CN105385660A - Hybridoma cell strain, antibody excreted by hybridoma cell strain, preparation method and application - Google Patents
Hybridoma cell strain, antibody excreted by hybridoma cell strain, preparation method and application Download PDFInfo
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Abstract
The invention provides a hybridoma cell strain MET1.0 producing a methylamphetamine-resistant monoclonal antibody, the methylamphetamine-resistant monoclonal antibody, a method for preparing the methylamphetamine-resistant monoclonal antibody excreted and produced by the methylamphetamine-resistant hybridoma cell strain MET1.0 with the preservation number being CCTCC NO:C2013160, a reagent kit for detecting methylamphetamine in saliva, and application of the methylamphetamine-resistant monoclonal antibody excreted and produced by the methylamphetamine-resistant hybridoma cell strain MET1.0 with the preservation number being CCTCC NO:C2013160 to detecting methylamphetamine. The high-affinity methylamphetamine-resistant monoclonal antibody can be easily prepared and obtained, and the effective yield of methylamphetamine artificial antigens is also increased. Only 5 minutes are needed for detection of methylamphetamine, detection sensitivity can reach 50 ng/ml, and specificity is high.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of anti-meth hybridoma cell strain and anti-meth monoclonal antibody.
Also relate to anti-meth monoclonal antibody as detecting the application of meth, and prepare this anti-meth hybridoma cell strain and secrete the method for anti-meth monoclonal antibody.
Background technology
Meth (Methamphetamine, MET), chemistry N-methyl isophthalic acid-phenyl-propan-2-amine by name, its structural formula is:
Meth (Methamphetamine), also known as methyl amphetamine, is commonly called as ice, it belongs to Psychopathic Drugs, be a kind of sympathomimetic amine medicine, be medically used for the treatment of the not enough Attention Deficit Hyperactivity Disorder (ADHD) of attention, lethargy, extreme obesity, rear encephalitis, parkinsonism etc., excessively take and very easily cause psychotic state, show hyperactivity hyperkinesia, affective impulse, barbarous, vain hope, paranoia, illusion is even killed a person tendency.After this kind of state, there is a kind of extreme exhaustion and depressive state, also have the self-slayer because of major depression.Long-term abuse can cause chronic poisoning, weight loss, becomes thin, ulcer, abscess, nail are brittle and bruxism.Meth tolerance increases with life-time service, for the people not producing tolerance, uses meth 30 milligrams just can cause poisoning.And have report, long-term misuser, in order to reach euphoriant effect during initial stage use, unexpectedly dosage is increased to 2000 milligrams, this very easily causes acute poisoning, and frightened fault, stupor can be caused even dead.There is great social harm.
In the detection of meth, immunochromatographic method, enzyme-linked immunosorbent assay (ELISA), gas chromatography-mass spectrography, high performance liquid chromatography, radioimmunoassay is mainly taked to detect urine, blood, tissue and hair equal samples both at home and abroad.Urine specimen relates to ethics and privacy, and fidelity sampling being existed to certain difficulty and sample exists certain suspection; Blood sample relates to ethics and wound, has certain infection risk for for sample people; Tissue and hair equal samples need to carry out pre-treatment, and detect and need large-scale instrument and equipment, technical requirements is high, is not suitable for field quick detection.
Wherein saliva is uniquely successfully used for alternative blood to carry out the body fluid of kinetics and the research of some drug toxicologies, comprising the research of Drug abuse.The salivary concentrations of medicine and ratio (S/P ratio) relative constancy of haemoconcentration, aberration rate is little, has great importance to estimation medicine metaboilic level in vivo.Meth saliva testing mechanism: meth enters into saliva from blood, former medicine has more lipotropy than metabolite, easilier enters saliva by panniculus barrier, and therefore the illicit drugs inspection thing of saliva is drugs substance; And the urine detection mechanism of meth is by blood by the metabolism of liver kidney, wherein 40% is still former medicine meth, and other excretes from urine with Amphetamine derivative usually.What therefore saliva was detected is the former medicine of meth, and urine detection is meth and Amphetamine derivative, and saliva detection is more better than urine detection.
Meth diagnostic threshold international is at present 1000ng/ml, is mainly used in urine detection.And the content of meth is lower in saliva, the anti-meth monoclonal antibody being applied to saliva detection requires higher sensitivity and avidity, whether meth saliva detection kit of the present invention utilizes in colloidal gold immunochromatographimethod technology qualitative detection saliva containing meth, its detection threshold can reach 50ng/ml, can meet the needs that saliva is detected completely.
Summary of the invention
An object of the present invention is to provide a kind of hybridoma cell strain MET1.0 producing anti-meth monoclonal antibody, be preserved in China typical culture collection center, deposit number is CCTCCNO:C2013160, hand over China typical culture collection center (CCTCC) preservation on November 12nd, 2013, preservation address: in the Wuhan University of Wuchang District, Wuhan City, Hubei Province, deposit number: CCTCCNO:C2013160.
Second object of the present invention is to provide a kind of anti-meth monoclonal antibody, it is that the anti-meth hybridoma cell strain MET1.0 being CCTCCNO:C2013160 by deposit number secretes generation, energy specific binding meth, oozy anti-meth monoclonal antibody belongs to IgG1 hypotype, and light chain is kappa.
3rd object of the present invention is to provide a kind of method preparing the anti-meth monoclonal antibody that the described anti-meth hybridoma cell strain MET1.0 secretion being CCTCCNO:C2013160 by deposit number produces; in the process preparing meth artificial antigen, utilize trifluoroacetic anhydride to protect amino group; Pyroglutaric acid is made can be accurately combined in therebetween on position; enhance the immunogenicity of antigen, be conducive to the anti-meth monoclonal antibody preparing high-affinity.
Wherein, meth artificial antigen is preparation like this:
In 50mL single port bottle, 500mg methyl amphetamine, with liquid ammonia alkalinization to PH=9, extract by ethyl acetate and obtain 390mg pale yellow oil B, 400ul trifluoroacetic anhydride is added under ice bath, be warming up to 80 DEG C of backflows after stirring 2h to spend the night, next day extracts by ethyl acetate after evaporated under reduced pressure, concentrate and obtain 600mg yellow oil C, the Pyroglutaric acid of quality such as to add again, extraction agent 200mg, be warming up to 120 DEG C of reaction 48h, extract by ethyl acetate again and obtain 750mgD, dissolve with under 38mLDMF, add 380mgNHS and 675mgDCC, stirring is spent the night, centrifuging and taking supernatant activation solution, obtain (concentration is 20mg/ml) stirring in the BSA of 200ml to spend the night, with the Na of PH=12
2cO
3dialyse after three days and dialyse three days with the PBS of PH=7.4, the supernatant liquor after centrifugal is artificial antigen.Purify meth artificial antigen with high performance liquid chromatograph, antigen purity can reach more than 95%.
Further, be in the method for the anti-meth monoclonal antibody of the anti-meth hybridoma cell strain MET1.0 secretion generation of CCTCCNO:C2013160 by deposit number in preparation, immunologic adjuvant is adopted to combine mice immunized with antigen in immunologic process, have without the need to emulsification, antigen amount be few, immunization time is short (only needing 5 weeks), do not destroy antigen conformation, nontoxic, impel bone-marrow-derived lymphocyte to increase, be conducive to the advantages such as fusion.
Wherein, mouse immune process is such:
The male mouse of BALB/c in 8-12 age in week is selected to carry out immunity with the injection of meth artificial antigen near lymphoglandula subcutaneous location, this experiment concrete steps are the male mouse of BALB/c of getting 8 week age, meth artificial antigen (taking MET-BSA as immunizing antigen) injection is near lymphoglandula subcutaneous location, inject 4 points, totally four immunity, each immunization interval two weeks: meth artificial antigen 20ug/ is only diluted to 150ul by first time PBS damping fluid, obtains dilution antigen and isopyknic immunologic adjuvant (main component is DNA of bacteria); Meth 10ug/ artificial antigen immunity amount is diluted to 150ul by second time PBS damping fluid, obtains dilution antigen and isopyknic immunologic adjuvant; Booster immunization for the third time, with PBS damping fluid, meth 20ug/ artificial antigen immunity is diluted to 150ul, combine isopyknic immunologic adjuvant immune mouse, after raising one week, put to death mouse, put to death and only do a booster immunization with meth artificial antigen 20ug/ in first 3 days.
4th object of the present invention is to provide the test kit for detecting meth in saliva, it comprises upholder, sample pad, the Radioactive colloidal gold scraps of paper, nitrocellulose filter and absorbent pad, upholder is pasted with sample pad successively from application of sample end, the Radioactive colloidal gold scraps of paper, nitrocellulose filter and absorbent pad, nitrocellulose filter there are detection zone and check plot, the anti-meth hybridoma cell strain MET1.0 that it is CCTCCNO:C2013160 that detection zone is coated with by deposit number secretes the anti-meth monoclonal antibody produced, check plot bag is by sheep anti mouse polyclonal antibody.This test kit minimum detectability is 50ng/ml, by commonly using Drug abuse and molecular structure analogue with kind more than 40, and common anti-additive medicament, sleeping medicine, anaesthetic, cough suppressing medicine, cold medicine cross reaction experiment show, the present invention to these material no cross reactions, high specificity.
5th object of the present invention is to provide a kind of anti-meth hybridoma cell strain MET1.0 being CCTCCNO:C2013160 by deposit number and secretes the application of anti-meth monoclonal antibody in detection meth produced.
The beneficial effect that the present invention has:
1, meth does not have immunogenic small-molecule substance; the present invention is in meth artificial antigen preparation process; trifluoroacetic anhydride is utilized to protect the amino of meth; the Pyroglutaric acid selecting side chain longer is as linking agent; make Pyroglutaric acid can accurately be combined between meth on site, position; both the immunogenicity of meth had been improved; be conducive to the anti-meth monoclonal antibody preparing high-affinity, turn improve the useful output of meth artificial antigen.
2, the present invention is in the anti-meth monoclonal antibody process of preparation, adopt immunologic adjuvant associating mice immunized with antigen, prepare the monoclonal antibody of high-affinity, compared with routine immunization method, have without the need to emulsification, antigen amount be few, immunization time is short (only needing 5 weeks), do not destroy antigen conformation, nontoxic, impel bone-marrow-derived lymphocyte to increase, be conducive to the advantages such as fusion.
3, the present invention adopts high performance liquid chromatograph purifying meth artificial antigen purifying meth artificial antigen, antigen purity can reach 95%, in the process of the anti-meth monoclonal antibody of purifying, adopt n-caprylic acid-saturated ammonium sulphate method and G-albumen affinity chromatography in conjunction with the anti-meth monoclonal antibody of purifying, substantially increase anti-meth antibody purity, antibody purity reaches more than 95%, by the efficiently purifying to antigen and antibody, be conducive to the anti-meth monoclonal antibody obtaining high-affinity.
4, the present invention only needs 5 minutes to the detection of meth, detection sensitivity can reach 50ng/ml, the present invention takes appropriate means in the setting of limit of identification, decrease the generation of false negative and false positive rate, by the invention of anti-meth monoclonal antibody, prepare the Radioactive colloidal gold series products that applicable saliva is detected, by commonly using Drug abuse and molecular structure analogue with kind more than 40, and common anti-additive medicament, sleeping medicine, anaesthetic, the cross reaction experiment of cold medicine shows, the present invention is to these material no cross reactions, high specificity.
Accompanying drawing explanation
UV scanning figure prepared by Fig. 1 meth artificial antigen.
The reactive behavior figure of the anti-meth monoclonal antibody of Fig. 2.
The relative affinity measurement result figure of the anti-meth monoclonal antibody of Fig. 3.
Fig. 4 meth saliva test strip assembly drawing.
Embodiment
Be further analyzed below in conjunction with embodiment 1 couple of the present invention that (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; Immune mouse selects BALB/c male mice, and male mice immunizing power is strong, and ascites generation is many; Close lymphoglandula subcutaneous location is four limbs oxter, four limbs forearm or neck; The DMEM nutrient solution of 1 × HAT contains the FBS of volume fraction 15 ﹪; The DMEM nutrient solution of 1 × HT contains the FBS that volume fraction is 15 ﹪).
The present invention's anti-meth hybridoma cell strain called after hybridoma cell strain MET1.0, hand over China typical culture collection center (CCTCC) preservation on November 12nd, 2013, preservation address: in the Wuhan University of Wuhan City, Hubei Province, deposit number: CCTCCNO:C2013160.
The deposit number that the present invention relates to is that the anti-meth hybridoma cell strain MET1.0 of CCTCCNO:C2013160 can secrete a kind of anti-meth monoclonal antibody.
Prepare anti-meth hybridoma cell strain MET1.0 that the deposit number that the present invention relates to is CCTCCNO:C2013160 and secrete the method for anti-meth monoclonal antibody, it is as follows that embodiment 1 comprises step:
1. the preparation of meth artificial antigen:
In 50mL single port bottle, 500mg methyl amphetamine A ', with liquid ammonia alkalinization to PH=9, extract by ethyl acetate and obtain 390mg pale yellow oil B ', 400ul trifluoroacetic anhydride is added under ice bath, be warming up to 80 DEG C of backflows after stirring 2h to spend the night, next day extracts by ethyl acetate after evaporated under reduced pressure, concentrate and obtain 600mg yellow oil C ', the Pyroglutaric acid of quality such as to add again, extraction agent 200mg, be warming up to 120 DEG C of reaction 48h, extract by ethyl acetate again and obtain 750mgD ', dissolve with under 38mLDMF, add 380mgNHS and 675mgDCC, stirring is spent the night, centrifuging and taking supernatant activation solution, obtain (concentration is 20mg/ml) stirring in the BSA of 200ml to spend the night, with the Na of PH=12
2cO
3dialyse after three days and dialyse three days with the PBS of PH=7.4, the supernatant liquor after centrifugal is artificial antigen.Purify meth artificial antigen with high performance liquid chromatograph, antigen purity can reach more than 95%.Wherein, guarantee the adding of trifluoroacetic anhydride that Pyroglutaric acid to be accurately combined between meth on site, position, avoid Pyroglutaric acid and its amino group to react, both improve the useful output of meth artificial antigen, also enhanced the immunogenicity of meth.
Fig. 1 is the UV scanning figure that the preparation of meth artificial antigen is fitted together to, as can be seen from the figure the ultraviolet wavelength at bovine serum albumin maximum absorption band place is 278nm, the ultraviolet wavelength at meth haptens maximum absorption band place is 295nm, the ultraviolet wavelength at meth artificial antigen maximum absorption band place is 281nm, meth artificial antigen has obviously different at the wavelength of maximum absorption band from meth artificial antigen and bovine serum albumin, thus can think and successfully synthesize meth artificial antigen.
2. mouse immune:
The BALB/c male mouse meth artificial antigen injection in 8-12 ages in week is selected to carry out immunity near lymphoglandula subcutaneous location, this experiment concrete steps are the male mouse of BALB/c of getting 8 week age, meth artificial antigen (taking MET-BSA as immunizing antigen) injection is near lymphoglandula subcutaneous location, inject 4 points, totally four immunity, each immunization interval two weeks: meth artificial antigen 20ug is diluted to 150ul by first time PBS damping fluid, obtains dilution antigen and isopyknic immunologic adjuvant (main component is DNA of bacteria); Meth 10ug/ artificial antigen immunity amount is diluted to 150ul by second time PBS damping fluid, obtains dilution antigen and isopyknic immunologic adjuvant; Booster immunization, is diluted to 150ul with PBS damping fluid by meth 20ug/ artificial antigen immunity, combines isopyknic immunologic adjuvant immune mouse, put to death mouse after raising one week for the third time; Put to death and only do a booster immunization with meth artificial antigen 20ug/ in first 3 days.Whole immunologic process shares meth artificial antigen amount 70ug, and antigen amount ratio common immunological adjuvant associating antigen uses, and wherein antigen amount saves half.
3. the fusion of splenocyte and myeloma cell:
3.1 feeder cell preparations: mouse is plucked eyeball and puts to death, and is soaked in 75 ﹪ alcohol and sterilizes, tear mouse abdomen outer skin, expose its peritonaeum, inject the DMEM serum free medium of 5ml37 DEG C of preheating with the asepsis injector of 5ml, gently rub mouse peritoneal 1 minute, suspension abdominal cavity cell, sucking-off peritoneal fluid; Cut off mouse thoracic cavity and get thymus gland, collect suspension with after serum free medium cleaning with the grinding of syringe handle, merge the centrifugal 3min of 1500r/min with peritoneal fluid, throw out is resuspended to 80ml with the DMEM nutrient solution of 1 × HAT, obtains feeder cell suspension.
The cultivation of 3.2 murine myeloma cell SP2/0: the murine myeloma cell SP2/0 DMEM substratum containing volume fraction being 10 ﹪ FBS is carried out Secondary Culture, cytogamy carries out going down to posterity to ensure that murine myeloma cell SP2/0 growth conditions is well in logarithmic phase the day before yesterday, during cytogamy by myeloma cell SP2/0 with the centrifugal 3min of 1500r/min, serum-free medium repeated centrifugation is used once after abandoning supernatant liquor, stand-by.
3.3 splenocyte preparations: the male mouse of BALB/c of step of learning from else's experience (2) immunity is put to death, and cuts open the belly in the alcohol of bubble 75% after sterilization, aseptic taking-up spleen.Clean with serum free medium, again spleen is placed on the stainless steel filtering net being connected to 50ml centrifuge tube and removes unnecessary tissue with operation surgical forceps, add the DMEM nutrient solution of 5ml serum-free, shred spleen with eye scissors, then gently grind spleen with aseptic syringe handle, make spleen cell filter in centrifuge tube through mesh, add serum-free medium to 30ml, the centrifugal 3min of 1500r/min, abandons the spleen tissue serum-free medium repeated centrifugation of cleer and peaceful bulk once, stand-by.
3.4 splenocytes and myeloma cell fusion: adopt polyoxyethylene glycol fusion method.After splenocyte and murine myeloma cell SP2/0 being counted, according to splenocyte: SP2/0=4:1 mixes, the centrifugal 6min washed cell of 1000r/min, removes supernatant liquor, flicks at the bottom of pipe, make two kinds of cells be mixed into pasty state, be placed in 37 DEG C of water-baths with finger.The PEG4000 slowly adding 0.7mL37 DEG C of pre-temperature rotates centrifuge tube simultaneously gently, adds, then static 30 seconds in one minute.Stop PEG effect with the DMEM serum-free medium that first slow rear fast method adds 35ml37 DEG C of pre-temperature again, within the 1st minute, add 1ml, within the 2nd minute, add 3ml, 5ml, 7ml successively; The centrifugal 3min of 1500r/min, gets precipitation; Then in precipitation, add the feeder cell suspension of step 3-1, be inoculated in 96 porocyte culture plates, be placed in 37 DEG C, 5 ﹪ CO
2cultivate in incubator.
4. the fusion screening of hybridoma:
Fused cell is at 5 ﹪ CO
2cultivate in incubator after 3 days, partly liquid is changed with the DMEM nutrient solution of 1 × HAT, HAT nutrient solution is stopped using after 7 days, use HT nutrient solution instead, observe the fused cell growing state in 96 porocyte culture plates, treat 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 (about 10-11 days), adopt indirect ELISA method screening positive clone (Positive control wells and negative control hole are set simultaneously), with fused cell culture supernatant OD450 value/negative control OD450 value >3 for positive colony, further ELISA method is suppressed to carry out multiple sieve with indirect competition the positive colony screened, select strong positive, strong specificity, subclone is carried out in the hole that Growth of Cells is good.
5. the cloning of hybridoma cell strain MET:
Colonized culture 5 times of dilution methods of hybridoma cell strain MET, with the 1 × HT substratum dilution containing volume fraction being 15 ﹪ FBS, first by the whole sucking-off of cell in 96 holes after multiple sieve, add 6ml substratum, then the cell suspension inoculation after diluting with every hole 200ul is in 96 porocyte culture plates, 5ml1 × HT substratum is mended after adding three rows, three rows are added with every hole 200ul after mixing, continue to mend 5ml1 × HT substratum, three rows are added with every hole 200ul after mixing, finally mend 5ml1 × HT substratum again, add three rows.4, after 5 days, observation of cell growing state is (at 16 times of object lens and 10 times of order Microscopic observations, cell size takes 1/3 visual field) time, detect the antibody horizontal of cell culture supernatant in Tissue Culture Plate, select 3 mono-clonals that antibody titer is the highest, again do limiting dilution assay colonized culture, 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, until antibody test positive rate in mono-clonal hole reaches 100 ﹪, select by ELISA method the hybridoma cell strain the highest, avidity is the strongest of wherein tiring, called after MET1.0, it is monoclonal antibody-purified to carry out anti-meth after enlarged culturing.
6. anti-meth monoclonal antibody preparation and purifying
6.1 anti-meth monoclonal antibody preparation
Adopt in animal body and induce method-ascites preparation method.Using containing volume fraction by the monoclonal cell strain MET1.0 screened is that the DMEM culture medium inoculated of 10 ﹪ FBS is in 24 porocyte culture plates, cultivating was inoculated in small size Tissue Culture Flask after 1-2 days, at 16 times of object lens and 10 times of order Microscopic observations, when cell size takes 1/2 visual field, be inoculated in large size Tissue Culture Flask, cultivate after 1-2 days, observation of cell grows in logarithmic phase, collected by centrifugation hybridoma.With the normal saline dilution of 0.9%, be expelled in the mouse peritoneal in injecting fluid paraffin 1-2 week, every only injection 1 × 10
6individual hybridoma.After about 7-10 days, the obvious swell of mouse web portion, now uses the ethanol disinfection belly of 75%, collects ascites with injection needles.
6.2 anti-meths are monoclonal antibody-purified
N-caprylic acid-saturated ammonium sulphate method of purification and G-albumen affinity column method of purification
1) get 10ml ascites, dilute 2 times with the HAc-NaAc damping fluid of 0.06mol/LpH4.8;
2) dropwise add required n-caprylic acid (volume computing by solution before dilution), and stir 30min;
3) under room temperature, 12000r/min, centrifugal 30min, gets supernatant;
4) add the PBS damping fluid that volume is the 0.1mol/LpH7.4 of supernatant 10%, and adjust pH to 7.4 with the NaOH of 1.0mol/L;
5) under stirring, drip the saturated ammonium sulphate solution of equal-volume pH7.4, leave standstill 2h;
6) the centrifugal 30min of low-temperature centrifugation 12000r/min, abandons supernatant;
7) 1.5gProteinG-SepharoseCL-4B dry powder 6-7ml tri-distilled water is dissolved, use 0.02M again, the phosphate buffered saline buffer (sample-loading buffer) of PH7.4 soaks 15min, then load in chromatography column, post is crossed with the sample-loading buffer of 10 times of column volumes, flow velocity is 1ml/min, and flowing liquid PH measured by test paper is 7.4;
8) carry out stream with sample-loading buffer to wash, 10 times of column volumes, flow velocity is 1ml/min, uses 0.02M subsequently, the citrate buffer solution antibody elution of PH4.0, applying AKTAexplorer to monitor simultaneously, starting to rise, when namely there is elution peak when observing baseline, get clean 4ml centrifuge tube to collect, after often collecting 3ml, use 1M immediately, the Tris-HCl damping fluid adjustment PH to 7.0 of PH9.0;
9), after collection elutriant to elution peak gets back to baseline, the gentle 5-10 of continuation sample-loading buffer times column volume, flow velocity is adjusted to 1ml/min.10 times of column volumes are balanced again with tri-distilled water;
10) dialysate filter.Test proteins concentration censorship. protein concentration (mg/ml)=OD
280value * extension rate/1.35=protein concentration.
Anti-meth monoclonal antibody obtained by embodiment 1 is namely for by deposit number being the anti-meth monoclonal antibody that the anti-meth hybridoma cell strain MET1.0 of CCTCCNO:C2013160 secretes generation.
The Performance Detection of the anti-meth monoclonal antibody of embodiment 2
1. the qualification of anti-meth monoclonal antibody Ig type
By Double diffusion in gel immunoprecipitation assay, hypotype qualification is carried out to anti-meth monoclonal antibody prepared by embodiment 1.
1) agarose plate is prepared: 1% sepharose is placed in boiling water bath and makes it melt.Horizontal stand is recorded agarose plate, and after to be solidified, with the punching of gel punch tool, there is the agarose plate of a hole, around 6 holes (in quincunx arrangement) at the center of making.
2) application of sample: join in centre hole by the ascites (diluting 40 times through PBS) of monoclonal antibody, surrounding hole adds the antiserum(antisera) of the inhomogeneity such as anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3a, IgA and subclass respectively.
3) incubation: after application of sample, agarose plate is moved in wet box, observations after left at room temperature 24h to 48h.
4) if see, precipitation line occurs, can go out the type of monoclonal antibody according to the sero-fast type identification in corresponding hole.
Result display after testing: anti-meth monoclonal antibody belongs to IgG1 hypotype, and light chain is kappa.
2. anti-meth monoclonal antibody reactive determination of activity
Indirect ELISA method detects anti-meth monoclonal antibody prepared by embodiment 1: 1. dilute meth artificial antigen (MET-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 meth artificial antigen after 100ul dilution respectively, 4 DEG C of bags are spent the night; Then wash plate with the PBS damping fluid containing 0.1 ﹪ tween-20 to pat dry for 3 times; 2. add the 0.01mol/LpH7.4 phosphate buffered saline buffer of 200 μ l containing 1% bovine serum albumin in every hole, put 37 DEG C 60 minutes, then wash plate with the PBS damping fluid of 0.1 ﹪ tween-20 and pat dry for 3 times; 3. add the anti-meth 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, are washed plate with the PBS damping fluid containing 0.1 ﹪ tween-20 and are patted dry for 1 time; 4. PBS damping fluid is joined sheep anti-mouse igg-HRP and be diluted to 2000 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 are reacted 30 minutes, wash plate pat dry for 3 times with the PBS damping fluid containing 0.1 ﹪ tween-20; 5. every hole added 100ul substrate TMB37 DEG C reaction after 10 minutes, and concentration is the H of 2M
2sO
4termination reaction, 450nm measures its OD value, with anti-methamphetamine monoclonal antibody solution OD450 value/negative control OD450 value >3 for the positive is worth.As seen from Figure 2, the anti-meth monoclonal antibody reactive activity that prepared by embodiment 1 can arrive 0.001ug/ml.
3. anti-meth monoclonal antibody avidity measures
As shown in Figure 3, be CCTCCNO:C2013160 by deposit number anti-meth hybridoma cell strain MET1.0 of the present invention is secreted other companies B on the anti-meth monoclonal antibody (representing with A) and market produced, the anti-meth monoclonal antibody that C produces carries out the qualitative comparison of avidity, according to ELISA method, concrete operations can with reference to once large " a kind of qualitative enzyme-linked immunosorbent assay for measuring comparing antibody relative affinity " waiting people to deliver, antigen coated, close, washing, color operation is identical with general ELISA method, gradient dilution antibody is pressed by PBS solution, dilute 8 concentration.One incomplete antibody reacts as antigen Salmonella method, measures D
492mm/ D
630mm, half is reacted as antibody indirect ELISA method, measures D
492mm/ D
630mm, test result is shown in Fig. 3.Fig. 3 take absorbancy as X-axis, reflection be antibody concentration, what the absorbancy of Y-axis then reflected is antibody antigen response intensity, and the colour developing value that under same concentrations, the ELISA of antibody and equivalent amount of antigen reacts is higher, then affinity of antibody degree is higher.
As can be seen from Figure 3 the like product avidity that the anti-meth monoclonal antibody that the anti-meth hybridoma cell strain MET1.0 being CCTCCNO:C2013160 by deposit number secretes generation is produced than other companies on market is high.
4. anti-meth monoclonal antibody specificity, cross reactivity measure
By detecting following 44 kinds of common interfering substances that may affect this reagent detected result, when drug level is equal to or less than 100 μ g/ml, display negative findings, therefore there is not cross reaction:
| Dihydroetorphine | Papaverine | Noscapine | Fentanyl | Propoxyphene | U-26225A |
| Methadone | Naloxone | TREXUPONT | Buprenorphine | Lofexidine | Diazepam |
| Phenylethyl barbituric acid | Tetrahydro-cannabinolic acid | Pseudoephedrine | Lignocaine | Ranitidine HCL | Gatifloxacin |
| Cocaine | Caffeine | Scopolamine | Phenacetin | Amobarbital | Amphetamine |
| Yi'an oral liquor | Paracetamol | Acetylsalicylic acid | Ibuprofen BP/EP | Amitriptyline | Imipramine |
| Chloral Hydrate | Triazolam | Alprazolam | Chlorpromazine | Ofloxacin | Somatarax |
| Norxin | Ketamine | Pioneer IV | Berberine | Somedon | Lactose |
| PROCAINE HCL, PHARMA GRADE | Morphine |
The foundation of embodiment 3 meth colloidal gold strip method and assessment
One, the preparation of Radioactive colloidal gold
Get 1ml1% hydrochloro-auric acid (HAuCl
4) solution, join in 100ml water, be heated to boil, then add 1.5ml1% trisodium citrate, mixing boils 5 minutes, until color no longer changes.Now obtained colloid gold particle is 30nm.
Two, gold mark preparation
1, antibody purification
Get a certain amount of morphine monoclonal antibody (MET-Mab), after dialysis, centrifugal treating, be diluted to 1mg/mL with pH8.01MPB, 4 DEG C save backup.
2, antibody coupling
Get the 30nm Radioactive colloidal gold that 100ml prepares, regulate pH to 8.5 with 1MNaOH, then add above-mentioned antibody 1mL, magnetic stirring apparatus stirs 1 hour, then adds a certain amount of BSA to final concentration 1%, continue stirring 0.5 hour.
3, the preparation of gold mark working fluid
By centrifugal for the colloidal gold solution of good for above-mentioned coupling antibody (4000rpm, 15 minutes), discard precipitation, retain solution.Then carry out secondary centrifuging (12000rpm, 30 minutes), abandoning supernatant, retain precipitation.Precipitation is redissolved to 10mL with the golden labeling antibody diluent (PH8.00.02MTris+1%BSA) of the sucrose containing 20%, and proceed in brown bottle, 4 DEG C save backup.
4, the preparation of colloidal-gold strip
Get above-mentioned gold mark working fluid 10mL, be loaded to metal spraying machine, it is 1.5ul/cm that setup parameter chooses discharge rate.Open air pump, after stable gas pressure, start metal spraying machine, gold is marked working fluid and be evenly sprayed onto on the gold mark pad of 30cm*6.5cm.The gold mark pad of spray good gold mark working fluid was put into 37 DEG C of thermostat container inner dryings after 12 hours take out, being cut into the wide bar of 8mm puts into valve bag, then encloses the aluminium foil bag of in-built siccative, and 20 DEG C save backup.
Three, the selection of nitrocellulose filter
Choose the nitrocellulose filter of three higher companies of rate of utilization on market: PALLVivid170, Millipore135, SartoriusCN140, specification is 2.5*30cm, tape backing, and aperture is that 8um contrasts, require the width of film each point and thickness homogeneous; Performance of metalling run out meets the requirements: the time of chromatography 4cm at 130s-140s, and without inclination and blank; Performance test meets the requirements: wrap three kinds of nitrocellulose filters by the antigen of 0.5mg/mL respectively and match corresponding colloidal-gold strip, test a negative sample, a critical negative sample (-50%cutoff, require within 3-5 minute, develop the color) and a critical positive sample (cutoff requires not develop the color within 5 minutes)
(note :-represent negative findings ,+represent positive findings, +/-represents not obvious negative findings) interpretation of result, we select Millipore135 as the production nitrocellulose filter of morphine saliva detection kit.
Four, the bag quilt of nitrocellulose filter
1, antigen and many anti-purifying
By a certain amount of meth antigen conjugates (MET-BSA), after dialysis, centrifugal treating, be diluted to final concentration 0.5mg/mL with pH7.40.01MPBS, 4 DEG C save backup.
By a certain amount of sheep anti mouse polyclonal antibody (GAM), after dialysis, centrifugal treating, be diluted to final concentration 2.0mg/mL with pH7.40.01MPBS, 4 DEG C save backup.
2, antigen coated
By above-mentioned antigen and how be anti-ly loaded in T, C post of a film machine respectively, setup parameter selects discharge rate to be 1.0ul/cm.The position of film head is drawn in adjustment, makes T, C line in the middle part of NC, and spacing 5mm.Start-up point film machine, by T, C line solution bag by nitrocellulose filter.The gold mark pad of spray good gold mark working fluid was put into 37 DEG C of thermostat container inner dryings after 24 hours take out, put into valve bag, then enclose the aluminium foil bag of in-built siccative, 20 DEG C save backup.
Five, the process of glass
Glass fibre element film is cut into 17*300mm bar, and put into glass treatment solution and soak taking-up after 1 hour, put into 37 DEG C of thermostat container inner dryings and take out after 12 hours, put into valve bag, then enclose the aluminium foil bag of in-built siccative, 20 DEG C save backup.
Glass prescription for the treatment of liquid
| Material | Content |
| Tris | 0.02M |
| Triton X-100 | 0.5-1％ |
| Tween-80 | 0.5-1％ |
| BSA | 1-5％ |
| PVP-10 | 0.1-1％ |
| Trehalose | 0.1-0.5％ |
| LowCross-Buffer | 0.1-0.5％ |
| NaCl | 1-3％ |
| Ultrapure water | 90％ |
Six, the assembling of reagent strip
As shown in Figure 4, upholder 1 is comprised for detecting the test kit of meth in saliva, sample pad 5, the Radioactive colloidal gold scraps of paper 4, nitrocellulose filter 2 and absorbent pad, nitrocellulose filter 2 is above-mentioned ready nitrocellulose filter, upholder 1 is PVC board, upholder 1 is pasted with sample pad 5 successively from application of sample end, the Radioactive colloidal gold scraps of paper 1, nitrocellulose filter 2 and absorbent pad 3, nitrocellulose filter 2 there are detection zone and check plot, the hybridoma cell strain MET1.0 that it is CCTCCC2013160 that detection zone is coated with by deposit number secretes the anti-meth monoclonal antibody produced, check plot bag is by sheep anti mouse polyclonal antibody.
Seven, sensitivity determination
With PBS damping fluid, meth standard substance are mixed with 300ng/ml, 150ng/ml, 100ng/ml, 50ng/ml, 25ng/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 monomethyl Amphetamine ELISA test strip different concns standard substance judges
| Standard concentration | 300ng/ml | 150ng/ml | 100ng/ml | 50ng/ml | 25ng/ml | 0ng/ml |
| Result is strong and weak | - | - | - | +/- | ++ | +++ |
| Result judges | Positive | Positive | Positive | Positive | Negative | Negative |
Claims (6)
1. can produce an anti-meth hybridoma cell strain MET1.0 for anti-meth monoclonal antibody, be preserved in China typical culture collection center, deposit number is CCTCCNO:C2013160.
2. anti-meth monoclonal antibody, is characterized in that, it is that the anti-meth hybridoma cell strain MET1.0 being CCTCCNO:C2013160 by deposit number secretes generation, can specific binding meth.
3. the anti-meth hybridoma cell strain MET1.0 that preparation is CCTCCNO:C2013160 by deposit number as claimed in claim 2 secretes the method for the anti-meth monoclonal antibody produced, and it is characterized in that in the process preparing meth artificial antigen, utilize trifluoroacetic anhydride to protect amino group.
4. the anti-meth hybridoma cell strain MET1.0 that preparation as claimed in claim 3 is CCTCCNO:C2013160 by deposit number secretes the method for the anti-meth monoclonal antibody produced, and it is characterized in that adopting immunologic adjuvant to combine mice immunized with antigen in immunologic process.
5. for detecting the test kit of meth in saliva, it is characterized in that it comprises upholder, sample pad, the Radioactive colloidal gold scraps of paper, nitrocellulose filter and absorbent pad, upholder is pasted with sample pad, the Radioactive colloidal gold scraps of paper, nitrocellulose filter and absorbent pad successively from application of sample end, nitrocellulose filter there are detection zone and check plot, the anti-meth hybridoma cell strain MET1.0 that it is CCTCCNO:C2013160 that detection zone is coated with by deposit number secretes the anti-meth monoclonal antibody produced, and check plot bag is by sheep anti mouse polyclonal antibody.
6. be the application of anti-meth monoclonal antibody in detection meth of the anti-meth hybridoma cell strain MET1.0 secretion generation being CCTCCNO:C2013160 by deposit number by deposit number.
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| CN114409796A (en) * | 2022-01-27 | 2022-04-29 | 无锡迪腾敏生物科技有限公司 | Hybridoma cell strain of anti-fenfluramine monoclonal antibody and application thereof |
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