CN109280646A - One plant of anti-diclazuril monoclonal antibody specific hybridoma cell strain and its application - Google Patents
One plant of anti-diclazuril monoclonal antibody specific hybridoma cell strain and its application Download PDFInfo
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- CN109280646A CN109280646A CN201811275585.2A CN201811275585A CN109280646A CN 109280646 A CN109280646 A CN 109280646A CN 201811275585 A CN201811275585 A CN 201811275585A CN 109280646 A CN109280646 A CN 109280646A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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Abstract
One plant of anti-diclazuril monoclonal antibody specific hybridoma cell strain and its application, belong to food safety technical field of immunological detection.One plant of anti-diclazuril monoclonal antibody specific hybridoma cell strain SS0711, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.14689.Anti- diclazuril monoclonal antibody specific is that the anti-diclazuril monoclonal antibody specific hybridoma cell strain SS0711 secretion of CGMCC No.14689 generates by the deposit number.It is applied to the remaining analysis detection of diclazuril in food safety detection.The anti-diclazuril cell strain of monoclonal antibody that the present invention obtains has preferable detection sensitivity and affinity, the IC of monoclonal antibody of the present invention to diclazuril50=0.49 ng/mL, the detection range of linearity are 0.16-1.55 ng/mL, and lowest detection limit value (LOD) is 0.08 ng/mL.
Description
Technical field
The present invention relates to one plant of anti-diclazuril monoclonal antibody specific hybridoma cell strain and its applications, belong to food
Security immunization detection technique field.
Background technique
Diclazuril (Diclazuril) is a kind of polyethers drug, while being also ideal coccidiostat, has height
Imitate less toxic feature.Diclazuril was researched and developed in 1986 by Belgian Janssen company, with sterilization spirit in Britain, Germany etc.
43 country's listings.Diclazuril belongs to triazines broad-spectrum anticoccidial drug, can effectively prevent and kill to exist including coccidia
Interior a variety of protozoons.Wherein the anticoccidial of 6 kinds of most important eimeria tenellas such as tender, heap-type, Bu Shi, huge, gentle is referred to
Number belongs to efficient coccidiostat, is widely used in chicken coccidiasis more than 180.
Diclazuril detection at present mostly uses high performance liquid chromatography (HPLC), liquid chromatogram and Mass Spectrometry (LC-
MS), gas chromatography mass spectrometry method (GC-MS) etc..Above-mentioned detection method can carry out quantitative analysis and have lower detection limit, but usually
Expensive instrument and complicated operation are needed, pre-treatment and detection time are long, seriously constrain the popularization of these detection methods, nothing
Method reaches the requirement that live high-volume quickly detects.And immunoassay method have it is inexpensive, high-throughput, highly sensitive, to technology
The features such as personnel's relative requirement is low, therefore it is suitable for the rapid screening of a large amount of samples.
Summary of the invention
The purpose of the present invention is to provide one plant of anti-diclazuril monoclonal antibody specific hybridoma cell strain and its answer
With laying the foundation for the research and development popularization of indirect competitive ELISA kit and colloidal gold strip.
Technical solution of the present invention, one plant of anti-diclazuril monoclonal antibody specific hybridoma cell strain SS0711 are protected
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is BeiChen West Road, Chaoyang District, BeiJing City
No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute, deposit number are CGMCC No.14689, and classification naming is monoclonal cell
Strain, the deposit date is on September 5th, 2017.
In one embodiment of the invention, anti-diclazuril monoclonal antibody specific, by the deposit number
For CGMCC No.14689, anti-diclazuril monoclonal antibody specific hybridoma cell strain SS0711 secretion is generated.
In one embodiment of the invention, the application of the anti-diclazuril monoclonal antibody specific, is answered
For the remaining analysis detection of diclazuril in food safety detection.
In one embodiment of the invention, a kind of kit, contain the hybridoma cell strain SS0711 or
Anti- diclazuril monoclonal antibody specific described in person.
In one embodiment of the invention, the kit, the kit is for detecting diclazuril.
The preparation basic step of cell strain provided by the invention are as follows:
(1) preparation and identification of immunogene: sub- by carbonization two using the diclazuril derivative with carboxyl as initial reactant
Amine method is connected with protein carrier, and later by small haptens dialysis separation comlete antigen and be not coupled, comlete antigen is logical
Cross the identification of UV absorption scan method；
(2) mouse is immune: after immunogene and freund adjuvant emulsification completely, it is small that BALB/c is immunized by subcutaneous multi-point injection
Mouse.First immunisation uses Freund's complete adjuvant, and booster immunization uses freund 's incomplete adjuvant, before immunizing dose is when spurt is immune
The half of primary immunization dosage is directly injected intraperitoneally after mixing with physiological saline；Each secondary immunization interval is three weeks.The
After being immunized three times, interval blood sampling in one week detection serum titer and inhibition；
(3) cell fusion is established with cell strain: being merged mouse boosting cell and bone marrow cells in mice by polyethylene glycol method, is cultivated
Positive cell hole is detected, and measures the inhibitory effect of positive cell hole, by limiting dilution assay to there is the positive preferably inhibited thin
Hilum is subcloned three times, finally screens acquisition hybridoma cell strain；
(4) it the identification of hybridoma cell strain property: is set with and is measured with ELIAS secondary antibody using mouse monoclonal Ig class/subgroup identification；
IC50The measurement of value and affinity passes through ELISA method.
In one embodiment of the invention, the polyethylene glycol is using PEG 2000.
In one embodiment of the invention, the culture is using HAT culture medium.
In one embodiment of the invention, positive cell hole is detected using indirect ELISA method, positive cell
The inhibitory effect in hole is detected using indirect competitive ELISA method.
Beneficial effects of the present invention: the anti-diclazuril cell strain of monoclonal antibody that the present invention obtains has diclazuril
Preferable detection sensitivity and affinity additionally provide a kind of method of new synthesis diclazuril immunogene, the conjunction of haptens
More simplify at step, effectively, provides the thinking and method of synthetic immunogen for the research of people from now on.Monoclonal of the present invention
The IC of antibody50=0.49 ng/mL, the detection range of linearity are 0.16-1.55 ng/mL, and lowest detection limit value (LOD) is 0.08
ng/mL。
Biological material specimens preservation: one plant of anti-diclazuril monoclonal antibody specific hybridoma cell strain SS0711,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, address are as follows: Beijing's southern exposure
The institute 3 of area North Star West Road 1, Institute of Microorganism, Academia Sinica, preservation date on September 5th, 2017, deposit number was
CGMCC No.14689, classification naming are monoclonal cell strain.
Detailed description of the invention
The ultra-violet absorption spectrum of Fig. 1 immunogene characterizes.
Standard suppression curve of Fig. 2 monoclonal antibody to diclazuril.
Specific embodiment
The present invention passes through cell fusion, the training of HAT selective medium by the way that mouse is immunized in diclazuril comlete antigen
It supports, cell conditioned medium is screened by indirect ELISA and indirect competitive ELISA, having finally obtained has preferable affinity to diclazuril
With the monoclonal antibody hybridoma cell strain of sensitivity.
The configuration of solution:
Carbonate buffer solution (CBS): Na is weighed2CO31.59g NaHCO32.93g is mixed after being dissolved in a small amount of distilled water respectively,
Add distilled water to mix to about 800mL, adjusts pH value to 9.6, add distilled water to be settled to 1000mL, 4 DEG C of storages are spare；
Phosphate buffer (PBS): 8.00g NaCl, 0.2g KCl, 0.24g KH2PO4, 3.62g Na2HPO4·12H2O, it is molten
In 800mL pure water, with NaOH or HCl tune pH to 7.2~7.4, it is settled to 1000 mL；
PBST: the PBS containing 0.05% Tween20；
TMB developing solution: A liquid: Na2HPO4·12H2O 18.43g, citric acid 9.33g, pure water are settled to 1000 mL；B liquid: 60
Mg TMB is dissolved in 100 mL ethylene glycol.A, B liquid 1 ︰ 5 mixing by volume is TMB developing solution, current existing mixed.
Embodiment 1: the preparation of hybridoma cell strain
(1) synthesis of comlete antigen:
The synthesis of haptens: joined Dic(1g in the concentrated sulfuric acid (50mL) and the mixture solution of deionized water (10 mL),
Solution 2.5mmol).Mixed liquor reacts for 24 hours at 110 DEG C.After reaction, reaction mixture is diluted with the deionized water of 1L,
To terminate reaction, then extracted three times with the ethyl acetate of 20mL.Organic layer is cleaned with NaCl, uses MgSO4Drying, then true
Evaporative air obtains haptens Dic-hapten-1.Specific synthetic route is as follows:
The synthesis of comlete antigen: the above-mentioned haptens Dic-hapten-1 of 5mg is taken, 5.0mg EDC(1- (3- dimethylamino is added
Propyl) -3- ethyl-carbodiimide hydrochloride) and 3.0mg NHS(N- HOSu NHS), use DMF(N, N- dimethyl methyl
Amide) dissolution, it is stirred at room temperature, activates 6h；Separately take 10mg KLH(keyhole limpet hemocyanin) it is dissolved in the CB of 3mL, 0.05M, pH9.6
In (carbonate buffer solution) solution, above-mentioned activating solution is added dropwise in KLH solution, is stirred at room temperature after reaction overnight, 4 DEG C thoroughly
Analysis three days, -20 DEG C of packing save.
(2) animal immune: the BALB/c mouse of 6~8 week old of health is selected to be immunized.Take diclazuril comlete antigen
After the emulsification uniformly of (1mg/mL) and equivalent freund adjuvant, BALB/c mouse, every 100 μ L are immunized by subcutaneous multi-point injection.It is first
It is secondary it is immune use Freund's complete adjuvant, booster immunization uses freund 's incomplete adjuvant, and immunizing dose is preceding primary when spurt is immune
The half of immunizing dose is directly injected intraperitoneally after mixing with physiological saline；Each secondary immunization interval is three weeks.For the third time
After immune, interval blood sampling in one week detection serum titer and inhibition；Selection inhibits best mouse, and spurt in 21 days is exempted from after exempting from five
Epidemic disease prepares fusion.
(3) cell fusion: after spurt immune three days, according to conventional PEG(polyethylene glycol, molecular weight 2000) method into
Row cell fusion, the specific steps are as follows:
A, sterile to take mouse spleen, it grinds and obtains splenocyte suspension by 200 mesh cell screen clothes, and carry out cell count；
B, SP2/0 cell is collected, is suspended in RPMI-1640 basic culture solution, cell count is carried out；
C, splenocyte and SP2/0 cell are mixed according to the ratio counted than 5-10:1, are merged after centrifugation with PEG, time 1min,
Later according to from slowly to fast, RPMI-1640 basic culture solution is added, be suspended in after centrifugation containing 20% fetal calf serum, 2% 50 ×
In the RPMI-1640 screening and culturing liquid of HAT, 96 porocyte culture plates are added to, 37 DEG C, 5%CO are placed in2Incubator in cultivate.
(4) cell screening and cell strain are established: carrying out RPMI-1640 screening to fused cell in the third day of cell fusion
Culture solution partly changes liquid, carries out within the 5th day being carried out entirely with the RPMI-1640 transition culture solution of 100 × HT containing 20% fetal calf serum, 1%
Liquid is changed, took cell conditioned medium to be screened at the 7th day.Screen in two steps: the first step first filters out positive cell with indirect ELISA
Hole, it is standard items that second step, which selects diclazuril, carries out inhibitory effect measurement to positive cell with indirect competitive ELISA.Selection
There is the cell hole preferably inhibited to diclazuril, be subcloned using limiting dilution assay, is detected with same method.Weight
Again three times, cell strain is obtained.
(5) preparation and identification of monoclonal antibody: taking 8-10 week old BALB/c mouse, and every mouse peritoneal injects paraffin oil
1mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma collected ascites since the 7th day, ascites was passed through pungent
Acid-saturated ammonium sulfate method purifying, the monoclonal antibody of acquisition are placed in -20 DEG C of preservations.
It is sub- that immunoglobulin is carried out using the monoclonal antibody that mouse monoclonal subtype identification kit obtains ascites purifying
Type identification, hypotype are IgG2a type.
The foundation of 2 enzyme linked immuno sorbent assay of embodiment
Hybridoma cell strain is applied to enzyme linked immuno sorbent assay side by monoclonal antibody prepared by internal ascites
The foundation of method, the specific steps are as follows:
1, the 0.1 μ g/mL for using carbonate buffer solution (CBS) to dilute as coating 96 hole elisa Plates of primordial covering, every 100 μ L of hole,
After 37 DEG C of coating 2h, three times with PBST washing lotion board-washing, each every 200 μ L of hole, 3 min, is patted dry every time；
2, it is closed with the CBS containing 0.2% gelatin, every hole 200 μ L, 37 DEG C of closing 2h, three times with PBST washing lotion board-washing, every time
Every 200 μ L of hole, 3 min, pats dry every time；
3, the diclazuril standard for 0,2,5,10,20,50,100,200 μ g/L being respectively configured with phosphate buffer (PBS) is molten
Liquid.Standard solution is added separately in the ELISA Plate closed, every 50 μ L of hole, each standard solution repeats 3 holes, then
Every hole is added the diluted anti-diclazuril monoclonal antibody of 50 μ L, 1 ︰ 32000, and after 37 DEG C of reaction 0.5h, board-washing is patted dry；
4,100 μ L of the every hole addition sheep anti-mouse igg secondary antibody of the diluted HRP label of 1 ︰ of PBS 3000 containing 0.1% gelatin, 37
DEG C reaction 0.5 h after, board-washing pats dry；
5, every hole is added 100 μ L TMB developing solutions, after 37 DEG C of 15 min of colour developing, 50 μ L 2M H of every hole addition2SO4Terminate liquid,
450 nm survey light absorption value；
6, Specification Curve of Increasing: standard suppression curve is drawn with origin 8.5.
Using the light absorption value of enzyme linked immuno sorbent assay detection as ordinate, built using diclazuril concentration as abscissa
Anti- diclazuril monoclonal antibody is found to the standard suppression curve of diclazuril, as shown in Fig. 2.It can be with by drawn standard curve
Calculate the IC of this monoclonal antibody50=0.49 ng/mL, the detection range of linearity are 0.16-1.55 ng/mL, minimum detection limit
Being worth (LOD) is 0.08 ng/mL.
3 monoclonal antibody application of embodiment
Sample pre-treatments and addition recovery experiment: it to the addition recovery experiment of actual sample, is tested by taking chicken as an example.It is first
First, three portions of 1.0g chicken are weighed, 100 μ L, 200 μ L and 500 μ L diclazuril standard solutions (100ng/mL) are separately added into,
It mixes, 10mL acetonitrile is added, high speed homogenization 2min, ultrasonic extraction 10min take 200 μ L supernatant PBS after standing 5 min
After 4 times of dilution, as ELISA sample extracting solution, recovery test, rate of recovery difference are added using indirect competitive ELISA
It is 91%, 97%, 95%.
The subtype identification of 1. monoclonal antibody of table
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this technology
People can do various change and modification, therefore protection scope of the present invention is answered without departing from the spirit and scope of the present invention
This is subjected to the definition of the claims.
Claims (5)
1. one plant of anti-diclazuril monoclonal antibody specific hybridoma cell strain SS0711 is preserved in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center is hidden, preservation address is that the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences is micro-
Biological study institute, deposit number are CGMCC No.14689, and classification naming is monoclonal cell strain, and the deposit date is 2017 9
The moon 5.
2. anti-diclazuril monoclonal antibody specific, it is characterised in that: its deposit number as described in claim 1 is CGMCC
No.14689, anti-diclazuril monoclonal antibody specific hybridoma cell strain SS0711 secretion generate.
3. the application of anti-diclazuril monoclonal antibody specific described in claim 2, it is characterised in that: be applied to food
The remaining analysis detection of diclazuril in safety detection.
4. a kind of kit, it is characterised in that: it contains hybridoma cell strain SS0711 described in claim 1 or right is wanted
Anti- diclazuril monoclonal antibody specific described in asking 2.
5. kit according to claim 4, it is characterised in that: the kit is for detecting diclazuril.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022367A (en) * | 2018-08-16 | 2018-12-18 | 江南大学 | The anti-sibutramine monoclonal antibody specific hybridoma cell strain of one plant of secretion and its application |
| CN110045108A (en) * | 2019-05-28 | 2019-07-23 | 江南大学 | A kind of gutter oil enzyme-linked immunologic detecting kit and its detection method based on capsaicine index |
| CN110724192A (en) * | 2019-12-12 | 2020-01-24 | 江南大学 | Hybridoma cell strain secreting serpentine monoclonal antibody and application thereof |
-
2018
- 2018-10-30 CN CN201811275585.2A patent/CN109280646A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022367A (en) * | 2018-08-16 | 2018-12-18 | 江南大学 | The anti-sibutramine monoclonal antibody specific hybridoma cell strain of one plant of secretion and its application |
| CN110045108A (en) * | 2019-05-28 | 2019-07-23 | 江南大学 | A kind of gutter oil enzyme-linked immunologic detecting kit and its detection method based on capsaicine index |
| CN110724192A (en) * | 2019-12-12 | 2020-01-24 | 江南大学 | Hybridoma cell strain secreting serpentine monoclonal antibody and application thereof |
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Application publication date: 20190129 |