CN107513522A - A kind of hybridoma cell strain for secreting anti-ochratoxin monoclonal antibody and its application - Google Patents
A kind of hybridoma cell strain for secreting anti-ochratoxin monoclonal antibody and its application Download PDFInfo
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Abstract
The invention provides a kind of hybridoma cell strain F 33 for secreting anti-ochratoxin monoclonal antibody, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, deposit number is CGMCC No.14303.The anti-ochratoxin monoclonal antibody high sensitivity of this cell line secretion, specificity is good, anti-interference is good, to 50% inhibition concentration IC of ochratoxin A50For 0.990 μ g/L, preferable anti-interference is respectively provided with to the auxiliary material used in mycotoxin that may be present, agricultural chemicals, heavy metal, concocting process in Chinese medicine and contaminated bacteria etc., immune detection for ochratoxin A in Chinese medicine provides condition, has actual application value.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of hybridoma for secreting anti-ochratoxin monoclonal antibody
Cell line and its application.
Background technology
Chinese medicine is possible to because of self property and extraneous factor from processes such as production, harvesting, processing, transport, storages
Influence and contaminant, and then the simultaneously contaminant toxin that goes mouldy.The quality of Chinese medicine can not only be influenceed by going mouldy, and lose medicinal material
Original medical value is removed, causes huge economic loss, and during going mouldy, caused a variety of mycotoxins can be to suffering from
Liver, kidney, nerve and hemopoietic system of person etc. cause serious infringement, in some instances it may even be possible to induce cancer.Meanwhile with current traditional Chinese medicine
The popularization of health care's idea, large Chinese medicine of more and more " integration of drinking and medicinal herbs " is used for the daily diet of people and health care is worked as
In, the demand in market is big, if these Chinese medicines are polluted by mycotoxin, will certainly endanger the strong of more crowds
Health, personal and medical system financial burden is aggravated, more likely further influence society and harmonious stabilization.
Ochratoxin (ochratoxin) is that secondary metabolism caused by the toxigenic bacterium strain such as Eurotium and Penicillium produces
Thing, including ochratoxin A (OTA), ochratoxin B (OTB), ochratoxin C (OTC), ochratoxin D (OTD),
The similar compound of 7 kinds of structures such as OTA methyl esters and OTB methyl esters and ethyl ester, is that isocoumarin is coupled L-phenylalanine
Derivative.Pollution of the ochratoxin to crops is in the world all than more serious, wherein with ochratoxin A certainly
Right boundary is distributed most wide, level of pollution highest, and toxicity is most strong, and only teratogenesis, mutagenesis and the effect such as carcinogenic, does not have also immune
Toxicity, hepatotoxicity wind agitation and renal toxicity, and it is considered as relevant with Balkan region nephrosis, therefore it is the closeest with the relation of human health
Cut.International cancer research institution (IARC) was set to 2B class carcinogenic substances in 1993.Because the harm of ochratoxin is serious,
Cause the most attention of various countries.
OTA content is generally very low, so detection means requires higher.The method for being presently used for determining OTA has thin layer color
The chromatographies such as spectrometry (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), it is combined with mass spectrum and nuclear magnetic resonance
Technology is just being increasingly used for very special measure and is determining a variety of mycotoxins.The detection of these methods is accurate, test limit is low, but
Sample pretreatment process, special instrument and the professional staff training of complexity are needed, is received in actual applications very big
Limitation.
Immunoassay can make up all of above shortcoming, and immunoassay is that one kind is combined using antigen and antibody specific
The analysis method of the various materials of reaction detection, the key for establishing the immunoassay method of micromolecular compound are can to produce pair
Micromolecular compound has the antibody of high-affinity and high specific.
The content of the invention
It is an object of the invention to provide a kind of hybridoma cell strain for secreting anti-ochratoxin monoclonal antibody, the cell
Monoclonal antibody prepared by strain has preferably specific and higher detection sensitivity to OTA, to that may be present in Chinese medicine
Auxiliary material and contaminated bacteria for being used in mycotoxin, agricultural chemicals, heavy metal, concocting process etc. are respectively provided with preferable anti-interference, to build
OTA immunological detection method lays the foundation in vertical Chinese medicine.
A kind of technical scheme, hybridoma cell strain for secreting anti-ochratoxin monoclonal antibody, is named as
Ochratoxin monoclonal antibody hybridoma cell strain F-3-3, it is general to be preserved in China Committee for Culture Collection of Microorganisms
Logical microorganism center, deposit number are CGMCC No.14303, and preservation date is on 06 15th, 2017.
Anti- ochratoxin monoclonal antibody, it is the ochratoxin Dan Ke for CGMCC No.14303 by deposit number
Caused by grand antibody hybridoma cell strain F-3-3 secretions, the anti-ochratoxin monoclonal antibody is applied to reddish brown song in Chinese medicine
Mould toxin A analysis detection.
The present invention provides the basic preparation process of F-3-3 cell lines：
(1) OTA hapten synthesis and identification：8- hydroxy-3-methyl -1- oxochromen -7- carboxylic acid 0.5g are taken, are added
20mL dichloromethane dissolves, and takes oxalyl chloride 0.31g, adds 0.5mL DMFs (DMF) dissolved dilution, be added drop-wise to two
In chloromethanes solution, 2h is stirred at room temperature, adds 4- nitrophenylalanine 0.52g, adds triethylamine 0.3mL, 2h is stirred at room temperature, stops
Reaction, adding water 20mL, concussion is stood, and separates aqueous phase, and organic phase concentration is evaporated, upper silicagel column, ethyl acetate/petroleum ether (V/V,
1/5) elution separation, obtains nitro ochracin 0.87g；Nitro ochracin 0.87g is taken, adds ethanol 50mL to dissolve, adds palladium carbon
0.2g, hydrogen being passed through, 4h is stirred at room temperature, stopped reaction, filtering, remove palladium carbon, revolving removes ethanol, obtains red oil,
Recrystallized in 30mL dichloromethane/petroleum ether (V/V, 1/1), obtain amino ochracin 0.76g, as OTA haptens, yield
95%, proton nmr spectra qualification result；
(2) comlete antigen synthesizes：OTA haptens is coupled with bovine serum albumin(BSA) (BSA), ovalbumin (OVA) respectively
Obtain immunizing antigen (OTA-BSA) and envelope antigen (OTA-OVA)；
(3) animal immune：6~8 weeks female Balb/c mouse 10 (be divided into two groups of A and B, every group 5) of health are taken, just
Secondary immune Freund's complete adjuvant emulsification collare dorsal sc multi-point injection, every mouse immune dosage is 200 μ g OTA-BSA；
Afterwards booster immunization every two weeks the subcutaneous multi-point injection of nape part once, emulsification incomplete Freund's adjuvant；Last time is immune to be made
Incomplete Freund's adjuvant is replaced with physiological saline, using intraperitoneal injection, injection dosage and above identical several times；By indirect
ELISA detects serum titer；
(4) cell fusion is screened with cell line：, will be small by polyethylene glycol (PEG4000) method the 3rd day after booster immunization
Mice spleen cell and murine myeloma cell fusion, by complete culture solution culture, detect secretion OTA's using indirect elisa method
Cell line, the inhibition of the cell line is determined using indirect competitive ELISA method, screen the positive cell strain preferably suppressed and carry out
It is subcloned three times, it is final to obtain hybridoma cell strain F-3-3；
(5) Antibody preparation purifying and CHARACTERISTICS IDENTIFICATION：Atoleine is injected into 6~8 weeks Balb/c mouse, 500 μ L/ are only；10
Every mouse 0.5mL hybridoma CGMCC No.14303 is expelled to abdominal cavity after it, ascites is collected since the 7th day, by abdomen
Water is purified by caprylic acid-ammonium, and PAGE gel electrophoresis analysis purification effect, the monoclonal antibody of acquisition is imitated
Valency, hypotype, cross reactivity measure, are placed in -20 DEG C of preservations；
(6) application of antibody：The anti-ochratoxin monoclonal antibody that hybridoma cell strain F-3-3 secretes is used to prepare
Ochratoxin A enzyme linked immunological kit, for the analysis detection of ochratoxin A in Chinese medicine.
The beneficial effects of the present invention are：
(1) hybridoma cell strain F-3-3 provided by the invention can be used for preparing the anti-ochratoxin monoclonal of high-titer
Antibody, potency >=20000 that anti-ochratoxin mouse ascites antibody ELISA method measures.
(2) anti-ochratoxin monoclonal antibody high sensitivity provided by the invention, specificity is good, anti-interference is good, right
50% inhibition concentration IC of ochratoxin A50For 0.990 μ g/L, to mycotoxin that may be present, agricultural chemicals, a huge sum of money in Chinese medicine
Auxiliary material and contaminated bacteria for being used in category, concocting process etc. are respectively provided with preferable anti-interference.
(3) anti-ochratoxin monoclonal antibody provided by the invention can be applied to determine ochratoxin A in Chinese medicine and contain
Amount.
Biological material specimens preservation：The hybridoma cell strain of one plant of anti-ochratoxin monoclonal antibody of secretion, preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, address：The Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica, preservation date 2017 year 06 month 15 days, deposit number CGMCC
No.14303。
Brief description of the drawings
Fig. 1 ochratoxin A hapten synthesis route maps
Fig. 2 ochratoxin A competitive ELISA canonical plottings
Fig. 3 ochratoxin A competitive ELISA Logit/Log canonical plottings
Embodiment
Further explanation of the following examples of the present invention only as present invention, it is impossible in the restriction as the present invention
Perhaps scope.Below by embodiment, the present invention will be further described.
Embodiment 1：Hybridoma cell strain F-3-3 screening
1. hapten synthesis and identification
The synthetic route of OTA haptens is as shown in Figure 1.
8- hydroxy-3-methyl -1- oxochromen -7- carboxylic acid 0.5g are taken, adds 20mL dichloromethane to dissolve, takes oxalyl chloride
0.31g, add 0.5mL DMFs (DMF) dissolved dilution, be added drop-wise in dichloromethane solution, 2h is stirred at room temperature,
4- nitrophenylalanine 0.52g are added, add triethylamine 0.3mL, 2h is stirred at room temperature, stop reaction, add water 20mL, concussion is stood,
Aqueous phase is separated, organic phase concentration is evaporated, upper silicagel column, ethyl acetate/petroleum ether (V/V, 1/5) elution separation, it is reddish brown to obtain nitro
Aspergillin 0.87g；Nitro ochracin 0.87g is taken, adds ethanol 50mL to dissolve, adds palladium carbon 0.2g, be passed through hydrogen, be stirred at room temperature
4h, stop reaction, filtering removes palladium carbon, and revolving removes ethanol, red oil obtained, in 30mL dichloromethane/petroleum ether
(V/V, 1/1) is recrystallized, and obtains amino ochracin 0.76g, as OTA haptens, yield 95%.
Above-mentioned haptens is taken to be identified through proton nmr spectra,1H-NMR(CDCl3,300MHz)δ：11.0 (1H, s, COOH),
8.45 (1H, s, NH), 8.01 (1H, d, ArH), 7.14 (1H, d, ArH), 7.04 (2H, d, ArH), 6.58 (2H, d, ArH),
6.27(2H,s,NH2), 5.35 (1H, s, OH), 4.72 (1H, dd, CH), 4.52 (1H, dd, CH), 2.85-3.10 (4H, dd,
CH2), 1.37 (3H, s, CH3).In collection of illustrative plates, the resonance absorbing peak for amino hydrogen on phenyl ring of chemical shift δ=6.27, the absorption
The presence at peak proves that spacerarm is coupled successfully, and OTA haptens structures are correct.
2. comlete antigen synthesizes
Immunizing antigen synthesizes --- and OTA haptens is coupled with BSA
OTA haptens 15mg are taken, add 1mL ethanol to dissolve, adds 0.05mol/L carbonate buffer solutions to dilute, adds 10%
Glutaraldehyde water solution 0.2mL, is stirred at room temperature 1h, obtains A liquid；BSA 50mg are taken, add 0.05mol/L carbonate buffer solutions to dissolve,
Obtain B liquid；A liquid is added in B liquid, 4h is stirred at room temperature, adds 2mg sodium borohydrides, continues to stir 1h, 0.02mol/L phosphate
Buffer solution dialysis purification 3 days, changes liquid 3 times daily, obtains immunizing antigen, packing, -20 DEG C of preservations, standby.
Envelope antigen synthesizes --- and OTA haptens is coupled with OVA
OTA haptens 8mg are taken, add water 0.5mL, adds 1mol/L watery hydrochloric acid 0.2mL, is sufficiently stirred, be allowed to dissolve, 0~5 DEG C
20min is stirred, takes 0.2mg natrium nitrosums, adds 1mL water to dissolve, is added drop-wise in haptens solution, 0~5 DEG C is continued to stir 1h, is obtained
To haptens activating solution A liquid；OVA 50mg are taken, add 0.1mol/L carbonate buffer solutions to dissolve, 0~5 DEG C of stirring balance 10min,
A liquid is added, continues to stir 2h, 0.02mol/L phosphate buffers dialysis purification 3 days changes liquid 3 times daily, and it is anti-to obtain coating
Original, packing, -20 DEG C of preservations are standby.
3. animal immune
Take 6~8 weeks female Balb/c mouse 10 (be divided into two groups of A and B, every group 5) of health, initial immunity Freund
Freund's complete adjuvant emulsifies collare dorsal sc multi-point injection, and every mouse immune dosage is 200 μ g OTA-BSA；Booster immunization afterwards
Every two weeks the subcutaneous multi-point injection of nape part once, emulsification incomplete Freund's adjuvant；Last time is immune to use physiological saline generation
For incomplete Freund's adjuvant, using intraperitoneal injection, injection dosage and above identical several times.Specifically immune step is shown in Table 1.
The mouse immune program of table 1
For the third time, four times, 7d after booster immunization, blood, ELISA method measure mice serum potency, tool are taken to mouse docking
Body step is as follows：
(1) envelope antigen (OTA-OVA) is done 1 with 0.05mol/L pH9.6 carbonate buffer solution:1000 dilutions, often
The μ L coated elisa plates of hole 100,37 DEG C of incubation 2h, are got rid of coating buffer, are washed 1 time, patted dry with PBST；
(2) 150 μ L confining liquids are added per hole, 37 DEG C of reaction 2h hypsokinesis deblocking liquid, are patted dry；
(3) 50 μ L are added per hole with the antiserum of PBS doubling dilutions, 25 DEG C of reaction 30min hypsokinesis dereaction liquid, with PBST
Washing 3~5 times, per minor tick 30s, pat dry；
(4) ELIAS secondary antibody (1 of PBS dilutions is added:1000) 100 μ L/ holes, 25 DEG C of reaction 30min, 3~5 are washed with PBST
It is secondary, per minor tick 30s, pat dry；
(5) substrate nitrite ion A liquid and each 50 μ L of B liquid are added per hole, 25 DEG C of lucifuges react 15min, 50 μ are added per hole
L2mol/L H2SO4Solution terminating reaction；
(6) ELIASA determines OD value of the wavelength in 450nm, with sample well OD450Extension rate close to 1 is used as the positive
The potency of serum.
4. cell fusion
(1) prepared by feeder cells：Disconnected neck puts to death 8~10 week old Balb/c mouse, is immersed in 5min in 75% alcohol, immediately
It is put into superclean bench, belly is put in plate or be fixed on dissection plate upward.Mouse web portion skin is played with ophthalmic tweezers sub-folder
Skin, an osculum is cut with scissors, pay attention to being sure not to break peritonaeum, in order to avoid peritoneal fluid outflow and pollution.Then scissors is used to upper and lower both sides
Blunt separation is done, fully exposes peritonaeum.Peritonaeum is wiped with cotton ball soaked in alcohol to sterilize.5mL RPMI-1640 bases are drawn with syringe
Nutrient solution, mouse peritoneal is injected, syringe is gently drawn back, rocks mouse leg and afterbody several times.Abdominal cavity is drawn back with original annotation emitter
Interior liquid, inject centrifuge tube.So operate 3~4 times repeatedly.1000r/min centrifuges 10min, abandons supernatant.It is complete with 20~50mL
Cell is resuspended in nutrient solution, and 100 μ L/ holes are added drop-wise to culture plate, it is standby to put incubator.
(2) prepared by splenocyte：3d after booster immunization, immune Balb/c mouse are taken, dislocates and puts to death after eye socket blood sampling, 75%
Spleen is taken after being sterilized in alcohol, connective tissue is removed, prepares splenocyte suspension, be transferred in 50mL centrifuge tubes, add RPMI-1640
To 30mL, 1500~2000r/min centrifugation 5min, supernatant is abandoned, adds RPMI-1640 to 30mL, is counted stand-by.
(3) prepared by myeloma cell：Take the good (viable count of 3 bottles of growth conditions>95%) myeloma cell, by it is complete
Full blow down, be transferred in 50mL centrifuge tubes, add RPMI-1640 to 30mL, 1500~2000r/min centrifugation 5min, abandon supernatant, add
RPMI-1640 to 30mL, count stand-by.
(4) mixing with cells：Splenocyte:Myeloma cell=8:1, mixing, 1500~2000r/min centrifugations 5min.
(5) cell fusion：The cell mixed is centrifuged, dry supernatant, sedimentation cell block bullet into pasty state, puts 37 DEG C of water
Bath, 1mL fusion agents being added in 1min, fusion agent is polyethylene glycol (PEG) 4000, acts on 2min, and is gently mixed cell,
The PEG nutrient solutions of 20mL serum-frees are added in subsequent 4min, 1000r/min centrifugation 10min, abandon supernatant.It is complete with 20~50mL
Cell is resuspended in nutrient solution, and cover plant, per the μ L of hole 100, is put in incubator in 96 porocyte culture plates containing feeder cells.
5. cell line screens
When the 1/2~1/3 of cell length to bottom hole, you can carry out antibody test.It is thin to there is hybridoma using ELISA method
The culture hole of intracellular growth is screened, and screening is in two steps：The first step first filters out positive cell hole, second step with indirect ELISA
It is standard items from OTA, inhibition measure is carried out to positive cell with indirect competitive ELISA.Select has to OTA standard items
The hole preferably suppressed, is subcloned using limiting dilution assay, is detected with same method.In triplicate, you can obtain
The cell line F-3-3 of the energy anti-ochratoxin monoclonal antibody of stably excreting.The cell line is in preservation on the 15th in 06 month in 2017
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：Chaoyang District, Beijing City great Tun
Road, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.14303.
Embodiment 2：The preparation purifying of anti-ochratoxin monoclonal antibody and CHARACTERISTICS IDENTIFICATION
1. prepared by ascites
Atoleine is injected into 6~8 weeks Balb/c mouse, 500 μ L/ are only.By the hybridization in exponential phase after 10 days
Oncocyte CGMCC No.14303 are collected with RPMI-1640 basal mediums, dense with blood counting chamber and microscopic counting, cell
Degree is 1.0 × 106~1.5 × 106In the range of individual/mL.Every mouse 0.5mL hybridoma CGMCC No.14303 is expelled to
Abdominal cavity.Notice that observation mouse web portion after one week expands, ascites is gathered in mouse peritoneal with asepsis injector, every one to two days
Collection once, so repeatedly gathers until mouse natural death repeatedly.5000r/min centrifuges 5min at 4 DEG C, collects supernatant, and
Remove the fat and albuminous membranae of ascites upper strata floating.
2. antibody purification
Monoclonal antibody is comprised the following steps that using octanoic acid-ammonium sulfate method purifying：
(1) ascites is taken out into thaw at RT from -20 DEG C of refrigerators.Ascites is filtered with double-layer filter paper, tentatively removes fatty piece, thin
Born of the same parents' fragment and other impurities.12000r/min centrifuges 15min, takes supernatant, abandons precipitation.Precise volume ascites volume；
The acetate buffer magnetic agitation of the ascites of (2) 1 parts of volumes and 3 parts of volumes is mixed, and pH is adjusted with 2mol/L HCl
To 4.5~4.8；
(3) caprylic acid is slowly added under magnetic agitation, 1mL ascites adds 33 μ L caprylic acids, adds rear room temperature magnetic agitation
30min, rearmounted 4 DEG C of standings 2h；
(4) 12000r/min centrifuges 5min, takes supernatant, double-layer filter paper filtering, collects filtrate；
(5) filtrate volume is measured, adds the 0.1mol/L pH7.4 of 1/10 volume PBS, with 2mol/L NaOH (records
NaOH volumes) adjust pH to 7.4；
(6) by supernatant ice bath precooling, ammonium sulfate solids are added to 0.277g/mL, it is stirring while adding, and in being added in 30min,
Put 4 DEG C overnight；
(7) 12000r/min centrifuges 15min, abandons supernatant.Precipitation is dissolved with the 0.01mol/L PBS of certain volume.Use PB
Dialysis changes 0.01mol/L PBSs two days two days later, collects dialyzate, 12000r/min centrifugation 15min, takes supernatant, put -20
DEG C preserve；
(8) antibody of purifying analyzes purification effect with PAGE gel electrophoresis.
3. antibody titer determines
Using indirect ELISA method measure antibody titer, the serum titer of 3. animal immunes is surveyed in step reference implementation example 1
It is fixed.As a result show, potency >=20000 of anti-ochratoxin monoclonal antibody.
4. antibody subtype is identified
With the anti-ochratoxin Dan Ke of commercially available SIGMA hypotypes identification kit identification hybridoma cell strain F-3-3 secretions
The hypotype of grand antibody is IgG1Type.
5. antibody cross reaction determines
Determined, concretely comprised the following steps using indirect competitive ELISA method：
(1) envelope antigen (OTA-OVA) is done 1 with 0.05mol/L pH9.6 carbonate buffer solution:1000 dilutions, often
The μ L coated elisa plates of hole 100,37 DEG C of incubation 2h, are got rid of coating buffer, are washed 1 time, patted dry with PBST；
(2) 150 μ L confining liquids are added per hole, 37 DEG C of reaction 2h hypsokinesis deblocking liquid, are patted dry；
(3) first added per hole ochratoxin A standard working solution that 50 μ L are serially diluted (concentration is respectively 0,0.4,0.8,
1.6th, 3.2,6.4 μ g/L), then add 50 μ L and do 1 with PBS:The monoclonal antibody of 20000 dilutions, 25 DEG C of reaction 30min hypsokinesis
Dereaction liquid, washed 3~5 times with PBST, per minor tick 30s, patted dry；
(4) ELIAS secondary antibody (1 of PBS dilutions is added:1000) 100 μ L/ holes, 25 DEG C of reaction 30min, 3~5 are washed with PBST
It is secondary, per minor tick 30s, pat dry；
(5) substrate nitrite ion A liquid and each 50 μ L of B liquid are added per hole, 25 DEG C of lucifuges react 15min, 50 μ are added per hole
L2mol/L H2SO4Solution terminating reaction；
(6) OD value of the ELIASA measure wavelength in 450nm.
Using the logarithm value of ochratoxin A concentration as abscissa, with percentage absorbance (each concentration standards OD values with not
The percentage of the quasi- sample wells OD values of mark-on) it is that ordinate draws standard curve.Following interfering material is delayed with standard items respectively simultaneously
The add value that fliud flushing is diluted to listed by form is used to analyze, and the OD values according to corresponding to developing the color medicine, combined standard tracing analysis goes out
Alluvial calculate cross reacting rate (cross reacting rate=alluvial/add value × 100%), the results are shown in Table 2.
Anti-interference data of the table 2 to other materials common in Chinese medicine
As shown in Table 2, the auxiliary material that is used in Chinese medicine in mycotoxin that may be present, agricultural chemicals, heavy metal, concocting process and
The property of contaminated bacteria confrontation ochratoxin monoclonal antibody does not interfere with.
Embodiment 3：The application of anti-ochratoxin monoclonal antibody
The anti-ochratoxin monoclonal antibody that hybridoma cell strain F-3-3 secretes is used to prepare ochratoxin A enzyme
Linked immunoassay reagent kit, for the analysis detection of ochratoxin A in Chinese medicine.
1. the composition of enzyme linked immunological kit
(1) it is coated with the ELISA Plate of OTA-OVA conjugates；
(2) anti-ochratoxin monoclonal antibody；
(3) the sheep anti mouse antiantibody of horseradish peroxidase-labeled；
(4) ochratoxin A standard items：Standard solution concentration is respectively 0 μ g/L, 0.4 μ g/L, 0.8 μ g/L, 1.6 μ g/
L、3.2μg/L、6.4μg/L；
(5) substrate nitrite ion：It is made up of A liquid and B liquid, A liquid is the aqueous solution of 2% urea peroxide, and B liquid is 1% tetramethyl
The aqueous solution of benzidine；
(6) terminate liquid：2mol/L sulfuric acid solutions；
(7) cleaning solution：Prepared as follows per 1L cleaning solutions：By 10mL Tween-20s, 5g sodium azide and 990mL
Phosphate buffer mixes；Wherein, the concentration of phosphate buffer is 0.02mol/L, pH value 7.4.
2. the preparation of reagent constituents
(1) it is coated with the preparation of the ELISA Plate of OTA-OVA conjugates
OTA-OVA conjugates are diluted to best effort concentration with coating buffer solution, are coated with 96 hole polystyrene ELISA Plates,
Per the μ L of hole 100,37 DEG C of incubation 2h, coating buffer is got rid of, is washed 1 time, each 30s, patted dry with PBST, then adds 150 μ per hole again
L confining liquids, 37 DEG C of incubation 2h, liquid in hole of inclining, are preserved after drying with aluminium film vacuum sealing.
It is coated with buffer solution：PH9.6,0.05mol/L carbonate buffer solution；
Confining liquid：Prepared as follows per 1L confining liquids：5mL horse serums, 1g sodium azide, 30g caseins are mixed
Close, dissolved with phosphate buffer and be settled to 1000mL；Wherein, the concentration of phosphate buffer is 0.02mol/L, and pH value is
7.2。
(2) preparation of the sheep anti mouse antiantibody of horseradish peroxidase-labeled
Sheep anti mouse antiantibody is coupled with horseradish peroxidase (HRP) using the Over-voltage protection after improvement.Pass
The Over-voltage protection of system requires that the molar concentration rate of enzyme and antibody is 4 in reaction system:1, because horseradish peroxidase is strong
Many sites with antibody binding are produced under oxidation, the horseradish peroxidase molecule so activated act as connecting each point
The bridge of son, reduces the enzymatic activity of enzyme marker, makes to be mixed with many condensates in the conjugate of preparation.Asked to solve this
Topic, we are improved traditional method, i.e.,：
(1) closed process of amino is eliminated, because the amino that can produce the connection of itself amino is actually seldom；
(2) molar concentration ratio of horseradish peroxidase and antibody is reduced to 2:1, the method after improvement is than traditional side
Method is easy, and the loss to enzymatic activity is reduced.
3. kit test method
(1) sample pre-treatments
The Chinese medicine sample of 5.0g ± 0.05g crushing is weighed into 50mL polystyrene centrifuge tubes, adds 25mL deionized waters,
5min, more than 3000g room temperatures (20-25 DEG C/68-77 ℉) centrifugation 5min are acutely vibrated with oscillator；Specimen extraction liquid is spent
Ionized water is according to 1:After 3 volume ratio dilution, 50 μ L are taken to be used to analyze.
(2) detected with kit
OTA standard solutions/μ L/ holes of sample liquid 20 are added into plate hole, the sheep anti mouse of horseradish peroxidase-labeled resists
The μ L/ holes of antibody 50, the anti-μ L/ holes of ochratoxin monoclonal antibody 80, gently vibration are mixed, kept away for rearmounted 25 DEG C with cover plate membrane cover plate
20min is reacted in luminous environment, pours out liquid in hole, liquid in hole is poured out after adding cleaning solution 250 μ L, 30s per hole, is so repeated
Operation board-washing 5 times altogether, are patted dry with blotting paper；Substrate nitrite ion A liquid and each 50 μ L of B liquid are added per hole, gently vibration mixes, with lid
10min is reacted in the rearmounted 25 DEG C of light protected environments of plate membrane cover plate；The μ L of terminate liquid 50 are added per hole, gently vibration mixes, and sets enzyme mark
Instrument determines the absorbance per hole at 450nm.
(3) result calculates
Calculate percentage absorbance：With the standard solution or sample liquid absorbance obtained and blank solution absorbance
The ratio of value is calculated, and sees below formula：
In formula：W --- percentage absorbance, %；
The mean absorbance values of A --- standard solution or sample liquid；
A0--- the mean absorbance values of blank solution (concentration is 0 μ g/L standard solution).
Draw standard curve：With the percentage absorbance (%) of calculating for ordinate, the logarithm value of standard solution concentration
(log10) standard curve is drawn for abscissa.
Sample results calculate：The percentage absorbance of sample liquid is substituted into standard curve, sample is read from standard curve
After concentration corresponding to this, the content of ochratoxin A is calculated as follows in sample：
X=C × n
In formula：X --- the content of ochratoxin A in sample, μ g/kg；
C --- the concentration of ochratoxin A in the sample checked in from standard curve, μ g/L；
N --- Sample Dilution multiple.
It can also be calculated with the data processing software of various ELIASAs.
Note：Result of calculation deducts blank value, and acquired results retain a decimal.
4. Detection results are evaluated
(1) linear relationship
Concentration of standard solution is respectively 0,0.4,0.8,1.6,3.2,6.4 μ g/L, and the OD values of measure are shown in Table 3.Inhaled with percentage
Shading value (W, %) is ordinate, and the logarithm value (log10) of concentration of standard solution draws standard curve (see accompanying drawing for abscissa
2).Using logitW as ordinate, the logarithm value of concentration of standard solution is abscissa, is understood after accompanying drawing 2 is changed, in 0.4 μ g/L
In~6.4 μ g/L concentration ranges, good linear relationship (see accompanying drawing 3) is presented with ochratoxin A log concentration in logitW, obtains
Regression equation Y=-2.890X+0.091, R2=0.9923.
The OD value measurement results of the standard liquid of table 3
(2) pattern detection limits
Using the blank malt of separate sources, peach kernel, spina date seed, the sterculia seed, stiff silkworm, centipede, coix seed, scorpio, leech,
Lotus seeds, the fruit of Rangoon creeper, dried orange peel, polygala, cassia seed, jujube, betel nut, earthworm, each 20 parts of seed of Oriental arborvitae sample are measured, test limit with
Measure average value adds 3 times of standard deviations to determine.As a result show, the equal μ g/kg of ＜ 5 of test limit of 18 kinds of matrix, it is thus determined that reagent
Detection of the box to traditional Chinese medicine sample is limited to 5 μ g/kg.
(3) degree of accuracy (rate of recovery) and precision (repeatability)
With the malt without OTA, peach kernel, spina date seed, the sterculia seed, stiff silkworm, centipede, coix seed, scorpio, leech, lotus seeds, make
Gentleman, dried orange peel, polygala, cassia seed, jujube, betel nut, earthworm, the seed of Oriental arborvitae are blank sample matrix, carry out three concentration level (5 μ
G/kg, 10 μ g/kg, 20 μ g/kg) addition recovery test, respectively with three batch kit measurements, calculate sample TIANZHU XINGNAO Capsul
With batch interior, interassay coefficient of variation.As a result show, the TIANZHU XINGNAO Capsul of 18 kinds of matrix is 70%~110%, in batch, batch anaplasia
The different equal ＜ 15% of coefficient.
(4) kit storage life
Kit preservation condition is 2~8 DEG C, by the measure of 12 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, OTA TIANZHU XINGNAO Capsuls are in normal range (NR).In view of having improper preservation during transport and use
Condition occurs, and kit is placed 8 days at 37 DEG C and carries out accelerated aging tests, as a result the indices of the kit accord with completely
Close and require；Situation is freezed in view of kit, kit is placed 8 days in -20 DEG C of refrigerators, as a result the kit is each
Item index is also completely normal.It can show that kit can at least preserve more than 12 months at 2~8 DEG C from result above.
Claims (3)
- A kind of 1. hybridoma cell strain for secreting anti-ochratoxin monoclonal antibody, it is characterised in that：It is named as Aspergillus ochraceus poison Plain monoclonal antibody hybridoma cell strain F-3-3, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms Center, deposit number are CGMCC No.14303, and preservation date is on 06 15th, 2017.
- A kind of 2. anti-ochratoxin monoclonal antibody, it is characterised in that：It is that deposit number is as described in claim 1 Caused by CGMCC No.14303 ochratoxin monoclonal antibody hybridoma cell strain F-3-3 secretions.
- 3. the application of anti-ochratoxin monoclonal antibody described in claim 2, it is characterised in that：For Aspergillus ochraceus poison in Chinese medicine Plain A analysis detection.
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| CN108828205A (en) * | 2018-04-09 | 2018-11-16 | 国家食品安全风险评估中心 | Ochratoxin A haptens, artificial antigen and preparation method thereof, kit and ochratoxin A detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103215231A (en) * | 2013-04-03 | 2013-07-24 | 中国农业科学院油料作物研究所 | Hybridoma cell strain 1H2, anti-ochratoxin A monoclonal antibody produced from same and application thereof |
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| CN103215231A (en) * | 2013-04-03 | 2013-07-24 | 中国农业科学院油料作物研究所 | Hybridoma cell strain 1H2, anti-ochratoxin A monoclonal antibody produced from same and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108828205A (en) * | 2018-04-09 | 2018-11-16 | 国家食品安全风险评估中心 | Ochratoxin A haptens, artificial antigen and preparation method thereof, kit and ochratoxin A detection method |
| CN108828205B (en) * | 2018-04-09 | 2021-11-02 | 国家食品安全风险评估中心 | Ochratoxin A hapten, artificial antigen, preparation method and kit of artificial antigen, and ochratoxin A detection method |
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