CN102585009A - Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting chloramphenicol residues - Google Patents
Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting chloramphenicol residues Download PDFInfo
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Abstract
The invention discloses a specific monoclonal antibody capable of identifying chloramphenicol and an enzyme-linked immunosorbent assay (ELISA) method and kit for detecting chloramphenicol. The monoclonal antibody is secreted by a hybridoma cell HSBSA/4B7 and the hybridoma cell is collected in the China Center for Type Culture Collection, with collection number being CCTCC NO:C201152. The ELISA method comprises the steps of preparation of immunogen, coating antigen and the antibody, treatment and detection of samples, and the like. The monoclonal antibody has strong chloramphenicol identification specificity and high sensitivity. The ELISA method and the kit have good precision and high accuracy.
Description
Technical field
The present invention relates to a kind of enzyme-linked immune analytic method and the test kit that can discern the monoclonal antibody of paraxin and be used for the chlorine detection mycin.
Background technology
Paraxin has good antibacterial effect and cheap price as a kind of Broad spectrum antibiotics, is widely used in the various sensitive organisms of treatment and infects.Yet paraxin has serious adverse reaction to hemopoietic system, can cause irreversible aplastic anemia.The use that relevant law is controlled paraxin has all been formulated in many countries and regions, and China Ministry of Agriculture also classifies paraxin as the forbidding medicine, in all animal derived foods, must not detect (No. 235 bulletins of the Ministry of Agriculture, 2002).
The method for detecting residue of paraxin mainly contains instrument analytical method and immunological analysis method both at home and abroad at present.Instrumental method mainly is to adopt liquid phase-mass spectrometry and gas phase-mass spectrometry; Have the result of mensuration accurately and reliably, separating size is good, can carry out advantages such as multi-residue analysis; But need expensive equipment, complicated pre-treatment and the operator of specialty, to a great extent limit its use.Enzyme-linked immune detection method (ELISA) is a kind of immune analysis method of classics, has advantages such as specificity is good, highly sensitive, simple to operate, testing cost is low, is fit to batch detection in the residue of veterinary drug supervisory system.
In recent years, the report of relevant residual chloromycetin ELISA detection method is more, as shown in table 1.
Table 1 is the research of relevant residual chloromycetin ELISA detection method in recent years
It is thus clear that the existing ELISA method that detects residual chloromycetin is comparatively ripe, and business-like residual chloromycetin ELISA detection kit is also arranged both at home and abroad, visit like Germany send out, all there are related prods in companies such as Beijing Wang Er, U.S. Charles Bell.But these detection methods and test kit are unfavorable for keeping the different batches performance of products stable mostly based on polyclonal antibody.Because paraxin belongs to zero left drug, require increasingly high to various performance index such as the purity of related antigen antibody, sensitivity, homogeneity.Therefore, study a kind of residual chloromycetin ELISA method for quick and test kit, detect significant residual chloromycetin in the batch samples based on the highly sensitive monoclonal antibody specific.
Summary of the invention
First purpose of the present invention is to provide a kind of monoclonal antibody of ability specific recognition paraxin.
Second purpose of the present invention is to utilize this monoclonal antibody, sets up a kind of enzyme-linked immunoassay method that can be used for animal tissues's residual chloromycetin detection.
The 3rd purpose of the present invention provides the enzyme linked immunological kit that is applicable to that residual chloromycetin detects.
The 4th purpose of the present invention provides the application of said monoclonal antibody in the enzyme linked immunological kit of preparation chlorine detection mycin.
The 5th purpose of the present invention provide contain said monoclonal antibody test kit in animal tissues's Determination of chloramphenicol residues.
The present invention realizes through following technical scheme:
A kind of monoclonal antibody that can discern paraxin, it is to be that the hybridoma HSBSA/4B7 of CCTCC NO:C201152 is secreted by preserving number.
Above-mentioned hybridoma 4B7 is deposited in the Chinese typical culture collection center (CCTCC) that is positioned at Wuhan City, Hubei Province Wuhan University, and its preserving number is CCTCC NO:C201152.
Further, the invention provides a kind of enzyme-linked immunoassay method that is applicable to that residual chloromycetin detects, this method comprises the steps such as processing and detection of preparation and the sample of immunogen, coating antigen, antibody, and is specific as follows:
(1) haptin paraxin hemisuccinic acid ester (CAP-HS) and bovine serum albumin (BSA) coupling are obtained immunogen CAP-HS-BSA;
(2) paraxin hemisuccinic acid ester and ovalbumin (OVA) coupling are obtained coating antigen CAP-HS-OVA;
(3) utilize the immunogen immune mouse of step (1), obtain the hybridoma HSBSA/4B7 that preserving number is CCTCC NO:C201152 through cytogamy and screening;
(4) use preserving number to prepare monoclonal antibody as the hybridoma HSBSA/4B7 of CCTCC NO:C201152;
(5) coating antigen with step (2) encapsulates solid phase carrier (like enzyme plate);
(6) with testing sample with ethyl acetate extraction, nitrogen dry up, normal hexane purifies and sample diluting liquid dissolves again and obtains determinand;
(7) determinand to step (6) carries out enzyme linked immunosorbent detection,
The component and the proportioning of step (6) sample diluting liquid are: NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, KCl 0.2g adds distilled water to 1000mL.
The present invention with said monoclonal antibody and coating antigen as core reagent and other conventional agent combination, processed can the chlorine detection mycin enzyme linked immunological kit, in conjunction with above-mentioned enzyme-linked immunoassay method, realized enzyme linked immunosorbent detection to paraxin.
Monoclonal antibody of the present invention is strong, highly sensitive to the identification specificity of paraxin, and detection method precision is good, accuracy is high.
Description of drawings
Fig. 1 is technological line figure of the present invention.
Fig. 2 prepares the indirect competitive ELISA reaction normal curve of paraxin monoclonal antibody for the present invention, and the X axle is a paraxin concentration of standard solution logarithmic value, and the Y axle is that OD value is divided by " zero " hole OD value (B/B
0).
Embodiment
Through embodiment the present invention is described further below.
The preparation of embodiment 1 immunogen and coating antigen
Immunogenic synthetic: accurately take by weighing paraxin hemisuccinic acid sodium salt 0.5g, be dissolved in the 2mL zero(ppm) water, regulate pH=2 with the hydrochloric acid soln of 1mol/L, 5000r/ minute centrifugal 10 minutes, vacuum-drying obtained about 300mg white solid matter paraxin hemisuccinic acid.
Accurately extracting chloromycetin hemisuccinic acid 39.2mg is dissolved in 1mL N, in the dinethylformamide (DMF), adds 20 μ L triethylamines and 20 μ L isobutyl chlorocarbonates, at room temperature reacts activation 1 hour, filters then, obtains A liquid.
Accurately get BSA 67.1mg again, be dissolved in the 9mL phosphate buffered saline buffer (PBS, pH=7.4) in, this is a B liquid, above-mentioned A drop is gone in the B liquid lucifuge reaction 17 hours.After reaction finishes, will react and change over to night in the dialysis tubing, and the PBS of pH=7.4 stirred dialyse, and change liquid every day and do not have uv-absorbing until extracellular fluid dialysis three times.Product is paraxin hemisuccinic acid ester-BSA conjugate (CAP-HS-BSA).With the product packing, lyophilize, subsequent use.
Synthesizing of coating antigen: accurate extracting chloromycetin hemisuccinic acid 37.3mg, be dissolved in 2mL N, in the dinethylformamide (DMF), add 60 μ L triethylamines and 60 μ L isobutyl chlorocarbonates, at room temperature reacted activation 1 hour, filter then, obtain A liquid.
Accurately get OVA 85.1mg again, be dissolved among the PBS of 10mL pH=7.4, this is a B liquid, and above-mentioned A drop is gone in the B liquid, and stirring reaction is 20 hours under ice bath.After reaction finishes, will react and change over to night in the dialysis tubing, and the PBS of pH=7.4 stirred dialyse, and change liquid every day and do not have uv-absorbing until extracellular fluid dialysis three times.Product is paraxin hemisuccinic acid ester-OVA conjugate (CAP-HS-OVA).With the product packing, lyophilize, subsequent use.
The preparation of hybridoma: with reference to the method in Xue Qingshan " philosophy and technique of vitro culture " the Science Press calendar year 2001 version: the conjugate CAP-HS-BSA with embodiment 1 preparation is an immunogen; Immunity Balb/C female mice, fundamental immunity of immune programme for children employing and several booster immunization.Use during first immunisation with isopyknic Freund's complete adjuvant emulsive to contain the subcutaneous fundamental immunity of carrying out of nape portion that the immunogenic protein emulsion of 100 μ g is injected in mouse, whenever contained the immunogenic protein emulsion of 100 μ g with the Freund's incomplete adjuvant emulsive later on and carry out booster immunization at a distance from 15 days.From immunity for the third time, 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.
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).Merge and got a preparation of non-immune Balb/C mouse feeder cell in preceding 1 day.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.1500r/ minute centrifugal 5 minutes, abandon supernatant, the back-off centrifuge tube, the control solid carbon dioxide drips, and raps the pipe end then, makes cell become pasty state.Centrifuge tube is placed 37 ℃ of water-baths, have the preparatory temperature of 0.8mL to the transfer pipet of 37 ℃ 50%PEG to be inserted into the pipe end suction then, in 60 seconds, PEG is added on the cell mixing, the limit edged stirs gently, leaves standstill after adding 90 seconds; Slowly be added on the fused cell along tube wall with the warm in advance RPMI 1640 basic culture solution 10mL to 37 ℃ of pipette, extract then, the limit edged rocks centrifuge tube gently.Dropwise add 1mL (3 seconds/drip) on the 1st minute, added 2mL again on the 2nd minute, add remaining 7mL (adding in 5 minutes) at last.After adding first 10mL, then add RPMI 1640 substratum to 50mL, tighten lid after adding, slowly put upside down several times mixing along tube wall.1500r/ minute centrifugal 5 minutes, abandon supernatant, stir fused cell resuspended gently with the HAT perfect medium 72mL that contains feeder cell.
The amount of fused cell suspension with 2/hole is inoculated in the 96 porocyte culture plates, puts 5%CO
237 ℃ of cultivations in the incubator.To count the same day be 0 day from merging, and the preceding 3 days kinetocyte plates of trying not keep the incubator homeostasis.Every hole was added 1 of HAT perfect medium and was observed the colony growing state in the 3rd day; The 5th day every hole sucking-off 1/2 culture supernatant (100 μ L) adds 1 of HT perfect medium again; Whenever go 1/2 culture supernatant at a distance from 3 days by last method suction later on, change to the HT perfect medium.
To account for hole floorage about 1/4 be that desirable cells and supernatant detects when cell grows to.Promptly the conjugate CAP-HS-OVA with contriver's preparation is a coating antigen, utilizes the ELISA method to filter out the positive cell hole of secretion chloramphenicol resistance antibody.To the positive cell hole that screens, adopt continuous 3 time cloningizations of limiting dilution assay, finally set up the hybridoma of strain ability stably excreting chloramphenicol resistance antibody.Chromosome counting is the result show; 96.5 of this hybridoma chromosome number average out to; (SP2/0 myeloma cell's karyomit(e) mean number is 58 to be higher than the chromosome number of parental cell; Splenocyte karyomit(e) is 40), explain that this cell is the hybridization product of SP2/0 myeloma cell and splenocyte.The applicant is this hybridoma called after HSBSA/4B7, and delivers the Chinese typical culture collection center preservation that is positioned at Wuhan City, Hubei Province Wuhan University on June 29th, 2011, and its preserving number is CCTCC NO:C201152.
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.Use RPMI 1640 basic mediums to suspend, and cell count is transferred to 1 * 10 by the cell of preserving number as the hybridoma HSBSA/4B7 enlarged culturing of CCTCC NO:C201152
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 a mouse IgGl 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.
Washings (pH 7.4): accurately take by weighing NaCl 8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O 2.90g, KCl0.20g, a small amount of ultrapure water dissolving adds polysorbas20 0.50mL, is settled to 1000mL.
Phosphate buffered saline buffer (pH 7.4): accurately take by weighing NaCl 8.00g, KH
2PO
40.20g, Na
2HPO
412H
2O2.90g, 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 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 is joined existing usefulness at present.
Stop buffer: accurately measure vitriol oil 100mL, slowly be added drop-wise in the 800mL ultrapure water.
3.2 the preliminary of the concentration of encapsulating and antibody dilution confirmed
Encapsulating the preliminary of concentration and antibody dilution confirms: with the working concentration combination of square formation volumetry initial option coating antigen and antibody.Use carbonate buffer solution that the low coupling ratio coating antigen doubling dilution of CAP-HS-OVA is become the horizontal coated elisa plate of 40,20,10,5,2.5,1.25,0.625,0.3125 μ g/mL; Antibody uses the phosphate buffered saline buffer doubling dilution to become 1: 4000, vertically added enzyme plate in 1: 8000,1: 16000,1: 32000.The square formation titration results is seen table 2, following coating antigen concentration of initial option and antibody dilution combination: (2.5,16000) and (1.25,8000).
The titration of table 2 antibody square formation
3.3 encapsulating concentration, the best confirms
The best encapsulates concentration and confirms: the concentration that encapsulates so that the square formation titration is selected is made the inhibition curve respectively with the antibody dilution combination, and paraxin standard substance concentration is set to 0,0.1,0.2,0.4,0.8,1.6 μ g/L, its " 0 " hole and IC
50Value is seen table 3.The ratio of antigen-antibody is the key that influences its sensitivity, if antigen or antibody excess all will cause IC
50Higher, visible by data, it is 2.5 μ g/mL that the best encapsulates concentration, and two kinds of antibody dilutions are tentatively confirmed to be respectively and 1: 16000.
Table 3 the best encapsulates concentration optimization
3.4 the optimum antibody extent of dilution is confirmed
The optimum antibody extent of dilution is confirmed: encapsulating the concentration coated elisa plate with the best, was 3 dilutions of reference concentration equal difference design gradient with antibody with 1: 12000, its 0 hole and IC
50Value is seen table 4.Along with the increase of antibody dilution, IC
50Value changes not obvious, and under the prerequisite that guarantees 0 hole OD value, selecting 1: 15000 is the optimum antibody extent of dilution.
Table 4 optimum antibody extent of dilution is optimized
3.5 the foundation of typical curve
The CAP standard substance are mixed with 6 concentration gradients such as 0,0.1,0.2,0.4,0.8,1.6 μ g/L, measure the drawing standard curve according to top definite indirect competitive ELISA method.Shown in accompanying drawing 2, the regression equation and the index of correlation of racing ELISA detecting method of the present invention are respectively: y=-0.4177x+1.282, r=0.9981, IC
50Value is 0.75 ± 0.09 μ g/L (n=5), and linearity range is 0.1～1.6 μ g/L.
3.6 specificity
Become concentration gradient to carry out indirect competitive ELISA each chloromycetin pharmaceutical standards article doubling dilution respectively, calculate IC
50Value is with CAP standard substance IC
50The value contrast obtains cross reacting rate, and the result sees table 5.The result shows, the indirect competitive ELISA method that the present invention sets up has certain cross reaction to paraxin hemisuccinic acid sodium, and with the equal no cross reaction of other chloromycetin medicine.
The specificity of table 5 residual chloromycetin ELISA detection method
| The competition thing | IC 50(μg/L) | Cross reacting rate (%) |
| Paraxin | 0.75 | 100 |
| Paraxin hemisuccinic acid sodium | 1.28 | 58.7 |
| Thiamphenicol | ＞1000 | ＜0.1 |
| Florfenicol | ＞1000 | ＜0.1 |
The assembling of embodiment 4 ELISA detection kit of the present invention
4.1 test kit moity
(1) is coated with the enzyme plate of CAP-HS-OVA coating antigen;
(2) the paraxin standard solution is 6 bottles, and concentration is respectively 0,0.1,0.2,0.4,0.8,1.6 μ g/L;
(3) paraxin HSBSA/4B7 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
2O29.00g, KCl2.00g adds distilled water to 1000mL;
(6) concentrated cleaning solution: NaCl80.00g, KH
2PO
42.00g, Na
2HPO
412H
2O 29.00g, KCl2.00g, polysorbas20 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 is joined existing usefulness at present.
(8) stop buffer: 2mol/L sulphuric acid soln.
4.2 enzyme plate preparation
(1) encapsulates: with carbonate buffer solution the CAP-HS-OVA coating antigen is diluted to 2.5 μ g/mL coating antigen solution, accurately draws 100 μ L coating antigen solution, be placed horizontally at wet box, hatched 12 hours for 4 ℃ in each enzyme mark hole.
(2) wash plate: throw away enzyme plate endoperidium original solution, clap to do, accurately draw 250 μ L washingss in each enzyme mark hole, leave standstill 30 seconds after, throw away washings, do at the thieving paper arsis; 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 were hatched 2 hours.
(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, do at the thieving paper arsis; Repeated washing 3 times.
(5) oven dry: after the thieving paper arsis is done; Enzyme plate is inverted oven dry 0.5 hour 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.
The mensuration program of embodiment 5 enzyme linked immunological kits of the present invention
5.1 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 preparation: NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, KCl 0.2g adds distilled water to 1000mL.
Substrate mixed solution preparation: according to each institute expense, get an amount of substrate A liquid and B liquid, join existing usefulness at present in 1: 100 ratio mixing.
5.2 tissue sample is handled
(3g ± 0.1g) adds ETHYLE ACETATE 6mL in centrifuge tube, vibrated room temperature 4000r/ minute (2000g) centrifugal 10 minutes strongly 2 minutes to take by weighing sample behind the homogeneous; Take out 4mL supernatant liquid (sample that is equivalent to 2g) 50-60 ℃ of drying under nitrogen gas stream then; Add sample diluting liquid 0.5mL and normal hexane 1mL again, thermal agitation 1 minute, room temperature 4000r/ minute (2000g) centrifugal 10 minutes; Abandon upper organic phase, take off layer water 50 μ L and analyze.
Annotate: present method is 4 to the extension rate of pig muscle.
5.3ELISA mensuration 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) add paraxin standard solution 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, thorough mixing; Level is put in the wet box, hatches 60 minutes for 37 ℃.
(3) get rid of liquid in the clear opening, do at the thieving paper arsis.Accurately draw washings 250 μ L in each hole, leave standstill about 30 seconds, get rid of clean washings, do at the thieving paper arsis.Repeated washing 4 times.
(4) the sheep anti-mouse igg antibody working fluid 100 μ L that add horseradish peroxidase (HRP) mark are to each hole, thorough mixing; Level is put in the wet box, hatches 60 minutes for 37 ℃.
(5) get rid of liquid in the clear opening, do at the thieving paper arsis.Accurately draw washings 250 μ L in each hole, leave standstill about 30 seconds, get rid of clean washings, do repeated washing 5 times at the thieving paper arsis.
(6) add substrate mixed solution 100 μ L to each hole, thorough mixing; Level is put in the wet box, hatches 15 minutes for 37 ℃.
(7) add stop buffer 50 μ L to each hole; Thorough mixing; Light absorption value was measured at inherent 450nm place in 30 minutes.
5.4 the result judges
The MV of reference liquid or sample liquid light absorption value multiply by 100% again divided by the light absorption value of " 0 " standard orifice; Being inhibiting rate, in 0.1-1.6 μ g/L scope, is ordinate zou with the inhibiting rate; The logarithm of concentration of standard solution is an X-coordinate drawing standard curve, obtains regression equation.According to the inhibiting rate of formula calculation sample, with inhibiting rate substitution regression equation, calculate mensuration concentration, multiply by dilution factor, be the residual concentration of paraxin in the sample.
Sensitivity, precision, the accuracy of embodiment 6 test kits of the present invention
6.1 the sensitivity of test kit of the present invention
With the sensitivity index of LDL as test kit of the present invention.Get 20 parts of blank control group tissue samples; After sample preparation, carrying out ELISA detects; Measure the OD value; The MV
that calculates blank sample OD value is with the concentration (C) of finding correspondence on
substitution typical curve, and base of calculation poor (SD).Calculate the Z value according to formula Z=C+3 * SD, this is the LDL (LOD) of method for organizing, and the result sees table 6.
The sensitivity of table 6 indirect competitive ELISA pork method for organizing
6.2 the precision of test kit of the present invention
Respectively that CAP standard substance concentration such as 0.1,0.2,0.4,0.8,1.6 μ g/L is corresponding its typical curve equation of OD value substitution 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, the result sees table 7.The result shows, reaches Variation Lines number average＜15% between plate in the plate of typical curve, explains that the indirect competitive ELISA method of setting up has better precision.
The variation coefficient in the plate of table 7 typical curve and between plate
6.3 the accuracy of test kit of the present invention
In the good pig muscle tissue of even matter, add the paraxin standardized solution, make its final concentration be respectively 0.05 and 0.1 μ g/kg, 5 parallel appearance of each concentration; Carry out sample preparation then, ELISA measures drug level, repeats 3 batches; Calculate recovery rate; The recovery is calculated by following formula, and calculates batch interior and interassay coefficient of variation, and the result sees table 8.
CAP adds the recovery and the variation coefficient in the table 8 pork tissue
Claims (7)
1. the monoclonal antibody that can discern paraxin is characterized in that, it is to be that the hybridoma HSBSA/4B7 of CCTCC NO:C201152 is secreted by preserving number.
2. the described hybridoma HSBSA/4B7 of claim 1 is deposited in Chinese typical culture collection center, and preserving number is CCTCC NO:C201152.
3. the test kit that comprises the described monoclonal antibody of claim 1.
4. test kit according to claim 3, this test kit are the enzyme linked immunological kits that is used for the chlorine detection mycin.
5. the enzyme-linked immunoassay method of a chlorine detection mycin comprises the preparation of immunogen, coating antigen, antibody and the processing and the detection of sample, and its step is following:
(1) paraxin hemisuccinic acid ester and bovine serum albumin coupling are obtained immunogen;
(2) paraxin hemisuccinic acid ester and ovalbumin coupling are obtained coating antigen;
(3) utilize the immunogen immune mouse of step (1), obtain the hybridoma HSBSA/4B7 that preserving number is CCTCC NO:C201152 through cytogamy and screening;
(4) use preserving number to prepare monoclonal antibody as the hybridoma HSBSA/4B7 of CCTCC NO:C201152;
(5) coating antigen with step (2) encapsulates solid phase carrier;
(6) testing sample is used ethyl acetate extraction, through nitrogen dry up purify with normal hexane after, residue obtains determinand with the sample diluting liquid dissolving;
(7) determinand to step (6) carries out enzyme linked immunosorbent detection,
Wherein:
The component of sample diluting liquid and proportioning are: NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O2.9g, KCl 0.2g adds distilled water to 1000mL.
6. the application of the described monoclonal antibody of claim 1 in the enzyme linked immunological kit of preparation chlorine detection mycin.
7. claim 3 or 4 described test kits are in animal tissues's Determination of chloramphenicol residues.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971727A (en) * | 2019-03-19 | 2019-07-05 | 北京望尔生物技术有限公司 | It is a kind of secrete chloramphenicol resistance monoclonal antibody hybridoma cell strain and its application |
| CN109971727B (en) * | 2019-03-19 | 2022-05-13 | 北京望尔生物技术有限公司 | Hybridoma cell strain secreting monoclonal antibody against chloramphenicol and application thereof |
| CN112239751A (en) * | 2020-10-13 | 2021-01-19 | 苏州大学 | Florfenicol/thiamphenicol monoclonal antibody hybridoma cell strain, monoclonal antibody secreted by same and application |
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