CN106380520A - Furazolidone-metabolite-resistant monoclonal antibody and application thereof - Google Patents
Furazolidone-metabolite-resistant monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention relates to a furazolidone-metabolite-resistant monoclonal antibody and application thereof, and belongs to the technical field of immunology and the technical field of veterinary drug residue analysis. An anti-monoclonal antibody is generated through being secreted by hybridoma cell strains with the preservation number of CCTCC NO: C2016140; the valence of the antibody is 1 to 1024000; the subtype is IgG1; the affinity constant Ka is 1.6*10<9>L/mol; the inhibition rate IC50 on the furazolidone metabolites is 1.7 mu g/L; no cross reaction exists on other furazolidone metabolite analogues. The monoclonal antibody can be used for preparing an enzyme linked immunosorbent assay kit and a colloidal gold trail chromatography test paper strip for testing the furazolidone metabolites so as to achieve the goal of fast and sensitively detecting the furazolidone medicine metabolites in animal tissues, urine and milk.
Description
Technical field
The present invention relates to a kind of anti-Furaxone metabolite monoclonal antibody, producing the hybridoma of this monoclonal antibody
Strain, and application on detection Furaxone metabolite for this antibody, belong to immunological technique field and wild animal resources skill
Art field.
Background technology
Furazolidone (Furazolidone, FZD), also known as furazolidone, are a kind of Nitrofuran metabolites, to common leather
Lan Shi negative bacterium and positive bacteria have inhibitory action, act on microbial enzyme system, suppress S-acetyl-coenzyme-A, interference microorganism sugar generation
Thank, thus playing bacteriostasis.It is widely used in Aquatic product and birdss, in order to treat bacillary dysentery, enteritis, coccidiosiss etc., and cultivate fish
Scabies, red fin fish disease, ulcer etc..Because its sterilizing ability is strong, has a broad antifungal spectrum, it is not likely to produce drug resistance, oral absorption is rapid, with other
Antibiotic no cross resistance the advantages of, be once widely used in clinic.Furazolidone has very strong side effect, and in animal
Internal metabolism soon is 3- amino -2- oxazolidone (AOZ), and AOZ is directly metabolized into having under the conditions of gastric acid and strongly causes to dash forward
The beta-hydroxyethyl hydrazine of degeneration and carcinogenecity, has potential threat to health and ecological balance.At present, European Union, the U.S., day
Basis, China etc. provide against using Nitrofuran antibiotics in edible animal, and the file agriculture and animal husbandry of the Ministry of Agriculture of China sends out [2002] 1
Number file specifies that the detection of furazolidone in food animal must not be limited to and detects.
Detection technique about furazolidone residual mainly has high performance liquid chromatography (HPLC), liquid chromatograph-matter at present
Spectrum combination method (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS/MS) and immuno analytical method, first three methods are sensitive, accurate
Really, but complex operation, higher to experimental facilitiess and technical requirements, be not suitable for the quick detection of batch samples.Immunoassay side
Method includes enzyme linked immunosorbent assay (ELISA) and colloidal gold immunity chromatography (GICA), has that sensitivity is high, specificity is good, becomes
The features such as this is low, easy to operate, is suitable to batch samples screening.Therefore, the preparation of anti-Furaxone metabolite monoclonal antibody
Foundation and guarantee human health for animal food immunity method for quick are significant.
Content of the invention
The technical problem to be solved is that the defect overcoming prior art provides a kind of anti-Furaxone metabolite
Monoclonal antibody, described monoclonal antibody has specificity to Furaxone metabolite, and described anti-monoclonal antibody is by deposit number
For CCTCC NO:The hybridoma cell strain AOZ-4G3 of C2016140 produces.Further, the present invention should by this monoclonal antibody
For detecting Furaxone metabolite.
Technical problem of the present invention is realized by technical scheme below.
A kind of anti-Furaxone metabolite monoclonal antibody, this monoclonal antibody is CCTCC NO by preserving number:
The hybridoma cell strain of C2016140 produces.This hybridoma cell strain was named as hybridoma cell strain AOZ-4G3, in 2016
July 31 delivered China typical culture collection center (CCTCC) preservation, and preserving number is CCTCC NO:C2016140.
Above-mentioned anti-Furaxone metabolite antibody titer is 1:1024000, hypotype is IgG1, affinity constant Ka
=1.6 × 109L/mol, to Furaxone metabolite suppression ratio IC50For 1.7 μ g/L, the cross reaction with Furaxone metabolite
Rate is 100.00%, with other several homologue (furazolidone, nitrofurantoin, furaltadone, nitrofural, nitrofurantoin generations
Thank thing, AMOZ, Furacilin metabolite) no cross reaction；
Above-mentioned Furaxone metabolite monoclonal antibody is for preparing the Furaxone metabolite in detection biological sample
Non-diagnostic purpose detect product in application.
Above-mentioned application, described non-diagnostic purpose detection product is enzyme linked immunological kit or colloid gold chromatographic test paper strip.
Further, a kind of enzyme linked immunological kit of detection Furaxone metabolite, contains described in this test kit
The monoclonal antibody of anti-Furaxone metabolite.
A kind of colloid gold chromatographic test paper strip of detection Furaxone metabolite, contains described anti-furan azoles in this test strips
The monoclonal antibody of bupropion metabolite thing.
A kind of method preparing above-mentioned Furaxone metabolite monoclonal antibody, comprises the steps：
(1) prepare Furaxone metabolite antigen working solution：
(a) Furaxone metabolite derivatization：Take 0.25g AOZ, with 2mL about methanol stir at room temperature and be allowed to molten
Solution；Take 0.15g 3- carboxyl benzaldehyde, be allowed to dissolve with 2mL methanol, be slowly dropped under agitation in above-mentioned AOZ solution, room temperature
Lower stirring reaction 5h, filters, and dries derivant CPAOZ that filtering residue obtains final product Furaxone metabolite；
(b) immunogenic synthesis：The bovine serum albumin (BSA) weighing 50mg is dissolved in the 1mL 0.1mol/L of pH value 7.4
PBS in, add N, N- dimethylformamide (DMF) 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in
In 1mLDMF solution, stirring is lower to add dicyclohexyl imines (DCC) 14mg and N-hydroxy-succinamide (NHS) 6mg, at 4 DEG C
Sealing is stirred overnight, and 8000r/min is centrifuged 15 minutes, collects supernatant and loads in bag filter, uses pH value 7.4 under the conditions of 4 DEG C
0.01mol/L PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-BSA solution；
The former synthesis of (c) detection：The ovalbumin (OVA) weighing 40mg is dissolved in the 1mL 0.1mol/L PBS of pH value 7.4
In, add N, N- dimethylformamide (DMF) 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in 1mLDMF molten
In liquid, stirring is lower to add dicyclohexyl imines (DCC) 14mg and N-hydroxy-succinamide (NHS) 6mg, sealing stirring at 4 DEG C
Overnight, 8000r/min is centrifuged 15 minutes, collects supernatant and loads the 0.01mol/L using pH value 7.4 in bag filter under the conditions of 4 DEG C
PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-OVA solution；
(2) prepare Furaxone metabolite monoclonal antibody：
(a) animal immune：Select the immunogen that carrier protein is bovine serum albumin, the female Blab/ of immune 6-8 week old
C mice, interval immunity in 2 weeks 1 time, docking after three immunity takes hematometry potency and suppression ratio, and the optimal mice of selection result is accurate
Standby fusion；
(b) cell fusion：The splenocyte of the selected mice of step (a) and mouse myeloma SP2/O cell is taken to be merged,
Indirect elisa method measures supernatant and chooses positive high hole, carries out sub-clone by limiting dilution assay to positive hole, until setting up
Produce the hybridoma cell strain of the monoclonal antibody of single anti-Furaxone metabolite；
A large amount of preparations of (c) monoclonal antibody：Choose individual larger female Blab/c mice, using inducing ascites in vivo
Method, prepares ascites in a large number, and passes through octanoic acid-ammonium sulfate precipitation purification ascites, is divided into tubule, -20 DEG C of preservations, obtains furazolidone
Metabolite monoclonal antibody.
A kind of method identifying above-mentioned Furaxone metabolite monoclonal anti bulk properties, comprises the steps：
(a) titration
With 1：40000 dilutions are coated primordial covering detection plate, and monoclonal antibody after purification is carried out 1:200,1:400,1:
800 ... ... 1:800000 dilutions, are added to ELISA Plate in the hole, add the sheep anti mouse two of HRP labelling to resist, finally use TMB after reaction
Colour developing, result shows that Furaxone metabolite MAb concentration after purification reaches 1 for potency during 1mg/ml：1024000.
B () hypotype measures
Carry out hypotype mensure using purchased from the Mus source monoclonal antibody hypotype identification kit of Sigma company, result shows furan azoles
Bupropion metabolite thing monoclonal antibody hypotype is IgG1.
C () affinity measures
Measure the affinity costant of Furaxone metabolite monoclonal antibody, result display parent using non-competing euzymelinked immunosorbent assay (ELISA)
With constant Ka=1.6 × 109L/mol.
D () suppression ratio measures
Suppression ratio is measured using indirect competitive ELISA, (B represents variable concentrations titer with absorbance percentage ratio B/B0%
A450, B0 represents the A450 of zero standard's liquid) be vertical coordinate, the logarithm [Lg (CPAOZ)] with variable concentrations titer is
Abscissa, draws the suppression curve of antibody.Result shows this antibody to Furaxone metabolite suppression ratio IC50For 1.7 μ g/L.
(f) specific assay
With Inhibition ELISA measure this antibody and Furaxone metabolite and the like (furazolidone, nitrofurantoin,
Furaltadone, nitrofural, Cistofuran metabolite, AMOZ, Furacilin metabolite) cross reacting rate,
This antibody of result and Furaxone metabolite CR (%) are 100%, are respectively less than 0.1% with its analog CR (%).
The monoclonal antibody that the present invention provides can be applicable to Furaxone metabolite in detection sample, is mainly applied
In the enzyme linked immunological kit preparing Furaxone metabolite detection and colloid gold test strip paper.The anti-furazolidone that the present invention provides
The quality of metabolite monoclonal antibody has very strong controllability and repeatability, and by preliminary point to gained antibody characteristic
Analysis and identification, foundation further and application for special, sensitive method of immunity provide experimental basis.
Brief description
Fig. 1 Furaxone metabolite of the present invention antibody titer measures figure
Fig. 2 Furaxone metabolite of the present invention monoclonal antibody hypotype measures figure
Fig. 3 Furaxone metabolite of the present invention monoclonal antibody affinity measures figure
Fig. 4 Furaxone metabolite of the present invention monoclonal antibody suppression ratio measures figure
The hybridoma cell strain AOZ-4G3 producing Furaxone metabolite monoclonal antibody of the present invention, in 2016
On July 31, in delivers China typical culture collection center (abbreviation CCTCC, address：Wuhan University, Chinese Typical Representative culture is protected
Tibetan center, postcode：430072) preservation, preserving number is CCTCC NO:C2016140.
Specific embodiment
With reference to specific embodiment, the present invention is described in further detail, cited embodiment is not to the present invention
Content and protection domain constitute any restriction.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
The preparation of embodiment one Furaxone metabolite antigen of the present invention
(1) Furaxone metabolite derivatization：Take 0.25g AOZ, with 2mL about methanol stir at room temperature and be allowed to molten
Solution；Take 0.15g 3- carboxyl benzaldehyde, be allowed to dissolve with 2mL methanol, be slowly dropped under agitation in above-mentioned AOZ solution, room temperature
Lower stirring reaction 5h, filters, and dries filtering residue and obtains final product furazolidone metabolite derivative CPAOZ；
(2) immunogenic synthesis：The bovine serum albumin (BSA) weighing 50mg is dissolved in the 1mL 0.1mol/L of pH value 7.4
PBS in, add N, N- dimethylformamide (DMF) 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in
In 1mLDMF solution, stirring is lower to add dicyclohexyl imines (DCC) 14mg and N-hydroxy-succinamide (NHS) 6mg, at 4 DEG C
Sealing is stirred overnight, and 8000r/min is centrifuged 15 minutes, collects supernatant and loads in bag filter, uses pH value 7.4 under the conditions of 4 DEG C
0.01mol/L PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-BSA solution；
(3) detect former synthesis：The ovalbumin (OVA) weighing 40mg is dissolved in the 1mL 0.1mol/L PBS of pH value 7.4
In, add N, N- dimethylformamide (DMF) 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in 1mLDMF molten
In liquid, stirring is lower to add dicyclohexyl imines (DCC) 14mg and N-hydroxy-succinamide (NHS) 6mg, sealing stirring at 4 DEG C
Overnight, 8000r/min is centrifuged 15 minutes, collects supernatant and loads the 0.01mol/L using pH value 7.4 in bag filter under the conditions of 4 DEG C
PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-OVA solution.
The preparation of embodiment two Furaxone metabolite monoclonal antibody of the present invention
(1) animal immune：Carrier protein is that the female Blab/c of the immunogen immune 6-8 week old of bovine serum albumin is little
Mus, interval immunity in 2 weeks 1 time, immune flow process is shown in Table 1, three exempt from after dock and take hematometry potency and suppression ratio, selection result is optimal
Mice prepares to merge；
The immune flow chart of table 1
(2) cell fusion：Merge mice and pluck eyeball blood-letting, using serum as positive control, after death aseptic condition at de- neck
Lower taking-up spleen, prepares splenocyte, presses 5 with SP2/0 cell:1 ratio is merged by PEG, and the cell suspension after merging is added
Enter to be covered with 96 orifice plates of feeder cells, put into 37 DEG C, cultivate in the incubator of 5%CO2；
(3) screening of positive hybridoma cell strain：Cell after fusion, next day checks for polluting, the 10th day after fusion
Use HT culture medium instead.After changing liquid, 2-3d indirect ELISA and indirect competitive ELISA carry out positive hole sizer choosing, pick out strong as far as possible
The hole that positive, suppression ratio is high, single clone, cell state are good, carries out sub-clone by limiting dilution assay, is enlarged training simultaneously
Foster, frozen, until setting up the monoclonal cell strain of single secretory antibody.
(4) a large amount of preparations of monoclonal antibody：Method using inducing ascites in mice body, takes the Balb/c female of health
Mice, every mice internal injection paraffin oil 0.5mL, adjust cloning positive hybridoma cell 10 after 7d6/ mL, every mice
Lumbar injection 1mL, takes ascites for 7-9 days, and fat is abandoned in centrifugation, through octanoic acid-ammonium sulfate, Protein A affinity chromatograph column purification after elder generation,
- 20 DEG C of preservations after lyophilizing, obtain Furaxone metabolite monoclonal antibody.
Embodiment three Furaxone metabolite monoclonal antibody CHARACTERISTICS IDENTIFICATION of the present invention
(1) titration
Using indirect elisa method, comprise the following steps that：It is coated：Being coated as far as concentration with carbonate buffer solution dilution is 2 μ
G/mL, 96 hole elisa Plates 100 μ L/ hole, 4 DEG C are overnight；Washing：It is coated plate to recover to room temperature, inclines and be coated liquid, every hole adds washing liquid
300 μ L, standing l min every time, wash 3 times, pat dry for the last time；Closing：Every hole adds 200 μ L and contains washing of 10% calf serum
Liquid, 37 DEG C of 1h；Incline deblocking liquid, washs 3 times, pats dry；It is anti-plus one：With washing liquid by monoclonal antibody with 1:2000 times start doubling dilution,
Every hole adds 100 μ L, and arranges blank control wells (PBS) and negative control (negative serum), 37 DEG C of placement 45min；Incline one
Anti-, wash 3 times, pat dry；Plus ELIAS secondary antibody：Every hole adds the l of 100 μ L:The mountain sheep anti mouse of the HRP enzyme labelling of 10000 times of dilutions
IgG, 37 DEG C of placement 30min；Incline two resist, wash 3 times, pat dry；Colour developing：Every hole adds substrate nitrite ion 100 μ L, and 37 DEG C of lucifuges are anti-
Answer 15min；Terminate：Every hole adds 50 μ L terminate liquids, terminating reaction；Detection：Mensure wavelength is the absorbance at 450nm
(A450nm).
Result judgement：(A sample well-A blank)/(A negative control-A blank) 2.1, and negative control
A450nm be less than 0.2 when, now the extension rate of antibody is antibody titer.Result is shown in Fig. 1, anti-Furaxone metabolite list
Clonal antibody 4G3 concentration is that potency during 1mg/ml reaches 1：1024000.
(2) hypotype measures
Hypotype mensure is carried out using the Mus source monoclonal antibody hypotype identification kit purchased from Sigma company.Hybridoma is secreted
Monoclonal antibody colour developing anti-from the two of different subclass have a notable difference, the A450nm value highest wherein resisting with IgG1 bis-, with IgM
Two anti-colour developing faint, and with IgG2a, IgG2b, IgG3 and IgA bis- resist hardly develop the color, this cell secretion Antibody types with
Based on IgG1.As shown in Figure 2, Furaxone metabolite monoclonal antibody hypotype is IgG1 to result.
(3) affinity measures
Affinity costant (Ka) is measured using non-competing ELISA method.Comprise the following steps that：It is coated：Dilute with carbonate buffer solution
Release that to be coated as far as concentration be 0.8,0.1,0.05,0.01 μ g/mL, 96 hole elisa Plates 100 μ L/ hole is coated respectively, and 4 DEG C overnight；Incline
Remove to be coated liquid, wash 3 times, pat dry；Closing：Every hole adds the washing liquid that 200 μ L contain 10% calf serum, 37 DEG C of 1h；Incline deblocking liquid,
Washing 3 times, pats dry；Plus monoclonal antibody：With washing liquid, monoclonal antibody is started doubling dilution with 100 μ g/mL, every hole adds 100 μ L, 37 DEG C of insulations
Moisturizing 45min；Incline one resist, wash 3 times, pat dry；Plus ELIAS secondary antibody：Every hole adds the l of 100uL:The HRP of 10000 times of dilutions
The mountain sheep anti-mouse igg of enzyme labelling, 37 DEG C of placement 30min；Incline two resist, wash 3 times, pat dry；Colour developing：Every hole adds substrate nitrite ion
100 μ L, 37 DEG C of lucifuges react 15min；Terminate：Every hole adds 50 μ L terminate liquids, terminating reaction；Detection：Mensure wavelength is 450nm
The absorbance (A450nm) at place.Calculate Ka by following equation
Ka=(n-1)/2 (n Ab '-Ab)
In formula：When Ab is Ag for antigen concentration, produce the antibody concentration of half absorbance；Ab ' is Ag ' for antigen concentration
When, produce the antibody concentration of half absorbance；N is that the extension rate between Ag and Ag ' adopts non-competing euzymelinked immunosorbent assay (ELISA) to survey
Determine the affinity costant of Furaxone metabolite monoclonal antibody, result is as shown in figure 3, affinity costant Ka=1.6 × 109L/mol.
(4) suppression ratio measures
Using indirect competitive ELISA method, comprise the following steps that：It is coated：It is coated as far as concentration with carbonate buffer solution dilution
For 2 μ g/mL, 96 hole elisa Plates 100 μ L/ hole, 4 DEG C overnight；Incline and be coated liquid, wash 3 times, pat dry；Closing：Every hole adds 200 μ L
Washing liquid containing 10% calf serum, 37 DEG C of 1h；Incline deblocking liquid, washs 3 times, pats dry；Plus monoclonal antibody and standard substance：Standard substance are female
Liquid PBS is diluted to 8.1,2.7,0.9,0.3,0.1,0 μ g/L, and every hole adds 50 μ L, adds the monoclonal antibody of certain extension rate,
Every hole 50 μ L, setting blank control wells (PBS) and negative control (50 μ L monoclonal antibody+50 μ L washing liquid) simultaneously, 37 DEG C of heat and moisture preservings
45min；Incline one resist, wash 3 times, pat dry；Plus ELIAS secondary antibody：Every hole adds the l of 100uL:The HRP enzyme labelling of 10000 dilutions
Mountain sheep anti-mouse igg, 37 DEG C placement 30min；Incline two resist, wash 3 times, pat dry；Colour developing：Every hole adds substrate nitrite ion 100 μ L,
37 DEG C of lucifuges react 15min；Terminate：Every hole adds 50 μ L terminate liquids, terminating reaction；Detection：Mensure wavelength is the suction at 450nm
Shading value (A450nm).
With absorbance percentage ratio B/B0%, (B represents the A450 of variable concentrations titer, and B0 represents zero standard's liquid
A450 it is) vertical coordinate, with the logarithm [Lg (CPAOZ)] of variable concentrations titer as abscissa, draw the suppression curve of antibody.Knot
Fruit sees Fig. 4, and this antibody is to Furaxone metabolite suppression ratio IC50For 1.7 μ g/L.
(5) specific assay
With Inhibition ELISA measure this antibody and Furaxone metabolite and the like (furazolidone, nitrofurantoin,
Furaltadone, nitrofural, Cistofuran metabolite, AMOZ, Furacilin metabolite) cross reacting rate,
The results are shown in Table 2, this antibody and Furaxone metabolite CR (%) are 100%, are respectively less than 0.01% with its analog CR (%).
The anti-Furaxone metabolite monoclonal antibody of table 2 and analog cross reaction result
The application of example IV Furaxone metabolite of the present invention monoclonal antibody
The present embodiment is that Furaxone metabolite monoclonal antibody of the present invention is setting up detection Furaxone metabolite residual
Enzyme-linked immunologic detecting kit or colloid gold chromatographic test paper strip method after applicating example, can be used for animal tissue, urine
Detection with Furaxone metabolite in milk.
(1) sample pre-treatments
The pre-treatment of (a) meat, liver or aquatic products sample tissue
After tissue homogenate, weigh 2g in 50ml centrifuge tube, add 4mL deionized water, 1mL 1mol/L hydrochloric acid solution, shake
Swing 1min, add 0.5mL p -carboxybenzaldehyde, vibrate 1min；0.5mol/L disodium phosphate soln 1mL is added after taking-up,
Adjust PH to 7.5 about with 1mol/L sodium hydroxide solution, vibrate 1min, add 5mL ethyl acetate, vibrate 1min, 5000rpm
Centrifugation 10min；Pipette upper strata ethyl acetate layer solution in another centrifuge tube, aqueous phase repeats to extract two with 5mL ethyl acetate again
Secondary, after merging, nitrogen dries up；Add 1ml normal hexane, add a cover vibration 1min, be subsequently adding 0.5mL PBST buffer, vibration
1min, 5000rpm are centrifuged 10min；Draw lower floor's aqueous phase solution to be used for analyzing.
The pre-treatment of (b) urine sample
By urine sample 5000rpm centrifugation 5min to limpid, supernatant 50ul is taken to be detected；
The pre-treatment of (c) milk sample
Take 5000rpm centrifugation 10min under milk sample 2mL room temperature condition, take off layer solution and add 4mL deionized water, 1mL
1mol/L hydrochloric acid solution, vibrates 1min, adds 0.5mL p -carboxybenzaldehyde, vibrates 1min；0.5mol/L phosphorus is added after taking-up
Sour disodium hydrogen solution 1mL, adjusts PH to 7.5 about with 1mol/L sodium hydroxide solution, vibrates 1min, adds 5mL ethyl acetate,
Vibration 1min, 5000rpm are centrifuged 10min；Pipette upper strata ethyl acetate layer solution in another centrifuge tube, aqueous phase uses 5mL second again
Acetoacetic ester repeats to be extracted twice, and after merging, nitrogen dries up；Add 1ml normal hexane, add a cover vibration 1min, be subsequently adding 0.5mL
PBST buffer, vibrates 1min, and 5000rpm is centrifuged 10min；Draw lower floor's aqueous phase solution to be used for analyzing.
(2) this antibody is applied to the detection of enzyme-linked immunologic detecting kit method
In the present invention, the Cleaning Principle of test kit is indirect competitive ELISA method.
Detection：Furaxone metabolite coating antigen is coated on microwell plate, takes out lath on demand, be placed on ELISA Plate
On；Furaxone metabolite monoclonal antibody working solution and enzyme labelling anti antibody working solution are pressed 5 by amount on demand:1 ratio is mixed
Close；Sequentially add standard substance or testing sample 50ul, Furaxone metabolite monoclonal antibody working solution and enzyme labelling in the hole
The mixed liquor 50ul of anti antibody working solution, gently vibration mixes, and 25 DEG C of lucifuges are incubated 30min；In the hole liquid is dried, with washing
Working solution (20 × concentrated cleaning solution is diluted for 20 times by deionized water) 250ul/ hole, washs 4 times, pats dry；By nitrite ion A and aobvious
Color liquid B presses 1:1 ratio mixing, 100ul/ hole adds colour developing, and 25 DEG C of lucifuges are incubated 15min；Plus terminate liquid 50ul terminating reaction, enzyme
Measured using dual wavelength 450nm/630nm under mark instrument 450nm, carried out according to standard curve quantitative or qualitative.
Result judgement：(a) quantitative analyses：Calculate the mean absorbance values of standard substance and testing sample respectively, standard substance or
The absorbance (B) of sample is multiplied by 100% again divided by the absorbance of 0 standard substance, as percentage absorbance, percentage absorbance
Value=(B/B0) × 100%.With percentage absorbance as vertical coordinate, the logarithm of normal concentration is abscissa, draws standard curve.
The percentage absorbance of sample to be tested is substituted into standard curve, you can obtain corresponding concentration, then be multiplied by extension rate and be sample
This actual content.(b) qualitative analyses：Compared with the absorbance of standard substance with the mean absorbance values of sample to be tested, that is,
The concentration range of sample to be tested can be drawn, test scope is 0.1ug/ml-8.1ug/ml.
(3) this antibody is applied to the detection of colloid gold chromatographic test paper strip method
Reaction principle carries out half-quantitative detection using competition law to Furaxone metabolite, furazolidone present in sample
Metabolite is first combined with the antibody 4G3 of gold grain labelling during moving along in test strips, be fixed on coating antigen on NC film and
Furaxone metabolite competition binding gold labeling antibody simultaneously, remains furan azoles in the colour developing power of T location (detection line) and sample
The content of bupropion metabolite thing is inversely proportional to, if no Furaxone metabolite residual in sample, gold labeling antibody is all reacted with coating antigen,
The T line colour developing of test strips.
Detection：Take out AOZ colloid gold chromatographic test paper strip, sample end is inserted in analyte sample fluid, insertion depth is less than
Mark line, takes out test strip, horizontal positioned for about 10-20 second, 3-5 minute is observed and judged testing result, after 10 minutes
Result is invalid.
Result judgement：On coated film, corresponding quality control region location of C (nature controlling line) shows a reddish brown colo(u)r streak, detection zone T position
Put and do not show reddish brown colo(u)r streak, represent that testing result is the positive, illustrate to contain AOZ in testing sample；T, location of C on coated film
Show two days reddish brown colo(u)r streaks, represent that result is feminine gender, illustrate not containing AOZ in testing sample；When quality control region C does not show palm fibre
Red stripes, then no matter detection zone T show brownish red band whether this reagent paper be all judged to invalid.
Above-described embodiment only technology design to illustrate the invention and advantage, the present invention can also have other forms and become
Change, as well known to the skilled person, above-described embodiment functions only as to the exemplary role in foregoing invention protection domain, right
For those of ordinary skill in the art, also has a lot of conventional deformation and other enforcement in the protection domain that the present invention is limited
Example, these deformation and embodiment are all by within the protection domain pending in the present invention.
Claims (8)
1. a kind of anti-Furaxone metabolite monoclonal antibody is it is characterised in that this monoclonal antibody is CCTCC by preserving number
NO:The hybridoma cell strain AOZ-4G3 of C2016140 produces.
2. anti-Furaxone metabolite monoclonal antibody according to claim 1 is it is characterised in that this antibody titer is 1:
1024000, hypotype is IgG1, affinity constant Ka=1.6 × 109L/mol, to Furaxone metabolite suppression ratio IC50For 1.7
μ g/L, the cross reacting rate with Furaxone metabolite is 100.00%, with furazolidone, nitrofurantoin, furaltadone, furan
XiLin, Cistofuran metabolite, AMOZ, Furacilin metabolite no cross reaction.
3. a kind of hybridoma cell strain producing anti-Furaxone metabolite monoclonal antibody as claimed in claim 1 or 2
AOZ-4G3 is it is characterised in that it is preserved in China typical culture collection center, deposit number CCTCC NO:C2016140,
Preservation date is on July 31st, 2016.
4. a kind of method preparing the Furaxone metabolite monoclonal antibody as described in claim asks 1 or 2, its feature exists
In comprising the steps：
(1) prepare Furaxone metabolite antigen working solution：
(a) Furaxone metabolite derivatization：Take 0.25g AOZ, with 2mL about methanol stir at room temperature and be allowed to dissolve；
Take 0.15g 3- carboxyl benzaldehyde, be allowed to dissolve with 2mL methanol, be slowly dropped under agitation in above-mentioned AOZ solution, stir under room temperature
Mix reaction 5h, filter, dry derivant CPAOZ that filtering residue obtains final product Furaxone metabolite；
(b) immunogenic synthesis：Weigh 50mg bovine serum albumin BSA be dissolved in pH value 7.4 1mL 0.1mol/L PBS
In, add N, N- dimethylformamide DMF 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in 1mLDMF molten
In liquid, stirring is lower to add dicyclohexyl imines DCC 14mg and N-hydroxy-succinamide (NHS) 6mg, seals stirred at 4 DEG C
At night, 8000r/min is centrifuged 15 minutes, collects supernatant and loads the 0.01mol/L using pH value 7.4 in bag filter under the conditions of 4 DEG C
PBS 3d, changes liquid 3-4 time daily, obtains CPAOZ-BSA solution；
The former synthesis of (c) detection：The ovalbumin OVA weighing 40mg is dissolved in the 1mL 0.1mol/L PBS of pH value 7.4, adds
N, N- dimethylformamide DMF 1mL, stirring and dissolving；Again the CPAOZ 8mg of step (a) gained is dissolved in 1mLDMF solution, stirs
Mix lower addition dicyclohexyl imines DCC 14mg and N-hydroxy-succinamide NHS 6mg, sealing at 4 DEG C is stirred overnight,
8000r/min is centrifuged 15 minutes, collects supernatant and loads the 0.01mol/L PBS using pH value 7.4 in bag filter under the conditions of 4 DEG C
Dialysis 3d, changes liquid 3-4 time daily, obtains CPAOZ-OVA solution；
(2) prepare Furaxone metabolite monoclonal antibody：
(a) animal immune：Select the immunogen that carrier protein is bovine serum albumin, the female Blab/c of immune 6-8 week old is little
Mus, interval immunity in 2 weeks 1 time, docking after three immunity takes hematometry potency and suppression ratio, and the optimal mice of selection result prepares to melt
Close；
(b) cell fusion：The splenocyte of the selected mice of step (a) and mouse myeloma SP2/O cell is taken to be merged, indirectly
ELISA method measures supernatant and chooses positive high hole, carries out sub-clone by limiting dilution assay to positive hole, until set up producing
The hybridoma cell strain of the monoclonal antibody of single anti-Furaxone metabolite；
A large amount of preparations of (c) monoclonal antibody：Choose individual larger female Blab/c mice, using inducing ascites method in vivo,
A large amount of preparation ascites, and pass through octanoic acid-ammonium sulfate precipitation purification ascites, it is divided into tubule, -20 DEG C of preservations, obtain furazolidone generation
Thank to thing monoclonal antibody.
5. the anti-Furaxone metabolite monoclonal antibody described in claim 1 or 2 is for preparing in detection biological sample
The non-diagnostic purpose of Furaxone metabolite detects the application in product.
6. application according to claim 5 is it is characterised in that described non-diagnostic purpose detection product is ELISA reagent
Box or colloid gold chromatographic test paper strip.
7. a kind of enzyme linked immunological kit of detection Furaxone metabolite is it is characterised in that contain such as right in this test kit
Require the monoclonal antibody of the anti-Furaxone metabolite described in 1 or 2.
8. a kind of colloid gold chromatographic test paper strip of detection Furaxone metabolite is it is characterised in that contain as weighed in this test strips
Profit requires the monoclonal antibody of the anti-Furaxone metabolite described in 1 or 2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112415204A (en) * | 2020-10-23 | 2021-02-26 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Method for detecting bacteria by using colloidal gold test strip containing bispecific antibody |
| CN112578110A (en) * | 2020-12-16 | 2021-03-30 | 西北农林科技大学 | Biological probe and kit for detecting furacilin and furazolidone and application |
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2016
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| 任海涛等: "呋喃唑酮代谢物单克隆抗体制备及酶联免疫吸附分析方法", 《分析化学》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112415204A (en) * | 2020-10-23 | 2021-02-26 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Method for detecting bacteria by using colloidal gold test strip containing bispecific antibody |
| CN112415204B (en) * | 2020-10-23 | 2023-10-13 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Method for detecting bacteria by using colloidal gold test strip containing bispecific antibody |
| CN112578110A (en) * | 2020-12-16 | 2021-03-30 | 西北农林科技大学 | Biological probe and kit for detecting furacilin and furazolidone and application |
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