infiltration.5 be to stamp detection trace with ovalbumin (OVA) solution of coupling chloramphenicol drug
" I ", 6 is stamp control trace " I " with goat anti-mouse igg solution, and trace band " II " is combined in the formation arranged in parallel of two traces.8-1
Test sample end white protective film above bed course 1 and gold labeling antibody fibrous layer 2 is absorbed to be covered on sample, in 1 and 2 intersections
Be partial to be printed at 0.5 cm of sample absorption pad side on the corresponding position protective film 8-1 mark line 9,9 right end be printed on arrow and
Max printed words, filter paper layer 4 are to be covered with other colors (such as yellow) protective film 8-2 on water absorption layer (handle end).
7. the preparation of test sample liquid:
(1) take 2g animal tissue homogenate in 50 mL polystyrene centrifuge tubes；
(2) -1% solution of trichloroacetic acid of 8 mL methanol is added, mixes well, room temperature is centrifuged 5 with the speed of 3000 g or more
min；
(3) it takes 1 mL supernatant liquid to be added in 9mL phosphate buffer, the sodium hydroxide solution tune PH of 1M is used after mixing well
Value mixes, room temperature is with 5 min of speed centrifugation centrifugation of 3000 g or more to neutrality；
(4) take upper liquid to be measured.
8. detection operating method: taking out test strips from packaging bag, draw solution to be checked with dropper, dripped in well
Enter 3 drops (about 100 μ L), start timing after sample-adding, as a result should be read at 3-5 minutes, other times interpretation is invalid.
9. testing result judges: if there is reddish brown color marker " I " display on cellulose membrane, inspection side result is in sun
Property, the drug containing chloramphenicol (as shown in Figure 3b) in being detected sample liquid is indicated, if chloramphenicol drug Rapid detection test strip
On cellulose membrane on occur two reddish brown color markers " II ", testing result is negative, indicate measuring samples in be free of chloramphenicol
Pharmaceutical compositions (as shown in Figure 3a) such as do not occur C line, and possible operation is improper or agent plate has failed (as shown in Fig. 3 .c).
Claims (8)
1. a kind of chloramphenicol medicament residue Rapid detection test strip, is supporting layer containing bottom, middle layer adsorption layer is fixed on branch
It supports on layer, outer protective layer is fixed on adsorption layer, it is characterized in that: adsorption layer is followed successively by fibrous layer, ADSORPTION OF GOLD mark from test lead
The absorbent material layer of antibody fibrous layer, cellulose film layer and handle end, wherein the useful coupling chloramphenicol medicine in cellulose film layer
It is linear right that the linear detection trace of the carrier protein solution printing of object, useful goat-anti or rabbit anti-mouse IgG solution are printed
According to trace, detects trace combination parallel with control trace and be arranged as " II ".
2. test strips according to claim 1, it is characterized in that: supporting layer is with the hard plastic item or cardboard item not absorbed water
It is made.
3. test strips according to claim 1, it is characterized in that: test lead fibrous layer mineral wool or nylon membrane or poly- inclined
Difluoride membranes or polyester film are made.
4. test strips according to claim 1, it is characterized in that: absorbent material layer is made of blotting paper.
5. test strips according to claim 1, it is characterized in that: cellulose film layer nitrocellulose filter or pure cellulose
Film or carboxylated cellulose film are made.
6. test strips according to claim 1, it is characterized in that: the gold mark of gold labeling antibody fibrous layer absorption chloramphenicol drug
Antibody mineral wool, chloramphenicol drug gold labeling antibody are the chloramphenicol anti-drug monoclonal antibody or polyclonal antibody of colloid gold label,
The carrier protein solution for being coupled chloramphenicol drug is the composite solution of chloramphenicol drug and carrier protein couplet.
7. test strips according to claim 1, it is characterized in that: test lead adsorbing fiber layer, gold labeling antibody fibrous layer and
It is covered with protective film on absorbent material layer, is printed on test lead fibrous layer protective film corresponding with gold labeling antibody fibrous layer intersection
It is formed with sample mark line, which is biased at about 0.5 cm of test lead fibrous layer side.
8. test strips according to claim 1, it is characterized in that: the carrier protein of coupling chloramphenicol drug is bovine serum albumin
White (BSA), or be chicken ovalbumin (OVA), or be ferritin, or be hemocyanin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711151019.6A CN109813896A (en) | 2017-11-18 | 2017-11-18 | Chloramphenicol medicament residue Rapid detection test strip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711151019.6A CN109813896A (en) | 2017-11-18 | 2017-11-18 | Chloramphenicol medicament residue Rapid detection test strip |
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| CN109813896A true CN109813896A (en) | 2019-05-28 |
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| CN201711151019.6A Withdrawn CN109813896A (en) | 2017-11-18 | 2017-11-18 | Chloramphenicol medicament residue Rapid detection test strip |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1403815A (en) * | 2002-01-10 | 2003-03-19 | 河南省农业科学院生物技术研究所 | Fast detection test paper strip for medicine residue in animal body and product |
| US20050214951A1 (en) * | 2002-01-23 | 2005-09-29 | Boditech Inc | Lateral flow quantitative assay method and strip and laser-induced fluoerescence detection device therefor |
| US20080176342A1 (en) * | 2005-04-14 | 2008-07-24 | Benoit Granier | In Vitro Method For the Simultaneous Detection and Identification of Antibiotics of Different Classes and Corresponding Diagnostic Kit |
| CN103424550A (en) * | 2012-05-18 | 2013-12-04 | 北京勤邦生物技术有限公司 | Detection kit for chloramphenicol and detection method thereof |
| CN104237521A (en) * | 2014-10-16 | 2014-12-24 | 江南大学 | Three-in-one colloidal gold chromatographic test strip for detecting thiamphenicol, chloramphenicol and florfenicol and preparation method thereof |
| CN206177961U (en) * | 2016-11-14 | 2017-05-17 | 苏州快捷康生物技术有限公司 | Chloramphenicol colloidal gold method immunity chromatography test paper card |
-
2017
- 2017-11-18 CN CN201711151019.6A patent/CN109813896A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1403815A (en) * | 2002-01-10 | 2003-03-19 | 河南省农业科学院生物技术研究所 | Fast detection test paper strip for medicine residue in animal body and product |
| US20050214951A1 (en) * | 2002-01-23 | 2005-09-29 | Boditech Inc | Lateral flow quantitative assay method and strip and laser-induced fluoerescence detection device therefor |
| US20080176342A1 (en) * | 2005-04-14 | 2008-07-24 | Benoit Granier | In Vitro Method For the Simultaneous Detection and Identification of Antibiotics of Different Classes and Corresponding Diagnostic Kit |
| CN103424550A (en) * | 2012-05-18 | 2013-12-04 | 北京勤邦生物技术有限公司 | Detection kit for chloramphenicol and detection method thereof |
| CN104237521A (en) * | 2014-10-16 | 2014-12-24 | 江南大学 | Three-in-one colloidal gold chromatographic test strip for detecting thiamphenicol, chloramphenicol and florfenicol and preparation method thereof |
| CN206177961U (en) * | 2016-11-14 | 2017-05-17 | 苏州快捷康生物技术有限公司 | Chloramphenicol colloidal gold method immunity chromatography test paper card |
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Application publication date: 20190528 |