CN102585007B - Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting beta-carotene pigments - Google Patents
Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting beta-carotene pigments Download PDFInfo
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Abstract
The invention discloses a specific monoclonal antibody capable of identifying beta-carotene pigments and an enzyme-linked immunosorbent assay (ELISA) method and kit for detecting beta-carotene pigments. The monoclonal antibody is secreted by a hybridoma cell DCC/C11 and the hybridoma cell is collected in the China Center for Type Culture Collection, with collection number being CCTCC NO:C201146. The ELISA method comprises the steps of preparation of immunogen, coating antigen and the antibody, treatment and detection of samples, and the like. The ELISA method and the kit can detect the total content of canthaxanthin, beta-carotene, beta-apo-8'-carotenal, xanthophyll, capsorubin and beta-ionone in the samples by one step, thus shortening the detection time and lowering the detection cost; and the ELISA method and the kit have the characteristics of high detection sensitivity, good precision and good accuracy.
Description
Technical field
The present invention relates to a kind of monoclonal antibody and a kind of enzyme-linked immunoassay method and the test kit for detection of β-carotene class pigment that can identify β-carotene class pigment.
Background technology
β-carotene class pigment has β-A Piao-8 '-carotenal, β-A Piao-8 '-carotene acetoacetic ester, xenthophylls, capsanthin, canthaxanthin, astaxanthin etc.
At present, to the detection method of β-carotene class material, be mainly the instrument detection method, as Paper Chromatography, thin layer chromatography, column chromatography, spectrophotometry, high performance liquid chromatography or Liquid Chromatography/Mass Spectrometry.These detection methods need to be equipped with expensive instrument and strongly professional operator, the complex pretreatment of sample, and detection time is long, is unfavorable for the screening of batch samples, can not detect fast at the scene.Enzyme-linked immune detection method (ELISA) is to utilize the high degree of specificity of immune response (being the Ag-Ab association reaction) and the high susceptibility of enzymatic reaction medicine to be carried out to a kind of comprehensive detection technique of qualitative and quantitative analysis.It has the advantages such as easy and simple to handle, high-throughput, highly sensitive, low expense, can overcome the deficiency of instrument detection method.At present, there is no the bibliographical information for the ELISA detection method of β-carotene class both at home and abroad.
For the foundation of micromolecular compound ELISA detection method, the preparation of the antibody of micromolecular compound is core, and the appropriate design haptens is crucial.Haptenic chemical structure difference, the chemical site be connected on carrier proteins is just different, and the chemical property of crosslinked arm is also different, and this is the deciding factor that affects micromolecular compound antibody character.Therefore micromolecular compound is carried out to the chemical structure transformation, the appropriate design haptens, be connected to haptens on carrier proteins, is the core of this invention.
The applicant retrieves the patent of invention that one piece of application number is 201010140887.6, and this patent provides a kind of method of oxidation α-ionone and β-ionone, but does not have it as the haptens synthetic immunogen and be applied to enzyme linked immunosorbent detection.
Summary of the invention
First purpose of the present invention is to provide a kind of monoclonal antibody that can identify β-carotene class pigment.
Second purpose of the present invention is to utilize this monoclonal antibody, sets up a kind of ELISA method that can detect β-carotene class pigment.
The 3rd purpose of the present invention is to provide a kind of enzyme linked immunological kit that detects β-carotene class pigment.
The 4th purpose of the present invention is to provide the application of described monoclonal antibody in the enzyme linked immunological kit of preparation detection β-carotene class pigment.
The 5th purpose of the present invention is to provide the application of test kit in animal tissues's β-carotene class pigment residue detection that contains described monoclonal antibody.
The present invention is achieved by the following technical solutions:
A kind of monoclonal antibody that can identify β-carotene class pigment, it is that CCTCC NO:C201146 hybridoma DCC/C11 is secreted by preserving number.
Described carotenoid pigment refers to canthaxanthin, β-carotene, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid.
Described hybridoma DCC/C11 is deposited in Chinese Typical Representative culture collection center (CCTCC), and preserving number is CCTCC NO:C201146.
Immunogen used prepares with the bovine serum albumin coupling by haptens β-ionone acid.
Further, the invention provides a kind of enzyme-linked immunoassay method that detects β-carotene class pigment, comprise the preparation of immunogen, coating antigen, antibody and processing and the detection of sample, its step is as follows:
(1) haptens β-ionone acid is obtained to immunogen with bovine serum albumin (BSA) coupling;
(2) haptens 4-oxo-beta-ionone acid is obtained to coating antigen with ovalbumin (OVA) coupling;
(3) utilize the immunogen immune mouse of step (1), by cytogamy and screening, obtain the hybridoma DCC/C11 that preserving number is CCTCC NO:C201146;
(4) the hybridoma DCC/C11 that is CCTCC NO:C201146 with preserving number prepares monoclonal antibody;
(5) with the coated solid phase carrier of the coating antigen of step (2);
(6) by testing sample, by volume ratio, be ethyl acetate: the mixing solutions of sodium hydroxide (2%)=2: 1 extracts, and after ethyl acetate extraction and nitrogen dry up, residue obtains determinand with the sample diluting liquid dissolving;
(7) determinand of step (6) carried out to enzyme linked immunosorbent detection,
Wherein:
The component of sample diluting liquid and proportioning are: NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, KCl 0.2g, N ', N '-N,N-DIMETHYLACETAMIDE 20mL, add distilled water to 1000mL.
The present invention is usingd said monoclonal antibody and coating antigen as core reagent and conventional other agent combination, made the enzyme linked immunological kit that can detect β-carotene class pigment, in conjunction with above-mentioned enzyme-linked immunoassay method, realized the enzyme linked immunosorbent detection to β-carotene class pigment.
The invention has the beneficial effects as follows:
1, canthaxanthin, β-carotene, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid in the monoclonal anti physical efficiency identification β-carotene class pigment that prepared by the present invention, specificity is strong, highly sensitive.
2, because the present invention adopts β-ionone acid as haptens, this haptens has retained canthaxanthin, β-carotene, β-A Piao-8 '-carotenal, xenthophylls, the common chemical structure of Capsorubin, and the monoclonal antibody prepared by this haptens can be identified canthaxanthin, β-carotene, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid simultaneously.
3, ELISA method of the present invention and the disposable total content of measuring canthaxanthin in determinand, β-carotene, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid of test kit energy, shorten detection time, reduced testing cost.
4, ELISA the method for the present invention and test kit detection sensitivity is high, precision good, accuracy is good.
The accompanying drawing explanation
Accompanying drawing 1 is Technology Roadmap of the present invention.
The indirect competitive ELISA response curve that accompanying drawing 2 is monoclonal antibody of the present invention and canthaxanthin (CTX) standard substance, X-axis is canthaxanthin (CTX) concentration of standard solution logarithmic value, and the optical density value that Y-axis is the canthaxanthin standard solution is divided by " zero " hole optical density value (B/B
0).
Embodiment
Below by embodiment, the present invention will be further described.
The preparation of embodiment 1 immunogen and coating antigen
1.1 β-ionone acid is synthetic
Accurately measure distilled water 30mL, join in the round-bottomed flask of precooling; Add bromine 20g in flask, after stirring 2 hours, form A liquid standby.Accurately β-ionone 6g, be dissolved in dioxane 20mL, forms B liquid.B liquid is added in A liquid, and under room temperature, reaction is 8 hours.After having reacted, add 20% sodium bisulfite until starch potassium iodide paper no longer becomes blue.Then add concentrated hydrochloric acid white solid matter to occur to solution.Filter, the filter residue white solid matter is dropped in methyl alcohol, put 60 ℃ of stirring in water bath to after dissolving fully, filter.Filtrate is placed in to-20 ℃ of rear filtrations of spending the night, it is dry that filter cake is placed in 50 ℃ of vacuum drying ovens, and it is β-ionone acid that the product obtained carries out Mass Spectrometric Identification.
1.2 β-ionone acid-BSA's is synthetic
Get β-ionone acid 93mg, 1,3-dicyclohexylcarbodiimide (DCC) 50mg and N-hydroxy-succinamide (NHS) 33mg are dissolved in dioxane 5mL, and the room temperature lucifuge stirs 8 hours, and reaction product forms A liquid after filtering.Separately get BSA80mg and be dissolved in phosphate buffered saline buffer (0.01mol/L, pH8.0) 20mL, form B liquid.Ice bath is added to B liquid by the A drop under stirring; Reaction solution by reaction after 12 hours centrifugal 10 minutes in 4000r/ minute; Get supernatant and pack in dialysis tubing, to normal saline dialysis 5-6 days, obtain conjugate β-ionone acid-BSA.Get the supernatant lyophilize after centrifugal, put-20 ℃ of preservations, as immunogen.
1.3 4-oxo-beta-ionone is synthetic
β-ionone 20g and methylene dichloride 60ml are joined in there-necked flask; Heating in water bath to 37 ℃, the aqueous solution that the aqueous solution that adds 15ml to contain the 2g potassiumiodide and 6ml contain the 3g sodium pyrosulfate.Under agitation condition, then add the 20ml aqueous solution that contains the 8g sodium bromate, after having reacted, separate organic phase.After this organic phase is used the 8g anhydrous magnesium sulfate drying, concentrated by rotary evaporation.Carry out column chromatography, isolation of target substances with sherwood oil and acetone.By after the target substance concentrated by rotary evaporation of separating, add the sherwood oil of same volume, put-20 ℃ of rear filtrations of spending the night.It is dry that filter cake is placed in 50 ℃ of vacuum drying ovens, and it is 4-oxo-beta-ionone that the product obtained carries out Mass Spectrometric Identification.
1.4 4-oxo β-ionone acid (4-keto-β-ionone acid) is synthetic
Accurately take NaOH4g, be dissolved in 20mL water, splash into bromine 2mL, continue to stir.Take 4-oxo-beta-ionone crude product 1.2g, be dissolved in dioxane 4mL, add in the reaction solution that above-mentioned activation is good, stopped reaction while to starch potassium iodide paper, no longer becoming indigo plant.Add 30% NaHSO
416mL, use the dichloromethane extraction removal of impurities, and water is added to the concentrated hydrochloric acid acidifying, standing to red precipitate occurring.After sedimentation and filtration, filter residue dissolves with methylene dichloride, adopts sherwood oil and ethanol column chromatography, isolation of target substances.By after the target substance concentrated by rotary evaporation of separating, add the methylene dichloride of same volume, be placed in-20 ℃ of rear filtrations of spending the night.It is dry that filter cake is placed in 50 ℃ of vacuum drying ovens, and it is 4-oxo-beta-ionone acid that the product obtained carries out Mass Spectrometric Identification.
1.5 4-oxo-beta-ionone acid-OVA's (4-keto-β-ionone acid-OVA) is synthetic
Get 4-oxo β-ionone acid 48mg, add DMF (DMF) 2mL, then add triethylamine 60 μ L, then add isobutyl chlorocarbonate 60 μ L, under room temperature, stirring reaction is 60 minutes, forms A liquid.Separately get in the phosphate buffer soln (pH7.4) that OVA100mg is dissolved in 30mL0.01mol/L, form B liquid.The A drop is added in B liquid to stirring reaction 16 hours.After having reacted, that reaction solution is centrifugal 10 minutes 5000r/ minute, 4 ℃.Get supernatant liquor physiological saline is thoroughly dialysed, obtain conjugate 4-oxo-beta-ionone acid-OVA.Get the supernatant lyophilize after centrifugal, put-20 ℃ of preservations, as coating antigen.
The preparation of embodiment 2 monoclonal antibodies
The preparation of hybridoma: with reference to the method in Xue Qingshan " philosophy and technique of vitro culture " (Science Press's calendar year 2001 version): with the conjugate β of embodiment 1 preparation-ionone acid-BSA immunity Balb/C mouse (purchased from Disease Prevention Control Center, Hubei Prov's Experimental Animal Center).After immune programme for children is the protein solution and isopyknic Freund's complete adjuvant (purchased from sigma company) emulsification of getting containing conjugate β-ionone acid-BSA 125 μ g, in the subcutaneous multi-point injection of mouse back.Strengthened once at interval of 2 weeks later, use Freunds incomplete adjuvant (purchased from sigma company) emulsification instead.Finally in merging first three day (preferably and immunity last time be separated by more than 4 weeks), abdominal injection, reinforced immunological, the antigen amount doubles, and does not add adjuvant.
During fusion, one of the Balb/C mouse of the last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum) is soaked sterilization in 5 minutes in 75% alcohol.Aseptic taking-up mouse spleen, isolate splenocyte, with the SP2/0 myeloma cell (SP2/0 myeloma cell is from this laboratory) of fresh preparation, presses 1-2 * 10
7Individual SP2/0 and 10
8The ratio of individual immune spleen cell (1: 10～1: 15) mixes in the 50mL centrifuge tube, 1500r/ minute, centrifugal 10 minutes.Evacuation supernatant (filter paper of available sterilizing blots), knock the pipe end gently, makes cell precipitation loosening slightly.The centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths.Then slowly splashed into 50% polyoxyethylene glycol (PEG) 0.8mL (purchased from sigma company product) of pre-temperature to 37 ℃ in 1 minute, the limit edged stirs with pipette tip gently, continues to stir 1 minute.Then 1640 (purchased from Hyclone company business substratum) the basic medium 10mL that slowly adds 37 ℃ of pre-temperature.Concrete grammar is: within first minute, dropwise splash into 1mL, within second minute, add 1mL, within 3rd～4 minutes, add 3mL, within the 5th minute, add remaining 5mL, each added-time need slowly add, and constantly stirs lightly.Finally add 30mL 1640 liquid, also need slowly to add.Centrifugal 5 minutes of 800r/m, remove supernatant, in 37 ℃, places 5～8 minutes.With HAT (purchased from Sigma company) substratum, suspend, simultaneously also with the HAT substratum suspend the raising splenocyte for preparing and with merge after cytomixis, add as required appropriate HAT substratum, minute plant in 96 well culture plates, approximately 250 μ L/ holes.Single cell fusion can be inoculated 4～8 96 orifice plates.Also can plant less as required, generally press the cell count of SP2/0 and calculate, every hole inoculum size is approximately containing 10
4A left and right SP2/0 cell.In 37 ℃, 5%CO
2In incubator, cultivate.After merging, second day starts to observe has pollution-freely, in the 4th day, adds 1 HAT substratum, within 8th～10 days, sucks 100 μ L substratum and changes HT (purchased from sigma company) substratum 100 μ L.Treat that the fused cell colony grows to culture hole 1/4, when substratum omits flavescence, carry out antibody test.Adopt 4-oxo-beta-ionone acid-OVA as screening antigen, utilize the ELISA method to filter out the positive hole of the anti-canthaxanthin antibody of secretion.To the positive hole screened, use at once limiting dilution assay (with reference to Xue Qingshan " philosophy and technique of vitro culture " Science Press calendar year 2001 version) to be cloned, screen.Through 3～4 time clonings, finishing screen is selected the monoclonal hybridoma of secretion anti-β-Radix Dauci Sativae class pigment antibody.This clone has been carried out to chromosome counting, and result shows, the chromosomal mean number of SP2/0 is 58, and splenocyte karyomit(e) is 40, and the chromosome number of hybridoma is between 92～104, all higher than the chromosome number of two parent's cells.The chromosome number of hybridoma, obviously more than the karyomit(e) of myeloma cell SP2/0, illustrates the cell of SP2/0 really of fused cell and the hybridization product of splenocyte.To this monoclonal hybridoma filtered out, the applicant is by its called after DCC/C11, and deliver Chinese Typical Representative culture collection center (CCTCC) preservation that is positioned at Wuhan City, Hubei Province Wuhan University on June 29th, 2011, its preserving number is CCTCC NO:C201146.
The preparation of ascites monoclonal antibody and evaluation: only within first 7 days, get the Balb/c number of mice in inoculation, every mouse peritoneal injection 0.5ml Freund's incomplete adjuvant carries out pre-treatment.The cell of the hybridoma DCC/C11 enlarged culturing that is CCTCC NO:C201146 by preserving number with RPMI 1640 basic mediums suspensions, 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 obtains monoclonal antibody.Employing is carried out the hypotype evaluation purchased 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 result is mouse IgG
1Hypotype.
The foundation of embodiment 3 ELISA detection methods
3.1 the preparation of reagent (reagent that the present embodiment is used all adopts the following methods preparation except another indicating)
Phosphate buffered saline buffer: NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, KCl 0.2g, add ultrapure water to 1000mL, regulates pH to 7.4;
Coating buffer: get Na
2CO
31.5g, NaHCO
32.9g, add tri-distilled water to 1000mL, regulate pH value to 9.6;
Washings: NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, KCl 0.2g, polysorbas20 0.5mL, add ultrapure water to 1000mL, regulates pH to 7.4;
Confining liquid: ovalbumin 0.1g is dissolved in the 100mL phosphate buffered saline buffer;
Substrate solution: by Wuhan, Fei Yuan Science and Technology Ltd. provides.
Stop buffer: 2mol/L sulphuric acid soln.
3.2 the preliminary of coating antigen concentration and antibody working concentration determined
Working concentration combination with square formation volumetry initial option coating antigen and antibody.Use coating buffer that coating antigen 4-keto-β-ionone acid-OVA doubling dilution is become to the horizontal coated elisa plate of 0.25,0.5,1,2,4,8,16,32 μ g/mL; The DCC/C11 monoclonal antibody is used antibody diluent (purchased from Wuhan Fei Yuan Science and Technology Ltd.) doubling dilution to become 1: 500, within 1: 1000,1: 2000,1: 4000,1: 8000,1: 16000,1: 32000,1: 64000,1: 128000, vertically add enzyme plate.The square formation titration results is in Table 1, and the following coating antigen concentration of initial option and antibody dilution combine: (2,1000), (4,4000), (8,8000) and (16,16000).
Result, as table 1, tentatively determines that the coated concentration of coating antigen 4-keto-β-ionone acid-OVA is 8 μ g/mL, and the antibody working concentration is 1: 8000.
The titration of table 1 DCC/C11 monoclonal antibody square formation
3.3 determining of best coating antigen concentration and antibody working concentration
The coated concentration of selecting with the square formation titration and antibody dilution combination are done respectively to suppress curve, and CTX standard substance concentration is set to 0,1,2,3,4,5 μ g/mL, its " 0 " hole and IC
50Value is in Table 2.The ratio of antigen-antibody is the key that affects its sensitivity, if there is antigen or antibody excess, all will cause IC
50Higher, from data, best coated concentration is 8 μ g/mL, and antibody dilution tentatively is defined as 1: 8000.
The optimum antibody extent of dilution is determined: with the coated concentration coated elisa plate of the best, by antibody 4 dilution gradients of concentration equal difference design centered by 1: 8000, 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: 8000 for the optimum antibody extent of dilution.
The best coated concentration optimization of table 2
| Coating antigen concentration (μ g/mL) | Antibody dilution multiple (1: X) | 0 hole OD value | IC 50Value (μ g/mL) |
| 2 | 1000 | 1.77 | 5.3 |
| 4 | 4000 | 1.5 | 4.7 |
| 8 | 8000 | 1.63 | 2.7 |
| 16 | 16000 | 1.44 | 4.8 |
Table 3 optimum antibody extent of dilution is optimized
| Antibody dilution multiple (1: X) | 0 hole OD value | IC50 value (μ g/mL) |
| 6000 | 1.755 | 2.93 |
| 7000 | 1.712 | 3.46 |
| 8000 | 1.6 | 2.93 |
| 9000 | 1.48 | 3.17 |
| 10000 | 1.38 | 3.58 |
3.4 the foundation of typical curve
The CTX standard substance are mixed with to 6 concentration gradients such as 0,1,2,3,4,5 μ g/mL, measure the drawing standard curve according to top definite indirect competitive ELISA method.As shown in Figure 2, the regression equation of racing ELISA detecting method of the present invention and the index of correlation are: y=-0.7076+0.8366, r=0.996, IC
50Value is 3.05 ± 0.274 μ g/mL, and linearity range is 1-5 μ g/mL.
3.5 specificity
Become concentration gradient to carry out indirect competitive ELISA the standard of physical product doubling dilution of the medicine of various β-carotene classes and structural similitude thereof respectively, calculate IC
50Value, with CTX standard substance IC
50The value contrast obtains cross reacting rate, the results are shown in Table 4.Result shows, the indirect competitive ELISA method that this research is set up has fine cross reaction to β-carotene, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid, with β-ionone, certain cross reaction is arranged, and with 4-oxo-beta-ionone acid, vitamin A acid, the equal no cross reaction of Vogan-Neu.
The specificity of table 4 ELISA detection method of the present invention
| The competition thing | IC 50(μg/mL) | Cross reacting rate (%) |
| Canthaxanthin | 2.99 | 100 |
| β-carotene | 3.22 | 92.9 |
| β-A Piao-8 '-carotenal | 3.06 | 97.7 |
| Xenthophylls | 3.12 | 95.8 |
| Capsorubin | 3.32 | 90.1 |
| β-ionone acid | 2.13 | 140.4 |
| β-ionone | 44.33 | 6.7 |
| 4-oxo-beta-ionone acid | ＞10000 | ＜0.03 |
| Vitamin A acid | ＞10000 | ＜0.03 |
| Vogan-Neu | ＞10000 | ＜0.03 |
The assembling of embodiment 4 ELISA detection kit
4.1 test kit moiety
(1) be coated with the enzyme plate of coating antigen 4-keto-β-ionone acid-OVA;
(2) the CTX standard solution is 6 bottles, and concentration is respectively 0,1,2,3,4,5 μ g/mL;
(3) the hybridoma DCC/C11 monoclonal antibody working fluid that preserving number is CCTCC NO:C201146;
(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
44.00g, Na
2HPO
412H
2O 58.00g, KCl 2.00g, add distilled water to 1000mL;
(6) concentrated cleaning solution: NaCl 80.00g, KH
2PO
42.00g, Na
2HPO
412H
2O 29.00g, KCl 2.00g, Tween 20 5mL, add 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.
(8) stop buffer: 2mol/L sulphuric acid soln.
4.2 the preparation of enzyme plate
(1) coated: as with coating buffer, coating antigen 4-keto-β-ionone acid-OVA to be diluted to 8 μ g/mL, accurately to draw 100 μ L coating antigen solution in each enzyme mark hole, be placed horizontally at wet box, hatch 12 hours for 4 ℃.
(2) wash plate: throw away enzyme plate endoperidium original solution, pat dry, accurately draw 250 μ L washingss in each enzyme mark hole, after standing 30 seconds, throw away washings, pat dry on thieving paper, repeated washing 3 times, pat dry.
(3) sealing: accurately draw 250 μ L confining liquids in each enzyme mark hole, level is placed in wet box, and 37 ℃ of incubators are hatched 2 hours.
(4) wash plate: throw away confining liquid, accurately draw 250 μ L washingss in each enzyme mark hole, after standing 30 seconds, throw away washings, pat dry on thieving paper; Repeated washing 3 times, pat dry.
(5) dry: enzyme plate is inverted in 37 ℃ of incubators and is dried 0.5 hour.
(6) encapsulation: enzyme plate is dried the rear aluminium foil bag of packing into together with siccative, with vacuum packaging machine, encapsulates.
In embodiment 5 animal tissuess, the enzyme linked immunological kit of β-carotene class pigment is measured program
5.1 reagent preparation
Washings preparation: NaCl 80.00g, KH
2PO
42.00g, Na
2HPO
412H
2O 29.00g, KCl 2.00g, polysorbas20 5mL, add distilled water to 1000mL.
The component of sample diluting liquid and proportioning are: NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, KCl 0.2g, N ', N '-N,N-DIMETHYLACETAMIDE 20mL, add distilled water to 1000mL.
Substrate mixed solution preparation: 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.
5.2 tissue sample is processed
Take the homogeneous sample 2.00 ± 0.02g of chicken muscle in the 50mL centrifuge tube, add ethyl acetate 10mL, whirlpool mixing immediately makes sample dispersion complete in 1 minute.Add 2% sodium hydroxide solution 5mL, whirlpool mixes 5 minutes; 4000r/ minute centrifugal 5 minutes, get supernatant liquor 5mL 40-50 ℃ of nitrogen in the 10mL centrifuge tube and dry up.Residue dissolves with N,N-dimethylacetamide 0.5ml, then gets sample detection after 4 times of sample diluting liquid dilutions for 50 μ L.
Annotate: present method is 2 to the extension rate of chicken muscle.
5.3 ELISA measures program
(1) take out test kit, balance, to room temperature, is inserted the micropore frame by the hole bar of enough standard substance and sample quantity used.
(2) first add CTX standard solution or sample liquid 50 μ L in each micropore; Standard substance and sample do two parallel, the position of record standard product and sample.The hybridoma DCC/C11 monoclonal antibody working fluid 50 μ L that to add preserving number be CCTCC NO:C201146 again, to each hole, fully mix; Level is put in wet box, hatches 30 minutes for 37 ℃.
(3) get rid of liquid in clear opening, pat dry on thieving paper.Accurately draw washings 250 μ L in each hole, standing about 30 seconds, get rid of clean washings, pat dry repeated washing 3 times on thieving paper.
(4) the sheep anti-mouse igg antibody working fluid 100 μ L that add horseradish peroxidase (HRP) mark, to each hole, fully mix; Level is put in wet box, hatches 30 minutes for 37 ℃.
(5) get rid of liquid in clear opening, pat dry on thieving paper.Accurately draw washings 250 μ L in each hole, standing about 30 seconds, get rid of clean washings, on thieving paper, pat dry.Repeated washing 3 times.
(6) add substrate mixed solution 100 μ L to each hole, fully mix; Level is put in wet box, hatches 15 minutes for 37 ℃.
(7) add stop buffer 50 μ L to each hole; Within 30 minutes, light absorption value is measured at inherent 450nm place.
5.4 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.In 1-5 μ g/mL scope, take inhibiting rate as ordinate zou, the logarithm of concentration of standard solution is X-coordinate drawing standard curve, obtains regression equation.According to the inhibiting rate of formula 1 calculation sample, by inhibiting rate substitution regression equation, calculate mensuration concentration, be multiplied by corresponding extension rate, be the residual concentration of β-carotene class in sample.
Sensitivity, precision, the accuracy of embodiment 6 test kits
6.1 the sensitivity of test kit of the present invention
Get 20 parts of blank tissue samples, carry out the ELISA detection, measure the OD value, calculate the mean value of blank sample OD value
Will
Find corresponding concentration (C) on the substitution typical curve, and calculate standard deviation (SD).Calculate the Z value according to formula Z=C+3 * SD, this is the lowest detectable limit (LOD) of method for organizing.As shown in table 5, the lowest detection of canthaxanthin in chicken muscle is limited to 1.29 μ g/g.
The lowest detectable limit of table 5 canthaxanthin in chicken muscle
6.2 the Precision Experiment of test kit of the present invention
Respectively its typical curve equations of OD value substitution corresponding to CTX standard substance concentration such as 1,2,3,4,5 μ g/mL are 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 6.Result shows, all<15%, between plate, the variation coefficient all<20%, illustrates that the indirect competitive ELISA method of this research foundation has precision preferably to the plate within variance coefficient of typical curve.
The precision of table 6 test kit of the present invention
6.3 the accuracy of test kit of the present invention
With the accuracy of adding rate of recovery reflection test kit.Get blank tissue sample, the maximum residue limit(MRL) according to medicine in various tissues (MRL) is added respectively experiment, and making the concentration of interpolation medicine in tissue is 0.5 * MRL, MRL, 2 * MRL, and each sample concentration arranges 5 Duplicate Samples.After sample preparation, ELISA measures drug level, repeats 3 batches, and calculate recovery rate is criticized interior and interassay coefficient of variation.As shown in table 7, the medicine rate of recovery, between 77.05%～122.12%, all is less than 25% with differences between batches in batch.
The accuracy of table 7 test kit of the present invention
Claims (7)
1. the monoclonal antibody that can identify β-carotene class pigment, is characterized in that, it is that CCTCC NO:C201146 hybridoma DCC/C11 is secreted by preserving number.
2. the hybridoma DCC/C11 described in claim 1, be deposited in Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:C201146.
3. the test kit that comprises monoclonal antibody claimed in claim 1.
4. test kit according to claim 3, this test kit is the enzyme linked immunological kit for detection of β-carotene class pigment.
5. the enzyme-linked immunoassay method for detection of β-carotene class pigment, comprise the preparation of coating antigen, antibody and processing and the detection of sample, and its step is as follows:
(1) haptens 4-oxo-beta-ionone acid is obtained to coating antigen with the ovalbumin coupling;
(2) the hybridoma DCC/C11 that is CCTCC NO:C201146 with preserving number prepares monoclonal antibody;
(3) with the coated solid phase carrier of the coating antigen of step (1);
(4) by testing sample, by volume ratio, be ethyl acetate: the mixing solutions of 2% sodium hydroxide=2:1 extracts, and after ethyl acetate extraction and nitrogen dry up, residue obtains determinand with the sample diluting liquid dissolving;
(5) determinand of step (4) carried out to enzyme linked immunosorbent detection,
Wherein:
The component of sample diluting liquid and proportioning are: NaCl8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O2.9g, KCl0.2g, N ', N '-N,N-DIMETHYLACETAMIDE 20mL, add distilled water to 1000mL,
Described β-carotene class pigment is canthaxanthin, β-carotene, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid.
6. the application of monoclonal antibody claimed in claim 1 in the enzyme linked immunological kit of preparation detection β-carotene class pigment, described β-carotene class pigment is canthaxanthin, β-carotene, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid.
7. the application of the described test kit of claim 3 or 4 in animal tissues's β-carotene class pigment residue detection, described β-carotene class pigment is canthaxanthin, β-carotene, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid.
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| CN110749688A (en) * | 2019-11-07 | 2020-02-04 | 贵州省兽药饲料监察所(贵州省兽药残留监测中心) | Method for measuring β -apo-8' -ethyl carotenoate colorant content in feed |
| CN111175499A (en) * | 2020-02-28 | 2020-05-19 | 江南大学 | Preparation method of ELISA kit for detecting testosterone |
| CN114133326A (en) * | 2022-01-20 | 2022-03-04 | 中国科学院成都生物研究所 | Preparation and application of cantharis yellow colloidal gold lateral flow immunochromatographic card |
| CN114671761B (en) * | 2022-04-18 | 2024-05-31 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Method for preparing beta-ionone hapten, artificial antigen and antibody |
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| CN1668754A (en) * | 2002-05-14 | 2005-09-14 | 马泰克生物科学公司 | Carotene synthase gene and uses therefor |
| CN101979601A (en) * | 2010-09-21 | 2011-02-23 | 山东省农业科学院高新技术研究中心 | Method for improving carotenoid content of tomato |
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| CN101979601A (en) * | 2010-09-21 | 2011-02-23 | 山东省农业科学院高新技术研究中心 | Method for improving carotenoid content of tomato |
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