CN105255838B - Secrete the hybridoma cell strain of T-2 toxin monoclone antibodies - Google Patents
Secrete the hybridoma cell strain of T-2 toxin monoclone antibodies Download PDFInfo
- Publication number
- CN105255838B CN105255838B CN201510740455.1A CN201510740455A CN105255838B CN 105255838 B CN105255838 B CN 105255838B CN 201510740455 A CN201510740455 A CN 201510740455A CN 105255838 B CN105255838 B CN 105255838B
- Authority
- CN
- China
- Prior art keywords
- toxin
- monoclonal antibody
- cell strain
- hybridoma cell
- cgmcc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of hybridoma cell strains of secretion 2 toxin monoclone antibodies of T, additionally provide the 2 toxin monoclonal antibodies of T of cell secretion.Monoclonal antibody provided by the invention, titer of ascites ratio reach 1:50000, it is better than existing monoclonal antibody.Meanwhile the cross reaction of the monoclonal antibody and 2 toxin of HT reaches 92.8%, the cross reaction with other a variety of antigens is less than 0.1%.Include the affine in immunity liquid column chromatography of the monoclonal antibody, 2 toxin of T and 2 toxin of HT extracted in sample can be efficiently separated simultaneously, it is accurate, economical, effective.
Description
Technical field
The present invention relates to a kind of hybridoma cell strains of secretion T-2 toxin monoclone antibodies, further relate to cell strain secretion
T-2 toxin monoclone antibodies, including the immunosorbent of the antibody, immune affinity column, kit, and utilize the immune parent
With the method for column extraction purification T-2 toxin and HT-2 toxin.
Background technology
T-2 toxin and HT-2 toxin are the Trichothecenes chemical combination that mainly fusarium tricinctum generates by a variety of fungies
Object (trichothecenes, TS), HT-2 toxin are the metabolite of T-2 toxin.They are distributed widely in nature, are common
Pollution field crops and inventory's cereal primary toxins, to people, animal endanger it is larger.T-2 and HT-2 toxin mainly acts on carefully
Born of the same parents divide vigorous histoorgan, such as thymus gland, marrow, liver, spleen, lymph node, gonad and gastrointestinal mucosa, inhibit these organs
Cell protein and DNA synthesis.Moreover, it has been found that the toxin can cause the mono- chain breaks of DNA in lymphocyte.T-2 and HT-2
Toxin may also act to multiple positions of oxidative phosphorylation and mitochondrial respiratory caused to inhibit.FAO (Food and Agriculture Organization of the United Nation) in 1973
(FAQ) and the World Health Organization (WHO) is in the joint conference that Geneva is held, using this toxoid with aflatoxins equally as
The most dangerous food pollution source of naturally occurring.
Currently, the detection method of T-2 toxin, HT-2 toxin mainly has thin layer chromatography, enzyme-linked immunization (ELISA).
Thin layer chromatography will contact a large amount of standard items, be unfavorable for the health of experimenter, and sensitivity is very low.It is enzyme-linked
Immunization is only applicable to qualitative detection, it is easy to the phenomenon that false positive and false negative occurs.And immunoaffinity chromatography-liquid phase color
Spectrometry be by immunoaffinity chromatography pretreatment sample separation and Extraction substance to be checked, then with liquid chromatography detect method,
Economic, quick, accurate, safety, but the immune affinity chromatographic column of separation and Extraction substance to be checked but not a duck soup is prepared, most
It is to find simultaneously effectively in conjunction with T-2 toxin, the antibody of HT-2 toxin for crucial place.
Wang Xiong et al. (preparation and identification of anti-T-2 toxin monoclone antibodies, Chinese Journal of Health Laboratory Technology, 2 months 2010,
20(2)：301-304) report a kind of monoclonal antibody of T-2 toxin.Although the monoclonal antibody and aflatoxins no cross reaction, with
The cross reaction of HT-2 toxin is 65.9%.Therefore, when being used for immunoaffinity chromatography, which can effectively extract T-2 toxin,
But it can not effectively extract HT-2 toxin.Meanwhile the potency of the monoclonal antibody is only 1:12000, it is not strong with the binding force of T-2 toxin, hold
Legibility is from causing final testing result inaccurate.
Therefore, need a kind of and other toxin that cross reaction does not occur at present, potency is high, and can effectively extract simultaneously
The antibody of T-2 toxin and HT-2 toxin.
Invention content
To solve the above problems, the present invention provides a kind of hybridoma cell strain, which is the micro- life of China
The hybridoma cell strain that the number of object culture presevation administration committee common micro-organisms center preservation is CGMCC NO.11181.
Hybridoma cell strain of the present invention is named as anti-T2 toxin, HT-2 toxin monoclone antibody cells, in August, 2015
It was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center in 21st, is referred to as CGMCC, address is：
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preserving number are：CGMCC NO.11181.
The present invention also provides a kind of T-2 toxin monoclone antibodies, which is by above-mentioned hybridoma
What strain was prepared.
Include solid phase carrier and antibody the present invention also provides the immunosorbent, the antibody includes of the present invention
Monoclonal antibody.
Preferably, the antibody further includes following monoclonal antibody any one or more ofs：
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO.5504
The monoclonal antibody that is prepared of hybridoma cell strain；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO.5505
The monoclonal antibody that is prepared of hybridoma cell strain；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO.5506
The monoclonal antibody that is prepared of hybridoma cell strain；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO.9204
The monoclonal antibody that is prepared of hybridoma cell strain.
It is highly preferred that the antibody includes following monoclonal antibodies：
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO.5504
The monoclonal antibody that is prepared of hybridoma cell strain；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO.5505
The monoclonal antibody that is prepared of hybridoma cell strain；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO.5506
The monoclonal antibody that is prepared of hybridoma cell strain；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO.9204
The monoclonal antibody that is prepared of hybridoma cell strain.
It is further preferred that the antibody is combined with solid phase carrier by coupling mode.
The present invention also provides a kind of immune affinity columns being mounted with above-mentioned immunosorbent.
The present invention also provides a kind of kits of detection T-2 toxin and HT-2 toxin, it includes that monoclonal above-mentioned is anti-
Body, immunosorbent above-mentioned or/and immune affinity column above-mentioned.
The present invention also provides a kind of extraction T-2 toxin and HT-2 toxin methods, it is characterised in that：
(1) sample is taken, immune affinity column above-mentioned is passed through；
(2) it washs, elution obtains eluent, you can；In the elution, preferred eluant, eluent is methanol.
Preferably, the fluid sample is feed extract sample.
And the T-2 toxin monoclonal antibodies of the present invention being prepared, titer of ascites ratio reach 1:50000, existing monoclonal antibody is compared,
It is strong with the binding ability of antigen with higher potency ratio.Meanwhile the Percentage bound of the monoclonal antibody and T-2 toxin is 100%, with HT-
The Percentage bound of 2 toxin reaches 92.8%, and the cross reaction with other a variety of antigens is less than 0.1%.That is, monoclonal antibody of the present invention
T-2 toxin and HT-2 toxin can be specifically bound, without being combined with other substances.Therefore, immunosorbent is prepared as with it,
T-2 toxin and HT-2 toxin can be precisely separating out from sample.
Therefore, on the basis of T-2 toxin monoclonal antibody provided by the invention, immunoaffinity chromatography-liquid chromatogram of the invention
Method can accurate, efficiently, economically detect T-2 toxin and HT-2 toxin in many commodity.
In addition to this, it in specific embodiments of the present invention, is avenged with the monoclonal antibody of the present invention and known 3- acetyl deoxidation
The immune affinity column that rotten sickle-like bacteria enol, aflatoxin, ochratoxin, zearalenone monoclonal antibody are prepared, can be with
T-2 toxin in sample, HT-2 toxin, 3- acetyl deoxynivalenol, Huang are simultaneously accurately detected in conjunction with liquid chromatogram
Aspertoxin B1, B2, G1, G2, ochratoxin A and zearalenone, further demonstrating monoclonal antibody of the present invention can be with T-2 poison
Element, HT-2 toxin efficiently, specific bond, without combine other toxin.
Obviously, the above according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific implementation mode of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on the above of the present invention
The technology realized all belongs to the scope of the present invention.
Description of the drawings
Fig. 1 is the chromatogram of 3- acetyl deoxynivalenols in sample.
Fig. 2 is aflatoxin B1 in sample, B2, G1, the chromatogram of G2.
Fig. 3 is the chromatogram of ochratoxin A in sample.
Fig. 4 is the chromatogram of zearalenone in sample.
Fig. 5 is the chromatogram of T-2 toxin and HT-2 toxin in sample.
Specific implementation mode
Anti- 3- acetyl deoxynivalenol, aflatoxin, ochratoxin, zearalenone monoclonal are anti-
Body is respectively CGMCC by the number of China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
NO.9204, CGMCC NO.5506, CGMCC NO.5505 and CGMCC NO.5504 hybridoma be prepared.It is above-mentioned miscellaneous
It hands over tumor cell strain to be disclosed in patent 201510002847.8, is disclosed in 104707362 A of patent CN.Its corresponding monoclonal antibody
The preparation method for being prepared as this field routine.
Embodiment 1 prepares T-2 toxin monoclone antibodies
1, animal immune
Immune animal is 8 week old or so female BAl BIc/c mouse.By antigen T-2-BSA, (inspection dimension health is biological in Beijing respectively
Technology Co., Ltd. makes by oneself) 10 mouse are immunized.Appropriate immunogene (100mg/ is only) plus equivalent Freund's complete adjuvant are taken, breast is made
Agent is immunized, and is immunized 4-6 times altogether, every minor tick 2 weeks.In addition to being immunized for the last time as intraperitoneal injection, remaining is nape
The subcutaneous multi-point injection in portion.
2, the preparation of hybridoma
1. the culture of myeloma cell
SP2/0 myeloma cell cultivates in the complete culture solution containing 10% fetal calf serum.When cell is in logarithm
When growth period, by 1:3-1:10 dilution proportions, passage are general to carry out 1 passage per 3-4 days or expand culture.It is given birth in logarithm
Long-term cell answer perfectly round, bright, size is uniform, marshalling, in half fine and close distribution.It chooses in growth is vigorous, form is good
Good exponential phase cell is for fusion.For 24 hours by SP2/0 cell expansion cultures before fusion.The fusion same day, selection are in logarithm
Cell is blown down with pipette, is collected in 50mL centrifuge tubes by the cell in growth period, and 1000rpm centrifuges 10min, then uses
DMEM culture solutions suspend again, and cell survival rate is counted and calculated under microscope, are more than 90%, can be used for merging.
2. the preparation of immune spleen cell suspension
After being measured to serum titer and suppression level using ELISA, the good mouse of potency height, suppression level is chosen in last
Eyeball bloodletting is extractd in third day after primary immunization, and is detached positive serum and compared.
A) mouse is put to death, is placed in 75% alcohol after impregnating 5min and moves into super-clean bench, open abdominal cavity, sterile working is taken out small
Mouse spleen is put into the plate for filling 5-10mL DMEM culture solutions and rinses.
B) spleen is moved into another plate for filling the endless full nutrient solutions of 5mLDMEM, with the note for being filled DMEM culture solutions
Emitter syringe needle is inserted into spleen, is rinsed repeatedly to splenocyte and is washed out completely.
C) splenocyte suspension is transferred in 50mL centrifuge tubes, 1000rpm centrifuges 5min, supernatant is abandoned, with 10mL DMEM
Liquid suspends, and is counted under microscope.
3, cell fusion
A) it draws respectively and contains 6 × 107A splenocyte and contain 6 × 106A SP2/0 cells cell suspension (two kinds of cell numbers it
Than for 10: 1), merging in merging 50mL plastic centrifuge tubes, 1000rpm centrifuges 5min, abandons supernatant；
B) tube bottom is flicked with finger, makes precipitation loosely at paste；
C) 50%PEG 15001mL, left hand uniform rotation centrifuge tube, the right hand is taken to hold 1mL pipette, extract 50%PEG solution
0.7mL, the tube wall (as possible close at cell) along rotation slowly instills, and control time adds in 60s, then hangs cell
Liquid slowly sucks in pipette, and control time just sucks in 30s, after standing 30s, then cell suspension is slowly blown back into from
In heart pipe, time control is in 30s；
D) the endless full nutrient solutions of 25mL DMEM are added in 5min immediately, terminate reaction.Speed, which is added, is：In 1min
Add 1mL, adds 4mL, remaining 20mL to be added in 3min in 2min.When liquid feeding should gently uniform rotation centrifuge tube, operate at this time
It should be soft；
E) 1000rpm centrifuges 7min, abandons supernatant, and 20mL HAT culture solutions are added, and gently pressure-vaccum makes sedimentation cell suspend.
Operation at this time should be soft, is sure not firmly to blow and beat；
F) cell suspension addition has been covered in 96 porocyte culture plates of feeder cells, the holes 0.1mL/ are set 37 DEG C, contained
5%CO2Incubator in cultivate；
G) cell growth status is observed and recorded daily.It is measured with HAT complete culture solutions half per hole within the 3rd day after fusion and changes liquid,
It uses within the 7th, 10 day after fusion half amount of HT complete culture solutions instead and changes liquid, then change liquid with complete culture solution.
4, the screening of hybridoma
When cell clone after fusion is grown to the 1/4-1/3 of hole floor space, you can with indirect competitive ELISA method to all grams
The culture supernatant in grand growth hole is detected, and the cell hole that OD values are high, inhibiting rate is big, colony counts are less is selected to be cloned,
And expand culture.
5, the clone of hybridoma
Cell clone is carried out using limiting dilution assay, is as follows：
A) cell in positive hole to be cloned is suspended, suspension is sucked out to the sterile test tube containing 1mL HT culture solutions
In；
B) 0.1mL cell suspensions is taken to count.With HAT complete culture solutions diluting cells to 100/mL.Each clone cell
3 dilutions (100/mL, 10/mL, 5/mL) are done, are separately added into 96 porocyte plates, it is per hole 0.1mL, i.e., average
Cell quantity be respectively 10/hole, 1/hole, 0.5/hole.
C) culture plate is placed in 37 DEG C, contains 5%CO2Incubator in cultivate 7-10 days, centre changes the liquid once.When clone is thin
When born of the same parents are grown to the 1/4-1/3 of hole floor space, indirect competitive ELISA detection is carried out to its supernatant.Taking strong positive, (OD values are high, press down
Rate processed is maximum) clone's expansion culture, it freezes, while continuing to be cloned into positive rate up to 100% by upper method；
D) strong positive monoclonal cell being expanded into culture to 12 orifice plates, expanded to 10mL cell bottles after covering with, a part freezes,
A part continues to cultivate, and for producing ascites, prepares monoclonal antibody.
Hybridoma of the present invention is the finally obtained strong positive monoclonal cell of preceding method, is named as anti-T2 poison
Element, HT-2 toxin monoclone antibody cells, China Committee for Culture Collection of Microorganisms is deposited on 21st in August in 2015
Common micro-organisms center, is referred to as CGMCC, and address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preserving number
For：CGMCC NO.11181.
Hybridoma of the present invention and abbreviation hybridoma 11181.
6, the preparation of monoclonal antibody
Monoclonal antibody is prepared using ascites method is induced in vivo.Select 8-10 weeks BALB/c female mice 10, every abdomen
Intracavitary administration sterile paraffin oil 0.5mL.After 10-14 days, (preserving number is inoculation hybridoma：CGMCC NO.11181).It will
The hybridoma of culture is blown and beaten, and 1000rpm centrifuges 10min, discards supernatant liquid, cannots be used up full nutrient solution by its mixing,
And cell number is adjusted to 1-4 × 105/mL, every mouse peritoneal injects 0.5mL.It is implanted into after cell from the 7th day, observes daily
Mouse web portion waits for that mouse web portion obviously expands, and abdominal cavity is punctured with 9# syringe needles, collects ascites, 5000rpm centrifuges 10min, in collection
Clear liquid, packing, -20 DEG C save backup.
7, antibody performance is tested
The measurement of 7.1 antiserum titres
It is detected according to conventional indirect elisa method, with coating buffer by 0.2 μ g/mL coatings of coating antigen, 100 holes μ L/ add
Enter ELISA Plate, 4 DEG C of coatings are overnight；Coating buffer is abandoned, washing lotion, 200 holes μ L/ is added to be washed 3 times in vibrating 3min on oscillator；It is added
6% gelatin confining liquid, 200 holes μ L/, 37 DEG C of closing 2h；Washing 3 times, is added the test serum of doubling dilution, 100 holes μ L/, most
Latter row plus negative serum control group, 37 DEG C of reaction 30min；HRP- sheep anti-mouse iggs (1 are added in washing 3 times:4000 times of dilutions),
100 holes μ L/, 37 DEG C of reaction 30min；Washing 3 times, is added tmb substrate liquid, 100 holes μ L/, and room temperature avoid light place 10min is added
2mol/L H2SO4Terminate reaction, 50 holes μ L/.OD is measured with microplate reader450nmValue.And P/N values are calculated according to the following formula：P/N values
=Positive control wells OD450nm mean values/negative control hole OD450nmMean value.When P/N values>When 2, i.e. positive control OD450nmValue is big
In 2 times of negative control hole OD450nmWhen value, ELISA potency of the positive serum highest extension rate as serum.
After measured, the potency for the T-2 toxin monoclone antibodies that prepared by this experiment reaches 1：50000, the knot with antigen
Conjunction ability is strong.
The identification of 7.2 monoclonal antibodies specificity
The most suitable working concentration of antigen-antibody is determined according to indirect elisa method and square formation titration.In best operating condition
Under, the cross reacting rate of drug and different pharmaceutical to be measured is detected using indirect competitive ELISA method.Blank control light absorption value is B0,
The light absorption value of different quality concentration drug is B, and using B/B0 as ordinate, the logarithm of mass concentration is abscissa, draws standard items
Suppression curve obtains 50% inhibition mass concentration.Cross reacting rate is calculated according to formula (1.1) simultaneously, the results are shown in Table 1.
(1.1)
1 T-2 toxin antibody specificity identifications of table
| Medicament categories | IC50μg/L | Cross reacting rate % |
| T-2 toxin | 6.5 | 100 |
| HT-2 toxin | 7.0 | 92.8 |
| Aflatoxin | >1000 | <0.1 |
| Vomitoxin | >1000 | <0.1 |
| Zearalenone | >1000 | <0.1 |
| Fumonisin | >1000 | <0.1 |
The results show that the Percentage bound of T-2 toxin monoclone antibodies of the present invention and T-2 toxin is 100%, with HT-2 toxin
Percentage bound is 92.8%, and to other toxin, the no cross reaction if aflatoxin etc. illustrates T-2 toxin list of the present invention
Clonal antibody can with T-2 toxin, HT-2 toxin efficiently, specific bond, without combine other toxin, can be used for T-2 poison
It is plain, HT-2 toxin to isolate and purify.
To sum up, the hybridoma of secretion T-2 toxin monoclone antibodies has been prepared in the present invention, also uses the cell system
Standby to have obtained T-2 toxin monoclone antibodies, the potency of the antibody is high, has good combination energy with T-2 toxin and HT-2 toxin
Power, and with other toxin then no cross reaction, can effectively combine simultaneously, extract T-2 toxin and HT-2 toxin.
2 T-2 toxin of embodiment, 3- acetyl deoxynivalenol, aflatoxin, ochratoxin, corn are red
The preparation of mould ketenes immune affinity column
1. prepared by matrix
Weighing the Sepharose matrix powder of required 1g, (every gram of lyophilized matrix powder can form the swelling of 3.5ml final volumes
Matrix), it is dissolved in 1mM HCl.Matrix will be swollen immediately, be subsequently placed in fritted glass filter and washed using 1mM HCl
15min。
2. ligand (antibody) is coupled
A uses coupling buffer 0.2M NaHCO3PH8.3 dissolves hybridoma 9204 to be coupled, and 5506,5505,
5504 and 11181 secretion 3- acetyl deoxynivalenol, aflatoxin, ochratoxin, zearalenone,
T-2 toxin antibodies (antibody prepared by embodiment 1), antibody concentration 12.5mg/ml, the antibody of dissolving, which is placed in ice bath, keeps in.
The above-mentioned coupling buffer containing antibody is added in a container being fully sealed with cover.Rapidly by CNBr activation
Sepharose is transferred in antibody-solutions.It is mixed well by the way of end-over-end under room temperature condition (20-25 DEG C)
The mixture 2-4h stated,
The calculating of b Conjugate ratios：Sepharose is centrifuged to tube bottom, supernatant is shifted by centrifugation, 2,000RMP × 1min
Into new centrifuge tube, the protein content value of supernatant is measured.Calculating Conjugate ratio was 98.3% (illustrating to be coupled very successful).It takes
It centrifuges to the sepharose of tube bottom, is washed using coupling buffer, remove extra ligand.
C is closed：Close all remaining active groups.In transfer medium to 0.1M Tris-HCl buffer solutions.Room temperature condition
Lower standing 2-4h
D is to remove the extra ligand not being coupled after coupling, is carried out successively to matrix with the buffer solution of low, high two kinds of pH
3 cycles, usage amount at least 5 times of matrix volumes of each buffer solution are at least washed in washing.
Each wash cycle step：It is first washed with 0.1M acetic acid/sodium-acetate buffer, then uses 0.1M Tris-HCl again
Buffer solution is washed.
The 0.01%NaN that e is accumulated with 5 times of colloids3- PBS is washed, and uses 0.01%NaN3- PBS is preserved.
3. filling column
Slurries are prepared using combination buffer, are mixed with the ratio of 75% sedimentation matrix and 25% buffer solution.With even
The operation of continuous property is poured into slurries into column.The glass bar leant against on column wall using one carries out filling out column operation, it will help
Reduce the generation of bubble.After filling out column, the opening of affinity column lower end is closed, and removes the tip part of affinity column.Carefully operation, makes
The remaining part of filling affinity column is added with buffer solution, to form a upward meniscus on the top of affinity column.By top
Sieve plate is inserted into affinity column at an angle, it is ensured that does not have air in the lower section of sieve plate.Sieve plate is locked in stromal surface
On position appropriate, the opening below affinity column is opened, with the 0.01%NaN of the aseptic filtration of 5 times of bed volumes3- PBS mistakes
Column, and being preserved using 0.01%NaN3-PBS, so far T-2 toxin, 3- acetyl deoxynivalenol, aflatoxin,
Ochratoxin, zearalenone affinity column have been loaded and have been balanced and finished, can be directly for using.
T-2 toxin, HT-2 toxin, 3- acetyl deoxynivalenol, aflatoxin in 3 feed of embodiment
The detection of B1, B2, G1, G2, ochratoxin A, zearalenone
T-2 toxin, HT-2 toxin, 3- acetyl deoxynivalenol, aflatoxin B1, B2 in 1.0 feeds,
The detection of G1, G2, ochratoxin, zearalenone
Feed adds recovery experiment, adds 20 μ g/kg, 50 μ g/kg, 100 concentration gradients of μ g/kg tri- respectively.It is each real
It tests and does five groups of parallel tests.
T-2 toxin, HT-2 toxin, 3- acetyl deoxynivalenols in 1.1 feeds, aflatoxin B1, B2,
G1, G2, the extraction of ochratoxin A, zearalenone：
It is accurate to weigh the sample 50.0g by levigate (granularity is less than 2mm) in 250mL conical flask with cover, 5.0g is added
Sodium chloride and accurate addition 100.0mL methanol-waters (8+2) extract 2min with homogenizer high-speed stirred or shaking table shake 30min.
Quantitative filter paper filters, and accurately pipettes 5.0mL filtrates and the dilution of 45.0mL PH7.0PBS solution is added, with glass fiber filter paper mistake
Filter 1~2 time, until filtrate is clarified.
Immune affinity column is connected under 10.0mL glass syringes.Accurately pipette 10.0mL sample extracting solution implantation glass
In syringe, air pressure pump is connect with glass syringe, adjusting pressure makes solution slow transit through with about 6mL/min flow velocitys to exempt from
Epidemic disease affinity column, until 2~3mL air passes through cylinder.With 10.0mL water wash pillar 2 times, whole effluxes are discarded, and make 2mL
~3mL air passes through cylinder.Accurate that the elution of 2.0mL hplc grade methanols is added, flow velocity is 1mL/min~2mL/min, is collected all
Eluent is in teat glass, for detection.
2.0 high-efficient liquid phase chromatogram condition
3- acetyl deoxynivalenols
A mobile phases：Acetonitrile：Water (10：90)
B detectors：UV detector wavelength 218nm
C chromatographic columns：C-18 columns (column length 250mm, internal diameter 4.6mm, 5 μm of filler diameter)
D flow velocitys：0.8mL/min
Aflatoxin B1, B2, G1, G2：
A mobile phases：Methanol-water (45+55)
B detectors：Fluorescence detector excitation wavelength 360nm, launch wavelength 440nm
C chromatographic columns：C-18 columns (column length 150mm, internal diameter 4.6mm, 5 μm of filler diameter)
D flow velocitys：0.8mL/min
E photochemical derivatization systems：Photochemical derivatization pond (after being connected to chromatographic column, then leads to fluorescence detector)
Ochratoxin A：
A mobile phases：Acetonitrile：Water：Acetic acid (49.5：49.5：1)
B detectors：Fluorescence detector excitation wavelength 333nm, launch wavelength 477nm
C chromatographic columns：C-18 columns (column length 250mm, internal diameter 4.6mm, 5 μm of filler diameter)
D flow velocitys：0.8mL/min
Zearalenone
A mobile phases：Acetonitrile-water (60：40)
B detectors：Fluorescence detector excitation wavelength 274nm, launch wavelength 440nm
C chromatographic columns：C-18 columns (column length 150mm, internal diameter 4.6mm, 5 μm of filler diameter)
D flow velocitys：0.8mL/min
T-2 toxin, HT-2 toxin
A mobile phases：Methanol-water (60：40)
B detectors：UV detector 208nm
C chromatographic columns：C-18 columns (column length 150mm, internal diameter 4.6mm, 5 μm of filler diameter)
D flow velocitys：0.8mL/min
3.0 quantitative
50 μ L standard working solutions, which are drawn, with injector injects high performance liquid chromatograph, the bioassay standard under above-mentioned chromatographic condition
The response (peak height or peak area) of solution, respectively obtains standard solution high-efficient liquid phase chromatogram.Reference spectrum 3- acetyl deoxidations
Nivalenol is shown in Fig. 1, aflatoxin B1, B2, G1, and G2 is shown in that Fig. 2, ochratoxin A are shown in Fig. 3, zearalenone
See that Fig. 4, T-2 toxin and HT-2 toxin are shown in Fig. 5
4.0 result
For peanut TIANZHU XINGNAO Capsul result all between 80-100%, RSD is respectively less than 10%.The result shows that this method is completely full
3- acetyl deoxynivalenol in sufficient feed, aflatoxin, point of ochratoxin A and zearalenone detection
Analysis requires.As a result it is shown in Table 2-7 respectively.
3- acetyl deoxynivalenol TIANZHU XINGNAO Capsul result in 2 feed of table
Aflatoxin TIANZHU XINGNAO Capsul result in 3 feed of table
Ochratoxin A TIANZHU XINGNAO Capsul result in 4 feed of table
Zearalenone TIANZHU XINGNAO Capsul result in 5 feed of table
T-2 toxin TIANZHU XINGNAO Capsul result in 6 feed of table
HT-2 toxin TIANZHU XINGNAO Capsul result in 7 feed of table
As it can be seen that T-2 toxin monoclonal antibody provided by the invention, can efficiently separate and extract T-2 toxin and HT-2 toxin simultaneously,
Will not also be to other substances --- 3- acetyl deoxynivalenol, aflatoxin B1, B2, G1, G2, ochratoxin
A, the measurement of zearalenone generates interference, and it includes T-2 toxin and HT-2 toxin to go out in sample so as to Accurate Determining
Many kinds of substance content inside.
In conclusion the present invention secretes the hybridoma cell strain of T-2 toxin monoclone antibodies, the ascites of the monoclonal antibody of secretion
Potency ratio reaches 1:50000, it is better than existing monoclonal antibody.Meanwhile the cross reaction of the monoclonal antibody and HT-2 toxin reaches 92.8%, with
The cross reaction of other a variety of antigens is less than 0.1%.Include immune affinity column-liquid chromatography of the monoclonal antibody, it can be high simultaneously
Effect separates and extracts T-2 toxin and HT-2 toxin, also interference will not be generated to the measurement of other substances, to which Accurate Determining goes out sample
Include T-2 toxin and many kinds of substance content including HT-2 toxin in product.
Claims (11)
1. a kind of hybridoma cell strain, it is characterised in that：The hybridoma cell strain is Chinese microorganism strain preservation conservator
The hybridoma cell strain that the number of meeting common micro-organisms center preservation is CGMCC NO.11181.
2. a kind of T-2 toxin monoclone antibodies, it is characterised in that：The monoclonal antibody is by hybridoma described in claim 1
What cell strain was prepared.
3. a kind of immunosorbent, it is characterised in that：The immunosorbent includes solid phase carrier and antibody, and the antibody includes power
Profit requires the monoclonal antibody described in 2.
4. immunosorbent according to claim 3, it is characterised in that：The antibody further includes in following monoclonal antibodies
It is any one or more：
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO. 5504
The monoclonal antibody that hybridoma cell strain is prepared；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO. 5505
The monoclonal antibody that hybridoma cell strain is prepared；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO. 5506
The monoclonal antibody that hybridoma cell strain is prepared；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO. 9204
The monoclonal antibody that hybridoma cell strain is prepared.
5. immunosorbent according to claim 4, it is characterised in that：The antibody includes following monoclonal antibodies：
Monoclonal antibody described in claim 2；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO. 5504
The monoclonal antibody that hybridoma cell strain is prepared；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO. 5505
The monoclonal antibody that hybridoma cell strain is prepared；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO. 5506
The monoclonal antibody that hybridoma cell strain is prepared；
Number by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation is CGMCC NO. 9204
The monoclonal antibody that hybridoma cell strain is prepared.
6. according to claim 3-5 any one of them immunosorbent, it is characterised in that：The antibody is logical with solid phase carrier
Cross coupling mode combination.
7. a kind of immune affinity column being mounted with any one of claim 3-6 immunosorbent.
8. a kind of kit of detection T-2 toxin and HT-2 toxin, it is characterised in that：It includes the Dan Ke described in claim 2
Immune affinity column described in grand antibody, claim 3-6 any one of them immunosorbent or/and claim 7.
9. a kind of extraction T-2 toxin and HT-2 toxin methods, it is characterised in that：
（1）Sample is taken, the immune affinity column described in claim 7 is passed through；
（2）Washing, elution, obtains eluent, you can.
10. according to the method described in claim 9, it is characterized in that：Step（2）In, the eluant, eluent used is eluted as methanol.
11. according to the method described in claim 10, it is characterized in that：The sample is feed extract sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510740455.1A CN105255838B (en) | 2015-11-02 | 2015-11-02 | Secrete the hybridoma cell strain of T-2 toxin monoclone antibodies |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510740455.1A CN105255838B (en) | 2015-11-02 | 2015-11-02 | Secrete the hybridoma cell strain of T-2 toxin monoclone antibodies |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105255838A CN105255838A (en) | 2016-01-20 |
| CN105255838B true CN105255838B (en) | 2018-09-25 |
Family
ID=55095768
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510740455.1A Active CN105255838B (en) | 2015-11-02 | 2015-11-02 | Secrete the hybridoma cell strain of T-2 toxin monoclone antibodies |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105255838B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106977600B (en) * | 2017-03-07 | 2019-08-13 | 中国农业科学院油料作物研究所 | Purify fumonisin B1, aflatoxin, ochratoxin A, zearalenone immunosorbent and compound affinity column |
| CN107942062A (en) * | 2017-11-29 | 2018-04-20 | 洛阳现代生物技术研究院有限公司 | Test card, preparation and the detection method of synchronous detection 2 toxin of ochracin, vomitoxin and T |
| CN109030437B (en) * | 2018-07-04 | 2020-12-11 | 中山出入境检验检疫局检验检疫技术中心 | Detection device and detection method for aflatoxin |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080066B (en) * | 2009-11-26 | 2012-07-04 | 北京维德维康生物技术有限公司 | Method for detecting T-2 toxin and special reagent kit thereof |
| CN102766208A (en) * | 2012-05-31 | 2012-11-07 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting T-2 and HT-2 toxin |
| FI126746B (en) * | 2014-04-07 | 2017-05-15 | Teknologian Tutkimuskeskus Vtt Oy | Antibody to HT-2 Toxin-HT-2 Toxin Antibody Complex |
| CN104707362A (en) * | 2015-01-05 | 2015-06-17 | 重庆出入境检验检疫局检验检疫技术中心 | Composite affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone |
-
2015
- 2015-11-02 CN CN201510740455.1A patent/CN105255838B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105255838A (en) | 2016-01-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102174474B (en) | Sildenafil monoclonal antibody and colloidal gold chromatography test strip used for detecting sildenafil | |
| CN106970223A (en) | Purify five kinds of mycotoxin immunosorbent and the compound affinity columns such as fumonisin B1, aflatoxin B1 | |
| CN102618502B (en) | Hybridoma cell strain capable of secreting monoclonal antibodies to quinolones and application of monoclonal antibodies thereof | |
| CN105255838B (en) | Secrete the hybridoma cell strain of T-2 toxin monoclone antibodies | |
| CN205506834U (en) | Short -term test aflatoxin B1 and zearalenone's colloidal gold test paper | |
| CN105200013B (en) | One plant of anti-vancocin monoclonal antibody hybridoma cell strain and its application | |
| CN102768278A (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant | |
| CN105087498B (en) | One plant of benzene thiophene cyanogen monoclonal antibody hybridoma cell strain and its application | |
| CN100406115C (en) | Method of purifying alpha corn gibberellol and its metabolic substance and immune affinity chromatographic column | |
| CN106589124A (en) | Application of CD146 monoclonal antibody in detection and separation and identification of glioma perivascular cells | |
| CN104560886B (en) | One plant of anti-strain of natamycin monoclonal antibody hybridoma cell and its application | |
| CN101620231A (en) | ELISA testing kit for detecting norketamine and preparation method thereof | |
| CN108456661A (en) | One plant of anti-BaP monoclonal antibody specific hybridoma cell strain and its application | |
| CN107058242A (en) | The anti-human CD61 monoclonal antibody hybridoma cells strain of mouse, monoclonal antibody and its preparation method and application, flow cytometer detection reagent | |
| CN102676459B (en) | Monoclonal antibody resistant to thermostable direct hemolysin of vibrio parahaemolyticus and preparation method of monoclonal antibody | |
| CN102778564B (en) | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting ractopamine | |
| CN101565464A (en) | ELISA kit for rapidly testing melamine content and method thereof | |
| CN109022366A (en) | One plant of hybridoma cell strain for secreting anti-levamisol monoclonal antibody and its application | |
| CN106636006A (en) | Papaverine monoclonal antibody hybridoma cell strain YH3 and application thereof | |
| CN105907723A (en) | Monoclonal antibody hybridoma cell strain YH4 resisting furaltadone metabolite AMOZ and application thereof | |
| CN102608320A (en) | Monoclonal antibody, ELISA method and kit used for detecting malachite green, leuco malachite green, and leuco crystal violet | |
| CN107189987B (en) | One plant of hybridoma cell strain YY for secreting anti-silaenafil monoclonal antibody and its application | |
| CN107188961A (en) | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof | |
| CN107121552B (en) | One plant of hybridoma cell strain ZY-2G5-3 for secreting 3,4- methylene benzylene chloride propylamine monoclonal antibodies and its application | |
| CN101498728B (en) | Reagent kit for detecting o-allyl phenol and its special antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |