CN101308139B - Norketamine gold mark detection test paper box and method for making same - Google Patents
Norketamine gold mark detection test paper box and method for making same Download PDFInfo
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- CN101308139B CN101308139B CN 200810131795 CN200810131795A CN101308139B CN 101308139 B CN101308139 B CN 101308139B CN 200810131795 CN200810131795 CN 200810131795 CN 200810131795 A CN200810131795 A CN 200810131795A CN 101308139 B CN101308139 B CN 101308139B
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Abstract
The invention relates to a norketamine gold-labeled test paper detection kit, which can rapidly, easily and sensitively detect the norketamine, therefore, the invention also provides a preparation method of the detection kit. The detection kit includes a test strip which is packaged in a box shell body, and a sample injection hole and an observation hole are arranged on the box shell body, the test paper detection kit is characterized in that: the test strip takes polyvinyl chloride or polyethylene as a back lining, a sample injection area at one end of the test strip is affixed and adsorbed with a glass fiber film of a conjugate of colloidal gold and anti- norketamine monoclonal antibody; a cellulose nitrate film is affixed to a testing area in the middle of the test strip, a small section of overlapping area is arranged at the connecting part of the cellulose nitrate film and the glass fiber film, and a water absorption area at the other end of the test strip is affixed with a piece of absorbent paper; a detection line with material of norketamine-egg white albumin and a quality control line with material of goat anti-mouse are arranged in the direction from the sample injection area to the water absorption area sequentially, and the cellulose nitrate film is covered with a bovine serum albumin layer with the concentration of 1 percent; and the sample injection hole is corresponding to the glass fiber film in the sample injection area of the test strip, and the observation hole is corresponding to the testing area of the test strip.
Description
(1) technical field
The present invention relates to the detection of illegal drug, belong to biological technical field, be specially a kind of Norketamine gold mark detection test paper box and preparation method thereof.
(2) background technology
Ketamine (Ketamine) is a kind of fugitive anesthetic through intravenously administrable, and major part got into brain tissue after it got into blood circulation, and then was distributed in the body tissue, and liver, lung and intrafat drug concentration are also higher.This drug main will bio-transformation become Norketamine (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 anesthetic of great majority, the spinoff of respiratory function, in clinic trial for many years.
As far back as June calendar year 2001, ketamine has been included in second type of psychotropic substances of country and has been managed.Because ketamine is the principal ingredient 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 criminal case relevant with ketamine rises and the day by day strictness of country to the ketamine management year by year, have higher requirement to the detection of Norketamine in ketamine and the drug abuse person's biological sample in this area.Because the half life period of ketamine metabolism is 3-4 hour; After taking in the body; Form demethyl ketamine (norketamine), the dehydrogenation demethyl ketamine (Dehydronorketamine) that can produce by means of cytochrome enzyme P450 through the N Demethylation, see Fig. 3 with the ketamine same effect.
The content of metabolin is than higher in biological material (like blood, urine, hair etc.); Therefore as detecting index; The detection of ketamine metabolin (mainly being the demethyl ketamine) is more important than the detection of ketamine, and is also more effective to the affirmation of whether sucking ketamine.At present, the analytical approach of detection ketamine and metabolin thereof mainly contains: (one) vapor-phase chromatography (GC); (2) gas-matter coupling method (GC-MS); (3) high performance liquid chromatography (HPLC); (4) liquid-matter coupling method (LC-MS); (5) high performance capillary electrophoresis (HPCE) etc.These chromatographic processes have good sensitivity and specificity, but complex operation needs expensive instrument and equipment, and length consuming time.Therefore, this area presses for a kind of detection method and detectable that can detect Norketamine quicker, simple and easy, delicately.
(3) summary of the invention
To the problems referred to above, the invention provides a kind of Norketamine gold mark detection test paper box, it can detect Norketamine quick, simple and easy, delicately, and for this reason, the present invention also provides the preparation method of Norketamine gold mark detection test paper box.
Technical scheme of the present invention:
Norketamine gold mark detection test paper box; Comprise test strips; Said test strips is packaged in the box housing, has above the said box housing to annotate appearance hole, observation port; It is characterized in that: test strips is made backing with PVC or tygon, pastes the glass fibre membrane of absorption collaurum-anti-Norketamine monoclonal antibody bond in the notes appearance district of test strips one end; Nitrocellulose filter is pasted in test section in the middle of test strips, and there is a bit of overlay region nitrocellulose filter and glass fibre membrane junction, and a bit of of glass fibre membrane overlaps on the nitrocellulose filter, pastes thieving paper in test strips other end suction zones; On nitrocellulose filter, from annotating the direction that appearance is distinguished suction zones, set gradually that material is made detection line for the Norketamine oralbumin and material is the sheep anti mouse nature controlling line, being coated with concentration on the said nitrocellulose filter and being 1% is bovine serum albumin(BSA); The glass fibre membrane in appearance district is annotated corresponding to test strips in the said appearance hole site of annotating, and said observation port is corresponding to the test section of test strips;
It is further characterized in that: said cellulose nitrate film thickness is 120 μ m; The protein load amount is 5 μ g/cm
2～20 μ g/cm
2PVC or tygon backing thickness are 100 μ m; Test strips is of a size of (55～65) mm * (3～5) mm; The width of detection line and nature controlling line is 0.5mm～1mm;
The preparation method of Norketamine gold mark detection test paper box is characterized in that:
(1) preparation of Norketamine gold mark detection test paper bar; Collaurum-anti-Norketamine monoclonal antibody bond is diluted to 0.5 μ g/mL～2 μ g/mL; Put into 10mm * 300mm glass fibre membrane, soaked 37 ℃ of oven dry 10 minutes～20 minutes; 4 ℃～8 ℃ preservations stick on the notes appearance district of backing one end; The material of backing is PVC or tygon; PVC or tygon backing thickness are 100 μ m; Paste nitrocellulose filter in backing intermediate detection district; The cellulose nitrate film thickness is 120 μ m, and spraying concentration is that 100 μ g/mL～500 μ g/mL NKET-OA bonds are made detection line on nitrocellulose filter, and spraying concentration is that 50 μ g/mL～200 μ g/mL sheep anti-mouse iggs are made nature controlling line; The width of detection line and nature controlling line is 0.5mm～1mm, again with 1% bovine serum albumin(BSA) sealing nitrocellulose filter; The protein load amount is 5 μ g/cm
2～20 μ g/cm
2, paste thieving paper in backing other end suction zones;
(2) have above gained Norketamine (NKET) gold mark detection test paper bar is encapsulated in the box housing of annotating appearance hole and observation port.
It is further characterized in that:
The preparation of collaurum: the chlorauric acid solution of getting 50ml 0.01%; Under the 1000r/min magnetic agitation, be heated to 110 ℃, add 1% sodium citrate aqueous solution 2ml rapidly; Keep temperature of reaction and speed of agitator constant; Boiling reaction 5min, solution colour stop reaction after just having become limpid Chinese red, and the collaurum mean grain size of acquisition is 10.9nm;
The preparation of anti-Norketamine monoclonal antibody and solution thereof
50 μ g NKET-BSA coupled antigens are made into 1 μ g/ μ l antigenic solution with 50 μ l physiological saline, and are fully emulsified with the complete freund adjuvant mixing of equal-volume, supply first immunisation to use; Replace complete freund adjuvant with incomplete freund adjuvant during booster immunization with amount; First immunisation adopts directly injection in the mouse peritoneal; Immunizing dose is 50 a μ g/ mouse, and later on whenever at a distance from 2 all booster immunizations once, booster immunization adopts tail vein injection; Immunizing dose 10 μ g/ only; Intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with 10: 1 mixed; Centrifugal, remove supernatant; In 50～90S, 1ml 50% polyglycol (molecular weight is 1500) is added in the cell, fully mixing makes its fusion, adds 20ml DEM nutrient solution behind the 1min, stops merging; Centrifugal behind the static 10min of water-bath, remove supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 20% calf serum, be 1 * 10 with final concentration
4Feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 5%CO
2, cultivate under 37 ℃ of conditions, after 8 days, every culture hole is changed 2/3 HT nutrient solution; After 10 days～20 days, begin microscopy is had the culture hole of hybridoma clonal growth, get supernatant and screen, the cell that testing result is positive is cloned with limiting dilution assay immediately; The hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out; With NKET-OVA is envelope antigen, with the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A
Test-A
Blank)/(A
Contrast-A
Blank)＞2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope, dilution is that 70/ml gets 20 times of 1ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone; Till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.3～0.5ml/ of 10～13 all age, after 8 days～10 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only; Note after 5 days observing, collect ascites, centrifugal removal deposition, glycerol adding is in-20 ℃ of preservations;
Freund adjuvant is to be formed whiteruss by following material mixture ratio fully: sheep oil: BCG vaccine=12g: 20ml: 0.105g; Not exclusively freund adjuvant is to be formed whiteruss by following material mixture ratio: sheep oil=12g: 20ml;
The preparation of collaurum-anti-Norketamine monoclonal antibody bond: in the colloidal gold solution of certain volume, slowly drip 0.1mg/ml Norketamine monoclonal anti liquid solution, be dissolved in the PBS damping fluid of 0.01M pH7.4; Ultrasound wave mixing 2min carries out purifying through 4 ℃ of refrigerated centrifuges after leaving standstill 0.5h, first centrifugal rotational speed 10000r/min; Time 45min; Abandon supernatant, deposition is dissolved with 10%BSA solution, and it is centrifugal to carry out next time; For the second time with 10%～30% glycerine density gradient centrifugation, rotating speed 7000r/min, time 45min is used to remove unlabelled albumen and gold grain aggregation, and collection centre bronzing partly gets final product.
Beneficial effect of the present invention: the present invention is used for detecting the content of sample Norketamine; Only need during detection test sample is splashed in the notes appearance hole, slightly, can in the area of observation coverage, observe the variable color situation of detection line and nature controlling line; Confirm whether Norketamine content exceeds standard in the sample; Compare with existing method of testing, do not need large-scale detecting instrument, detect quick, accurate, obvious, highly sensitive.
(4) description of drawings
Fig. 1 is that Norketamine (NKET) gold mark detection test paper box detects synoptic diagram;
Fig. 2 Norketamine (NKET) gold mark detection test paper bar structural drawing;
Fig. 3 is ketamine and metabolic product figure thereof.
(5) embodiment
See Fig. 1, Fig. 2; Norketamine (NKET) gold mark detection test paper box, it comprises test strips 2, test strips 2 is packaged in box housing 1; Have above the box housing 1 and annotate appearance hole 8; Observation port 9, test strips 2 usefulness PVC or tygon are made backing, paste the glass fibre membrane 3 of absorption collaurum-anti-Norketamine (NKET) monoclonal antibody bond in the notes appearance district of test strips 2 one ends; Nitrocellulose filter 4 is pasted in test section in the middle of test strips; Nitrocellulose filter 4 has a bit of overlay region with glass fibre membrane 3 junctions; The a bit of of glass fibre membrane 3 overlaps on the nitrocellulose filter 4, pastes thieving paper 7 in test strips 2 other end suction zones; On nitrocellulose filter 4 from annotating the direction that appearance is distinguished suction zones; Setting gradually material is that Norketamine (NKET)-oralbumin is made detection line 5; With material be sheep anti-mouse igg nature controlling line 6, being coated with concentration on the nitrocellulose filter 4 and being 1% is bovine serum albumin(BSA); Annotate appearance 8 positions, hole and annotate the glass fibre membrane 3 in appearance district corresponding to test strips 2, observation port 9 is corresponding to the test section of test strips 2.Nitrocellulose filter 4 thickness are 120 μ m; The protein load amount is 5 μ g/cm
2-20 μ g/cm
2PVC or tygon backing thickness are 100 μ m; Test strips is of a size of (55～65) mm * (3～5) mm; The width of detection line 5 and nature controlling line 6 is 0.5mm～1mm;
The preparation method of Norketamine (NKET) gold mark detection test paper box
The preparation of collaurum: the chlorauric acid solution of getting 50ml 0.01%; Under the 1000r/min magnetic agitation, be heated to 110 ℃, add 1% sodium citrate aqueous solution 2ml rapidly; Keep temperature of reaction and speed of agitator constant; Boiling reaction 5min, solution colour stop reaction after just having become limpid Chinese red, and the collaurum mean grain size of acquisition is 10.7nm;
The preparation of anti-Norketamine (NKET) monoclonal antibody and solution thereof
50 μ g NKET-BSA coupled antigens are made into 1 μ g/ μ l antigenic solution with 50 μ l physiological saline, and are fully emulsified with the complete freund adjuvant mixing of equal-volume, supply first immunisation usefulness, replace complete freund adjuvant with the incomplete freund adjuvant with amount during booster immunization; First immunisation adopts directly injection in the mouse peritoneal, and immunizing dose is 50 a μ g/ mouse, and later on whenever at a distance from 2 all booster immunizations once, booster immunization adopts tail vein injection; Immunizing dose 10 μ g/ only, intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with 10: 1 mixed; Centrifugal, remove supernatant, in 50S～90S, 1ml 50% polyglycol (molecular weight is 1500) is added in the cell; Fully mixing makes its fusion, adds 20ml DEM nutrient solution behind the 1min; Stop merging, centrifugal behind the static 10min of water-bath, remove supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 20% calf serum, be 1 * 10 with final concentration
4Feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 5%CO
2, cultivate under 37 ℃ of conditions, after 8 days, every culture hole is changed 2/3 HT nutrient solution; After 10 days～20 days; Begin microscopy is had the culture hole of hybridoma clonal growth, get supernatant and screen, the cell that testing result is positive is cloned with limiting dilution assay immediately; The hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out; With NKET-OVA is envelope antigen, with the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A
Test-A
Blank)/(A
Contrast-A
Blank)＞2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope, dilution is that 70/ml gets 20 times of 1ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone; Till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.3ml/ of 10～13 all age～0.5ml/, after 8 days～10 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only, note after 5 days observing is collected ascites, centrifugal removal deposition, and glycerol adding is in-20 ℃ of preservations;
Freund adjuvant is to be formed whiteruss by following material mixture ratio fully: sheep oil: BCG vaccine=12g: 20ml: 0.105g; Not exclusively freund adjuvant is to be formed whiteruss by following material mixture ratio: sheep oil=12g: 20ml;
The preparation of collaurum-anti-Norketamine (NKET) monoclonal antibody bond: in the colloidal gold solution of certain volume, slowly drip 0.1mg/ml Norketamine (NKET) monoclonal anti liquid solution (being dissolved in the PBS damping fluid of 0.01M pH7.4), ultrasound wave mixing 2min; Carry out purifying through 4 ℃ of refrigerated centrifuges after leaving standstill 0.5h; Centrifugal rotational speed 10000r/min first, time 45min abandons supernatant; Deposition is dissolved with 10%BSA solution, and it is centrifugal to carry out next time; For the second time with 10%～30% glycerine density gradient centrifugation, rotating speed 7000r/min, time 45min is used to remove unlabelled albumen and gold grain aggregation, and collection centre bronzing partly gets final product;
The preparation of Norketamine (NKET) gold mark detection test paper bar is diluted to 0.5 μ g/mL～2 μ g/mL with collaurum-anti-Norketamine (NKET) monoclonal antibody bond, and the best is 1.25 μ g/mL; Put into 10mm * 300mm glass fibre membrane, soaked 10 minutes～20 minutes, Best Times is 15 minutes; 37 ℃ of oven dry; 4 ℃～8 ℃ preservations, optimum temperature is 6 ℃, sticks on the notes appearance district of backing one end; The material of backing is PVC or tygon; PVC or tygon backing thickness are 100 μ m; Paste nitrocellulose filter in backing intermediate detection district; The cellulose nitrate film thickness is 120 μ m, and spraying concentration is that 100 μ g/mL～500 μ g/mL NKET-OVA bonds are made detection line (best spraying concentration is 300 μ g/mL) on nitrocellulose filter, and spraying concentration is that 50 μ g/mL～200 μ g/mL sheep anti-mouse iggs are made nature controlling line (best spraying concentration is 125 μ g/mL); The width of detection line and nature controlling line is 0.5mm～1mm (optimum width 0.75mm), again with 1% bovine serum albumin(BSA) sealing nitrocellulose filter; The protein load amount is 5 μ g/cm
2-20 μ g/cm
2, (optimum load amount is 12.5 μ g/cm
2) paste thieving paper in backing other end suction zones;
Above being encapsulated in, gained Norketamine (NKET) gold mark detection test paper bar has in the box housing of annotating appearance hole and observation port.
Detection principle of the present invention is: the anti-Norketamine monoclonal antibody reactive that the Norketamine in the sample is at first surperficial with colloid gold particle; If the content of Norketamine surpasses limit value in the sample; The antibody sites of collaurum will no longer include residue, and when colloid gold particle chromatography process detection line, colloid gold particle will can not rest on the position of this line; Continue when up and control line on the reaction of the sheep anti mouse that sprays, demonstrate the redness of collaurum.If do not contain Norketamine in the sample or Norketamine content is lower than limit value, the antibody on collaurum surface will demonstrate redness with the compound reaction on the detection line, and nature controlling line also presents redness;
During the present invention implemented, relevant production of antibodies adopted following method:
Haptens
Norketamine (Norketamine) molecular weight very little (223.6 dalton) is the haptens material, only possesses immunoreactivity, does not have immunogenicity, can not directly be used for immune animal and obtains antibody.Therefore,, Norketamine has been carried out activation, and made haptens of the present invention in order to prepare comlete antigen of the present invention.
As used herein, " haptens " of the present invention or " Norketamine activated derivatives " is meant the material with structural formula 1 that obtains through derivatization reaction of the present invention, and its structure is suc as formula shown in 1:
Formula 1
Comlete antigen
Usually, haptens need with big molecule such as KLH (hemocyanin) or BSA (bovine serum albumin(BSA)) with the covalent coupling, become and both have immunoreactivity, have immunogenic comlete antigen again.
As used herein, the product after " comlete 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 comlete antigen, it for example can be, hemocyanin, bovine serum albumin(BSA), ovalbumin or gamma Globulin etc.
The present invention be used for that Norketamine detects and the structure of the comlete antigen of Antibody Preparation suc as formula shown in 2:
Formula 2
Wherein, X is a protein carrier, preferred hemocyanin (KLH) or bovine serum albumin(BSA) (BSA) among the present invention; With the part of X carrier covalent cross-linking be the derivant 1-carboxymethoxyl ammonia-Norketamine of Norketamine
In a preferred scheme of the present invention, X is a hemocyanin, and comlete antigen is Norketamine-KLH, uses antigen as immunity, is used to prepare the hybridoma of secreting anti-Norketamine monoclonal antibody.
In another preferred scheme of the present invention, X is a bovine serum albumin(BSA), and comlete antigen is Norketamine-BSA, uses antigen as detecting, and is used to prepare the monoclonal antibody immunity check-out console that detects Norketamine.
The preparation method of comlete antigen of the present invention is following:
At first, obtain its derivant with the Norketamine activation: 1-carboxymethoxyl ammonia-Norketamine, again itself and the protein carrier (for example, KLH, BSA) that is fit to are connected, obtain comlete antigen.Wherein X is a protein carrier, preferred hemocyanin (KLH) or bovine serum albumin(BSA) (BSA) among the present invention; With the part of X carrier covalent cross-linking be the derivant 1-carboxymethoxyl ammonia-Norketamine of Norketamine;
The preparation of 1-carboxymethoxyl ammonia-Norketamine: get 260mg NKET+400mg Omixe, be dissolved in the 40ml reaction dissolvent [reaction dissolvent is: pyridine+methyl alcohol+water=1: 4: 1], magnetic agitation is muddy even, reflux 3hr, ambient temperature overnight; 40 ℃ of water-baths, evaporated under reduced pressure.With the water-soluble solid of separating of 30mL, and use 1N NaHCO3 solution to transfer pH to be 8.5, to be transferred in the separating funnel, use 80mL CHCl
3Extract 3 times, discard CHCl
3Layer.Using 1.2N HCl solution to transfer pH again is 3, extracts 4 times with 100mL CHCl3, collects CHCl
3Layer; Add 20mL water washing CHCl3 layer, collect the CHCl3 layer; Add anhydrous Na 2SO4 solution-off water 10min, filter, CHCl3 liquid is evaporated to dried to cucurbit.
Being connected of haptens of the present invention and protein carrier can be used any connected mode known in the art.Such as but not limited to: carbodlimide method (EDC), glutaraldehyde method etc.
The comlete antigen of the present invention's preparation, Norketamine-KLH has good immunogenicity, can stimulate mouse to produce strong immune response, and through 1 fundamental immunity, 3 booster immunizations, antiserum titre can reach 1: 6400; Comlete antigen Norketamine-BSA has well kept the immunoreactivity of Norketamine.
Anti-NKET MONOCLONAL ANTIBODIES SPECIFIC FOR
Term used herein " monoclonal antibody " is meant available from the antibody of the antibody population of homology basically; Promptly; The antibody individuality of forming this colony is all identical; Except there being a small amount of possible white hair sudden change, therefore, modifier " monoclonal " is meant that the character of this antibody is not the potpourri of discrete antibody.
Detection principle of the present invention is: anti-Norketamine (NKET) monoclonal antibody reactive that the Norketamine in the sample (NKET) is at first surperficial with colloid gold particle; If the content of Norketamine in the sample (NKET) surpasses limit value; The antibody sites of collaurum will no longer include residue, and when colloid gold particle chromatography process detection line, colloid gold particle will can not rest on the position of this line; Continue when up and control line on the reaction of the sheep anti-mouse igg that sprays, demonstrate the redness of collaurum.If do not contain Norketamine (NKET) in the sample or Norketamine (NKET) content is lower than limit value, the antibody on collaurum surface will demonstrate redness with the compound reaction on the detection line, and nature controlling line also presents redness.
Claims (1)
1. the preparation method of Norketamine gold mark detection test paper box is characterized in that, it may further comprise the steps:
(1) preparation of Norketamine gold mark detection test paper bar: collaurum-anti-Norketamine monoclonal antibody bond is diluted to 0.5 μ g/mL～2 μ g/mL; Put into 10mm * 300mm glass fibre membrane; Soaked 10-20 minute; 37 ℃ of oven dry, 4 ℃～8 ℃ preservations stick on the notes appearance district of backing one end; The material of backing is PVC or tygon; PVC or tygon backing thickness are 100 μ m; Paste nitrocellulose filter in backing intermediate detection district; The cellulose nitrate film thickness is 120 μ m, and spraying concentration is that 100 μ g/mL～500 μ g/mL NKET-OVA bonds are made detection line on nitrocellulose filter, and spraying concentration is that 50 μ g/mL～200 μ g/mL sheep anti-mouse iggs are made nature controlling line; The width of detection line and nature controlling line is 0.5 mm～1mm, again with 1% bovine serum albumin(BSA) sealing nitrocellulose filter; The protein load amount is 5 μ g/cm
2～20 μ g/cm
2, paste thieving paper in backing other end suction zones;
(2) have above gained Norketamine gold mark detection test paper bar is encapsulated in the box housing of annotating appearance hole and observation port; The preparation of said collaurum: the chlorauric acid solution of getting 50ml 0.01%; Under the 1000r/min magnetic agitation, be heated to 110 ℃, add 1% sodium citrate aqueous solution 2ml rapidly; Keep temperature of reaction and speed of agitator constant; Boiling reaction 5min, solution colour stop reaction after just having become limpid Chinese red, and the collaurum mean grain size of acquisition is 10.9nm;
The preparation of anti-Norketamine monoclonal antibody and solution thereof:
50 μ g NKET-BSA coupled antigens are made into 1 μ g/ μ l antigenic solution with 50 μ l physiological saline, and are fully emulsified with the complete freund adjuvant mixing of equal-volume, supply first immunisation to use; Replace complete freund adjuvant with incomplete freund adjuvant during booster immunization with amount; First immunisation adopts directly injection in the mouse peritoneal; Immunizing dose is 50 a μ g/ mouse, and later on whenever at a distance from 2 all booster immunizations once, booster immunization adopts tail vein injection; Immunizing dose 10 μ g/ only; Intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with the 10:1 mixed; Centrifugal, remove supernatant; In 50～90 S, be that 1500 polyglycol adds in the cell with 1 ml, 50% molecular weight, fully mixing makes its fusion, adds 20 ml DEM nutrient solutions behind 1 min, stops merging; Centrifugal behind static 10 min of water-bath, remove supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 20% calf serum, be 1 * 10 with final concentration
4Feeder cells/0.1 ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 5%CO
2, cultivate under 37 ℃ of conditions, after 8 days, every culture hole is changed 2/3 HT nutrient solution; After 10 days～20 days, begin have the culture hole of hybridoma clonal growth to get supernatant and screen, the cell that testing result is positive is cloned with limiting dilution assay immediately microscopy; The hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out; With NKET-OVA is envelope antigen, with the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A
Test-A
Blank)/(A
Contrast-A
Blank)>2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope, dilution is 70/m1, gets 20 times of 1 ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone; Till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.3～0.5 ml/ of 10～13 all age, after 8 days～10 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only; Note after 5 days observing, collect ascites, centrifugal removal deposition, glycerol adding is in-20 ℃ of preservations;
Freund adjuvant is to be formed whiteruss by following material mixture ratio fully: sheep oil: BCG vaccine=12 g:20 ml:0.105g;
Not exclusively freund adjuvant is to be formed whiteruss by following material mixture ratio: sheep oil=12 g:20 ml;
The preparation of collaurum-anti-Norketamine monoclonal antibody bond: in the colloidal gold solution of certain volume, slowly drip 0.1 mg/ml Norketamine monoclonal anti liquid solution, be dissolved in the PBS damping fluid of 0.01M pH7.4; Ultrasound wave mixing 2 min carry out purifying through 4 ℃ of refrigerated centrifuges after leaving standstill 0.5 h, first centrifugal rotational speed 10000 r/min; Times 45 min; Abandon supernatant, deposition is dissolved with 10%BSA solution, and it is centrifugal to carry out next time; For the second time with 10%～30% glycerine density gradient centrifugation, rotating speed 7000 r/min, times 45 min is used to remove unlabelled albumen and gold grain aggregation, and collection centre bronzing partly gets final product.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810131795 CN101308139B (en) | 2008-06-30 | 2008-06-30 | Norketamine gold mark detection test paper box and method for making same |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810131795 CN101308139B (en) | 2008-06-30 | 2008-06-30 | Norketamine gold mark detection test paper box and method for making same |
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| Publication Number | Publication Date |
|---|---|
| CN101308139A CN101308139A (en) | 2008-11-19 |
| CN101308139B true CN101308139B (en) | 2012-12-12 |
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| US20150210626A1 (en) * | 2014-01-29 | 2015-07-30 | Dennis Michael Godek | Cycloalkyl-Diamines |
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| CN102128930A (en) * | 2010-12-08 | 2011-07-20 | 沈阳大学 | Colloidal gold drug test card for morphine-ketamine |
| CN102636647B (en) * | 2012-03-31 | 2014-05-21 | 戴国华 | Ketamine-collaurum test paper for detection of saliva |
| CN103808932A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Maduramicin colloidal gold detection card |
| CN103808930A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Zilpaterol immune colloidal gold detection card and preparation method thereof |
| KR101439790B1 (en) * | 2013-09-12 | 2014-09-12 | 유승국 | Apparatus for manufacturing diagnostic kit and diagnostic kit |
| CN112174851A (en) * | 2020-11-09 | 2021-01-05 | 广州万孚生物技术股份有限公司 | Fluoroaminoketone hapten, fludrominoketone antigen and preparation method and application thereof |
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| CN1570642A (en) * | 2004-04-29 | 2005-01-26 | 江苏省微生物研究所有限责任公司 | Gold mark detection test paper box for algal toxin and preparation method thereof |
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| US9345673B2 (en) * | 2013-01-30 | 2016-05-24 | MediSynergics, LLC | Anti-cancer cycloalkyl diamines |
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