CN102768278A - Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant - Google Patents
Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody capable of identifying a beta-receptor stimulant. The monoclonal antibody is secreted by a hybridoma cell line Sal, which is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO:C201187. The invention also discloses an enzyme-linked immunosorbent assay method and a kit for detecting the beta-receptor stimulant, and application thereof to detection of the beta-receptor stimulant. Compared with prior art, the monoclonal antibody prepared by the invention can identify 8 beta-receptor stimulant drugs, has a wide range of identification; and the monoclonal antibody, the ELISA kit and the ELISA method provided by the invention have high sensitivity, and can detect beta-receptor stimulant residues with lower concentration.
Description
Technical field
The invention belongs to residue of veterinary drug analysis and immunological technique field, be specifically related to a kind ofly can discern 8 kinds of anti-depressant monoclonal antibodies of beta-receptor and enzyme-linked immunoassay method and kit.
Background technology
The beta-receptor excitant is the phenolethanolamine analog derivative of one group of structure and similar adrenaline of physiological function and norepinephrine; Can combine with the B-adrenergic receptor on most of histocytes in the animal body; And activation B-adrenergic receptor; Thereby the decomposition of fat and the oxidation of free fatty acid in the acceleration cell. therefore, the anti-depressant main effect of beta-receptor is the growth that promotes animal muscle tissue, reduces the content of trunk fat; Improve lean meat percentage. meanwhile, the raising speed of growth and efficiency of feed utilization that can be in various degree.European Union and the legislation of some other countries and regions ban use of the beta-receptor excitant, and No. 235 bulletin of China Ministry of Agriculture provides against in animal food and use.
Effectively control beta-receptor stimulants is abused and is residual, and detection method is crucial.In the domestic and foreign literature about beta-receptor excitant method for detecting residue; Though the sensitivity of instrument detecting and accuracy are more and more optimized and are improved, its defective that is difficult to avoid is arranged, the method for distilling that has is loaded down with trivial details; The rare outfit of instrument and equipment common laboratory (like supercritical extract) of the employing that has; And it is long to detect required time, is unfavorable for the screening of batch samples, can not detect at the scene fast.Enzyme-linked immune analytic method (ELISA) can overcome the defective of instrumental analysis, is a kind of effectively residual screening technique in enormous quantities, and the application on this type of medicine has very big development prospect.Enzyme-linked immunosorbent assay is with antigen-antibody reaction and the ultramicro method that modern means of testing combines, and integrates the high sensitivity of modern test method and the strong specificity (Li Junsuo etc., 2002) of anti-former – antibody response.With respect to instrumental method, advantage such as that the 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.
Guy etc. (1993) combine the preparation comlete antigen with salbutamol with bovine serum albumin; Obtain polyclonal antibody behind the immunity new zealand white rabbit to 5 kinds of beta-receptor stimulants idols cross reactions such as salbutamols; The enzyme immunization method of its foundation detects salbutamol content in the urine sample, and lowest detection is limited to 0.14ng.Lei etc. (2008) use salbutamol as the monoclonal antibody of haptens preparation salbutamol, Clenbuterol, Terbutaline, adrenal gland to be have cross reaction, and the ELISA method LDL of foundation can reach 0.052ng/mL.The sensitivity of polyclonal antibody is higher in the above-mentioned research, but is unfavorable for standardization; Though monoclonal antibody is beneficial to standardized production, but sensitivity is not enough again, and the drug kinds that detects is limited.Therefore, prepare a kind of highly sensitive being used to and detect anti-depressant monoclonal antibody of multiple beta-receptor and enzyme linked immunological kit, and it is significant to set up corresponding ELISA detection method.
Summary of the invention
The purpose of this invention is to provide a kind of monoclonal antibody and enzyme-linked immunoassay method and the kit that can discern eight kinds of beta-receptor excitants (Clenbuterol, salbutamol, Cimaterol, Mabuterol, gram human relations third sieve, horse are sprayed special sieve, Terbutaline and tulobuterol), the present invention also provides said monoclonal antibody to detect application and enzyme-linked immunoassay method and the application of kit in the beta-receptor drug-testing in the anti-depressant enzyme linked immunological kit of beta-receptor in preparation.
Above-mentioned purpose realizes through following technical scheme:
A kind ofly can discern the anti-depressant monoclonal antibody of beta-receptor, it is to be that the hybridoma Sal of CCTCC NO:C201187 is secreted by preserving number.
Described hybridoma Sal is deposited in Chinese typical culture collection center, and preserving number is CCTCC NO:C201187.
Described monoclonal antibody detects the application in the anti-depressant enzyme linked immunological kit of beta-receptor in preparation.
The kit that comprises described monoclonal antibody.
Said kit is to be used to detect the anti-depressant enzyme linked immunological kit of beta-receptor.
The application of described kit in the beta-receptor drug-testing.
Further, the invention provides the anti-depressant enzyme-linked immunoassay method of a kind of detection beta-receptor, may further comprise the steps:
(1) haptens succinic anhydride salbutamol (SAL-SA) and bovine serum albumin(BSA) (BSA) coupling are obtained immunogene (SAL-SA-BSA);
(2) haptens p-aminobenzoic acid salbutamol (SAL-ABA) and ovalbumin (OVA) coupling are obtained coating antigen (SAL-ABA-OVA);
(3) utilize the immunogen preparing of step (1) to obtain the hybridoma Sal that preserving number is CCTCC NO:C201187;
(4) utilize preserving number to prepare monoclonal antibody for the hybridoma Sal of CCTCC NO:C201187;
(5) coating antigen with step (2) encapsulates solid phase carrier;
(6) sample pre-treatments: the pig urine samples adds acetonitrile treatment and gets supernatant after centrifugal; The pork sample is used the ethyl acetate back extraction after acid treatment, get ethyl acetate layer nitrogen again and dry up, and centrifuging and taking supernatant after residue redissolves gets determinand;
(7) determinand to step (6) detects.
The application of described enzyme-linked immunoassay method in the beta-receptor drug-testing.
The invention has the beneficial effects as follows:
1. the monoclonal antibody of the present invention's preparation can be discerned 8 kinds of beta-receptor stimulants, and identification range is wide;
2. the enzyme-linked immunoassay method of monoclonal antibody, enzyme linked immunological kit and the foundation of the present invention preparation is highly sensitive, and the beta-receptor excitant class that can detect lower concentration is residual, has precision height, characteristics that sensitivity is good simultaneously.
Description of drawings
Fig. 1 is technology path figure of the present invention.
Fig. 2 for of the present invention be the indirect competitive ELISA reaction normal curve of standard items with the Clenbuterol, the X axle is Clenbuterol (CL) concentration of standard solution logarithm value, the Y axle is that the OD value of Clenbuterol standard solution is divided by " zero " hole OD value (B/B
0).
Embodiment
Through embodiment the present invention is described further below, but does not limit the present invention.
The preparation of embodiment 1 immunogene and coating antigen
1.1 salbutamol immunity haptens (SAL-SA) is synthetic
Salbutamol immunity haptens (SAL-SA) is synthetic according to following route: take by weighing salbutamol alkali 80mg (0.33 mmol) and use the 10mL dissolve with methanol; Rotary evaporation is a yellow oil; Add the 16mL anhydrous alcohol solution again; And constantly stir, dropwise add succinic anhydride 36mg then and (be dissolved in the pyridine solution, 0.36mmol).After white casse occurring, detect the structure of succinic anhydride with thin-layer chromatography.After reacting 3h at ambient temperature, stop to stir, the centrifugal 15min of 2750r/min, abandoning supernatant, with absolute ethanol washing sediment 3 times, solvent evaporated gained white solid is haptens SAL-SA.
1.2 the preparation of salbutamol immunogene (SAL-SA-BSA)
SAL-SA 40mg is dissolved in 2mL N, dinethylformamide (DMF), 1mL 1, in the mixed solution of 4-dioxane, 30 μ L tri-n-butylamines, stirs 30min under the room temperature, adds chloro-carbonic acid isobutyl fat 30 μ L in the ice bath, and 4 ℃ of stirring reaction 5h obtain A liquid.Accurately take by weighing BSA 86mg, it fully is dissolved in is B liquid in the 25mL borate buffer solution (pH8.5).In the ice bath, under the magnetic agitation A liquid slowly is added drop-wise in the B liquid, 4 ℃ of following stirring reactions spend the night, the bag filter of packing into afterwards, the 72h that in the pH7.4 phosphate buffer, dialyses, during change dislysate 6 times.Lyophilized promptly gets conjugate SAL-SA-BSA, puts-20 ℃ of preservations, uses as immunogene.
1.3 salbutamol encapsulates the synthetic of haptens (SAL-ABA)
It is synthetic according to following route that salbutamol encapsulates haptens (SAL-ABA).Take by weighing p-aminobenzoic acid 137mg, be dissolved among the HCl of 3mL 1mol/L, ice bath stirs.Take by weighing NaNO
2160mg is dissolved in the 1mL ultrapure water, under the stirring at low speed state, with NaNO
2The each 100 μ L of solution are every to be added drop-wise in the p-aminobenzoic acid solution at a distance from 15s, uses the TLC monitoring reaction.Take by weighing salbutamol 240mg, be dissolved in the 5mL borate buffer solution (0.05mol/L), treat that above-mentioned reaction finishes, salbutamol solution is dropwise added, reaction is spent the night, and obtains peony haptens solution.
1.4 salbutamol coating antigen (SAL-ABA-OVA) is synthetic
Get SAL-ABA 3mL, add 40 μ L tri-n-butylamines, ice bath adds 40 μ L isobutyl chlorocarbonates after stirring 15min, and priming reaction 5.5h obtains A liquid.Taking by weighing OVA 46mg is dissolved in and is B liquid in the carbonate buffer solution of 20 mL pH9.6.A liquid is slowly added in the B liquid, ice bath stirring reaction 8h, the bag filter of packing into afterwards, the 72h that in the pH7.4 phosphate buffer, dialyses, during change dislysate 6 times.Lyophilized had both got conjugate SAL-ABA-OVA, put-20 ℃ of preservations.Use as coating antigen.
2.1 mouse immune
With reference to the method in Xue Qingshan " philosophy and technique of in vitro culture " the Science Press calendar year 2001 version: the SAL-SA – BSA conjugate with embodiment 1 preparation is an immunogene, immune Balb/C female mice.Fundamental immunity of immune programme for children employing and several booster immunization.Use the subcutaneous fundamental immunity of carrying out of nape portion that the immunogenic protein emulsion of 100 μ g is injected in mouse that contains with isopyknic Freund's complete adjuvant emulsification during first immunisation, whenever carry out booster immunization later at a distance from 15 days immunogenic protein emulsions of 100 μ g that contain with incomplete Freund emulsification.From immunity three times, tail blood was adopted on the 8th day in each immunity back, separation of serum, and indirect elisa method detects serum antibody titer.The qualified mouse of immunity (height of tiring, sensitivity good) stops immunity in order to merging.
2.2 Fusion of Cells and screening
Qualified mouse peritoneal injection contains the immunogenic protein solution of 100 μ g (not adding adjuvant), reinforced immunological to give immunity in preceding 3 days of fusion (best and immunity last time was separated by more than 3 weeks).According to myeloma cell's count results, get 3～5 * 10
7Individual myeloma cell mixes with immune spleen cell that (ratio is 1:10～5:10).The centrifugal 5min of 1500r/min after emptying to the greatest extent on the centrifuge tube, tips upside down on the thieving paper, and the control solid carbon dioxide drips.Knock the pipe end lightly, make the pipe floor cells become pasty state, it is positioned in 37 ℃ of water-baths; With suction have 0.8mL in advance temperature to 37 ℃ 50% polyglycol (PEG; Available from Amersco) the lmL calibrated pipet be inserted into the pipe end, slowly add PEG in the 60sec to cell mixing, the limit edged stirs gently; Leave standstill 90sec after adding, centrifuge tube is inserted on the centrifuge tube shelf.Remove the water-bath cup; Drawing warm in advance RPMI – 1640 basic culture solutions (available from Hyclone) to 37 ℃ of 10mL with suction pipe slowly is added on the fused cell along tube wall; The limit edged rocks centrifuge tube gently; Dropwise add 1mL (3sec/ drips) on the 1st minute, added 2mL again on the 2nd minute, add remaining 7mL (adding in the 5min) at last.After adding first 10mL, then add RPMI – 1640 nutrient culture media to 50mL, tighten lid after adding, slowly put upside down several times mixing along tube wall.The centrifugal 5min of 1500r/min, supernatant discarded stirs fused cell resuspended with the 72mL HAT complete medium that contains feeder cells gently.With the fused cell suspension inoculation in 96 porocyte culture plates, 2/hole.Once merge and to inoculate 5 96 porocyte culture plates, place 37 ℃ of 5%CO
2Cultivate in the incubator.Be 0d, the front 3d kinetocyte plate of trying not from merging to count the same day, keep the incubator homeostasis.3d adds in every hole 1 HAT complete medium (available from Amersco) and observes the colony growing state; The every hole sucking-off of 5d l/2 culture supernatant (100 μ L) adds 1 HT complete medium (available from Amersco) again; Every later on the suction at a distance from the same method of 3d gone the l/2 culture supernatant, changes to the HT complete medium.Growing state according to cell; To account for hole floorage about 1/4 be desirable cells and supernatant when cell grows to; SAL-ABA – OVA conjugate with inventor's preparation is a coating antigen, utilizes the ELISA method to filter out the positive cell hole of excreting beta-receptor agonist antibody-like.The positive cell hole that screens is utilized continuous 3 time cloningizations of limiting dilution assay; Finally set up the hybridoma cell strain of the anti-beta-receptor excitant of strain stably excreting antibody-like; It is 103.3 that this hybridoma cell strain is carried out chromosome counting mean value; Be higher than the chromosome number (SP2/0 myeloma cell's chromosome average is 58, and splenocyte chromosome is 40) of parental cell, the SP2/0 myeloma cell really of fused cell and the hybridization product of splenocyte are described.The applicant is this hybridoma called after Sal, and delivers Chinese typical culture collection center (CCTCC) preservation that is positioned at Wuhan City, Hubei Province Wuhan University on September 8th, 2011, and its preserving number is CCTCC NO: C201187.
2.3 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 incomplete Freund carries out pre-service.Transfer to 1 * 10 with RPMI – 1640 basal medium suspension cells and with cell number
6Individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spiritual variation, the dying ascites of gathering when motionless, purifying obtains monoclonal antibody.Confirm that with the quick ELISA homotype of mouse monoclonal antibody detection kit monoclonal antibody is an IgG1 κ hypotype.
The foundation of embodiment 3 racing ELISA detecting methods
3.1 reagent preparation
Carbonate buffer solution (pH9.6): accurately take by weighing Na
2CO
31.59g, NaHCO
32.93g a small amount of ultrapure water dissolving is settled to 1000mL.
Cleansing solution (pH7.4): accurately take by weighing NaCl 8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O 2.90g, KCl 0.20g, a small amount of ultrapure water dissolving adds Tween 20 0.50mL, is settled to 1000mL.
Phosphate buffer (pH7.4): accurately take by weighing NaCl 8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O 2.90g, KCl 0.20g, a small amount of ultrapure water dissolving is settled to 1000mL.
Confining liquid: accurately take by weighing ovalbumin 10.00g, add phosphate buffer 1 000mL, stirring and evenly mixing dissolves until albumen fully.
The substrate mixed liquor: 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 is joined existing usefulness at present.
Stop buffer: accurately measure concentrated sulphuric acid 100mL, slowly be added drop-wise in the 800mL ultrapure water.
3.2 square formation titrimetry
Adopt square formation titration initial option coating antigen concentration and antibody dilution.Use carbonate buffer solution that SAL-ABA-OVA coating antigen doubling dilution is become 80,40,20,10,5,2.5,1.25 μ g/mL, from first to the 7th row laterally adds 96 hole ELISA Plates successively, and 4 ℃ are spent the night; Wash 3 times, clap and do, add confining liquid 250 μ L, 37 ℃ of sealing 2h; Wash 3 times; Clap and do; Monoclonal antibody uses the phosphate buffer doubling dilution to become 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000, and from first to the 8th leu vertically adds 96 hole ELISA Plates and adds ELISA Plate, hatches 30min for 37 ℃; Wash 3 times; Clap and do; Each hole adds uses phosphate buffer 1: (be called for short two resists the sheep anti-mouse igg antibody of the horseradish peroxidase-labeled of 5000 dilutions; The following indication two anti-sheep anti-mouse igg antibodies that are horseradish peroxidase-labeled fly Yi, a legendary monarch of Youqiong State in the xia Dynasty Bioisystech Co., Ltd available from Wuhan) 100 μ L, hatch 30min for 37 ℃; Wash 4 times, clap and do, each hole adds substrate mixed liquor 100 μ L, lucifuge colour developing 15min; Add stop buffer 50 μ L; Measure OD value (OD value) with automatic ELIASA in the 450nm wavelength.The square formation titration results is seen table 1, and the several OD values of initial option are near 2.0, and adjacent two hole OD values have the antibody dilution combination that encapsulates concentration and correspondence of bigger variation.The result shows, can select the combination of following coating antigen concentration and antibody dilution: (5,16000) and (10,32000).
The titration of table 1 monoclonal antibody square formation
3.3 the best selection that encapsulates concentration
With the Clenbuterol is the competition thing, is set to 0,0.05,0.1,0.4,0.8,1.6 μ g/L concentration gradients, and the antibody dilution of selecting with 3.2 square formation titrimetrys respectively that encapsulates concentration and correspondence is combined into and connects competitive ELISA in the ranks.Logarithm value with the competition substrate concentration is drawn the inhibition curve as horizontal ordinate, B/B0 (the OD value when suppressing with no medicine is B0, and the OD value when the respective concentration medicine suppresses is the B value) as ordinate.The result sees table 2, with " 0 " hole OD value and IC
50Value is confirmed best coating antigen concentration as judging index.Visible by data, it is 10 μ g/mL that the best encapsulates concentration, and antibody dilution is tentatively confirmed as 1:32000.
Table 2 the best encapsulates concentration optimization
| Coating antigen concentration (μ g/mL) | Antibody coefficient multiple (1:X) | 0 hole OD value | IC 50Value (μ g/L) |
| 5 | 16000 | 2.324 | 0.78 |
| 10 | 32000 | 1.773 | 0.49 |
3.4 the dilution selection of optimum antibody
Encapsulate the concentration coated elisa plate with the best, with antibody with 1:30000 for centre concentration equal difference designs several dilution gradients, its 0 hole and IC
50Value is seen table 3.Along with the increase of antibody dilution, IC
50Value reduces, and still " 0 " hole value is near 2.0, when 0 hole be worth its IC when hanging down
50Value raises on the contrary, and therefore selecting 1:28000 is the optimum antibody dilutability.
Table 3 optimum antibody dilutability is optimized
| Antibody coefficient multiple (1:X) | 0 hole OD value | IC 50Value (μ g/L) |
| 28000 | 1.894 | 0.47 |
| 30000 | 1.654 | 0.43 |
| 32000 | 1.488 | 0.38 |
3.5 the foundation of typical curve
The Clenbuterol standard items are mixed with 6 concentration gradients such as 0,0.05,0.1,0.4,0.8,1.6 μ g/L, measure according to the indirect competitive ELISA condition after optimizing, drawing standard curve (seeing accompanying drawing 2) repeats 5 times.The regression equation of racing ELISA detecting method and the index of correlation are: y=-2.2231x+2.7328, r
2=0.9962, IC
50Value is 0.46 ± 0.075 μ g/L (n=5), and the range of linearity is 0.05～1.6 μ g/L.
3.6 cross reaction experiment
Become concentration gradient to carry out indirect competitive ELISA various beta-receptor stimulants standard items doubling dilutions respectively, the drawing standard curve calculates IC
50Value is with Clenbuterol standard items IC
50The value contrast obtains cross reacting rate, and the result sees table 4.This monoclonal antibody has recognition capability to 8 kinds of beta-receptor excitant classes.
Table 4 monoclonal antibody is to the cross reacting rate of beta-receptor stimulants
| Medicine | IC 50(μg/L) | Cross reacting rate (%) |
| Clenbuterol | 0.46 | 100 |
| Salbutamol | 0.86 | 53.5 |
| Cimaterol | 0.55 | 83.6 |
| Mabuterol | 0.46 | 100 |
| Gram human relations third sieve | 0.64 | 71.8 |
| Horse is sprayed special sieve | 1.04 | 44.2 |
| Tulobuterol | 7.3 | 6.3 |
| Terbutaline | 20.0 | 2.3 |
| Ractopamine | ＞10000 | ＜0.005 |
| Dopamine | ＞10000 | ＜0.005 |
| Isoprel | ＞100 | ＜0.46 |
| Sharp holder monarch | ＞100 | ＜0.46 |
| Horse baud sieve | ＞10000 | ＜0.005 |
| Fenoterol | ＞100 | ＜0.46 |
Embodiment 4: the assembling of ELISA detection kit of the present invention
4.1 kit is formed:
⑴ be coated with the ELISA Plate of coating antigen SAL-ABA – OVA;
⑵ 6 bottles of Clenbuterol standard solutions, concentration are respectively 0,0.05,0.1,0.4,0.8,1.6 μ g/L;
⑶ hybridoma Sal monoclonal antibody working fluid;
⑷ the sheep anti-mouse igg antibody working fluid of horseradish peroxidase (HRP) mark;
⑸ 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;
⑹ 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
⑺ substrate mixed liquor: 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 is joined existing usefulness at present.
⑼ stop buffer: 2mol/L sulfuric acid solution.
4.2 the preparation of ELISA Plate:
⑴ encapsulate: with carbonate buffer solution SAL-ABA – OVA is diluted to 10 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 ℃.
⑵ wash plate: throw away ELISA Plate endoperidium original solution, clap to do, accurately draw 250 μ L cleansing solutions in each enzyme mark hole, leave standstill 30s after, throw away cleansing solution, do at the thieving paper arsis; Repeated washing 3 times.
⑶ 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.
⑷ wash plate: throw away confining liquid, accurately draw 250 μ L cleansing solutions in each enzyme mark hole, leave standstill 30s after, throw away cleansing solution, do at the thieving paper arsis; Repeated washing 3 times.
⑸ oven dry: after the thieving paper arsis is done; ELISA Plate is inverted oven dry 0.5h in 37 ℃ of incubators.
⑹ encapsulation: ELISA Plate oven dry back and the drying agent aluminium foil bag of packing into together encapsulates with vacuum packaging machine.Described drying agent is wherein a kind of of silica gel or anhydrous calcium chloride.
Embodiment 5: kit is the application in the beta-receptor excitant residue detection in edible animal tissue
5.1 reagent preparation
0.05mol/L hydrochloric acid: accurately measure concentrated hydrochloric acid 4.1 mL, add mixing in the appropriate amount of deionized water, be settled to 1000mL, be 0.05 mol/L hydrochloric acid.
Sample extracting solution: 0.05 mol/L hydrochloric acid: acetonitrile=6:4.
Cleansing solution: the concentrated cleaning solution liquid that provides in the kit with 10 times of dilutions of deionized water, is put 4 ℃ of refrigerators and preserved subsequent use.
Sample dilution: the concentrated sample dilution that provides in the kit with 5 times of dilutions of deionized water, is put 4 ℃ of refrigerators and preserved subsequent use.
5.2 tissue sample is handled
The pig urine samples is got 1mL clarification pig urine (if cloudy urine is taken a sample behind the centrifugal 10min of 4000r/min again) and in the 10mL centrifuge tube, is added acetonitrile 50uL, whirlpool 1min; The centrifugal 5min of 4000r/min gets supernatant and detects.
The pork sample takes by weighing homogeneous pork 2.0 ± 0.01g in the 50mL plastic centrifuge tube, adds the 6mL sample extracting solution, vortex 1min; The centrifugal 20min of 4000r/min gets 3mL upper strata liquid in the 10mL plastic centrifuge tube, adds 4mL ethyl acetate in the 10mL plastic centrifuge tube, vortex 30S, the centrifugal 10min of 4000r/min.Getting ethyl acetate layer dries up in 30-50 ℃ of nitrogen.Add the 1mL sample diluting liquid, vortex 30S adds 1mL normal hexane vortex 30S again, and the centrifugal 10min of 4000r/min takes off layer and detects.
This method is 1 to pig urine and pork dilution of sample multiple.
5.3 ELISA measures program
⑴ take out kit, and balance is to room temperature, with the hole bar insertion micropore frame of enough standard items and the used quantity of sample.
⑵ add Clenbuterol standard solution or sample liquid 50 μ L in micropore separately; Standard items and sample are done two parallel laboratory tests, note the position of standard items and sample.Add antibody liquid 50 μ L to each hole, fully mix; Level is put in the wet box, hatches 60min for 37 ℃.
⑶ get rid of liquid in the clear opening, does at the thieving paper arsis.Accurately draw cleansing solution 250 μ L in each hole, leave standstill about 30 seconds, get rid of clean cleansing solution, do at the thieving paper arsis.Repeated washing 4 times.
⑷ add the sheep anti-mouse igg antibody working fluid 100 μ L of horseradish peroxidase (HRP) mark to each hole, fully mixes; Level is put in the wet box, hatches 60min for 37 ℃.
⑸ get rid of liquid in the clear opening, does at the thieving paper arsis.Accurately draw cleansing solution 250 μ L in each hole, leave standstill about 30 seconds, get rid of clean cleansing solution, do at the thieving paper arsis.Repeated washing 5 times.
⑹ add substrate mixed liquor 100 μ L to each hole, fully mixes; Level is put in the wet box, hatches 15min for 37 ℃.
⑺ add stop buffer 50 μ L to each hole; Fully mix; The inherent 450nm of 30min measures light absorption value in the place.
5.4 the result judges
The mean value of titer or sample liquid light absorption value multiply by 100 % again divided by the light absorption value of " 0 " gauge orifice, be inhibiting rate.In 0.05 ~ 1.6 μ g/L scope, be ordinate with the inhibiting rate, the logarithm of concentration of standard solution is a horizontal ordinate 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 anti-depressant residual concentration of beta-receptor in the sample.
Embodiment 6: the sensitivity of kit of the present invention, precision, accuracy
6.1 the sensitivity of kit of the present invention
With the sensitivity index of LDL as kit of the present invention.Get each 20 parts of homogeneous blank control group tissue samples (pig urine and pork) respectively, ELISA measures after the sample preparation.With the OD value substitution typical curve of measuring, calculate mensuration concentration, mean value and the standard deviation of blank sample.According to formula
+3SD; Obtain the LDL of Clenbuterol at each tissue sample; Salbutamol can be calculated according to cross reacting rate at the LDL of each tissue, and the result sees table 5.This kit is respectively 0.13 μ g/kg and 0.16 μ g/kg to the LDL of Clenbuterol in pig urine and pork, and salbutamol is respectively 0.24 μ g/kg and 0.30 μ g/kg at the LDL in pig urine and pork.
Table 5 kit is to the LDL of each tissue
6.2 the precision of kit of the present invention
Respectively that 0,0.05,0.1,0.4,0.8,1.6 μ g/L Clenbuterol standard items concentration are corresponding its typical curve equation of OD value substitution is obtained the measured value that ELISA detects; With the coefficient of variation between the plate inner panel of standard items concentration determination value calculating indirect competitive ELISA typical curve, the result sees table 6.The result shows, reaches Variation Lines number average＜15% between plate in the plate of typical curve, explains that the indirect competitive ELISA method that this research is set up has better precision.
The coefficient of variation in the plate of table 6 typical curve and between plate
6.3 the accuracy of kit of the present invention
The accuracy of kit is represented with the interpolation recovery.Clenbuterol and salbutamol are added respectively in pork and pig urine, make its concentration reach 0.5 μ g/kg and 1.0 μ g/kg, measure every batch of 5 repetitions, 3 batches of replications, calculate recovery rate and variability after the sample pre-treatments with this kit.Result's (see table 7-table 10) shows, this kit in the recovery of each tissue all in 69.8%～118.6% scope, in batch with interassay coefficient of variation＜22.3%.
Clenbuterol adds the recovery and the coefficient of variation in table 7 pork
Salbutamol adds the recovery and the coefficient of variation in table 8 pork
Clenbuterol adds the recovery and the coefficient of variation in the table 9 pig urine
Salbutamol adds the recovery and the coefficient of variation in the table 10 pig urine
Claims (8)
1. can discern the anti-depressant monoclonal antibody of beta-receptor for one kind, it is characterized in that: it is to be that the hybridoma Sal of CCTCC NO:C201187 is secreted by preserving number.
2. the described hybridoma Sal of claim 1 is deposited in Chinese typical culture collection center, and preserving number is CCTCC NO:C201187.
3. the described monoclonal antibody of claim 1 detects the application in the anti-depressant enzyme linked immunological kit of beta-receptor in preparation.
4. the kit that comprises the described monoclonal antibody of claim 1.
5. kit according to claim 4 is characterized in that: said kit is to be used to detect the anti-depressant enzyme linked immunological kit of beta-receptor.
6. claim 4 or 5 application of described kit in the beta-receptor drug-testing.
7. one kind is detected the anti-depressant enzyme-linked immunoassay method of beta-receptor, it is characterized in that: may further comprise the steps:
(1) haptens succinic anhydride salbutamol and bovine serum albumin(BSA) coupling are obtained immunogene;
(2) haptens p-aminobenzoic acid salbutamol and ovalbumin coupling are obtained coating antigen;
(3) utilizing the immunogen preparing preserving number of step (1) is the hybridoma Sal of CCTCC NO:C201187;
(4) utilize preserving number to prepare monoclonal antibody for the hybridoma Sal of CCTCC NO:C201187;
(5) coating antigen with step (2) encapsulates solid phase carrier;
(6) sample pre-treatments: the pig urine samples adds acetonitrile treatment and gets supernatant after centrifugal; The pork sample is used the ethyl acetate back extraction after acid treatment, get ethyl acetate layer nitrogen again and dry up, and centrifuging and taking supernatant after residue redissolves gets determinand;
(7) determinand to step (6) detects.
8. the application of the described enzyme-linked immunoassay method of claim 7 in the beta-receptor drug-testing.
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