CN101580544B - Colloidal gold labelled monoclonal antibody immunity detection Plate for detecting cannabis and tetrahydrocannabinol - Google Patents

Colloidal gold labelled monoclonal antibody immunity detection Plate for detecting cannabis and tetrahydrocannabinol Download PDF

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CN101580544B
CN101580544B CN2009100047365A CN200910004736A CN101580544B CN 101580544 B CN101580544 B CN 101580544B CN 2009100047365 A CN2009100047365 A CN 2009100047365A CN 200910004736 A CN200910004736 A CN 200910004736A CN 101580544 B CN101580544 B CN 101580544B
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thc
monoclonal antibody
complete antigen
check
out console
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CN101580544A (en
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曾立波
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Shanghai Eight Way Biological Polytron Technologies Inc
SHANGHAI INSTITUTE OF CRIMINAL SCIENCE AND TECHNOLOGY
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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 isused 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

Colloid gold label hemp, THC monoclonal antibody plate for detecting immunity
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 THC.
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 smoke cannabis more than at least 1.4 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 junkies 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 property is strong.Hemp is the general name of a kind of phenol derivatives of from cannabis plants, extracting; It mainly contains THC, cannabinol, CBD, tetrahydro-cannabinolic acid etc.; Be main wherein with THC (Tetrahydrocannabinol, Δ 9THC or THC) and 11-nor-Δ 8-THC-9-carboxylic acid (11-nor-Δ 8-Tetrahydrocannabinol-9-carboxylic acid).THC (THC) is the small molecules haptin 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 that for a long time do not smoke cannabis because accumulate poisoning causes the behavioral toxicity reaction, is 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 high (sample needs more than 400 yuans) is difficult to promote in vast grass-roots unit.
Though external (like 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 THC (THC) in the sample; Yet the hemp monoclonal antibody of external research and development can only be discerned minimum metabolite 11-nor-Δ 8-THC-9 carboxylic acid of content in the urine sample, can't detect THC (THC); (2) hemp monoclonal antibody plate for detecting immunity sensitivity at present all more than 100.0ng, detect 11-nor-Δ 8-THC-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, the monoclonal antibody that therefore prepares is tired lower, poor specificity.
Along with the criminal case relevant with THC rises and the day by day strictness of country to the hemp management year by year, this area is to hemp, and especially the detection of hemp and THC is had higher requirement in THC 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
The object of the invention just providing a kind of quick, simple and easy, detect the detection method and the detection reagent of THC delicately.
In first aspect of the present invention, a kind of complete antigen is provided, said complete antigen has the structure shown in the formula 1:
Figure G2009100047365D00021
Formula 1
Wherein, A is a hemocyanin.
In second aspect of the present invention, a kind of method for preparing formula 1 described complete antigen is provided, said method comprises step:
(a) compound of hydroxyl is at first reacted with succinyl oxide, generate the midbody of band carboxyl;
(b) make the midbody 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 following: 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 following: 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 following: 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 the third aspect of the invention, the purposes of aforesaid haptin of a kind of the present invention or complete antigen is provided, it is used to prepare the THC monoclonal antibody specific.
In a preference, said method comprises step:
Figure G2009100047365D00031
In the third aspect of the invention, the purposes of complete antigen of the present invention is provided, it is used to prepare the THC monoclonal antibody specific.
In fourth aspect of the present invention, a kind of monoclonal antibody is provided, said monoclonal antibody specificity is incorporated into THC.In a preference of the present invention, said monoclonal antibody is that THC-KLH-CH1 produces by mouse hybridoma cell.
In another preference, described monoclonal antibody combines to tire greater than 1: 6000 with THC, 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-THC-9 carboxylic acid with hemp, 11-hydroxyl-Δ 9-THC; Δ 8-THC, Δ 9-THC, cannabinol; Immune association reaction takes place in CBD.
In aspect the of the present invention the 5th; A kind of hybridoma cell line that produces monoclonal antibody of the present invention is provided; Said hybridoma cell line is that mouse hybridoma cell is that CCTCC NO.C200915 (is hemp monoclonal antibody cell strain THC10A, is deposited in Chinese typical culture collection center (CCTCC, China on February 20th, 2009; Wuhan, Wuhan University)).
In aspect the of the present invention the 6th, 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 THC.
In a preference of the present invention, said sample is a biological sample, and is preferably blood sample or urine sample.
In aspect the of the present invention the 7th, the method that whether has THC in a kind of detection of biological sample is provided, said 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 THC.
In a preference of the present invention, said monoclonal antibody has detectable.More preferably, described affinity tag is selected from down group: colloid gold label thing, coloured affinity tag or fluorescent marker.
In a preference of the present invention, said detection method is Radioactive colloidal gold detection method, colorimetric detection method or fluorescence detection.
In eight 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 by filter appearance paper, chromatographic material, nitrocellulose filter and thieving paper successively to be formed.
In another preference, said 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 said 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 said 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 said 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 aspect the of the present invention the 9th, 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 THC-10A of the present invention (having another name called THC-KLH-CH1) is deposited in Chinese typical culture collection center (CCTCC, Chinese Wuhan, Wuhan University) on February 20th, 2009, and deposit number is CCTCC NO.C200915.
Description of drawings
Fig. 1: the THC complete antigen prepares synoptic diagram.Wherein step 1 is 1 small molecules activation; 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; THC is connected with suitable protein carrier produced complete antigen; As immunogen immune Balb/C mouse, obtain the monoclonal cell strain of the anti-THC of specific secretion, and prepare purifying thus and obtained the THC monoclonal antibody; Thereafter further with said complete antigen and THC Antibody Preparation have highly sensitive THC colloid gold immune check-out console, thereby accomplished the present invention.
Particularly, structural simulation calculating is at first carried out in this research, and THC is carried out activation, adopts the chemical structure guard method, on its phenyl ring, introduces reactive group-carboxyl (THC-carboxyl-KLH), make it have active chemical property.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 accomplish 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 confirms 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) with identifying 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) encapsulates the anti-THC monoclonal antibody of colloid gold label in advance; Be attached on the PVC polyester offset plate with materials such as filter appearance 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
THC (THC) molecular weight very little (314Da) is the haptin material, only possesses immunoreactivity, does not have immunogenicity, can not directly be used for immune animal and obtains antibody.Usually, haptin 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 is merely 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-THC-9 carboxylic acid, can not detect the difficult problem of 11-hydroxyl-Δ 9-THC, Δ 8-THC, Δ 9-THC, cannabinol, CBD.
For this reason; The inventor calculates and the structural chemistry simulated experiment through structural simulation; THC is carried out activation, adopt the chemical structure guard method, and reactive 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 haptin 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 following: 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 haptin 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) is at first reacted the compound of hydroxyl with succinyl oxide; Generate the midbody 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 NSC 57182 (DCC).Between small molecules haptin 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 carried out under following condition: 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 carried out under following condition: 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 following condition: 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 haptin 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; THC-KLH has good immunogenicity; Can stimulate mouse to produce the intensive immunoreation, the antiserum(antisera) titre can reach more than 1: 8000, and complete antigen THC-KLH has well kept the immunoreactivity of THC.
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 prepare through 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, hybridoma technology capable of using prepares 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, supports the stable high level of antibody to produce and those myeloma cells responsive to substratum (HAT medium matrix) through the antibody produced cell of selecting; Comprise myeloma cell line, for example the myeloma cell line of muroid comprises 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, 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)].
Substratum to hybridoma grows in is wherein analyzed the generation that has required specific monoclonal antibody with detection, as, through 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; Can the hybridoma clone be formed subclone (subcloned) through the limiting dilution step; And through standard method growth (Goding; Monoclonal antibody (Monoclonal Antibodies): principle with 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 through 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 accomplish 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 confirms 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) with identifying 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 again is through affinity column (Protein G-Sephrose) purifying.
The monoclonal antibody of the present invention of mark
In a preference of the present invention, said monoclonal antibody has detectable.More preferably, described affinity tag is selected from down group: colloid gold label thing, coloured affinity tag or fluorescent marker.
Through cultivating hybridoma or mouse ascites method manufacture order clonal antibody, the antibody of production is used colloid gold label.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-THC (CCTCC NO.C200915) uses colloid gold label, obtains the THC-KLH-CH1 monoclonal antibody of colloid gold label.
Anti-THC 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 THC-KLH is not handed over the justice reaction.Monoclonal antibody THC-KLH-CH1 of the present invention has very high tiring, and in the enzyme plate that THC-KLH encapsulates detected, tiring reached 1: 16000.
THC detects with colloid gold label-plate for detecting immunity
Competition inhibition method is adopted in the detection of THC.The present invention is fixed in the detection zone (solid phase antigen) on the nitrocellulose membrane with THC-KLH, the anti-THC monoclonal antibody (traget antibody) of THC in the sample solution to be checked (free antigen) and solid phase antigen competition association colloid gold mark.The THC 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 THC; Otherwise, do not form colour band, then the result is positive, and test sample contains THC.
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-, on chromatography carrier glass fiber paper, is surrounded by by colloid gold label in advance or the THC monoclonal antibody of color marker is arranged.No matter whether contain THC in the sample to be checked; Colloid gold label that encapsulates in advance on the chromatography carrier spun glass or the THC monoclonal antibody that color marker is arranged 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 to judge the whether normal standard that whether goes bad with check-out console of chromatography process.
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 process.
The present invention detects the plate for detecting immunity of THC, comprises test strip and the back up pad that supports test strip, as adopting PVC polyester offset plate etc.; Described test strip is overlapped by filter appearance paper, chromatographic material, nitrocellulose filter and thieving paper successively to be formed, and overlapping part can adopt conventional method, like fixed connections such as adhesive tapes; Wherein: chromatographic material encapsulates colloid gold label in advance or the THC monoclonal antibody or the polyclonal antibody of color marker is arranged; Preferably, adsorb detection line and nature controlling line on the nitrocellulose filter by the THC 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: the THC monoclonal antibody (THC-KLH-CH1), detection line and the nature controlling line that encapsulate colloid gold label in advance;
In a preferred scheme: the THC monoclonal antibody (THC-KLH-CH1) that encapsulates colloid gold label on the chromatographic material in advance is that employing concentration is 0.1-5mg/ml; THC monoclonal antibody (THC-KLH-CH1) solution of preferred 0.5-1.5mg/ml colloid gold label encapsulates 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 concentrations 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 concentrations 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 concentrations 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 LDL is merely 100.0-300.0ng/ml.Comparatively speaking, check-out console of the present invention can detect THC, and highly sensitive, and LDL is between 5.0ng～10.0ng/ml.
The performance of plate for detecting immunity
Colloid gold label THC 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 that encapsulates in advance on the reagent strip, the amount of THC-KLH complete antigen, the THC that contains in the test urine sample carries out sensitivity test.The result shows, the check-out console of the THC monoclonal antibody of colloid gold label of the present invention and complete antigen preparation, and the lowest detection amount of THC can reach 5.0ng～10.0ng/ml.
Good stability: the THC 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 THC monoclonal antibody of colloid gold label has satisfactory stability property tolerating more than 5 days under 65 ℃.
Specificity is good: detect more than 60 kind of drugs with THC monoclonal antibody immunity check-out console, the result shows that it only with THC and verivate 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-THC-9 carboxylic acid, 11-hydroxyl-Δ 9-THC, Δ 8-THC, Δ 9-THC, cannabinol, CBD, methyl amphetamine, amphetamine, MDMA; MDA, morphine, morphine monomethyl ether, heroine, pholcodine, dihydroetorphine, thunder rice is for fourth, Pethidine, fentanyl, U-26225A; Propoxyphene, nx, TREXUPONT, nalorphine, clonidine, Luo Feixiding, Scopolamine, benefit peace oral liquid, just logical peaceful sheet; Paracetamol USP23,BP98, Asprin, Ibuprofen BP/EP, amitriptyline, imipramine, CHLORPROMAZINE HCL, promethazine, Chloral Hydrate, stable; Triazolam, alprazolam, veronal, Somatarax, Amobarbital, theine, Norxin, pioneer IV, Umbellatine; 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 THC 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 THC, whole test can be accomplished 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 appearance paper, observe tomographic results in 1.5～5min.Fringe position according to occurring comes judged result.
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 THC monoclonal antibody behind the colloid gold label of the present invention and complete antigen preparation, the lowest detection amount of THC can reach 5.0ng/mL.Consider in some normal medicine that uses and also contain the THC 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 that encapsulates in advance 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 THC; Preferred 5.0-20.0ng/ml, more preferably 5.0-10.0ng/ml.When the THC concentration in the urine during less than the lowest detection amount, the anti-THC monoclonal antibody of colloid gold label moves up through the chromatography effect, combines with the complete antigen of solid phase on nitrocellulose filter, forms colour band, and test result is negative; THC concentration in urine is greater than the lowest detection amount; The anti-THC monoclonal antibody of colloid gold label fully with urine in THC combine; 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 THC;
(2) the excellent and height of tiring of the specificity of anti-THC monoclonal antibody THC-KLH-CH1 of the present invention;
(3) colloid gold label THC 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.That check-out console of the present invention has is quick, special, the advantage of sensitivity and reliable and stable, and detection speed was accomplished 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 the restriction scope of the present 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: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
The preparation of embodiment 1 THC-KLH complete antigen
Take by weighing THC 100.0mg, be dissolved in the 10.0mL pyridine solution, and in this solution, add the succinyl oxide of 300.0mg, 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 among the 1.0mL DMF (N, N-dimethylformamide), add 10.0mg N-hydroxy-succinamide (NHS) and 65.0mg DCC therein; React 3h under the room temperature, the centrifugal insolubles of removing of 10000.0rpm dropwise splashes into supernatant in the KLH protein solution of 10.0mL, 10.0mg/mL; Room temperature reaction 2h, reaction product is used 0.01M at 4 ℃; PH 7.4 phosphate buffered saline buffers dialysis, during exchange buffering liquid 3～4 times, to remove unreacted small-molecule substance; Identify with ultraviolet spectroscopy at last, promptly get complete antigen THC-KLH.
The preparation of embodiment 2 THC monoclonal antibodies (CCTCC NO.C200915)
The operation of Balb/C mouse immune
Earlier with antigen and freund's adjuvant (Freund ' s adjuvant) emulsification; Be mixed with complete antigen THC-KLH with PBS the solution of 1.0mg/mL; 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.In cytogamy reaction the last week, with 100.0ug/100.0uL antigen saline water booster immunization once more.Blood sampling is after measure, and the gained antiserum titre was at 1: 16000.
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) through the trypan blue dyeing counting, behind the inspection cell viability (should be higher than 95%), is suspended in RPMI or the DMEM substratum.
To use washed mouse boosting cell of RPMI-1640 and myeloma cell by 5: 1 mixed, with 108 splenocytes and 2 * 107 myeloma cells.1000.0rpm/min centrifugal 5.0min exhausts supernatant.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.RPMI1640 with containing HAT and 20% foetal calf serum is made into the 100.0mL suspension with cell, with even shop 10 96 well culture plates (add peritoneal macrophage in advance and make feeder layer) of cell, culture plate is placed in 37 ℃, the incubator of 5%CO2 cultivate.
The selectivity of fused cell is cultivated
The cytogamy operation is inspection cytogamy situation after 2～3 days, not fused cell mass mortality this moment, and having fused cell only could become to live.
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, the elisa plate (ELISA method) that encapsulates with THC-KLH filters out the hybridoma of anti-THC.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)
In 96 well culture plates, added the scavenger cell feeder layer in preceding 1 day 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, select positive hole, carry out enlarged culturing again and clone again.
Draw liquid in the hole, the ELISA method enzyme plate that encapsulates with THC-KLH again filters out the specific hybrid oncocyte of anti-THC.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, a kind of damping fluid balance again before using 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 liquid 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 level and divides the collection effluent.Begin several pipes and be not have proteic, if strong positive, antibody just combines with albumin A; If the weak positive possibly be that upward appearance 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 one-level is divided.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 through CFSE green fluorescence preliminary making lymphocyte and anti-mouse B220PE 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 again is through affinity column (ProteinG-Sephrose) purifying, 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 encapsulated 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 4 ℃.Sealed 2 hours at 37 ℃ 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 37 ℃ 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; Hatch after 1 hour for 37 ℃ and wash plate 3 times, add the TMB/H2O2 substrate 10.0min that develops the color, with stop buffer (0.1N sulfuric acid) color development stopping with the PBS-Tween-20 washing lotion
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 of Sp2/0 cell cultures, the criterion in positive cell hole is: (OD450 test-OD450 is blank)/(OD450 contrast-OD450 is 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, confirm the cross reaction that 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 have no.We use complete antigen THC-KLH and KLH coated elisa plate respectively; THC monoclonal antibody with dilution in 1: 2000 combines with it; Carry out integrated enzyme reaction and detect, the result shows that the THC monoclonal antibody can detect the THC-KLH of 20.0ng, and has no cross reaction with KLH.(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-THC-9 carboxylic acid, 11-hydroxyl-Δ 9-THC, Δ 8-THC, Δ 9-THC, cannabinol, CBD, methyl amphetamine, amphetamine, MDMA; MDA, morphine, morphine monomethyl ether, heroine, pholcodine, dihydroetorphine, thunder rice is for fourth, Pethidine, fentanyl, U-26225A; Propoxyphene, nx, TREXUPONT, nalorphine, clonidine, Luo Feixiding, Scopolamine, benefit peace oral liquid, just logical peaceful sheet; Paracetamol USP23,BP98, Asprin, Ibuprofen BP/EP, amitriptyline, imipramine, CHLORPROMAZINE HCL, promethazine, Chloral Hydrate, stable; Triazolam, alprazolam, veronal, Somatarax, Amobarbital, theine, Norxin, pioneer IV, Umbellatine; 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.
The enzyme plate that encapsulates with kind material to be detected surplus above-mentioned 60 antibody response therewith carries out ELISA and measures, the result confirm this antibody only with phenol derivatives 11-nor-Δ 8-THC-9 carboxylic acid of hemp, 11-hydroxyl-Δ 9-THC; Δ 8-THC; Δ 9-THC, cannabinol, CBD; 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.Get the 247.5mL deionized water and boil, 1.0% chlorauric acid solution of dissolving 2.5mL, 90 ℃ of insulation 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, draw preservation with the BB damping fluid (borate buffer) that contains the few stable agent deposition 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 spun glass 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 on nitrocellulose membrane, be respectively detection line and nature controlling line, 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 appearance 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, test strip is packed in the plastic clip and encapsulated 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) encapsulates the anti-THC monoclonal antibody of colloid gold label in advance; Be attached on the PVC polyester offset plate with materials such as filter appearance 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 the mensuration result through the viewing window detection zone.
At the detection zone (T) of nitrocellulose membrane with Quality Control district (C) encapsulates complete antigen respectively and how anti-sheep anti-mouse igg is, on chromatography carrier spun glass, encapsulate the anti-THC monoclonal antibody of colloid gold label.THC concentration in urine＜50.0 ng/mL; The anti-THC monoclonal antibody of colloid gold label moves up through 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), explained that detected result is negative.THC concentration＞50.0ng/mL in urine; The anti-THC monoclonal antibody of colloid gold label fully with urine in THC combine; 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 method of use 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 appears in (C) in the Quality Control district, and does not have purplish red colour band at detection zone (T), is shown the positive.Quality Control district (C), the no any colour band of detection zone (T) or in the Quality Control district (C) colour band do not occur and purplish red colour band occurs at detection zone (T), show that detection method mistake or check-out console are rotten or lost efficacy, 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/mL 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 property 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-THC-9 carboxylic acid, 11-hydroxyl-Δ 9-THC, Δ 8-THC, Δ 9-THC, cannabinol, CBD, methyl amphetamine, amphetamine, MDMA; MDA, morphine, morphine monomethyl ether, heroine, pholcodine, dihydroetorphine, thunder rice is for fourth, Pethidine, fentanyl, U-26225A; Propoxyphene, nx, TREXUPONT, nalorphine, clonidine, Luo Feixiding, Scopolamine, benefit peace oral liquid, just logical peaceful sheet; Paracetamol USP23,BP98, Asprin, Ibuprofen BP/EP, amitriptyline, imipramine, CHLORPROMAZINE HCL, promethazine, Chloral Hydrate, stable; Triazolam, alprazolam, veronal, Somatarax, Amobarbital, theine, Norxin, pioneer IV, Umbellatine; 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 verivate 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
Figure G2009100047365D00221
Figure G2009100047365D00231
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
Figure G2009100047365D00232
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 through 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 junkies does not admit the urine sample of taking drugs, adopt GC/MS to detect to check, the result is consistent.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (11)

1. a complete antigen is characterized in that, said complete antigen has the structure shown in the formula 1:
Figure FDA0000134576080000011
Formula 1
Wherein, A is a hemocyanin.
2. a method for preparing formula 1 described complete antigen is characterized in that, said method comprises step:
(a) compound of hydroxyl is at first reacted with succinyl oxide, generate the midbody of band carboxyl;
(b) midbody of step (a) gained and N-hydroxy-succinamide are reacted, generate ester bond;
(c) step (b) products therefrom is connected with hemocyanin, thereby makes the complete antigen of formula 1.
3. the method for preparing formula 1 described complete antigen as claimed in claim 2 is characterized in that said method comprises the steps:
Figure FDA0000134576080000012
4. the purposes of the described complete antigen of claim 1 is characterized in that, is used to prepare the THC monoclonal antibody specific.
5. a monoclonal antibody is characterized in that, its specificity is incorporated into THC, and described monoclonal antibody is that CCTCC NO.C200915 produces 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 THC.
8. whether there is the method for THC 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 THC.
9. a check-out console is characterized in that, described check-out console comprises substrate and test strip, and described test strip contains the described monoclonal antibody of claim 5.
10. check-out console as claimed in claim 9 is characterized in that said substrate is a back up pad.
11. 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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