CN107238713A - The detection method of trace hemp and the SPR chips used in a kind of saliva sample - Google Patents
The detection method of trace hemp and the SPR chips used in a kind of saliva sample Download PDFInfo
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- CN107238713A CN107238713A CN201710487526.0A CN201710487526A CN107238713A CN 107238713 A CN107238713 A CN 107238713A CN 201710487526 A CN201710487526 A CN 201710487526A CN 107238713 A CN107238713 A CN 107238713A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/948—Sedatives, e.g. cannabinoids, barbiturates
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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Abstract
The present invention relates to a kind of detection method of trace hemp in saliva sample and its SPR chips used, the detection method is to detect the THC contents in sample to be tested on SPR instrument using SPR chips, SPR chips are anti-THC antibody SPR chips, and anti-THC antibody SPR chips are made by the way that anti-THC Fab section is coated in after 3D epoxy SPR chips.SPR chips prepared by the present invention are the specific antibodies that surface has been coupled anti-THC, can specifically bind the THC molecules in complex samples, thus can be applied to the detection of trace hemp in saliva sample.Simultaneously, when the anti-THC antibody SPR chips prepared using the present invention detect the THC in the personnel's saliva sample that smokes cannabis on SPR instrument, quick, accurate, sensitive effect is can reach, its detection sensitivity can at least reach 0.3 3ng/mL, meet the demand of scene of a crime quick detection.
Description
Technical field
The present invention relates to a kind of detection method of marijuana hemp, more particularly in a kind of saliva sample trace hemp detection
Method and its SPR chips used, belong to detection technique field.
The meaning of following expression formula is in the present invention：
THC：THC
KLH：Hemocyanin
OVA：Chicken ovalbumin
Background technology
Hemp, its main effectively chemical analysis is THC, spiritedness and physiological active function, quilt after sucking or be oral
It is considered one of big drugs in the world three, social harm is very serious.
At present, the method for inspection for hemp mainly includes conventional chemistry, capillary electrophoresis, thin-layered chromatography, height
Effect liquid phase chromatogram method, gas chromatography and Gas Chromatography-mass Spectrometry, immunodetection and bioassay method etc..Wherein inspection in vivo
Material analysis is the focus of forensic science research instantly, and urine is to test and analyze the internal sample that drug abuse is most widely used.
But, existing urine examination technology can not meet the demand of scene of a crime quick detection, in this context, seek one kind and can reach soon
Fast, accurate, sensitive, economic scene of a crime detection method becomes scientific worker's research emphasis.
Surface plasma resonance technology (SPR) is a kind of new technology grown up from 1990s, and it applies SPR
Interaction situation on principle detection bio-sensing chip between ligand and analyte, is widely used in every field,
Through a kind of important research tool as life science and pharmaceutical field.Compared with traditional detection method, with high sensitivity,
Respond fast, small volume, mechanical strength is big, detection process is fast, detects in real time, monitor to dynamic the complete of bio-molecular interaction
Process and real time data, sample need not be marked, molecular activity, quantitative determination not balance of disturbing reaction etc. to compound is kept
Advantage, becomes more preferably selecting for detection hemp.At present, on the side using surface plasma resonance technology quick detection hemp
There is not been reported for method.Chinese invention patent (publication number：CN102057277A the detection that a kind of hemp uses) is disclosed, the patent
Use the Time resolution FRET (TR- of the noncompetitive homology immunoassays based on binding partners
FRET) analyze；Being immunized between THC (THC) and anti-thc-antibody can be combined by being prepared using gene engineering method
The antibody fragment of compound.Binding partners must carry out fluorescence labeling, to realize the TR-FRET fluoroscopic examinations of fluorescence photometer, inspection
Survey sensitive.In addition, Chinese invention patent (publication number：CN101580544A a kind of colloid gold label hemp, tetrahydrochysene) are disclosed big
Numb phenol monoclonal antibody plate for detecting immunity, that patent describes THC-KLH preparation；Using THC-KLH as antigen, using Freund's adjuvant method
(Freund ' s adjuvant) immune balb/c mice；Hybridoma is prepared with monoclonal antibody technique；It is coated with THC-KLH
Elisa plate (ELISA methods) filters out anti-THC hybridoma.
The content of the invention
The purpose of the present invention is to be directed to the above-mentioned problems in the prior art, it is proposed that one kind can using saliva sample
Reach the detection method of fast and accurately trace hemp.
The purpose of the present invention can be realized by following technical proposal：The detection side of trace hemp in a kind of saliva sample
Method, the detection method is to detect the THC contents in sample to be tested on SPR instrument using SPR chips, and the SPR chips are anti-
THC antibody SPR chips, the anti-THC antibody SPR chips are made by the following method：
S1, THC is coupled to KLH and OVA respectively, obtains THC-KLH and THC-OVA；
S2, with above-mentioned THC-KLH and tachysynthesis adjuvant immunity BALB/C mice, detect that immune mouse resists with THC-OVA
THC antiserum titre；
S3, take antiserum ELISA potency in more than 6K mouse, take the bone-marrow-derived lymphocyte and murine myeloma cell of spleen
SP2/0 is merged, and the anti-THC antibody hybridoma cells strain for obtaining antiserum ELISA potency in more than 10K is screened through monoclonal；
S4, above-mentioned hybridoma cell strain is seeded to BALB/C mice abdominal cavity, antibody purification is prepared from ascites；
S5, with this melon protease above-mentioned antibody purification is hydrolyzed into Fc sections and Fab section, Fab section is coated in 3D epoxies SPR
Chip, obtains anti-THC antibody SPR chips.
In a kind of above-mentioned saliva sample in the detection method of trace hemp, the tachysynthesis assistant described in step S2
Agent is QuickAntibody-Mouse5W.
In a kind of above-mentioned saliva sample in the detection method of trace hemp, the THC-OVA detections described in step S2
Carried out after THC-KLH and tachysynthesis adjuvant immunity BALB/C mice 5 weeks, progress after further preferred 5-8 week, to obtain
Obtain more efficient subcloned cells strain.
In a kind of above-mentioned saliva sample in the detection method of trace hemp, the anti-THC antibody described in step S3 is miscellaneous
The sensitivity for handing over tumor cell strain is 0.3-3ng.The anti-THC antibody hybridoma cells strain that sensitivity is 0.3-3ng is by with lower section
Method screening is obtained：THC-OVA point samples are coupled to SPRi carboxyl chips, THC-OVA amount be respectively 30ng, 3ng, 0.3ng,
0.03ng；In addition, same OVA point samples, are used as negative control.With between surface plasma resonance SPRi high flux biomolecule mutually
Effect instrument analysis antiserum ELISA potency is combined instead in the antibody of more than 10K strain of hybridoma strain with the sample on chip
Situation is answered, 0.3ng-3ng antibody hybridoma cell strain can be reached by filtering out detection sensitivity.This sensitivity makes the antibody can
Saliva sample applied at least 1 week interior drugs personnel that smoke cannabis of detection.
In a kind of above-mentioned saliva sample in the detection method of trace hemp, being prepared from ascites described in step S4
The detailed process of antibody purification is：Antibody is obtained from ascites, then antibody purification is obtained with Protein A post adsorption and purifications.
Step S5 of the present invention, directly will with this melon protease before step S4 is eluted with Protein A posts adsorption and purification
Antibody on Protein A- antibody columns is hydrolyzed into Fc sections and Fab section.Because Protein A are the Fc sections specificity with antibody
, thus after digestion, Fab section, which can dissociate, to be purified, and is collected, desalination and concentration by ultrafiltration this anti-THC Fab, is freezed and is preserved.
The present invention anti-THC Fab section is coated in 3D epoxy SPR chips, while using normal mouse IgG Fab fragments as
Control, the Fab fragments for obtaining the Fab section for including anti-THC on anti-THC antibody SPR chips, chip and normal mouse IgG are (negative right
According to).Because the molecule of Fab section is the 1/3 of complete antibody molecule size, adding does not have Y steric hindrances, can greatly improve Fab
Section coated density on chip, at least effective density of 3-5 times than complete antibody raising, can for this micromolecular as drugs
Greatly improve its detection sensitivity.
The present invention utilizes the Fab antibody SPR chips of the anti-THC, is detected on SPR instrument in the personnel's saliva sample that smokes cannabis
THC, sampling, detection etc. operation more rapidly, conveniently, testing result can be learnt at 1-2 minutes or so, detection sensitivity at least may be used
0.3-3ng/mL is reached, with quick, accurate, sensitive effect.Compared with current conventional urine examination method, of the invention is excellent
Gesture is：
(1) it is convenient to sample, do not limited by place, reduce tested personnel be unworthy of possibility that is right and avoiding its cheating,
Make it possible driving with poison visiting.
(2) can interpretation be negative and positive findings (urine examination at least 3-8 minutes) in 1-2 minutes.
(3) detect that accurate, sensitivity is high, detection threshold value can be applied to saliva quickly real-time at least up to 0.3-3ng/mL
Detection.
Compared with prior art, the present invention is same using THC-KLH as antigen, but using new better than traditional Freund's adjuvant
Tachysynthesis adjuvant (QuickAntibody-Mouse5W) method immune balb/c mice；It is thin in screening monoclonal antibody hybridoma
Surface plasma resonance SPRi high throughput analysis roguings are used during born of the same parents' strain：THC-OVA point samples are coupled to SPRi carboxyl chips,
Same OVA point samples, are used as negative control.Analyzed with surface plasma resonance SPRi high flux biological dispersers instrument
Sample association reaction situation of the antiserum ELISA potency on the antibody and chip of more than 10K strain of hybridoma strain, screening
0.3ng-3ng/mL antibody hybridoma cell strain can be reached by going out detection sensitivity.With following advantage：1) using THC-KLH as
Antigen immune, the screening that ensure that anti-THC specific hybrids tumor cell strain is screened with THC-OVA；2) SPRi screenings not only can be real
It is existing high flux, quick, accurate, and can more importantly obtain association rate constant Ka in real time, it is dissociation rate constant Kd, flat
The thermokinetic parameters such as the dissociation constant that weighs KD, and sensitivity, can quickly obtain high-quality monoclonal antibody hybridoma cell
Strain.
And on Cleaning Principle, the present invention uses different SPR principles detections, the method is quick, sensitive, nothing can be achieved
Mark detection, is not disturbed by fluorescent impurity in complex samples, can both realize non-competitive immunoassay, can also be realized non-competing
Property immunoassays.The Fab fragments that the antibody of use can directly be obtained with the monoclonal antibody of high-affinity with digestion.
Another object of the present invention is to provide the SPR cores used in above-mentioned saliva sample in the detection method of trace hemp
Piece, described SPR chips are anti-THC antibody SPR chips, and the anti-THC antibody SPR chips are made by the following method：
S1, THC is coupled to KLH and OVA respectively, obtains THC-KLH and THC-OVA；
S2, with above-mentioned THC-KLH and tachysynthesis adjuvant immunity BALB/C mice, detect that immune mouse resists with THC-OVA
THC antiserum titre；
S3, take antiserum ELISA potency in more than 6K mouse, take the bone-marrow-derived lymphocyte and murine myeloma cell of spleen
SP2/0 is merged, and the anti-THC antibody hybridoma cells strain for obtaining antiserum ELISA potency in more than 10K is screened through monoclonal；
S4, above-mentioned hybridoma cell strain is seeded to BALB/C mice abdominal cavity, antibody purification is prepared from ascites；
S5, with this melon protease above-mentioned antibody purification is hydrolyzed into Fc sections and Fab section, Fab section is coated in 3D epoxies SPR
Chip, obtains anti-THC antibody SPR chips.
In the SPR chips used in a kind of above-mentioned saliva sample in the detection method of trace hemp, institute in step S2
The tachysynthesis adjuvant stated is QuickAntibody-Mouse5W.
In the SPR chips used in a kind of above-mentioned saliva sample in the detection method of trace hemp, institute in step S2
The THC-OVA detections stated are carried out after THC-KLH and tachysynthesis adjuvant immunity BALB/C mice 5 weeks, further preferred 5-8 weeks
After carry out.
In the SPR chips used in a kind of above-mentioned saliva sample in the detection method of trace hemp, institute in step S3
The sensitivity for the anti-THC antibody hybridoma cells strain stated is 0.3-3ng.
In the SPR chips used in a kind of above-mentioned saliva sample in the detection method of trace hemp, institute in step S4
That states prepares the detailed process of antibody purification from ascites is：Antibody is obtained from ascites, it is then pure with the absorption of ProteinG posts
Change obtains antibody purification.
Compared with prior art, the SPR sensorgram chip that prepared by the present invention is the specific antibody that surface has been coupled anti-THC,
The THC molecules in complex samples can be specifically bound, thus can be applied to the detection of trace hemp in saliva sample.Meanwhile, profit
The anti-THC antibody SPR chips prepared with the present invention detect the crystal methamphetamine in the personnel's saliva sample that smokes cannabis on SPR instrument
When, quick, accurate, sensitive effect is can reach, its detection sensitivity can at least reach 0.3-3ng/mL, meet scene of a crime
The demand of quick detection.
Brief description of the drawings
Fig. 1 is the SPR result figures for filtering out the antibody hybridoma cell strain that detection sensitivity can reach 0.3ng.
Embodiment
The following is the present invention specific embodiment, and be described with reference to the drawings to technical scheme make further retouch
State, but the present invention is not limited to these embodiments.
Embodiment 1：
THC is coupled to protein carrier KLH and OVA respectively by phenolic hydroxyl group, THC-KLH and THC-OVA is obtained.
After THC-KLH and tachysynthesis adjuvant QuickAntibody-Mouse5W immune balb/c mices 15,5 weeks
The antiserum titre of the anti-crystal methamphetamine of immune mouse is detected with THC-OVA；High specific and high-affinity antibody can be produced by taking
The mouse 5 of (antiserum ELISA potency is in more than 6K).
Take the bone-marrow-derived lymphocyte of spleen to be merged with murine myeloma cell SP2/0 respectively, obtain high special through monoclonal screening
Property and high-affinity the strain of anti-crystal methamphetamine antibody hybridoma cell, ELISA potency is in 100 plants of more than 10K.Then by THC-
OVA point samples are coupled to SPRi carboxyl chips, and THC-OVA amount is respectively 30ng, 3ng, 0.3ng, 0.03ng；In addition, same
OVA point samples, are used as negative control.With surface plasma resonance SPRi high flux biological dispersers instrument analysis primary dcreening operation institute
Sample association reaction situation on the antibody and chip of the 100 strain of hybridoma strain obtained, filters out detection spirit as shown in Figure 1
Sensitivity can reach 0.3ng antibody hybridoma cell strain.
The antibody hybridoma cell strain that above-mentioned sensitivity can reach 0.3ng is seeded to BALB/C mice abdominal cavity, from ascites
In it is a large amount of obtain antibody, and with Protein G post adsorption and purifications.Before elution, directly with this melon protease by Protein G-
Antibody on antibody column is hydrolyzed into Fc sections and Fab section.Because Protein G are, thus digestions specific with the Fc sections of antibody
Afterwards, Fab section can dissociate and be purified, and collect, desalination and concentration by ultrafiltration this anti-THC Fab section, and be coated in 3D epoxy SPR cores
Piece, with the bright Fab fragments using normal mouse IgG as control, obtains anti-THC antibody SPR chips.
Application Example 1-5：
The anti-THC antibody SPR chips that embodiment 1 is prepared are used to detecting 4 on SPR instrument smokes cannabis 1 respectively
My god, 3 days, 5 days and the personnel after 7 days, the THC in 1 normal personnel's saliva sample.
Comparison study example 1-5：
Above-mentioned Application Example 1-5 is carried out to conventional urine examination simultaneously.
Application Example 1-5 and Comparison study example 1-5 testing result are contrasted, as shown in table 1.
Table 1：The present invention is compared with conventional urine examination
As known from Table 1, with conventional urine examination ratio, the present invention more rapidly, it is accurate, sensitive.
In embodiment and its alternative, the sensitivity of the anti-MA antibody hybridoma cells strain filtered out can also be
0.4ng、0.5ng、0.6ng、0.7ng、0.8ng、0.9ng、 1ng、1.1ng、1.2ng、1.3ng、1.4ng、1.5ng、1.6ng、
1.7ng、1.8ng、 1.9ng、2ng、2.1ng、2.2ng、2.3ng、2.4ng、2.5ng、2.6ng、2.7ng、 2.8ng、
2.9ng、3ng。
In view of the present invention program embodiment is numerous, each embodiment experimental data is huge numerous, is not suitable for arranging one by one herein
Explanation is lifted, but the content of checking required for each embodiment and obtained final conclusion are approached.So herein not to each reality
The checking content for applying example is illustrated one by one, only excellent using embodiment 1 and Application Example 1-5 as explanation the present patent application is represented
Different part.
Specific embodiment described herein is only to spirit explanation for example of the invention.Technology neck belonging to of the invention
The technical staff in domain can be made various modifications or supplement to described specific embodiment or be substituted using similar mode, but simultaneously
Do not deviate by the spirit of the present invention or surmount scope defined in appended claims.
It is skilled to this area although having been made a detailed description to the present invention and being cited some specific embodiments
For technical staff, as long as it is obvious that can make various changes or correct without departing from the spirit and scope of the present invention.
Claims (10)
1. a kind of detection method of trace hemp in saliva sample, the detection method is to be detected using SPR chips on SPR instrument
THC contents in sample to be tested, it is characterised in that the SPR chips are anti-THC antibody SPR chips, the anti-THC antibody SPR
Chip is made by the following method：
S1, THC is coupled to KLH and OVA respectively, obtains THC-KLH and THC-OVA；
S2, with above-mentioned THC-KLH and tachysynthesis adjuvant immunity BALB/C mice, detect the anti-THC's of immune mouse with THC-OVA
Antiserum titre；
S3, antiserum ELISA potency is taken in more than 6K mouse, take the bone-marrow-derived lymphocyte and murine myeloma cell SP2/0 of spleen
Fusion, the anti-THC antibody hybridoma cells strain for obtaining antiserum ELISA potency in more than 10K is screened through monoclonal；
S4, above-mentioned hybridoma cell strain is seeded to BALB/C mice abdominal cavity, antibody purification is prepared from ascites；
S5, with this melon protease above-mentioned antibody purification is hydrolyzed into Fc sections and Fab section, Fab section is coated in 3D epoxy SPR cores
Piece, obtains anti-THC antibody SPR chips.
2. the detection method of trace hemp in a kind of saliva sample according to claim 1, it is characterised in that in step S2
Described tachysynthesis adjuvant is QuickAntibody-Mouse5W.
3. the detection method of trace hemp in a kind of saliva sample according to claim 1, it is characterised in that in step S2
Described THC-OVA detections are carried out after 5 weeks in THC-KLH and tachysynthesis adjuvant immunity BALB/C mice.
4. the detection method of trace hemp in a kind of saliva sample according to claim 1, it is characterised in that in step S3
The sensitivity of described anti-THC antibody hybridoma cells strain is 0.3-3ng.
5. the detection method of trace hemp in a kind of saliva sample according to claim 1, it is characterised in that in step S4
The described detailed process that antibody purification is prepared from ascites is：Antibody is obtained from ascites, is then adsorbed with Protein G posts
Purifying obtains antibody purification.
6. the SPR chips used in a kind of saliva sample as described in claim 1-5 is any in the detection method of trace hemp,
Characterized in that, described SPR chips are anti-THC antibody SPR chips, the anti-THC antibody SPR chips are made by the following method
：
S1, THC is coupled to KLH and OVA respectively, obtains THC-KLH and THC-OVA；
S2, with above-mentioned THC-KLH and tachysynthesis adjuvant immunity BALB/C mice, detect the anti-THC's of immune mouse with THC-OVA
Antiserum titre；
S3, antiserum ELISA potency is taken in more than 6K mouse, take the bone-marrow-derived lymphocyte and murine myeloma cell SP2/0 of spleen
Fusion, the anti-THC antibody hybridoma cells strain for obtaining antiserum ELISA potency in more than 10K is screened through monoclonal；
S4, above-mentioned hybridoma cell strain is seeded to BALB/C mice abdominal cavity, antibody purification is prepared from ascites；
S5, with this melon protease above-mentioned antibody purification is hydrolyzed into Fc sections and Fab section, Fab section is coated in 3D epoxy SPR cores
Piece, obtains anti-THC antibody SPR chips.
7. the SPR chips used in a kind of saliva sample according to claim 6 in the detection method of trace hemp, it is special
Levy and be, the tachysynthesis adjuvant described in step S2 is QuickAntibody-Mouse5W.
8. the SPR chips used in a kind of saliva sample according to claim 6 in the detection method of trace hemp, it is special
Levy and be, THC-OVA described in step S2 detection is laggard in THC-KLH and tachysynthesis adjuvant immunity BALB/C mice 5 weeks
OK.
9. the SPR chips used in a kind of saliva sample according to claim 6 in the detection method of trace hemp, it is special
Levy and be, the sensitivity of the anti-THC antibody hybridoma cells strain described in step S3 is 0.3-3ng.
10. the SPR chips used in a kind of saliva sample according to claim 6 in the detection method of trace hemp, its
It is characterised by, the detailed process that antibody purification is prepared from ascites described in step S4 is：Antibody is obtained from ascites, then
Antibody purification is obtained with Protein G post adsorption and purifications.
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| US20100184082A1 (en) * | 2006-06-29 | 2010-07-22 | Daniel Wang | Antibody and immunoassays for determining the presence of delta9-tetrahydrocannabinol |
| CN101580544B (en) * | 2008-02-21 | 2012-10-10 | 曾立波 | Colloidal gold labelled monoclonal antibody immunity detection Plate for detecting cannabis and tetrahydrocannabinol |
| CN101556248A (en) * | 2009-05-18 | 2009-10-14 | 中国科学院长春应用化学研究所 | Method for detecting time resolution of surface plasmon resonance spectroscopy |
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Application publication date: 20171010 |