CN100473641C - Monoclonal antibody for ketamine detection and immune detection board - Google Patents
Monoclonal antibody for ketamine detection and immune detection board Download PDFInfo
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- CN100473641C CN100473641C CNB2006100946875A CN200610094687A CN100473641C CN 100473641 C CN100473641 C CN 100473641C CN B2006100946875 A CNB2006100946875 A CN B2006100946875A CN 200610094687 A CN200610094687 A CN 200610094687A CN 100473641 C CN100473641 C CN 100473641C
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Abstract
The invention discloses a hapten and complete antigen used for ketamine detection and antibody preparation. The invention also discloses an anti-ketamine monoclonal antibody prepared by the complete antigen and a colloidal gold-labeled ketamine monoclonal antibody immunoassay plate used for detecting ketamine in drugs or urine, etc., human samples. Compared with HPLC method, the invented detection plate is simple, portable and easy to carry, and can be used for spot detection without need of expensive equipment. The whole detection for ketamine by using the detection plate can be completed in 10 minutes with sensitivity up to 50 ng and with no cross reaction with 39 kinds of common pharmaceuticals, drugs, and ketamine metabolites in vivo.
Description
Technical field
The present invention relates to the detection of illegal drug, relate in particular to the enzyme linked immunosorbent detection of ketamine.
Background technology
Ketamine (Ketamine) is a kind of fugitive narcotic by intravenously administrable, and major part entered cerebral tissue after it entered circulation of blood, and then was distributed in the body tissue, and liver, lung and intrafat drug level are also higher.This drug main will carry out bio-transformation and become norketamine in liver, the compound that progressively is metabolized to non-activity is again discharged through kidney, only has 2.5% to discharge with urine with original shape.This medicine just can the degree of depth ease pain when the liquor-saturated dosage of flax, and does not have the relevant inhibition heart of other conventional narcotic of great majority, the side effect of respiratory function, in clinic trial for many years.
As far back as June calendar year 2001, ketamine has been included into the national second class psychotropic substances and has managed.Because ketamine is the main component of drugs " K powder ", in recent years, it is serious that it illegally abuses phenomenon.Abuse ketamine to 70 milligram will cause poisoning, and 200 milligrams of meetings are hallucinated, and excessively then can cause death.Suck and peddle ketamine and be developmenting spread situation in part provinces and cities, the criminal phenomena that the abuse ketamine causes is more outstanding.Therefore, at present in first kind control of psychochemicals is listed ketamine and salt thereof and preparation by State Food and Drug Administration.
Along with the strictness day by day that the criminal case relevant with ketamine rises year by year and country manages ketamine, have higher requirement to the detection of ketamine in ketamine and the drug abuse person's biological sample in this area.
At present, the detection of ketamine mainly is based on chromatogram analysis method, for example gas chromatography mass spectrometry stratographic analysis (GC-MS) and efficient liquid phase chromatographic analysis (HPLC).These chromatographic processes have good sensitivity and specificity, but complex operation needs expensive plant and instrument, and length consuming time.
Specific association reaction, for example antigen-antibody reaction has been widely used in the immunoassay of the various materials that exist in the detection of biological sample.Wherein, the colloidal gold immunochromatographimethod technology is the immune diagnostic technique of development in recent years a kind of uniqueness of getting up, have the specific of immune response and chromatography, with GC-MS relatively, the colloidal gold chromatographic technology have high specificity, highly sensitive, simple fast, easy handling, the easy interpretation of result, need not advantage such as any plant and instrument.
Disclosed in the U.S. Pat 2003/224447 shown in a kind of formula A in the N position of norketamine with the haptens of linking agent preparation, and with immunogen, antigen, antibody and the conjugate of its preparation:
Formula A
Yet studies have shown that the coupled efficient of this haptens and carrier proteins is lower, difficulty is prepared into complete antigen.In addition, by its complete antigen that makes can not with the reaction that is at war with of ketamine standard substance, thereby can not be used to prepare the Radioactive colloidal gold fast diagnosis reagent.
Therefore, this area presses for a kind of detection method and detection reagent that can detect ketamine quicker, simple and easy, delicately.
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 ketamine delicately.
In a first aspect of the present invention, a kind of haptens is provided, described haptens has the structure shown in the formula 1:
In a second aspect of the present invention, a kind of complete antigen is provided, described complete antigen has the structure shown in the formula 2:
Wherein, X is a protein carrier.
In a preference of the present invention, described protein carrier is any protein that is selected from down in the group: hemocyanin, bovine serum albumin, ovalbumin or gamma Globulin.
In another preference of the present invention, described protein carrier is hemocyanin or bovine serum albumin.
In a third aspect of the present invention, a kind of method of preparation formula 2 described complete antigens is provided, described method comprises step:
(a) ketamine is carried out nitrated, insert nitryl group in its contraposition, to form to nitro-ketamine;
(b) will be amino to the nitroreduction in nitro-ketamine, to make p-NH2-Ketamine;
(c) p-NH2-Ketamine is connected with protein carrier, to make the complete antigen shown in the formula 2.
In a preference of the present invention, the condition of step (a) is as follows: temperature of reaction is-and 10-10 ℃, preferred-5-0 ℃; 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 a room temperature-100 ℃, preferred 50-70 ℃, and more preferably 60 ℃; Reaction times is 5-24 hour, preferred 8-12 hour, and more preferably 8 hours.
In another preference, the condition of step (c) is as follows: temperature of reaction is 0-40 ℃, preferred 4-25 ℃; Reaction pH is 3.0-6.0, preferred 4.0; Reaction times is 3-24 hour, preferred 6-12 hour, and more preferably 6 hours.
In a fourth 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 ketamine specific immunoglobulin.
In a fifth aspect of the present invention, a kind of immunoglobulin (Ig) is provided, described immunoglobulin (Ig) specificity is incorporated into ketamine.
In a preference of the present invention, described immunoglobulin (Ig) is that CGMCCNo.1404 is produced by mouse hybridoma cell.
In another preference, described immunoglobulin (Ig) is tired greater than 1:2000 with combining of ketamine, more preferably greater than 1:3000.
In another preference, described immunoglobulin (Ig) also is incorporated into norketamine.
In another preference, described immunoglobulin (Ig) debond is in the cylinder metabolism-ure of ketamine.
In another preference, described immunoglobulin (Ig) is a monoclonal antibody.
In a sixth aspect of the present invention, a kind of hybridoma cell line that produces aforesaid immunoglobulin (Ig) is provided, described hybridoma cell line is that mouse hybridoma cell is CGMCC No.1404.
In a seventh aspect of the present invention, a kind of purposes of aforesaid immunoglobulin (Ig) is provided, it is used for preparing reagent, check-out console or the test kit of test sample ketamine.
In a preference of the present invention, described sample is a biological sample, and is preferably blood sample or urine sample.
In a eighth aspect of the present invention, the method that whether has ketamine in a kind of detection of biological sample is provided, described method comprises step:
(a) sample is contacted with aforesaid immunoglobulin (Ig);
(b) detect whether form antigen-antibody complex, wherein form mixture and just represent to exist in the sample ketamine.
In a preference of the present invention, described immunoglobulin (Ig) 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 ninth 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 immunoglobulin (Ig).
In another preference, described test strip also contains complete antigen point sample district, and the complete antigen shown in the immobilized formula 2 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 immunoglobulin (Ig) 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 2;
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 1/cm
2Through colloid gold label or the immunoglobulin (Ig) 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 2 shown in complete antigen.
In a tenth aspect of the present invention, a kind of test kit is provided, the aforesaid immunoglobulin (Ig) that described test kit contains container and is positioned at container, perhaps described test kit contains aforesaid check-out console and working instructions.
Description of drawings
Fig. 1: ketamine-KLH immune mouse antiserum titre (titre) is measured.
Fig. 2: fusion rate is measured synoptic diagram.
The anti-mouse B220 monoclonal antibody preliminary making SP2/0 cell of 13:PE mark
14:CFSE green fluorescence preliminary making lymphocyte
15: two fluorocytes
Fig. 3: the polyacrylamide gel electrophoresis collection of illustrative plates of antibody purification after the 2-ME reduction.
Fig. 4: ketamine monoclonal antibody ket-5 titration.
Fig. 5: the mensuration of ketamine monoclonal antibody Ket-5 and BSA cross reaction.
Fig. 6: strip is formed synoptic diagram in the check-out console.
1: filter sample paper 2: chromatographic material 3: nitrocellulose filter 4: thieving paper
11: detection line 12: nature controlling line
Wherein 2: chromatographic material has the ket-5 monoclonal antibody of colloid gold label
Fig. 7: check-out console building block principle synoptic diagram.
1: filter sample paper 2: chromatographic material 3: nitrocellulose filter 4: thieving paper
5: colloid gold particle 6:ket-5 monoclonal antibody 7: the ket-5 monoclonal antibody of colloid gold label
8: the how anti-9:BSA-ketamine 10 of sheep anti mouse sheep anti-mouse igg: back up pad
Fig. 8: drugs monoclonal antibody immunity rapid detection plate result of determination synoptic diagram.
11: detection line 12: nature controlling line
16: viewing window 17: well
Fig. 9 A: according to the complete antigen of the antibody of U.S. Pat 2003/224447 preparation and the competing reaction test-results of ketamine standard substance.
Fig. 9 B: the competing reaction test-results of complete antigen of the present invention and ketamine standard substance.
Embodiment
The inventor has synthesized ketamine activated derivatives p-NH2-Ketamine through long-term and deep research, and it is connected with suitable protein carrier has produced complete antigen, as immunogen immune Balb/C mouse, with its splenocyte and mouse myeloma SP20 cytogamy, obtain the monoclonal cell strain (CGMCC No.1404) of the anti-ketamine of specific secretion, and the preparation and purifying obtained the ketamine monoclonal antibody, thereafter further with described complete antigen and ketamine Antibody Preparation have the plate for detecting immunity of highly sensitive and ketamine, thereby finished the present invention.
Haptens
Ketamine (Ketamine) molecular weight very little (237.72 dalton) is the haptens material, only possesses immunoreactivity, does not have immunogenicity, can not be directly used in immune animal and obtains antibody.Therefore, in order to prepare complete antigen of the present invention, ketamine has been carried out activation and made haptens of the present invention.
As used herein, " haptens " of the present invention or " ketamine activated derivatives " is meant the p-NH2-Ketamine (p-NH with structural formula 1 that obtains through derivatization reaction of the present invention
2-Ketamine), its structure as shown in Equation 1:
Complete antigen
Usually, haptens need and macromole such as KLH (hemocyanin) or BSA (bovine serum albumin) with the covalent coupling, become and both have immunoreactivity, have immunogenic complete antigen again.
As used herein, the product after " complete antigen " of the present invention is meant haptens of the present invention and suitable protein carrier combines.
As used herein, " protein carrier " among the present invention be meant any on immunology the acceptable protein that is used to form complete antigen, it for example can be, hemocyanin, bovine serum albumin, ovalbumin or gamma Globulin etc.
The present invention be used for that ketamine detects and the structure of the complete antigen of Antibody Preparation as shown in Equation 2:
Wherein, X is a protein carrier, preferred hemocyanin (KLH) or bovine serum albumin (BSA) among the present invention; With the part of X carrier covalent cross-linking be the derivative p-NH2-Ketamine (p-NH of ketamine
2-Ketamine).
In a preferred scheme of the present invention, X is a hemocyanin, and complete antigen is ketamine-KLH, as immunity antigen, is used to prepare the hybridoma of secreting anti-ketamine monoclonal antibody.
In another preferred scheme of the present invention, X is a bovine serum albumin, and complete antigen is ketamine-BSA, uses antigen as detecting, and is used to prepare the monoclonal antibody immunity check-out console that detects ketamine.
The preparation method that complete antigen of the present invention is is as follows:
At first, obtain its derivative with the ketamine activation: p-NH2-Ketamine, again p-NH2-Ketamine and the protein carrier (for example, KLH, BSA) that is fit to are connected, obtain complete antigen.Wherein X is a protein carrier, preferred hemocyanin (KLH) or bovine serum albumin (BSA) among the present invention; With the part of X carrier covalent cross-linking be the derivative p-NH2-Ketamine (p-NH of ketamine
2-Ketamine).
Wherein said nitrated and reduction reaction can any method well known by persons skilled in the art, any suitable condition is carried out.For example, nitration reaction of the present invention can be carried out under the following conditions: temperature of reaction is-10-10 ℃, preferred-5-0 ℃; Reaction times is 1-24 hour, preferred 2-12 hour, and more preferably 2-6 hour; Reduction reaction conditions of the present invention can carry out under the following conditions: temperature of reaction is a room temperature-100 ℃, preferred 50-70 ℃, and more preferably 60 ℃; Reaction times is 5-24 hour, preferred 8-12 hour, and more preferably 8 hours; Carrier ligation of the present invention can be carried out under the following conditions: temperature of reaction is 0-40 ℃, preferred 4-25 ℃; Reaction pH is 3.0-6.0, preferred 4.0; Reaction times is 3-24 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, ketamine-KLH has good immunogenicity, can stimulate mouse to produce the intensive immune response, and through 1 fundamental immunity, 3 booster immunizations, antiserum titre can reach 1:6400; Complete antigen ketamine-BSA has well kept the immunoreactivity of ketamine.
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 immunoglobulin purification technology 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.
The invention provides a kind of monoclonal antibody Ket-5 of anti-ketamine, through being accredited as the IgG1 type, κ (kappa) hypotype.In the preferred scheme of the present invention, monoclonal antibody Ket-5 adopts and cultivates the preparation of hybridoma method.Get the supernatant liquor of Hybridoma Cell Culture, slightly propose IgG through the saturated ammonium sulphate method, the antibody that will slightly carry is through affinity column (Protein G-Sephrose) purifying again.
In the preferred scheme of the present invention, monoclonal antibody Ket-5 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 present invention is when adopting ketamine-KLH mice immunized splenocyte and mouse myeloma S/P20 cell to prepare hybridoma according to a conventional method, screen the monoclonal cell strain (depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center of the anti-ketamine of a strain specific secretion, preserving number is CGMCC No.1404, and preservation date is on June 30th, 2005).
The immunoglobulin (Ig) of the present invention of mark
In a preference of the present invention, described immunoglobulin (Ig) 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 Ket-5 colloid gold label of anti-ketamine obtains the Ket-5 monoclonal antibody of colloid gold label.
Anti-ketamine monoclonal antibody ket-5 of the present invention has excellent specificity, with 38 kinds of common medicines, drugs, there is not cross reaction; The carrier B SA of ket-5 and ketamine-BSA does not have cross reaction.
Monoclonal antibody ket-5 of the present invention has very high tiring, and in the enzyme plate of ketamine-BSA bag quilt detected, tiring reached 1:3200.
Ketamine detects with colloid gold label-plate for detecting immunity
Detect principle
Competition inhibition method is adopted in the detection of ketamine.The present invention is fixed in detection zone (solid phase antigen) on the nitrocellulose membrane with ketamine-BSA, the anti-ketamine monoclonal antibody (traget antibody) of the ketamine in the sample solution to be checked (free antigen) and solid phase antigen competition association colloid gold mark.The ketamine 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 ketamine; Otherwise, do not form colour band, then the result is positive, and test sample contains ketamine.
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 ketamine monoclonal antibody of color marker is arranged.No matter whether contain ketamine in the sample to be checked, on the chromatography carrier glass fibre colloid gold label of pre-bag quilt or have the ketamine 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 ketamine, 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 ketamine monoclonal antibody or the polyclonal antibody of color marker are arranged, preferably by the ketamine monoclonal antibody (ket-5) of colloid gold label, and absorption detection line and nature controlling line on the nitrocellulose filter;
Described detection line is complete antigen ketamine-BSA, 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 ketamine monoclonal antibody (Ket-5), detection line and the nature controlling line of colloid gold label;
In a preferred scheme: pre-bag is that to adopt concentration be that ketamine monoclonal antibody (Ket-5) solution of 0.5-1.5mg/ml colloid gold label wraps quilt in advance by the ketamine monoclonal antibody (Ket-5) of colloid gold label on the chromatographic material, and package amount is 50 μ l/cm
2Preferred concentration is 0.5 or 1.5mg/ml, 50 μ l/cm
2
Complete antigen ketamine-the BSA that adsorbs on the nitrocellulose membrane is that to adopt concentration be that complete antigen ketamine-BSA solution of 0.5～1mg/ml adsorbs, and adsorptive capacity is 10 μ l/cm
2Preferred concentration is 0.5 or 1mg/ml, 10 μ l/cm
2
How anti-employing concentration is that the how anti-solution of 0.8～1.2mg/ml sheep anti-mouse igg adsorbs to the sheep anti-mouse igg that adsorbs on the nitrocellulose membrane, and adsorptive capacity is 10 μ l/cm
2Preferred concentration is 0.8 or 1.2mg/ml, 10 μ 1/cm
2
The sensitivity of check-out console is between 50ng～1000ng.
The performance of plate for detecting immunity
Colloid gold label ketamine monoclonal antibody plate for detecting immunity of the present invention has following performance:
Highly sensitive: regulate the ket-5 monoclonal antibody solution of the colloid gold label of pre-bag quilt on the reagent strip, the amount of ketamine-BSA complete antigen, the ketamine that contains in the test urine sample carries out sensitivity test.The result shows, the check-out console of the ketamine monoclonal antibody of colloid gold label of the present invention and complete antigen preparation, and the lowest detection amount of ketamine can reach 50ng/ml.
Good stability: with the ketamine monoclonal antibody reagent bar of colloid gold label, sensitivity is that 1000ng/ml places and is incubated 0.5d, 1d, 1.5d, 2d, 2.5d, 3d, 4d, 5d under 65 ℃ respectively, carries out the sensitivity test of 1000ng/ml by above-mentioned sensitivity test method then.The result shows that the ketamine monoclonal antibody of colloid gold label can tolerate 5 days under 65 ℃, have satisfactory stability.
Specificity is good: detect totally 40 kinds of drugs with ketamine monoclonal antibody immunity check-out console, result only ketamine and norketamine is positive, and other are negative.The drugs and the medicine that are used to detect are: ketamine, norketamine, morphine, methadone, pethidine, heroine, racephedrine, Sanedrine, the dextrorotation racephedrine, MDMA, morphine monomethyl ether, hemp, Cocaine, caffeine, chlorpromazine, Ibuprofen BP/EP, thebaine, tetrahydrocannabinol, lignocaine, Noscapine, alprazolam, but Fu Er pyridine, Ftorafur, Somigran, stable, triazolam, bisacodyl, diphenoxylate, nifedipine, Carbamzepine, the fast pyridine of sulphur azoles, coromegine, triamterene, haloperidol, the two Ecbolines of methylsulfonic acid, demethyl pethidine; Lepetan; totally 40 kinds of Phenylpropanolamine and phenylethylamines.
In sum, colloid gold label ketamine 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.Use check-out console of the present invention to detect ketamine, whole test can be finished in the 10min clock, and the sensitivity of detection can reach 50ng, with 38 kinds of common medicines, 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 the about 120 μ l of sample, 3～5min.Come judged result according to the fringe position that occurs, synoptic diagram is seen Fig. 8.
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 ketamine monoclonal antibody behind the colloid gold label of the present invention and complete antigen preparation, the lowest detection amount of ketamine can reach 50ng/mL.Consider in some normal medicine that uses and also contain the ketamine composition, in the real work for avoiding false-positive appearance, with reference to the concentration value of the current international practice, the threshold value of setting check-out console with 1000ng/mL for well.
Regulate the ket-5 monoclonal antibody solution of the colloid gold label of pre-bag quilt on the reagent strip, the amount of ketamine-BSA complete antigen, make that check-out console is 1000ng/ml to the lowest detection amount of ketamine.Ketamine concentration＜1000ng/ml in urine, the anti-ketamine 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; Ketamine concentration in urine〉1000ng/ml, the anti-ketamine monoclonal antibody of colloid gold label ketamine 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 immunoglobulin (Ig) 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 ketamine;
(2) the excellent and height of tiring of the specificity of anti-ketamine monoclonal antibody ket-5 of the present invention;
(3) colloid gold label ketamine 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.
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 activation of embodiment 1 ketamine and haptenic preparation
The vitriol oil that in reaction flask, adds ketamine raw material 0.52g and 10ml 98wt%, stirring makes it dissolving, place cryosel to bathe and be cooled to-5 ℃, in 1 hour, divide to add the mix acid liquor of forming by the sulfuric acid of the nitric acid of 0.25ml 70wt% and 0.75ml98wt% for 4 times.After adding, reaction is 2 hours under 0 ℃ of condition, pours into then in the 10g trash ice, after treating that trash ice melts, under 0 ℃ of condition, react half an hour again, when white solid is separated out, suction filtration gets white solid thing 0.62g (crude product, needn't purifying can be directly used in next step reaction).The reaction yield: 53%, reaction product is analyzed through MNR and is target compound: to nitro-N-ketamine.
0.5g is dissolved in the 1.4ml water with reaction product (promptly to the nitro ketamine), stirs the concentrated hydrochloric acid that adds 0.55ml 38wt% down, heats up gradually, begins to add zinc powder (dividing 4 batches) 0.62g altogether in the time of 40 ℃, until 55 ℃.After adding, reaction is 2 hours under this temperature condition, filter, filtrate is neutralized to weakly alkaline (pH=8) with the NaOH aqueous solution of 20wt%, and suction filtration, the white solid (for zinc hydroxide) that leaches is washed with warm water, merge the filtrate of collecting, use 30ml dichloromethane extraction 3 times, the combined dichloromethane solvent, be washed to neutrality with saturated common salt, use anhydrous sodium sulfate drying, volatilize solvent, get the semi-solid yellow substance, (the developping agent ratio is an ethyl acetate: sherwood oil=1:2), obtain light yellow solid 252mg with silica gel column chromatography.The reaction yield: 56%, reaction product is analyzed through MNR and is target compound, p-NH2-Ketamine (p-NH
2-Ketamine).
1H?NMR(DMSO)δ?6.40～6.97(m，4H，ArH)5.17(s，2H，-NH2)5.12(s，1H，-NH)1.98(s，3H，-NCH3)1.60～2.51(m，8H，CH2)
The preparation of embodiment 2 ketamines-KLH complete antigen
Take by weighing the 100mg p-NH2-Ketamine, under agitation it is dissolved in the 10ml water, slowly add the EDC (carbodiimide) of 400mg then, the limit edged shakes, and with 0.1M hydrochloric acid the pH value is adjusted to 4.5, continues reaction 10min at 25 ℃.Get 100mg hemocyanin (KLH) and be dissolved in the 5ml distilled water, join in the p-NH2-Ketamine solution, 25 ℃ are continued reaction 3 hours.Reaction product is dialysed under 4 ℃ with the 0.01M phosphate buffered saline buffer, removes unreacted p-NH2-Ketamine and EDC, exchange buffering liquid 3～4 times.Obtain ketamine-KLH complete antigen 120mg.
The preparation of embodiment 3 ketamines-BSA complete antigen
The preparation method just changes KLH into bovine serum albumin (BSA) with embodiment 2, makes ketamine-BSA complete antigen 120mg.
The operation of Balb/C mouse immune
Earlier with antigen and freund's adjuvant (Freund ' s adjuvant) emulsification, complete antigen ketamine-KLH is mixed with the solution of 1mg/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 μ L.The 14th day, again to every mouse peritoneal injection incomplete Freund's adjuvant emulsification antigen 1 00 μ L.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 μ l once more.In cytogamy reaction the last week, with 100 μ g/100 μ l antigen physiological saline booster immunization once more.After measured, the gained antiserum titre is 1:6400 after the blood sampling, sees embodiment 5.
Cytogamy and cloning
Press the method for document " Ed Harlow, David Lane, Using Antibodies:A LaboratoryManual.1999 " description and carry out cytogamy and cloning operation.
The cytogamy situation is checked in the cytogamy operation after 3 days, add HT after 8 days
+Perfect medium, every hole adds 1ml.
Between 12 days, just can observe the clone of hybrid cell.Long during to about 1mm when clone's diameter, the elisa plate that wraps quilt with ketamine-BSA filters out the hybridoma of anti-ketamine.The positive colony that screens carries out cloning with limiting dilution assay, screens the specific hybrid oncocyte of anti-ketamine with the ELISA method enzyme plate of ketamine-BSA bag quilt.Behind five time clonings, obtain a strain and can secrete the anti-ketamine monoclonal cell of specificity antibody (depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number are CGMCCNo.1404).
Embodiment 5 ketamines-KLH immune mouse antiserum titre (titre) is measured
Get among the embodiment 4 through 4 mice immunized, measure sero-fast titre with the elisa plate of ketamine-BSA bag quilt.Envelope antigen is diluted in 10 μ g/ml in the 0.05M carbonate buffer solution of pH9.6, and 100 μ l/ holes join 96 hole enzyme plates and spend the night in 4 ℃.Sealed 2 hours down at 37 ℃ with 1% gelatin/PBS after discarding coating buffer, wash plate 3 times with the PBS-Tween-20 washing lotion then, add the ketamine antiserum(antisera) 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 the horseradish peroxidase-labeled of 1:2000 resists more, hatch after 1 hour for 37 ℃ and wash plate 3 times, add the TMB/H202 substrate 10min that develops the color with the PBS-Tween-20 washing lotion, and with stop buffer (0.1N sulfuric acid) color development stopping.
Under the 450nm optical wavelength, measure absorption value (0D),, adopt the t check of measurement data, get minimum extent of dilution the tiring of P＜0.05 as this antibody with the OD average comparison in blank hole.Through colorimetric estimation, sero-fast the tiring of gained ketamine is 1:6400, sees Fig. 1.
Press Hodgkin, P.D., J.H.Lee, and AB Lyons (1996) B cell differentiationand isotype switching is related to division cycle number.J.Exp.Med.184, the method for describing in 277. is carried out the fusion efficiencies analysis.
Briefly, apparent green with the lymphocyte of CFSE green fluorescence preliminary making, use the SP2/0 cell of the anti-mouse B220 monoclonal antibody preliminary making of PE mark to show red, the hybridoma of two kinds of cytogamy formation has Two Colour Fluorescence.Analyze two fluorocyte ratios through cells were tested by flow cytometry, be used to reflect fusion efficiencies.As shown in Figure 2,13 represent SP2/0 (myelomatosis) cell of the anti-mouse B220 monoclonal antibody preliminary making of PE mark, and 14 represent CFSE green fluorescence preliminary making lymphocyte, and 15 representatives have the hybridoma of Two Colour Fluorescence.
The initial fusion efficiencies of splenocyte and SP2/0 is about 34% in this experimental system, and it is 13.8% that the ELISA method is measured positive rate.
Wherein CFSE is a green fluorescence dyestuff carboxyethyl germanium sesquioxide, purchases the RENOVAR.INC company in the U.S.; PE is phycoerythrin (a commercialization fluorescin), and B220 is the mouse cell surface antigen, and the anti-mouse B220 monoclonal antibody of PE mark is purchased the company in RENOVAR.INC.
The mass preparation of embodiment 7 Ket-5 monoclonal antibodies
The ascites preparation
Cultivate the monoclonal cell strain of embodiment 4 preparations, get 10
6-10
7Individual hybridoma is inoculated in the mouse peritoneal of sensitization.Visible belly obviously swells in 3 weeks.Extraction ascites is centrifugal, adds the sodium azide of 0.02wt%, preserves in 4 ℃ of refrigerators.
Purifying antibody (Sepharose-G albumen affinity chromatography)
Get 10ml ascites, use earlier saturated ammonium sulphate precipitation, again to the phosphoric acid buffer dialysis of pH=8, and with the protein concentration under the ultraviolet absorption method mensuration 280nm.
Get 1g CL-4B Sepharose-Protein G, be suspended in the phosphate buffer solution (pH=8.0) of 200ml, at least imbibition 30min.Get about 4ml dress post, successively wash post, use preceding a kind of damping fluid balance again then with the phosphoric acid buffer 100ml of pH=8.0 and the 0.1M Trisodium Citrate 100ml of pH=3.
The CL-4B Sepharose-Protein G glue of every 1ml imbibition can combine with the IgG of 23mg, and controlling column flow rate is 8.5ml/min, detects the A of effluent liquid
280nm10mM phosphoric acid buffer (pH=8) with 5ml is washed post; Repeat once, most of IgA, IgM, IgG3 will flow out in this fraction.With 0.1M Trisodium Citrate (pH=6) the wash-out IgG1 antibody of 5ml,, use the wash-out IgG2b of the 0.1M sodium citrate buffer solution (pH=3.5) of 5ml with 0.1M sodium citrate buffer solution (pH=4.5) the wash-out IgG2a of 5mL.
Make antibody 20mg, called after Ket-5.
The evaluation of embodiment 8 Ket-5 antibody
Molecular weight determination
Reduce with 2-ME (beta-mercaptoethanol) by the antibody of " molecular cloning " (Science Press, second edition, 2002) method to embodiment 7 preparations, carry out polyacrylamide gel electrophoresis, recording Ket-5 antibody molecule amount is 55Kd, sees Fig. 3.
Type is measured
(monoclonal antibodyisotyping kit identifies that PIERCE) working method case test kit specification sheets carries out to the specific antibody parting kit that Ket-5 antibody is produced through PIERCE company.Qualification result is the IgG1 type for Ket-5 antibody, the kappa hypotype.
The titration of embodiment 9 Ket-5 antibody
Measure the titre of antibody according to the method for embodiment 5, tiring of Ket-5 monoclonal antibody is 1:3200, sees Fig. 4.
With complete antigen ketamine-BSA and BSA coated elisa plate, ketamine monoclonal antibody Ket-5 combination with it with the 1:2000 dilution, carrying out integrated enzyme reaction detects, the result shows that ketamine monoclonal antibody Ket-5 can detect ketamine-BSA of 50ng, and with BSA without any cross reaction.(see figure 5)
The colloid gold label of embodiment 11 Ket-5 antibody
Radioactive colloidal gold (Gold Colloidal) granules preparation
The preparation of employing trisodium citrate reduction method.Get the 247.5ml water boil, the 1wt% chlorauric acid solution of dissolving 2.5ml, in 90 ℃ of insulation 2min, 1% trisodium citrate aqueous solution that adds 7.5ml, continue to boil 5min, 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.
Radioactive colloidal gold-monoclonal antibody binding substances preparation
Get above-mentioned Radioactive colloidal gold 100ml, the pH that slowly is added dropwise to 10ml is 9.0 borate buffer, dropwises the back and add the anti-ketamine antibody of 1.5mg rapidly under magnetic force stirs fast, continues to stir 15min.The bovine serum albumin that adds the 10wt% of 2ml, behind the restir 15min, with 12000rpm/min, centrifugal 30min under 4 ℃ inhales and removes supernatant liquor, and the borate buffer suspension precipitation that contains 0.1% sodium azide with 10ml is drawn preservation.
The preparation of embodiment 12 check-out consoles
(1) preparation of Radioactive colloidal gold-monoclonal antibody binding substances glass fibre bar
With the Ket-5 antibody of the colloid gold label of embodiment 11 preparation and pH is that 7.4 10mM phosphate buffer soln mixes, and is mixed with the solution of 0.5mg/ml concentration, is coated on equably on the glass fibre element paper, and glue spread is 50 μ l/cm
2, vacuum-drying.
(2) detection line 12 and nature controlling line 11
Detection line 12: with ketamine-BSA complete antigen and pH is that 7.4 10mM phosphate buffer soln mixed preparing becomes the mixing solutions that concentration is 0.5mg/ml, is sprayed on the nitrocellulose membrane, and glue spread is 10 μ l/cm
2Nature controlling line 11: with goat anti-rabbit igg how anti-with pH be 7.4 10mM phosphate buffer soln mixed preparing to become concentration be the 0.8mg/ml mixing solutions, be sprayed on the nitrocellulose membrane, glue spread is 10 μ l/cm
2
Then 15～35 ℃ of dryings.
(3) test strip assembling
With filter sample paper 1, be coated with the Ket-5 antibody of colloid gold label glass fiber paper 2, be coated with ketamine-BSA (detection line) and goat anti-rabbit igg how nitrocellulose filter 3, the thieving paper of anti-(nature controlling line) overlap composition test strip 4 successively, as shown in Figure 6; Test strip is placed back up pad 10, fix, as shown in Figure 7 with adhesive tape; With the cover plate coverage test bar and the back up pad that are provided with well and viewing window, well is positioned at filter sample paper 1 place, and viewing window is positioned at nitrocellulose filter 3 places that have detection line and nature controlling line, as Fig. 8.
The preparation of embodiment 13 check-out consoles
The preparation method is with embodiment 12, wherein:
With the Ket-5 antibody of the colloid gold label of embodiment 11 preparation and pH is that 7.4 10mM phosphate buffer soln mixes, and is mixed with the solution of 1.5mg/ml concentration;
The concentration of ketamine-BSA complete antigen solution is 1mg/ml;
The concentration of the how anti-solution of goat anti-rabbit igg is 1.2mg/ml.
The sensitivity test of embodiment 14 colloid gold label ketamine monoclonal antibody plate for detecting immunity
The reagent preparation
According to test request, in blank urine sample, add ketamine, be mixed with respectively and contain the serial sample that ketamine concentration is 1ng/ml, 2ng/ml, 3ng/ml, 4ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, 300ng/ml, 500ng/ml, 1000ng/ml, 1500ng/ml and 2000ng/ml.
Detect and the result
Above-mentioned series concentration is made quantitative gradient point sample, and sensitivity test the results are shown in Table 1.
The sensitivity test result of table 1 plate for detecting immunity
| Point sample amount (ng/ml) | 1 | 2 | 3 | 4 | 5 | 10 | 20 | 50 |
| |
- | - | - | - | - | - | - | |
| Embodiment | ||||||||
| 13 check-out console test-results | - | - | - | - | - | - | - | - |
| Point sample amount (ng/ml) | 100 | 150 | 200 | 300 | 500 | 1000 | 1500 | 2000 |
| |
+ | + | + | + | + | + | + | + |
| |
- | - | - | - | - | + | + | + |
Annotate: "+" expression is positive; "-" expression is negative.
Test-results shows that the plate for detecting immunity lowest detection amount of embodiment 12 preparations is 50ng/ml, and the plate for detecting immunity lowest detection amount of embodiment 13 preparations is 1000ng/ml.
The stability test of embodiment 15 colloid gold label ketamine monoclonal antibody plate for detecting immunity
With the sensitivity of embodiment 13 preparation is that the test strip of the check-out console of 1000ng/ml places under 65 ℃ and is incubated 0.5,1,1.5,2,2.5,3,4,5 day respectively, carries out the sensitivity test of 1000ng/ml by above-mentioned sensitivity test method then.
Test result under the above-mentioned condition sees Table 2
Table 2 stability test result
| Shelf time (my god) | 0.5 | 1 | 1.5 | 2 | 2.5 | 3 | 3.5 | 4 | 5 |
| The result | + | + | + | + | + | + | + | + | + |
Annotate: "+" expression is positive
The result shows that the ketamine monoclonal antibody of colloid gold label can tolerate 5 days under 65 ℃, have satisfactory stability.
The specific reaction test of embodiment 16 colloid gold label ketamine monoclonal antibody plate for detecting immunity
The material of selecting for use to be detected
Ketamine, the demethyl ketamine, morphine, amphetamine, methadone, pethidine, heroine, racephedrine, Sanedrine, the dextrorotation racephedrine, MDMA, morphine monomethyl ether, hemp, Cocaine, caffeine, chlorpromazine, Ibuprofen BP/EP, thebaine, tetrahydrocannabinol, lignocaine, Noscapine, alprazolam, but Fu Er pyridine, Ftorafur, Somigran, stable, triazolam, bisacodyl, diphenoxylate, nifedipine, Carbamzepine, the fast pyridine of sulphur azoles, coromegine, triamterene, haloperidol, the two Ecbolines of methylsulfonic acid, demethyl pethidine; Lepetan; Phenylpropanolamine and phenylethylamine be totally 40 kinds of drugs and medicine.
Detect and the result
Above-mentioned detection material is mixed with the solution of series concentration, ketamine monoclonal antibody colloid gold label check-out console with embodiment 12 preparations detects, the result shows that the ketamine monoclonal antibody colloid gold label check-out console with this antibody producing only with ketamine and demethyl ketamine 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 ketamine monoclonal antibody plate for detecting immunity of preparation is functional, and specificity is strong.Test result sees Table 3.
The specific reaction test result of table 3 check-out console
| Sample concentration (ng/ml) | 50 | 100 | 150 | 200 | 300 | 400 | 500 | 1000 | 1500 | 2000 |
| Ketamine | + | + | + | + | + | + | + | + | + | + |
| Norketamine | + | + | + | + | + | + | + | + | + | + |
| Amphetamine | - | - | - | - | - | - | - | - | - | - |
| Morphine | - | - | - | - | - | - | - | - | - | - |
| Methadone | - | - | - | - | - | - | - | - | - | - |
| Pethidine | - | - | - | - | - | - | - | - | - | - |
| Heroine | - | - | - | - | - | - | - | - | - | - |
| Racephedrine | - | - | - | - | - | - | - | - | - | - |
| Sanedrine | - | - | - | - | - | - | - | - | - | - |
| The dextrorotation racephedrine | - | - | - | - | - | - | - | - | - | - |
| MDMA | - | - | - | - | - | - | - | - | - | - |
| Morphine monomethyl ether | - | - | - | - | - | - | - | - | - | - |
| Hemp | - | - | - | - | - | - | - | - | - | - |
| Cocaine | - | - | - | - | - | - | - | - | - | - |
| Caffeine | - | - | - | - | - | - | - | - | - | - |
| Chlorpromazine | - | - | - | - | - | - | - | - | - | - |
| Ibuprofen BP/EP | - | - | - | - | - | - | - | - | - | - |
| Thebaine | - | - | - | - | - | - | - | - | - | - |
| Tetrahydrocannabinol | - | - | - | - | - | - | - | - | - | - |
| Lignocaine | - | - | - | - | - | - | - | - | - | - |
| Noscapine | - | - | - | - | - | - | - | - | - | - |
| Alprazolam | - | - | - | - | - | - | - | - | - | - |
| But Fu Er pyridine | - | - | - | - | - | - | - | - | - | - |
| Ftorafur | - | - | - | - | - | - | - | - | - | - |
| Somigran | - | - | - | - | - | - | - | - | - | - |
| Stable | - | - | - | - | - | - | - | - | - | - |
| Triazolam | - | - | - | - | - | - | - | - | - | - |
| Bisacodyl | - | - | - | - | - | - | - | - | - | - |
| Diphenoxylate | - | - | - | - | - | - | - | - | - | - |
| Nifedipine | - | - | - | - | - | - | - | - | - | - |
| Carbamzepine | - | - | - | - | - | - | - | - | - | - |
| The fast pyridine of sulphur azoles | - | - | - | - | - | - | - | - | - | - |
| Coromegine | - | - | - | - | - | - | - | - | - | - |
| Triamterene | - | - | - | - | - | - | - | - | - | - |
| Haloperidol | - | - | - | - | - | - | - | - | - | - |
| The two Ecbolines of methylsulfonic acid | - | - | - | - | - | - | - | - | - | - |
| The demethyl pethidine | - | - | - | - | - | - | - | - | - | - |
| Lepetan | - | - | - | - | - | - | - | - | - | - |
| Phenylpropanolamine | - | - | - | - | - | - | - | - | - | - |
| Phenylethylamine | - | - | - | - | - | - | - | - | - | - |
In the present embodiment, preparation method in the employing U.S. Pat 2003/224447 has produced the haptens (to call comparison A in the following text) of formula A, and this haptens and haptens of the present invention are compared with the coupled efficient of carrier proteins and the competing reaction of corresponding complete antigen and ketamine standard substance separately.
Coupled efficient with carrier proteins
Take by weighing 100mg respectively to amino-ketamine and comparison A, under agitation be dissolved in the 10ml distilled water, slowly add 400mgEDC then, the limit edged shakes, and with 0.1M hydrochloric acid the pH value is adjusted to 4.5, and at room temperature (20～25 ℃) continue reaction 10 minutes.Get 100mg bovine serum albumin (BSA) and be dissolved in the 5ml distilled water, join in amino-ketamine solution or the comparison A solution, (4～25 ℃) continue reaction 2-12 hour under the room temperature.Reaction product is dialysed for 4 ℃ to the 0.01M phosphate buffered saline buffer, remove unreacted to amino-ketamine or comparison A and EDC, exchange buffering liquid 3～4 times.The reaction product that obtains is ketamine-BSA complete antigen or comparison A-BSA complete antigen.
Test-results shows that the solvability of comparison A is not good, and is lower to the coupled efficient of carrier proteins, only is about 12%.And that the prepared haptens of this patent method is easy to carry out is coupled, and coupled efficient is 54%.
Competing reaction with the ketamine standard substance
The ketamine standard substance that add 1mg/ml respectively in the ketamine of the present invention-BSA complete antigen of different extension rates and comparison A-BSA complete antigen are measured the OD value and are assessed the various complete antigens of each extension rate and the competing reaction degree between the ketamine standard substance with this under 450nm.
Test-results is shown in Fig. 9 A and 9B.The complete antigen of comparison A is when adding the ketamine standard substance of 1mg/ml, and association reaction is unaffected substantially.And complete antigen of the present invention is when adding the ketamine standard substance of 1mg/ml, and association reaction is suppressed by 100%.
The result show comparison A complete antigen can not with the reaction that is at war with of ketamine standard substance, therefore can not be used for preparing the Radioactive colloidal gold fast diagnosis reagent.
Culture presevation
Hybridoma cell line of the present invention is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC, China, Beijing) on June 30th, 2005, and preserving number is CGMCC No.1404.
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 (11)
1. a haptens is characterized in that, described haptens has the structure shown in the formula 1:
Formula 1.
2. a complete antigen is characterized in that, described complete antigen has the structure shown in the formula 2:
Formula 2
Wherein, X is a protein carrier.
3. the method for preparation formula 2 described complete antigens is characterized in that, described method comprises step:
(a) ketamine is carried out nitrated, insert nitryl group in its contraposition, to form to nitro-ketamine;
(b) will be amino to the nitroreduction in nitro-ketamine, to make p-NH2-Ketamine;
(c) p-NH2-Ketamine is connected with protein carrier, to make the complete antigen shown in the formula 2.
4. the purposes of described haptens of claim 1 or the described complete antigen of claim 2 is characterized in that, is used to prepare the ketamine monoclonal antibody specific.
5. a monoclonal antibody is characterized in that, its specificity is incorporated into ketamine, and described monoclonal antibody is that CGMCC No.1404 is produced by mouse hybridoma cell.
6. one kind produces the hybridoma cell line that specificity is incorporated into the monoclonal antibody of ketamine, it is characterized in that, it is that mouse hybridoma cell is CGMCC No.1404.
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 ketamine.
8. whether there is the method for ketamine 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 ketamine.
9. a check-out console is characterized in that, described check-out console comprises: substrate and test strip, described test strip contain the described monoclonal antibody of claim 5.
10. check-out console as claimed in claim 9 is characterized in that, described 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 claim 9 or 10 described check-out console and working instructions.
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| CN101949925B (en) * | 2010-10-22 | 2013-07-31 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatographic test strip for rapidly testing prednisone acetate and derivative thereof and preparation method |
| CN101980021A (en) * | 2010-10-22 | 2011-02-23 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatography test strip for quickly detecting sibutramine and derivative thereof and preparation method |
| CN101957376A (en) * | 2010-10-22 | 2011-01-26 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatography test paper for rapidly detecting guanidineacetic acid and derivatives thereof and preparation method thereof |
| CN101986156A (en) * | 2010-10-22 | 2011-03-16 | 南通市伊士生物技术有限责任公司 | Colloidal gold chromatography test paper for detecting glibenclamide and derivatives of glibenclamide quickly and preparation method thereof |
| CN102128930A (en) * | 2010-12-08 | 2011-07-20 | 沈阳大学 | Colloidal gold drug test card for morphine-ketamine |
| CN103159669B (en) * | 2011-12-15 | 2015-09-30 | 曾立波 | Pethidine monoclonal antibody and plate for detecting immunity and test kit |
| CN102539773B (en) * | 2011-12-31 | 2014-07-16 | 上海凯创生物技术有限公司 | KET (Ketamine) colloid gold detection kit and preparation method thereof |
| CN102636647B (en) * | 2012-03-31 | 2014-05-21 | 戴国华 | Ketamine-collaurum test paper for detection of saliva |
| CN103060275B (en) * | 2012-12-29 | 2014-07-09 | 杭州傲锐生物医药科技有限公司 | Ketamine hybridoma cell strain and preparation method and application thereof |
| CN104558140A (en) * | 2013-10-29 | 2015-04-29 | 艾博生物医药(杭州)有限公司 | Preparation method of artificial antigen of ketamine |
| CN110981949B (en) * | 2019-11-26 | 2020-11-06 | 杭州隆基生物技术有限公司 | Preparation method of ketamine antigen |
| CN114057881B (en) * | 2021-12-28 | 2023-10-13 | 杭州贤至生物科技有限公司 | Anti-ketamine specific antibodies, plasmid vectors and methods |
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