CN109856393A - A kind of detection method and its detection kit of the opioid activity substance based on cell dopamine D_2 receptors effect - Google Patents

A kind of detection method and its detection kit of the opioid activity substance based on cell dopamine D_2 receptors effect Download PDF

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CN109856393A
CN109856393A CN201910306028.0A CN201910306028A CN109856393A CN 109856393 A CN109856393 A CN 109856393A CN 201910306028 A CN201910306028 A CN 201910306028A CN 109856393 A CN109856393 A CN 109856393A
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cell
detection
mor1
opioid activity
sample
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范春雷
程向荣
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Zhejiang Nuojia Biological Technology Co Ltd
Zhejiang Neogene Biotechnology Co Ltd
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Zhejiang Nuojia Biological Technology Co Ltd
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Abstract

The invention discloses the detection methods and its detection kit of a kind of opioid activity substance based on cell dopamine D_2 receptors effect, the detection kit includes detection two kinds of ingredients of group reagent box and control group kit, the detection group reagent box includes the monoclonal cell system MOR1/SK-N-SH and ELISA detection kit for stablizing expression source of people μ type opiate receptor MOR1 gene, and the control group kit includes blank control cell line Neo/SK-N-SH and ELISA detection kit.Accurate, the highly sensitive quick detection of all opioid activity substances can be achieved in the present invention, either natural opiate class product or synthesis opiates molecule, including the novel synthesis opioid activity material to emerge one after another, such as fentanyl and its various derivatives, it can solve the problems, such as that novel synthesis opiates psychoactive drug substance supervision is difficult.

Description

A kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect And its detection kit
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of opioid activity based on cell dopamine D_2 receptors effect The detection method and its detection kit of substance.
Background technique
Novel opiates spirit substance is sucked for drug addict, if the detection and supervision of fentanyl are supervision departments, various countries Problem.Because current detection means depends on the immunization of antibody (such as anti-fentanyl antibody), including chromatography, ELISA method etc.；And the large-scale instruments such as gas chromatography mass spectrometry, LC-MS analysis.But fentanyl is very fast in human body metabolism, causes to examine Target detection component content is extremely low in test sample sheet, can not detect.On the other hand, the fentanyl of novel synthesis emerges one after another, and Molecular structure multiplicity, is difficult to realize the almost impossible immunodetection based on antibody or even gas chromatography mass spectrometry, LC-MS also Supervision detection.
Summary of the invention
For above-mentioned technical problem of the existing technology, the purpose of the present invention is to provide one kind to be based on cell dopamine The detection method and its detection kit of the opioid activity substance of release effects.
Present inventive concept are as follows: either natural opiods or various synthesis opiates spirit substances, such as Fentanyl and its derivatives can all be applied to this target spot of opiate receptor in body, to generate its biological effect.Ah Piece receptor includes at least μ type, four kinds of K-type, δ type and σ type opiate receptors, and μ type opiate receptor is the opiates such as morphine, fentanyl Spiritual substance affinity and the strongest receptor of effect.Opiate receptor belongs to GPCR class, acts on when by opiates molecular agonist Signal path activates afterwards, i.e., makes the Ca stored in endoplasmic reticulum by IP3-DAG signal2+The cytoplasm being promptly released into, in cytoplasm Free calcium ion concentration increases, so that cell response is generated, as nerve cell discharges dopamine.Since the measurement of dopamine has into Ripe ELISA detection kit, thus we surely turn the Human Neuroblastoma Cell Line MOR1/SK-N-SH of MOR1 by establishing, Establish a kind of opioid activity substance detecting method based on cell dopamine D_2 receptors effect, be applied to quickly, efficiently, precisely, Opioid activity substance in the high-throughput various samples of detection.
A kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect, it is characterised in that The following steps are included:
1) opioid receptor gene is cloned under CMV promoter, constructs eukaryon expression plasmid；
2) by step 1) eukaryotic expression plasmids to neuroblastoma cell, and it is female thin to establish the nerve that opiate receptor surely turns Born of the same parents' oncocyte system carries out culture amplification and obtains detection group cell culture fluid；The nerve of a transfection control empty carrier is established simultaneously Blastoma cell line carries out culture amplification and obtains cellular control unit culture solution；
3) standard items are added in step 2 detection group cell culture fluid and sufficiently react, standard items are morphine or fentanyl, are prepared A series of titer of various criterion product concentration；After titer is centrifugated, takes supernatant and examined with ELISA detection kit The absorbance OD value under DOPA amine effect is surveyed, using the absorbance OD value measured as ordinate, with the standard concentration in titer Standard curve, digital simulation regression equation are drawn for abscissa；
4) sample to be tested is added in step 2 detection group cell culture fluid, sufficiently reacts, supernatant is taken after centrifuge separation, to reaction The supernatant of detection group cell culture fluid afterwards detects the absorbance OD under DOPA amine effect with ELISA detection kit1Value；Step Sample to be tested is added in rapid 2) cellular control unit culture solution, sufficiently reacts, supernatant is taken after centrifuge separation, to the control after reaction The supernatant of group cell culture fluid detects the absorbance OD under DOPA amine effect with ELISA detection kit2Value；Calculate absorbance OD1Value and absorbance OD2The difference of value, and bring step 3) regression equation into, the opioid activity object in sample to be tested can be extrapolated The effect content of matter.
A kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect, it is characterised in that In step 1), the opioid receptor gene is source of people μ type opiate receptor MOR1 gene；The eukaryon expression plasmid is pCMV- MOR1 plasmid, building process are as follows: by source of people μ type opiate receptor MOR1 gene cloning to Lentiviral pCDH-CMV- Under the CMV promoter of MCS-EF1-Neo, connection restriction enzyme site is EcoR I and Not I, constructs eukaryon expression plasmid pCMV- MOR1。
A kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect, it is characterised in that In step 2, neuroblastoma cell is SK-N-SH cell；Establish the Human Neuroblastoma Cell Line that opiate receptor surely turns Process are as follows: by pCMV-MOR1 plasmid, pH1 plasmid, pH2 plasmid co-transfection to slow virus incasing cells 293V, CMV- is prepared MOR1 slow virus；CMV-MOR1 slow-virus infection SK-N-SH cell is obtained into MOR1/SK-N-SH and surely turns cell, with containing neomycin The conditioned medium screening MOR1/SK-N-SH of G418 surely turns cell, and is obtained by the method for picked clones and stablize expression source of people The monoclonal cell system MOR1/SK-N-SH of μ type opiate receptor MOR1 gene.
A kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect, it is characterised in that In step 2, the process of the Human Neuroblastoma Cell Line of a transfection control empty carrier is established are as follows: by pCDH-CMV-MCS- Empty carrier slow virus is prepared to slow virus incasing cells 293V in EF1-Neo empty carrier, pH1 plasmid, pH2 plasmid co-transfection； Empty carrier slow-virus infection SK-N-SH cell is obtained into Neo/SK-N-SH and surely turns cell, with the CMC model of the G418 containing neomycin Base screening Neo/SK-N-SH surely turns cell, and obtains blank control cell line Neo/SK-N-SH by the method for picked clones.
A kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect, it is characterised in that In step 3), it is added and standard items and sufficiently reacts into detection group cell culture fluid, preparation standard concentration is respectively 0.01, 0.05,5 kinds of titers of 0.25,1.25,6.25ng/ml, the standard items are morphine.
A kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect, it is characterised in that In step 3), be added and standard items and sufficiently react into detection group cell culture fluid, preparation standard concentration is respectively 1,3,9, 27,5 kinds of titers of 81pg/ml, the standard items are fentanyl.
A kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect, it is characterised in that In step 4), the sample to be tested is urine, blood, hair, dandruff, sweat or the saliva of drug addict, or is natural Hemp, natural hemp products, synthesis hemp, synthesis hemp products, sewage, soil, any one in pool.
The detection kit of a kind of opioid activity substance, it is characterised in that the detection kit includes detection Two kinds of ingredients of group reagent box and control group kit, the detection group reagent box include stablizing expression source of people μ type opiate receptor The monoclonal cell system MOR1/SK-N-SH and ELISA detection kit of MOR1 gene, the control group kit includes blank Control cell lines Neo/SK-N-SH and ELISA detection kit.
Compared with the existing technology, the beneficial effect that the present invention obtains is:
(1) limitation that immunodetection depends on antibody is avoided, general inspection directly can be carried out to all opioid activity substances It surveys, without nonspecific binding problem, without expensive manpower and material resources and complicated macrocyclic antibody R&D process；It also avoids simultaneously The sample pre-treatments work of gas chromatography mass spectrometry, Liquid Chromatography/Mass Spectrometry dependent on the limitation of reference substance, limited detection limit and complexity Make.Technical solution of the present invention can realize accurate, the highly sensitive quick detection of all opioid activity substances, either naturally Opiates product or synthesis opiates molecule, including the novel synthesis opioid activity material to emerge one after another, such as fentanyl and its Various derivatives can solve the problems, such as that novel synthesis opiates psychoactive drug substance supervision is difficult.
(2) using existing dopamine ELISA detection kit quantitative detecting analysis.
(3) it can be measured with general fluorescence microplate reader, can realize and detect in most common labs.
(4) by being then based on the detection of the biological effect of receptor signaling pathways, as a result not only can correctly judge whether be Opioid activity substance, and its activity being quantitatively evaluated, for supervision and medical intervention provide reliable experiment according to According to.
(5) present invention provide it is a kind of surely turn cell line, can be by the IP3-DAG signal path of MOR1 to including naturally All opioid activity substances including opiates product and synthesis opiates molecule generate response, and cell discharges dopamine, from And it can be applied to the highly sensitive general detection of opioid activity substance.This surely turns in Human Neuroblastoma Cell Line comprising one Cance high-expression gene MOR1 is started by CMV, when opioid activity material effect is when MOR1, IP3-DAG signal path can be started, Nerve cell is set to discharge rapidly dopamine；Thus using existing dopamine detection kit quantitative detecting analysis.Another party Face, the present invention provides a kind of simple, quick, efficient, sensitive (pieck stage), accurate, high-throughput opioid activity substance are logical With detection kit, IP3-DAG signal path of the detection kit based on MOR1 receptor is discharged rapidly by detection cell Dopamine detects to realize quick, sensitive, accurate quantitative detecting analysis.
Detailed description of the invention
Fig. 1 is pCMV-MOR1 plasmid map；
Fig. 2 is using morphine standard concentration as abscissa, and the absorbance OD value of detection is the standard curve that ordinate is drawn；
Fig. 3 is the testing result using opioid activity substance universal testing kit of the invention to morphine sample of hair；
Fig. 4 is Inhibition ELISA to the testing result of the morphine of 0-12.8ng/ml concentration, curve show morphine detectable limit > 0.2ng/ml；
Fig. 5 is morphine examination criteria curve of the Inhibition ELISA to 0.2-6.4ng/ml concentration；
Fig. 6 is testing result of the Inhibition ELISA to sample of hair；
Fig. 7 is using fentanyl standard concentration as abscissa, and the absorbance OD value of standard item group is that ordinate draws standard curve Mark；
Fig. 8 is the testing result using opioid activity substance universal testing kit of the invention to fragrant too class sample；
Fig. 9 is testing result of the Inhibition ELISA to the fentanyl standard items of 0-12.8ng/ml concentration, and curve shows fentanyl Detectable limit > 0.4ng/ml；
Figure 10 is fentanyl examination criteria curve of the Inhibition ELISA to 0.4-12.8ng/ml concentration；
Figure 11 is the testing result of Inhibition ELISA remifentanil sample.
Specific embodiment
The present invention is further explained in the light of specific embodiments, but the scope of protection of the present invention is not limited thereto.
In following embodiment, slow virus packaging is cell 293V purchased from Inovogen Tech Co., Ltd..
1 pCMV-MOR1 plasmid construction of embodiment
By connecting restriction enzyme siteEcoRI andNotSource of people μ type opiate receptor MOR1 gene cloning to slow virus is expressed and is carried by I Under the CMV promoter of body pCDH-CMV-MCS-EF1-Neo, construct eukaryon expression plasmid pCMV-MOR1 (as shown in Figure 1).
Embodiment 2 establishes that MOR1/SK-N-SH surely turns cell line and blank control Neo/SK-N-SH surely turns cell line
It is cell 293V by pCMV-MOR1 plasmid, pH1 plasmid, pH2 plasmid co-transfection to slow virus packaging, preparation MOR1 is sick slowly Poison, and SK-N-SH cell is transfected, G418 screening, clone establish MOR1/SK-N-SH and surely turn cell line.By pCDH-CMV-MCS- EF1-Neo plasmid, pH1 plasmid, pH2 plasmid co-transfection to slow virus packaging are cell 293V, prepare empty carrier slow virus, and turn SK-N-SH cell is contaminated, G418 screening, clone establish blank control Neo/SK-N-SH and surely turn cell line.Specific step is as follows:
1) packaging is that cell prepares: the day before transfection will use the DMEM-H complete culture solution (mould containing 10%FBS and 100U/ml Element, 100 μ g/ml streptomysins it is dual anti-) by slow virus packaging be that cell 293V is made into 1 × 106A/ml concentration is inoculated in one The Tissue Culture Dish of D19cm, 37 DEG C, 5% CO2Overnight incubation.
2) it transfects: when the degrees of fusion of cell length in culture dish to 70%-80%, with PEI transfection reagent (referring to its standard Transfection procedure regulation) by the pH2 plasmid co-transfection culture of the pCMV-MOR1 plasmid of 20 μ g, the pH1 plasmid of 14 μ g and 4.7 μ g Cell 293V in ware.
3) collect virus: step 2 culture collected supernatant after 72 hours；In 4 DEG C of temperature and 8000g relative centrifugal force Under the conditions of be centrifugated 15 minutes, go to precipitate, viral supernatant liquid taken to cross 0.45 μm of filter membrane, the viral supernatant liquid filtered.
4) viral concentration: the viral supernatant liquid (about 30ml) for taking step 3) to filter, in 4 DEG C of temperature and 90000g it is opposite from It is centrifugated 90 minutes under conditions of mental and physical efforts, removes supernatant, precipitating is CMV-MOR1 slow virus, is trained completely with the DMEM-H of 1ml Nutrient solution is resuspended, the CMV-MOR1 slow virus after being concentrated.
5) SK-N-SH cell prepares: in advance by 1 × 105The 12 hole cell culture of SK-N-SH cell inoculation of a/ml concentration 1 hole of plate, 37 DEG C, 5% CO2It cultivates to 50% cell fusion degree.
6) SK-N-SH cell transfecting: the culture solution of the culture SK-N-SH cell in 12 orifice plates of step 5) is sopped up, is added The CMV-MOR1 slow virus of 1ml after entering step 4) concentration；37℃,5% CO2Culture 22-24 hours, it is complete to renew fresh DMEM-H Culture solution.
7) MOR1/SK-N-SH surely turns cell screening: above-mentioned steps 6) transfection after SK-N-SH cell culture one week or more Afterwards, it is made into 1 × 104A/ml concentration is inoculated in 6 porocyte culture plates, the hole 2ml/；Culture solution is sucked after cell is adherent within second day, The conditioned medium (the DMEM-H complete culture solution of the G418 containing 600 μ g/ml) of 2ml is added；1 fresh condition is changed every three days Culture solution (the DMEM-H complete culture solution of the G418 containing 600 μ g/ml), changes the G418's containing 200 μ g/ml after 21-30 days DMEM-H complete culture solution.
8) monoclonal MOR1/SK-N-SH surely turns Establishment of Cell Line: by above-mentioned steps 7) MOR1/SK-N-SH surely turns cell It is made into 1 × 103A/ml concentration is inoculated in 6 porocyte culture plates, the hole 2ml/；Under the microscope with the shifting of 50 μ l after culture 10 days Liquid rifle picking, which is individually cloned, moves to 6 new porocyte culture plates cultures, 1 clone/hole；After amplification with PCR detection MOR1 gene, WB detects MOR1 albumen, to obtain the MOR1/SK-N-SH cell clone system of high, medium and low different expressions, is used for different work Property effect degree opioid activity substance detection.
Blank control Neo/SK-N-SH surely turns Establishment of Cell Line: according to above-mentioned 1) -7) the step of, establish blank control Neo/ SK-N-SH surely turns cell line, the difference is that: the pCDH-CMV- of the pCMV-MOR1 plasmid equal quality in step 2 The replacement of MCS-EF1-Neo plasmid, finally obtains blank control Neo/SK-N-SH and surely turns cell line.
The application of 3 opioid activity substance universal testing kit of embodiment
Opioid activity substance universal testing kit based on technical solution of the present invention research and development can be applied to containing opioid activity The various pattern detections of substance.We are for sucking the hair of morphine personnel in the present embodiment.Specific step is as follows:
1) sample of hair handle: take 5 portions of morphines suck personnel sample of hair and 5 parts of nothings suck history normal person hair sample This, number such as table 1.
20mg/ parts of sample of hair within clip root of hair 3cm is put into the EP pipe of 5ml, adds 2ml containing 1% keratin after shredding A small amount of zirconium pearl and quartz sand is added in the HBSS buffer (pH7.4) of enzyme, and oscillation crushes 1 minute on crusher, respectively obtains corresponding Sample liquid.
2) cell prepares: mentioning the previous day for 5 × 105The monoclonal MOR1/SK-N-SH cell and blank pair of a/ml concentration Respectively it is inoculated in 96 porocyte culture plates, 200 holes μ l/ respectively according to Neo/SK-N-SH cell；37℃,5% CO2It is small to cultivate 24 When.
3) sample-adding is handled: being divided into standard item group, sample group, processing method is as follows.
Standard item group: 6 hole of MOR1/SK-N-SH cell, each hole adds final concentration of 0 respectively, 0.01,0.05,0.25,1.25, Morphine (MOP) standard items of 6.25ng/ml, 100 holes μ l/；Blank control Neo/SK-N-SH cell is equally handled.
Sample group: MOR1/SK-N-SH cell totally 42 holes (3 repeating holes of every sample) are separately added into sample obtained by step 1) Liquid, 100 holes μ l/；Blank control Neo/SK-N-SH cell is equally handled.
4) it detects: to the standard item group and sample group of step 3) processing, carrying out dopamine concentration test, standard item group respectively It is identical with the detection method step of sample group.The detection process of standard item group are as follows: step 3) treated standard item group reacts 5- After ten minutes, it is centrifugated 5 minutes under conditions of 800g relative centrifugal force, Aspirate supernatant is added to dopamine ELISA detection The orifice plate of kit carries out quantitative determination analysis.
5) result: using morphine standard concentration as abscissa, the absorbance OD value of dopamine detection is that ordinate draws mark Directrix curve, y=0.4551x+0.46, R2=0.9814, as shown in Figure 2.The absorbance OD value of sample group dopamine detection Result such as Fig. 3, can obviously distinguish positive sample and negative sample；It is calculated according to calibration curve equation is drawn, negative sample is equal It can't detect morphine ingredient, morphine content is respectively (ng/mg): M1: 0.0429, M2: 0.0845, M3 in positive sample: 0.0404、M4 : 0.0544、M5 : 0.0096。
Comparative example 1: Inhibition ELISA detection morphine sucks the sample of hair of personnel
Opiates detection at present depends on immunization detection, including colloidal gold immunity chromatography and Inhibition ELISA.Colloid Golden method is suitble to fast inspection, but ELISA method 1 order of magnitude higher than colloidal gold method in sensitivity.This comparative example is examined with ELISA method It surveys for the sample of hair that morphine sucks personnel.Specific step is as follows:
1) sample of hair is handled: with processing sample same in embodiment 3, respectively obtaining corresponding sample liquid, (sample of hair is compiled It is number identical as table 1).
2) MOP-BSA antigen protein envelope antigen: is configured to 1 μ g/ with 0.05mol/L carbonate buffer solution (pH9.6) ML concentration, and be coated with 96 orifice plates, 100 holes μ l/, 4 DEG C overnight after, remove non-spy as far as possible board-washing 5 times with PBS buffer solution (pH7.4) The opposite sex combines.
3) experimental group: standard curve group, sample group, processing method are as follows:
Standard curve group: by MOP standard items with PBS buffer solution (pH7.4) be made into final concentration be respectively 0,0.10,0.20,0.40, 0.80,1.60,3.20,6.40,9 groups of 12.80ng/ml, each group contains 5 μ g/ml, and (selection of concentration is determined by the potency of antibody Each 1 hole of coated 96 orifice plate of MOP-BSA, 100 holes μ l/ are added in MOP-Mab monoclonal antibody calmly) after mixing.
Sample group: MOP-Mab monoclonal antibody is added in the sample liquid that hair extracts, the final concentration of 5 μ g/ml of MOP-Mab monoclonal antibody is mixed Each 1 hole of coated 96 orifice plate of MOP-BSA, 100 holes μ l/, 3 multiple holes of each sample are added after even.
4) competitive binding and measurement: 37 DEG C of above-mentioned each group are incubated for 30 minutes, solution are removed, with PBS buffer solution (pH7.4) Board-washing 5 times；HRP is added to mark sheep anti-Mouse secondary antibody, 37 DEG C are incubated for 30 minutes, solution are removed, with PBS buffer solution (pH7.4) board-washing 5 It is secondary；Colour developing uses microplate reader to measure absorbance (OD) value under 450nm wavelength after terminating reaction.
5) result: as shown in Figure 4, the results showed that detectable limit > 0.2ng/ml of the Inhibition ELISA to MOP；MOP is dense Degree be 0.2-6.4ng/ml under absorbance (OD) value standard curve such as Fig. 5, curvilinear equation be y=- 0.2458x+ 1.6281 R2= 0.9444.The result of sample group is as shown in fig. 6, detect 4,1 is not detected in 5 positive samples.According to drawing Calibration curve equation processed calculates, and negative sample can't detect morphine ingredient, and morphine content is distinguished in 4 positive samples of detection For (ng/mg): M1:0.049, M2:0.078, M3:0.063, M4:0.059.
From embodiment 3 and comparative example 1 as can be seen that the Inhibition ELISA detection morphine sample of hair of sucking personnel is sensitive Degree, accuracy can not show a candle to system of the present invention.Moreover, ELISA method depend on specific antibody, can only detect it is single at Point, it is then helpless for all kinds of synthesis opiums to emerge one after another, and technology of the invention has well solved this problem.
The application of 4 opioid activity substance universal testing kit of embodiment-fentanyl class detection
Opioid activity substance universal testing kit based on technical solution of the present invention research and development can be applied to containing opioid activity The various pattern detections of substance.We are by taking the sample of class containing fentanyl as an example in the present embodiment.Specific step is as follows:
1) rabbit hair sample process: taking the galley proof sheet and 3 parts of normal rabbit galley proof sheets of 3 parts of rabbit for being injected intravenously fentanyl, number Such as table 2.
20mg/ parts of rabbit hair sample are taken, the EP pipe of 5ml is put into after shredding, 2ml is added to contain the HBSS buffer of 1% keratinase (pH7.4), a small amount of zirconium pearl and quartz sand is added, oscillation crushes 1 minute on crusher.Each sample sterile PBS buffer (pH7.4) 10 times of use are diluted, corresponding sample liquid is respectively obtained.
2) municipal sewage: taking municipal sewage, is divided into 3 parts, wherein 2 parts add fentanyl and Carfentanil to make final concentration point respectively It Wei not 3pg/ml and 0.3pg/ml, the respectively F4 and F5 of the sample-adding of table 2 municipal sewage；Another 1 part is the blank that any drug is not added The municipal sewage (NF4) of control.Each sample takes supernatant mistake through being centrifugated 15 minutes under conditions of 1200g relative centrifugal force Sterile PBS buffer (pH7.4) dialysed overnight is used after 0.22 μm of filter membrane degerming, respectively obtains corresponding sample liquid.
3) cell prepares: mentioning the previous day for 5 × 105The monoclonal MOR1/SK-N-SH cell inoculation of a/ml concentration is in one A 96 porocyte culture plates, 200 holes μ l/；37℃,5% CO2Culture 24 hours.
4) sample-adding is handled: being divided into standard item group, sample group, processing method is as follows.
Standard item group: totally 5 hole, each hole standard items containing fentanyl, fentanyl (fentanyl) are whole for MOR1/SK-N-SH cell Concentration is respectively 1,3,9,27,81pg/ml, 100 holes μ l/.
Sample group: totally 30 holes (3 repeating holes of every sample), 100 holes μ l/ are separately added into step 1) to MOR1/SK-N-SH cell Or sample liquid obtained by step 2, it separately sets blank control and PBS buffer solution (pH7.4) is added, same 3 repeating holes, 100 holes μ l/；It is empty White control Neo/SK-N-SH cell is equally handled.
5) detect: step 4) each group sample-adding acts on 5-10 minutes, and 5 points are centrifugated under conditions of 800g relative centrifugal force Clock, the orifice plate that Aspirate supernatant is added to dopamine ELISA detection kit carry out quantitative determination analysis.
6) result: using fentanyl standard concentration as abscissa, the OD value of dopamine detection is that ordinate draws standard song Line, the regression equation of standard curve are y=0.036x+0.7015, R2=0.9871, as shown in Figure 7.The detection of sample group As a result such as Fig. 8, positive sample and negative sample can obviously be distinguished；It is calculated according to calibration curve equation is drawn, negative sample is examined Fentanyl ingredient is not detected, fentanyl content is respectively (pg/mg): F1:19.87, F2:32.09, F3 in positive rabbit hair sample: 44.00.It being loaded in municipal sewage sample, the mean OD value (n=3) of the sewage sample of the fentanyl containing 3pg/ml is 0.8112 ± 0.2820, it is 4.09 times of negative sample；The mean OD value (n=3) of the sewage sample of the Carfentanil containing 0.3pg/ml is 1.7043 ± 0.4354, it is 8.91 times of negative sample.
Comparative example 2: Inhibition ELISA detects the ingredient of substance containing fentanyl sample
Opioid detection at present depends on immunization detection, including colloidal gold immunity chromatography and Inhibition ELISA. Colloidal gold method is suitble to fast inspection, but ELISA method 1 order of magnitude higher than colloidal gold method in sensitivity.This comparative example is i.e. with ELISA Method detects for the ingredient of substance containing fentanyl sample.Specific step is as follows:
1) sample process: with processing sample same in example 4.But do not have to sterile PBS (pH7.4) after rabbit hair sample system to dilute, Stoste is directly used, and corresponding sample liquid is respectively obtained (rabbit hair sample number is identical as table 2).
2) envelope antigen: Fentanly-BSA antigen protein is prepared with 0.05mol/L carbonate buffer solution (pH9.6) At 1 μ g/mL concentration, and be coated with 96 orifice plates, 100 holes μ l/, 4 DEG C overnight after, removed as far as possible for board-washing 5 times with PBS buffer solution (pH7.4) Go non-specific binding.
3) experimental group: standard curve group, sample group, processing method are as follows:
Standard curve group: by fentanyl standard items with PBS buffer solution (pH7.4) be made into final concentration be respectively 0,0.1,0.2,0.4, 0.8,1.6,3.2,6.4,9 groups of 12.8ng/ml, each group contains the Fentanly-Mab monoclonal antibody of 5 μ g/ml, is added after mixing Each 1 hole of coated 96 orifice plate of Fentanly-BSA, 100 holes μ l/.
Sample group: Fentanly-Mab monoclonal antibody, Fentanly-Mab monoclonal antibody final concentration is added in the sample liquid that the rabbit hair extracts For 5 μ g/ml, each 1 hole of coated 96 orifice plate of Fentanly-BSA, 100 holes μ l/, 3 multiple holes of each sample are added after mixing.
4) competitive binding and measurement: 37 DEG C of above-mentioned each group are incubated for 30 minutes, solution are removed, with PBS buffer solution (pH7.4) Board-washing 5 times；HRP is added to mark sheep anti-Mouse secondary antibody, 37 DEG C are incubated for 30 minutes, solution are removed, with PBS buffer solution (pH7.4) board-washing 5 It is secondary；Colour developing uses microplate reader to measure absorbance (OD) value under 450nm wavelength after terminating reaction.
5) result: as shown in Figure 9, the results showed that the detectable limit of the Inhibition ELISA remifentanil (fentanyl) > 0.4ng/ml；Fentanyl concentration is standard curve such as Figure 10 of absorbance (OD) value under 0.4-12.8ng/ml, and curvilinear equation is Y=- 0.1465x+1.7861, R2 = 0.9031.The result of sample group is as shown in figure 11,5 positive samples and 5 feminine genders Fentanyl ingredient is not detected in sample standard deviation.
From embodiment 4 and comparative example 2 as can be seen that Inhibition ELISA detects the sensitivity of fentanyl sample, accuracy far not System as described herein.Moreover, ELISA method depends on specific antibody, single component can only be detected, for emerging one after another All kinds of synthesis fentanyls it is then helpless, and technology of the invention has well solved this problem, and with analyte Bioactivity degree is detection foundation, can really reflect the biological effect of analyte.In addition, method of the present invention is Direct method has the methods of sensibility, ease-to-operate, the range of linearity of dose-effect and quantitative accuracy to be all substantially better than competition ELISA method.
Content described in this specification is only to enumerate to inventive concept way of realization, and protection scope of the present invention is not answered When the concrete form for being seen as limited by embodiment and being stated.

Claims (8)

1. a kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect, it is characterised in that including following Step:
1) opioid receptor gene is cloned under CMV promoter, constructs eukaryon expression plasmid；
2) by step 1) eukaryotic expression plasmids to neuroblastoma cell, and it is female thin to establish the nerve that opiate receptor surely turns Born of the same parents' oncocyte system carries out culture amplification and obtains detection group cell culture fluid；The nerve of a transfection control empty carrier is established simultaneously Blastoma cell line carries out culture amplification and obtains cellular control unit culture solution；
3) standard items are added in step 2 detection group cell culture fluid and sufficiently react, standard items are morphine or fentanyl, are prepared A series of titer of various criterion product concentration；After titer is centrifugated, takes supernatant and examined with ELISA detection kit The absorbance OD value under DOPA amine effect is surveyed, using the absorbance OD value measured as ordinate, with the standard concentration in titer Standard curve, digital simulation regression equation are drawn for abscissa；
4) sample to be tested is added in step 2 detection group cell culture fluid, sufficiently reacts, supernatant is taken after centrifuge separation, to reaction The supernatant of detection group cell culture fluid afterwards detects the absorbance OD under DOPA amine effect with ELISA detection kit1Value；Step Sample to be tested is added in rapid 2) cellular control unit culture solution, sufficiently reacts, supernatant is taken after centrifuge separation, to the control after reaction The supernatant of group cell culture fluid detects the absorbance OD under DOPA amine effect with ELISA detection kit2Value；Calculate absorbance OD1Value and absorbance OD2The difference of value, and bring step 3) regression equation into, the opioid activity object in sample to be tested can be extrapolated The effect content of matter.
2. a kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect as described in claim 1, It is characterized in that the opioid receptor gene is source of people μ type opiate receptor MOR1 gene in step 1)；The eukaryon expression plasmid For pCMV-MOR1 plasmid, building process are as follows: by source of people μ type opiate receptor MOR1 gene cloning to Lentiviral Under the CMV promoter of pCDH-CMV-MCS-EF1-Neo, connection restriction enzyme site is EcoR I and Not I, constructs eukaryotic expression matter Grain pCMV-MOR1.
3. a kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect as described in claim 1, It is characterized in that neuroblastoma cell is SK-N-SH cell in step 2；Establish the neuroblast that opiate receptor surely turns The process of oncocyte system are as follows: by pCMV-MOR1 plasmid, pH1 plasmid, pH2 plasmid co-transfection to slow virus incasing cells 293V, system It is standby to obtain CMV-MOR1 slow virus；CMV-MOR1 slow-virus infection SK-N-SH cell is obtained into MOR1/SK-N-SH and surely turns cell, Surely turn cell with the conditioned medium screening MOR1/SK-N-SH of the G418 containing neomycin, and is obtained surely by the method for picked clones Surely the monoclonal cell system MOR1/SK-N-SH of source of people μ type opiate receptor MOR1 gene is expressed.
4. a kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect as described in claim 1, It is characterized in that establishing the process of the Human Neuroblastoma Cell Line of a transfection control empty carrier in step 2 are as follows: by pCDH- Zero load is prepared to slow virus incasing cells 293V in CMV-MCS-EF1-Neo empty carrier, pH1 plasmid, pH2 plasmid co-transfection Body slow virus；Empty carrier slow-virus infection SK-N-SH cell is obtained into Neo/SK-N-SH and surely turns cell, with G418 containing neomycin Conditioned medium screening Neo/SK-N-SH surely turn cell, and blank control cell line is obtained by the method for picked clones Neo/SK-N-SH。
5. a kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect as described in claim 1, It is characterized in that standard items are added into detection group cell culture fluid and sufficiently react, prepare standard concentration point in step 3) Not Wei 0.01,5 kinds of titers of 0.05,0.25,1.25,6.25ng/ml, the standard items are morphine.
6. a kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect as described in claim 1, It is characterized in that standard items are added into detection group cell culture fluid and sufficiently react, prepare standard concentration point in step 3) Not Wei 1,5 kinds of titers of 3,9,27,81pg/ml, the standard items are fentanyl.
7. a kind of detection method of the opioid activity substance based on cell dopamine D_2 receptors effect as described in claim 1, It is characterized in that the sample to be tested is urine, blood, hair, dandruff, sweat or the saliva of drug addict in step 4), It or is any one in natural hemp, natural hemp products, synthesis hemp, synthesis hemp products, sewage, soil, pool.
8. a kind of detection kit of opioid activity substance, it is characterised in that the detection kit includes detection group reagent box With two kinds of ingredients of control group kit, the detection group reagent box includes stablizing expression source of people μ type opiate receptor MOR1 gene Monoclonal cell system MOR1/SK-N-SH and ELISA detection kit, the control group kit include blank control cell line Neo/SK-N-SH and ELISA detection kit.
CN201910306028.0A 2019-04-16 2019-04-16 A kind of detection method and its detection kit of the opioid activity substance based on cell dopamine D_2 receptors effect Pending CN109856393A (en)

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