CN103288964B - Albendazole-2-amino sulfone residue detection monoclonal antibody as well as preparation method and application thereof - Google Patents
Albendazole-2-amino sulfone residue detection monoclonal antibody as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an albendazole-2-amino sulfone residue detection monoclonal antibody as well as a preparation method and an application thereof, and belongs to a hybridoma cell GA/2A11 with the collection number of CCTCCNO:C201142. The preparation method comprises the following steps: a, coupling hapten albendazole-2-amino sulfone with bovine serum albumin, thereby obtaining an immunogen; b, coupling the hapten albendazole-2-amino sulfone with ovalbumin, thereby obtaining a coating antigen; c, preparing a hybridoma cell strain by using the immunogen; d, coating a solid phase carrier with the coating antigen; e, extracting a sample to be detected by using ethyl acetate under the action of potassium hydroxide, thereby obtaining an object to be detected; f, performing enzyme-linked immunity detection on the object to be detected. A kit comprises: (1) an elisa plate coated with the coating antigen ABZSO2NH2-GA-OVA, (2) an ABZSO2NH2 standard substance solution, (4) a Goat anti Mouse IgG antibody working solution marked by a horse radish peroxidase, (5) a concentrated phosphate buffering solution, (6) a concentrated washing solution, (7) a substrate mixing solution and (9) a stop solution. The invention further discloses the application of the kit in the detection of albendazole-2-amino sulfone residue detection. The kit has the characteristics of being simple, convenient, rapid, sensitive and accurate.
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 identify the amino sulfone of the residual marker albendazole of albendazole-2-, also relate to a kind of preparation method of the monoclonal antibody that can identify the amino sulfone of the residual marker albendazole of albendazole-2-simultaneously, also relate to a kind of purposes of the monoclonal antibody that can identify the amino sulfone of the residual marker albendazole of albendazole-2-.
Background technology
Albendazole is widely used benzimidazoles insect repellent on veterinary clinic, has the feature of wide spectrum, efficient, low toxicity.More in ruminating animal uses such as cattle and sheep, common nematode, fluke and tapeworm are all killed to effect.The rapid metabolism of first pass effect by liver or gastrointestinal tract mucous cell after albendazole administration is albendazole-sulfoxide and Albendazole sulfone, is finally converted into the amino sulfone of albendazole-2-.Albendazole original shape is difficult to detect in blood plasma, but its metabolite is extensively distributed to in-vivo tissue and body fluid, with maximum in liver, is secondly kidney.Albendazole and metabolite thereof have a toxicology meaning animal tissues is residual, and experimental study confirms that albendazole or accumulating of its metabolite have the danger of the toxicology such as teratogenesis and embryotoxicity; In addition, when Albendazole In Treatment people's parasitosis, there is the danger of potential generation untoward reaction, more easily there is tardy serious adverse reaction in long-term prescription, relate to blood system, Digestive tract, tetter, neural system etc., especially tardy taking encephalitis syndromes as main nervous system disorders, there is potential lethal, the danger that disables.The residual of albendazole and metabolite thereof produces and also has promoter action parasite resistance.
Various countries have classified albendazole as food residue key monitoring object, have specified its maximum residue limit (MRL) and residual marker (MR).No. 235 bulletins of The Ministry of Agriculture of the People's Republic of China, MOA " animal food herbal medicine maximum residue limit(MRL) " are to albendazole residual maximum residue limit(MRL) of also having formulated in animal tissues.At present about the method for detecting residue of albendazole and metabolite thereof mainly comprises biological assay, immune analysis method and instrumental method etc., wherein liquid phase chromatography (HPLC) is that in edibility animal product, albendazole and metabolite residue thereof detect most widely used method.Although instrument analytical method is sensitive, accurate, resolution is high and can carry out the qualitative and quantitative study of many residue detection, needs expensive instrument, loaded down with trivial details pre-treatment, skilled professional operator.If adopt instrumental method to carry out the detection of batch samples, its cost will be very high; And major part only has provincial, and municipal level to be just equipped with accurate analytical instrument in current national feeler mechanism.Therefore need to set up one from screening the detection system of confirmation, about the comparatively perfect of residual confirmation method of albendazole, but it is less to can be used for the detection method research of batch samples primary dcreening operation.Microorganism identification method and immunoassay are the common methods of residue detection screening.The benzimidazoles medicines such as albendazole have the activity of expelling parasite, the biological assay growing up based on parasite Vitro Culture Techniques is existing use in the residue detection of the medicines such as albendazole, but exists the low and result of poor specificity, sensitivity to judge the inherent defects such as difficulty.
Enzyme-linked immunosorbent assay (ELISA) is the ultramicro method that antigen antibody reaction is combined with modern means of testing, integrates the highly sensitive of modern test method and the strong specificity of antigen-antibody reaction.With respect to instrumental method, the advantage such as that immune analysis method has is simple to operate, quick, highly sensitive, high specificity, testing cost are low, is applicable to the primary dcreening operation of batch samples.But the immunological detection method of albendazole that up to the present, only had several sections of direct or relevant bibliographical informations.Johnsson etc. (2002) are combined albendazole to prepare complete antigen with human serum proteins, obtain 8 kinds of benzimidazoles medicines such as albendazole to have the polyclonal antibody of cross reaction after immune sheep.Brandon etc. (1994) utilize benzimidazoles medicine great majority to have the feature of Urethylane side chain, prepare monoclonal antibody with 5 (6)-carboxylic amyl group-sulphur-benzoglyoxaline Urethylane haptens, and set up indirect competitive ELISA method, lowest detectable limit can reach 10 μ g/kg.The antibody obtaining can be identified the benzimidazoles medicine that albendazole, albendazole-sulfoxide, Albendazole sulfone, fenbendazole etc. contain Urethylane side chain.More than the ELISA method of report all fails to form commercial test kit, and this is because its antibody of preparing exists defect, can not identify albendazole--the amino sulfone of 2-.The amino sulfone of albendazole--2-is the important component part of the residual marker of albendazole, there is proportionlity (being about 20%) in its residual quantity in tissue and the total residue of albendazole, the U.S. detects the residual of albendazole using it as residual marker even separately.In addition Brandon etc. (1994; 1998) think that liver organization be can be used for to ELISA after simple pure water or citric acid solution (pH 3.0) step extraction detects, but from the result of its report, the rate of recovery is between 14%～129%, general on the low side in testing requirement (60%～120%).Visible, research is a kind of based on albendazole--and the ELISA rapid screening method of the amino sulfone antibody of 2-is significant to albendazole residue detection in batch samples.
Summary of the invention
The object of the invention is to overcome prior art and can not identify the defect of the amino sulfone of the residual reach-2-of marker acetysalicylic acid phenobarbital of albendazole, a kind of monoclonal antibody that can the amino sulfone of the residual reach-2-of marker acetysalicylic acid phenobarbital of specific recognition albendazole is provided.This antibody is except having 68% cross reaction, with the equal no cross reaction of other benzimidazoles medicines with Albendazole sulfone.
Another object of the present invention is the preparation method who has been to provide a kind of monoclonal antibody that can identify the amino sulfone of the residual marker albendazole of albendazole-2-, the method is the immunogen immune mouse of being prepared by the amino sulfone of haptens albendazole-2-and bovine serum albumin coupling, after cytogamy and colony screening, obtain the cell strain hybridoma cell strain GA/2A11 of the monoclonal antibody of the amino sulfone of energy specific secretion albendazole-2-, CCTCC NO:C201142.
The 3rd object of the present invention is to be to provide a kind of test kit that can identify the amino sulfone of the residual marker albendazole of albendazole-2-, this test kit can be widely used in the residue detection of albendazole in edible animal tissue, and albendazole in edible animal tissue-2-residual quantity information of amino sulfone is accurately provided.
The 4th object of the present invention is to be to provide a kind of test kit application in albendazole residue detection in edible animal tissue that can identify the amino sulfone of the residual marker albendazole of albendazole-2-, for the residual monitoring of albendazole in animal derived food provides technique means reliably.
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 the amino sulfone of albendazole-2-, it is characterized in that, it is secreted by hybridoma cell strain GA/2A11.
Above-mentioned hybridoma GA/2A11, is deposited in the Chinese Typical Representative culture collection center (CCTCC) that is positioned at Wuhan City, Hubei Province Wuhan University, and its preserving number is CCTCC, NO:C201142.
Immunogen used is prepared by the amino sulfone of haptens albendazole-2-and bovine serum albumin coupling.
Further, the present invention proposes a kind of enzyme linked immunological (ELISA) method that is applicable to the amino sulfone residue detection of albendazole-2-, the method comprises the step such as the preparation of immunogen, coating antigen and antibody and the pre-treatment of sample, the preparation method that can identify the monoclonal antibody of the amino sulfone of the residual marker albendazole of albendazole-2-, the steps include:
A, amino haptens albendazole-2-sulfone and bovine serum albumin coupling are obtained to immunogen;
B, amino haptens albendazole-2-sulfone and ovalbumin coupling are obtained to coating antigen;
C, utilize the immunogen of steps A to prepare preserving number for the secreted monoclonal antibody of CCTCC NO:C201142 hybridoma cell strain GA/2A11;
The coated solid phase carrier (as enzyme plate) of coating antigen of D, use step B;
E, by testing sample with ethyl acetate under the alkalization such as potassium hydroxide or sodium carbonate solution condition, extract, nitrogen dries up, normal hexane purifies and sample diluting liquid again dissolves and obtains determinand;
F, the determinand of step e is carried out to enzyme linked immunosorbent detection;
The component of step e sample diluting liquid and proportioning are: NaCl 8.00g, KH
2pO4 0.20g, Na
2hPO412H
2o 2.90g, KCl 0.20g, adds distilled water and is settled to 1000mL.
The test kit that can identify the monoclonal antibody of the amino sulfone of the residual marker albendazole of albendazole-2-, it is composed as follows:
(1) be coated with coating antigen ABZSO
2nH
2the enzyme plate of-GA-OVA;
(2) ABZSO
2nH
26 bottles of standard solutions, concentration is respectively 0,20,40,80,160,320 μ g/L;
(3) GA/2A11 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 80.00g, KH
2pO
42.00g, Na
2hPO
412H
2o 29.00g, KCl 2.00g, adds distilled water to 1000mL;
(6) concentrated cleaning solution: NaCl 80.00g, KH2PO4 2.00g, Na
2hPO
412H
2o 29.00g, KCl 2.00g, Tween205mL, adds distilled water to 1000mL
(7) substrate mixed solution: 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.), mix, now with the current.
(9) stop buffer: 2mol/L sulphuric acid soln.
A preparation method for enzyme plate, the steps include:
(1) coated: with carbonate buffer solution by ABZSO
2nH
2-GA-OVA is diluted to 0.25 μ g/mL coating antigen 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, leave standstill after 30s, throw away washings, on thieving paper, pat dry; Repeated washing 3 times.
(3) sealing: accurately draw 250 μ L confining liquids in each enzyme mark hole, level is placed in wet box, and 37 DEG C 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 after 30s, throw away washings, on thieving paper, pat dry; Repeated washing 3 times.
(5) dry: after patting dry on thieving paper; Enzyme plate is inverted in 37 DEG C of incubators and is dried 0.5h.
(6) encapsulation: enzyme plate packs aluminium foil bag into after drying together with siccative (silica gel, Calcium Chloride Powder Anhydrous), encapsulates with vacuum packaging machine.
The test kit that can identify the amino sulfone of the residual marker albendazole of albendazole-2-, detecting the application of the amino sulfone of albendazole in edible animal tissue-2-in residual, the steps include:
(1) take out test kit, balance, to room temperature, is inserted micropore frame by the hole bar of enough standard substance and sample quantity used.
(2) with reference liquid or sample liquid 50 μ L in micropore separately; 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, on thieving paper, pat dry.Accurately draw washings 250 μ L in each hole, leave standstill 30s left and right, get rid of clean washings, on thieving paper, pat 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, on thieving paper, pat dry.Accurately draw washings 250 μ L in each hole, leave standstill 30s left and right, get rid of clean washings, on thieving paper, pat dry.Repeated washing 5 times.
(6) add substrate mixed solution 100 μ L to each hole, fully mix; Level is put in wet box, hatches 15min for 37 DEG C.
(7) add stop buffer 50 μ L to each hole; Fully mix; Light absorption value is measured at the inherent 450nm of 30min place.
Contriver dresses up and can detect the residual enzyme linked immunological kit of albendazole in many animals tissue (muscle (pig, chicken, ox, sheep, fish), liver (ox, sheep, pig, chicken), milk and egg) simultaneously as core reagent and other conventional reagent set using said monoclonal antibody and coating antigen, enzyme-linked immunoassay method is verified, realize task of the present invention, thereby completed the present invention.
Major advantage of the present invention is:
Monoclonal antibody prepared by the present invention can be identified the amino sulfone of albendazole-2-, and the amino sulfone of albendazole-2-is the residual marker of albendazole, does not also have bibliographical information to have antibody capable to identify this material;
The enzyme-linked immunoassay method that the present invention sets up and test kit can detect the residual, applied widely of the amino sulfone of albendazole in many animals tissue (muscle (pig, chicken, ox, sheep, fish), liver (ox, sheep, pig, chicken), milk and egg)-2-;
The sample treatment the present invention relates to is easy, easy to operate, and sample preparation main organic reagent used is ethyl acetate and normal hexane, less to the healthy harm of operator; And the rate of recovery is high, accuracy and precision are good.
Brief description of the drawings
Fig. 1 is a kind of preparation method's block diagram of the monoclonal antibody that can identify the amino sulfone of the residual marker albendazole of albendazole-2-.
Fig. 2 is a kind of indirect competitive ELISA reaction normal curve synoptic diagram taking the amino sulfone of albendazole-2-as standard substance of the present invention.
X-axis is the amino sulfone (ABZSO of albendazole-2-
2nH
2) concentration of standard solution logarithmic value, Y-axis is that the optical density value of the amino sulfone standard solution of albendazole-2-is divided by " zero " hole optical density value (B/B
0).
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, but does not limit the present invention.
Embodiment 1: the preparation of immunogen and coating antigen
Accurately take ABZSO
2nH
218.00mg, being dissolved in completely in 5mL DMF is A liquid.Accurately take BSA90.00mg, it is fully dissolved in 40mL phosphate buffered saline buffer (pH 7.2) is B liquid.Room temperature (20-25 DEG C, below identical), dropwise joins A liquid in B liquid under magnetic agitation, after both mix, slowly drips volume ratio and be 15% glutaraldehyde solution (GA) 1.5mL.Room temperature magnetic agitation reaction 2h.After having reacted, reaction solution is proceeded in dialysis tubing, the 3d that dialyses in 4 DEG C of physiological saline, changes dialyzate every day 3 times.The centrifugal 5min of 4000r/min, gets supernatant liquor lyophilize, obtains conjugate ABZSO
2nH
2-GA-BSA, puts-20 DEG C of preservations, uses as immunogen.
In above-mentioned steps, change BSA (purchased from sigma company) into OVA (purchased from sigma company), obtain conjugate ABZSO
2nH
2-GA-OVA, uses as coating antigen.
Embodiment 2: 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 ABZO preparing with embodiment 1
2nH
2-GA-BSA conjugate is immunogen, immune Balb/C female mice.Immune programme for children adopts a fundamental immunity and 3～5 booster immunizations.When first immunisation, use with isopyknic Freund's complete adjuvant emulsification be injected in the subcutaneous fundamental immunity of carrying out of nape portion of mouse containing the immunogenic protein emulsion of 100 μ g, carried out booster immunization with Freund's incomplete adjuvant emulsification containing the immunogenic protein emulsion of 100 μ g every 15 days later.From immunity three times, after each immunity, within the 8th day, adopt tail blood, separation of serum, indirect elisa method detects serum antibody titer.Contain the immunogenic protein solution of 100 μ g (not adding adjuvant), reinforced immunological in first 3 days of fusion (best and immunity last time is separated by more than 3 weeks) to the qualified mouse peritoneal injection of immunity.Merge and within first 1 day, get one of non-immune Balb/C mouse and prepare feeder cell.According to myeloma cell's count results, get 3～5 × 10
7individual myeloma cell mixes (ratio is 1: 10～5: 10) with immune spleen cell.The centrifugal 5min of 1500r/min, after emptying to the greatest extent on centrifuge tube, tips upside down on thieving paper, and control solid carbon dioxide drips.Knock lightly the pipe end, make pipe floor cells become pasty state, be positioned in 37 DEG C of water-baths, there is the pre-temperature of 0.8mL to the 1mL calibrated pipet of the 50%PEG (purchased from Amersco) of 37 DEG C to be inserted into the pipe end suction, in 60sec, slowly add PEG to cell mixing, limit edged stirs gently, adds rear standing 90sec, and centrifuge tube is inserted on centrifuge tube shelf.Remove water-bath cup, drawing the pre-temperature of 10mL to the RPMI-1640 basic culture solution (purchased from Hyclone) of 37 DEG C with suction pipe is slowly added on fused cell along tube wall, limit edged rocks centrifuge tube gently, within the 1st minute, dropwise add 1mL (3sec/ drips), within the 2nd minute, add again 2mL, finally add remaining 7mL (adding in 5min).Add after first 10mL, then add RPMI-1640 substratum to 50mL along tube wall, after adding, tighten lid, slowly put upside down 3～5 times, mix.The centrifugal 5min of 1500r/min, supernatant discarded, uses the 72mL HAT perfect medium that contains feeder cell gently fused cell to be stirred resuspended.By fused cell suspension inoculation in 96 porocyte culture plates, 2/hole.Single cell fusion can be inoculated 5 96 porocyte culture plates, is placed in 37 DEG C of 5%CO2 incubators and cultivates.Counted as 0d the same day from merging, before the 3d kinetocyte plate of trying not, keep incubator homeostasis.3d adds in every hole 1 HAT perfect medium (purchased from Amersco) and observes colony growing state; 5d every hole sucking-off 1/2 culture supernatant (100 μ L), then add 1 HT perfect medium (purchased from Amersco); Suck 1/2 culture supernatant every the same method of 3d later, change to HT perfect medium.According to the growing state of cell, when Growth of Cells is desirable cells and supernatant to accounting for hole floorage 1/4 left and right, the ABZSO preparing with contriver
2nH
2-GA-OVA conjugate is coating antigen, utilizes ELISA method to filter out the positive cell hole of the amino sulfone antibody of secretion anti-albendazole-2-.The positive cell hole screening is utilized to continuous 3 time clonings of limiting dilution assay, finally set up the hybridoma cell strain of the amino sulfone antibody of the anti-albendazole of a strain stably excreting-2-, it is 94.4 that this hybridoma cell strain is carried out to chromosome counting mean value, higher than the chromosome number of parental cell, (SP2/0 myeloma cell's karyomit(e) mean number is 58, splenocyte karyomit(e) is 40), the SP2/0 myeloma cell really of fused cell and the hybridization product of splenocyte are described.Applicant was this hybridoma cell strain called after GA/2A11, and delivered Chinese Typical Representative culture collection center (CCTCC) preservation that is positioned at Wuhan City, Hubei Province Wuhan University on June 29th, 2011, and its preserving number is CCTCC NO:C201142.
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.Suspending with RPMI-1640 basic medium is the cell of the hybridoma cell strain GA/2A11 enlarged culturing of CCTCC NO:C201142 by preserving number, and cell count is adjusted to 1 × 10
6individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spiritual variation, the dying ascites that gathers when motionless.According to literature method (Zhu Liping, Chen Xueqing. immunology common experimental method. Beijing: People's Medical Officer Press, 2000), purifying obtain monoclonal antibody.Adopt monoclonal antibody the present invention being obtained purchased from the mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit) of ROCKLAND company to carry out hypotype qualification, result is mouse IgG 2a hypotype.
Embodiment 3: the foundation of racing ELISA detecting method
1 reagent preparation
Carbonate buffer solution (pH 9.6): accurately take Na
2cO
31.59g, NaHCO
32.93g, a small amount of ultrapure water dissolves, and is settled to 1000mL.
Washings (pH 7.4): accurately take NaCl 8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o 2.90g, KCl 0.20g, a small amount of ultrapure water dissolves, and adds Tween 20 0.50mL, is settled to 1000mL.
Phosphate buffered saline buffer (pH 7.4): accurately take NaCl 8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o 2.90g, KCl 0.20g, a small amount of ultrapure water dissolves, and is settled to 1000mL.
Confining liquid: accurately take ovalbumin 10.00g, add phosphate buffer 1 000mL, stirring and evenly mixing until albumen dissolve completely.
Substrate mixed solution: 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), mix, now with the current.
Stop buffer: accurately measure vitriol oil 100mL, be slowly added drop-wise in 800mL ultrapure water.
2 square formation volumetrys
Adopt square formation volumetry initial option coating antigen concentration and antibody dilution.Use carbonate buffer solution by ABZSO
2nH
-2-gA-OVA coating antigen doubling dilution becomes 1,0.5,0.25,0.125,0.0625,0.03125 μ g/mL, and the from first to the 6th row laterally adds 96 hole enzyme plates successively, and 4 DEG C are spent the night; Wash 3 times, pat dry, add confining liquid 250 μ L, 37 DEG C of sealing 1h; Wash 3 times, pat dry, monoclonal antibody use phosphate buffered saline buffer doubling dilution become 1: 128000,1: 256000,1: 512000,1: 1024000,1: 2048000,1: 4096000, the from first to the 6th leu longitudinally adds 96 hole enzyme plates to add enzyme plate, hatches 1h for 37 DEG C; Wash 3 times, pat dry, each hole adds with phosphate buffer 1: (be called for short two resists the sheep anti-mouse igg antibody of the horseradish peroxidase-labeled of 5000 dilutions, the anti-sheep anti-mouse igg antibody that is horseradish peroxidase-labeled of following indication two, purchased from Wuhan Fei Yi Bioisystech Co., Ltd) 100 μ L, hatch 1h for 37 DEG C; Wash 4 times, pat dry, each hole adds substrate mixed solution 100 μ L, lucifuge colour developing 15min; Add stop buffer 50 μ L; Measure optical density value (OD value) at 450nm wavelength place by automatic microplate reader.Square formation titration results is in table 1, and the several OD values of initial option approach 1.0, and adjacent two hole OD values have the coated concentration of larger variation and corresponding antibody dilution combination.Result shows, can select following coating antigen concentration and antibody dilution combination: (0.5,512000), (0.25,512000), (0.125,256000) and (0.0625,128000).
Table 1 GA/2A
11the titration of monoclonal antibody square formation
A coating antigen is ABZSO
2nH
-2-gA-OVA coating antigen; The antibody that the hybridoma cell strain GA/2A11 that b antibody is is CCTCC NO:C201142 by preserving number prepares.
The selection of 3 best coated concentration
With ABZSO
2nH
2for competition thing, be set to 0,20,40,80,160,320 μ g/L concentration gradients, the coated concentration of selecting with 3.1 square formation volumetrys respectively and corresponding antibody dilution are combined into row indirect competitive ELISA.Using the logarithmic value of competition substrate concentration as X-coordinate, B/B0, (the OD value when suppressing without medicine is as B0, and OD value when respective concentration medicine suppresses is B value) is as ordinate zou drafting inhibition curve.The results are shown in Table 2, with " 0 " hole OD value and IC
50value, as judging index, is determined best coating antigen concentration.The ratio of antigen-antibody is the key that affects its sensitivity, all will cause IC50 higher if there is antigen or antibody excess, and from data, best coated concentration is 0.25 μ g/mL, and antibody dilution is tentatively defined as 1: 256000.
The best coated concentration optimization of table 2
| Coating antigen concentration (μ g/mL) | Antibody coefficient multiple (1: X) | 0 hole OD value | IC 50Value (μ g/L) |
| 0.5 | 256000 | 1.169 | 130.6 |
| 0.25 | 256000 | 0.938 | 91.2 |
| 0.125 | 128000 | 0.885 | 110.5 |
| 0.0625 | 64000 | 0.876 | 114.3 |
The dilution selection of 4 optimum antibody
With the coated concentration coated elisa plate of the best, by antibody 4 dilution gradients of concentration equal difference design centered by 1: 256000, its 0 hole and IC
50value is in table 3.Along with the increase of antibody dilution, IC
50value reduces, and still " 0 " hole value also reduces, when 0 hole is worth its IC when too low
50value raises on the contrary, therefore selects 1: 200000 for optimum antibody extent of dilution.
Table 3 optimum antibody extent of dilution is optimized
| Antibody coefficient multiple (1: X) | 0 hole OD value | IC 50Value (μ g/L) |
| 312000 | 0.688 | 79.4 |
| 256000 | 0.885 | 80.2 |
| 200000 | 1.028 | 69.2 |
| 144000 | 1.213 | 83.2 |
The selection of 5 best competition times
It is 30min, 45min and 60min that the competition time is set, and makes to suppress curve, its " 0 " hole and IC
50value is in table 4.The speed of antibody and pharmaceutical standards product and coating antigen competition combination determined by the affinity constant of antibody, and therefore investigating the impact that the competition time reacts ELISA is the important factor of optimum detection condition.From data in table, along with the shortening of competition time, " 0 " hole OD value reduces, but IC
50value also reduces, and sensitivity improves.Consider saturability and the stability of competing reaction, finally selecting 60min is the best competition time.
The best competition of table 4 is time-optimized
| The competition time (min) | 0 hole OD value | IC 50Value (μ g/L) |
| 30 | 0.850 | 50.1 |
| 45 | 0.939 | 75.5 |
| 60 | 1.076 | 74.1 |
The selection of 6 best concentration of substrate
The Urea Peroxide that adds respectively different concns in substrate mixed solution, does indirect competitive ELISA, its " 0 " hole and IC
50value is in table 5.Result shows, along with the increase of hydrogen peroxide urea concentration in substrate solution, " 0 " hole OD value obviously improves, and IC
50change little; But when hydrogen peroxide urea concentration is increased to after certain value, " 0 " hole OD value no longer raises.Consider the stability of test kit, " 0 " hole value is arranged on to 1.8～2.0 to extend the validity period of test kit, finally determine that the concentration of Urea Peroxide in substrate solution is 3.0mmol/L.
The best concentration of substrate optimization of table 5
| Hydrogen peroxide urea concentration (mmol/L) | 0 hole OD value | IC 50Value (μ g/L) |
| 0.25 | 1.037 | 71.8 |
| 0.50 | 1.613 | 77.1 |
| 1.00 | 1.895 | 78.3 |
| 3.00 | 1.827 | 84.3 |
The foundation of 7 typical curves
By ABZSO
2nH
2standard substance are mixed with 6 concentration gradients such as 0,20,40,80,160,320 μ g/L, and 3 parallel holes of each concentration are measured drawing standard curve according to indirect competitive ELISA method.Replication 5 times.As shown in Figure 2, the regression equation of racing ELISA detecting method and the index of correlation are: y=-0.494x+1.448, r=0.997, IC
50value is 90.61 ± 8.29 (n=5), and linearity range is 20～320 μ g/L.
8 specificitys
By the specificity of cross reacting rate reflection ELISA detection method of the present invention.Become concentration gradient to carry out indirect competitive ELISA various benzimidazoles medicines or metabolite standard substance doubling dilution respectively, calculate IC
50value, by formula 1 and haptens ABZSO
2nH
2standard substance IC
50value calculates cross reacting rate, the results are shown in Table 6.Result shows, the indirect competitive ELISA method that this research is set up has certain cross reaction to Albendazole sulfone, and with other benzimidazoles medicine or the equal no cross reaction of metabolite or very low.
Cross reacting rate=IC
50(haptens)/IC
50(other drug) × 100% (formula 1)
The specificity of table 6 ELISA detection method of the present invention
| Competition thing | IC 50(μg/L) | Cross reacting rate (%) |
| The amino sulfone of albendazole-2- | 85 | 100％ |
| Albendazole sulfone | 125 | 68.00 |
| Albendazole-sulfoxide | 2241.38 | 3.7 |
| Albendazole | ＞10000 | ＜0.85 |
| Fenbendazole | ＞10000 | ＜0.85 |
| Oxibendazole | ＞10000 | ＜0.85 |
| Vermox | ＞10000 | ＜0.85 |
| Flubendazole | ＞10000 | ＜0.85 |
| Thiabendazole | ＞10000 | ＜0.85 |
Embodiment 4: the assembling of ELISA detection kit of the present invention
Test kit moiety
(1) be coated with coating antigen ABZSO
2nH
2the enzyme plate of-GA-OVA;
(2) ABZSO
2nH
26 bottles of standard solutions, concentration is respectively 0,20,40,80,160,320 μ g/L;
(3) GA/2A11 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 80.00g, KH
2pO
42.00g, Na
2hPO
412H
2o 29.00g, KCl 2.00g, adds distilled water to 1000mL;
(6) concentrated cleaning solution: NaCl 80.00g, KH2PO4 2.00g, Na
2hPO
412H
2o 29.00g, KCl 2.00g, Tween205mL, adds distilled water to 1000mL
(7) substrate mixed solution: 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), mix, now with the current.
(9) stop buffer: 2mol/L sulphuric acid soln.
A preparation method for enzyme plate, the steps include:
(1) coated: with carbonate buffer solution by ABZSO
2nH
2-GA-OVA is diluted to 0.25 μ g/mL coating antigen 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, leave standstill after 30s, throw away washings, on thieving paper, pat dry; Repeated washing 3 times.
(3) sealing: accurately draw 250 μ L confining liquids in each enzyme mark hole, level is placed in wet box, and 37 DEG C 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 after 30s, throw away washings, on thieving paper, pat dry; Repeated washing 3 times.
(5) dry: after patting dry on thieving paper; Enzyme plate is inverted in 37 DEG C of incubators and is dried 0.5h.
(6) encapsulation: enzyme plate packs aluminium foil bag into after drying together with siccative, encapsulates with vacuum packaging machine.
Embodiment 5:
Can identify the test kit of the amino sulfone of the residual marker albendazole of albendazole-2-in the application detecting in the amino sulfone residual quantity of albendazole in edible animal tissue-2-, the steps include:
Reagent preparation
Washings: by the concentrated cleaning solution providing in test kit with using after 10 times of dilutions of distilled water.
Sample diluting liquid preparation: accurately take NaCl 8.0g, KH
2pO
40.2g, Na
2hPO
4.12H
2o 2.9g, KCl 0.2g, in 500mL beaker, adds a small amount of ultrapure water and dissolves, and distilled water is settled to 1000mL.
Mass percent is the preparation of 5% potassium hydroxide solution: accurately take potassium hydroxide 5g, distilled water dissolves, cooling and be settled to 100mL.
0.1M sodium carbonate solution preparation: accurately weighing sodium carbonate 1.06g, ultrapure water dissolves and is settled to 100mL.
The preparation of substrate mixed solution: according to each institute expense, get appropriate substrate A liquid and B liquid and mix in the ratio of 1: 100, now with the current.
Tissue sample processing
(1) processing of muscle (ox, sheep, pig, chicken, fish) sample: take the homogeneous sample 3.00 ± 0.02g of muscle in 50mL centrifuge tube, add ethyl acetate 15mL, the 1min of whirlpool mixing immediately makes sample dispersion complete.Adding mass percent is 5% potassium hydroxide solution 50 μ L, whirlpool mixing 5min; Add again anhydrous sodium sulphate 1g, whirlpool mixing 30s.Whirlpool mixing 30s after standing 30min, the centrifugal 5min of 4000r/min, gets supernatant liquor 5mL 50～60 DEG C of nitrogen in 10mL centrifuge tube and dries up.In residue, add normal hexane 2mL, whirlpool mixing 30s; Add again phosphate buffer 1 mL, the centrifugal 5min of 4000r/min after whirlpool mixing 1min.Remove upper strata normal hexane and middle impurity layer, take off layer water 150 μ L for analysis.
(2) processing of liver (ox, sheep, pig, chicken) sample: take the homogeneous sample 2.00 ± 0.02g of liver in 50mL centrifuge tube, add ethyl acetate 20mL, the 1min of whirlpool mixing immediately makes sample dispersion complete.Adding 5% mass percent is 5% potassium hydroxide solution 100 μ L, whirlpool mixing 5min; Add again anhydrous sodium sulphate 1g, whirlpool mixing 30s.Whirlpool mixing 30s after standing 30min, the centrifugal 5min of 4000r/min, draws supernatant liquor 2.5mL in 10mL centrifuge tube, and 50～60 DEG C of nitrogen dry up.In residue, add normal hexane 2mL, whirlpool mixing 30s, then add phosphate buffer 1 mL, the centrifugal 5min of 4000r/min after whirlpool mixing 1min.Remove upper strata normal hexane and middle impurity layer, take off layer water 150 μ L for analysis.
(3) processing of milk sample: take milk sample 1.00 ± 0.02g in 10mL centrifuge tube, add 0.1mol/L sodium carbonate solution 20 μ L, whirlpool mixing 10s; Add again ethyl acetate 7mL, the 2min of whirlpool mixing immediately.The centrifugal 10min of 4000r/min, draws supernatant liquor 4mL in another 10mL centrifuge tube, adds 1mL ultrapure water, whirlpool mixing 5s.The centrifugal 1min of 4000r/min, proceeds to organic phase in another 10mL centrifuge tube, and 50～60 DEG C of nitrogen dry up.In residue, add normal hexane 2mL, whirlpool mixing 30s, then add phosphate buffer 1 mL, the centrifugal 5min of 4000r/min after whirlpool mixing 1min.Remove upper strata normal hexane and middle impurity layer, take off layer water 150 μ L for analysis.
(4) processing of egg sample: take the equal quality sample 3.00 ± 0.02g of egg in 50mL centrifuge tube, add 0.1mol/L sodium carbonate solution 50 μ L, whirlpool mixing 10s; Add ethyl acetate 15mL, the 5min of whirlpool mixing immediately, adds anhydrous sodium sulphate 3g again, whirlpool mixing 30s.The centrifugal 5min of 4000r/min, draws supernatant liquor 5mL in another 10mL centrifuge tube, and 50～60 DEG C of nitrogen dry up.In residue, add normal hexane 2mL, whirlpool mixing 30s, then add phosphate buffer 1 mL, the centrifugal 5min of 4000r/min after whirlpool mixing 1min.Remove upper strata normal hexane and middle impurity layer, take off layer water 150 μ L for analysis.
Note: while adopting this kit measurement, being 1 to the dilution factor of muscle and egg sample, is 4 to the dilution factor of liver sample, and the extension rate of milk sample is 0.875.
ELISA measures program
(1) take out test kit, balance, to room temperature, is inserted micropore frame by the hole bar of enough standard substance and sample quantity used.
(2) with reference liquid or sample liquid 50 μ L in micropore separately; 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, on thieving paper, pat dry.Accurately draw washings 250 μ L in each hole, leave standstill 30s left and right, get rid of clean washings, on thieving paper, pat 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, on thieving paper, pat dry.Accurately draw washings 250 μ L in each hole, leave standstill 30s left and right, get rid of clean washings, on thieving paper, pat dry.Repeated washing 5 times.
(6) add substrate mixed solution 100 μ L to each hole, fully mix; Level is put in wet box, hatches 15min for 37 DEG C.
(7) add stop buffer 50 μ L to each hole; Fully mix; Light absorption value is measured at the inherent 450nm of 30min place.
Result is judged
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 20～320 μ g/L, taking 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 4 calculation samples, by inhibiting rate substitution regression equation, calculate mensuration concentration, be multiplied by dilution factor, be the residual concentration of the amino sulfone of albendazole in sample-2-.
(formula 4)
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
on substitution typical curve, find corresponding concentration (C), and calculate standard deviation (SD).Calculate 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 7.
Table 7 ABZSO
2nH
2lowest detectable limit in various animal tissuess sample
The precision of test kit of the present invention
Amino albendazole-2-sulfone standard substance are diluted to 6 concentration of 0,20,40,80,160,320 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 corresponding each standard substance concentration is obtained to 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, the results are shown in Table 8.
The precision of table 8 test kit of the present invention
The accuracy of test kit of the present invention
In homogeneous good various muscle, milk or egg tissue, add the amino sulfone standardized solution of albendazole-2-, make its final concentration be respectively 25,50 or 100 μ g/kg; In homogeneous good various liver organizations, add the amino sulfone standardized solution of albendazole-2-, make its final concentration be respectively 125,250 or 500 μ g/kg (beef liver is 100,200 or 400 μ g/kg).5 Duplicate Samples of each concentration, then carry out sample preparation, and ELISA measures drug level, repeat 3 batches, calculate recovery rate, and the rate of recovery is calculated by formula 3, and calculates batch interior and interassay coefficient of variation, the results are shown in Table 9～19.
(formula 3)
ABZSO in table 9 ox muscle
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 10 cattle liver
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 11 sheep muscle
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 12 sheep liver
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 13 chicken muscle
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 14 chicken liver
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 15 pig muscle
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 16 pig liver
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 17 fish muscle
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 18 milk
2nH
2add the rate of recovery and the variation coefficient
ABZSO in table 19 egg
2nH
2add the rate of recovery and the variation coefficient
Claims (5)
1. can identify the monoclonal antibody that Ah benzene reaches the amino sulfones of azoles – 2 –, it is characterized in that, it is to be that the hybridoma cell strain GA/2A11 of CCTCC NO:C201142 is secreted by preserving number.
2. a method that detects the amino sulfone of albendazole-2-, the steps include:
(1) amino haptens albendazole-2-sulfone and ovalbumin coupling are obtained to coating antigen;
(2) prepare monoclonal antibody with the hybridoma cell strain GA/2A11 that preserving number is CCTCC NO:C201142;
(3) be coated with solid phase carrier with the coating antigen of step 1;
By testing sample with ethyl acetate under the alkalization such as potassium hydroxide or sodium carbonate solution condition, extract, nitrogen dries up, normal hexane purifies and sample diluting liquid again dissolves and obtains determinand;
(5) the determinand of step (4) is carried out to enzyme linked immunosorbent detection;
Step (5) component and the proportioning of sample diluting liquid is: NaCl 8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o 2.90g, KCl 0.20g, adds distilled water and is settled to 1000mL.
3. the test kit of a kind of monoclonal antibody that can identify the amino sulfone of the residual marker albendazole of albendazole-2-claimed in claim 1, this test kit comprises:
(1) be coated with coating antigen ABZSO
2nH
2the enzyme plate of – GA – OVA;
(2) ABZSO
2nH
26 bottles of standard solutions, concentration is respectively 0,20,40,80,160,320 μ g/L;
(3) GA/2A11 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 80.00g, KH
2pO
42.00g, Na
2hPO
412H
2o 29.00g, KCl 2.00g, adds distilled water to 1000mL;
(6) concentrated cleaning solution: NaCl 80.00g, KH
2pO
42.00g, Na
2hPO
412H
2o 29.00g, KCl 2.00g, Tween20 5mL, adds distilled water to 1000mL;
(7) substrate mixed solution: accurately draw substrate B liquid 10mL, add 100 μ L substrate A liquid, mix;
(9) stop buffer: 2mol/L sulphuric acid soln;
Described substrate A liquid and substrate B liquid are all purchased from Wuhan Fei Yuan Science and Technology Ltd..
4. a kind of test kit that can identify the amino sulfone of the residual marker albendazole of albendazole-2-according to claim 3, is characterized in that: the preparation method of described a kind of enzyme plate, the steps include:
(1) coated: with carbonate buffer solution by ABZSO
2nH
2-GA-OVA is diluted to 0.25 μ g/mL coating antigen solution, 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, draw 250 μ L washingss in each enzyme mark hole, leave standstill after 30s, throw away washings, on thieving paper, pat dry; Repeated washing 3 times;
(3) sealing: draw 250 μ L confining liquids in each enzyme mark hole, level is placed in wet box, and 37 DEG C of incubators are hatched 2h;
(4) wash plate: throw away confining liquid, draw 250 μ L washingss in each enzyme mark hole, leave standstill after 30s, throw away washings, on thieving paper, pat dry; Repeated washing 3 times;
(5) dry: after patting dry on thieving paper; Enzyme plate is inverted in 37 DEG C of incubators and is dried 0.5h;
(6) encapsulation: enzyme plate packs aluminium foil bag into after drying together with siccative, encapsulates with vacuum packaging machine;
Described carbonate buffer solution pH9.6: accurately take Na
2cO
31.59g, NaHCO
32.93g, a small amount of ultrapure water dissolves, and is settled to 1000mL;
Washings pH7.4: accurately take NaCl 8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o 2.90g, KCl 0.20g, a small amount of ultrapure water dissolves, and adds Tween20 0.50mL, is settled to 1000mL;
Phosphate buffered saline buffer pH7.4: accurately take NaCl 8.00g, KH
2pO
40.20g, Na
2hPO
412H
2o 2.90g, KCl 0.20g, a small amount of ultrapure water dissolves, and is settled to 1000mL;
Confining liquid: accurately take ovalbumin 10.00g, add phosphate buffer 1 000mL, stirring and evenly mixing until albumen dissolve completely.
5. a kind of test kit that can identify the amino sulfone of the residual marker albendazole of albendazole-2-claimed in claim 3 is detecting the application of the amino sulfone of albendazole in edible animal tissue-2-in residual.
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| 猪牛羊可食性组织中阿苯达唑残留检测方法研究;范盛先;《中国农业科学》;20041231;第37卷(第8期);1229-1234 * |
| 范盛先.猪牛羊可食性组织中阿苯达唑残留检测方法研究.《中国农业科学》.2004,第37卷(第8期),1229-1234. |
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