CN102213719A - Flunitrazepam detection kit and preparation method thereof - Google Patents
Flunitrazepam detection kit and preparation method thereof Download PDFInfo
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- CN102213719A CN102213719A CN2010101384848A CN201010138484A CN102213719A CN 102213719 A CN102213719 A CN 102213719A CN 2010101384848 A CN2010101384848 A CN 2010101384848A CN 201010138484 A CN201010138484 A CN 201010138484A CN 102213719 A CN102213719 A CN 102213719A
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Abstract
The invention belongs to the technical field of determination of biological immune methods, and particularly relates to a flunitrazepam detection kit and a preparation method thereof. The kit comprises a sample pad (1), a colloidal gold pad (2), a nitrocellulose membrane (3), a sample sucking pad (4) and a PVC support plate (5). The colloidal gold pad is a colloidal gold-labeled anti-flunitrazepam multi-antibody glass fiber (or non-woven fabric), the nitrocellulose membrane is sequentially coated with a flunitrazepam coupling antigen as a detection line (T line), and a goat anti-rabbit IgG antibody as a quality control line (C line). The invention adopts the colloidal gold immunochromatography technology to prepare the flunitrazepam detection kit, has simple preparation method, can be used for detecting the flunitrazepam possibly existing in a sample, and has the characteristics of convenient use, simple operation, rapid reaction, economy, practicality and the like.
Description
Technical field
The present invention relates to the determination techniques field of biology immunization method, particularly relates to a kind of with colloidal gold immunity chromatography fast detecting Flunitrazepam detection kit and preparation method thereof.
Background technology
Flunitrazepam (Flunitrazepam) belongs to this diazepine hypnotic sedative agent, is commonly called as " cross ", has another name called flunitrazepan; Fluorine first intrazepam; The fluorine Nitrazepam.Be stronger hypnotic sedative agent, its effect is similar to intrazepam, but stronger than it, and stronger myorelaxant effects is also arranged.Be used for preceding calmness of art and various insomnia.Syngignoscism begins soon, sustainable 5～7 hours; As intravenous anesthetics (single using or induced anesthesia), induction time is longer, and about 135 seconds, but effect is satisfied; With muscle relaxant---curare share, and can stablize anesthesia and reach 1～2 hour.Suck Flunitrazepam, have stronger calmness, syngignoscism, induced hypnotic is rapid, sustainable sleep 5-7 hour.Common and the alcohol merging abuse of flunitrazepan can make victim's impotentia under drug effect revolt and be raped and plunder, and institute's occurrence is lost memory behind the abuse.Therefore be subjected to special concern in recent years, the U.S. classified Flunitrazepam as forbidden drug in 1992.After share, flunitrazepan and alcohol and other hypnotic sedative agent can cause being poisoned to death.Rise to 540 examples in 1999 according to U.S.'s emergency ward report by 13 examples in 1994 because of the dead case of abuse Flunitrazepam.
Flunitrazepam is a medicine commonly used in the crime case of anesthesia back, is the character of determining case, the detection of body fluid Chinese traditional medicine such as often need be injured human blood, urine or its metabolin.At present, the detection of relevant Flunitrazepam both at home and abroad mainly contains high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), high performance thin layer chromatography methods such as (HPTLC), though the sensitivity that these methods detect is higher, testing result is accurate, but need expensive instrument and equipment, also high to the requirement of test material, need just can carry out after the purification processes, and need the special experimenter of training, these methods all are not suitable for the fast detecting at high flux, scene in addition.Therefore the detection method of research with advantage such as quick, portable has realistic meaning.
The present invention adopts the colloidal gold immunochromatographimethod technology, prepares a kind of kit of fast detecting Flunitrazepam.This method starts from eighties of last century the mid-90, is to grow up on the basis of immunity percolation method, is the combination of immune affine technology, printing technology, immunolabelling technique and chromatographic technique.This method not only has sensitivity, characteristics such as special, convenient, quick, and suitable basic unit and on-the-spot the use.
Summary of the invention
The purpose of this invention is to provide a kind of portable, quick, be suitable for the on-the-spot kit that detects Flunitrazepam, be used for detecting body fluid Flunitrazepams such as blood, urine.
The invention provides a kind of Flunitrazepam detection kit.Comprise sample pad (1), closely be connected in the collaurum pad (2) that containing of sample pad one end, underlined anti-Flunitrazepam resisted, more with the tight nitrocellulose filter (3) that links to each other of the other end of collaurum pad and the suction sample pad (4) that closely is connected in the nitrocellulose filter other end.The nitrocellulose filter bag is by two lines, and wherein a bag is by the detection line of Flunitrazepam coupled antigen (T line) (6), and a bag is by the nature controlling line of goat anti-rabbit igg antibody (C line) (7).Sample pad, collaurum pad, nitrocellulose filter and suction sample pad stick on PVC back up pad (5) and go up to form reagent strip, and reagent strip also can be packed into and be formed card type packing in the plastic clip.
The present invention also provides a kind of preparation method of Flunitrazepam detection kit, may further comprise the steps:
(1) preparation Flunitrazepam coupled antigen
Flunitrazepam and bovine serum albumin molecule are carried out coupling, as the Flunitrazepam coupled antigen.
(2) how anti-the anti-Flunitrazepam of preparation is
How anti-adopt the conventional method immunity to make anti-Flunitrazepam with the Flunitrazepam coupled antigen.
(3) preparation colloid gold particle
Get 0.01% aqueous solution of chloraurate 100mL, heated and boiled.Add 1% citric acid, three sodium water solution 0.75mL as required rapidly, continue to boil about 5min, occur orange red.The size of the colloid gold particle of making like this is 20-40nm.
(4) preparation collaurum pad
Getting grain size is the colloidal gold solution 5ml of 20-40nm, uses 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 9.0, and room temperature was placed 10 minutes; Dropwise adding protein concentration is the how anti-0.04ml of anti-Flunitrazepam of 0.2mg/ml, mixes, and room temperature was placed 30 minutes; Add 0.075ml 10% bovine serum albumin (BSA) solution, mix, room temperature was placed 10 minutes; 11000 left the heart 30 minutes, carefully drew supernatant, discarded, and the borate buffer solution 5mL redissolution with the 0.002mol/L pH 9.0 that contains 5% sucrose repeats 2 times; Be dissolved to 3mL with borate buffer solution at last, obtain anti-Flunitrazepam and resist-the colloid gold label thing more; How anti--colloid gold label thing is spread 56cm by 1mL will to resist Flunitrazepam
2Ratio evenly be layered on the nonwoven fabrics, put drying room again, 38 ℃ of temperature, humidity was made the collaurum pad less than dry 2-4 under 30% the condition hour.
(5) bag is by Flunitrazepam coupled antigen, goat anti-rabbit igg
Set and draw film instrument coating parameters 1 μ L/cm, get 0.2mL Flunitrazepam coupled antigen, goat anti-rabbit igg with microsyringe respectively, receive A, the B pipe joint of drawing the film instrument in order.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film instrument, open and draw the film instrument, on nitrocellulose filter, apply Flunitrazepam coupled antigen (T line), goat anti-rabbit igg (C line).After the line with in the nitrocellulose filter baking oven, 38 ℃ of temperature, dry 24 hours, standby.
(6) assembling of kit
Sample pad, collaurum pad, nitrocellulose filter, suction sample pad are attached on the PVC back up pad successively by an end, can form the reagent strip that detects Flunitrazepam, reagent strip also can be packed into and be formed card type packing in the plastic clip.
The present invention adopts one step of chromatography type competition law, and the Flunitrazepam that may contain in utilization Flunitrazepam coupled antigen and sample competition joining gold is marked anti-Flunitrazepam, and how anti-principle detects Flunitrazepam.During test sample, sample because of capillarity to inhaling sample pad one end chromatography.If contain Flunitrazepam in the sample, they will and detection line (T line) go up the limited antibody combining site of Flunitrazepam coupled antigen competition of bag quilt, when the Flunitrazepam in the sample reaches finite concentration, resist generation immune response and saturated fully with the anti-Flunitrazepam of colloid gold label more, this moment, colloidal gold composite did not have the Flunitrazepam coupled antigen combination of bag quilt on vacant site and the detection line, this moment, the T line did not develop the color this positive result.If do not contain Flunitrazepam in the sample, mark the how anti-colloid gold particles of anti-Flunitrazepam will be to the T line position in company with the sample chromatography, immune association reaction takes place with the Flunitrazepam coupled antigen of bag quilt on the T line, colloid gold particle is piled up at the T line position and is made the T line present a macroscopic red stripes, this negative result.No matter whether contain Flunitrazepam in the sample, the colloid gold label thing all can combine with the goat anti-rabbit igg on being coated on nature controlling line (C line) and develop the color, C line colour developing is to judge whether enough samples are arranged, and whether the chromatography process normal standard, simultaneously also as the inner quality standard of reagent.
The detection method of kit of the present invention is: with the test sample balance to room temperature; Take out the Flunitrazepam pick-up unit, horizontal positioned; In sample pad, add 2-3 and drip sample, observe and write down the colour developing situation of C, T line in the time of 10 minutes, judge testing result.
Kit of the present invention adopts colloidal gold immunochromatographimethod technical measurement Flunitrazepam, during detection, sample is added on the sample pad on the test strips (card), can observe directly immunoreactive result, finishes sample detection.The present invention can be used for detecting the Flunitrazepam that may exist in the sample, have easy to use, simple to operate, be swift in response, characteristics such as economical and practical.
Description of drawings
Fig. 1 Flunitrazepam detection kit structural representation;
The reference numeral explanation:
1: sample pad;
2: contain the collaurum pad that underlined anti-Flunitrazepam resists more;
3: (T: bag is by the detection line of Flunitrazepam coupled antigen for nitrocellulose filter; C: bag is by the nature controlling line of goat anti-rabbit igg);
4: inhale the sample pad;
The 5:PVC back up pad;
The testing result synoptic diagram of Fig. 2 kit of the present invention.
Be followed successively by line positive test symbol of C from left to right, two line positive test symbol of T, C; Two line negative result of T, C; Invalid.
Embodiment:
Embodiment 1: the preparation of Flunitrazepam detection kit
(1) preparation Flunitrazepam coupled antigen
Flunitrazepam and bovine serum albumin molecule are carried out coupling, as the Flunitrazepam coupled antigen.
(2) how anti-the anti-Flunitrazepam of preparation is
How anti-adopt the conventional method immunity to make anti-Flunitrazepam with the Flunitrazepam coupled antigen.
(3) preparation colloid gold particle
Get 0.01% aqueous solution of chloraurate 100mL, heated and boiled.Add 1% citric acid, three sodium water solution 1.0mL as required rapidly, continue to boil about 5min, occur orange red.The size of the colloid gold particle of making like this is 20-40nm.
(4) preparation collaurum pad
Getting grain size is the colloidal gold solution 5ml of 20-40nm, uses 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 9.0, and room temperature was placed 10 minutes; Dropwise adding protein concentration is the how anti-0.04ml of anti-Flunitrazepam of 0.2mg/ml, mixes, and room temperature was placed 30 minutes; Add 0.075ml 10% bovine serum albumin (BSA) solution, mix, room temperature was placed 10 minutes; 11000 left the heart 30 minutes, carefully drew supernatant, discarded, and the borate buffer solution 5mL redissolution with the 0.002mol/L pH 9.0 that contains 5% sucrose repeats 2 times; Be dissolved to 3mL with borate buffer solution at last, obtain anti-Flunitrazepam and resist-the colloid gold label thing more; How anti--colloid gold label thing is spread 56cm by 1mL will to resist Flunitrazepam
2Ratio evenly be layered on the nonwoven fabrics, put drying room again, 38 ℃ of temperature, humidity was made the collaurum pad less than dry 2-4 under 30% the condition hour.
(5) bag is by Flunitrazepam coupled antigen, goat anti-rabbit igg
Set and draw film instrument coating parameters 1 μ L/cm, get 0.2mL Flunitrazepam coupled antigen, goat anti-rabbit igg with microsyringe respectively, receive A, the B pipe joint of drawing the film instrument in order.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film instrument, open and draw the film instrument, on nitrocellulose filter, apply Flunitrazepam coupled antigen (T line), goat anti-rabbit igg (C line).After the line with in the nitrocellulose filter baking oven, 38 ℃ of temperature, dry 24 hours, standby.
(6) assembling of kit
Sample pad, collaurum pad, nitrocellulose filter, suction sample pad are attached on the PVC back up pad successively by an end, can form the reagent strip that detects Flunitrazepam, reagent strip also can be packed into and be formed card type packing in the plastic clip.
Embodiment 2: the requirement of Flunitrazepam detection kit
(1) negative reference material coincidence rate
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is that the amphetamine standard solution of 100ng/mL carries out 10 parallel testings, observes testing result in the time of 10 minutes, and T, C line all present red stripes and colour developing degree homogeneous, and the result is negative.
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is that the barbital standard solution of 100ng/mL carries out 10 parallel testings, observes testing result in the time of 10 minutes, and T, C line all present red stripes and colour developing degree homogeneous, and the result is negative.
(2) positive reference material coincidence rate
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration be 20,50, the Flunitrazepam standard items of 100ng/mL, each concentration is carried out 10 parallel testings respectively, observes testing result in the time of 10 minutes, and the T line does not develop the color, the C line all presents redness, and the result is positive.
(3) limit of identification
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration be 0,5,10,15,20, the Flunitrazepam standard items of 25ng/mL, each concentration is carried out 10 parallel testings respectively, observe testing result in the time of 10 minutes, 0, the testing result of the Flunitrazepam standard items of 5ng/mL is that T, C line all develop the color and colour developing degree homogeneous negative result; 10, the testing result of the Flunitrazepam standard items of 15ng/mL is that T, C line all develop the color and the T line color is shallower than the C line color, is judged to be positive test symbol; 20, the testing result of the Flunitrazepam standard items of 25ng/mL is that the T line does not develop the color, the colour developing of C line, positive result.Limit of identification is not higher than 10ng/mL.
(4) repeatability
With phosphate buffer solution (PBS:0.01mol/L, pH 7.5) compound concentration is the Flunitrazepam standard items of 20ng/mL, carries out 10 parallel testings, observes testing result in the time of 10 minutes, and the result is all positive, colour developing degree homogeneous.
(5) stability
Place after 10 days for 37 ℃, every index all meets above requirement.
Embodiment 3: the detection method of Flunitrazepam detection kit
(1) specimen preparation
Serum: extract serum, centrifugal or leave standstill after get transparent supernatant and use; If the excessive haemolysis of serum, serum is experimentized with behind one times of the distilled water diluting again, otherwise the analoids redness is too dark, influences test result.
Urine: directly test with urine, if urine is visible muddy shape, need centrifugal earlier, filter or treat that its post precipitation gets supernatant and detect.
(2) operation
Take out the Flunitrazepam pick-up unit, horizontal positioned; On sample pad, splash into 2-3 and drip sample, observe and write down the colour developing situation of C, T line in the time of 10 minutes, judge testing result.
(3) result judges
Positive:
The T line does not develop the color, and the C line presents red stripes, is judged to be positive findings, illustrates that the content of Flunitrazepam is higher than 20ng/mL in the sample;
T line, C line all develop the color, and the T line color is shallower than the C line color, and the content that Flunitrazepam in the sample is described is between 10-20ng/mL.
Negative:
T line, C line all present red stripes, and the T line color equals or be deeper than the C line color, and the content of Flunitrazepam is lower than 10ng/mL in the interpret sample.
Invalid:
The C line does not develop the color, and the rotten damage of maloperation or kit is described.
Claims (7)
1. Flunitrazepam detection kit and preparation method thereof, it is characterized in that forming by sample pad, collaurum pad, nitrocellulose filter, suction sample pad and PVC back up pad, sample pad, collaurum pad, nitrocellulose filter, suction sample pad stick on the PVC back up pad successively, the collaurum pad is the how anti-glass fibre of anti-Flunitrazepam or the nonwoven fabrics of colloid gold label, on the nitrocellulose filter successively the bag by the Flunitrazepam coupled antigen as detection line (T line), goat anti-rabbit igg antibody is as nature controlling line (C line).
2. described Flunitrazepam detection kit of claim 1 and preparation method thereof is characterized in that described sample pad is glass fibre or nonwoven fabrics, and inhaling the sample pad is absorbent filter.
3. described Flunitrazepam detection kit of claim 1 and preparation method thereof is characterized in that described Flunitrazepam coupled antigen is the conjugate of Flunitrazepam and bovine serum albumin, and how anti-by Flunitrazepam coupled antigen immunity acquisition anti-Flunitrazepam is.
4. described Flunitrazepam detection kit of claim 1 and preparation method thereof is characterized in that described collaurum is by gold chloride (HAuCl
4) under the effect of reductive agent citric acid trisodium, make size and be the colloid gold particle of 20-40nm.
5. described Flunitrazepam detection kit of claim 1 and preparation method thereof, it is characterized in that the preparation method of described collaurum pad is: getting grain size is the colloidal gold solution 5ml of 20-40nm, uses 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 9.0, and room temperature was placed 10 minutes; Dropwise adding protein concentration is the how anti-0.04ml of anti-Flunitrazepam of 0.2mg/ml, mixes, and room temperature was placed 30 minutes; Add 0.075ml 10% bovine serum albumin (BSA) solution, mix, room temperature was placed 10 minutes; 11000 left the heart 30 minutes, carefully drew supernatant, discarded, and the borate buffer solution 5mL redissolution with the 0.002mol/L pH 9.0 that contains 5% sucrose repeats 2 times; Be dissolved to 3mL with borate buffer solution at last, obtain anti-Flunitrazepam and resist-the colloid gold label thing more; How anti--colloid gold label thing is spread 56cm by 1mL will to resist Flunitrazepam
2Ratio evenly be layered on the nonwoven fabrics, put drying room again, 38 ℃ of temperature, humidity was made the collaurum pad less than dry 2-4 under 30% the condition hour.
6. described Flunitrazepam detection kit of claim 1 and preparation method thereof, the method for coating that it is characterized in that two lines on the described nitrocellulose filter is: set and draw film instrument coating parameters 1 μ L/cm, get 0.2mL Flunitrazepam coupled antigen, goat anti-rabbit igg with microsyringe respectively, receive A, the B pipe joint of drawing the film instrument in order.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film instrument, open and draw the film instrument, on nitrocellulose filter, apply Flunitrazepam coupled antigen (T line), goat anti-rabbit igg (C line).After the line with in the nitrocellulose filter baking oven, 38 ℃ of temperature, dry 24 hours, standby.
7. described Flunitrazepam detection kit of claim 1 and preparation method thereof, the assemble method that it is characterized in that described Flunitrazepam detection kit is: sample pad, collaurum pad, nitrocellulose filter, suction sample pad are attached on the PVC back up pad successively by an end, can form the reagent strip that detects Flunitrazepam, reagent strip also can be packed into and be formed card type packing in the plastic clip.
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| CN2010101384848A CN102213719A (en) | 2010-04-02 | 2010-04-02 | Flunitrazepam detection kit and preparation method thereof |
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| CN2010101384848A CN102213719A (en) | 2010-04-02 | 2010-04-02 | Flunitrazepam detection kit and preparation method thereof |
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Application publication date: 20111012 |