CN102585007A - Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting beta-carotene pigments - Google Patents

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

Be used to detect monoclonal antibody and the enzyme-linked immunoassay method and the test kit of β-Hu Luobusu class pigment

Technical field

The present invention relates to a kind of monoclonal antibody and a kind of enzyme-linked immunoassay method and the test kit that is used to detect β-Hu Luobusu class pigment that can discern β-Hu Luobusu class pigment.

Background technology

β-Hu Luobusu class pigment has β-A Piao-8 '-carotenal, β-A Piao-8 '-Serlabo acetoacetic ester, xenthophylls, capsanthin, canthaxanthin, astaxanthin etc.

At present, mainly be the instrument detecting method to the detection method of β-Hu Luobusu class material, like Paper Chromatography, thin layer chromatography, column chromatography, spectrophotometry, performance liquid chromatography or LC-MS method.These detection methods need 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 susceptibility of high degree of specificity and the enzymatic reaction of immunoreation (being the Ag-Ab association reaction) medicine to be carried out a kind of comprehensive detection technique of qualitative and quantitative analysis.It has advantages such as easy and simple to handle, high-throughput, highly sensitive, low expense, can overcome the deficiency of instrument detecting method.At present, be not directed against the bibliographical information of the ELISA detection method of β-Hu Luobusu class both at home and abroad.

For the foundation of micromolecular compound ELISA detection method, the Antibody Preparation of micromolecular compound is the core, and the appropriate design haptin is crucial.Haptenic chemical structure is different, and the chemical site that is connected on the carrier proteins is just different, and the chemical property of crosslinked arm is also different, and this is the deciding factor that influences micromolecular compound antibody character.Therefore micromolecular compound is carried out the chemical structure transformation, the appropriate design haptin is connected to haptin on the carrier proteins, is the core of this invention.

It is 201010140887.6 patent of invention that the applicant retrieves one piece of application number, and this patent provides the method for a kind of oxidation α-Zi Luotong and β-ionone, but not with it as the haptin synthetic immunogen and be applied to enzyme linked immunosorbent detection.

Summary of the invention

First purpose of the present invention provides a kind of monoclonal antibody that can discern β-Hu Luobusu 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-Hu Luobusu class pigment.

The 3rd purpose of the present invention provides a kind of enzyme linked immunological kit that detects β-Hu Luobusu class pigment.

The 4th purpose of the present invention provides the application of said monoclonal antibody in the enzyme linked immunological kit of preparation detection β-Hu Luobusu class pigment.

The 5th purpose of the present invention provides the application of test kit in animal tissues's β-Hu Luobusu class pigment residue detection that contains said monoclonal antibody.

The present invention realizes through following technical scheme:

A kind of monoclonal antibody that can discern β-Hu Luobusu class pigment, it is that CCTCC NO:C201146 hybridoma DCC/C11 is secreted by preserving number.

Said carotenoid pigment refers to canthaxanthin, β-Hu Luobusu, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid.

Said hybridoma DCC/C11 is deposited in Chinese typical culture collection center (CCTCC), and preserving number is CCTCC NO:C201146.

Used immunogen prepares with the bovine serum albumin coupling through haptin β-ionone acid.

Further, the invention provides a kind of enzyme-linked immunoassay method that detects β-Hu Luobusu class pigment, comprise the preparation of immunogen, coating antigen, antibody and the processing and the detection of sample, its step is following:

(1) haptin β-ionone acid is obtained immunogen with bovine serum albumin (BSA) coupling;

(2) haptin 4-oxo-beta-ionone acid is obtained coating antigen with ovalbumin (OVA) coupling;

(3) utilize the immunogen immune mouse of step (1), obtain the hybridoma DCC/C11 that preserving number is CCTCC NO:C201146 through cytogamy and screening;

(4) use preserving number to prepare monoclonal antibody as the hybridoma DCC/C11 of CCTCC NO:C201146;

(5) coating antigen with step (2) encapsulates solid phase carrier;

(6) use volume ratio to be ETHYLE ACETATE testing sample: the mixing solutions of sodium hydroxide (2%)=2: 1 extracts, and after ethyl acetate extraction and nitrogen dried up, residue dissolved with sample diluting liquid and obtains determinand;

(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 ₂O 2.9g, KCl 0.2g, N ', N '-N,N-DIMETHYLACETAMIDE 20mL adds distilled water to 1000mL.

The present invention with said monoclonal antibody and coating antigen as core reagent and other conventional agent combination; Processed the enzyme linked immunological kit that can detect β-Hu Luobusu class pigment; In conjunction with above-mentioned enzyme-linked immunoassay method, realized enzyme linked immunosorbent detection to β-Hu Luobusu class pigment.

The invention has the beneficial effects as follows:

1, canthaxanthin, β-Hu Luobusu, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, the β-ionone acid in the monoclonal anti physical efficiency identification β-Hu Luobusu class pigment of the present invention's preparation, specificity is strong, and is highly sensitive.

2, because the present invention adopts β-ionone acid as haptin; This haptin has kept canthaxanthin, β-Hu Luobusu, β-A Piao-8 '-carotenal, xenthophylls, the common chemical structure of Capsorubin, can discern canthaxanthin, β-Hu Luobusu, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid simultaneously by the monoclonal antibody of this haptin preparation.

3, ELISA method of the present invention and test kit can disposablely be measured the total content of canthaxanthin in the determinand, β-Hu Luobusu, β-A Piao-8 '-carotenal, xenthophylls, Capsorubin, β-ionone acid; Shorten detection time, reduced the detection cost.

4, high, good, the accuracy good of precision of ELISA method of the present invention and test kit detection sensitivity.

Description of drawings

Accompanying drawing 1 is technological line figure of the present invention.

Accompanying drawing 2 is the indirect competitive ELISA response curve of monoclonal antibody of the present invention and canthaxanthin (CTX) standard substance, and the X axle is canthaxanthin (CTX) concentration of standard solution logarithmic value, and the Y axle is that the OD value of canthaxanthin standard solution is divided by " zero " hole OD value (B/B ₀).

Embodiment

Through embodiment the present invention is further specified below.

The preparation of embodiment 1 immunogen and coating antigen

1.1 β-ionone acid is synthetic

Accurately measure zero(ppm) water 30mL, join in the round-bottomed flask of precooling; In flask, add liquid bromine 20g, stir after 2 hours, it is subsequent use to form A liquid.Accurately β-ionone 6g is dissolved among the dioxane 20mL, forms B liquid.B liquid is added in the A liquid, and reaction is 8 hours under the room temperature.After reaction is accomplished, add 20% sodium sulfite anhy 96 and no longer become blue up to starch potassium iodide paper.Add then in concentrated hydrochloric acid to the solution and white solid matter occurs.Filter, the filter residue white solid matter dropped in the methyl alcohol, put 60 ℃ of stirring in water bath to dissolving fully after, filter.Filtrating is placed-20 ℃ of after-filtration that spend the night, filter cake places 50 ℃ of vacuum drying ovens dry, and the product that obtains carries out mass spectrum and is accredited as β-ionone acid.

1.2 β-ionone acid-BSA's is synthetic

Get β-ionone acid 93mg, 1,3-NSC 57182 (DCC) 50mg and N-hydroxy-succinamide (NHS) 33mg are dissolved in dioxane 5mL, and the room temperature lucifuge stirred 8 hours, and reaction product is filtered the back and is formed A liquid.Other gets BSA80mg and is dissolved in phosphate buffered saline buffer (0.01mol/L pH8.0) among the 20mL, forms B liquid.Ice bath stirs down, and the A drop is added to B liquid; With the reaction solution of reaction after 12 hours centrifugal 10 minutes in 4000r/ minute; Get supernatant and pack in the dialysis tubing,, promptly get conjugate β-ionone acid-BSA normal saline dialysis 5-6 days.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 the there-necked flask; Heating in water bath to 37 ℃ adds 15ml and contains the aqueous solution of 2g potassiumiodide and the aqueous solution that 6ml contains the 3g sodium pyrosulfate.Under the agitation condition, add the 20ml aqueous solution that contains the 8g sodium bromate again, after reaction is accomplished, separate organic phase.This organic phase with the 8g anhydrous magnesium sulfate drying after, revolve inspissation and contract.Carry out column chromatography, isolation of target substances with sherwood oil and acetone.The target substance of separating is revolved after inspissation contracts, add sherwood oil, put-20 ℃ of after-filtration that spend the night with volume.Filter cake places 50 ℃ of vacuum drying ovens dry, and the product that obtains carries out mass spectrum and is accredited as 4-oxo-beta-ionone.

1.4 4-oxo β-ionone acid (4-keto-β-ionone acid) is synthetic

Accurately take by weighing NaOH4g, be dissolved in the 20mL water, splash into bromine 2mL, continue to stir.Take by weighing 4-oxo-beta-ionone bullion 1.2g, be dissolved in dioxane 4mL, add in the good reaction solution of above-mentioned activation, stopped reaction when starch potassium iodide paper no longer becomes indigo plant.The NaHSO of adding 30% ₄16mL uses the dichloromethane extraction removal of impurities, and water is added the concentrated hydrochloric acid acidifying, leaves standstill to red precipitate occurring.Behind the sedimentation and filtration, filter residue dissolves with methylene dichloride, adopts sherwood oil and ethanol column chromatography, isolation of target substances.The target substance of separating is revolved after inspissation contracts, add methylene dichloride, place-20 ℃ of after-filtration that spend the night with volume.Filter cake places 50 ℃ of vacuum drying ovens dry, and the product that obtains carries out mass spectrum and is accredited as 4-oxo-beta-ionone acid.

1.5 4-oxo-beta-ionone acid-OVA (4-keto-β-ionone acid-OVA) is synthetic

Get 4-oxo β-ionone acid 48mg, add N, dinethylformamide (DMF) 2mL adds triethylamine 60 μ L then, adds isobutyl chlorocarbonate 60 μ L again, and stirring reaction is 60 minutes under the room temperature, forms A liquid.Other gets in the phosphate buffer soln (pH7.4) that OVA100mg is dissolved in 30mL0.01mol/L, forms B liquid.The A drop is added in the B liquid stirring reaction 16 hours.After reaction is accomplished, that reaction solution is centrifugal 10 minutes 5000r/ minute, 4 ℃.Get supernatant saline water is thoroughly dialysed, promptly get conjugate 4-oxo-beta-ionone acid-OVA.Get the supernatant lyophilize after centrifugal, put-20 ℃ of preservations, as coating antigen.

Embodiment 2 MONOCLONAL ANTIBODIES SPECIFIC FOR

The preparation of hybridoma: with reference to the method among the Xue Qingshan " philosophy and technique of vitro culture " (Science Press's calendar year 2001 version): with conjugate β-ionone acid-BSA immunity Balb/C mouse (available from Disease Prevention Control Center, Hubei Prov's Experimental Animal Center) of embodiment 1 preparation.Immune programme for children is after getting the protein solution and isopyknic Freund's complete adjuvant (available from sigma company) emulsification that contains conjugate β-ionone acid-BSA 125 μ g, in the subcutaneous multi-point injection of mouse back.2 weeks of later every interval are strengthened once, use Freund (available from sigma company) emulsification instead.At last 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 is isolated splenocyte, presses 1-2 * 10 with the SP2/0 myeloma cell (SP2/0 myeloma cell is from this laboratory) of prepared fresh ⁷Individual SP2/0 and 10 ⁸Ratio mixing in the 50mL centrifuge tube of individual immune spleen cell (1: 10～1: 15), 1500r/ minute, centrifugal 10 minutes.Evacuation supernatant (filter paper of available sterilization blots) knocks the pipe end gently, makes cell precipitation loosening slightly.The centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths.In 1 minute, slowly splash into 50% polyoxyethylene glycol (PEG) 0.8mL (available from the sigma Company products) of preparatory temperature to 37 ℃ then, the limit edged stirs with pipette tip gently, continues to stir 1 minute.Slowly add 37 ℃ of 1640 (available from the commercial substratum of Hyclone company) basic medium 10mL of temperature in advance then.Concrete grammar is: dropwise splashed into 1mL in first minute, added 1mL in second minute, added 3mL on the 3rd～4 minute, added remaining 5mL on the 5th minute, each added-time needs slowly to add, and constantly stirs lightly.Add 30mL 1640 liquid at last, also need slowly to add.Centrifugal 5 minutes of 800r/m removes supernatant, places 5～8 minutes in 37 ℃.Suspend with HAT (available from Sigma company) substratum, simultaneously also with the HAT substratum suspend the raising splenocyte for preparing and with merge after cytomixis, add an amount of HAT substratum as required, divide and plant in 96 well culture plates, about 250 μ L/ holes.Once merge and to inoculate 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 contains 10 approximately ⁴About SP2/0 cell.In 37 ℃, 5%CO ₂Cultivate in the incubator.Merging to begin in back second day to observe had pollution-freely, added 1 HAT substratum in the 4th day, and suction in the 8th～10 day goes 100 μ L substratum to change HT (available 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.Use limiting dilution assay (with reference to Xue Qingshan " philosophy and technique of vitro culture " Science Press calendar year 2001 version) to clone, screen at once to the positive hole that screens.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 chromosome counting, and the result shows that the chromosomal mean number of SP2/0 is 58, and splenocyte karyomit(e) is 40, and the chromosome number of hybridoma all is higher than the chromosome number of two parent's cells between 92～104.The chromosome number of hybridoma is explained the cell of SP2/0 really of fused cell and the hybridization product of splenocyte obviously more than the karyomit(e) of myeloma cell SP2/0.To this monoclonal hybridoma that filters out; The applicant is with its called after DCC/C11; And deliver Chinese typical 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.

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 DCC/C11 enlarged culturing of CCTCC NO:C201146 ⁶Individual/mL, every mouse peritoneal inoculation 0.5ml.Treat that mouse web portion obviously expands, spiritual variation, the dying ascites of gathering when motionless.According to literature method (Zhu Liping, Chen Xueqing. immunology common experimental method. Beijing: People's Medical Officer Press, 2000), purifying obtains monoclonal antibody.Employing is carried out the hypotype evaluation available from the mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit) of ROCKLAND company to the resulting monoclonal antibody of the present invention, and the result is mouse IgG ₁Hypotype.

The foundation of embodiment 3 ELISA detection methods

3.1 the preparation of reagent (reagent that present embodiment uses all adopts following method preparation except that other indicates)

Phosphate buffered saline buffer: NaCl 8.0g, KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, KCl 0.2g adds 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 adds 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: flying Science and Technology Ltd. far away by Wuhan provides.

Stop buffer: 2mol/L sulphuric acid soln.

3.2 the preliminary of coating antigen concentration and antibody working concentration confirmed

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 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 (flying Science and Technology Ltd. far away available from Wuhan) doubling dilution to become 1: 500, was vertically added enzyme plate in 1: 1000,1: 2000,1: 4000,1: 8000,1: 16000,1: 32000,1: 64000,1: 128000.The square formation titration results is seen table 1, following coating antigen concentration of initial option and antibody dilution combination: (2,1000), (4,4000), (8,8000) and (16,16000).

Result such as table 1 confirm that tentatively the concentration that encapsulates 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 confirming of best coating antigen concentration and antibody working concentration

The concentration that encapsulates so that the square formation titration is selected is made the inhibition curve respectively with the antibody dilution combination, and CTX standard substance concentration is set to 0,1,2,3,4,5 μ g/mL, its " 0 " hole and IC ₅₀Value is seen table 2.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 8 μ g/mL that the best encapsulates concentration, and antibody dilution is tentatively confirmed as 1: 8000.

The optimum antibody extent of dilution is confirmed: encapsulating the concentration coated elisa plate with the best, was 4 dilutions of centre concentration equal difference design gradient with antibody with 1: 8000, 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 also reduces, when 0 hole be worth its IC when hanging down ₅₀Value raises on the contrary, and therefore selecting 1: 8000 is the optimum antibody extent of dilution.

Table 2 the best encapsulates concentration optimization

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 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 medicine of various β-Hu Luobusu classes and the standard of physical article doubling dilution of structural similitude thereof respectively, calculate IC ₅₀Value is with CTX standard substance IC ₅₀The value contrast obtains cross reacting rate, and the result sees table 4.The result shows; The indirect competitive ELISA method that this research is set up has fine cross reaction to β-Hu Luobusu, 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-Hu Luobusu	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 moity

(1) is 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) preserving number is the hybridoma DCC/C11 monoclonal antibody working fluid of 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 adds distilled water to 1000mL;

(6) concentrated cleaning solution: NaCl 80.00g, KH ₂PO ₄2.00g, Na ₂HPO ₄12H ₂O 29.00g, KCl 2.00g, Tween 20 5mL add distilled water to 1000mL

(7) substrate mixed solution: accurately draw substrate B liquid (flying Science and Technology Ltd. far away available from Wuhan) 10mL, add 100 μ L substrate A liquid (flying Science and Technology Ltd. far away available from Wuhan), mixing is joined existing usefulness at present.

(8) stop buffer: 2mol/L sulphuric acid soln.

4.2 the preparation of enzyme plate

(1) encapsulates: with coating buffer coating antigen 4-keto-β-ionone acid-OVA is diluted to 8 μ g/mL, 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 is clapped and is done.

(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 is clapped and is done.

(5) oven dry: 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 enzyme linked immunological kit of β-Hu Luobusu class pigment is measured program in embodiment 5 animal tissuess

5.1 reagent preparation

Washings preparation: NaCl 80.00g, KH ₂PO ₄2.00g, Na ₂HPO ₄12H ₂O 29.00g, KCl 2.00g, polysorbas20 5mL adds 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 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

Take by weighing the even quality sample 2.00 ± 0.02g of chicken muscle in the 50mL centrifuge tube, add ETHYLE ACETATE 10mL, whirlpool mixing immediately made sample dispersion complete in 1 minute.Add 2% sodium hydroxide solution 5mL, whirlpool mixed 5 minutes; 4000r/ minute centrifugal 5 minutes, get supernatant 5mL 40-50 ℃ of nitrogen in the 10mL centrifuge tube and dry up.Residue is got and is gone up the appearance detection after 50 μ L dilute 4 times with sample diluting liquid with DMAC N,N 0.5ml dissolving.

Annotate: present method is 2 to the extension rate of chicken muscle.

5.3 ELISA measures program

(1) take out test kit, balance is to room temperature, with the hole bar insertion micropore frame of enough standard substance and the used quantity of sample.

(2) add CTX standard solution or sample liquid 50 μ L earlier in each micropore; Standard substance and sample do two parallel, the position of record standard article and sample.The hybridoma DCC/C11 monoclonal antibody working fluid 50 μ L that add preserving number again and be CCTCC NO:C201146 are to each hole, thorough mixing; Level is put in the wet box, hatches 30 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 repeated washing 3 times at the thieving paper arsis.

(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 30 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 at the thieving paper arsis.Repeated washing 3 times.

(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; 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, be inhibiting rate.In 1-5 μ g/mL scope, be 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 1 calculation sample, with inhibiting rate substitution regression equation, calculate mensuration concentration, multiply by corresponding extension rate, be the residual concentration of β-Hu Luobusu class in the 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 control group tissue samples; 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.As shown in table 5, the lowest detection of canthaxanthin in chicken muscle is limited to 1.29 μ g/g.

The LDL of table 5 canthaxanthin in chicken muscle

6.2 the precision of test kit of the present invention experiment

Respectively that CTX standard substance concentration such as 1,2,3,4,5 μ g/mL 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 6.The result shows, the plate within variance coefficient of typical curve all＜15%, Variation Lines number average＜20% between plate explains that the indirect competitive ELISA method of this research foundation has better precision.

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 recovery reflection test kit.Get the blank control group tissue samples, add experiment respectively according to the MRL (MRL) of medicine in various tissues, make in the tissue that to add drug concentrations be 0.5 * MRL, MRL, 2 * MRL, each sample concentration is provided with 5 parallel appearance.ELISA measures drug level after the sample preparation, repeats 3 batches, and calculate recovery rate is criticized interior and interassay coefficient of variation.As shown in table 7, the medicine recovery between 77.05%～122.12%, batch in differences between batches all less than 25%.

The accuracy of table 7 test kit of the present invention

Claims (7)

1. the monoclonal antibody that can discern β-Hu Luobusu class pigment is characterized in that, it is that CCTCC NO:C201146 hybridoma DCC/C11 is secreted by preserving number.

2. the described hybridoma DCC/C11 of claim 1 is deposited in Chinese typical culture collection center, and preserving number is CCTCC NO:C201146.

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 to detect β-Hu Luobusu class pigment.

5. an enzyme-linked immunoassay method that is used to detect β-Hu Luobusu class pigment comprises the preparation of immunogen, coating antigen, antibody and the processing and the detection of sample, and its step is following:

(1) haptin β-ionone acid is obtained immunogen with the bovine serum albumin coupling;

(2) haptin 4-oxo-beta-ionone acid is obtained coating antigen with the ovalbumin coupling;

(3) utilize the immunogen immune mouse of step (1), obtain the hybridoma DCC/C11 that preserving number is CCTCC NO:C201146 through cytogamy and screening;

(4) use preserving number to prepare monoclonal antibody as the hybridoma DCC/C11 of CCTCC NO:C201146;

(5) coating antigen with step (2) encapsulates solid phase carrier;

(6) use volume ratio to be ETHYLE ACETATE testing sample: the mixing solutions of sodium hydroxide (2%)=2: 1 extracts, and after ethyl acetate extraction and nitrogen dried up, residue dissolved with sample diluting liquid and obtains determinand;

(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 ₂O 2.9g, KCl 0.2g, N ', N '-N,N-DIMETHYLACETAMIDE 20mL adds distilled water to 1000mL.

6. the application of the described monoclonal antibody of claim 1 in the enzyme linked immunological kit of preparation detection β-Hu Luobusu class pigment.

7. claim 3 or the 4 described test kits application in animal tissues's β-Hu Luobusu class pigment residue detection.

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