CN106053798A - Doxycycline field test paper and methods of preparing and using same - Google Patents
Doxycycline field test paper and methods of preparing and using same Download PDFInfo
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- CN106053798A CN106053798A CN201610596849.9A CN201610596849A CN106053798A CN 106053798 A CN106053798 A CN 106053798A CN 201610596849 A CN201610596849 A CN 201610596849A CN 106053798 A CN106053798 A CN 106053798A
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- doxycycline
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention relates to the improvement for antibiotic detection techniques, in particular to field test paper and methods of preparing and using the same, and belongs to the field of immunology. The field test paper includes: anti-doxycycline and colloidal gold labeled monoclonal antibody C secreted by hybridoma DC-C preserved under CCTCC-C201680, the monoclonal antibody is provided with a support board with non-setting adhesive, two ends of the support board are provided with a sampling end water-absorbing layer and a handheld end water-absorbing layer respectively, the middle of the support board is provided with a detection layer made with nictrocellulose membrane, a glass fiber layer-block supported with the colloidal gold labeled monoclonal antibody C is disposed at the junction of the detection layer and the sampling end water-absorbing layer, one end of the glass fiber layer-block is arranged below the sampling end water-absorbing layer, the other end is arranged on the detection layer, the detection layer continuous with the glass fiber layer-block is provided with a detection line and a quality control line, the detection line and the quality control line are coated with DC-OVA and goat anti-mouse IgG respectively. The field test paper enables quick, simple and accurate field detection for doxycycline and can be used to detect residue of doxycycline on the field.
Description
Technical field
The present invention relates to the improvement of antibiotic detection technique, be specifically related to Site Detection reagent paper and preparation, using method,
Belong to field of immunology.
Background technology
Mechanism is identical with tetracycline, mainly disturbs the protein synthesis of sensitive organism.It can specifically with antibacterial core
Sugar body 30S subunit combines at location A, thus suppresses aminoacyl-tRNA connection on this position, stops the prolongation of peptide chain；By force
Power mycin can also change the permeability of bacterial cell membrane, makes intracellular nucleotide and other important component spill, thus presses down
The duplication of DNA of bacteria processed.Doxycycline is to staphylococcus aureus, streptococcus pneumoniae, micrococcus scarlatinae, gonococcus, meninges
There is stronger antibacterial activity scorching coccus, escherichia coli, aerobacteria, Shigella, yersinia, mononuclear cell Listerella etc.,
Rickettsia, mycoplasma, chlamydia, actinomycetes etc. also there is certain effect.Antimicrobial spectrum and tetracycline, oxytetracycline are essentially identical.
The inside and outside antimicrbial power of body is all strong compared with tetracycline.
Doxycycline is distributed widely in each tissue and body fluid after being absorbed, distribution volume is about 0.7L/kg.Because of fat-soluble relatively
Height, doxycycline is relatively strong, in Thoracic duct lymph, seroperitoneum, intestinal tissue, eye and prostata tissue to the penetration power of tissue
Drug level the highest, about the 60%～75% of blood drug level, the drug level in bile up to blood drug level 10～
20 times；Also higher drug level can be reached in milk；Doxycycline can also be distributed in liver, spleen, bone marrow, skeleton,
In dentin and enamel.Doxycycline protein binding rate in vivo is 80%～95%, and the removing half-life is 12～22h, kidney
Hypothyroid's Increased Plasma Half-life is inconspicuous.Drug main to inactivate at liver intracellular metabolite, by glomerular filtration with urine drains,
When renal function injury, doxycycline increases from gastrointestinal excretion, becomes main metabolic pathway.Doxycycline can not be through
Dialysis is removed.
Doxycycline uses as the most universal a kind of anti-infectives in aquaculture industry at present, has well
Therapeutical effect, but the situation of doxycycline excess often occurs in actual production, cause aquatic products and breeding water body are deposited
In residual.According to " animal food herbal medicine MRL " clear stipulaties of the Ministry of Agriculture's in December, 2002 issue, animal
In property food muscle, residual quantity is 100 μ g/kg, and in sebum, maximum residue limit is 300 μ g/kg, and in liver, maximum residue limit is
300 μ g/kg, in kidney, maximum residue limit is 600 μ g/kg.
Summary of the invention
The present invention is directed to the present situation of doxycycline residual, and its harm to human body, design invention offer one can
On-the-spot Test paper quick, easy, accurately detection doxycycline and preparation, using method.
Technical scheme realizes as follows
Doxycycline Rapid detection test strip, it is characterized in that and includes: preserving number is CCTCC C201680 hybridization
Anti-doxycycline secreted by oncocyte DC-C, monoclonal antibody C (being called for short: gold mark monoclonal antibody C) of colloid gold label, coating antigen is strong
Power mycin-chicken egg white (DC-OVA) and sheep anti-mouse igg, with the carrier board 3 of adhesive sticker, at the both ends of carrier board 3, point
It is not provided with sample-adding end water accepting layer 1 and handheld terminal water accepting layer 7, is provided with, at this carrier board 3 middle part, the inspection being made up of nitrocellulose membrane
Survey layer 6, be provided with the glass layer block 2 being loaded with gold mark monoclonal antibody C, glass fibers at this detection layers 6 and sample-adding end water accepting layer 1 intersection
Dimension layer block 2 one end be arranged on sample-adding end water accepting layer 1 under, the other end is arranged on detection layers 6, with glass layer block
The detection layers 6 of 2 continuities is provided with detection line 4 and nature controlling line 5, is coated with DC-OVA and goat-anti respectively at detection line 4 and nature controlling line 5
Mus IgG；
Described gold mark monoclonal antibody C is to be passed through penta 2 by small haptens doxycycline (DC) with bovine serum albumin (BSA)
Doxycycline-bovine serum albumin (DC-BSA) complete antigen of aldehyde (GA) method coupling synthesis, prepares as immunogen
Anti-doxycycline monoclonal antibody, cause after colloid gold label；
The preparation process of monoclonal antibody C of described colloid gold label is as follows:
1, preparing colloidal gold solution with the preparation technology of known gold colloidal, the granular size of prepared gold colloidal is 18-
20nm；
2, by monoclonal antibody C (3g/L antibody protein), above-mentioned 100ml colloidal gold solution is joined by 200 μ l-3ml
In, mix even slowly；
3, in mixed solution, add PEG20000 so that it is final concentration of 1-5%, stand overnight；
4, centrifugal 8000-10000 rev/min, 1-1.5 hour, supernatant is abandoned；
5, centrifugation is taken, with containing 1-5%PEG20000,0.05-0.25% tween 20,0.02% sodium azide
0.01mol/L phosphate buffer hangs, and makes gold mark monoclonal antibody C；
The pH value of described phosphate buffer is 7.4, wherein contains KCI 0.2g, NaCI 8.0g, KH2PO4 0.2g、
Na2HPO412H2O 2.9g, distilled water 1000ml；
In described step 1, the preparation method of colloidal gold solution is to be mixed with trisodium citrate by gold chloride by certain proportioning
And heating is prepared as colloidal gold solution；
In described step 2, monoclonal antibody C and colloidal gold solution mix even 30-60min slowly.
The using method of doxycycline Rapid detection test strip, it is characterized in that and comprises the following steps:
Being inserted by the sample-adding end water accepting layer 1 of this reagent paper or the detected sample of dropping thereon, 5-10min is being positioned at detection layers
Not developing the color at the detection line 4 of 6 lower ends, the nature controlling line 5 of detection layers upper end shows redness, and representing residual in sample has doxycycline；Inspection
Showing redness at survey line 4, nature controlling line 5 develops the color, and does not has doxycycline residual or doxycycline concentration is less than in expression sample
0.2ug/ml。
It is an advantage of the current invention that: the present invention, with the actual features of prior art, have developed a kind of doxycycline residual fast
Speed Site Detection reagent paper and preparation, using method, use animal blood or internal organs such as liver to add appropriate normal saline and grind
Broken being prepared as detects sample liquid, detects it, and principle is to use competitive ELISA principle, i.e. by gold mark monoclonal antibody C with detecting sample
Antigen-reactive in product liquid, formed gold mark monoclonal antibody C-doxycycline complex, now, gold mark monoclonal antibody C with doxycycline
In conjunction with, cannot be combined by the DC-OVA at detection line 4, therefore, gold mark monoclonal antibody C and DC-OVA competition binding doxycycline,
Gold mark monoclonal antibody C-doxycycline is by chromatography effect at nature controlling line 5, and sheep anti-mouse igg can be combined with gold mark monoclonal antibody C, gold colloidal
Granule is fixed at this and accumulates the macroscopic redness of appearance；If without doxycycline in liquid to be measured, gold mark monoclonal antibody C can be with detection
DC-OVA at line 4 combines there is redness, and it is effective that nature controlling line colour developing represents this test strips.
The invention have the characteristics that 1, the doxycycline Site Detection reagent paper of the present invention, from actual cultivation needs, general
The cultivation site that the detection of drug residue is moved to, without specialized facilities and operating technology during use, is suitable for common raiser cultivation
Site Detection, application is simple.2, there is the detection feature such as quick, easy, accurate, sensitive, and there is higher stability, at 5-
Just can show testing result within 10min, once detection exceeds standard and drug metabolism can be made straight how foster for cultivated animals a period of time
To residual less than national standard, can list safely, be a kind of method solving the residual problem of medicine of most convenient, decrease because of medicine
Remaining exceeds standard because medicine is residual to the injury of human body and raiser is caused huge loss by the return of goods.Improve because of medicine residual censorship fiber crops
Tired, the cycle is long, and high reason of charging causes more than 99% raiser to be the situation that not censorship medicine remains before cultivated animals lists.
Accompanying drawing explanation
Fig. 1: the structural representation of doxycycline Rapid detection test strip of the present invention；
Fig. 2: the testing result schematic diagram of doxycycline Rapid detection test strip of the present invention.
Hybridoma cell strain DC-C, its deposit number is: CCTCC NO:C201680；Depositary institution's full name and abbreviation: in
State's Type Tissue Collection (CCTCC)；Depositary institution address: in the preservation of Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University
The heart；Preservation date: on May 20th, 2016.
Detailed description of the invention
Embodiment 1
The preparation of doxycycline complete antigen: weigh 100mg BSA and join in 10mL PBS (pH 7.4), weigh 30mg
DC is dissolved in 10ml PBS, is added dropwise over BSA solution under DC solution is slowly stirred, and is slowly added dropwise the GA (concentration of 200 μ L subsequently
25%), to be stirred continuously while addition, react 24 hours under the conditions of lucifuge and 4 DEG C, be centrifuged 5min with 4000rpm rotating speed,
Taking supernatant and load in the bag filter that advanced processing is good, PBS is that dialysis solution dialysis 3d, every 8h change a dialysis solution, has dialysed
Quan Hou, coupled product subpackage ,-20 DEG C of preservations.Identical method prepares coating antigen DC-OVA.Composition principle is as follows:
Embodiment 2
The preparation of the monoclonal antibody of anti-doxycycline: the hybridization of the monoclonal antibody secreting anti-doxycycline of the present invention
Oncocyte, its preparation is as follows with the method selected:
(1) doxycycline complete antigen is synthesized, including immunogen DC-BSA, coating antigen DC-OVA with glutaraldehyde method；
(2) with immunogen DC-BSA immunity Balb/c white mice；
(3) by the fusion of the splenocyte of immune mouse Yu myeloma cell, 166 strain of hybridoma are cultivated to obtain；
(4) positive hybridoma cell strain is screened with coating antigen DC-OVA wrapper sheet indirect elisa method；
(5) limiting dilution assay the strongest hybridoma positive to wherein 1 strain is used to be cloned；
(6) monoclonal antibody 1B7-1F9 bis-time cloning thin of the highest anti-doxycycline of titer is filtered out by indirect elisa method
Antibody prepared by born of the same parents' strain, preparation gold mark monoclonal antibody.It is shown in Table 1
Table 1: monoclonal antibody the selection result
Embodiment 3
The specificity identification of the doxycycline monoclonal antibody C of the present invention: in order to determine whether monoclonal antibody can specificity knot
Conjunction doxycycline, it is necessary to gained antibody is done specific detection and cross-over experiment, determines whether monoclonal antibody exists with other antibiotic
Crossover phenomenon.It is respectively configured doxycycline, oxytetracycline, sulfamethoxazole, norfloxacin hydrochloride and the florfenicol of 0.1 μ g/ml, uses
Various medicines are carried out cross-over experiment by the detection method of competitive ELISA.With DC-OVA as coating antigen, monoclonal antibody after purification is
One antibody, adds above-mentioned several drugs after addition one is anti-immediately, and every hole adds 100 μ l, using PBS as comparison, adds for the two anti-and ends
After thing buffer, measuring the OD value in each hole. result display doxycycline monoclonal antibody C does not the most exist with these four medicine and intersects now
As, doxycycline compares with positive control, there are differences, and illustrates that monoclonal antibody prepared by this experiment is tied with doxycycline generation specificity
Close, can not specific bond with other antibiotic.Experimental result sees table 2.
Table 2: the experiment of anti-doxycycline monoclonal antibody specificity (N=6)
Note: * doxycycline group is compared with the positive, and its P value is 0.0232 ＜ 0.05, illustrates to there is significant difference.
Embodiment 4
Doxycycline Rapid detection test strip, sees accompanying drawing 1, comprising: preserving number is CCTCC C201680 hybridoma
Anti-doxycycline secreted by cell DC-C, monoclonal antibody C (being called for short: gold mark monoclonal antibody C) of colloid gold label, coating antigen (DC-
OVA) and goat anti-mouse igg (be called for short: sheep anti-mouse igg), with the carrier board 3 of adhesive sticker, at the both ends of carrier board 3, respectively
It is provided with sample-adding end water accepting layer 1 and handheld terminal water accepting layer 7, is provided with, at this carrier board 3 middle part, the detection being made up of nitrocellulose membrane
Layer 6, is provided with the glass layer block 2 being loaded with gold mark monoclonal antibody C, glass fibre at this detection layers 6 and sample-adding end water accepting layer 1 intersection
Layer block 2 one end be arranged on sample-adding end water accepting layer 1 under, the other end is arranged on detection layers 6, with glass layer block 2
The detection layers 6 of continuity is provided with detection line 4 and nature controlling line 5, is coated with DC-OVA (360ug/ respectively at detection line 4 and nature controlling line 5
And sheep anti-mouse igg (1 μ g/ml) ml).
Embodiment 5
The preparation process of monoclonal antibody C of colloid gold label is as follows:
(1) preparation of gold colloidal: prepared by 18-20nm colloid gold particle, by concentration be 0.01% chlorauric acid solution 250ml with
1% sodium citrate 6.65ml mixing, is heated to 100 degrees Celsius, makes colloidal gold solution, the pH value of this solution: use 0.2M carbonic acid
Potassium is adjusted to 8.2, standby；
(2) preparation of gold mark monoclonal antibody C:
When anti-doxycycline monoclonal antibody concentration is 3g/L, the suitableeest monoclonal antibody amount of 100ml colloid gold label is 1.5ml, compares by this
Anti-doxycycline monoclonal antibody is joined in colloidal gold solution by example, stirs 50min slowly, adds 5%PEG20000, and 4 DEG C overnight；Then will
They are at 4 DEG C, and 8000r is centrifuged 60min；Take centrifugation, with containing containing 5%PEG20000,0.25% polysorbas20
0.01mol/L phosphate buffer (KCL 0.2g, NaCL 8.0g, KH2PO4 0.2g, Na2HPO412H2O 2.9g, distilled water
1000ml, pH 7.4) hang, then add sodium azide concentration is adjusted to 0.02%, i.e. make gold mark monoclonal antibody C.
Embodiment 6
The doxycycline Site Detection detection paper layer preparation method of the present invention is: by DC-OVA360ug/ml Membrane jetter
It is sprayed on nitrocellulose membrane, is formed detection line 4；Equally, goat anti-mouse igg is made into 200 μ g/ml, with Membrane jetter by it
It is sprayed on nitrocellulose membrane, forms nature controlling line 5.Two lines are closely loaded at a distance of 5mm, the nearly handheld terminal water accepting layer of nature controlling line 5, detection line 4
End water accepting layer.Dry under room temperature, then close 30min, PBS with pH 7.4, the 0.01mol/LPBS containing 5,%PE,G20,000 37 DEG C
Rinsing is dried.
Embodiment 7
The minimum residual quantity of the doxycycline quick detector bar detection of the present invention was 0.2ug/ml, less than the Ministry of Agriculture 2002
In the sebum that " the animal food herbal medicine MRL " that December is issued specifies, maximum residue limit is 300 μ g/kg, liver
Middle maximum residue limit is 300 μ g/kg, and in kidney, maximum residue limit is 600 μ g/kg；Higher than residual quantity in animal food muscle
It is 100 μ g/kg, reaches the target that pool side quickly detects.It is shown in Table 3
Table 3: the mensuration that doxycycline Site Detection reagent paper is sensitive
Note :+representing that detection line color is red, testing result is positive；-representing that detection line is colourless, testing result is
Negative.
Embodiment 8
The using method of the doxycycline Rapid detection test strip of the present invention, detecting step is as follows: first, from the pond of cultivation
In take live fish one, take liver, by weight/volume (w/v) adds normal saline or the phosphate-buffered of pH 6.0-8.0 than 1:2
Liquid, pulverizes liver, is prepared as detecting sample；Then, sample-adding end 1 water accepting layer of reagent paper is immersed in the detection sample of test tube
(liquid level must not exceed the 2/3 of absorbent paper 1), 5min reads result.
Positive findings: inspected layer 6 bottom detection line 4 does not develops the color, and inspected layer 6 top nature controlling line 5 shows red line, represents sample
Product are residual in having doxycycline；
Negative findings: inspected layer 6 bottom detection line 4 shows that red line, inspected layer 6 top nature controlling line 5 develop the color, and represents sample
Represent and can't detect doxycycline or doxycycline concentration less than 0.2ug/ml.Refer to accompanying drawing 2.
Claims (6)
1. doxycycline field quick detection reagent paper, it is characterised in that including: preserving number is that CCTCC C201680 hybridoma is thin
Anti-doxycycline secreted by born of the same parents DC-C, monoclonal antibody C (being called for short gold mark monoclonal antibody C) of colloid gold label, coating antigen DC-OVA
And sheep anti-mouse igg, with the carrier board (3) of adhesive sticker, at the both ends of carrier board (3), it is respectively equipped with sample-adding end water accepting layer (1)
With handheld terminal water accepting layer (7), it is provided with the detection layers (6) being made up of nitrocellulose membrane, in this detection layers at this carrier board (3) middle part
(6) it is provided with is loaded with the golden glass layer block (2) marking monoclonal antibody C, glass layer block (2) with sample-adding end water accepting layer (1) intersection
One end be arranged under sample-adding end water accepting layer (1), the other end is arranged on detection layers (6), with glass layer block (2)
The detection layers (6) of continuity is provided with detection line (4) and nature controlling line (5), and detection line (4) and nature controlling line (5) place are coated with DC-respectively
OVA and sheep anti-mouse igg.
Doxycycline field quick detection reagent paper the most according to claim 1, it is characterised in that described gold mark monoclonal antibody C be by
Small haptens doxycycline (DC) and bovine serum albumin (BSA) are by glutaraldehyde method coupling complete antigen synthesis DC-BSA
The anti-doxycycline monoclonal antibody prepared as immunogen, prepares after colloid gold label.
Doxycycline field quick detection reagent paper the most according to claim 1, it is characterised in that the system of described gold mark monoclonal antibody C
Preparation Method is as follows:
(1) preparing colloidal gold solution with the preparation technology of known gold colloidal, the granular size of prepared gold colloidal is 18-
20nm；
(2) by monoclonal antibody C (3g/L antibody protein), join in above-mentioned 100ml colloidal gold solution by 200 μ l-3ml,
Slowly mix even；
(3) in mixed solution, add PEG20000 so that it is final concentration of 1-5%, stand overnight；
(4) centrifugal 8000-10000 rev/min, 1-1.5 hour, supernatant is abandoned；
(5) take centrifugation, delay with the 0.01mol/L phosphate containing 0.05-0.25% tween 20,0.02% Hydrazoic acid,sodium salt
Rush liquid to hang, make gold mark monoclonal antibody C.
4. according to the florfenicol field quick detection reagent paper described in claim 3, it is characterised in that colloid described in step (1)
The preparation method of gold solution is that gold chloride and trisodium citrate be mixed and heated by certain proportioning to be prepared as gold colloidal molten
Liquid.
5. according to the doxycycline field quick detection reagent paper described in claim 3, it is characterised in that monoclonal anti in step (2)
Body C and colloidal gold solution mix even 30-60min slowly.
6. the using method of doxycycline Rapid detection test strip, it is characterised in that comprise the following steps:
Being inserted by sample-adding end water accepting layer (1) of this reagent paper or the detected sample of dropping thereon, 5-10min is being positioned at detection layers
(6) do not develop the color in lower end detection line (4), inspected layer (6) top nature controlling line (5) display red line, and expression sample is residual in having strength
Mycin；Inspected layer (6) bottom detection line (4) display red line, developing the color in inspected layer (6) top nature controlling line (5), represents that sample represents
Can't detect doxycycline or doxycycline concentration less than 0.2ug/ml, at nature controlling line, do not show redness, represent this reagent paper without
Effect.
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| CN201610596849.9A CN106053798A (en) | 2016-07-26 | 2016-07-26 | Doxycycline field test paper and methods of preparing and using same |
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| CN201610596849.9A CN106053798A (en) | 2016-07-26 | 2016-07-26 | Doxycycline field test paper and methods of preparing and using same |
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Application publication date: 20161026 |