CN104789535B - Anti- methadone metabolin EDDP hybridoma cell strains and its preparation method and application - Google Patents
Anti- methadone metabolin EDDP hybridoma cell strains and its preparation method and application Download PDFInfo
- Publication number
- CN104789535B CN104789535B CN201510208740.9A CN201510208740A CN104789535B CN 104789535 B CN104789535 B CN 104789535B CN 201510208740 A CN201510208740 A CN 201510208740A CN 104789535 B CN104789535 B CN 104789535B
- Authority
- CN
- China
- Prior art keywords
- methadone
- eddp
- metabolin
- hybridoma cell
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of anti-methadone metabolin EDDP hybridoma cell strains, it is characterized in that the anti-methadone metabolin EDDP hybridoma cell strains are named as hybridoma cell strain 9G2.5, China typical culture collection center is preserved on January 15th, 2015, preserving number is CCTCC NO:C201507.The hybridoma cell strain can produce the anti-methadone metabolin EDDP monoclonal antibodies of high specific, and production efficiency is high.The anti-methadone metabolin EDDP monoclonal antibodies of secretion have higher specificity, sensitiveness, and cross reacting rate is low, and methadone metabolin EDDP colloidal gold strips are detected available for preparing.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of anti-methadone metabolin EDDP hybridoma cell strains and its
The application of the monoclonal antibody of generation.
Background technology
Methadone metabolin EDDP be methadone in liver through bioconversion, formed by demethylation and lived without pharmacology
Property pyrrolidines (pyrrolidine) derivative, its entitled 2- ethylidene -1,5- dimethyl -3,3- Diphenyl Pyrrole alkane of chemistry
(EDDP)。
Methadone is a kind of artificial synthesized narcotics, belongs to one of narcotics of national strict supervision.Methadone
It can be used for treating heroin and opiate addiction.Methadone is a kind of long-acting opioid drug, different with the drugs such as heroin, is inhaled
After malicious personnel take methadone, the dependence to drugs will not be produced, that is to say, that the painful symptom after drug rehabilitation will not be produced,
Since methadone is special medicine, so the keeping to it is particularly severe, once someone illegally takes hospital out of, will be regarded as carrying poison
Product deliver public security department's processing.Determine whether to take methadone by detecting methadone metabolin EDDP.
In the prior art, methadone can use Instrumental Analysis.Instrumental Analysis dependence technical requirements are high, detection time is long,
Of high cost, testing result reliability is not high.
The content of the invention
It is an object of the present invention to provide a kind of anti-methadone metabolin EDDP hybridoma cell strains.
The anti-methadone metabolin EDDP hybridoma cell strains of the present invention were named as hybridoma cell strain 9G2.5, in 2015 1
The moon be preserved within 15th China typical culture collection center (China Center for Type Culture Collection,
Abbreviation CCTCC), preserving number is CCTCC NO:C201507；Preservation address is Wuhan, China Wuhan University.The hybridoma cell strain
The anti-methadone metabolin EDDP monoclonal antibodies of high specific can be produced, production efficiency is high, and cost is low.
It is a further object to provide the anti-methadone metabolin EDDP Dan Ke of hybridoma cell strain 9G2.5 secretions
Grand antibody.The monoclonal antibody has higher specificity, sensitiveness, and cross reacting rate is low.
Third object of the present invention is to provide the preparation method of anti-methadone metabolin EDDP monoclonal antibodies, including step
It is rapid as follows：
Step (1) mouse immunes
The BALB/c female mice methadone metabolin EDDP artificial antigens of 8 week old are selected to inject mouse gaskin muscle；Institute
The gaskin muscle sites stated have the place of bulk muscle for mouse leg exposed to outside, and the thigh of mouse is most of by abdomen
Portion's skin parcel, it is possible to locate that knee joint distinguishes shank thigh；
The BALB/c female mices of 8 week old are taken, mouse gaskin muscle is injected with methadone metabolin EDDP artificial antigens, every
Inject once within 1 week, co-injection 4 times；For 20 μ g/ only, first time is beautiful for the first time methadone metabolin EDDP artificial antigens amount of being immunized
Husky bupropion metabolite thing EDDP artificial antigens carry out antigen emulsifying step before being immunized, and antigen emulsifying step is by methadone metabolin
The EDDP artificial antigen amounts of being immunized are diluted in 20ulPBS buffer solutions, obtain dilution antigen, the Freund's complete adjuvant with equal volume amounts
Emulsification；The follow-up secondary methadone metabolin EDDP artificial antigens amount of being immunized is 20ug/, antigen emulsifying step and first time phase
Together；Last 1 methadone metabolin EDDP artificial antigen amounts of being immunized are changed to 5ug/ only, and antigen emulsifying step is identical with first time.
Step (2) lymphocytes are merged with myeloma cell's
It is prepared by 2-1. feeder cells：Mouse plucks eyeball execution, is soaked in 75 ﹪ alcohol and sterilizes, tears mouse abdomen crust
Skin, its peritonaeum of exposure, injects the IMDM serum free mediums of 37 DEG C of preheatings, gently rubs mouse peritoneal, suspension abdominal cavity cell, suctions out abdomen
Chamber liquid；Centrifugation, sediment are resuspended with the IMDM nutrient solutions of 1 × HAT, obtain feeder cells suspension.
The culture of 2-2. murine myeloma cells SP2/0：It is 10 ﹪ that murine myeloma cell SP2/0, which is used containing volume fraction,
The IMDM culture mediums of FBS carry out secondary culture, and cell fusion the previous day is passed on to ensure that murine myeloma cell SP2/0 is fitted
Symphysis long status is well used for cell fusion.
It is prepared by 2-3. lymphocytes：Lymph of the BALB/c female mices of step of learning from else's experience (2) processing close to lymph node subcutaneous location
Knot, grinding lymphocyte to suspension；Lymphocyte suspension centrifuges, and takes precipitation；IMDM culture mediums are added in precipitation, adjust lymph
The concentration of cell is to 1 × 107A/mL.
2-4. lymphocytes are merged with myeloma cell：Using polyethylene glycol fusion method.Lymph after adjustment concentration is thin
Born of the same parents are 2 by number of cells ratio with murine myeloma cell SP2/0：1 is uniformly mixed, and centrifuge washing cell, removes supernatant, be placed in
37 DEG C of water-baths, add the PEG 1450 of 37 DEG C of pre-temperatures of 1mL, and the IMDM serum free mediums for adding 37 DEG C of pre-temperatures of 25ml terminate
PEG is acted on；Centrifugation, takes precipitation；Then the feeder cells suspension of step 2-1 is added in precipitation, is inoculated into 96 hole cell culture
In plate, 37 DEG C are placed in, 5 ﹪ CO2Cultivated in incubator.
The screening of step (3) hybridomas
3-1. primary dcreening operation：
Fused cell is in 5 ﹪ CO2After being cultivated 3 days in incubator, the IMDM nutrient solutions for using 1 × HT instead continue after cultivating 4 days,
The fused cell growing state in 96 porocyte culture plates is observed, in cell growth to cell cluster (in 16 times of object lens and 10 times
Mesh Microscopic observation, cell size take 1/3 visual field) when, fused cell culture supernatant is drawn, is sieved using indirect ELISA method
Select positive colony.
The IMDM nutrient solutions of the 1 × HT contain the FBS that volume fraction is 15 ﹪.
3-2. secondary screening：
The positive colony screened is further subjected to secondary screening with Competitive assays ELISA method, filters out Competitive assays rate
Highest 9G2.5 hybridoma cell strains do clone's culture.
The cloning of 3-3. hybridoma cell strains 9G2.5：
The colonized culture of hybridoma cell strain 9G2.5 is carried out by limiting dilution assay, accurate counting cell, with containing volume integral
Number is diluted to the cell suspension of 4/mL for the IMDM culture mediums of 15 ﹪ FBS, the cell suspension after then being diluted with every hole 200ul
It is inoculated into 96 porocyte culture plates, cell growth status is observed after 7 days, and detects cell culture supernatant in Tissue Culture Plate
Antibody level, select 3 highest monoclonals of antibody titer, do colonized culture again, until the antibody test of monoclonal hole
Positive rate reaches 100 ﹪；Then picking monoclonal cell, is named as 9G2.5, and anti-methadone metabolin is carried out after expanding culture
EDDP is monoclonal antibody-purified.
The anti-methadone metabolin EDDP of step (4) are monoclonal antibody-purified
By the monoclonal cell strain 9G2.5 screened with containing the IMDM culture medium inoculateds that volume fraction is 10 ﹪ FBS to 24
In porocyte culture plates, it is inoculated into Tissue Culture Flask within second day, after cultivating 1 day, is inoculated into cell roller bottle, roller bottle culture 4
After it, culture supernatant is collected, is obtained after culture supernatant is concentrated containing anti-methadone metabolin EDDP monoclonal antibodies
Cell culture concentrate, then with the affine column purifications of HiTrap PROTEIN G HP, obtains anti-methadone metabolin after purification
EDDP monoclonal antibodies.
The PBS buffer is to contain 0.008M disodium hydrogen phosphates, 0.15M sodium chloride, bis- water phosphorus of 0.002M
The aqueous solution of acid dihydride sodium, pH 7.4；
The IMDM nutrient solutions of the 1 × HAT contain the FBS that volume fraction is 15 ﹪.
Last purpose of the invention is to provide the anti-methadone metabolin EDDP Dan Ke of hybridoma cell strain 9G2.5 secretions
Application of the grand antibody on detection methadone metabolin EDDP colloidal gold strips are prepared.Methadone metabolin EDDP colloidal golds try
Paper slip product has quick, simple operation and other advantages.A kind of effective live urine examination is provided for the detection of methadone metabolin
Means.Antibody secreted by EDDP hybridoma cell strains provided by the invention is used for the mark raw material on colloidal gold strip, urine
The detection threshold value of inspection is in 100ng/ml.
Beneficial effect possessed by the present invention：
1. the present invention is exempted from during anti-methadone metabolin EDDP monoclonal antibodies are prepared using low dose of intramuscular injection
Epidemic disease method, immunizing potency are high.
2. hybridoma cell strain 9G2.5 can efficiently, the anti-methadone metabolin EDDP monoclonal antibodies of stably excreting, and
The potency of the monoclonal antibody is high, specificity is good, sensitiveness is high, can largely produce, and is metabolized available for detection methadone is prepared
The immunology detection reagents such as the enzyme-linked immunosorbent assay kit of thing EDDP, colloidal gold strip.
3. can quick and spirit using colloidal gold strip of the present invention comprising anti-methadone metabolin EDDP monoclonal antibodies
Urine is detected quickly, there is high sensitivity, suitable for the detection of 100ng/ml threshold values.
Embodiment
The present invention is further analyzed with reference to embodiment (in example below PBS buffer be containing
0.008M disodium hydrogen phosphates, 0.15M sodium chloride, the aqueous solution of 0.002M sodium dihydrogen phosphate dihydrates, pH 7.4；It is small afterwards
Leg muscle position has the place of bulk muscle for mouse leg exposed to outside, and the thigh of mouse is most of by skin of abdomen bag
Wrap up in, it is possible to locate that knee joint distinguishes shank thigh；The IMDM nutrient solutions of 1 × HAT contain the FBS of 15 ﹪ of volume fraction；1 × HT's
IMDM nutrient solutions contain the FBS that volume fraction is 15 ﹪).
The anti-methadone metabolin EDDP hybridoma cell strains of the present invention were named as hybridoma cell strain 9G2.5, in 2015 1
The moon be preserved within 15th China typical culture collection center (China Center for Type Culture Collection,
Abbreviation CCTCC), preserving number is CCTCC NO:C201507；Preservation address is Wuhan, China Wuhan University.
Hybridoma cell strain 9G2.5 of the present invention can secrete a kind of anti-methadone metabolin EDDP monoclonal antibodies.
Prepare hybridoma cell strain 9G2.5 of the present invention and secrete anti-methadone metabolin EDDP monoclonal antibodies
Method, including step are as follows：
Step (1) mouse immunes：
The BALB/c female mices of 8 week old are taken, mouse gaskin muscle is injected with methadone metabolin EDDP artificial antigens, every
Inject once within 1 week, co-injection 4 times；For 20 μ g/ only, first time is beautiful for the first time methadone metabolin EDDP artificial antigens amount of being immunized
The husky bupropion metabolite thing EDDP artificial antigens amount of being immunized carries out antigen emulsifying step, and antigen emulsifying step is by methadone metabolin EDDP
The artificial antigen amount of being immunized is diluted in 20ulPBS buffer solutions, obtains dilution antigen, the Freund's complete adjuvant breast with equal volume amounts
Change；For 20ug/ only, antigen emulsifying step is identical with first time for the follow-up secondary methadone metabolin EDDP artificial antigens amount of being immunized；
Last 1 methadone metabolin EDDP artificial antigen amounts of being immunized are changed to 5ug/ only, and antigen emulsifying step is identical with first time.
Step (2) lymphocytes are merged with myeloma cell's：
It is prepared by 2-1. feeder cells：Mouse plucks eyeball execution, is soaked in 75 ﹪ alcohol and sterilizes 10min, tears mouse abdomen
Outer skin, its peritonaeum of exposure, with the IMDM serum free mediums of asepsis injector injection 37 DEG C of preheatings of 8mL, gently rubs mouse abdomen
Chamber, suspension abdominal cavity cell, suctions out peritoneal fluid；3min is centrifuged under 1500rpm, sediment is resuspended with the IMDM nutrient solutions of 1 × HAT,
Obtain feeder cells suspension.
The culture of 2-2. murine myeloma cells SP2/0：It is 10 ﹪ that murine myeloma cell SP2/0, which is used containing volume fraction,
The IMDM culture mediums of FBS carry out secondary culture, and cell fusion the previous day is passed on to ensure that murine myeloma cell SP2/0 is fitted
Symphysis long status is well used for cell fusion.
It is prepared by 2-3. lymphocytes：Lymph of the BALB/c female mices of step of learning from else's experience (2) processing close to lymph node subcutaneous location
Knot, grinding lymphocyte will centrifuge 5min under lymphocyte suspension 1500rpm, precipitation taken, then with IMDM culture mediums to suspension
The concentration of lymphocyte is to 1 × 10 after adjustment grinding7A/mL.
2-4. lymphocytes are merged with myeloma cell：By the lymphocyte and murine myeloma cell after adjustment concentration
SP2/0 is 2 by number of cells ratio：1 be uniformly mixed, centrifuge washing cell 1 time under 1500rpm, remove supernatant, gently attack from
Heart tube bottom, loosens cell precipitation, is placed in 37 DEG C of water-baths, and the 1mL PEG 1450 of 37 DEG C of pre-temperatures of 1mL are slowly added into 90s,
Side edged gentle agitation；The IMDM serum free mediums for adding 37 DEG C of pre-temperatures of 25ml terminate PEG effects；Centrifuged under 1000rpm
5min, takes precipitation；Then feeder cells suspension prepared by step 2-1 is added in precipitation, is inoculated into 96 porocyte culture plates,
37 DEG C are placed in, 5 ﹪ CO2Cultivated in incubator.
The fusion screening of step (3) hybridomas：
3-1. primary dcreening operation：
Fused cell is in 5 ﹪ CO in step 3-42After being cultivated 3 days in incubator, the IMDM nutrient solutions of 1 × HT are used instead, continue
After culture 4 days, the fused cell growing state in 96 porocyte culture plates is observed, in cell growth to cell cluster (at 16 times
Object lens and 10 times of mesh Microscopic observations, cell size are advisable with taking 1/3 visual field) when, fused cell culture supernatant is drawn, is used
Indirect ELISA method screening positive clone.The operating procedure of indirect ELISA method is：1. the carbon for being 0.05M with pH9.6 concentration
Phthalate buffer dilutes methadone metabolin EDDP artificial antigens to 1ug/mL, 96 hole elisa Plates and is separately added into 100ul in every hole
Methadone metabolin EDDP artificial antigens after dilution, 4 DEG C of coatings are stayed overnight, with the PBS buffer board-washing containing 0.05 ﹪ tween20
3 times；2. PBS buffer is added to fused cell culture supernatant is diluted to 25 times of volumes, then in 96 porocyte culture plates
In the fused cell culture supernatant after 100 μ l dilutions is added per hole, while set up negative control, 37 DEG C of reactions after sixty minutes,
With the PBS buffer board-washing 1 time containing 0.05 ﹪ tween20；3. PBS buffer is added to sheep anti-mouse igg-HRP to be diluted to
5000 times of volumes, then add the sheep anti-mouse igg-HRP after 100 μ l dilutions, 37 DEG C of reactions in 96 porocyte culture plates per hole
After 30 minutes, with the PBS buffer board-washing 3 times containing 0.05 ﹪ tween20；4. adding 50ul substrates TMB per hole, reacted at 37 DEG C
8 minutes；5. add the H that 50ul concentration is 2M2SO4Reaction is terminated, its OD value is measured at 450 nm, with fused cell culture supernatant
Liquid OD450 values/negative control OD450 values>2.5 be positive colony, and the positive colony screened is further used Competitive assays
ELISA method carries out secondary screening.
3-2. secondary screening：
The operating procedure of Competitive assays ELISA method is：1. take the methadone metabolin that 50ul concentration is 100ng/ml
After the positive colony culture supernatant mixing that EDDP standard items and 50ul above-mentioned steps 3-1 are obtained, it is added to and is metabolized with methadone
In the coated enzyme mark hole of thing EDDP artificial antigens, while blank control is designed, i.e. 50ulPBS buffer solutions and the training of 50ul positive colonies
Foster supernatant is mixed to join in enzyme mark hole, and 37 DEG C are incubated after sixty minutes, with the PBS buffer board-washing 1 containing 0.05 ﹪ tween20
It is secondary；2. PBS buffer is added to sheep anti-mouse igg-HRP is diluted to 5000 times of volumes, then every in 96 porocyte culture plates
Hole adds the sheep anti-mouse igg-HRP after 100 μ l dilutions, is incubated 30 minutes at 37 DEG C, with the PBS bufferings containing 0.05 ﹪ tween20
Liquid board-washing 3 times；3. adding 50ul substrates TMB per hole, reacted 8 minutes at 37 DEG C；4. add the H that 50ul concentration is 2M2SO4Terminate
Reaction, measures its OD value at 450 nm, and the highest hybridoma cell strain 9G2.5 of picking Competitive assays rate does clone's culture.
One Competitive assays ELISA of table
The cloning of 4-3. hybridoma cell strains 9G2.5：
The colonized culture of hybridoma cell strain 9G2.5 is carried out by limiting dilution assay, accurate counting cell, with containing volume integral
Number is diluted to the cell suspension of 4/mL for the IMDM culture mediums of 15 ﹪ FBS, is then inoculated into 96 hole cell trainings with every hole 200ul
Support in plate, cell growth status is observed after 7 days, and detect the antibody level of cell culture supernatant in Tissue Culture Plate, select 3
A highest monoclonal of antibody titer, does colonized culture again, until antibody test positive rate in monoclonal hole reaches 100 ﹪；
Then picking monoclonal cell, is named as 9G2.5, and it is pure to carry out anti-methadone metabolin EDDP monoclonal antibodies after expansion culture
Change.
The anti-methadone metabolin EDDP of step (5) are monoclonal antibody-purified
By the monoclonal cell strain 9G2.5 screened with containing the IMDM culture medium inoculateds that volume fraction is 10 ﹪ FBS to 24
In porocyte culture plates, it is inoculated into Tissue Culture Flask within second day, after cultivating 1 day, is inoculated into cell roller bottle, roller bottle culture 4
After it, culture supernatant is collected, is obtained after culture supernatant is concentrated containing anti-methadone metabolin EDDP monoclonal antibodies
Cell culture concentrate, then with the affine column purifications of HiTrap PROTEIN G HP, obtains anti-methadone metabolin after purification
EDDP monoclonal antibodies.The operating procedure of the affine column purifications of HiTrap PROTEIN G HP is：By HiTrap PROTEIN G
HP affinity columns are first fully washed with the level pad Binding Buffer of 5 times of bed volumes, until flowing through liquid A280 extinctions
Value is less than 0.1；Then by cell culture concentrate upper prop, flow velocity is controlled to be dripped/2 seconds for 1；After loading, with 5 times of column bed bodies
Long-pending level pad Binding Buffer are fully washed, until flowing through liquid A280 light absorption values less than 0.1；Then eluent is used
The monoclonal antibody of Elution Buffer elution of bound, controls elution speed to be dripped/2 seconds for 1, collects A280 light absorption values and is more than
0.2 eluent, the Tris solution that 1M pH9.0 are added dropwise in the eluent of collection adjust pH value to 7；After pH value is adjusted
Eluent is placed in bag filter, is dialysed with PBS buffer, replaces dialyzate for every eight hours 1 time, is dialysed 3 times altogether；After dialysis
Antibody-solutions measure protein content in 280nm.
The performance detection of the anti-methadone metabolin EDDP monoclonal antibodies of embodiment 2
2.1 anti-methadone metabolin EDDP monoclonal antibody reactive active testings
Anti- methadone metabolin EDDP monoclonal antibodies prepared by indirect ELISA method detection embodiment 1：It is dense with pH9.6
The carbonate buffer solution spent for 0.05M dilutes methadone metabolin EDDP artificial antigens (KET-BSA) to 1ug/mL, then 96
The methadone metabolin EDDP artificial antigens after 100ul dilutions are separately added into the every hole of hole elisa Plates, 4 DEG C of coatings are overnight；Then
With the PBS buffer board-washing 3 times containing 0.05 ﹪ tween20；Then the anti-methadone metabolin that 100ul concentration is 1ug/ml is added
EDDP monoclonal antibody solutions, do doubling dilution, while set up negative control, and 37 DEG C are reacted after sixty minutes, with containing 0.05 ﹪
The PBS buffer board-washing of tween20 1 time；PBS buffer is added to sheep anti-mouse igg-HRP and is diluted to 5000 times of volumes, then
Sheep anti-mouse igg-the HRP after 100 μ l dilutions is added per hole in 96 porocyte culture plates, 37 DEG C are reacted 30 minutes, with containing
The PBS buffer board-washing of 0.05 ﹪ tween20 3 times；Then after 37 DEG C of 50ul substrates TMB reactions 8 minutes are added per hole, concentration
For the H of 2M2SO4Terminate reaction, 450nm measures its OD value, with anti-methadone metabolin EDDP monoclonal antibody solution OD450 values/
Negative control OD450 values>2.5 be positive value.From table two, it can be seen that, anti-methadone metabolin EDDP prepared by embodiment 1 is mono-
Clonal antibody reactivity can reach 0.001ug/ml.
The OD450 values of anti-methadone metabolin EDDP monoclonal antibodies prepared by embodiment 1 under two various concentrations of table
| Antibody concentration | OD450 values | Antibody concentration | OD450 values |
| 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 control) | 0.074 |
The foundation and assessment of 3 methadone metabolin EDDP colloidal gold strip methods of embodiment
1. prepared by colloidal gold
The chlorauric acid solution 20ml of 0.1g/L is prepared first, is heated to seethe with excitement with thermostatic electromagnetic blender, adds 1 ﹪ lemons
Three sodium solution 0.4ml of acid, continue heating stirring 10min, add to original volume with distilled water after being cooled to room temperature, obtain colloidal gold
Solution；
2. colloidal gold labeled monoclonal antibody albumen
With 0.1mol/L K2CO3Colloidal gold solution pH value is adjusted to 9.0, it is mono- that the anti-methadone metabolin EDDP of 0.5mg are added dropwise
Clonal antibody albumen；Continue after stirring 3min, dropwise addition mass volume ratio is 5 ﹪ BSA solution, until BSA solution quality volume ratios
For 1 ﹪, continue to stir 10min, obtain antibody-solutions；Its moderate resistance methadone metabolin EDDP monoclonal antibody protein is anti-U.S. husky
The advance desalination of bupropion metabolite thing EDDP monoclonal antibodies, is diluted to the antibody protein obtained after 1mg/ml；
3. the purifying of gold labeling antibody
15min will be centrifuged under the antibody-solutions 2000r/min marked, discard precipitation；Supernatant again through 10000r/min from
Heart 15min, abandoning supernatant；Precipitation is washed 2 times with the borate buffer of PH9.0, and the boric acid for being finally resuspended in 2ml PH9.0 delays
In fliud flushing；
4. test strips assemble
According to a conventional method by methadone metabolin EDDP artificial antigens (EDDP-BSA) point in detecting line position on film, goat-anti
Then mouse secondary antibody point is assembled into gold mark detection test paper bar in C line positions on film, gold labeling antibody point in gold-labelled pad；
5. methadone metabolin EDDP test strips sensitivity tests
With PBS buffer by methadone metabolin EDDP standard items be configured to 300ng/ml, 150ng/ml, 100ng/ml,
50ng/ml, 0ng/ml, gold mark detection test paper bar is inserted into above-mentioned prepared solution respectively, result is observed after 5min.
The result judgement of three methadone metabolin EDDP ELISA test strip various concentrations standard items of table
| Standard concentration | 300ng/ml | 150ng/ml | 100ng/ml | 50ng/ml | 0ng/ml |
| As a result it is strong and weak | - | - | +/- | ++ | +++ |
| Result judgement | It is positive | It is positive | It is positive | It is negative | It is negative |
Methadone metabolin EDDP colloidal gold strips product has quickly, and simple operation and other advantages are provided by the invention
Antibody secreted by EDDP hybridoma cell strains is used for the mark raw material on colloidal gold strip, and the detection threshold value of urine examination exists
100ng/ml。
Claims (3)
- A kind of 1. anti-methadone metabolin EDDP hybridoma cell strains, it is characterised in that the anti-methadone metabolin EDDP hybridization Tumor cell strain is named as hybridoma cell strain 9G2.5, and China typical culture collection center is preserved on January 15th, 2015, Preserving number is CCTCC NO:C201507.
- A kind of 2. anti-methadone metabolin EDDP monoclonal antibodies of the secretion of the hybridoma cell strain as described in claim 1.
- 3. anti-methadone metabolin EDDP monoclonal antibodies described in claim 2 are preparing detection methadone metabolin EDDP colloids Application on gold test paper strip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510208740.9A CN104789535B (en) | 2015-04-28 | 2015-04-28 | Anti- methadone metabolin EDDP hybridoma cell strains and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510208740.9A CN104789535B (en) | 2015-04-28 | 2015-04-28 | Anti- methadone metabolin EDDP hybridoma cell strains and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104789535A CN104789535A (en) | 2015-07-22 |
| CN104789535B true CN104789535B (en) | 2018-04-13 |
Family
ID=53554730
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510208740.9A Active CN104789535B (en) | 2015-04-28 | 2015-04-28 | Anti- methadone metabolin EDDP hybridoma cell strains and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104789535B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2229341T3 (en) * | 1997-05-27 | 2005-04-16 | Microgenics Corporation | CONJUGATES AND SPECIFIC IMMUNO TESTS FOR METABOLITE METADONA 2-ETILIDINA-1,5-DIMETIL-3,3-DIFENIL-PIRROLIDINA. |
| US8771964B2 (en) * | 2012-02-02 | 2014-07-08 | Siemens Healthcare Diagnostics Inc. | Compositions and methods for detection of methadone metabolite |
| CN102590520B (en) * | 2012-02-27 | 2014-04-09 | 上海凯创生物技术有限公司 | EDDP (ethylenediamine-dimethylphosphinic acid) colloidal gold detection kit and preparation method thereof |
| CN103060275B (en) * | 2012-12-29 | 2014-07-09 | 杭州傲锐生物医药科技有限公司 | Ketamine hybridoma cell strain and preparation method and application thereof |
| CN103360271B (en) * | 2013-06-19 | 2015-09-16 | 广州万孚生物技术股份有限公司 | Methadone haptens and preparation method thereof, methadone antigen and methadone monoclonal antibody and application thereof |
-
2015
- 2015-04-28 CN CN201510208740.9A patent/CN104789535B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104789535A (en) | 2015-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104031144B (en) | Specific bond HEV 3, antibody of 4 types and application thereof | |
| CN111732664B (en) | Novel coronavirus recombinant protein, rabbit-human chimeric antibody, preparation method and application thereof | |
| CN101921731A (en) | Monoclonal antibody of fluoroquinolone medicines as well as preparation method and application thereof | |
| CN105112398A (en) | Preparation of hybridoma cell, monoclonal antibody secreted by hybridoma cell and application of monoclonal antibody | |
| CN104312978B (en) | A kind of TOB monoclonal antibody and preparation method and application | |
| CN104789535B (en) | Anti- methadone metabolin EDDP hybridoma cell strains and its preparation method and application | |
| CN110713986A (en) | Vitamin B1Monoclonal antibody hybridoma cell strain CBDD and application thereof | |
| CN112574957B (en) | Hybridoma cell strain secreting clomazone monoclonal antibody and application thereof | |
| CN101921730B (en) | Monoclonal antibody of ractopamine and preparation method and application thereof | |
| CN110950962B (en) | Hybridoma cell strain A11S for secreting bimesomepheniul monoclonal antibody and application thereof | |
| CN108752471A (en) | The preparation method and applications of anti-PCV2 monoclonal antibodies | |
| CN110724192B (en) | Hybridoma cell strain secreting serpentine monoclonal antibody and application thereof | |
| CN112375744A (en) | Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof | |
| CN112625135A (en) | Dicofol monoclonal antibody and application thereof | |
| CN108866006A (en) | A kind of preparation method and applications of anti-buprenorphine hybridoma cell strain, antibody | |
| CN101717447A (en) | Method for preparing antihuman recombinant tissue factor monoclonal antibody | |
| CN111057149A (en) | Anti-human D-dimer monoclonal antibody and application thereof | |
| CN114752568B (en) | Furosemide monoclonal antibody, hybridoma cell strain and application | |
| CN107058240B (en) | One strain of hybridoma strain AB1 and its 2,4,5 trichlorophenoxyacetic acid monoclonal antibody of generation | |
| CN102818896B (en) | Detection method of nitration modification sites based on specific antibodies and antibody capable of specifically recognizing succinyl-CoA: 3-oxoacid CoA transferase (SCOT) nitration sites | |
| CN111748528B (en) | Hybridoma cell strain secreting monoclonal antibody against fipronil and metabolite thereof and application of hybridoma cell strain | |
| CN105567643B (en) | Hybridoma cell capable of secreting anti-EV 71 virus protein VP1 monoclonal antibody, monoclonal antibody and application | |
| CN107056941A (en) | A kind of specific recognition goat IFN γ method for preparing monoclonal antibody | |
| CN102936584A (en) | Semicarbazide derivative monoclonal antibody and applications thereof | |
| CN110669736A (en) | Analgin monoclonal antibody hybridoma cell strain CBD and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 311215 workshop No. 550, workshop No. 550, Yinhai street, Baiyang street, Hangzhou economic and Technological Development Zone, Zhejiang Province, 2 and third, fourth floors Applicant after: Hangzhou Aotai biotechnology Limited by Share Ltd Address before: No. 3, building No. 2, No. 550, Yinhai street, Zhejiang economic and Technological Development Zone, Hangzhou, Zhejiang Applicant before: Hangzhou Ao Tai Bioisystech Co., Ltd |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 311215 Building 5, building 4, building 3, No. 550, Yinhai street, Baiyang street, Hangzhou Economic and Technological Development Zone, Hangzhou City, Zhejiang Province Patentee after: Hangzhou Aotai biotechnology Limited by Share Ltd Address before: 311215 workshop No. 550, workshop No. 550, Yinhai street, Baiyang street, Hangzhou economic and Technological Development Zone, Zhejiang Province, 2 and third, fourth floors Patentee before: Hangzhou Aotai biotechnology Limited by Share Ltd |