CN109735503A - One plant of Diclofenac monoclonal antibody hybridoma cell strain and its application - Google Patents
One plant of Diclofenac monoclonal antibody hybridoma cell strain and its application Download PDFInfo
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- CN109735503A CN109735503A CN201910026689.8A CN201910026689A CN109735503A CN 109735503 A CN109735503 A CN 109735503A CN 201910026689 A CN201910026689 A CN 201910026689A CN 109735503 A CN109735503 A CN 109735503A
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Abstract
One plant of Diclofenac monoclonal antibody hybridoma cell strain and its application, belong to food safety technical field of immunoassay.The strain of Diclofenac monoclonal antibody hybridoma cell A of the present invention, China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.17301 have been preserved in it.The present invention is by high-titer, low IC50The splenocyte of mouse is merged by PEG method with murine myeloma cell, the hybrid cell using Selective agar medium, after filtering out two kinds of cell fusions；Cell is screened using Indirect cELISA and is subcloned three times, and one plant of monoclonal antibody hybridoma cell strain A is finally obtained.The monoclonal antibody of this cell strain secretion has preferable specificity and detection sensitivity (IC to Diclofenac50It is worth for 2ng/mL) detection, it can be achieved that Diclofenac residual quantity in the liver and feed of chicken, chicken, provides raw material for the remaining immune detection of Diclofenac in food, there is practical application value.
Description
Technical field
The present invention relates to one plant of Diclofenac monoclonal antibody hybridoma cell strain and its applications, and it is immune to belong to food safety
Detection technique field.
Background technique
Diclofenac (Diclofenac, DCF) belongs to non-steroidal anti-inflammatory drugs.With anti-inflammatory, analgesia and refrigeration function.For
Rheumatic arthritis, ankylosing spondylitis, non-inflammatory arthralgia, arthritis, nonarticular rheumatism, non-non-articular inflammatory cause
Pain, fever caused by various neuralgias, cancer pain, post-traumatic pain and various inflammation etc..Its analgesia, anti-inflammatory and antipyretic work
It is 26 ~ 50 times stronger than aspirin with 2 ~ 2.5 times stronger than Indomethacin.Main mechanism is to inhibit prostaglandin synthetase, is made
Prostaglandin biosynthesis is obstructed.Feature is strong drug action, and adverse reaction is few, and dosage is small, and individual difference is small.
Diclofenac has been widely used in the fields such as agricultural, livestock and poultry and feed.Therefore Diclofenac is one in tap water
As have certain residual, and human body intake Diclofenac excessively can the organs such as liver kidney to the mankind cause metabolic burden, if
In tap water, there is excessive Diclofenac to remain, be then harmful to human health.Standard GB/T/T 29691-2013 is by double chlorine
Fragrant acid (DCF) as allow using feed addictive, while also specify its highest allow using limitation (0.02mg/kg) and
Highest maximum permission quantity (0.1mg/kg).But these standards all use efficient liquid-phase chromatography method to detect, detection method is more
It is cumbersome, complicated, in order to safeguard the interests of the majority of consumers, it is necessary to establish a kind of efficient, quick detection side for DCF
Method, and enzyme-linked immunization (ELISA) pre-treatment is simple, it is at low cost, it can be achieved that a large amount of samples quick detection, and to sample when detecting
This purity requirement is not high.Therefore, it is necessary to establish efficient immunological detection method, and one for establishing the method is important
Premise is the highly specific monoclonal antibody that need to be filtered out for Diclofenac.
Summary of the invention
The object of the present invention is to provide one plant of Diclofenac monoclonal antibody hybridoma cell strain and its applications, by the cell
The antibody of strain preparation has preferably specificity and detection sensitivity to Diclofenac, can be used to establish the immunology of Diclofenac
Detection method.
It is micro- to be preserved in China for technical solution of the present invention, one plant of Diclofenac monoclonal antibody hybridoma cell strain A
Biological inoculum preservation administration committee common micro-organisms center CGMCC, in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 of address
Institute of microbiology, the academy of sciences, state, classification naming are monoclonal cell strain, preservation date on May 24th, 2018, deposit number
CGMCC No.17301。
Diclofenac monoclonal antibody, it by the deposit number CGMCC No.17301 Diclofenac monoclonal antibody
No. A secretion of hybridoma cell strain generates.
The application of the Diclofenac monoclonal antibody, for the remaining analysis inspection of Diclofenac in food safety detection
It surveys.
The strain of Diclofenac monoclonal antibody hybridoma cell A preparation basic step provided by the invention are as follows:
(1) preparation of haptens:
Raw medicine Mol. Wt:278.13
Due to containing reactive group (carboxyl) in DCF chemical structural formula, the present invention is by raw medicine directly as haptens.
(2) preparation of comlete antigen DCF-BSA: the present invention uses carbodiimide (EDC) by the carboxyl and load of DCF haptens
Amino coupled on body protein is to prepare comlete antigen.Diclofenac 1.4mg is weighed, 500 μ L DMF dissolution is added, it is sufficiently molten
EDC 2.5mg and NHS 1.6mg is added after solution thereto, and in 30 DEG C of stirring 6h, as A liquid.The BSA of 6mg is weighed, is added
5mL CB dissolution, as B liquid.A drop is added in B liquid, 12h is coupled, mixed liquor is dialysed with 0.01M PBS solution, is removed not
The small haptens of reaction obtain comlete antigen DCF-BSA, and are identified by UV absorption scan method；
(3) mouse is immune: after DCF-BSA comlete antigen and equivalent Freund's adjuvant mixing and emulsifying, carrying out to BALB/c mouse
The subcutaneous multi-point injection of the nape of the neck is immune (except spurt is immune).First immunisation complete Freund's adjuvant, dosage are 100 μ g/；It is more
Secondary booster immunization cannots be used up full Freund's adjuvant and dosage halves as 50 μ g/ only；Spurt is immune not to have to adjuvant, directly uses physiology salt
It is injected intraperitoneally after water dilution, dosage halves again as 25 μ g/ only.One is spaced between first immunisation and second of booster immunization
Month, it is spaced 21 days between multiple booster immunization, spurt is immune to be spaced 18-21 days between last time booster immunization.By
Connect potency and inhibition that competitive enzyme-linked immune method (ic-ELISA) observation mouse immune effect detects mice serum；
(4) cell fusion and cell strain are established: by polyethylene glycol (PEG 4000) method by mouse boosting cell and mouse myeloma
Cell is merged, and filters out hybridoma using selective medium (HAT culture medium), and carry out cell with HT culture medium
Culture.Fusion detects positive cell hole using ic-ELISA method after a week, and further positive thin using the measurement of ic-ELISA method
The inhibitory effect of hilum, by limiting dilution assay to inhibiting preferable positive cell hole to be subcloned, detect again after a week,
Choose hole, subclone.The Monoclonal hybridomas that the hypersecretion specific antibody of DCF is obtained after being subcloned three times according to the above method is thin
Born of the same parents' strain A；
(5) identification of hybridoma cell strain property: pass through ic-ELISA measurement sensitivity and specificity.
Beneficial effects of the present invention: the monoclonal antibody of cell strain A secretion provided by the invention has DCF preferable
Specificity and detection sensitivity (IC50Value is 2.0 ng/mL), it can be achieved that DCF residual quantity in the liver of chicken and chicken, feed
Detection, provide raw material for the remaining immune detection of DCF in food, have practical application value.
Biological material specimens preservation: it is micro- to be preserved in China for one plant of Diclofenac monoclonal antibody hybridoma cell strain A
Biological inoculum preservation administration committee common micro-organisms center CGMCC, in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 of address
Institute of microbiology, the academy of sciences, state, classification naming are monoclonal cell strain, preservation date on May 24th, 2018, deposit number
CGMCC No.17301。
Detailed description of the invention
Fig. 1 is the inhibition standard curve of Diclofenac monoclonal antibody hybridoma cell strain A monoclonal antibody.
Specific embodiment
The following examples of the present invention are only used as the further explanation of the content of present invention, not as a limitation of the present invention interior
Perhaps range.Below by embodiment, the invention will be further described.
The present invention passes through cell fusion, the training of HAT selective medium by the way that mouse is immunized in Diclofenac comlete antigen
It supports, cell conditioned medium is screened by ic-ELISA, has finally obtained the hybridization that there is hypersecretion specific antibody for Diclofenac
Tumor cell strain.
The preparation of 1 hybridoma cell strain of embodiment A
(1) synthesis of comlete antigen: weighing 1.4 mg of Diclofenac, 500 μ L DMF dissolution is added, after completely dissolution thereto
EDC 2.5mg and NHS 1.6mg is added, and in 30 DEG C of stirring 6h, as A liquid.The BSA of 6mg is weighed, 5mL CB dissolution is added,
As B liquid.A drop is added in B liquid, 12h is coupled, is then dialysed with 0.01M PBS solution, unreacted small molecule half is removed
Antigen obtains comlete antigen DCF-BSA, and is identified by UV absorption scan method；
(2) after DCF-BSA comlete antigen and equivalent Freund's adjuvant mixing and emulsifying, neck animal immune: is carried out to BALB/c mouse
Dorsal sc multi-point injection is immune (except spurt is immune).First immunisation complete Freund's adjuvant, dosage are 100 μ g/；Repeatedly
Booster immunization cannots be used up full Freund's adjuvant and dosage halves as 50 μ g/ only；Spurt is immune not to have to adjuvant, directly uses physiological saline
It is injected intraperitoneally after dilution, dosage halves again as 25 μ g/ only.It is spaced one month between first immunisation and second of booster immunization,
It is spaced 21 days between multiple booster immunization, spurt is immune to be spaced 18-21 days between last time booster immunization.By indirectly competing
Strive potency and inhibition that enzyme-linked immunization (ic-ELISA) observation mouse immune effect detects mice serum；
(3) cell fusion: after spurt is three days immune, according to conventional PEG(polyethylene glycol, molecular weight 4000) method progress is carefully
Born of the same parents' fusion, the specific steps are as follows:
A, mouse plucks eyeball and takes blood, after cervical dislocation puts to death mouse, is immediately placed in 75% alcohol and sterilizes, and it is left to impregnate 5min
The spleen of mouse is taken out in the right side, sterile working, is moderately ground with syringe rubber head and obtains splenocyte by 200 mesh cell screen clothes and hanged
Liquid is collected, and is centrifuged (1200rpm, 8min), is washed splenocyte three times with RPMI-1640 culture medium, will after last time is centrifuged
Splenocyte is diluted to certain volume, counts, spare；
B, collect SP2/0 cell: 7-10 days before fusion, SP2/0 oncocyte being used and contains 10% FBS(fetal calf serum) RPMI-
1640 culture mediums are in 5% CO2It is cultivated in incubator.SP2/0 oncocyte quantity is required to reach (1-4) × 10 before fusion7, guarantee
SP2/0 oncocyte is in logarithmic growth phase before merging.When fusion, oncocyte is collected, RPMI-1640 basic culture solution is suspended in
In, carry out cell count；
C, the PEG 4000 of 1mL is added drop-wise in cell by the 7min: the 1min of fusion process from slow to fast；2min is stood.
1mL RPMI-1640 culture medium is added dropwise in 3min and 4min in 1min；5min and 6min is added dropwise in 1min
2mL RPMI-1640 culture medium；The RPMI-1640 culture medium of 1mL is added dropwise in 7min, every 10s.Then 37 DEG C of warm bath 5
min.It is centrifuged (800 rpm, 10 min), abandons supernatant, cell gently strikes scattered, and is added into it containing 20% fetal calf serum, 2% 50
The RPMI-1640 selective medium (HAT culture medium) of × HAT is added to 96 porocyte plates according to 200 holes μ L/, is placed in 37
℃、5% CO2It is cultivated in incubator；
(4) cell screening and cell strain are established: partly being changed with HAT culture medium fused cell within the 3rd day after cell fusion
Liquid；It is changed entirely with the RPMI-1640 transition culture solution (HT culture medium) of 100 × HT containing 20% fetal calf serum, 1% within 5th day
Liquid；Cell conditioned medium is taken to be screened within 7th day.Screen in two steps: the first step first filters out positive cell hole with ic-ELISA method, the
It is standard items that two steps, which select Diclofenac, carries out inhibitory effect measurement to positive cell with ic-ELISA method.Selection is fragrant to double chlorine
Sour standard items have the cell hole preferably inhibited, are subcloned using limiting dilution assay, are examined after seven days with same method
It surveys.It is subcloned three times according to the above method, it is final to obtain Diclofenac cell strain of monoclonal antibody A；
(5) preparation and identification of monoclonal antibody: taking 8-10 week old BALB/c mouse, and every mouse peritoneal injects sterile paraffin oil
1mL；Every mouse peritoneal injection 1 × 10 after 7 days6 Diclofenac hybridoma collected ascites, by ascites since the 7th day
Antibody purification is carried out by octanoic acid-saturated ammonium sulfate method.Under the conditions of meta-acid, caprylic acid can be precipitated in ascites except IgG is immune
Other foreign proteins outside globulin, are then centrifuged for, and abandon precipitating；Again with the list of the ammonium sulfate precipitating IgG type of equivalent saturation degree
Supernatant is abandoned in clonal antibody, centrifugation, and after 0.01M PBS solution (pH7.4) dissolution, dialysis desalting finally obtains list after purification
Clonal antibody is placed in -20 DEG C of preservations.
Add recovery test:
5.1 coating: by coating antigen DCF-BSA with 0.05M pH9.6 carbonate buffer solution 3 doubling dilutions since 1 μ g/mL,
100 holes μ L/, 37 DEG C of reaction 2h；
5.2 washings: solution in plate is inclined, and is washed 3 times with cleaning solution, each 3min；
5.3 closings: after patting dry, 200 hole μ L/ confining liquids, 37 DEG C of reaction 2h are added.It is dried for standby after washing.
5.4 sample-addings: by antiserum since 1:1000 doubling dilution, and be added in the coating hole of each dilution, 100 μ
The hole L/, 37 DEG C of reaction 30min；Sufficiently after washing, the diluted HRP- sheep anti-mouse igg of 1:3000,100 holes μ L/, 37 DEG C of reactions are added
30min；
5.5 colour developings: ELISA Plate is taken out, and sufficiently after washing, the TMB developing solution of 100 μ L is added in every hole, and 37 DEG C are protected from light
15min；
5.6 terminate and measure: 50 μ L terminate liquids are added to terminate reaction in every hole, and the OD in each hole is then measured with microplate reader450Value.
With the IC of ic-ELISA measurement monoclonal antibody Diclofenac50Are as follows: 2.0ng/mL illustrates have to Diclofenac very well
Sensitivity, can be used for Diclofenac immunoassay detection.
The configuration of solution:
Carbonate buffer solution (CBS): Na is weighed2CO31.59 g, NaHCO32.93 g are mixed after being dissolved in a small amount of distilled water respectively
It closes, adds distilled water to mix to about 800mL, adjust 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, 0.2g KH2PO4, 2.9g Na2HPO4·12 H2O,
It is dissolved in 800mL pure water, with NaOH or HCl tune pH to 7.2-7.4, is settled to 1000mL；
PBST: the PBS containing 0.05 % polysorbas20；
TMB developing solution: A liquid: Na2HPO4 .12H2O 18.43g, citric acid 9.33g, pure water are settled to 1000mL；B liquid: 60mg
TMB is dissolved in 100mL ethylene glycol.A, B liquid 5:1 mixing by volume is TMB developing solution, current existing mixed.
Claims (3)
1. one plant of Diclofenac monoclonal antibody hybridoma cell strain A has been preserved in Chinese microorganism strain preservation management committee
Member meeting common micro-organisms center CGMCC, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microbe research
Institute, classification naming are monoclonal cell strain, preservation date on May 24th, 2018, deposit number CGMCC No.17301.
2. Diclofenac monoclonal antibody, it is characterised in that: it by the deposit number CGMCC No.17301 Diclofenac
No. A secretion of monoclonal antibody hybridoma cell strain generates.
3. the application of Diclofenac monoclonal antibody described in claim 2, it is characterised in that: for chlorine double in food safety detection
The fragrant remaining analysis detection of acid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923208A (en) * | 2019-12-18 | 2020-03-27 | 江南大学 | Polymyxin B sulfate monoclonal antibody hybridoma cell strain and application thereof |
| CN111334479A (en) * | 2020-04-16 | 2020-06-26 | 江南大学 | Chlorhydroxypyridine monoclonal antibody hybridoma cell strain TYL and application thereof |
-
2019
- 2019-01-11 CN CN201910026689.8A patent/CN109735503A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923208A (en) * | 2019-12-18 | 2020-03-27 | 江南大学 | Polymyxin B sulfate monoclonal antibody hybridoma cell strain and application thereof |
| CN111334479A (en) * | 2020-04-16 | 2020-06-26 | 江南大学 | Chlorhydroxypyridine monoclonal antibody hybridoma cell strain TYL and application thereof |
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Application publication date: 20190510 |