CN103288962B - Monoclonal antibody of furacilin residue marker semicarbazide, and preparation method and application thereof - Google Patents
Monoclonal antibody of furacilin residue marker semicarbazide, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody of furacilin residue marker semicarbazide, and a preparation method and application thereof. The monoclonal antibody is secreted by a hybridoma cell SEM/4G6 of which the collection number is CCTCC NO:C201150. The preparation method comprises the following steps: A, coupling hapten CPSEM and bovine serum albumin to obtain immunogen; B, coupling hapten CPSEM and ovalbumin to obtain coating antigen; C, preparing the monoclonal antibody from the immunogen in the step A, wherein the monoclonal antibody is secreted by a hybridoma cell strain SEM/4G6 of which the collection number is CCTCC NO:C201150; D, coating a solid-phase carrier with the coating antigen in the step B; E, treating a sample to be detected with acid, adding benzaldehyde, performing ultrasonic derivation, extracting with ethyl acetate, taking nitrogen gas at the ethyl acetate layer, performing blow-drying, purify n-hexane, and redissolving a sample diluent to obtain a substance to be detected; and F, performing ELISA (enzyme-linked immunosorbent assay) detection on the substance to be detected. The invention also discloses application of a kit in furacilin residue detection of animal edible tissues. The method is convenient, quick, sensitive and accurate, and can be used for developing an ELISA kit capable of detecting semicarbazide residue in animal edible tissues.
Description
Technical field
The invention belongs to wild animal resources and immunological technique field, be specifically related to the monoclonal antibody that one can detect monoconal antibody mediated furacilinum residue marker Urea,amino-(SEM), also relate to the preparation method that one can detect the monoclonal antibody of monoconal antibody mediated furacilinum residue marker Urea,amino-(SEM) simultaneously, also relate to the purposes that one can detect the monoclonal antibody of monoconal antibody mediated furacilinum residue marker Urea,amino-(SEM).
Background technology
Nitrofural belongs to Nitrofuran metabolites, has the basic structure of 5-nitrofuran ring, is a kind of broad spectrum antibiotic, all effective to gram-positive microorganism, Gram-negative bacteria, fungi, protozoon etc., is once widely used in aquaculture.Toxicology test proves that nitrofural and metabolite thereof all have the serious toxicity effects such as carcinogenic, mutagenesis significantly.In 1993, European Union forbade that nitrofural uses in aquaculture, No. 193 bulletins " veterinary drug of food animal forbidding and other compound inventories " that China issues list Nitrofuran metabolites in forbidding inventory.
Because nitrofural low price, drug effect are fast, still have the violated use of a lot of raiser at present.The residual monitoring situation of China to Nitrofuran metabolites is still severe, must improve selective mechanisms system as early as possible, to ensure the life and health safety of the public and to promote carrying out smoothly of domestic and international economic trade.
HPLC-MS/MS is the confirmation means that the Nitrofuran metabolites drug residue of generally acknowledging at present detects, and can reach 0.1 ~ 0.3 μ g/kg to the detectability of the samples such as milk, eggs, pork, poultry and shrimp.Although the sensitivity of the method and tolerance range are all very high, need to be equipped with expensive instrument and professional and technical personnel, be not suitable for field operation and high flux screening.
Be generally used for the biologically of immunological detection method based on antigen-antibody of high flux screening, highly sensitive and easy handling.The existing immunology detection report of such medicine is polyclonal antibody, and the authors such as Cooper (2007) and Vass (2008a, b) prepare rabbit polyclonal antibody, the IC of antibody
50value (with SEM calculating concentration) is respectively 0.86 μ g/L and 0.14 μ g/L, adopts direct or indirect competitive ELISA method to remain to SEM in the samples such as chicken, pork, eggs the lowest detectable limit detected and is respectively 0.21 μ g/kg, 0.11 μ g/kg and 0.13 μ g/kg.Though polyclonal antibody is highly sensitive, rabbit individual difference causes its existing defects in standardized production process, can not realize long-continued standardized production, bring difficulty to the standardized construcion of detection technique.
Summary of the invention
The object of the invention is to there are provided a kind of monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody (monoclonal antibody of monoconal antibody mediated furacilinum residue marker Urea,amino-(SEM) can be detected), this antibody capable specific recognition SEM, with other analogue no cross reactions.
Another object of the present invention is the preparation method that there are provided a kind of monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody (can detect the monoclonal antibody of monoconal antibody mediated furacilinum residue marker Urea,amino-(SEM)), monoclonal antibody identifiable design Urea,amino-prepared by the present invention, method has easy, fast, sensitive, the advantage such as accurately, can be used for developing and can detect the enzyme linked immunological kit that in edible animal tissue, Urea,amino-is residual, the method is the immunogen immune mouse of being prepared by haptens SEM derivative (CPSEM) and bovine serum albumin coupling, after cytogamy and colony screening, obtain the cell strain hybridoma cell strain SEM/4G6 of energy specific secretion SEM monoclonal antibody, CCTCC NO:C201150.
3rd object of the present invention is the test kit that there are provided a kind of monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody (can identify monoconal antibody mediated furacilinum residue marker SEM), this test kit can be widely used in the residue detection of nitrofural in edible animal tissue, accurately provides the residual quantity information of SEM in edible animal tissue.
4th object of the present invention there are provided the application of test kit in edible animal tissue in monoconal antibody mediated furacilinum residue detection providing a kind of monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody (can identify monoconal antibody mediated furacilinum residue marker SEM), for the residual monitoring of nitrofural in animal derived food provides reliable technique means.
The present invention is achieved through the following technical solutions:
In order to realize task of the present invention, contriver has prepared a kind of monoclonal antibody that can identify SEM, it is characterized in that, it is secreted by hybridoma SEM/4G6.
Above-mentioned hybridoma SEM/4G6, be deposited in the China typical culture collection center (CCTCC) being positioned at Wuhan, China Wuhan University, its preserving number is CCTCC NO:C201150.
Immunogen used is prepared by haptens CPSEM and bovine serum albumin coupling.
Further, the present invention proposes a kind of enzyme linked immunological (ELISA) method being applicable to SEM residue detection, the method comprises the step such as the preparation of immunogen, coating antigen and antibody and the pre-treatment of sample, a preparation method for the monoclonal antibody of monoconal antibody mediated furacilinum residue marker SEM can be identified, the steps include:
A, active ester method is adopted to carry out coupling synthetic immunogen haptens CPSEM and bovine serum albumin;
B, adopt active ester method to carry out coupling haptens CPSEM and ovalbumin to synthesize coating antigen;
C, the immunogen of steps A is utilized to prepare the monoclonal antibody of preserving number secreted by CCTCC NO:C201150 hybridoma cell strain SEM/4G6;
The coating antigen bag of D, use step B is by solid phase carrier (as enzyme plate);
E, by testing sample acid treatment, add phenyl aldehyde ultrasonic derivative after, be extracted with ethyl acetate, get that ethyl acetate layer nitrogen dries up, normal hexane purification and sample diluting liquid again dissolve and obtain determinand;
F, enzyme linked immunosorbent detection is carried out to the determinand of step e;
The component of step e sample diluting liquid and proportioning are: NaCl 8.0g, KH
2pO
40.2g, Na
2hPO412H
2o 2.9g, KCl 0.2g, adds distilled water and is settled to 1000mL.
A kind of test kit of monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody (monoclonal antibody of monoconal antibody mediated furacilinum residue marker SEM can be identified), it is composed as follows:
(1) enzyme plate of coating antigen CPSEM-OVA is coated with;
(2) CPSEM standard solution 6 bottles, concentration is respectively 0,0.25,0.5,1,2,4 μ g/L;
(3) SEM/4G6 cell strain monoclonal antibody working fluid;
(4) the sheep anti-mouse igg antibody working fluid that marks of horseradish peroxidase (HRP);
(5) concentrated phosphoric acid salt buffer: NaCl 80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o 29.0g, KCl 2.0g, adds distilled water to 1000mL;
(6) concentrated cleaning solution: NaCl 80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o 29.0g, KCl 2.0g, Tween-205mL, add distilled water to 1000mL
(7) Substrate cocktail: accurately draw substrate B liquid (purchased from Wuhan Fei Yuan Science and Technology Ltd.) 10mL, add 100 μ L substrate A liquid (purchased from Wuhan Fei Yuan Science and Technology Ltd.), mixing, now with the current.
(9) stop buffer: 2mol/L sulphuric acid soln.
A preparation method for enzyme plate, the steps include:
(1) bag quilt: CPSEM-OVA is diluted to 0.2 μ g/mL coating antigen solution with carbonate buffer solution, accurately draws 100 μ L coating antigen solution in each enzyme mark hole, is placed horizontally at wet box, hatches 12h for 4 DEG C.
(2) wash plate: throw away enzyme plate endoperidium original solution, pat dry, accurately draw 250 μ L washingss in each enzyme mark hole, after leaving standstill 30s, throw away washings, thieving paper pats dry; Repeated washing 3 times.
(3) close: accurately draw 250 μ L confining liquids in each enzyme mark hole, level is placed in wet box, and 37 DEG C of incubators hatch 2h.
(4) wash plate: throw away confining liquid, accurately draw 250 μ L washingss in each enzyme mark hole, after leaving standstill 30s, throw away washings, thieving paper pats dry; Repeated washing 3 times.
(5) dry: after patting dry on thieving paper; Enzyme plate is inverted in 37 DEG C of incubators and dries 0.5h.
(6) encapsulate: enzyme plate loads aluminium foil bag after drying together with siccative (silica gel, Calcium Chloride Powder Anhydrous), encapsulates with vacuum packaging machine.Described siccative is silica gel or Calcium Chloride Powder Anhydrous one wherein.
A kind of test kit of monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody (monoconal antibody mediated furacilinum residue marker SEM can be identified) detect SEM in edible animal tissue remain in application, the steps include:
(1) take out test kit, balance to room temperature, the hole bar of enough standard substance and sample quantity used is inserted micropore frame.
(2) with reference liquid or sample liquid 50 μ L in respective micropore; Standard substance and sample do two parallel laboratory tests, record the position of standard substance and sample.Add antibody liquid 50 μ L to each hole, fully mix; Level is put in wet box, hatches 60min for 37 DEG C.
(3) get rid of liquid in clear opening, thieving paper pats dry.Accurate absorption washings 250 μ L, in each hole, leaves standstill about 30 seconds, gets rid of clean washings, thieving paper pats dry.Repeated washing 4 times.
(4) add enzyme labelled antibody liquid 100 μ L to each hole, fully mix; Level is put in wet box, hatches 60min for 37 DEG C.
(5) get rid of liquid in clear opening, thieving paper pats dry.Accurate absorption washings 250 μ L, in each hole, leaves standstill about 30 seconds, gets rid of clean washings, thieving paper pats dry.Repeated washing 5 times.
(6) add Substrate cocktail 100 μ L to each hole, fully mix; Level is put in wet box, hatches 15min for 37 DEG C.
(7) stop buffer 50 μ L is added to each hole; Abundant mixing; Light absorption value is measured at the inherent 450nm place of 30min.
Contriver dresses up using said monoclonal antibody and coating antigen as core reagent with other conventional reagent set can detect the enzyme linked immunological kit that in many animals tissue, SEM remains simultaneously, enzyme-linked immunoassay method is verified, achieve task of the present invention, thus complete the present invention.
Major advantage of the present invention is:
1. the monoclonal antibody that prepared by the present invention can identify CPSEM, and CPSEM is the derivative of the residual marker SEM of nitrofural;
2. the hapten synthesis simple process of the present invention's design, combined coefficient is high, and reagent used is the terephthalaldehydic acid that toxicity is less, less to the healthy harm of operator.
3. the derivative reagent that kit sample pre-treatment of the present invention uses is phenyl aldehyde, and the derivative reagent Ortho Nitro Benzaldehyde conventional compared with other is compared, and has the advantage that toxicity is little.
Accompanying drawing explanation
Fig. 1 is that a kind of monoconal antibody mediated furacilinum residue marker SEM remains ELISA canonical plotting.
Wherein, x-axis is added with 100 the logarithmic value adding concentration doubly and is drawn, and y-axis is to measure the ratio (B/B of the detection OD value in hole and zero medicine hole under each drug level
0) draw.
Embodiment
Below by embodiment, the invention will be further described, but do not limit the present invention.
Embodiment 1: the synthesis of haptens SEM derivative (CPSEM)
By monoconal antibody mediated furacilinum residue marker SEM and p-carboxybenzaldehyde (4-CBA) at tri-distilled water and N, react in the medium of dinethylformamide, detailed process is as follows: take SEMHCl 0.15g and 4-CBA 0.23g, adds 4mL tri-distilled water and DMF dissolving respectively, uses Na
2cO
3after regulating the SEM aqueous solution to neutrality, slowly add in 4-CBA solution, room temperature (20-25 DEG C, identical below) reaction 3h.Reaction terminating, suction filtration, washes 3 times respectively with tri-distilled water and dehydrated alcohol, obtains faint yellow solid, and stored refrigerated after dry, this is haptens CPSEM.
Embodiment 2: the synthesis of complete antigen
CPSEM and bovine serum albumin adopt active ester method to carry out coupling synthetic immunogen by immunogenic synthesis, detailed process is as follows: take CPSEM 20.7mg and be dissolved in 2mL N, in dinethylformamide, stirring adds N-hydroxy-succinamide (NHS) 14.4mg and N, N-dicyclohexylcarbodiimide (DCC) 27.5mg, room temperature (20-25 DEG C, identical below) stirring reaction spends the night.The centrifugal 10min of 10000r/min, gets supernatant for subsequent use, and this is A liquid.Taking bovine serum albumin (BSA) 167mg is dissolved in phosphate buffered saline buffer (pH 7.4) 10mL for subsequent use, and this is B liquid.Dropwise join in B liquid by A liquid under magnetic agitation, the centrifugal 15min of ice bath reaction 10h, 10000r/min, collects supernatant liquor.Supernatant liquor is used at 4 DEG C phosphate buffered saline buffer (pH 7.4) dialysis 3d.Packing, freeze-drying, obtain immunogen, called after CPSEM-BSA, in-20 DEG C of preservations.
The synthesis of coating antigen: adopt active ester method to carry out coupling CPSEM and ovalbumin and synthesize coating antigen, detailed process is as follows: take CPSEM 20.7mg and be dissolved in 2mL N, in dinethylformamide, stirring adds N-hydroxy-succinamide (NHS) 14.4mg and N, N-dicyclohexylcarbodiimide (DCC) 27.5mg, stirring at room temperature reaction is spent the night.The centrifugal 10min of 10000r/min, gets supernatant for subsequent use, and this is C liquid.Taking ovalbumin (OVA) 200mg is dissolved in phosphate buffered saline buffer (pH 7.4) 10mL for subsequent use, and this is D liquid.Dropwise join in D liquid by C liquid under magnetic agitation, the centrifugal 15min of ice bath reaction 10h, 10000r/min, collects supernatant liquor.Supernatant liquor is used at 4 DEG C phosphate buffered saline buffer (pH 7.4) dialysis 3d.Packing, freeze-drying, obtain coating antigen, called after CPSEM-OVA, in-20 DEG C of preservations.
Embodiment 3: the preparation of monoclonal antibody
The preparation of hybridoma cell strain: the method with reference in Xue Qingshan " philosophy and technique of vitro culture " Science Press calendar year 2001 version: the CPSEM-BSA conjugate immunity Balb/C mouse of preparing with embodiment 2, immune programme for children is: fundamental immunity is by after immunogen and isopyknic Freund's complete adjuvant emulsification, in the subcutaneous multi-point injection of mouse back, once at interval of 2 weeks booster immunizations later, use Freund's incomplete adjuvant emulsification instead, finally in fusion first three day abdominal injection, reinforced immunological, antigen amount doubles, and does not add adjuvant.During fusion, the Balb/C mouse of last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum) is soak 5min sterilization in the alcohol of 75% in volume ratio.
By 3 ~ 5 × 10
7sP2/0 myeloma cell and immune mouse spleen cell suspension join in 50mL centrifuge tube, mixing, the centrifugal 5min of 1500r/min.Abandon supernatant, and blot with the filter paper of sterilizing.Knock gently at the bottom of pipe, pipe floor cells is loosened.Centrifuge tube is placed in 37 DEG C of water-baths, slowly add the 50%PEG 0.8mL of pre-temperature to 37 DEG C in 1min, limit edged stirs with pipette tip gently, adds rear continuation and stirs 30sec, leave standstill 1min.
Slowly add the RPMI-1640 basal liquid 10mL of 37 DEG C of pre-temperature.Concrete grammar is: 1min dropwise adds 1mL, and 2min adds 2mL, slowly adds remaining RPMI-1640 basal liquid afterwards, limit edged jog centrifuge tube.Slowly add 40mL RPMI-1640 basal liquid, after adding, mixing of slowly turning upside down, the centrifugal 5min of 1500r/min.Abandon supernatant, 10mL dropper is drawn containing feeder cell HAT substratum, slowly drips, by the slow mechanical stirring of dropper, slowly draw fused cell along tube wall, drips close to feeder cell liquid level, and mechanical stirring mixes.Be inoculated in 6 piece of 96 well culture plate, about 150 μ L/ holes, are placed in 37 DEG C, 5%CO
2cultivate in cell culture incubator.
From fusion the same day count 0d, after 3d, each culture hole drips 1 HAT substratum, sucks the substratum of 1/2 volume after 5d regularly every 2d, changes to equivalent HT substratum.4d after fusion, starts to carry out tracing observation to fused cell, marks and record the culture hole of Growth of Hybridoma Cell, and calculate fusion rate.6 ~ 7d after fusion, when hole inner cell to grow at the bottom of hole 1/10 ~ 1/5, gets culture supernatant, with indirect competitive ELISA method screening positive cell hole.Coating antigen concentration is 10mg/L.Each cell culture well arranges 0 hole and 200 μ g/L medicine holes, adds 50 μ L culture supernatant and carry out indirect competitive ELISA detection in every hole.
Select 4 ~ 6 supernatants to detect in strong positive and only have the hole of 1 ~ 2 good colony growth of form, carrying out subclone with limiting dilution assay.Through 3 ~ 4 time clonings, finishing screen selects the monoclonal hybridoma strain of secretion SEM specific antibody..Applicant by this hybridoma called after SEM/4G6, and delivers on June 29th, 2011 the China typical culture collection center preservation being positioned at Wuhan, China Wuhan University, and its preserving number is CCTCC NO:C201150.
The preparation of ascites monoclonal antibody and qualification: only within first 7 days, get Balb/c number of mice in inoculation, every mouse peritoneal injection 0.5ml Freund's incomplete adjuvant carries out pre-treatment.The cell of the hybridoma cell strain SEM/4G6 enlarged culturing being CCTCCNO:C201150 that suspends by preserving number with RPMI-1640 basic medium, and cell count is adjusted to 1 × 10
6individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spirit be deteriorated, dying motionless time gather ascites.According to literature method (Zhu Liping, Chen Xueqing. immunology common experimental method. Beijing: People's Medical Officer Press, 2000), purifying obtain monoclonal antibody.Adopt the mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit) purchased from ROCKLAND company to carry out hypotype qualification to the monoclonal antibody that the present invention obtains, result is mouse IgG 1 hypotype.
The foundation of embodiment 4:ELISA detection method
The optimization of ELISA condition is according to the antigen coated concentration of square formation titration initial option and antibody dilution.Coating antigen carbonate buffer solution successively doubling dilution is become 8 concentration, each concentration bag is by 1 row.Ascites antibody primary antibodie diluent successively doubling dilution is become 8 gradients.During mensuration, first every hole adds PBS 50 μ L, then adds 8 dilution antibody by row, 50 μ L/ holes.Hatch 30min, wash plate, add ELIAS secondary antibody, 100 μ L/ holes.Continue to hatch 30min, wash plate, add substrate, 100 μ L/ holes.Colour developing 15min, termination reaction, with the absorbance (OD at microplate reader determined wavelength 450nm place
450nm).Select OD value close to 2.0, the combination of the adjacent holes OD value the most significant coating antigen concentration of difference and antibody dilution, for the determination of best coating antigen concentration provides foundation.Square formation titration results is as shown in table 1, and the OD value of (0.1,16000) and (0.2,32000) holes and the difference of adjacent holes are comparatively large, are the more excellent combinations of coating antigen concentration and antibody dilution.
The titration of table 1SEM/4G6 monoclonal antibody square formation
Respectively coating antigen is diluted to 0.1 μ g/mL and 0.2 μ g/mL, coated elisa plate.Be corresponding antibodies extent of dilution with 1: 16000 and 1: 32000, carry out indirect competitive ELISA, drawing standard curve, calculate IC
50(see table 2).Because 0.2 μ g/mL wraps by IC corresponding to concentration
50be worth less, the sensitivity of antibody exhibits is higher, therefore selects bag and is wrapped by concentration as antigen the best by concentration 0.2 μ g/mL.
Table 2 the best bag is by concentration optimization
Wrap by concentration coated elisa plate with 0.2 μ g/mL, antibody dilution equal difference is designed to 1: 26000,1: 28000,1: 30000,1: 32000, carries out indirect competitive ELISA, drawing standard curve, calculate IC
50(see table 3).From table 13, determine that antibody optimum dilution degree is 1: 30000.
Table 3 optimum antibody extent of dilution is optimized
CPSEM Pharmaceutical formulations is become the mother liquor of 1mg/mL by the foundation DMF of typical curve, then with PBS, it is diluted to 5 concentration such as 4,2,1,0.5,0.25,0 μ g/L successively, carries out indirect competitive ELISA, drawing standard curve, calculates IC
50.As shown in Figure 1, the regression equation of typical curve of the present invention is y=-0.516x+1.4938, index of correlation r=0.9942, IC
50value is 0.848 ± 0.0757 μ g/L (n=5), and linearity range is 0.25 ~ 4 μ g/L.
The specificity specificity of cross reacting rate reflection method.Respectively Nitrofuran metabolites, the residual competitor doubling dilution such as marker and derivative, p-carbamylaminophenylarsonic acid, paraxin, sulphamethazine, Ciprofloxacin are become gradient concentration, carry out indirect competitive ELISA, drawing standard curve, calculate IC
50.By the IC of CPSEM
50the IC of value and competitor
50value is compared, and namely obtains the cross reacting rate (see table 4) of competitor.Result shows, the standby monoclonal antibody of this institute system has comparatively high specific to Urea,amino-derived products CPSEM, to similar other drug and the equal no cross reaction of other class medicines.
The cross reacting rate of table 4 test kit of the present invention
Embodiment 5: the assembling of ELISA detection kit of the present invention
Test kit moiety
(1) enzyme plate of coating antigen CPSEM-OVA is coated with;
(2) CPSEM standard solution 6 bottles, concentration is respectively 0,0.25,0.5,1,2,4 μ g/L;
(3) SEM/4G6 cell strain monoclonal antibody working fluid;
(4) the sheep anti-mouse igg antibody working fluid that marks of horseradish peroxidase (HRP);
(5) concentrated phosphoric acid salt buffer: NaCl 80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o 29.0g, KCl 2.0g, adds distilled water to 1000mL;
(6) concentrated cleaning solution: NaCl 80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o 29.0g, KCl 2.0g, Tween-205mL, add distilled water to 1000mL
(7) Substrate cocktail: accurately draw substrate B liquid (purchased from Fei Yuan Science and Technology Ltd. of Wuhan City) 10mL, add 100 μ L substrate A liquid (purchased from Fei Yuan Science and Technology Ltd. of Wuhan City), mixing, now with the current.
(9) stop buffer: 2mol/L sulphuric acid soln.
A preparation method for enzyme plate, the steps include:
(1) bag quilt: CPSEM-OVA is diluted to 0.2 μ g/mL coating antigen solution with carbonate buffer solution, accurately draws 100 μ L coating antigen solution in each enzyme mark hole, is placed horizontally at wet box, hatches 12h for 4 DEG C.
(2) wash plate: throw away enzyme plate endoperidium original solution, pat dry, accurately draw 250 μ L washingss in each enzyme mark hole, after leaving standstill 30s, throw away washings, thieving paper pats dry; Repeated washing 3 times.
(3) close: accurately draw 250 μ L confining liquids in each enzyme mark hole, level is placed in wet box, and 37 DEG C of incubators hatch 2h.
(4) wash plate: throw away confining liquid, accurately draw 250 μ L washingss in each enzyme mark hole, after leaving standstill 30s, throw away washings, thieving paper pats dry; Repeated washing 3 times.
(5) dry: after patting dry on thieving paper; Enzyme plate is inverted in 37 DEG C of incubators and dries 0.5h.
(6) encapsulate: enzyme plate loads aluminium foil bag after drying together with siccative, encapsulates with vacuum packaging machine.Described siccative is silica gel or Calcium Chloride Powder Anhydrous one wherein.
Embodiment 6:
Can identify that the test kit of monoconal antibody mediated furacilinum residue marker SEM is detecting the application in edible animal tissue in SEM residual quantity, the steps include:
Preparation of reagents
Washings: use after the concentrated cleaning solution distilled water 10 times dilution provided in test kit.
Sample diluting liquid: accurately take NaCl 8.0g, KH
2pO
40.2g, Na
2hPO
4.12H
2o 2.9g, KCl 0.2g is in 500mL beaker, and add a small amount of distilled water and dissolve, distilled water is settled to 1000mL.
1M hydrochloric acid: accurately draw concentrated hydrochloric acid 8.2ml, distilled water is settled to 100mL.
10mM benzaldehyde solution is prepared: accurately take phenyl aldehyde 1.06g, methanol constant volume is to 1000mL.
0.1M K
2hPO
4solution: accurately take K
2hPO
43H
2o 22.8g, add a small amount of distilled water and dissolve, distilled water is settled to 1000mL.
1M NaOH: accurately take NaOH 4g, add a small amount of distilled water and dissolve, distilled water is settled to 100mL.
Substrate cocktail is prepared: according to each institute expense, get appropriate substrate A liquid and B liquid in 1: 100 ratio mix, now with the current.
Tissue sample process
(1) tissue sample taking 2.00 ± 0.02g homogeneous, in 50mL tool plug centrifuge tube, adds 4mL distilled water, 1M hydrochloric acid soln 0.5mL; With 10mM phenyl aldehyde 200uL, whirlpool concussion 1min, 50 DEG C of ultrasonic 2h;
(2) 0.1M K is added respectively
2hPO
45mL, 1M NaOH 0.5mL; With the ethyl acetate of 6mL, vortex 2min.The at room temperature centrifugal 10min of (20-25 DEG C) more than 4000r/min, the ethyl acetate of taking out 1.5mL dries up in 50 DEG C of nitrogen in another clean container;
(3) with the dry thing of 1mL n-hexane dissolution, 1mL PBS is then added, vortex 2min, the centrifugal 10min of (20-25 DEG C) more than 4000r/min under room temperature;
(4) layer aqueous phase 50uL is taken off for analyzing.(extension rate: 2).
ELISA measures program
(1) take out test kit, balance to room temperature, the hole bar of enough standard substance and sample quantity used is inserted micropore frame.
(2) with reference liquid or sample liquid 50 μ L in respective micropore; Standard substance and sample do two parallel laboratory tests, record the position of standard substance and sample.Add antibody liquid 50 μ L to each hole, fully mix; Level is put in wet box, hatches 60min for 37 DEG C.
(3) get rid of liquid in clear opening, thieving paper pats dry.Accurate absorption washings 250 μ L, in each hole, leaves standstill about 30s, gets rid of clean washings, thieving paper pats dry.Repeated washing 4 times.
(4) add enzyme labelled antibody liquid 100 μ L to each hole, fully mix; Level is put in wet box, hatches 60min for 37 DEG C.
(5) get rid of liquid in clear opening, thieving paper pats dry.Accurate absorption washings 250 μ L, in each hole, leaves standstill about 30s, gets rid of clean washings, thieving paper pats dry.Repeated washing 5 times.
(6) add Substrate cocktail 100 μ L to each hole, fully mix; Level is put in wet box, hatches 15min for 37 DEG C.
(7) stop buffer 50 μ L is added to each hole; Abundant mixing; Light absorption value is measured at the inherent 450nm place of 30min.
Result judges
The mean value of reference liquid or sample liquid light absorption value is multiplied by 100% again divided by the light absorption value of " 0 " standard orifice, is inhibiting rate.Within the scope of 0.25 ~ 4 μ g/L, take inhibiting rate as ordinate zou, the logarithm of concentration of standard solution is X-coordinate drawing standard curve, obtains regression equation.According to the inhibiting rate of formula 1 calculation sample, inhibiting rate is substituted into regression equation, calculate mensuration concentration, be multiplied by dilution factor, be the residual concentration of SEM in sample.
(formula 1)
Embodiment 6: the sensitivity of test kit of the present invention, precision, accuracy
The sensitivity of test kit of the present invention
Sensitivity index using lowest detectable limit as test kit of the present invention.Get 20 parts of blank tissue samples, carry out ELISA detection, measure OD value, calculate the mean value of blank sample OD value
will
substitute on typical curve and find corresponding concentration (C), and calculate standard deviation (SD).Calculate Z value according to formula Z=C+3 × SD, this is the lowest detectable limit (LOD) of method for organizing, the results are shown in Table 5.
The lowest detectable limit of table 5SEM in various animal tissuess sample
The precision of test kit of the present invention
CPSEM standard substance are diluted to 0,0.25,0.5,1,2,4 μ g/L, 5 concentration, each concentration 3 parallel holes, according to indirect competitive ELISA method replication 5 times, OD value corresponding for each standard concentration is substituted into the measured value that its typical curve equation obtains ELISA detection, with the variation coefficient between the plate inner panel of standard concentration measured value calculating indirect competitive ELISA typical curve, judge the precision of test kit with this, the results are shown in Table 6.
The variation coefficient in the plate of table 6 test kit of the present invention and between plate
The accuracy of test kit of the present invention
In homogeneous good various tissue samples, add SEM standardized solution, make its final concentration be respectively 0.5 μ g/kg and 1 μ g/kg.Each concentration 5 Duplicate Samples, then carry out sample preparation, and ELISA measures drug level, repeat 3 batches, and calculate the rate of recovery, the rate of recovery calculates by formula 3, and calculates within-run and between-run analysis coefficient, the results are shown in Table 7 ~ 10.Visible, the interpolation of test kit of the present invention is reclaimed all 60% ~ 130%, and batch variation < 20% in crowd, shows that accuracy is good.
(formula 2)
SEM TIANZHU XINGNAO Capsul and the variation coefficient in table 7 pig muscle
SEM TIANZHU XINGNAO Capsul and the variation coefficient in table 8 flesh of fish
SEM TIANZHU XINGNAO Capsul and the variation coefficient in table 9 shrimp
SEM TIANZHU XINGNAO Capsul and the variation coefficient in table 10 honey
Claims (5)
1. a monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody, is characterized in that: this antibody is by hybridoma SEM/4G6, and CCTCC, CCTCC NO:C201150 secretes.
2. monoconal antibody mediated furacilinum residue marker Urea,amino-a detection method, the steps include:
A. haptens CPSEM and ovalbumin coupling are obtained coating antigen;
B. preserving number is utilized to prepare monoclonal antibody for CCTCC NO:C201150 hybridoma cell strain SEM/4G6;
C. use the coating antigen bag of steps A by solid phase carrier;
D. by testing sample acid treatment, add phenyl aldehyde ultrasonic derivative after, be extracted with ethyl acetate, get that ethyl acetate layer nitrogen dries up, normal hexane purification and sample diluting liquid again dissolve and obtain determinand;
E. enzyme linked immunosorbent detection is carried out to the determinand of step D;
In step D, the treatment step of testing sample is specific as follows:
(1) tissue sample taking 2.00 ± 0.02g homogeneous, in 50mL tool plug centrifuge tube, adds 4mL distilled water, 1M hydrochloric acid soln 0.5mL; With 10mM phenyl aldehyde 200uL, whirlpool concussion 1min, 50 DEG C of ultrasonic 2h;
(2) 0.1M K is added respectively
2hPO
45mL, 1M NaOH0.5mL; With the ethyl acetate of 6mL, vortex 2min.The at room temperature centrifugal 10min of (20-25 DEG C) more than 4000r/min, the ethyl acetate of taking out 1.5mL dries up in 50 DEG C of nitrogen in another clean container;
(3) dissolve dry thing with 1mL normal hexane, then add 1mL PBS, vortex 2min, the centrifugal 10min of (20-25 DEG C) more than 4000r/min under room temperature;
(4) layer aqueous phase 50uL is taken off for analyzing.
The component of step D sample diluting liquid and proportioning are: NaCl8.0g, KH
2pO
40.2g, Na
2hPO412H
2o2.9g, KCl0.2g, add distilled water and be settled to 1000mL.
3. the test kit of a kind of monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody according to claim 1, is characterized in that:
(1) be coated with the enzyme plate of coating antigen CPSEM-OVA;
(2) CPSEM standard solution 6 bottles, concentration is respectively 0,0.25,0.5,1,2,4 μ g/L;
(3) SEM/4G6 cell strain monoclonal antibody working fluid;
(4) the sheep anti-mouse igg antibody working fluid of horseradish peroxidase-labeled;
(5) concentrated phosphoric acid salt buffer: NaCl80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o29.0g, KCl2.0g, add distilled water to 1000mL;
(6) concentrated cleaning solution: NaCl80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o29.0g, KCl2.0g, Tween-205mL, add distilled water to 1000mL;
(7) Substrate cocktail: accurately draw substrate B liquid 10mL, add 100 μ L substrate A liquid, mixing, now with the current;
(9) stop buffer: 2mol/L sulphuric acid soln.
4. the test kit of a kind of monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody according to claim 1, is characterized in that: the preparation method of described enzyme plate, the steps include:
(1) wrap quilt: with carbonate buffer solution, CPSEM-OVA is diluted to 0.2 μ g/mL coating antigen solution, draw 100 μ L coating antigen solution in each enzyme mark hole, be placed horizontally at wet box, hatch 12h for 4 DEG C;
(2) wash plate: throw away enzyme plate endoperidium original solution, pat dry, draw 250 μ L washingss in each enzyme mark hole, leave standstill after 30 seconds, throw away washings, thieving paper pats dry; Repeated washing 3 times;
(3) close: draw 250 μ L confining liquids in each enzyme mark hole, level is placed in wet box, and 37 DEG C of incubators hatch 2h;
(4) wash plate: throw away confining liquid, draw 250 μ L washingss in each enzyme mark hole, leave standstill after 30 seconds, throw away washings, thieving paper pats dry; Repeated washing 3 times;
(5) dry: after patting dry on thieving paper; Enzyme plate is inverted in 37 DEG C of incubators and dries 0.5h;
(6) encapsulate: enzyme plate loads aluminium foil bag after drying together with siccative, encapsulates with vacuum packaging machine;
Described siccative is silica gel or Calcium Chloride Powder Anhydrous one wherein.
5. the application of the test kit of a kind of monoconal antibody mediated furacilinum residue marker Urea,amino-monoclonal antibody according to claim 3 in edible animal tissue in monoconal antibody mediated furacilinum residue detection.
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| CN112578110B (en) * | 2020-12-16 | 2023-03-14 | 西北农林科技大学 | Biological probe and kit for detecting furacilin and furazolidone and application of biological probe and kit |
Citations (4)
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|---|---|---|---|---|
| CN1920573A (en) * | 2006-09-08 | 2007-02-28 | 蔡宝亮 | Method for detecting monoconal antibody mediated furacilinum residue |
| CN101013129A (en) * | 2007-02-13 | 2007-08-08 | 北京望尔生物技术有限公司 | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof |
| CN101012186A (en) * | 2007-01-19 | 2007-08-08 | 中国科学院广州生物医药与健康研究院 | Semicarbazide derivative, monoclonal antibody thereof and application |
| CN101349695A (en) * | 2008-08-25 | 2009-01-21 | 深圳市绿诗源生物技术有限公司 | Furacillin metabolite SEM fast detecting reagent kit and uses thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1920573A (en) * | 2006-09-08 | 2007-02-28 | 蔡宝亮 | Method for detecting monoconal antibody mediated furacilinum residue |
| CN101012186A (en) * | 2007-01-19 | 2007-08-08 | 中国科学院广州生物医药与健康研究院 | Semicarbazide derivative, monoclonal antibody thereof and application |
| CN101013129A (en) * | 2007-02-13 | 2007-08-08 | 北京望尔生物技术有限公司 | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof |
| CN101349695A (en) * | 2008-08-25 | 2009-01-21 | 深圳市绿诗源生物技术有限公司 | Furacillin metabolite SEM fast detecting reagent kit and uses thereof |
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