CN107189987B - One plant of hybridoma cell strain YY for secreting anti-silaenafil monoclonal antibody and its application - Google Patents
One plant of hybridoma cell strain YY for secreting anti-silaenafil monoclonal antibody and its application Download PDFInfo
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- CN107189987B CN107189987B CN201710580569.3A CN201710580569A CN107189987B CN 107189987 B CN107189987 B CN 107189987B CN 201710580569 A CN201710580569 A CN 201710580569A CN 107189987 B CN107189987 B CN 107189987B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
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- 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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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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Abstract
One plant of hybridoma cell strain YY for secreting anti-silaenafil monoclonal antibody and its application, belong to food safety field of immunodetection.The hybridoma cell strain YY of the anti-silaenafil monoclonal antibody of secretion of the invention, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.13100.The hybridoma cell strain YY, which can secrete generation, has preferable affinity and higher sensitivity to silaenafil, to 50% inhibition concentration IC of silaenafil50For 0.48 μ g/L, it can be used for preparing the immunity detection reagent and colloidal gold strip of silaenafil, the detection for silaenafil in Chinese patent drug and health food provides strong detection method and means.
Description
Technical field
The hybridoma cell strain YY for secreting anti-silaenafil monoclonal antibody the present invention relates to one plant and its application, belong to food
Product security immunization detection field.
Background technique
Silaenafil (Sildenafil, Sild), full name sildenafil citrate are viagra (also known as " vigours ")
Sole active pharmaceutical ingredient.Silaenafil is 5 type phosphodiesterases (PDE5) selectivity special to cyclic guanosine monophosphate (cGMP)
Inhibitor can be used for treating Erectile Dysfunction (ED).In recent years, chemical drugs are illegally added in Chinese patent drug and health food
Product event remains incessant after repeated prohibition, and tonifying kidney and strengthening yang class Chinese patent drug and health care product take crowd and the huge market sales volume because its is huge,
Even more become the severely afflicated area illegally added, therefore, how to establish and save money, is with strong points, is high-efficient fast detecting method, severe
Hit adulterated drug, control drug quality is to carry out the important leverage of Drug Administration.
Silaenafil detection at present mostly uses physico-chemical method such as ultraviolet spectroscopy, thin-layered chromatography and high performance liquid chromatography
Deng, ultraviolet spectroscopy and thin-layered chromatography because interference is difficult to exclude, method is restricted.Instrument detection method can quantitatively be divided
It analyses and there is lower detection to limit, but usually require expensive instrument and complicated operation, pre-treatment and detection time are long, sternly
The popularization of these detection methods is constrained again.And immunoassay method have it is inexpensive, high-throughput, highly sensitive, to technical staff
The features such as relative requirement is low, therefore it is suitable for the rapid screening of a large amount of samples.The purpose of the present invention is to provide one kind to western ground
The preparation method of that non-monoclonal antibody hybridoma cell strain with higher affinity and detection sensitivity.For indirect competition
The research and development popularization of ELISA kit and colloidal gold strip is laid a good foundation.
Summary of the invention
The purpose of the present invention is to provide the hybridoma cell strain YY of one plant of anti-silaenafil monoclonal antibody of secretion and its
Using the monoclonal antibody prepared by the cell strain, with preferable affinity and sensitivity, can be used to build to silaenafil
Vertical silaenafil enzyme-linked immune detection method, or establish colloidal gold immuno-chromatography test paper strip rapid detection method.
Technical solution of the present invention: the hybridoma cell strain YY of one plant of anti-silaenafil monoclonal antibody of secretion, preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, deposit number CGMCC
No.13100, preservation date on October 31st, 2016, classification naming monoclonal cell strain.
Anti- silaenafil monoclonal antibody is to be secreted to produce by deposit number CGMCC No.13100 hybridoma cell strain YY
It is raw.
The application of the anti-silaenafil monoclonal antibody, for the remaining inspection of silaenafil in Chinese patent drug and health food
It surveys.
The preparation basic step of YY cell strain provided by the invention are as follows:
(1) preparation and identification of immunogene: using silaenafil as raw material, by the amino phase for activating rouge method and protein carrier
Even, after reaction, by small haptens dialysis separation comlete antigen and be not coupled, comlete antigen passes through UV absorption
Scan method identification；
(2) mouse is immune: the BALB/c mouse for choosing 6~8 week old is immunized.Immunogene and freund adjuvant are emulsified
After completely, mouse is immunized by subcutaneous multi-point injection, first immunisation uses Freund's complete adjuvant, and booster immunization is endless using Fu Shi
Full adjuvant, immunizing dose is the half of a preceding immunizing dose when spurt is immune, is directly carried out after mixing with physiological saline
Intraperitoneal injection；Each secondary immunization interval is three weeks.After third time is immune, interval blood sampling in one week detection serum titer and inhibition；
(3) cell fusion and cell strain are established: by polyethylene glycol (PEG 4000) method, making mouse boosting cell and Mouse Bone
Myeloma cells fusion detects positive cell hole using indirect ELISA by HAT culture medium culture, and further using indirectly competing
Strive ELISA method measurement positive cell hole inhibitory effect, by limiting dilution assay to have the positive cell hole preferably inhibited progress
It is subcloned three times, finally screens and obtain hybridoma cell strain YY；
(4) it the identification of hybridoma cell strain property: is set with and is surveyed with ELIAS secondary antibody using mouse monoclonal Ig class/subgroup identification
It is fixed；IC50The measurement of value, cross reacting rate and affinity passes through ELISA method.
Beneficial effects of the present invention: (1) the anti-silaenafil monoclonal antibody that the present invention obtains has preferably silaenafil
Detection sensitivity and affinity；(2) a kind of method of new synthesis silaenafil immunogene, the synthesis step of haptens is more
Simplify, effectively, provides the thinking and method of synthetic immunogen for the research of people from now on.
Biological material specimens preservation: the hybridoma cell strain YY of one plant of anti-silaenafil monoclonal antibody of secretion, preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, deposit number CGMCC
No.13100, preservation date on October 31st, 2016, classification naming monoclonal cell strain.
Detailed description of the invention
Fig. 1 is the ultra-violet absorption spectrum characterization of immunogene.
Fig. 2 is the standard suppression curve of silaenafil 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 silaenafil comlete antigen
It supports, cell conditioned medium is screened by indirect ELISA and indirect competitive ELISA, having finally obtained has preferable affinity to silaenafil
With the hybridoma cell strain of the monoclonal antibody of sensitivity.
Embodiment 1: the preparation of the hybridoma cell strain YY of anti-silaenafil monoclonal antibody
1, the synthesis of silaenafil haptens: 500mg silaenafil and 90mg amion acetic acid is taken to be dissolved in the methanol of 20mL
In, the triethylamine of 236.4mg is added while stirring.Mixture reacts 4 hours at 70 DEG C.After reaction, 50ml is steamed
Distilled water, which is added in reactant, terminates reaction.It is extracted with ethyl acetate later three times, merges organic layer, cleaned with saturation NaCl solution
Three times, Na2SO4Remove the water in organic phase, cross silicagel column, be dried in vacuo yellow solid powder is anti-to get silaenafil half
It is former.
2, the synthesis of comlete antigen: taking 3mg silaenafil haptens, and 4.5mg EDC(1- (3- dimethylamino third is added
Base) -3- ethyl-carbodiimide hydrochloride) and 3.6mg NHS(N- HOSu NHS), use DMF(N, N- dimethyl formyl
Amine) dissolution, it is stirred at room temperature, activates 4h；Separately taking 10mg KLH(keyhole limpet hemocyanin) CB that is dissolved in 3mL, 0.05M, pH9.6 is molten
In liquid (carbonate buffer solution), above-mentioned activating solution is added dropwise in KLH solution, is stirred at room temperature after reaction overnight, 4 DEG C of dialysis
Three days, -20 DEG C of packing saved.
3, animal immune: the BALB/c mouse of 6~8 week old of health is selected to be immunized.Take silaenafil 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 18 days is exempted from after exempting from five
Epidemic disease prepares fusion.
4, cell fusion: after spurt immune three days, according to conventional PEG(polyethylene glycol, molecular weight 4000) method into
Row cell fusion, the specific steps are as follows:
(1) sterile to take mouse spleen, it grinds and obtains splenocyte suspension by 200 mesh cell screen clothes, and carry out cytometer
Number；
(2) SP2/0 cell is collected, is suspended in RPMI-1640 basic culture solution, cell count is carried out；
(3) splenocyte and SP2/0 cell are mixed according to the counting ratio of 2~10:1, is merged after centrifugation with PEG, the time 1
RPMI-1640 basic culture solution is added later according to from slowly to fast in min, is suspended in after centrifugation containing 20% fetal calf serum, 2%
In the RPMI-1640 screening and culturing liquid of 50 × HAT, 96 porocyte culture plates are added to, are placed in 37 DEG C, 5% CO2Incubator in
Culture.
5, cell screening and cell strain are established: carrying out RPMI-1640 screening training to fused cell within the 3rd day in cell fusion
Nutrient solution partly changes liquid, carries out within the 5th day being changed entirely with the RPMI-1640 transition culture solution of 100 × HT containing 20% fetal calf serum, 1%
Liquid took cell conditioned medium to be screened at the 7th day.Screen in two steps: the first step first filters out positive cell hole with indirect ELISA,
It is standard items that second step, which selects silaenafil, carries out inhibitory effect measurement to positive cell with indirect competitive ELISA.Selection is to west
Ground that it is non-have the cell hole preferably inhibited, be subcloned using limiting dilution assay, detected with same method.Repeat three
It is secondary, obtain cell strain YY.
6, the preparation and identification of monoclonal antibody: taking 8~10 week old BALB/c mouses, and every mouse peritoneal injects paraffin oil
1mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma collected ascites since the 7th day, and ascites is passed through octanoic acid-
The purifying of saturated ammonium sulfate method, 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, specific as shown in table 1.
The subtype identification of 1. silaenafil monoclonal antibody of table
Using indirect competitive ELISA method, monoclonal antibody is measured to the IC of silaenafil50For 0.48 μ g/L, and demonstrate
Its IC to Tadalafei etc.50And cross reacting rate, it is specific as shown in table 2.
2 silaenafil monoclonal antibody of table is to silaenafil, Vardenafil, Acctildenafil, bold and unconstrained not silaenafil, Ta Dala
Non- IC50And cross reacting rate
7, hybridoma cell strain YY antibody application: is applied to silaenafil by monoclonal antibody prepared by internal ascites
ELISA adds recovery test, the specific steps are as follows:
(1) silaenafil for the 0.1 μ g/mL for using carbonate buffer solution (CBS) to dilute is coated with as coating primordial covering 96
Hole elisa Plates, after every hole 100 μ L, 37 DEG C of 2 h of coating, three times with PBST washing lotion board-washing, each every 200 μ L of hole, every time 3 min,
It pats dry；
(2) it is closed with the CBS containing 0.2% gelatin, every hole 200 μ L, 37 DEG C of 2 h of closing, with PBST washing lotion board-washing three
Secondary, each every 200 μ L of hole, 3 min, pats dry every time；
(3) with phosphate buffer (PBS) be respectively configured 0,0.02,0.05,0.1,0.2,0.5,1,2 μ g/L west ground that
Non-standard solution.Standard solution and sample to be tested extracting solution are added separately in the ELISA Plate closed, every hole
50 μ L, 3 holes of each sample repetition, then every hole addition diluted anti-silaenafil monoclonal antibody of 50 μ L 1:16000,37 DEG C
After reacting half an hour, board-washing is patted dry；
(4) sheep anti-mouse igg two that 100 μ L use the diluted HRP label of the PBS 1:3000 containing 0.1% gelatin is added in every hole
Anti-, after 37 DEG C of reaction half an hour, board-washing is patted dry；
(5) every hole is added 100 μ L TMB developing solutions, after 37 DEG C of colour developing 15min, 50 μ L 2M H of every hole addition2SO4It terminates
Liquid, 450nm survey light absorption value；
(6) addition recycling and sample pre-treatments: 1g capsule sample content is taken to add the silaenafil of three various doses
Standard items, respectively 2ng, 5 ng, 10ng.Then content is added into 0.01M hydrochloric acid 20mL, ultrasonic extraction 30min, filtered
It crosses, filter residue extracts 2 times with method；Merge No. 3 extracting solutions, with ammonium hydroxide tune pH 9, chloroform is extracted 3 times, each 20mL, is closed
And chloroform solution, it sets and is evaporated in water-bath, residue is settled to 5mL with the PBS containing 10% methanol, shakes up, is to be checked.(pill or tablet take phase
Same amount, ibid method operates).Recovery test is added using indirect competitive ELISA, the rate of recovery is respectively 89%, 91%,
92%。
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
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): 8.00 g NaCl, 0.2 g KCl, 0.24 g KH2PO4, 3.62 g Na2HPO4·
12 H2O is dissolved in 800 mL pure water, with NaOH or HCl tune pH to 7.2~7.4, 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 are dissolved in 100 mL ethylene glycol.A, B liquid 1:5 mixing by volume is TMB developing solution, current existing mixed.
It is in summary only presently preferred embodiments of the present invention, practical range not for the purpose of limiting the invention.It is i.e. all
Equivalent changes and modifications made by content according to the present patent application range all should be technology scope of the invention.
Claims (3)
1. the hybridoma cell strain YY of one plant of anti-silaenafil monoclonal antibody of secretion, is preserved in Chinese microorganism strain preservation
Administration committee common micro-organisms center CGMCC, deposit number are CGMCC No.13100.
2. anti-silaenafil monoclonal antibody, it is characterised in that: it is the hybridoma for being CGMCC No.13100 by deposit number
Cell strain YY secretion generates.
3. the application of anti-silaenafil monoclonal antibody described in claim 2, it is characterised in that: be used for Chinese patent drug and health food
The middle remaining detection of silaenafil.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161662A (en) * | 2010-08-09 | 2011-08-24 | 江苏省南通药品检验所 | Artificially synthesized antigen of sildenafil and derivatives thereof as well as preparation method and application of antigen |
| CN102174474A (en) * | 2011-01-11 | 2011-09-07 | 南通市伊士生物技术有限责任公司 | Sildenafil monoclonal antibody and colloidal gold chromatography test strip used for detecting sildenafil |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161662A (en) * | 2010-08-09 | 2011-08-24 | 江苏省南通药品检验所 | Artificially synthesized antigen of sildenafil and derivatives thereof as well as preparation method and application of antigen |
| CN102174474A (en) * | 2011-01-11 | 2011-09-07 | 南通市伊士生物技术有限责任公司 | Sildenafil monoclonal antibody and colloidal gold chromatography test strip used for detecting sildenafil |
Non-Patent Citations (1)
| Title |
|---|
| Development of an immunoassay for rapid screening of vardenafil and its potential analogues in herbal products based on a group specific monoclonal antibody;Jie-Biao Guo等;《Analytica Chimica Acta》;20100125;第658卷(第2期);第197-203页，参见全文 * |
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