CN104789535A - Anti-methadone metabolite EDDP hybridoma cell strain as well as preparation method and application thereof - Google Patents
Anti-methadone metabolite EDDP hybridoma cell strain as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-methadone metabolite EDDP hybridoma cell strain. The anti-methadone metabolite EDDP hybridoma cell strain is characterized in that the anti-methadone metabolite EDDP hybridoma cell strain is called after hybridoma cell strain 9G2.5, wherein the strain is preserved in China Center for Type Culture Collection on 15, January, 2015 and the preservation number is CCTCC NO: C201507. The hybridoma cell strain can generate a high specific anti-methadone metabolite EDDP monoclonal antibody with high production efficiency. The secreted anti-methadone metabolite EDDP monoclonal antibody has high specificity and sensibility and low cross reaction rate and can be used for preparing a colloidal gold test strip for detecting the methadone metabolite EDDP.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the application of the monoclonal antibody of a kind of anti-methadone metabolite EDDP hybridoma cell strain and generation thereof.
Background technology
Methadone metabolite EDDP is methadone in liver through bio-transformation, tetramethyleneimine (pyrrolidine) derivative of parmacodynamics-less activity is formed by demethylation, its chemistry 2-ethylidene-1,5-dimethyl-3,3-Diphenyl Pyrrole alkane (EDDP) by name.
Methadone is a kind of narcotics of synthetic, belongs to one of narcotics of national strict supervision.Methadone can be used for treating heroine and opiate addiction.Methadone is a kind of long-acting opioid drug, different with drugs such as heroine, after drug addict takes methadone, the dependence to drugs can not be produced, that is the painful symptom after drug rehabilitation can not be produced, because methadone is special medicine, so strict especially to its keeping, once there be people illegally to take hospital out of, will be considered as carrying drugs and deliver public security department's process.Determine whether to take methadone by detecting methadone metabolite EDDP.
In prior art, methadone can adopt instrumental analysis.Instrumental analysis dependence technical requirements is high, detection time is long, cost is high, and detected result reliability is not high.
Summary of the invention
An object of the present invention is to provide a kind of anti-methadone metabolite EDDP hybridoma cell strain.
The present invention's anti-methadone metabolite EDDP hybridoma cell strain called after hybridoma cell strain 9G2.5, China typical culture collection center (China Center for Type Culture Collection is preserved on January 15th, 2015, be called for short CCTCC), preserving number is CCTCC NO:C201507; Preservation address is Wuhan, China Wuhan University.This hybridoma cell strain can produce the anti-methadone metabolite EDDP monoclonal antibody of high specific, and production efficiency is high, and cost is low.
Another object of the present invention is to provide the anti-methadone metabolite EDDP monoclonal antibody that hybridoma cell strain 9G2.5 secretes.This monoclonal antibody has higher specificity, susceptibility, and cross reacting rate is low.
3rd object of the present invention is to provide the preparation method of anti-methadone metabolite EDDP monoclonal antibody, comprises step as follows:
Step (1). mouse immune
Select the female mouse of BALB/c in 8 week age methadone metabolite EDDP artificial antigen injection mouse gaskin muscle; Described gaskin muscle sites is the place that mouse leg is exposed to that there is bulk muscle outside, and the thigh of mouse major part is wrapped up by skin of abdomen, and knee joint also can be found to distinguish shank thigh;
Get the female mouse of BALB/c in 8 week age, with methadone metabolite EDDP artificial antigen injection mouse gaskin muscle, every injection in 1 week once, inject 4 times altogether; First time methadone metabolite EDDP artificial antigen immunity amount is 20 μ g/, antigen emulsifying step is carried out before the immunity of first time methadone metabolite EDDP artificial antigen, antigen emulsifying step is diluted in 20ulPBS damping fluid by methadone metabolite EDDP artificial antigen immunity amount, obtain diluting antigen, with the Freund's complete adjuvant emulsification of equal volume amounts; Follow-up secondary methadone metabolite EDDP artificial antigen immunity amount is 20ug/, and antigen emulsifying step is identical with first time; Last 1 methadone metabolite EDDP artificial antigen immunity amount only changes 5ug/ into, and antigen emulsifying step is identical with first time.
Step (2). the fusion of lymphocyte and myeloma cell
Prepared by 2-1. feeder cell: mouse is plucked eyeball and puts to death, and is soaked in 75 ﹪ alcohol and sterilizes, tear mouse abdomen outer skin, expose its peritonaeum, injects the IMDM serum free medium of 37 DEG C of preheatings, gently rubs mouse peritoneal, suspension abdominal cavity cell, sucking-off peritoneal fluid; Centrifugal, throw out is resuspended with the IMDM nutrient solution of 1 × HAT, obtains feeder cell suspension.
The cultivation of 2-2. murine myeloma cell SP2/0: the murine myeloma cell SP2/0 IMDM substratum containing volume fraction being 10 ﹪ FBS is carried out Secondary Culture, and cytogamy carries out going down to posterity to ensure that murine myeloma cell SP2/0 is applicable to growth conditions well for cytogamy the day before yesterday.
Prepared by 2-3. lymphocyte: the female mouse of BALB/c that step of learning from else's experience (2) processes is near the lymphoglandula of lymphoglandula subcutaneous location, and grinding lymphocyte is to suspension; Lymphocyte suspension is centrifugal, gets precipitation; In precipitation, add IMDM substratum, adjust lymphocytic concentration to 1 × 10
7individual/mL.
2-4. lymphocyte and myeloma cell fusion: adopt polyoxyethylene glycol fusion method.Lymphocyte after adjustment concentration is mixed than for 2:1 by number of cells with murine myeloma cell SP2/0, centrifuge washing cell, remove supernatant liquor, be placed in 37 DEG C of water-baths, add the PEG 1450 of the pre-temperature of 1mL 37 DEG C, then the IMDM serum free medium adding the pre-temperature of 25ml 37 DEG C stops PEG effect; Centrifugal, get precipitation; Then in precipitation, add the feeder cell suspension of step 2-1, be inoculated in 96 porocyte culture plates, be placed in 37 DEG C, 5 ﹪ CO
2cultivate in incubator.
Step (3). the screening of hybridoma
3-1. primary dcreening operation:
Fused cell is at 5 ﹪ CO
2cultivate in incubator after 3 days, the IMDM nutrient solution using 1 × HT instead continued cultivation after 4 days, observe the fused cell growing state in 96 porocyte culture plates, at Growth of Cells to cell cluster (at 16 times of object lens and 10 times of order Microscopic observations, cell size takes 1/3 visual field) time, draw fused cell culture supernatant, adopt indirect ELISA method screening positive clone.
The IMDM nutrient solution of 1 described × HT contains the FBS that volume fraction is 15 ﹪.
3-2. sieves again:
The positive colony screened is carried out multiple sieve by Competitive assays ELISA method further, filters out the highest 9G2.5 hybridoma cell strain of Competitive assays rate and do clone's cultivation.
The cloning of 3-3. hybridoma cell strain 9G2.5:
The colonized culture of hybridoma cell strain 9G2.5 is undertaken by limiting dilution assay, accurate counting cell, the cell suspension of 4/mL is diluted to the IMDM substratum containing volume fraction being 15 ﹪ FBS, then the cell suspension inoculation after diluting with every hole 200ul is in 96 porocyte culture plates, observation of cell growing state after 7 days, and detect the antibody horizontal of cell culture supernatant in Tissue Culture Plate, select 3 mono-clonals that antibody titer is the highest, do colonized culture again, until antibody test positive rate in mono-clonal hole reaches 100 ﹪; Then picking monoclonal cell, called after 9G2.5, it is monoclonal antibody-purified to carry out anti-methadone metabolite EDDP after enlarged culturing.
Step (4). anti-methadone metabolite EDDP is monoclonal antibody-purified
Using containing volume fraction by the monoclonal cell strain 9G2.5 screened is that the IMDM culture medium inoculated of 10 ﹪ FBS is in 24 porocyte culture plates, within second day, be inoculated in Tissue Culture Flask, cultivate after 1 day, be inoculated in cell roller bottle, after roller bottle cultivates 4 days, collect culture supernatant, the cell cultures concentrated solution containing anti-methadone metabolite EDDP monoclonal antibody is obtained after culture supernatant being concentrated, then purify with HiTrap PROTEIN G HP affinity column, obtain the anti-methadone metabolite EDDP monoclonal antibody after purifying.
Described PBS damping fluid is the aqueous solution containing 0.008M disodium hydrogen phosphate, 0.15M sodium-chlor, 0.002M sodium dihydrogen phosphate dihydrate, and pH is 7.4;
The IMDM nutrient solution of 1 described × HAT contains the FBS that volume fraction is 15 ﹪.
Last object of the present invention is to provide the application of anti-methadone metabolite EDDP monoclonal antibody on preparation detection methadone metabolite EDDP colloidal gold strip that hybridoma cell strain 9G2.5 secretes.Methadone metabolite EDDP colloidal gold strip product has fast, simple operation and other advantages.Detection for methadone metabolite provides a kind of effective on-the-spot urine examination means.The mark raw material of antibody secreted by EDDP hybridoma cell strain provided by the invention on colloidal gold strip, the detection threshold of urine examination is at 100ng/ml.
The beneficial effect that the present invention has:
1. the present invention is in the anti-methadone metabolite EDDP monoclonal antibody process of preparation, and adopt low dose of intramuscular injection immunization method, immunizing potency is high.
2. hybridoma cell strain 9G2.5 can efficiently, stably excreting anti-methadone metabolite EDDP monoclonal antibody, and the height of tiring of this monoclonal antibody, specificity are good, susceptibility is high, can produce in a large number, can be used for preparing the immunology detection reagent such as enzyme-linked immunosorbent assay test kit, colloidal gold strip detecting methadone metabolite EDDP.
3. the colloidal gold strip adopting the present invention to comprise anti-methadone metabolite EDDP monoclonal antibody fast and detect urine delicately, can have highly sensitive feature, is applicable to the detection of 100ng/ml threshold value.
Embodiment
Below in conjunction with embodiment the present invention is further analyzed to (in embodiment, PBS damping fluid is the aqueous solution containing 0.008M disodium hydrogen phosphate, 0.15M sodium-chlor, 0.002M sodium dihydrogen phosphate dihydrate, and pH is 7.4 below; Gaskin muscle sites is the place that mouse leg is exposed to that there is bulk muscle outside, and the thigh of mouse major part is wrapped up by skin of abdomen, and knee joint also can be found to distinguish shank thigh; The IMDM nutrient solution of 1 × HAT contains the FBS of volume fraction 15 ﹪; The IMDM nutrient solution of 1 × HT contains the FBS that volume fraction is 15 ﹪).
The present invention's anti-methadone metabolite EDDP hybridoma cell strain called after hybridoma cell strain 9G2.5, China typical culture collection center (China Center for Type Culture Collection is preserved on January 15th, 2015, be called for short CCTCC), preserving number is CCTCC NO:C201507; Preservation address is Wuhan, China Wuhan University.
The hybridoma cell strain 9G2.5 that the present invention relates to can secrete a kind of anti-methadone metabolite EDDP monoclonal antibody.
Prepare the hybridoma cell strain 9G2.5 that the present invention relates to and secrete the method for anti-methadone metabolite EDDP monoclonal antibody, comprising step as follows:
Step (1). mouse immune:
Get the female mouse of BALB/c in 8 week age, with methadone metabolite EDDP artificial antigen injection mouse gaskin muscle, every injection in 1 week once, inject 4 times altogether; First time methadone metabolite EDDP artificial antigen immunity amount is 20 μ g/, first time methadone metabolite EDDP artificial antigen immunity amount carries out antigen emulsifying step, antigen emulsifying step is diluted in 20ulPBS damping fluid by methadone metabolite EDDP artificial antigen immunity amount, obtain diluting antigen, with the Freund's complete adjuvant emulsification of equal volume amounts; Follow-up secondary methadone metabolite EDDP artificial antigen immunity amount is 20ug/, and antigen emulsifying step is identical with first time; Last 1 methadone metabolite EDDP artificial antigen immunity amount only changes 5ug/ into, and antigen emulsifying step is identical with first time.
Step (2). the fusion of lymphocyte and myeloma cell:
Prepared by 2-1. feeder cell: mouse is plucked eyeball and puts to death, and is soaked in 75 ﹪ alcohol the 10min that sterilizes, tears mouse abdomen outer skin, expose its peritonaeum, inject the IMDM serum free medium of 8mL 37 DEG C of preheatings with asepsis injector, gently rub mouse peritoneal, suspension abdominal cavity cell, sucking-off peritoneal fluid; Centrifugal 3min under 1500rpm, throw out is resuspended with the IMDM nutrient solution of 1 × HAT, obtains feeder cell suspension.
The cultivation of 2-2. murine myeloma cell SP2/0: the murine myeloma cell SP2/0 IMDM substratum containing volume fraction being 10 ﹪ FBS is carried out Secondary Culture, and cytogamy carries out going down to posterity to ensure that murine myeloma cell SP2/0 is applicable to growth conditions well for cytogamy the day before yesterday.
Prepared by 2-3. lymphocyte: the female mouse of BALB/c that step of learning from else's experience (2) processes is near the lymphoglandula of lymphoglandula subcutaneous location, grinding lymphocyte is to suspension, by centrifugal 5min under lymphocyte suspension 1500rpm, get precipitation, then with lymphocytic concentration to 1 × 10 after IMDM substratum adjustment grinding
7individual/mL.
2-4. lymphocyte and myeloma cell fusion: the lymphocyte after adjusting concentration is mixed than for 2:1 by number of cells with murine myeloma cell SP2/0, centrifuge washing cell 1 time under 1500rpm, remove supernatant liquor, gently at the bottom of attack centrifuge tube, cell precipitation is loosened, be placed in 37 DEG C of water-baths, in 90s, slowly add the 1mL PEG 1450 of the pre-temperature of 1mL 37 DEG C, limit edged gentle agitation; The IMDM serum free medium adding the pre-temperature of 25ml 37 DEG C stops PEG effect; Under 1000rpm, centrifugal 5min, gets precipitation; Then in precipitation, add feeder cell suspension prepared by step 2-1, be inoculated in 96 porocyte culture plates, be placed in 37 DEG C, 5 ﹪ CO
2cultivate in incubator.
Step (3). the fusion screening of hybridoma:
3-1. primary dcreening operation:
In step 3-4, fused cell is at 5 ﹪ CO
2cultivate in incubator after 3 days, use the IMDM nutrient solution of 1 × HT instead, continue cultivation after 4 days, observe the fused cell growing state in 96 porocyte culture plates, at Growth of Cells to cell cluster (at 16 times of object lens and 10 times of order Microscopic observations, cell size is advisable to take 1/3 visual field) time, draw fused cell culture supernatant, adopt indirect ELISA method screening positive clone.The operation steps of indirect ELISA method is: 1. dilute methadone metabolite EDDP artificial antigen to 1ug/mL with the carbonate buffer solution that pH9.6 concentration is 0.05M, the methadone metabolite EDDP artificial antigen after 100ul dilution is added respectively in the 96 every holes of hole enzyme plate, 4 DEG C of bags are spent the night, and wash plate 3 times with the PBS damping fluid containing 0.05 ﹪ tween20; 2. PBS damping fluid is joined fused cell culture supernatant and be diluted to 25 times of volumes, then in 96 porocyte culture plates every hole add 100 μ l dilute after fused cell culture supernatant, set up negative control simultaneously, 37 DEG C of reactions, after 60 minutes, wash plate 1 time with the PBS damping fluid containing 0.05 ﹪ tween20; 3. PBS damping fluid is joined sheep anti-mouse igg-HRP and be diluted to 5000 times of volumes, then in 96 porocyte culture plates every hole add 100 μ l dilute after sheep anti-mouse igg-HRP, 37 DEG C of reactions, after 30 minutes, wash plate 3 times with the PBS damping fluid containing 0.05 ﹪ tween20; 4. every hole adds 50ul substrate TMB, reacts 8 minutes at 37 DEG C; 5. the H that 50ul concentration is 2M is added
2sO
4termination reaction, measures its OD value at 450 nm, with fused cell culture supernatant OD450 value/negative control OD450 value >2.5 for positive colony, the positive colony screened further is carried out multiple sieve by Competitive assays ELISA method.
3-2. sieves again:
The operation steps of Competitive assays ELISA method is: after 1. getting methadone metabolite EDDP standard substance that 50ul concentration is 100ng/ml and the positive colony culture supernatant that 50ul above-mentioned steps 3-1 obtains mixing, join in the enzyme mark hole with methadone metabolite EDDP artificial antigen bag quilt, design blank simultaneously, namely 50ulPBS damping fluid and 50ul positive colony culture supernatant are mixed to join in enzyme mark hole, 37 DEG C hatch 60 minutes after, wash plate 1 time with containing the PBS damping fluid of 0.05 ﹪ tween20; 2. PBS damping fluid is joined sheep anti-mouse igg-HRP and be diluted to 5000 times of volumes, then in 96 porocyte culture plates every hole add 100 μ l dilute after sheep anti-mouse igg-HRP, hatch 30 minutes at 37 DEG C, wash plate 3 times with the PBS damping fluid containing 0.05 ﹪ tween20; 3. every hole adds 50ul substrate TMB, reacts 8 minutes at 37 DEG C; 4. the H that 50ul concentration is 2M is added
2sO
4termination reaction, measures its OD value at 450 nm, and the hybridoma cell strain 9G2.5 that picking Competitive assays rate is the highest is clone and cultivates.
Table one Competitive assays ELISA
The cloning of 4-3. hybridoma cell strain 9G2.5:
The colonized culture of hybridoma cell strain 9G2.5 is undertaken by limiting dilution assay, accurate counting cell, the cell suspension of 4/mL is diluted to the IMDM substratum containing volume fraction being 15 ﹪ FBS, then be inoculated in 96 porocyte culture plates with every hole 200ul, observation of cell growing state after 7 days, and detect the antibody horizontal of cell culture supernatant in Tissue Culture Plate, select 3 mono-clonals that antibody titer is the highest, do colonized culture again, until antibody test positive rate in mono-clonal hole reaches 100 ﹪; Then picking monoclonal cell, called after 9G2.5, it is monoclonal antibody-purified to carry out anti-methadone metabolite EDDP after enlarged culturing.
Step (5). anti-methadone metabolite EDDP is monoclonal antibody-purified
Using containing volume fraction by the monoclonal cell strain 9G2.5 screened is that the IMDM culture medium inoculated of 10 ﹪ FBS is in 24 porocyte culture plates, within second day, be inoculated in Tissue Culture Flask, cultivate after 1 day, be inoculated in cell roller bottle, after roller bottle cultivates 4 days, collect culture supernatant, the cell cultures concentrated solution containing anti-methadone metabolite EDDP monoclonal antibody is obtained after culture supernatant being concentrated, then purify with HiTrap PROTEIN G HP affinity column, obtain the anti-methadone metabolite EDDP monoclonal antibody after purifying.The operation steps of HiTrap PROTEIN G HP affinity column purifying is: first fully washed with the level pad Binding Buffer of 5 times of column volumes by HiTrap PROTEIN G HP affinity column, until stream wears liquid A280 light absorption value be less than 0.1; Then by cell cultures concentrated solution upper prop, in control, flow velocity is 1/2 seconds; After loading, fully wash with the level pad Binding Buffer of 5 times of column volumes, until stream wears liquid A280 light absorption value be less than 0.1; Then use the monoclonal antibody of elutriant Elution Buffer elution of bound, controlling elution speed is 1/2 seconds, collects the elutriant that A280 light absorption value is greater than 0.2, drips the Tris solution adjust ph to 7 of 1M pH9.0 in the elutriant collected; Elutriant after adjust ph is placed in dialysis tubing, with the dialysis of PBS damping fluid, within every 8 hours, changes dialyzate 1 time, dialyse 3 times altogether; Antibody-solutions after dialysis is measured protein content in 280nm.
The Performance Detection of embodiment 2 anti-methadone metabolite EDDP monoclonal antibody
2.1 anti-methadone metabolite EDDP monoclonal antibody reactive active testings
Indirect ELISA method detects anti-methadone metabolite EDDP monoclonal antibody prepared by embodiment 1: dilute methadone metabolite EDDP artificial antigen (KET-BSA) to 1ug/mL with the carbonate buffer solution that pH9.6 concentration is 0.05M, then in the 96 every holes of hole enzyme plate, add the methadone metabolite EDDP artificial antigen after 100ul dilution respectively, 4 DEG C of bags are spent the night; Then plate is washed 3 times with the PBS damping fluid containing 0.05 ﹪ tween20; Then add the anti-methadone metabolite EDDP monoclonal antibody solution that 100ul concentration is 1ug/ml, do doubling dilution, set up negative control simultaneously, 37 DEG C of reactions, after 60 minutes, wash plate 1 time with the PBS damping fluid containing 0.05 ﹪ tween20; PBS damping fluid is joined sheep anti-mouse igg-HRP and is diluted to 5000 times of volumes, then in 96 porocyte culture plates every hole add 100 μ l dilute after sheep anti-mouse igg-HRP, 37 DEG C of reactions 30 minutes, wash plate 3 times with containing the PBS damping fluid of 0.05 ﹪ tween20; Then every hole added 50ul substrate TMB 37 DEG C reaction after 8 minutes, and concentration is the H of 2M
2sO
4termination reaction, 450nm measures its OD value, with anti-methadone metabolite EDDP monoclonal antibody solution OD450 value/negative control OD450 value >2.5 for the positive is worth.Can see from table two, anti-methadone metabolite EDDP monoclonal antibody reactive activity prepared by embodiment 1 can arrive 0.001ug/ml.
The OD450 value of anti-methadone metabolite EDDP monoclonal antibody prepared by embodiment 1 under table two different concns
| Antibody concentration | OD450 value | Antibody concentration | OD450 value |
| 1ug/ml | 2.599 | 0.008ug/ml | 0.651 |
| 0.5ug/ml | 2.371 | 0.004ug/ml | 0.496 |
| 0.25ug/ml | 2.010 | 0.002ug/ml | 0.314 |
| 0.125ug/ml | 1.862 | 0.001ug/ml | 0.232 |
| 0.0625ug/ml | 1.473 | 0.0005ug/ml | 0.179 |
| 0.032ug/ml | 1.194 | 0.00025ug/ml | 0.124 |
| 0.016ug/ml | 0.826 | PBS (blank) | 0.074 |
The foundation of embodiment 3 methadone metabolite EDDP colloidal gold strip method and assessment
1. Radioactive colloidal gold preparation
First prepare the chlorauric acid solution 20ml of 0.1g/L, be heated to boiling with thermostatic electromagnetic agitator, add 1 ﹪ citric acid three sodium solution 0.4ml, continue heated and stirred 10min, add to original volume with distilled water after being cooled to normal temperature, obtain colloidal gold solution;
2. colloidal gold labeled monoclonal antibody albumen
Use 0.1mol/L K
2cO
3regulate colloidal gold solution pH value to 9.0, drip 0.5mg anti-methadone metabolite EDDP monoclonal antibody protein; After continuing to stir 3min, dripping mass volume ratio is 5 ﹪ BSA solution, until BSA solution quality volume ratio is 1 ﹪, continues to stir 10min, obtains antibody-solutions; Wherein anti-methadone metabolite EDDP monoclonal antibody protein is the desalination in advance of anti-methadone metabolite EDDP monoclonal antibody, the antibody protein obtained after being diluted to 1mg/ml;
3. the purifying of gold labeling antibody
By centrifugal 15min under the antibody-solutions 2000r/min that marked, discard precipitation; Supernatant again through the centrifugal 15min of 10000r/min, abandoning supernatant; The borate buffer of precipitation PH9.0 washs 2 times, is finally resuspended in the borate buffer of 2ml PH9.0;
4. test strip assembling
According to a conventional method methadone metabolite EDDP artificial antigen (EDDP-BSA) is put detection line position on film, the anti-point of sheep anti mouse two C line position on film, golden labeling antibody point, on gold mark pad, is then assembled into gold mark detection test paper bar;
5. methadone metabolite EDDP test strip sensitivity test
With PBS damping fluid, methadone metabolite EDDP standard substance are mixed with 300ng/ml, 150ng/ml, 100ng/ml, 50ng/ml, 0ng/ml, gold mark detection test paper bar are inserted respectively in the above-mentioned solution prepared, observations after 5min.
The result of table three methadone metabolite EDDP ELISA test strip different concns standard substance judges
| Standard concentration | 300ng/ml | 150ng/ml | 100ng/ml | 50ng/ml | 0ng/ml |
| Result is strong and weak | - | - | +/- | ++ | +++ |
| Result judges | Positive | Positive | Positive | Negative | Negative |
Methadone metabolite EDDP colloidal gold strip product has fast, simple operation and other advantages, and the mark raw material of the antibody secreted by EDDP hybridoma cell strain provided by the invention on colloidal gold strip, the detection threshold of urine examination is at 100ng/ml.
Claims (4)
1. an anti-methadone metabolite EDDP hybridoma cell strain, it is characterized in that this anti-methadone metabolite EDDP hybridoma cell strain called after hybridoma cell strain 9G2.5, be preserved in China typical culture collection center on January 15th, 2015, preserving number is CCTCC NO:C201507.
2. an anti-methadone metabolite EDDP monoclonal antibody of being secreted by hybridoma cell strain described in claim 1.
3. the preparation method of monoclonal antibody described in claim 2, is characterized in that, comprises the following steps:
Step (1). mouse immune
Get the female mouse of BALB/c in 8 week age, with methadone metabolite EDDP artificial antigen injection mouse gaskin muscle, every injection in 1 week once, inject 4 times altogether; First time methadone metabolite EDDP artificial antigen immunity amount is 20 μ g/, antigen emulsifying step is carried out before the immunity of first time methadone metabolite EDDP artificial antigen, antigen emulsifying step is diluted in 20ulPBS damping fluid by methadone metabolite EDDP artificial antigen immunity amount, obtain diluting antigen, with the Freund's complete adjuvant emulsification of equal volume amounts; Follow-up secondary methadone metabolite EDDP artificial antigen immunity amount is 20ug/, and antigen emulsifying step is identical with first time; Last 1 methadone metabolite EDDP artificial antigen immunity amount only changes 5ug/ into, and antigen emulsifying step is identical with first time;
Step (2). the fusion of lymphocyte and myeloma cell
Prepared by 2-1. feeder cell: mouse is plucked eyeball and puts to death, and is soaked in 75 ﹪ alcohol and sterilizes, tear mouse abdomen outer skin, expose its peritonaeum, injects the IMDM serum free medium of 37 DEG C of preheatings, gently rubs mouse peritoneal, suspension abdominal cavity cell, sucking-off peritoneal fluid; Centrifugal, throw out is resuspended with the IMDM nutrient solution of 1 × HAT, obtains feeder cell suspension;
The cultivation of 2-2. murine myeloma cell SP2/0: the murine myeloma cell SP2/0 IMDM substratum containing volume fraction being 10 ﹪ FBS is carried out Secondary Culture, and cytogamy carries out going down to posterity to ensure that murine myeloma cell SP2/0 is applicable to growth conditions well for cytogamy the day before yesterday;
Prepared by 2-3. lymphocyte: the female mouse of BALB/c that step of learning from else's experience (2) processes is near the lymphoglandula of lymphoglandula subcutaneous location, and grinding lymphocyte is to suspension; Lymphocyte suspension is centrifugal, gets precipitation; In precipitation, add IMDM substratum, adjust lymphocytic concentration to 1 × 10
7individual/mL;
2-4. lymphocyte and myeloma cell fusion: adopt polyoxyethylene glycol fusion method; Lymphocyte after adjustment concentration is mixed than for 2:1 by number of cells with murine myeloma cell SP2/0, centrifuge washing cell, remove supernatant liquor, be placed in 37 DEG C of water-baths, add the PEG 1450 of the pre-temperature of 1mL 37 DEG C, then the IMDM serum free medium adding the pre-temperature of 25ml 37 DEG C stops PEG effect; Centrifugal, get precipitation; Then in precipitation, add the feeder cell suspension of step 2-1, be inoculated in 96 porocyte culture plates, be placed in 37 DEG C, 5 ﹪ CO
2cultivate in incubator;
Step (3). the screening of hybridoma
3-1. primary dcreening operation:
Fused cell is at 5 ﹪ CO
2cultivate in incubator after 3 days, the IMDM nutrient solution using 1 × HT instead continued cultivation after 4 days, observe the fused cell growing state in 96 porocyte culture plates, at Growth of Cells to cell cluster (at 16 times of object lens and 10 times of order Microscopic observations, cell size takes 1/3 visual field) time, draw fused cell culture supernatant, adopt indirect ELISA method screening positive clone; The IMDM nutrient solution of 1 described × HT contains the FBS that volume fraction is 15 ﹪;
3-2. sieves again:
The positive colony screened is carried out multiple sieve by Competitive assays ELISA method further, filters out the highest 9G2.5 hybridoma cell strain of Competitive assays rate and do clone's cultivation;
The cloning of 3-3. hybridoma cell strain 9G2.5:
The colonized culture of hybridoma cell strain 9G2.5 is undertaken by limiting dilution assay, accurate counting cell, the cell suspension of 4/mL is diluted to the IMDM substratum containing volume fraction being 15 ﹪ FBS, then the cell suspension inoculation after diluting with every hole 200ul is in 96 porocyte culture plates, observation of cell growing state after 7 days, and detect the antibody horizontal of cell culture supernatant in Tissue Culture Plate, select 3 mono-clonals that antibody titer is the highest, do colonized culture again, until antibody test positive rate in mono-clonal hole reaches 100 ﹪; Then picking monoclonal cell, called after 9G2.5, it is monoclonal antibody-purified to carry out anti-methadone metabolite EDDP after enlarged culturing;
Step (4). anti-methadone metabolite EDDP is monoclonal antibody-purified
Using containing volume fraction by the monoclonal cell strain 9G2.5 screened is that the IMDM culture medium inoculated of 10 ﹪ FBS is in 24 porocyte culture plates, within second day, be inoculated in Tissue Culture Flask, cultivate after 1 day, be inoculated in cell roller bottle, after roller bottle cultivates 4 days, collect culture supernatant, the cell cultures concentrated solution containing anti-methadone metabolite EDDP monoclonal antibody is obtained after culture supernatant being concentrated, then purify with HiTrap PROTEIN G HP affinity column, obtain the anti-methadone metabolite EDDP monoclonal antibody after purifying.
4. anti-methadone metabolite EDDP monoclonal antibody described in claim 2 detects the application on methadone metabolite EDDP colloidal gold strip in preparation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107344966A (en) * | 2017-07-05 | 2017-11-14 | 河南省农业科学院 | A kind of method that ZER monoclonal antibody is prepared based on analogue cross reactivity |
| CN108383910A (en) * | 2018-02-12 | 2018-08-10 | 山东华海力合创业投资管理有限公司 | A kind of monoclonal antibody and its preparation method and application of the single hydroxyl phenolic compound of identification |
| CN108383910B (en) * | 2018-02-12 | 2021-06-11 | 济南泰和医药科技有限公司 | Monoclonal antibody for identifying monohydroxyphenol compounds, and preparation method and application thereof |
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| CN104789535B (en) | 2018-04-13 |
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