CN103288961A - Monoclonal antibody of furantoin residue marker aminohydantoin, and preparation method and application thereof - Google Patents
Monoclonal antibody of furantoin residue marker aminohydantoin, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody of furantoin residue marker aminohydantoin, and a preparation method and application thereof. The monoclonal antibody is secreted by a hybridoma cell AHD/3D4 of which the collection number is CCTCC NO:C201151. The preparation method comprises the following steps: A, coupling hapten CPAHD and bovine serum albumin to obtain immunogen; B, coupling hapten CPAHD 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 AHD/3D4 of which the collection number is CCTCC NO:C201151; 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 furantoin 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 1-aminohydantoin residue in animal edible tissues.
Description
Technical field
The invention belongs to residue of veterinary drug analysis and immunological technique field, be specifically related to a kind of monoclonal antibody that can detect Nitrofurantoin residue marker 1-amido glycolyurea (AHD), also relate to a kind of MONOCLONAL ANTIBODIES SPECIFIC FOR method that can detect Nitrofurantoin residue marker 1-amido glycolyurea (AHD) simultaneously, also relate to a kind of purposes that can detect the monoclonal antibody of Nitrofurantoin residue marker 1-amido glycolyurea (AHD).
Background technology
Furadantin belongs to the itrofurans medicine, has the basic structure of 5-nitrofuran ring, is a kind of broad spectrum antibiotic, and is all effective to gram-positive microorganism, Gram-negative bacteria, fungi, protozoon etc., once is widely used in water industry.Toxicology test proves that it has serious toxicity effects such as carcinogenic, mutagenesis, is only second to nitrofural in itrofurans medicine toxic.European Union forbids that in 1993 furadantin uses in aquaculture, No. 193 bulletins of China's issue " veterinary drug and other compound inventories of food animal forbidding " are listed the itrofurans medicine in the forbidding inventory.
Because furadantin low price, drug effect are fast, still have a lot of aquacultures family to violate a ban at present and use.China must improve the screening detection architecture as early as possible to the residual monitoring situation sternness still of itrofurans medicine, to guarantee the public's life and health safety and carrying out smoothly of the domestic and international economic trade of promotion.
HPLC-MS/MS is the conclusive evidence means that the itrofurans medicine drug residue of generally acknowledging at present detects, to milk, eggs, pork, poultry, and the detectability of sample such as shrimp can reach 0.1～0.3 μ g/kg.Though the sensitivity of this 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 immunological detection method of high flux screening based on the biologically of anti-and antibody, highly sensitive and easy handling.The existing immunology detection report of such medicine is polyclonal antibody.Xu etc. (2009) are the IC of the used AHD polyclonal antibody of first-hand report not
50Value, its described colloidal gold colloidal gold detection test paper strip method detects the residual detectability 10 μ g/L of AHD in the pig urine, is higher than MRPL, can not reach zero residue detection requirement.In addition, though polyclonal antibody is highly sensitive, have individual difference, can not realize long-continued standardized production, make troubles for the stdn foundation of detection technique.
Summary of the invention
The objective of the invention is to be to provide a kind of monoclonal antibody (can detect the monoclonal antibody of Nitrofurantoin residue marker 1-amido glycolyurea (AHD)) of Nitrofurantoin residue marker amido glycolyurea, this antibody capable specific recognition AHD is with other analogue no cross reactions.
Another object of the present invention is the preparation method of the monoclonal antibody (can detect the monoclonal antibody of Nitrofurantoin residue marker 1-amido glycolyurea (AHD)) that has been to provide a kind of Nitrofurantoin residue marker amido glycolyurea, this method is by the immunogen immune mouse of haptens AHD derivative (CPAHD) with bovine serum albumin coupling preparation, behind cytogamy and colony screening, obtained the cell strain hybridoma cell strain AHD/3D4 of energy specific secretion AHD monoclonal antibody, CCTCC NO:C201151.
The 3rd purpose of the present invention is the test kit of the monoclonal antibody (can identify Nitrofurantoin residue marker AHD) that has been to provide a kind of Nitrofurantoin residue marker amido glycolyurea, this test kit can be widely used in the residue detection of furadantin in the edible animal tissue, and the residual quantity information of AHD in the edible animal tissue accurately is provided.
The 4th purpose of the present invention is the application during Nitrofurantoin residue detects in edible animal tissue of the test kit of the monoclonal antibody (can identify Nitrofurantoin residue marker AHD) that has been to provide a kind of Nitrofurantoin residue marker amido glycolyurea, for the residual monitoring of furadantin in the 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, the contriver has prepared a kind of monoclonal antibody that can identify the 1-amido glycolyurea, and it is to be that the hybridoma AHD/3D4 of CCTCC NO:C201151 is secreted by preserving number.
Above-mentioned hybridoma AHD/3D4 is deposited in the Chinese typical culture collection center (CCTCC) that is positioned at Chinese Wuhan Wuhan University, and its preserving number is CCTCC NO:C201151.
Used immunogen is by haptens CPAHD and bovine serum albumin coupling preparation.
Further, the present invention proposes a kind of enzyme linked immunological (ELISA) method of the AHD of being applicable to residue detection, this method comprises the preparation of immunogen, coating antigen and antibody and the steps such as pre-treatment of sample.
A kind of MONOCLONAL ANTIBODIES SPECIFIC FOR method of Nitrofurantoin residue marker amido glycolyurea the steps include:
A, adopt mixed anhydride method to carry out coupling haptens CPAHD and bovine serum albumin to obtain immunogen;
B, adopt mixed anhydride method to carry out coupling haptens CPAHD and ovalbumin to obtain coating antigen;
C, utilize the immunogen preparing of steps A to obtain preserving number to be the secreted monoclonal antibody of CCTCC NO:C201151 hybridoma cell strain AHD/3D4;
The coating antigen bag of D, usefulness step B is by solid phase carrier (as enzyme plate);
E, with the testing sample acid treatment, after adding that phenyl aldehyde is ultrasonic and deriving, use ethyl acetate extraction, get that ethyl acetate layer nitrogen dries up, normal hexane purification and sample diluting liquid dissolve again and obtain determinand;
F, the determinand of step e is carried out enzyme linked immunosorbent detection;
The component of step e sample diluting liquid and proportioning are: NaCl 8.0g, KH
2PO
40.2g, Na
2HPO412H
2O2.9g, KCl 0.2g adds distilled water and is settled to 1000mL.
A kind of test kit of monoclonal antibody of Nitrofurantoin residue marker amido glycolyurea, it is composed as follows:
(1) is coated with the enzyme plate of coating antigen CPAHD-OVA;
(2) the CPAHD standard solution is 6 bottles, and concentration is respectively 0,1,2,4,8,16 μ g/L;
(3) AHD/3D4 cell strain monoclonal antibody working fluid;
(4) the sheep anti-mouse igg antibody working fluid of horseradish peroxidase (HRP) mark;
(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-20 5mL adds distilled water to 1000mL;
(7) substrate mixed solution: accurately draw substrate B liquid (flying Science and Technology Ltd. far away available from Wuhan) 10mL, add 100 μ L substrate A liquid (flying Science and Technology Ltd. far away available from Wuhan), mixing, now with the current.
(9) stop buffer: 2mol/L sulphuric acid soln.
A kind of preparation method of enzyme plate the steps include:
(1) bag quilt: with carbonate buffer solution CPAHD-OVA is diluted to 0.1 μ g/mL coating antigen solution, accurately draws 100 μ L coating antigen solution in each enzyme mark hole, be placed horizontally at wet box, hatch 12h for 4 ℃.
(2) wash plate: throw away enzyme plate endoperidium original solution, pat dry, accurately draw 250 μ L washingss in each enzyme mark hole, leave standstill 30 seconds after, throw away washings, pat dry at thieving paper; Repeated washing 3 times.
(3) sealing: accurately draw 250 μ L confining liquids in each enzyme mark hole, level places in the wet box, and 37 ℃ of incubators are hatched 2h.
(4) wash plate: throw away confining liquid, accurately draw 250 μ L washingss in each enzyme mark hole, leave standstill 30 seconds after, throw away washings, pat dry at thieving paper; Repeated washing 3 times.
(5) oven dry: after thieving paper pats dry; Enzyme plate is inverted oven dry 0.5h in 37 ℃ of incubators.
(6) encapsulation: enzyme plate oven dry back and siccative (silica gel, the Calcium Chloride Powder Anhydrous) aluminium foil bag of packing into together encapsulates with vacuum packaging machine.Described siccative is wherein a kind of of silica gel or Calcium Chloride Powder Anhydrous.
Application during a kind of test kit of monoclonal antibody of Nitrofurantoin residue marker amido glycolyurea AHD in detecting edible animal tissue is residual the steps include:
(1) take out test kit, balance is inserted the micropore frame to room temperature (20-25 ℃, below identical) with the hole bar of enough standard substance and the used quantity of sample.
(2) with reference liquid or sample liquid 50 μ L in micropore separately; Standard substance and sample are done two parallel laboratory tests, note the position of standard substance and sample.Add antibody liquid 50 μ L to each hole, fully mix; Level is put in the wet box, hatches 60min for 37 ℃.
(3) get rid of liquid in the clear opening, pat dry at thieving paper.Accurately draw washings 250 μ L in each hole, leave standstill about 30 seconds, get rid of clean washings, pat dry at thieving paper.Repeated washing 4 times.
(4) add enzyme labelled antibody liquid 100 μ L to each hole, fully mix; Level is put in the wet box, hatches 60min for 37 ℃.
(5) get rid of liquid in the clear opening, pat dry at thieving paper.Accurately draw washings 250 μ L in each hole, leave standstill about 30 seconds, get rid of clean washings, pat dry at thieving paper.Repeated washing 5 times.
(6) add substrate mixed solution 100 μ L to each hole, fully mix; Level is put in the wet box, hatches 15min for 37 ℃.
(7) add stop buffer 50 μ L to each hole; Fully mix; The inherent 450nm of 30min measures light absorption value in the place.
The contriver dresses up as core reagent and other conventional reagent set with said monoclonal antibody and coating antigen can detect the residual enzyme linked immunological kit of AHD in the multiple animal tissues simultaneously, enzyme-linked immunoassay method is verified, realize task of the present invention, thereby finished the present invention.
Major advantage of the present invention is:
1. the monoclonal antibody of the present invention's preparation can be identified CPAHD, and CPAHD is the derivative of the residual marker AHD of furadantin;
2. the haptens synthesis technique of the present invention's design is easy, and combined coefficient height, used reagent are the less terephthalaldehydic acids of toxicity, and be less to the healthy harm of operator.
3. the derivative reagent of test kit sample pre-treatments use of the present invention is phenyl aldehyde, compares than other derivative reagent Ortho Nitro Benzaldehydes commonly used, has the little advantage of toxicity.
Description of drawings
Fig. 1 is the canonical plotting of a kind of Nitrofurantoin residue marker AHD.
Wherein, the x axle is added the logarithmic value that adds concentration doubly with 100 and is drawn, and the y axle is with the ratio (B/B of mensuration hole under each drug level with the detection OD value in zero medicine hole
0) draw.
Embodiment
The invention will be further described below by embodiment, but do not limit the present invention.
Embodiment 1: haptenic synthetic
Nitrofurantoin residue marker AHD with to carboxyl benzaldehyde (4-CBA) at tri-distilled water and N, react in the medium of dinethylformamide, detailed process is as follows: take by weighing AHDHCl 0.60g and 4-CBA 0.30g, add respectively 6mL tri-distilled water and DMF the dissolving, use Na
2CO
3Regulate the AHD aqueous solution to neutrality, slowly add in the 4-CBA solution, 60 ℃～70 ℃ reaction 4h.Reaction terminating, suction filtration is washed respectively 3 times with tri-distilled water and dehydrated alcohol, obtains faint yellow solid, dry back stored refrigerated, this is haptens CPAHD.
Embodiment 2: complete antigen synthetic
Immunogenic synthetic: as to adopt mixed anhydride method to carry out the coupling synthetic immunogen CPAHD and bovine serum albumin, detailed process is as follows: take by weighing CPAHD 20mg, be dissolved in 5mL N, in the dinethylformamide, stir and add 25 μ L tri-n-butylamines, room temperature (20-25 ℃, below identical) reaction 10min.Above-mentioned solution is put precooling in the ice bath, add 44 μ L isobutyl chlorocarbonates.Room temperature reaction 1～1.5h obtains active intermediate product.Take by weighing 132mg bovine serum albumin (BSA), be dissolved among phosphate buffered saline buffer (pH 7.4) 10mL.Active intermediate product is dropwise dropped in the bovine serum albumin solution room temperature reaction 4～5h.Use phosphate buffered saline buffer (pH 7.4) dialysis 3d down at 4 ℃.Packing, freeze-drying obtain immunogen, and called after CPAHD-BSA is in-20 ℃ of preservations.
Synthesizing of coating antigen: adopt mixed anhydride method to carry out the synthetic coating antigen of coupling CPAHD and ovalbumin, detailed process is as follows: take by weighing CPAHD 20mg, be dissolved in 5mL N, in the dinethylformamide, stir adding 25 μ L tri-n-butylamines, room temperature reaction 10min.Above-mentioned solution is put precooling in the ice bath, add 44 μ L isobutyl chlorocarbonates.Room temperature reaction 1～1.5h obtains active intermediate product.Take by weighing 137mg ovalbumin (OVA), be dissolved among phosphate buffered saline buffer (pH 7.4) 10mL.Active intermediate product is dropwise dropped in the bovine serum albumin solution room temperature reaction 4～5h.Use phosphate buffered saline buffer (pH 7.4) dialysis 3d down at 4 ℃.Packing, freeze-drying obtain immunogen, and called after CPAHD-OVA is in-20 ℃ of preservations.
Embodiment 3: MONOCLONAL ANTIBODIES SPECIFIC FOR
The preparation of hybridoma cell strain: with reference to the method in Xue Qingshan " philosophy and technique of vitro culture " the Science Press calendar year 2001 version: with the CPAHD-BSA conjugate immunity Balb/C mouse of embodiment 2 preparations, immune programme for children is: fundamental immunity with immunogen and isopyknic Freund's complete adjuvant emulsification after, in the subcutaneous multi-point injection of mouse back, later every interval 2 all booster immunizations once, use Freund emulsification instead, at last in merging first three day abdominal injection, reinforced immunological, the antigen amount doubles, and does not add adjuvant.During fusion, one of the Balb/C mouse of the last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum) is soaked the 5min sterilization in 75% alcohol.
With 3～5 * 10
7SP2/0 myeloma cell and immune mouse spleen cell suspension join in the 50mL centrifuge tube, mixing, the centrifugal 5min of 1500r/min.Abandon supernatant, and blot with the filter paper of sterilization.Knock the pipe end gently, make the pipe floor cells loosening.Centrifuge tube is placed 37 ℃ of water-baths, slowly add pre-temperature in the 1min to 37 ℃ 50%PEG 0.8mL, the limit edged stirs with pipette tip gently, adds the back and continues to stir 30sec, leaves standstill 1min.
The RPMI-1640 basal liquid 10mL that slowly adds 37 ℃ of pre-temperature.Concrete grammar is: 1min dropwise adds 1mL, and 2min adds 2mL, slowly adds remaining RPMI-1640 basal liquid, limit edged jog centrifuge tube afterwards.Slowly add 40mL RPMI-1640 basal liquid, after adding, the mixing that slowly turns upside down, the centrifugal 5min of 1500r/min.Abandon supernatant, the 10mL dropper is drawn and is contained feeder cell HAT substratum, slowly drips along tube wall, with the slow mechanical stirring of dropper, slowly draws fused cell, drips the mechanical stirring mixing near the feeder cell liquid level.Be inoculated in 6 96 well culture plates, about 150 μ L/ holes place 37 ℃, 5%CO
2Cultivate in the cell culture incubator.
Counted 0d the same day from merging, each culture hole drips 1 HAT substratum behind the 3d, inhales the substratum that removes the l/2 volume every 2d regularly behind the 5d, changes to equivalent HT substratum.4d after fusion begins fused cell is carried out tracing observation, mark and the culture hole that records the hybridoma growth, and calculate fusion rate.6～7d after fusion treats that the hole inner cell grows at the bottom of the hole at 1/10～1/5 o'clock, gets culture supernatant, with indirect competitive ELISA method screening positive cell hole.Coating antigen concentration is 10mg/L.Each cell cultures hole arranges 0 hole and 200 μ g/L medicine holes, adds 50 μ L culture supernatant in every hole and carries out the indirect competitive ELISA detection.
Subclone is carried out with limiting dilution assay in the hole of selecting 4～6 supernatants detections to be strong positive and to have only 1～2 good colony of form to grow.Through 3～4 time clonings, finishing screen is selected the monoclonal hybridoma strain of secretion SEM specific antibody..The applicant is this hybridoma cell strain called after AHD/3D4, and delivers the Chinese typical culture collection center preservation that is positioned at Chinese Wuhan Wuhan University on June 29th, 2011, and its preserving number is CCTCC NO:C201151.
The preparation of ascites monoclonal antibody and evaluation: only got the Balb/c number of mice in preceding 7 days in inoculation, every mouse peritoneal injection 0.5ml Freund's incomplete adjuvant carries out pre-treatment.Suspending by preserving number with the RPMI-1640 basic medium is the cell of the hybridoma cell strain 3D4 enlarged culturing of CCTCC NO:C201151, and cell count is transferred to 1 * 10
6Individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spiritual variation, the dying ascites of gathering when motionless.According to literature method (Zhu Liping, Chen Xueqing. immunology common experimental method. Beijing: People's Medical Officer Press, 2000), purifying obtains monoclonal antibody.Employing is carried out the hypotype evaluation available from the mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit) of ROCKLAND company to the resulting monoclonal antibody of the present invention, and the result is mouse IgG2a hypotype.
The foundation of embodiment 4:ELISA detection method
The optimization of ELISA condition: according to the antigen coated concentration of square formation titration initial option and antibody dilution.Coating antigen is become 8 concentration with carbonate buffer solution doubling dilution successively, and each concentration bag is by 1 row.Ascites antibody is become 8 gradients with primary antibodie diluent doubling dilution successively.During mensuration, at first every hole adds PBS 50 μ L, adds 8 dilution antibody, 50 μ L/ holes by row then.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 is with the absorbance (OD at microplate reader detection wavelength 450nm place
450nm).Select the OD value near 2.0, adjacent holes OD value differs the combination of the most significant coating antigen concentration and antibody dilution, determines to provide foundation for best coating antigen concentration.The result of square formation titration is as shown in table 1, and OD value and the adjacent holes comparing difference in (0.1,8000) and (0.2,16000) two holes are the most remarkable, is the more excellent combination of coating antigen concentration and antibody dilution therefore.
The titration of table 1 3D4 monoclonal antibody square formation
Respectively with the bag of 0.1 μ g/mL and 0.2 μ g/mL by the concentration coated elisa plate, with 1: 8000 and 1: 16000 be the corresponding antibodies extent of dilution, carry out indirect competitive ELISA, the drawing standard curve also calculates IC
50(seeing Table 2).0.1 the bag of μ g/mL is by the IC of concentration correspondence
50Be worth lower, select its as the best bag of antigen by concentration.
The best bag of table 2 is optimized by concentration
With 0.1 μ g/mL concentration coated elisa plate, antibody dilution equal difference is designed to 1: 8000,1: 10000 and 1: 12000, carry out indirect competitive ELISA, calculate IC
50Value (seeing Table 3).IC
50The sensitivity that is worth less hole representative is higher, and therefore the optimum dilution degree of final definite antibody is 1: 10000.
Table 3 optimum antibody extent of dilution is optimized
The foundation of typical curve is mixed with the mother liquor of 1mg/mL with DMF with the CPAHD medicine, and it is diluted to successively 6 concentration such as 0,1,2,4,8,16 μ g/L, drawing standard curve with PBS.As shown in Figure 1, the regression equation of typical curve of the present invention is y=-0.3758x+1.2254, index of correlation r=0.9956, IC
50Value is 8.13+0.1848 μ g/L (n=5), and linearity range is 1～16 μ g/L.
Specificity is with the specificity of cross reacting rate reflection method.Respectively competition thing doubling dilutions such as itrofurans medicine, residual marker and derivative thereof, paraxin, sulphamethazine, Ciprofloxacin are become gradient concentration, carry out indirect competitive ELISA, the drawing standard curve calculates IC
50IC with CPAHD
50Value is competed the IC of things with other
50Value is compared, and namely obtains competing the cross reacting rate (seeing Table 4) of thing.The result shows that the monoclonal antibody of this institute preparation has than high specific 1-amido glycolyurea derived products CPAHD, 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
The test kit moiety
(1) is coated with the enzyme plate of coating antigen CPAHD-OVA;
(2) the CPAHD standard solution is 6 bottles, and concentration is respectively 0,1,2,4,8,16 μ g/L;
(3) AHD/3D4 cell strain monoclonal antibody working fluid;
(4) the sheep anti-mouse igg antibody working fluid of horseradish peroxidase (HRP) mark;
(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-20 5mL adds distilled water to 1000mL
(7) substrate mixed solution: accurately draw substrate B liquid (flying Science and Technology Ltd. far away available from the Wuhan City) 10mL, add 100 μ L substrate A liquid (flying Science and Technology Ltd. far away available from the Wuhan City), mixing, now with the current.
(9) stop buffer: 2mol/L sulphuric acid soln.
A kind of preparation method of enzyme plate the steps include:
(1) bag quilt: with carbonate buffer solution CPAHD-OVA is diluted to 0.1 μ g/mL coating antigen solution, accurately draws 100 μ L coating antigen solution in each enzyme mark hole, be placed horizontally at wet box, hatch 12h for 4 ℃.
(2) wash plate: throw away enzyme plate endoperidium original solution, pat dry, accurately draw 250 μ L washingss in each enzyme mark hole, leave standstill 30s after, throw away washings, pat dry at thieving paper; Repeated washing 3 times.
(3) sealing: accurately draw 250 μ L confining liquids in each enzyme mark hole, level places in the wet box, and 37 ℃ of incubators are hatched 2h.
(4) wash plate: throw away confining liquid, accurately draw 250 μ L washingss in each enzyme mark hole, leave standstill 30s after, throw away washings, pat dry at thieving paper; Repeated washing 3 times.
(5) oven dry: after thieving paper pats dry; Enzyme plate is inverted oven dry 0.5h in 37 ℃ of incubators.
(6) encapsulation: enzyme plate oven dry back and the siccative aluminium foil bag of packing into together encapsulates with vacuum packaging machine.Described siccative is wherein a kind of of silica gel or Calcium Chloride Powder Anhydrous.
Embodiment 6:
A kind of test kit application in the AHD residual quantity in detecting edible animal tissue that can identify Nitrofurantoin residue marker AHD the steps include:
The reagent preparation
Washings: the concentrated cleaning solution that provides in the test kit is used after with 10 times of dilutions of distilled water.
Sample diluting liquid: accurately take by weighing NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
4.12H
2O 2.9g, KCl 0.2g add that a small amount of (dissolving of 200～300mL) distilled waters, distilled water is settled to 1000mL in the 500mL beaker.
1M hydrochloric acid: accurately draw concentrated hydrochloric acid 8.2ml, distilled water is settled to 100mL.
The preparation of 10mM benzaldehyde solution: accurately take by weighing phenyl aldehyde 1.06g, methanol constant volume is to 1000mL.
0.1M K
2HPO
4Solution: accurately take by weighing K
2HPO
43H
2O 22.8g adds that a small amount of (dissolving of 200～300mL) distilled waters, distilled water is settled to 1000mL.
1M NaOH: accurately take by weighing NaOH 4g, add that a small amount of (20～30mL) dissolved in distilled water, distilled water is settled to 100mL.
Substrate mixed solution preparation: according to each institute expense, get an amount of substrate A liquid and B liquid in 1: 100 ratio mixing, now with the current.
Tissue sample is handled
(1) tissue sample that takes by weighing 2.00 ± 0.02g homogeneous adds the 4mL distilled water, 1M hydrochloric acid soln 0.5mL in 50mL tool plug centrifuge tube; With 10mM phenyl aldehyde 200uL, whirlpool concussion 1min, 50 ℃ of ultrasonic 2h;
(2) add 0.1M K respectively
2HPO
45mL, 1M NaOH 0.5mL; With the ethyl acetate of 6mL, vortex 2min.The above centrifugal 10min of (20-25 ℃) 4000r/min at room temperature, the ethyl acetate of taking out 1.5mL dries up in 50 ℃ of nitrogen in another clean container;
(3) dissolve dry thing with the 1mL normal hexane, add 1mL PBS then, vortex 2min, the above centrifugal 10min of (20-25 ℃) 4000r/min under the room temperature;
(4) taking off layer water 50uL is used for analyzing.(extension rate: 2).
ELISA measures program
(1) take out test kit, balance is to room temperature, with the hole bar insertion micropore frame of enough standard substance and the used quantity of sample.
(2) with reference liquid or sample liquid 50 μ L in micropore separately; Standard substance and sample are done two parallel laboratory tests, note the position of standard substance and sample.Add antibody liquid 50 μ L to each hole, fully mix; Level is put in the wet box, hatches 60min for 37 ℃.
(3) get rid of liquid in the clear opening, pat dry at thieving paper.Accurately draw washings 250 μ L in each hole, leave standstill about 30 seconds, get rid of clean washings, pat dry at thieving paper.Repeated washing 4 times.
(4) add enzyme labelled antibody liquid 100 μ L to each hole, fully mix; Level is put in the wet box, hatches 60min for 37 ℃.
(5) get rid of liquid in the clear opening, pat dry at thieving paper.Accurately draw washings 250 μ L in each hole, leave standstill about 30 seconds, get rid of clean washings, pat dry at thieving paper.Repeated washing 5 times.
(6) add substrate mixed solution 100 μ L to each hole, fully mix; Level is put in the wet box, hatches 15min for 37 ℃.
(7) add stop buffer 50 μ L to each hole; Fully mix; The inherent 450nm of 30min measures light absorption value in the place.
The result judges
The mean value of reference liquid or sample liquid light absorption value be multiply by 100% again divided by the light absorption value of " 0 " standard orifice, be inhibiting rate.In 1～16 μ g/L scope, be ordinate zou with the inhibiting rate, 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, with inhibiting rate substitution regression equation, calculate mensuration concentration, multiply by dilution factor, be the residual concentration of AHD in the sample.
Embodiment 7: the sensitivity of test kit of the present invention, precision, accuracy
The sensitivity of test kit of the present invention
With the sensitivity index of lowest detectable limit as test kit of the present invention.Get 20 parts of blank tissue samples, carry out ELISA and detect, measure the OD value, calculate the mean value of blank sample OD value
Will
Find corresponding concentration (C) on the substitution typical curve, and calculate standard deviation (SD).Calculate the Z value according to formula Z=C+3 * SD, the lowest detectable limit (LOD) that this is method for organizing the results are shown in Table 5.
The lowest detectable limit of table 5 AHD in various animal tissuess sample
The precision of test kit of the present invention
The CPAHD standard substance are diluted to 5 concentration of 0,1,2,4,8,16 μ g/L, 3 parallel holes of each concentration, according to indirect competitive ELISA method replication 5 times, its typical curve equation of OD value substitution of each standard substance concentration correspondence is obtained the measured value that ELISA detects, with the variation coefficient between the plate inner panel of standard substance concentration determination value calculating indirect competitive ELISA typical curve, with this precision of judging test kit, the results are shown in Table 6.
The variation coefficient in the plate of table 6 typical curve and between plate
The accuracy of test kit of the present invention
In the good various tissue samples of even matter, add the AHD standardized solution, make its final concentration be respectively 5 μ g/kg and 10 μ g/kg.5 parallel samples of each concentration carry out sample preparation then, and ELISA measures drug level, repeat 3 batches, calculate recovery rate, and the rate of recovery by formula 3 is calculated, and calculates batch interior and interassay coefficient of variation, the results are shown in Table 7～10.As seen, the interpolation of test kit of the present invention is reclaimed all 60%～130%, and batch variation in batch＜20% shows that accuracy is good.
(formula 2)
SEM adds the rate of recovery and the variation coefficient in table 7 pig muscle
AHD adds the rate of recovery and the variation coefficient in table 8 flesh of fish
AHD adds the rate of recovery and the variation coefficient in table 9 shrimp
AHD adds the rate of recovery and the variation coefficient in table 10 honey
Claims (5)
1. the monoclonal antibody of a Nitrofurantoin residue marker amido glycolyurea is characterized in that, hybridoma AHD/3D4, CCTCC NO:C201151.
2. the MONOCLONAL ANTIBODIES SPECIFIC FOR method of the described a kind of Nitrofurantoin residue marker amido glycolyurea of claim 1 the steps include:
A, haptens CPAHD and bovine serum albumin coupling are obtained immunogen;
B, haptens CPAHD and ovalbumin coupling are obtained coating antigen;
C, utilize the immunogen preparing of steps A to obtain preserving number to be the secreted monoclonal antibody of CCTCC NO:C201151 hybridoma AHD/3D4;
The coating antigen bag of D, usefulness step B is by solid phase carrier;
E, with the testing sample acid treatment, after adding that phenyl aldehyde is ultrasonic and deriving, use ethyl acetate extraction, get that ethyl acetate layer nitrogen dries up, normal hexane purification and sample diluting liquid dissolve again and obtain determinand;
F, the determinand of step e is carried out enzyme linked immunosorbent detection;
The component of described 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.
3. the test kit of the monoclonal antibody of the described a kind of Nitrofurantoin residue marker amido glycolyurea of claim 1, it is characterized in that: it contains following component:
(1) is coated with the enzyme plate of coating antigen CPAHD-OVA;
(2) the CPAHD standard solution is 6 bottles, and concentration is respectively 0,1,2,4,8,16 μ g/L;
(3) AHD/3D4 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: 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-20 5mL adds distilled water to 1000mL;
(7) substrate mixed solution: 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 the monoclonal antibody of a kind of Nitrofurantoin residue marker amido glycolyurea according to claim 1, it is characterized in that: the preparation method of described enzyme plate the steps include:
(1) bag quilt: with carbonate buffer solution CPAHD-OVA is diluted to 0.1 μ g/mL coating antigen solution, draws 100 μ L coating antigen solution in each enzyme mark hole, be placed horizontally at wet box, hatch 12h for 4 ℃;
(2) wash plate: throw away enzyme plate endoperidium original solution, pat dry, accurately draw 250 μ L washingss in each enzyme mark hole, leave standstill 30 seconds after, throw away washings, pat dry at thieving paper; Repeated washing 3 times;
(3) sealing: accurately draw 250 μ L confining liquids in each enzyme mark hole, level places in the wet box, and 37 ℃ of incubators are hatched 2h;
(4) wash plate: throw away confining liquid, accurately draw 250 μ L washingss in each enzyme mark hole, leave standstill 30 seconds after, throw away washings, pat dry at thieving paper; Repeated washing 3 times;
(5) oven dry: after thieving paper pats dry; Enzyme plate is inverted oven dry 0.5h in 37 ℃ of incubators;
(6) encapsulation: enzyme plate oven dry back and the siccative aluminium foil bag of packing into together encapsulates with vacuum packaging machine;
Described siccative is wherein a kind of of silica gel or Calcium Chloride Powder Anhydrous.
5. the test kit of the monoclonal antibody of the described a kind of Nitrofurantoin residue marker amido glycolyurea of claim 3 application in the Nitrofurantoin residue detection in edible animal tissue.
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| CN115286667A (en) * | 2022-08-02 | 2022-11-04 | 华中农业大学 | Alpha-galactosyl ceramide analogue and preparation method and application thereof |
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Application publication date: 20130911 |