CN101580544A - Plate for detecting immunity of cannabis and tetrahydrocannabinol monoclonal antibody through collaurum tag - Google Patents
Plate for detecting immunity of cannabis and tetrahydrocannabinol monoclonal antibody through collaurum tag Download PDFInfo
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Abstract
The invention discloses a complete antigen for detecting tetrahydrocannabinol and preparing an antibody. The invention also discloses an anti-tetrahydrocannabinol monoclonal antibody prepared by the complete antigen and a tetrahydrocannabinol monoclonal antibody immunity detection plate through a collaurum tag for detecting the tetrahydrocannabinol in medicine, a urine specimen or other human specimens. Compared with HPLC and other methods, the detection plate is simple, portable and easy to carry, can carry out field detection and does not need expensive equipment. When the detection plate is used to detect the tetrahydrocannabinol, the whole test can be completed within 1.5 minutes; the detecting sensitivity can reach 0.5ng; and the detection plate has no crossed reaction with more than 60 kinds of common medicaments, drugs and internal metabolins of the tetrahydrocannabinol.
Description
Technical field
The present invention relates to molecular immunology and toxicology field.Particularly, the present invention relates to the detection of illegal drug, relate in particular to the enzyme linked immunosorbent detection of tetrahydrocannabinol.
Background technology
Current, the Drug-related crimes situation is very severe.Drug species is many, and only according to United Nations's statistics in 2004, whole world drug addict has surpassed 200,000,000 people, wherein at least l.4 smoke cannabis more than hundred million people, suck heroine more than 9,000,000 people, suck Cocaine more than 1,300 ten thousand people, suck other artificial synthetic drugs such as amphetamine and be difficult to counting especially.China's number of taking drugs continues to rise at present, and the drug addict that China registers on the books surpasses 1,000,000, and teenager drug addict sharply increases.Constantly enlarged by malicious scope, the counties and cities that system poison, traffic in drugs, drug abuse phenomenon occur account for more than 3/4ths of national counties and cities sum.
Hemp (Cannabis or Marijuana) is the narcotics of international and domestic strict control, and habituation is strong.Hemp is the general name of a kind of phenol derivatives of extracting from cannabis plants, it mainly contains tetrahydrocannabinol, cannabinol, cannabidiol, tetrahydro-cannabinolic acid etc., be main wherein with tetrahydrocannabinol (Tetrahydrocannabinol, Δ 9THC or THC) and 11-nor-Δ 8-tetrahydrocannabinol-9-carboxylic acid (11-nor-Δ 8-Tetrahydrocannabinol-9-carboxylic acid).Tetrahydrocannabinol (THC) is the small molecules haptens material that a molecular weight is 314Da, and itself does not have immunogenicity and only possesses immunoreactivity.
For a long time, what people were wrong always thinks that smoking cannabis is can not be easy to generate habituation as drugs such as heroine to rely on, and the idea of this mistake also is the one of the main reasons that causes cannabis abuse today.The person does not for a long time smoke cannabis, because accumulate poisoning causes the behavioral toxicity reaction, be drunk shape, excited, conscious glad, produce fantasy euphoria, illusion, illusion and the disturbance of thought take place simultaneously, the people is produced hostile idea, violence and introgression very easily take place, have great social harm.The hemp drugs have caused the great attention of the Chinese government now, must deliver powerful attack on.
The method of laboratory routine inspection drugs is instrumental analysis, but instrument testing speed is slow, and the analysis operation cycle is longer, buys instrument and costs an arm and a leg, need special training, instrument testing cost height (sample needs more than 400 yuans) is difficult to promote in vast grass-roots unit.
Though external (as the U.S. etc.) has developed hemp monoclonal antibody plate for detecting immunity, but wherein have following two problems: (1) hemp both at home and abroad identifies it mainly is according to whether containing tetrahydrocannabinol (THC) in the sample, yet the hemp monoclonal antibody of external research and development can only be discerned minimum metabolite 11-nor-Δ 8-tetrahydrocannabinol-9 carboxylic acid of content in the urine sample, can't detect tetrahydrocannabinol (THC); (2) hemp monoclonal antibody plate for detecting immunity sensitivity at present all more than 100.0ng, detect 11-nor-Δ 8-tetrahydrocannabinol-9 carboxylic acid accurately, and the sensitivity of hemp monoclonal antibody plate for detecting immunity must be below 10.0ng.Therefore, the urine sample of using the hemp monoclonal antibody plate for detecting immunity of external import to detect the drugs personnel that smoke cannabis all is a false negative generally, makes a large amount of hemp drug abuse cases to track down.
The reason that causes these problems mainly is to use BSA (bovine serum albumin) to prepare complete antigen (hemp-BSA) also prepare monoclonal antibody with its immune mouse abroad.Because the BSA molecular weight has only 40-60 ten thousand Da, source and the more approaching animal of people's kind, therefore the monoclonal antibody of preparation tire lower, poor specificity.
Along with the strictness day by day that the criminal case relevant with tetrahydrocannabinol rises year by year and country manages hemp, this area is to hemp, and especially the detection of hemp and tetrahydrocannabinol is had higher requirement in tetrahydrocannabinol and the drug abuse person biological sample.This area press for research and development a kind of easy fast, accurately and reliably, sensitive, low price is applicable to that the crime scene is to instant qualitative screening of a large amount of samples and the wield hemp drug ingredient check of laboratory quick test and grass-roots unit novel method.
Summary of the invention
Purpose of the present invention just providing a kind of quick, simple and easy, detect the detection method and the detection reagent of tetrahydrocannabinol delicately.
In a first aspect of the present invention, a kind of complete antigen is provided, described complete antigen has the structure shown in the formula 1:
Wherein, A is a hemocyanin.
In a second aspect of the present invention, a kind of method of preparation formula 1 described complete antigen is provided, described method comprises step:
(a) compound of hydroxyl is at first reacted with succinyl oxide, generate the intermediate of band carboxyl;
(b) make the intermediate of step (a) gained and N-hydroxy-succinamide react the ester bond of production;
(c) step (b) products therefrom is connected with hemocyanin, thereby makes the complete antigen of formula 1.
In a preference of the present invention, the condition of step (a) is as follows: temperature of reaction is 0-100 ℃, preferred room temperature-80 ℃, more preferably 30-60 ℃; Reaction times is 1-24 hour, preferred 2-12 hour, and more preferably 2-6 hour.
In another preference, the condition of step (b) is as follows: temperature of reaction is 0-60 ℃, preferred room temperature-60 ℃; Reaction times is 1-24 hour, preferred 2-12 hour, and more preferably 3 hours.
In another preference, the condition of step (c) is as follows: temperature of reaction is 0-40 ℃, preferred 4-30 ℃; Reaction pH is 3.0-8.0; Reaction times is 1-24 hour, preferred 6-12 hour, and more preferably 6 hours.
In a third aspect of the present invention, the purposes of aforesaid haptens of a kind of the present invention or complete antigen is provided, it is used to prepare the tetrahydrocannabinol monoclonal antibody specific.
In a preference, described method comprises step:
In a third aspect of the present invention, the purposes of complete antigen of the present invention is provided, it is used to prepare the tetrahydrocannabinol monoclonal antibody specific.
In a fourth aspect of the present invention, a kind of monoclonal antibody is provided, described monoclonal antibody specificity is incorporated into tetrahydrocannabinol.In a preference of the present invention, described monoclonal antibody is that THC-KLH-CH1 is produced by mouse hybridoma cell.
In another preference, described monoclonal antibody is tired greater than 1: 6000 with combining of tetrahydrocannabinol, more preferably greater than 1: 8000, most preferably greater than 1: 10000.In another preference, described monoclonal antibody only is incorporated into phenol derivatives 11-nor-Δ 8-tetrahydrocannabinol-9 carboxylic acid with hemp, 11-hydroxyl-Δ 9-tetrahydrocannabinol, Δ 8-tetrahydrocannabinol, Δ 9-tetrahydrocannabinol, cannabinol, immune association reaction takes place in cannabidiol.
In a fifth aspect of the present invention, a kind of hybridoma cell line that produces monoclonal antibody of the present invention is provided, described hybridoma cell line is that mouse hybridoma cell is that CCTCC NO.C200915 (is hemp monoclonal antibody cell strain THC10A, be deposited in Chinese typical culture collection center (CCTCC on February 20th, 2009, China, Wuhan, Wuhan University)).
In a sixth aspect of the present invention, a kind of purposes of monoclonal antibody of the present invention is provided, it is used for preparing reagent, check-out console or the test kit of test sample tetrahydrocannabinol.
In a preference of the present invention, described sample is a biological sample, and is preferably blood sample or urine sample.
In a seventh aspect of the present invention, the method that whether has tetrahydrocannabinol in a kind of detection of biological sample is provided, described method comprises step:
(a) sample is contacted with monoclonal antibody of the present invention;
(b) detect whether form antigen-antibody complex, wherein form mixture and just represent to exist in the sample tetrahydrocannabinol.
In a preference of the present invention, described monoclonal antibody has detectable.More preferably, described marker is selected from down group: colloid gold label thing, coloured marker or fluorescent marker.
In a preference of the present invention, described detection method is Radioactive colloidal gold detection method, colorimetric detection method or fluorescence detection.
In a eighth aspect of the present invention, a kind of check-out console is provided, described check-out console comprises substrate (back up pad) and test strip, described test strip contains aforesaid monoclonal antibody.
In another preference, described test strip also contains complete antigen point sample district, and the complete antigen shown in the immobilized formula 1 is contained in described complete antigen point sample district.
In another preference, described test strip is overlapped successively by filter sample paper, chromatographic material, nitrocellulose filter and thieving paper to be formed.
In another preference, described chromatographic material is coated with through colloid gold label in advance or the monoclonal antibody of the present invention of color marker is arranged;
Be adsorbed with detection line and nature controlling line on the described nitrocellulose filter, described detection line is the complete antigen shown in the formula 1;
Described nature controlling line is how anti-sheep anti-mouse igg is.
It is 0.01-20.0mg/ml that described chromatographic material is coated with concentration range in advance, preferred 0.1-10.0mg/ml, and 0.2-2.0mg/ml more preferably, and package amount is 5-150 μ l/cm
2, preferred 10-100 μ l/cm
2, more preferably 20-70 μ l/cm
2Through colloid gold label or the monoclonal antibody of the present invention of color marker is arranged.
It is 0.01-20.0mg/ml that described detection line has adopted concentration range, preferred 0.1-10.0mg/ml, and 0.2-2.0mg/ml more preferably, and adsorptive capacity is 0.01-100 μ l/cm
2, preferred 0.5-50 μ l/cm
2, more preferably 1-20 μ l/cm
2Formula 1 shown in complete antigen.
In a ninth aspect of the present invention, a kind of test kit is provided, the aforesaid monoclonal antibody that described test kit contains container and is positioned at container, perhaps described test kit contains aforesaid check-out console and working instructions.
Biomaterial preservation explanation
Mouse monoclonal antibody hybridoma cell strain of the present invention, be hemp monoclonal antibody cell strain THC10A (having another name called THC-KLH-CH1 or THC-10A), be deposited in Chinese typical culture collection center (CCTCC, Chinese Wuhan on February 20th, 2009, Wuhan University), deposit number is CCTCC NO.C200915.
Description of drawings
Fig. 1: the THC complete antigen prepares synoptic diagram.Wherein step 1 is the activation of 1 small molecules; Step 2 is activatory small molecules and macromole KLH, with the covalent coupling; Step 3 has the immunogenic complete antigen of drugs small molecules for making after the coupling.
Fig. 2: fusion rate is measured synoptic diagram.
Fig. 3: antibody polyacrylamide gel electrophoresis coomassie brilliant blue staining collection of illustrative plates after the 2-ME reduction behind the purifying.
Fig. 4: THC antiserum titre (titre) is measured.
Fig. 5: the THC monoclonal antibody tire (titre) measure.
Fig. 6: the mensuration of THC monoclonal antibody and KLH cross reaction.
Fig. 7: strip is formed synoptic diagram in the check-out console of the present invention.
Fig. 8: check-out console building block principle synoptic diagram.
Fig. 9: hemp monoclonal antibody immunity rapid detection plate result of determination synoptic diagram.
Embodiment
The inventor is through long-term and deep research, tetrahydrocannabinol is connected with suitable protein carrier produced complete antigen, as immunogen immune Balb/C mouse, obtain the monoclonal cell strain of the anti-tetrahydrocannabinol of specific secretion, and prepare purifying thus and obtained the tetrahydrocannabinol monoclonal antibody, thereafter further with described complete antigen and tetrahydrocannabinol Antibody Preparation have highly sensitive tetrahydrocannabinol colloid gold immune check-out console, thereby finished the present invention.
Particularly, structural simulation calculating is at first carried out in this research, and THC is activated, and adopts the chemical structure guard method, introduces active group-carboxyl (THC-carboxyl-KLH), make it have active chemical property on its phenyl ring.Adopt the difunctional amino coupling agent of homology with activated T HC and macromole KLH albumen coupling, so just make THC become the immunogenicity that complete antigen has.
The contriver has further prepared anti-THC monoclonal antibody: adopt operation stepss such as complete antigen immune animal, cytogamy, screening and cloning, antibody purification, hybridoma enlarged culturing to finish the preparation of hybridoma cell strain, obtain the high hybridoma cell strain of the anti-THC monoclonal antibody of specificity of tiring, secrete at last.The hybridoma of THC monoclonal antibody is purified and identify and to be defined as the IgG1 type, kappa hypotype, called after THC-KLH-CH1 (being that preserving number is the mouse monoclonal antibody hybridoma cell strain THC-10A of CCTCC NO.C200915) after fixed.Kind of drugs and medicine carry out the cross reaction test surplus selecting 60, and the result is a no cross reaction.
It is 30nm that the anti-THC studies on Monoclonal Antibody of Preparation of Colloidal Gold and mark Electronic Speculum method is selected the colloid gold particle size, carries out Radioactive colloidal gold-THC monoclonal antibody binding substances marks packets then and is produced.The preparation system of the research colloid gold label hemp monoclonal antibody rapid detection plate of colloid gold label hemp monoclonal antibody rapid detection plate preparation is solidificated on the nitrocellulose filter respectively by complete antigen and SPA or sheep anti-mouse igg, chromatographic material (carrier) wraps in advance by the anti-THC monoclonal antibody of colloid gold label, be attached on the PVC polyester offset plate with materials such as filter sample paper, glass, thieving paper, adhesive tapes respectively, mutual group is dressed up colloid gold label hemp monoclonal antibody immunity rapid detection plate.
Complete antigen and preparation thereof
Tetrahydrocannabinol (THC) molecular weight very little (314Da) is the haptens material, only possesses immunoreactivity, does not have immunogenicity, can not be directly used in immune animal and obtains antibody.Usually, haptens need and macromole with the covalent coupling, become and both have immunoreactivity, have immunogenic complete antigen again.
Though abroad prepared the THC complete antigen and use obtained monoclonal antibody, but owing to used molecular weight to have only 40-60 ten thousand Da, source and people's kind than the (hemp-BSA) for preparing complete antigen near the BSA (bovine serum albumin) of animal, prepared monoclonal antibody is tired lower, poor specificity, titre only are below 1: 5000.
The KLH molecular weight is 5,000,000 Da, much larger than BSA, and derives from the animal widely different with people's kind.This just make according to Chinese People's Anti-Japanese Military and Political College's fiber crops monoclonal antibody of the present invention's preparation tire and specificity increases substantially, solved in the past that hemp monoclonal antibody check-out console can only detect 11-nor-Δ 8-tetrahydrocannabinol-9 carboxylic acid, can not detect the difficult problem of 11-hydroxyl-Δ 9-tetrahydrocannabinol, Δ 8-tetrahydrocannabinol, Δ 9-tetrahydrocannabinol, cannabinol, cannabidiol.
For this reason; the inventor calculates and the structural chemistry simulated experiment by structural simulation; THC is activated; adopt the chemical structure guard method; active group-carboxyl of introducing on its phenyl ring (the THC-carboxyl-KLH); make it have active chemical property, solved location taking over a job property group and the key technical problem of protecting the THC primary formation on the hemp small molecule structure.On this basis, further adopt the difunctional amino coupling agent of homology, so just make THC become the immunogenicity that complete antigen has activated T HC and KLH albumen coupling.
As used herein, the product after " complete antigen " of the present invention is meant cannabinol haptens of the present invention and the hemocyanin carrier combines.As used herein, " protein carrier " among the present invention refers in particular to hemocyanin (KLH).
This research adopts succinyl oxide method, NHS method and DCC method to carry out the synthetic of hemp complete antigen.General step is as follows: the first step: use succinyl oxide, earlier THC carry out half esterization, introduce a pendant carboxylic group; Second step: use the NHS method, with the pendant carboxylic group esterification; The 3rd step: with the activatory haptens under the DCC condition with carrier proteins KLH on amino generation condensation reaction, form complete antigen.
The preparation principle of complete antigen and step are shown in following flow process: (1) at first reacts the compound of hydroxyl with succinyl oxide, generate the intermediate of band carboxyl, (2) and then with N-hydroxy-succinamide (NHS) react, the ester bond of producing forms amido linkage with proteinic amino reaction under the condition of fat-soluble dicyclohexylcarbodiimide (DCC).Between small molecules haptens and two cross-linking compounds of protein, insert an amber diacyl base.
The preparation feedback formula of THC complete antigen
Described esterification and condensation reaction can any methods well known by persons skilled in the art, any suitable condition is carried out.For example, half ester reaction of the present invention can be carried out under the following conditions: temperature of reaction is 0-100 ℃, preferred room temperature-80 ℃, more preferably 30-60 ℃; Reaction times is 1-24 hour, preferred 2-12 hour, and more preferably 2-6 hour; Esterification condition of the present invention can be carried out under the following conditions: temperature of reaction is 0-60 ℃, preferred room temperature-60 ℃; Reaction times is 1-24 hour, preferred 2-12 hour, and more preferably 3 hours; Carrier ligation of the present invention can be carried out under the following conditions: temperature of reaction is 0-40 ℃, preferred 4-30 ℃; Reaction pH is 3.0-8.0; Reaction times is 1-24 hour, preferred 6-12 hour, and more preferably 6 hours.Those of ordinary skills can suitably adjust these conditions according to concrete operations or to the requirement of product.
Being connected of haptens of the present invention and protein carrier can be used any mode of connection known in the art.Such as but not limited to: carbodlimide method (EDC), glutaraldehyde method etc.
The complete antigen of the present invention's preparation, tetrahydrocannabinol-KLH has good immunogenicity, can stimulate mouse to produce the intensive immune response, the antiserum(antisera) titre can reach more than 1: 8000, and complete antigen tetrahydrocannabinol-KLH has well kept the immunoreactivity of tetrahydrocannabinol.
MONOCLONAL ANTIBODIES SPECIFIC FOR
Term used herein " monoclonal antibody " is meant that promptly, the antibody individuality of forming this colony is all identical available from the antibody of homologous antibody population basically, except there being a small amount of possible spontaneous mutation.Therefore, modifier " monoclonal " is meant that the character of this antibody is not the mixture of discrete antibody.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, complete antigen of the present invention can be applied to animal to induce the generation of monoclonal antibody.For monoclonal antibody, can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or available recombinant DNA method (U.S. Patent number 4,816,567) preparation.
Representational myeloma cell is effective fusion, support the stable high level of antibody to produce by the antibody produced cell of selecting, and those myeloma cells responsive to substratum (HAT medium matrix), comprise myeloma cell line, the myeloma cell line of muroid for example, comprise that the myeloma cell line derived from MOPC-21 and MPC-11 mouse tumor (can be available from Salk Institute Cell Distribution Center, San Diego, California, the U.S.) and SP-2, NZ0 or X63-Ag8-653 cell (can be available from American Type Culture Collection, Luo Keweier, Maryland, the U.S.).Human myeloma and mouse-people's heterozygosis myeloma cell line also has been described and has been used to produce human monoclonal antibodies [Kozbor, J.Immunol., 133:3001 (1984); Brodeur etc., the production technology of monoclonal antibody and application (Monoclonal Antibodies Production Techniques and Applications), 51-63 page or leaf (Marcel Dekker, Inc., New York, 1987)].
The substratum that hybridoma grows in is wherein analyzed the generation that has required specific monoclonal antibody with detection, as, by external binding analysis for example, Enzyme Linked Immunoadsorbent Assay (ELISA) or radioimmunoassay (RIA).The position of the cell of expressing antibodies can be detected with FACS.Then, the hybridoma clone can be formed subclone (subcloned) by the limiting dilution step, and by standard method growth (Goding, monoclonal antibody (Monoclonal Antibodies): principle and put into practice (Principles and Practice), AcademicPress (1986) 59-103 page or leaf).The substratum that is fit to that uses in order to reach this purpose comprises, for example, and DMEM or RPMI-1640 substratum.In addition, hybridoma can be grown as ascitic tumor in animal body.
Suitably obtain separating by subclone excretory monoclonal antibody monoclonal antibody purifying process by routine from substratum, ascites or serum, these purifying process are for for example, albumin A-agarose method (proteinA-Sepharose), hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
In the present invention, adopt operation stepss such as complete antigen immune animal of the present invention, cytogamy, screening and cloning, antibody purification, hybridoma enlarged culturing to finish the preparation of hybridoma cell strain, obtain the high hybridoma cell strain of the anti-THC monoclonal antibody of specificity of tiring, secrete at last.The hybridoma of THC monoclonal antibody is purified and identify and to be defined as the IgG1 type, κ (kappa) hypotype, called after THC-KLH-CH1 (being that preserving number is the mouse monoclonal antibody hybridoma cell strain THC-10A of CCTCC NO.C200915) after fixed.
In the preferred scheme of the present invention, monoclonal antibody THC-KLH-CH1 (CCTCC NO.C200915) adopts the preparation of Balb/C mouse ascites production monoclonal antibody method.With about 10
6-10
7Individual hybridoma is inoculated in the mouse peritoneal of sensitization, and visible belly obviously swells in 2-4 week.Extract ascites, slightly propose IgG through the saturated ammonium sulphate method, the antibody that will slightly carry is through affinity column (Protein G-Sephrose) purifying again.
The monoclonal antibody of the present invention of mark
In a preference of the present invention, described monoclonal antibody has detectable.More preferably, described marker is selected from down group: colloid gold label thing, coloured marker or fluorescent marker.
By cultivating hybridoma or mouse ascites method manufacture order clonal antibody, the antibody colloid gold label of production.Concrete grammar is seen: " about setting up the pethidine studies on Monoclonal Antibody " the 3rd national toxicological analysis seminar selected thesess such as Ceng Libo, Chen Liankang, 289-294, China People's Public Security University Press in September, 2000; Zhu Liping, Chen Xueqing " immunology common experimental method " People's Medical Officer Press in March, 2000; Ed Harlow, David Lane, Using Antibodies:A Laboratory Manual.1999.
Colloid gold label can adopt method known to those skilled in the art to carry out.In a preferred scheme of the present invention, the monoclonal antibody THC-KLH-CH1 of anti-tetrahydrocannabinol (CCTCC NO.C200915) uses colloid gold label, obtains the THC-KLH-CH1 monoclonal antibody of colloid gold label.
Anti-tetrahydrocannabinol monoclonal antibody THC-KLH-CH1 of the present invention has excellent specificity, with the common medicine of kind more than 60, drugs, there is not cross reaction; The carrier of THC-KLH-CH1 and tetrahydrocannabinol-KLH does not have cross reaction.Monoclonal antibody THC-KLH-CH1 of the present invention has very high tiring, and in the enzyme plate of tetrahydrocannabinol-KLH bag quilt detected, tiring reached 1: 16000.
Tetrahydrocannabinol detects with colloid gold label-plate for detecting immunity
Competition inhibition method is adopted in the detection of tetrahydrocannabinol.The present invention is fixed in detection zone (solid phase antigen) on the nitrocellulose membrane with tetrahydrocannabinol-KLH, the anti-tetrahydrocannabinol monoclonal antibody (traget antibody) of the tetrahydrocannabinol in the sample solution to be checked (free antigen) and solid phase antigen competition association colloid gold mark.The tetrahydrocannabinol that contains in the sample to be checked with suppressing combining of traget antibody and immobilized antigen, is suppressed at the detection zone formation colour band of nitrocellulose filter.Form colour band if measure the back detection zone, then the result is negative, and testing sample does not contain tetrahydrocannabinol; Otherwise, do not form colour band, then the result is positive, and test sample contains tetrahydrocannabinol.
Usually, interior Quality Control is set in detection.The present invention is provided with sheep anti-mouse igg in the Quality Control district that the detection zone of nitrocellulose membrane closes on how anti-, is surrounded by by colloid gold label on chromatography carrier glass fiber paper in advance or the tetrahydrocannabinol monoclonal antibody of color marker is arranged.No matter whether contain tetrahydrocannabinol in the sample to be checked, on the chromatography carrier glass fibre colloid gold label of pre-bag quilt or have the tetrahydrocannabinol monoclonal antibody of color marker can be with the sheep anti-mouse igg on the nitrocellulose filter many resistive connections close and form a coloured quality control band, this colour band is a standard of judging that the chromatography process is whether normal and whether check-out console goes bad.
Check-out console and material thereof
Check-out console of the present invention can adopt this area check-out console material commonly used, adopts conventional check-out console preparation method to make.
The present invention detects the plate for detecting immunity of tetrahydrocannabinol, comprises the back up pad of test strip and support test strip, as adopting PVC polyester offset plate etc.; Described test strip is overlapped successively by filter sample paper, chromatographic material, nitrocellulose filter and thieving paper to be formed, and overlapping part can adopt conventional method, fixedly connected as adhesive tape etc.; Wherein: the pre-bag of chromatographic material is by colloid gold label or the tetrahydrocannabinol monoclonal antibody or the polyclonal antibody of color marker are arranged, preferably, adsorb detection line and nature controlling line on the nitrocellulose filter by the tetrahydrocannabinol monoclonal antibody (THC-KLH-CH1) of colloid gold label;
Described detection line is complete antigen THC-KLH, and the zone at detection line place is a detection zone;
Described nature controlling line is the sheep anti mouse polyclonal antibody, and the zone at nature controlling line place is the Quality Control district;
Therefore, the detection thing on the test strip is followed successively by: wrap in advance by tetrahydrocannabinol monoclonal antibody (THC-KLH-CH1), detection line and the nature controlling line of colloid gold label;
In a preferred scheme: pre-bag is that employing concentration is 0.1-5mg/ml by the tetrahydrocannabinol monoclonal antibody (THC-KLH-CH1) of colloid gold label on the chromatographic material, tetrahydrocannabinol monoclonal antibody (THC-KLH-CH1) solution of preferred 0.5-1.5mg/ml colloid gold label wraps quilt in advance, and package amount is 20-100 μ l/cm
2, preferred 30-80 μ l/cm
2, more preferably 50 μ l/cm
2Preferred concentration is 0.5 or 1.5mg/ml, 50 μ l/cm
2
The complete antigen THC-KLH that adsorbs on the nitrocellulose membrane is that employing concentration is 0.1-5mg/ml, and the complete antigen THC-KLH solution of preferred 0.5～1mg/ml adsorbs, and adsorptive capacity is 5-50 μ l/cm
2, preferred 8-20 μ l/cm
2, more preferably 10 μ l/cm
2Preferred concentration is 0.5 or 1mg/ml, 10 μ l/cm
2
It is that employing concentration is 0.5～5mg/ml that the sheep anti-mouse igg that adsorbs on the nitrocellulose membrane resists more, and the how anti-solution of preferred 0.8～1.2mg/ml sheep anti-mouse igg adsorbs, and adsorptive capacity is 5-50 μ l/cm
2, 10 μ l/cm
2Preferred concentration is 0.8 or 1.2mg/ml, 10 μ l/cm
2
Those skilled in the art also can suitably adjust above-mentioned package amount and adsorptive capacity according to concrete application.
External check-out console can't detect THC, and the detection lowest detectable limit only is 100.0-300.0ng/ml.Comparatively speaking, check-out console of the present invention can detect THC, and highly sensitive, and lowest detectable limit is between 5.0ng～10.0ng/ml.
The performance of plate for detecting immunity
Colloid gold label tetrahydrocannabinol monoclonal antibody plate for detecting immunity of the present invention has following performance:
Highly sensitive: regulate the THC-KLH-CH1 monoclonal antibody solution of the colloid gold label of pre-bag quilt on the reagent strip, the amount of THC-KLH complete antigen, the tetrahydrocannabinol that contains in the test urine sample is carried out sensitivity test.The result shows, the check-out console of the tetrahydrocannabinol monoclonal antibody of colloid gold label of the present invention and complete antigen preparation, and the lowest detection amount of tetrahydrocannabinol can reach 5.0ng～10.0ng/ml.
Good stability: the tetrahydrocannabinol monoclonal antibody reagent bar of colloid gold label placed be incubated 0.5d, 1d, 1.5d, 2d, 2.5d, 3d, 4d, 5d under 65 ℃ respectively, carry out sensitivity test by above-mentioned sensitivity test method then.The result shows that the tetrahydrocannabinol monoclonal antibody of colloid gold label has satisfactory stability tolerating more than 5 days under 65 ℃.
Specificity is good: detect more than 60 kind of drugs with tetrahydrocannabinol monoclonal antibody immunity check-out console, the result shows that it only with THC and derivative thereof immune association reaction takes place, and does not have cross reaction with above-mentioned other antigenic substances.The drugs and the medicine that are used to detect are: 11-nor-Δ 8-tetrahydrocannabinol-9 carboxylic acid, 11-hydroxyl-Δ 9-tetrahydrocannabinol, Δ 8-tetrahydrocannabinol, Δ 9-tetrahydrocannabinol, cannabinol, cannabidiol, methyl amphetamine, amphetamine, MDMA, MDA, morphine, morphine monomethyl ether, heroine, pholcodine, dihydroetorphine, thunder rice is for fourth, Pethidine, fentanyl, U-26225A, Propoxyphene, naloxone, TREXUPONT, nalorphine, clonidine, Luo Feixiding, Scopolamine, benefit peace oral liquid, just logical peaceful sheet, Paracetamol, Asprin, Ibuprofen BP/EP, amitriptyline, imipramine, chlorpromazine, promethazine, Chloral Hydrate, stable, triazolam, alprazolam, veronal, Somatarax, Amobarbital, caffeine, Norxin, pioneer IV, berberine, lactose, PROCAINE HCL, PHARMA GRADE, rehabilitation new capsule, Chloral Hydrate, Ofloxacin, Phenacetin, somedon, ketamine, methadone, pethidine, racephedrine, Cocaine, thebaine, lignocaine, Noscapine, buprenorphine, Phenylpropanolamine and phenylethylamine.
In sum, colloid gold label tetrahydrocannabinol monoclonal antibody plate for detecting immunity of the present invention is compared with HPLC isochromatic spectrum method, and check-out console of the present invention is simple, light, is easy to carry about with one, and can on-the-spotly detect, and does not need expensive equipment.Compare with external check-out console, use check-out console of the present invention to detect tetrahydrocannabinol, whole test can be finished in 1.5 minutes, and the sensitivity of detection can reach 5.0ng, with the common medicine of kind more than 60, drugs, there is not cross reaction.
Detection method and result judge:
Keep flat check-out console, sample is dropped on the filter sample paper, observe tomographic results in 1.5～5min.Come judged result according to the fringe position that occurs.
Negative: tangible colour band all appears in Quality Control district, detection zone, is shown feminine gender; Positive: as only obvious colour band to occur, and do not have colour band, be shown the positive at detection zone in the Quality Control district; Invalid: Quality Control district, detection zone do not have any colour band or colour band do not occur and colour band occurs at detection zone in the Quality Control district, show the rotten or inefficacy of detection method mistake or check-out console, should exchange check-out console again for and detect.
If detection line is shallower than that nature controlling line explanation measured sucked these drugs metabolism to the end or consumption less, so nature controlling line also is the standard of check-out console differentiation drug abuse situation.
The drug abuse threshold setting
With the check-out console of tetrahydrocannabinol monoclonal antibody behind the colloid gold label of the present invention and complete antigen preparation, the lowest detection amount of tetrahydrocannabinol can reach 5.0ng/mL.Consider in some normal medicine that uses and also contain the tetrahydrocannabinol composition, for avoiding false-positive appearance, can set required check-out console threshold value in the real work with reference to the concentration value of the current international practice.
Regulate the THC-KLH-CH1 monoclonal antibody solution of the colloid gold label of pre-bag quilt on the reagent strip, the amount of THC-KLH complete antigen, make that check-out console is 5.0-50.0ng/ml to the lowest detection amount of tetrahydrocannabinol, preferred 5.0-20.0ng/ml, more preferably 5.0-10.0ng/ml.When the tetrahydrocannabinol concentration in the urine during less than the lowest detection amount, the anti-tetrahydrocannabinol monoclonal antibody of colloid gold label moves up by the chromatography effect, combines with the complete antigen of solid phase on nitrocellulose filter, forms colour band, and test result is negative; Tetrahydrocannabinol concentration in urine is greater than the lowest detection amount, the anti-tetrahydrocannabinol monoclonal antibody of colloid gold label tetrahydrocannabinol complete and in the urine combines, can not combine with solid phase complete antigen of detection zone on nitrocellulose filter, detection zone can not form colour band, and test result is positive.
Test kit
Test kit of the present invention is meant the test kit that contains monoclonal antibody of the present invention or check-out console of the present invention.Described test kit can comprise container, working instructions, buffer reagent, immune auxiliaries etc. as required.
Compared with prior art, the invention has the advantages that:
(1) complete antigen of the present invention has high immunogenicity, and has kept the immunoreactivity of tetrahydrocannabinol;
(2) the excellent and height of tiring of the specificity of anti-tetrahydrocannabinol monoclonal antibody THC-KLH-CH1 of the present invention;
(3) colloid gold label tetrahydrocannabinol monoclonal antibody plate for detecting immunity of the present invention has highly sensitive, high specificity, simple and efficient, can carry out advantages such as scene detection, for the beat drugs crime provides strong weapon.
Compare with external similar check-out console, hemp monoclonal antibody check-out console lowest detection of the present invention is limited to 5.0-10.0ng/mL, and external check-out console lowest detection is limited to 100.0-300.0ng/mL.Check-out console of the present invention has quick, special, sensitive and reliable and stable advantage, and detection speed was finished in 1 minute and 30 seconds, and the quality guaranteed period is more than 2 years, and external check-out console, detection speed is about 3 minutes and 40 seconds, and the quality guaranteed period is short.
In addition, also adopt animal spleen direct immunization method among the present invention.Abandoned domestic and international employed freund's adjuvant, and adopted a kind of novel CPG immunological adjuvant that contains specific nucleotide sequence, facts have proved, used the adjuvant immunity animal that contains the CPG sequence, the titre of antibody will exceed 12 times than the freund's adjuvant of routine.Set up first strain in the world and had the hybridoma cell strain of secreting the high anti-THC monoclonal antibody of tiring.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The preparation of embodiment 1 tetrahydrocannabinol-KLH complete antigen
Take by weighing THC 100.0mg, be dissolved in the 10.0mL pyridine solution, and add the succinyl oxide of 300.0mg in this solution, 60 ℃ are reacted 4h down, steam and remove pyridine.Washing with alcohol obtains pressed powder.Take by weighing the above-mentioned pressed powder of 20.0mg and be dissolved in 1.0mL DMF (N, the N-dimethylformamide) in, add 10.0mg N-hydroxy-succinamide (NHS) and 65.0mg DCC therein, react 3h under the room temperature, 10000.0rpm the centrifugal insolubles of removing, supernatant liquor is dropwise splashed in the KLH protein solution of 10.0mL, 10.0mg/mL, room temperature reaction 2h, reaction product is used 0.01M at 4 ℃, the dialysis of pH 7.4 phosphate buffered saline buffers, exchange buffering liquid is 3～4 times during this time, to remove unreacted small-molecule substance, identify with ultraviolet spectroscopy at last, promptly get complete antigen THC-KLH.
The preparation of embodiment 2 tetrahydrocannabinol monoclonal antibodies (CCTCC NO.C200915)
The operation of Balb/C mouse immune
Earlier with antigen and freund's adjuvant (Freund ' s adjuvant) emulsification, complete antigen THC-KLH is mixed with the solution of 1.0mg/mL with PBS, then complete antigen solution is mixed with the freund's adjuvant equal-volume, form even emulsion with the concussion of high speed oscillator, this emulsion is used for the immunity of animal.
Select the mouse in 8 ages in week to carry out injecting immune.Get 12 of the healthy Balb/C mouse in 8 ages in week, at the 0th day, every abdominal injection complete Freund's adjuvant emulsification antigen 1 00.0uL.The 14th day, every mouse peritoneal was injected incomplete Freund's adjuvant emulsification antigen 1 00.0uL again.The 21st day,, detect antibody titer (titre) in the serum with the ELISA method to pluck the blood sampling of eyeball of mouse method.The 28th day, abdominal injection incomplete Freund's adjuvant emulsification antigen 1 00.0uL once more.React the last week in cytogamy, use 100.0ug/100.0uL antigen physiological saline booster immunization once more.After measured, the gained antiserum titre was at 1: 16000 after the blood sampling.
Merge programstep
To merge and all sterilize, foetal calf serum, HT and A stock solution will be thawed, place 37 ℃ of water-bath preheatings together with other medium component and PEG solution with equipment.
The SP2/0 murine myeloma cell that selection is in logarithmic phase merges.Counting (density range should at 108/mL), by the trypan blue dyeing counting, check cell viability (should be higher than 95%) after, be suspended in RPMI or the DMEM substratum.
To press 5: 1 mixed with washed mouse boosting cell of RPMI-1640 and myeloma cell, with 108 splenocytes and 2 * 107 myeloma cells.1000.0rpm/min centrifugal 5.0min exhausts supernatant liquor.Flick centrifuge tube and make cell precipitation loose, the 1.0min inner edge sways the 50%PEG4000 solution that the limit dropwise adds 1.5mL.With cell suspension mixing carefully, leave standstill 1.0min with suction pipe subsequently.Then, in 2.0min, jog test tube limit, limit slowly drips 10.0mL serum-free RPMI-1640 substratum.Leave standstill and place about 3.0min, the centrifugal 10.0min of 800.0rpm/min abandons supernatant liquor.With the RPMI1640 that contains HAT and 20% foetal calf serum cell is made into the 100.0mL suspension,, culture plate is placed in 37 ℃, the incubator of 5%CO2 cultivate even shop 10 96 well culture plates (add peritoneal macrophage in advance and make feeder layer) of cell.
The selectivity of fused cell is cultivated
The cytogamy situation is checked in the cytogamy operation after 2～3 days, not fused cell mass mortality this moment has fused cell only and just can survive.
Add the HT+ perfect medium after 7～10 days, every hole adds 1.0mL.Between 8～14 days, just can see the clone of hybrid cell.When the clone grows to the 1.0mm diameter approximately, get final product the anti-body contg of substratum in the inspection window.Take out 1.0mL from culture hole and measure antibody, filter out the hybridoma of anti-THC with the elisa plate (ELISA method) of THC-KLH bag quilt.Cell in the positive hole of selecting moved in 24 well culture plates cultivate, and get rid of the testing of cross-immune reaction, with further screening positive clone.
Hybridoma cloning (limiting dilution assay)
Added the scavenger cell feeder layer in preceding 1 day in 96 well culture plates in the clone, every hole adds 0.1mL, incubation in 37 ℃ of CO2 incubators.From positive culture hole, draw desire clone's cell, count a cloning and need 1000 cells approximately.Unnecessary cell is put back in 24 orifice plates.Become 30/mL with the perfect medium diluting cells, add in two 96 orifice plates, every hole adds 0.1mL (adding feeder layer in the hole in advance), remaining cell is diluted to 10/mL once again, and then gets two 96 orifice plates of having spread feeder layer, adds 0.1mL in every hole.The cell suspension of 3/mL of refabrication is added on the other two boards.
Culture plate put in 37 ℃ of CO2 incubators cultivate, 2-3 week can occur visible unicellular colony and form.Continue to cultivate, wait to cultivate the interior liquid color of plate hole and become orange.Detect the antibody activity of supernatant liquor, select positive hole, carry out enlarged culturing again and clone again.
Draw liquid in the hole, filter out the specific hybrid oncocyte of anti-THC again with the ELISA method enzyme plate of THC-KLH bag quilt.Behind five time clonings, obtain the monoclonal cell that a strain can be secreted the anti-THC of specificity antibody, hypotype is IgG1, the THC monoclonal antibody of this emiocytosis can be used for preparing colloid gold label THC monoclonal antibody plate for detecting immunity.
The hybridoma enlarged culturing
This project needs lot of antibodies because of preparing and producing the monoclonal antibody check-out console, adopts Balb/C mouse ascites production monoclonal antibody protein.About 106-107 hybridoma is inoculated in the mouse peritoneal of sensitization.Visible belly obviously swells in 2-4 week.Extraction ascites is centrifugal, adds 0.02% sodium azide, 4 ℃ or-20 ℃ of refrigerators preservations.
Purifying antibody (Sepharose-G albumen affinity chromatography)
Get 1g CL-4B Sepharose-Protein G, be suspended in the phosphate buffer solution (pH=8) of 200.0mL, at least imbibition 30.0min.Get about 4.0mL dress post, successively wash post, use preceding a kind of damping fluid balance again then with the phosphoric acid buffer 100.0mL of pH=8 and the 0.1M Trisodium Citrate 100.0mL of pH=3.Ascites or culture supernatant are to phosphoric acid buffer dialysis (earlier with behind the saturated ammonium sulphate precipitation, dialysis-effect the is better again) balance of pH=8.
Every mL glue can combine with the IgG of 20.0-25.0mg, and controlling column flow rate is 0.5-1.0mL/min.10.0mM phosphoric acid buffer (pH=8) with 5.0mL is washed post, and the A280.0nm of monitoring stream fluid presses the 0.5mL fraction and collects effluent liquid.Begin several pipes and be not have proteic, if strong positive, antibody just combines with albumin A; If the weak positive may be that upward sample is too much.Use above-mentioned damping fluid (being no less than 5.0mL) to wash post again, most of IgA, IgM, IgG3 will flow out in this fraction.0.1M Trisodium Citrate (pH=6) wash-out IgG1 antibody with 5.0mL.0.1M sodium citrate buffer solution (pH=4.5) wash-out IgG2a with 5.0mL.Similar buffer solution elution IgG2b with pH=3.5.
The mensuration of embodiment 3. embodiment 2 gained monoclonal antibodies
Fusion efficiencies is analyzed
Merge behind the monoclonal antibody preliminary making SP2/0 cell by CFSE green fluorescence preliminary making lymphocyte and anti-mouse B220 PE mark, and through the two fluorocyte ratio measurings of cells were tested by flow cytometry analysis, the initial fusion efficiencies of splenocyte and SP2/0 is about 35% in this experimental system.It is 14.2% that the ELISA method is measured positive rate.The synoptic diagram that fusion rate is measured is seen Fig. 2
The evaluation of anti-THC monoclonal antibody
Purifying antibody and type are measured
Ascites is earlier slightly proposed IgG through the saturated ammonium sulphate method, and the antibody that will slightly carry is through affinity column (ProteinG-Sephrose) purifying again, behind the SDS-PAGE electrophoresis, and the coomassie brilliant blue staining (see figure 3).
Antibody is identified through the specific antibody parting kit
Adopt monoclonal antibody parting kit (Monoclonal antibody isotyping kit, PIERCE), identify that the back is IgG1 type, kappa hypotype, called after THC-KLH-CH1 (being that preserving number is the mouse monoclonal antibody hybridoma cell strain THC-10A of CCTCC NO.C200915).
The titration of antibody
Sero-fast titer determination
The elisa plate that THC-KLH is wrapped quilt is used to measure this sero-fast titre, and envelope antigen 10.0ug/mL is diluted in the 0.05M carbonate buffer solution of pH9.6, and the 100.0uL/ hole joins 96 hole enzyme plates and spends the night in 4oC.Sealed 2 hours at 37oC with 1% gelatin/PBS after discarding coating buffer, wash plate 3 times with the PBS-Tween-20 washing lotion then, add THC antiserum(antisera) or monoclonal antibody after diluting again, place 37oC to hatch 1 hour.Wash plate 3 times with the PBS-Tween-20 washing lotion, the sheep anti-mouse igg that adds 1: 2000 horseradish peroxidase-labeled resists more, 37oC is hatched after 1 hour and is washed plate 3 times with the PBS-Tween-20 washing lotion, adds the TMB/H2O2 substrate 10.0min that develops the color, with stop buffer (0.1N sulfuric acid) color development stopping
Under the 450.0nm optical wavelength, measure absorption value (OD), compare with the OD average in blank hole, with the positive contrast of the serum of immune mouse, with the negative contrast of the supernatant liquor of Sp2/0 cell cultures, the criterion in positive cell hole is: (OD450 test-OD450 blank)/(OD450 contrast-OD450 blank) 〉=2.1, sero-fast the tiring of gained THC is 1: 16000 (see figure 4).
The mensuration of THC monoclonal antibody titre
Adopt the same method to measure the titre of antibody, recording tiring of THC monoclonal antibody is 1: 8000 (see figure 5).
Specific assay
Because in the process of preparation Radioactive colloidal gold rapid detection plate, used complete antigen is THC-KLH, therefore, if monoclonal antibody and KLH have cross reaction, will cause serious false negative result.So, determine THC monoclonal antibody (THC-KLH-CH1 (being that preserving number is the mouse monoclonal antibody hybridoma cell strain THC-10A of CCTCC NO.C200915)) and KLH without any cross reaction.We use complete antigen THC-KLH and KLH coated elisa plate respectively, THC monoclonal antibody combination with it with dilution in 1: 2000, carry out integrated enzyme reaction and detect, the result shows that the THC monoclonal antibody can detect the THC-KLH of 20.0ng, and with KLH without any cross reaction.(see figure 6)
Cross reaction method test THC monoclonal antibody specificity
Kind of drugs and medicine carried out the specificity test surplus this project selected 60 for use: 11-nor-Δ 8-tetrahydrocannabinol-9 carboxylic acid, 11-hydroxyl-Δ 9-tetrahydrocannabinol, Δ 8-tetrahydrocannabinol, Δ 9-tetrahydrocannabinol, cannabinol, cannabidiol, methyl amphetamine, amphetamine, MDMA, MDA, morphine, morphine monomethyl ether, heroine, pholcodine, dihydroetorphine, thunder rice is for fourth, Pethidine, fentanyl, U-26225A, Propoxyphene, naloxone, TREXUPONT, nalorphine, clonidine, Luo Feixiding, Scopolamine, benefit peace oral liquid, just logical peaceful sheet, Paracetamol, Asprin, Ibuprofen BP/EP, amitriptyline, imipramine, chlorpromazine, promethazine, Chloral Hydrate, stable, triazolam, alprazolam, veronal, Somatarax, Amobarbital, caffeine, Norxin, pioneer IV, berberine, lactose, PROCAINE HCL, PHARMA GRADE, rehabilitation new capsule, Chloral Hydrate, Ofloxacin, Phenacetin, somedon, ketamine, methadone, pethidine, racephedrine, Cocaine, thebaine, lignocaine, Noscapine, buprenorphine, Phenylpropanolamine and phenylethylamine.
With the enzyme plate of the detected material bag of kind quilt surplus above-mentioned 60 therewith antibody response carry out ELISA and measure, the result determine this antibody only with phenol derivatives 11-nor-Δ 8-tetrahydrocannabinol-9 carboxylic acid of hemp, 11-hydroxyl-Δ 9-tetrahydrocannabinol, Δ 8-tetrahydrocannabinol, Δ 9-tetrahydrocannabinol, cannabinol, cannabidiol, immune association reaction takes place, and does not have cross reaction with above-mentioned other drugs and medicine.
The preparation of embodiment 4. colloid gold label THC monoclonal antibody immunity check-out consoles
Colloid gold label THC MONOCLONAL ANTIBODIES SPECIFIC FOR
The preparation of employing trisodium citrate reduction method.Getting the 247.5mL deionized water boils, 1.0% chlorauric acid solution of dissolving 2.5mL, 90oC is incubated 2.0min, 1.0% trisodium citrate aqueous solution that adds 7.5mL, continue to boil 5.0min, be cooled to room temperature after, adopt spectrophotometer to detect uniform particles degree and globule size, maximum absorption band is at 521nm, and the Radioactive colloidal gold size is about 30nm.
Get above-mentioned Radioactive colloidal gold 100.0mL, add the 0.1M K2CO3 of 1.0mL, under magnetic agitation, stir then, the anti-THC antibody of disposable quick adding 1.0～2.0mg, after continue stirring 5.0min, add 10.0% the bovine serum albumin solution of 2.0mL again, continue to stir 5.0min.With supercentrifuge respectively with it at 12000rpm/min, 4 ℃ of following centrifugal 30.0min inhale and remove supernatant liquor, draw preservation with the BB damping fluid (borate buffer) that contains the few stable agent precipitation that suspends.The also available unnecessary antibody of albumen concentrating instrument flush away is also concentrated with its volume.
With after Radioactive colloidal gold-the monoclonal antibody binding substances is diluted to finite concentration, be coated on the plain paper of glass fibre 37 ℃ of dried overnight with the damping fluid that contains tensio-active agent and stablizer equably.
The design of detection line and nature controlling line
How anti-the THC-KLH complete antigen of preparation 0.5～1.0mg/mL and the sheep anti-mouse igg of 0.8～1.2mg/mL concentration be, sprays simultaneously and be respectively detection line and nature controlling line on nitrocellulose membrane, and put to 37 ℃ of dried overnight.
The test strip assembling
Thieving paper is placed in the lower grooves of pasting board, then one section film is placed on its top.The scraps of paper of offset plate opposite side are torn, the edge of complying with the pasting board chi with the Radioactive colloidal gold paper slip be attached to film above, will filter sample paper again and cover on the Radioactive colloidal gold paper slip.
" MAX " arrow adhesive tape is attached to the combining site of Radioactive colloidal gold paper slip and film, the Radioactive colloidal gold paper slip is covered fully, compresses, again masking tape is attached to thieving paper one side, the joint portion of thieving paper and film is covered fully, compresses, unnecessary masking tape is scratched with cutter.
With the test strip of the corresponding specification of cutting knife cutting, pack into test strip in the plastic clip and encapsulate with aluminium foil bag.Strip is formed the synoptic diagram (see figure 7) in the check-out console.
This strain is solidificated on the nitrocellulose filter respectively by complete antigen and SPA or sheep anti-mouse igg, chromatographic material (carrier) wraps in advance by the anti-THC monoclonal antibody of colloid gold label, be attached on the PVC polyester offset plate with materials such as filter sample paper, thieving paper, adhesive tapes respectively, mutual group is dressed up THC monoclonal antibody plate for detecting immunity, (see figure 8).
Detect principle and process
This check-out console is used immune competition law principle, and promptly the anti-THC monoclonal antibody of THC in the urine and the solid phase THC-KLH competition association colloid gold mark on nitrocellulose membrane has or not colour band to differentiate measurement result by the viewing window detection zone.
How anti-wrap by complete antigen and sheep anti-mouse igg respectively at the detection zone (T) of nitrocellulose membrane and Quality Control district (C), bag is by the anti-THC monoclonal antibody of colloid gold label on chromatography carrier glass fibre.THC concentration＜50.0ng/mL in urine, the anti-THC monoclonal antibody of colloid gold label moves up by the chromatography effect, combines with the complete antigen of solid phase on nitrocellulose filter, this moment, a purplish red colour band all appearred in detection zone (T) and Quality Control district (C), illustrated that detected result is negative.THC concentration＞50.0ng/mL in urine, the anti-THC monoclonal antibody of colloid gold label THC complete and in the urine combines, can not combine with solid phase complete antigen of detection zone on nitrocellulose filter, detection zone can not form purplish red colour band, and this moment, detected result was positive.
No matter whether contain THC in the urine, the THC monoclonal antibody of colloid gold label can close with the many resistive connections of the sheep anti-mouse igg of solid phase on nitrocellulose filter and form a purplish red colour band, this colour band be judge whether urine sample positive and negative, whether the chromatography process normal and whether check-out console rotten standard, be decided to be Quality Control district (C).
If detection line is shallower than that nature controlling line explanation measured sucked these drugs metabolism to the end or consumption less, so detection line also is the standard of check-out console differentiation drug abuse situation.
Check-out console using method and result judge
From sealing bag, take out check-out console, indicate test sample or control sample.Keep flat check-out console, in the point sample hole, splash into three samples (about 120uL), observe tomographic results in 3～5min.
The synoptic diagram that the result judges is seen Fig. 9.Tangible purplish red colour band all appears in Quality Control district (C), detection zone (T), two purplish red colour bands promptly occur at the viewing window place, and is negative.Only purplish red colour band occurs, and do not have purplish red colour band, be shown the positive at detection zone (T) at Quality Control district (C).Quality Control district (C), the no any colour band of detection zone (T) or colour band do not occur and purplish red colour band occurs at detection zone (T) at Quality Control district (C) show that detection method mistake or check-out console are rotten or lost efficacy, and should exchange check-out console again for and detect.
The performance test of embodiment 5. colloid gold label THC monoclonal antibody immunity check-out consoles
1. the sensitivity test of colloid gold label THC monoclonal antibody plate for detecting immunity
According to test request, in blank urine sample, add THC, be mixed with respectively and contain the serial sample that THC concentration is 1.0ng/mL, 2.0ng/mL, 3.0ng/mL, 4.0ng/mL, 5.0ng/mL, 10.0ng/mL, 15.0ng/mL, 20.0ng/mL, 25.0ng/mL, 50.0ng/mL, 75.0ng/mL, 100.0ng/mL, 150.0ng/mL and 200.0ng/mL.
Above-mentioned series concentration is made quantitative gradient point sample, sensitivity test result's (seeing Table 1).
Table 1 sensitivity test result
| Point sample amount ng/ |
1 | 2 | 3 | 4 | 5 | 10 | 15 |
| The result | - | - | - | - | - | - | - |
| Point sample amount ng/mL | 20 | 25 | 50 | 75 | 100 | 150 | 200 |
| The result | + | + | + | + | + | + | + |
Annotate: "+" expression is positive; "-" expression is negative.
Test-results shows, the THC monoclonal antibody lowest detection amount of carrying out behind the colloid gold label is 20.0ng/mL.
2. the stability experiment of colloid gold label THC monoclonal antibody plate for detecting immunity
Reagent strip in the THC monoclonal antibody check-out console of colloid gold label placed is incubated 0.5 day under 65 ℃ respectively, 1.0 days, 1.5 days, 2.0 days, 2.5 days, 3.0 days, 4.0 days, 5.0 days, carry out the sensitivity test of 50.0ng/mL by above-mentioned sensitivity test method then.
Test result under the above-mentioned condition (seeing Table 2).
Table 2 stability experiment result
| Shelf time (my god) | 0.5 | 1.0 | 1.5 | 2.0 | 2.5 | 3.0 | 3.5 | 4.0 | 5.0 |
| The result | + | + | + | + | + | + | + | + | + |
Annotate: "+" expression is positive
Experimental result shows that the THC monoclonal antibody of colloid gold label has satisfactory stability tolerating more than 5 days under 65 ℃.
3. the specificity of colloid gold label THC monoclonal antibody plate for detecting immunity test
Kind of drugs and medicine carry out the specificity test surplus selecting 60 for use: 11-nor-Δ 8-tetrahydrocannabinol-9 carboxylic acid, 11-hydroxyl-Δ 9-tetrahydrocannabinol, Δ 8-tetrahydrocannabinol, Δ 9-tetrahydrocannabinol, cannabinol, cannabidiol, methyl amphetamine, amphetamine, MDMA, MDA, morphine, morphine monomethyl ether, heroine, pholcodine, dihydroetorphine, thunder rice is for fourth, Pethidine, fentanyl, U-26225A, Propoxyphene, naloxone, TREXUPONT, nalorphine, clonidine, Luo Feixiding, Scopolamine, benefit peace oral liquid, just logical peaceful sheet, Paracetamol, Asprin, Ibuprofen BP/EP, amitriptyline, imipramine, chlorpromazine, promethazine, Chloral Hydrate, stable, triazolam, alprazolam, veronal, Somatarax, Amobarbital, caffeine, Norxin, pioneer IV, berberine, lactose, PROCAINE HCL, PHARMA GRADE, rehabilitation new capsule, Chloral Hydrate, Ofloxacin, Phenacetin, somedon, ketamine, methadone, pethidine, racephedrine, Cocaine, thebaine, lignocaine, Noscapine, buprenorphine, Phenylpropanolamine and phenylethylamine.
Above-mentioned detection material is mixed with certain density solution detects, the result shows that the monoclonal antibody plate for detecting immunity with this antibody producing only with THC and derivative thereof immune association reaction takes place, and does not have cross reaction with above-mentioned other antigenic substances.The result shows that the colloid gold label THC monoclonal antibody plate for detecting immunity of preparation is functional, and specificity is strong.Test result (seeing Table 3).
The specific reaction test result of table 3 check-out console
4. the comparison of check-out console and GC/MS detected result
Adopt check-out console of the present invention identical sample to be tested and its detected result is compared (seeing Table 4) with GC/MS.
The comparison of table 4 check-out console and GC/MS detected result
The detection threshold that we set is 50.0ng/mL, and susceptibility is 99.8%, and accuracy is 99.9%.
In addition, person-portion surplus hemp monoclonal antibody rapid detection plate had applied 90000 in 2005-2007, detect the drugs personnel that smoke cannabis by statistics and be (positive rate) 52%, the drugs personnel that do not smoke cannabis are (negative rate) 48%, and this result can judge that it is very fast that the current drugs personnel that smoke cannabis rise.For the urine sample positive and the drug addict does not admit the urine sample of taking drugs, adopt GC/MS to detect to check, as a result unanimity.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a complete antigen is characterized in that, described complete antigen has the structure shown in the formula 1:
Formula 1
Wherein, A is a hemocyanin.
2. the method for preparation formula 1 a described complete antigen is characterized in that, described method comprises step:
(a) compound of hydroxyl is at first reacted with succinyl oxide, generate the intermediate of band carboxyl;
(b) make the intermediate of step (a) gained and N-hydroxy-succinamide react the ester bond of production;
(c) step (b) products therefrom is connected with hemocyanin, thereby makes the complete antigen of formula 1.
3. the method for preparation formula 1 described complete antigen as claimed in claim 2 is characterized in that described method comprises the steps:
4. the purposes of the described complete antigen of claim 1 is characterized in that, is used to prepare the tetrahydrocannabinol monoclonal antibody specific.
5. a monoclonal antibody is characterized in that, its specificity is incorporated into tetrahydrocannabinol, and more preferably described monoclonal antibody is that CCTCC NO.C200915 is produced by mouse hybridoma cell.
6. a hybridoma cell line that produces the described monoclonal antibody of claim 5 is characterized in that, it is that mouse hybridoma cell is CCTCC NO.C200915.
7. the purposes of the described monoclonal antibody of claim 5 is characterized in that, is used for preparing reagent, check-out console or the test kit of test sample tetrahydrocannabinol.
8. whether there is the method for tetrahydrocannabinol in the detection of biological sample, it is characterized in that, comprise step:
(a) the described monoclonal antibody of sample and claim 5 is contacted;
(b) detect whether form antigen-antibody complex, wherein form mixture and just represent to exist in the sample tetrahydrocannabinol.
9. a check-out console is characterized in that, described check-out console comprises substrate (back up pad) and test strip, and described test strip contains the described monoclonal antibody of claim 5.
10. a test kit is characterized in that, the described monoclonal antibody of claim 5 that described test kit contains container and is positioned at container, and perhaps described test kit contains described check-out console of claim 9 and working instructions.
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| CN2009100047365A CN101580544B (en) | 2008-02-21 | 2009-02-23 | Colloidal gold labelled monoclonal antibody immunity detection Plate for detecting cannabis and tetrahydrocannabinol |
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| CN2009100047365A CN101580544B (en) | 2008-02-21 | 2009-02-23 | Colloidal gold labelled monoclonal antibody immunity detection Plate for detecting cannabis and tetrahydrocannabinol |
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| CN101580544B CN101580544B (en) | 2012-10-10 |
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| CN105925538A (en) * | 2016-05-17 | 2016-09-07 | 杭州奥泰生物技术有限公司 | Hybridoma cell strain, synthetic marijuana resisting monoclonal antibody generated by hybridoma cell strain and application of synthetic marijuana resisting monoclonal antibody |
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| CN115160135A (en) * | 2022-08-10 | 2022-10-11 | 郑州左安检测科技有限公司 | Preparation method and application of cannabidiol hapten and complete antigen |
| CN115160135B (en) * | 2022-08-10 | 2023-11-03 | 郑州左安检测科技有限公司 | Preparation method and application of cannabidiol hapten and complete antigen |
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| CN101580544B (en) | 2012-10-10 |
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Effective date of registration: 20151228 Address after: 200083 Hongkou District, Zhongshan, North Road, No. 803, No. Patentee after: Shanghai Institute of Criminal Science and Technology Patentee after: SHANGHAI VENTURE BIOTECHNOLOGY CO., LTD. Address before: 200083 No. 803, No. 1, North Road, Shanghai, Zhongshan Patentee before: Zeng Libo Patentee before: Chen Liankang Patentee before: Hu Xiaolong Patentee before: Zhang Yurong |
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Address after: 200083 Hongkou District, Zhongshan, North Road, No. 803, No. Co-patentee after: Shanghai eight way biological Polytron Technologies Inc Patentee after: Shanghai Institute of Criminal Science and Technology Address before: 200083 Hongkou District, Zhongshan, North Road, No. 803, No. Co-patentee before: SHANGHAI VENTURE BIOTECHNOLOGY CO., LTD. Patentee before: Shanghai Institute of Criminal Science and Technology |