CN117929722A - Multi-item combined detection kit for drug abuse in urine sample and saliva sample - Google Patents
Multi-item combined detection kit for drug abuse in urine sample and saliva sample Download PDFInfo
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of detection reagents, in particular to a multi-item combined detection kit for drug abuse in urine samples and saliva samples, which comprises a detection card, wherein the detection card comprises an upper plate block and a lower plate block, a sample adding hole and an observation port are arranged on the upper plate block, a plurality of test strips are arranged between the upper plate block and the lower plate block, and each test strip comprises a sample pad, a bonding pad, a reaction membrane, water absorbing paper and a pvc plate; the sample pad is formed by combining a filtering membrane and a glass fiber sample pad; the bonding pad is a multi-antibody fusion-labeled nano-gold bonding pad. The kit is suitable for screening of abuse of multiple drugs in urine and saliva, and has high sensitivity and high accuracy.
Description
Technical Field
The invention relates to the technical field of detection reagents, in particular to a multi-item combined detection kit for drug abuse in urine samples and saliva samples.
Background
Drug abuse refers to the repeated, massive use of drugs with dependency properties or dependency potential, which are not related to recognized medical needs, and which are non-medical purpose drugs. Drug abuse is largely divided into narcotics and psychotropic drugs, such as: opioids, cocaine, cannabis, and the like; psychotropic drugs such as: central inhibitors, such as sedative hypnotics; also central stimulants such as caffeine; in addition, hallucinogens such as mescalin, LSD, etc.
The drug detection methods mainly comprise 5 types, namely blood detection, hair detection, sweat detection, saliva detection and urine detection. Blood test: blood is also one of the test materials for determining whether to take a poison, and because of the high cost of such test equipment, it is generally only equipped in provincial hospitals at home. Hair inspection: hair analysis drugs may give biased results. Studies have shown that: black hair absorbs drugs more readily than yellow or bleached hair. In addition, the hair is used as a detection material for analysis, and the result is also subjected to potential factors that the detection material is easily polluted by the outside. At present, few such devices are used for detecting drugs in China. Sweat test: the advantage of using sweat as the detection material is that it is difficult to adulterate. The disadvantage is that the result does not provide a degree of damage to the body of the subject, and that certain substances (different substances produced by different persons) are produced during the sweat production process and interfere with the test results, but the specific situation is not completely clear. Urine testing: because the cost of using urine test is the lowest, the result is reliable, and convenient and quick, urine is the most commonly used tool for testing the components of human drugs at present. As can be seen from the above drug detection analysis, urine is currently the most commonly used tool for in vivo drug composition testing. At present, urine detection at home and abroad is widely applied to hospitals, investigation processes and court trial. The clinical drug test is also a method for detecting urine drugs. Saliva test: saliva can also be used as a sample for drug absorption detection, and saliva detection has the characteristics of easy operation, easy collection, high speed and high sensitivity, is not limited by sites, and can better protect the privacy of a tested person compared with urine detection. Therefore, the method is also very suitable for being used in entertainment places and driving poison, and can be also used in detection of hospitals, armies, units and the like.
At present, the domestic common drug abuse detection reagent is a qualitative detection reagent applying immune colloidal gold technology (ICT) and is used for primary screening of drug abuse. Other immunoassay methods such as Enzyme Immunoassay (EIA) including enzyme-linked amplified immunoassay (EMIT) and enzyme-linked immunosorbent assay (ELISA), fluorescence Polarization Immunoassay (FPIA) and the like are rarely used in China. Various clinical test items of medicines are various, so that the medicine abuse screening can be scientifically and reasonably implemented, the privacy and safety of a subject can be guaranteed, the test result is accurate and reliable, and the use of a plurality of medicine abuse combined detection kits (colloidal gold method) in urine and saliva drug screening is one of the necessary measures. Thus, in view of the above-mentioned problems, there is a need to develop a multiple-unit test kit for drug abuse in urine samples and saliva samples.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the defects of the prior art, the multi-item combined detection kit for the abuse of the drugs in the urine sample and the saliva sample is provided, and the kit is suitable for screening the abuse of the drugs in the urine and the saliva, and has high sensitivity and high accuracy.
In order to solve the technical problems, the technical scheme of the invention is as follows:
A multi-item joint detection kit for drug abuse in urine samples and saliva samples, the kit comprises a detection card, the detection card comprises an upper plate block and a lower plate block, a sample adding hole and an observation port are formed in the upper plate block, a plurality of test strips are arranged between the upper plate block and the lower plate block, and each test strip comprises a sample pad, a bonding pad, a reaction membrane, water absorbing paper and a pvc plate; the sample pad is formed by combining a filtering membrane and a glass fiber pad; the bonding pad is a multi-antibody fusion-labeled nano-gold bonding pad.
As an improved technical scheme, the preparation method of the sample pad comprises the following steps:
(1) Treatment of the filtration membrane: soaking a filtering membrane in a treatment solution containing 0.5% w/v of Tris-HCl, 0.5% -1.0% w/v of N-acetylneuraminic acid, 0.1-0.5% w/v of Tween20, 0.1-0.5% w/v of anhydrous magnesium sulfate and 1.5-2% w/v of NaCl for a period of time, and taking out and drying for later use;
(2) Preparation of a glass fiber mat: coating a treatment solution containing 0.5% w/v Tris-HCl, 0.1-0.5% w/v Triton X-100 and 0.5-1.0% w/v S17 on 0.5cm-0.59cm glass fiber, and drying to obtain a glass fiber mat for later use;
(3) Combination of sample pads: and (3) flatly paving the filtering membrane in the step (1) on the glass fiber pad in the step (2) to obtain the sample pad.
As an improved technical scheme, the filtering membrane in the step (1) is soaked in the treatment liquid for 10-14h, and is dried for 12-16h at 37 ℃.
As an improved technical scheme, the filtering membrane in the step (1) is made of polyester fiber, and the molecular weight cut-off of the filtering membrane is 100-1000Da.
As an improved technical scheme, the thickness of the treatment liquid coated on the glass fiber in the step (2) is 0.5cm-0.59cm, and the glass fiber is dried for 12-16 hours at 37 ℃ after being coated.
As an improved technical scheme, the number of the test strips is 2-5, and when the number of the test strips is 2, the multi-antibody fusion labeling nano-gold binding pad comprises a MET/COC/THC gold binding pad and an AMP/MOP/KET gold binding pad; when the number of the test strips is 3, the multi-antibody fusion labeling nano-gold binding pad comprises a MET/COC/THC gold binding pad, an AMP/MOP/KET gold binding pad and a BZO/TCA/BAR gold binding pad; when the number of the test strips is 4, the multi-antibody fusion labeling nano-gold binding pad comprises a MET/COC/THC gold binding pad, an AMP/MOP/KET gold binding pad, a BZO/TCA/BAR gold binding pad and an MDMA/OPI/PCP gold binding pad; when the number of the test strips is 5, the multi-antibody fusion labeling nano-gold binding pad comprises a MET/COC/THC gold binding pad, an AMP/MOP/KET gold binding pad, a BZO/TCA/BAR gold binding pad, an MDMA/OPI/PCP gold binding pad and a BUP/MTD/TRA gold binding pad.
As an improved technical scheme, the preparation of the multi-antibody fusion-labeled nano-gold binding pad adopts a plurality of antibody fusion labeling modes, and specifically comprises the following steps:
S1, taking a 20-40nm nano gold solution, regulating pH to be alkaline, simultaneously adding 3 antibodies at the same speed, standing at 4 ℃ for 24 hours, adding BSA (BSA) to reach a final concentration of 10g/L, continuously stirring, standing at 4 ℃ for 4 hours, and centrifuging at 4 ℃ at 10000rpm/min for 30 minutes to obtain precipitates, namely the multi-antibody fusion nano gold particles;
S2, re-dissolving the multi-antibody fusion nano-gold particles in the step S1 by using Jin Biaofu solution to prepare gold-labeled stock solution which is 20 times concentrated, then diluting by using Jin Biaofu solution, spraying on a polyester film according to 2.5 mu L/cm, and drying overnight at 37 ℃ to prepare the multi-antibody fusion-labeled nano-gold binding pad.
As an improved technical scheme, the nano-gold solution in the step S1 is adjusted to pH 8.0-9.5.
As an improved technical scheme, the pH of the gold-labeled complex solution in the step S2 is 8.0, and the gold-labeled complex solution contains 1.0% w/v of Tris-HCl, 0.5% w/v of casein, 0.5% w/v of Tween 20 and 0.1% w/v of Proclin 300.
As an improved technical scheme, the reaction film is coated with three corresponding drugs, namely BSA coupled antigen and goat anti-mouse IgG antibody, to form detection lines T1, T2, T3 and a quality control line C; the concentration of the BSA coupling antigen is 0.5-1.5mg/mL, the concentration of the goat anti-mouse IgG antibody is 1.0-1.2mg/mL, and the solid-phase antigen reaction membrane is prepared by drying the mixture for 4h at 37 ℃ after coating.
After the technical scheme is adopted, the invention has the beneficial effects that:
(1) In order to be suitable for saliva samples and urine samples at the same time, a layer of reticular filtering membrane (the reticular filtering membrane is polyester fiber non-woven fabric) is attached to a sample pad, mucin and food residues in saliva can be filtered through the reticular filtering membrane, and meanwhile, by adding N-acetylneuraminic acid, magnesium sulfate, surfactant and other components, the interference in the samples can be eliminated through the effects of regulating the balance of positive and negative charges, water salt and ion concentration, saliva dissociation treatment is not required in advance, the reagent detection sensitivity is improved, and the color of the bottom of a reagent strip fades more quickly. The buffer capacity enhancement in a double-pad mode can be suitable for urine sample detection with different pH values, so that the diversity of the detection materials is finally realized, and the detection materials can be used for different detection scenes.
(2) The multi-item combined detection kit for drug abuse in different detection material samples adopts a multi-antibody fusion labeling nano-gold mode, and a plurality of antibodies are labeled simultaneously to prepare a nano-gold binding pad, so that compared with the traditional method for preparing three gold binding pads by independently labeling three antibodies, the three gold binding pads are overlapped, the performance of the product sensitivity, the fading of ground color and the like is better than that of the traditional technology, the operation procedure of the technology is simplified, and the labor and the time cost are saved.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The multi-item combined detection kit for drug abuse in urine samples and saliva samples comprises a detection card, wherein the detection card comprises an upper plate block and a lower plate block, a sample adding hole and an observation port are formed in the upper plate block, 2 test strips are arranged between the upper plate block and the lower plate block, and each test strip comprises a sample pad, a combination pad, a reaction membrane, water absorbing paper and a pvc plate; the sample pad is formed by combining a filtering membrane and a glass fiber pad; the bonding pad is a multi-antibody fusion-labeled nano-gold bonding pad, and the multi-antibody fusion-labeled nano-gold bonding pad comprises a MET/COC/THC gold bonding pad and an AMP/MOP/KET gold bonding pad.
The preparation method of the sample pad comprises the following steps:
(1) Treatment of the filtration membrane: soaking a filtering membrane (made of polyester fiber) with a molecular weight cut-off of 100-1000Da in a treatment solution containing 0.5% w/v of Tris-HCl, 0.5% w/v of N-acetylneuraminic acid, 0.1% w/v of Tween20, 0.1% w/v of anhydrous magnesium sulfate and 1.5% w/v of NaCl for 10 hours, taking out, and drying at 37 ℃ for 12 hours to obtain the filtering membrane for standby;
(2) Preparation of a glass fiber mat: the treatment solution containing 0.5% w/v Tris-HCl, 0.1% w/v TritonX-100 and 0.5% w/v S17 is coated on 0.5cm glass fiber, the coating thickness is 0.5cm, and the glass fiber pad is dried for 12 hours at 37 ℃ for standby;
(3) Combination of sample pads: and (3) flatly paving the filtering membrane in the step (1) on the glass fiber pad in the step (2) to obtain the sample pad.
The preparation of the multi-antibody fusion-labeled nano-gold binding pad comprises the following steps:
(1) MET/COC/THC gold conjugate pad preparation: regulating the pH of 30nm nano gold solution to 8.0, simultaneously adding MET, COC, THC antibody with the final concentration of 5 mug/mL, standing for 24 hours at 4 ℃, adding BSA with the final concentration of 10g/L, continuously stirring for 20min, standing for 4 hours at 4 ℃, centrifuging for 30min at 4 ℃ under 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution with the pH of 8.0 (containing 1.0% w/v Tris-HCl, 0.5% w/v casein, 0.5% w/v tween 20 and 0.1% w/v Proclin 300) to prepare gold standard stock solution which is concentrated by 20 times. Before use, each multi-antibody fusion mark Jin Biaoye is diluted into 6 times of gold standard liquid by Jin Biaofu solutions, and sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and dried overnight at 37 ℃ to prepare the MET/COC/THC gold binding pad.
(2) AMP/MOP/key gold conjugate pad preparation: adjusting the pH of a 20nm nano gold solution to 8.0, simultaneously adding AMP, MOP, KET antibody with a final concentration of 5 mug/mL, standing for 24 hours at 4 ℃, adding BSA with a final concentration of 10g/L, continuously stirring for 20 minutes, standing for 4 hours at 4 ℃, centrifuging for 30 minutes at 4 ℃ under 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution with the pH of 8.0 (containing 1.0% w/v Tris-HCl, 0.5% w/v casein, 0.5% w/v tween 20 and 0.1% w/v Proclin 300) to prepare a gold standard stock solution which is 20 times concentrated. Each multi-antibody fusion tag Jin Biaoye was diluted to a 5-fold gold standard solution with Jin Biaofu solutions before use, sprayed onto a polyester film with a gold spraying instrument at 2.5. Mu.L/cm, and dried overnight at 37℃to prepare an AMP/MOP/KET gold conjugate pad.
Preparation of solid antigen reaction membrane:
(1) The nitrocellulose membrane is coated with MET/COC/THC-BSA coupling antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; the concentration of the MET/COC/THC-BSA coupling antigen is 0.5 mg/mL, 0.6mg/mL and the concentration of the goat anti-mouse IgG antibody is 1.0mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
(2) The nitrocellulose membrane is coated with AMP/MOP/KET-BSA coupled antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; AMP/MOP/KET-BSA coupled antigen concentration is 0.8, 1.0mg/mL, goat anti-mouse IgG antibody concentration is 1.0mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
Example 2
The multi-item combined detection kit for drug abuse in urine samples and saliva samples comprises a detection card, wherein the detection card comprises an upper plate block and a lower plate block, a sample adding hole and an observation port are formed in the upper plate block, 3 test strips are arranged between the upper plate block and the lower plate block, and each test strip comprises a sample pad, a combination pad, a reaction membrane, water absorbing paper and a pvc plate; the sample pad is formed by combining a filtering membrane and a glass fiber pad; the bonding pad is a multi-antibody fusion-labeled nano-gold bonding pad, and the multi-antibody fusion-labeled nano-gold bonding pad comprises a MET/COC/THC gold bonding pad, an AMP/MOP/KET gold bonding pad and a BZO/TCA/BAR gold bonding pad.
The preparation method of the sample pad comprises the following steps:
(1) Treatment of the filtration membrane: soaking a filtering membrane (made of polyester fiber) with a molecular weight cut-off of 100-1000Da in a treatment solution containing 0.5% w/v of Tris-HCl, 0.8% w/v of N-acetylneuraminic acid, 0.3% w/v of Tween20, 0.3% w/v of anhydrous magnesium sulfate and 1.8% w/v of NaCl for 12 hours, taking out, and drying for 14 hours at 37 ℃ to obtain the filtering membrane for standby;
(2) Preparation of a glass fiber mat: the treatment solution containing 0.5% w/v Tris-HCl, 0.3% w/v TritonX-100 and 0.8% w/v S17 is coated on 0.55cm glass fiber, the coating thickness is 0.55cm, and the glass fiber pad is dried for 14 hours at 37 ℃ for standby;
(3) Combination of sample pads: and (3) flatly paving the filtering membrane in the step (1) on the glass fiber pad in the step (2) to obtain the sample pad.
The preparation of the multi-antibody fusion-labeled nano-gold binding pad comprises the following steps:
(1) MET/COC/THC gold conjugate pad preparation: regulating the pH of 30nm nano gold solution to 8.5, simultaneously adding MET, COC, THC antibody, standing at 4 ℃ for 24 hours, adding BSA (BSA) to the final concentration of 10g/L, continuously stirring for 20 minutes, standing at 4 ℃ for 4 hours, centrifuging for 30 minutes at 4 ℃ under 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare 20 times concentrated gold standard stock solution. Before use, each multi-antibody fusion mark Jin Biaoye is diluted into 6 times of gold standard liquid by Jin Biaofu solutions, and sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and dried overnight at 37 ℃ to prepare the MET/COC/THC gold binding pad.
(2) AMP/MOP/key gold conjugate pad preparation: adjusting the pH of a 20nm nano gold solution to 8.5, simultaneously adding AMP, MOP, KET antibody, keeping stand at 4 ℃ for 24 hours, adding BSA (BSA) to reach the final concentration of 10g/L, continuously stirring for 20 minutes, keeping stand at 4 ℃ for 4 hours, centrifuging for 30 minutes under the condition of 4 ℃ and 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare the gold standard stock solution which is concentrated by 20 times. Each multi-antibody fusion tag Jin Biaoye was diluted to a 5-fold gold standard solution with Jin Biaofu solutions before use, sprayed onto a polyester film with a gold spraying instrument at 2.5. Mu.L/cm, and dried overnight at 37℃to prepare an AMP/MOP/KET gold conjugate pad.
(3) BZO/TCA/BAR gold conjugate pad preparation: regulating the pH of 30nm nano gold solution to 9.0, simultaneously adding BZO, TCA, BAR antibody, the final concentration is 8 mug/mL, standing for 24 hours at 4 ℃, adding BSA to the final concentration of 10g/L, continuously stirring for 20 minutes, standing for 4 hours at 4 ℃, centrifuging for 30 minutes at 4 ℃ under 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare 20 times concentrated gold standard stock solution. Before use, each multi-antibody fusion mark Jin Biaoye is diluted into 8 times of gold standard solution by Jin Biaofu solutions, and the solution is sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and is dried at 37 ℃ overnight, so that the BZO/TCA/BAR gold bonding pad is prepared.
Preparation of solid antigen reaction membrane:
(1) The nitrocellulose membrane is coated with MET/COC/THC-BSA coupling antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; the concentration of the MET/COC/THC-BSA coupling antigen is 0.5 mg/mL, 0.6mg/mL and the concentration of the goat anti-mouse IgG antibody is 1.0mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
(2) The nitrocellulose membrane is coated with AMP/MOP/KET-BSA coupled antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; AMP/MOP/KET-BSA coupled antigen concentration is 0.8, 1.0mg/mL, goat anti-mouse IgG antibody concentration is 1.0mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
(3) BZO/TCA/BAR-BSA coupling antigen and goat anti-mouse IgG antibody are coated on the nitrocellulose membrane to form detection lines T1, T2 and T3 and a quality control line; BZO/TCA/BAR-BSA coupling antigen concentration is 0.5, 0.8mg/mL, sheep anti-mouse IgG antibody concentration is 1.2mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
Example 3
The multi-item combined detection kit for drug abuse in urine samples and saliva samples comprises a detection card, wherein the detection card comprises an upper plate block and a lower plate block, a sample adding hole and an observation port are formed in the upper plate block, 4 test strips are arranged between the upper plate block and the lower plate block, and each test strip comprises a sample pad, a combination pad, a reaction membrane, water absorbing paper and a pvc plate; the sample pad is formed by combining a filtering membrane and a glass fiber pad; the bonding pad is a multi-antibody fusion-labeled nano-gold bonding pad, and the multi-antibody fusion-labeled nano-gold bonding pad comprises a MET/COC/THC gold bonding pad, an AMP/MOP/KET gold bonding pad, a BZO/TCA/BAR gold bonding pad, and an MDMA/OPI/PCP gold bonding pad.
The preparation method of the sample pad comprises the following steps:
(1) Treatment of the filtration membrane: soaking a filtering membrane (made of polyester fiber) with a molecular weight cut-off of 100-1000Da in a treatment solution containing 0.5% w/v of Tris-HCl, 1.0% w/v of N-acetylneuraminic acid, 0.5% w/v of Tween20, 0.5% w/v of anhydrous magnesium sulfate and 2% w/v of NaCl for 14 hours, taking out, and drying at 37 ℃ for 16 hours to obtain the filtering membrane for standby;
(2) Preparation of a glass fiber mat: the treatment solution containing 0.5% w/v Tris-HCl, 0.5% w/v TritonX-100 and 1.0% w/v S17 is coated on 0.59cm glass fiber, the coating thickness is 0.59cm, and the glass fiber pad is dried for 16 hours at 37 ℃ for standby;
(3) Combination of sample pads: and (3) flatly paving the filtering membrane in the step (1) on the glass fiber pad in the step (2) to obtain the sample pad.
The preparation of the multi-antibody fusion-labeled nano-gold binding pad comprises the following steps:
(1) MET/COC/THC gold conjugate pad preparation: regulating the pH of 30nm nano gold solution to 9.0, simultaneously adding MET, COC, THC antibody, the final concentration is 5 mug/mL, standing for 24 hours at 4 ℃, adding BSA, the final concentration is 10g/L, continuously stirring for 20 minutes, standing for 4 hours at 4 ℃, centrifuging for 30 minutes at 4 ℃ under 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare 20 times concentrated gold standard stock solution. Before use, each multi-antibody fusion mark Jin Biaoye is diluted into 6 times of gold standard liquid by Jin Biaofu solutions, and sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and dried overnight at 37 ℃ to prepare the MET/COC/THC gold binding pad.
(2) AMP/MOP/key gold conjugate pad preparation: adjusting the pH of a 20nm nano gold solution to 9.0, simultaneously adding AMP, MOP, KET antibody, keeping stand at 4 ℃ for 24 hours, adding BSA, keeping stirring for 20 minutes, keeping stand at 4 ℃ for 4 hours, centrifuging for 30 minutes under the condition of 4 ℃ and 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare a gold standard stock solution which is concentrated by 20 times. Each multi-antibody fusion tag Jin Biaoye was diluted to a 5-fold gold standard solution with Jin Biaofu solutions before use, sprayed onto a polyester film with a gold spraying instrument at 2.5. Mu.L/cm, and dried overnight at 37℃to prepare an AMP/MOP/KET gold conjugate pad.
(3) BZO/TCA/BAR gold conjugate pad preparation: regulating the pH of 30nm nano gold solution to 9.2, simultaneously adding BZO, TCA, BAR antibody, the final concentration is 8 mug/mL, standing for 24 hours at 4 ℃, adding BSA to the final concentration of 10g/L, continuously stirring for 20 minutes, standing for 4 hours at 4 ℃, centrifuging for 30 minutes at 4 ℃ under 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare 20 times concentrated gold standard stock solution. Before use, each multi-antibody fusion mark Jin Biaoye is diluted into 8 times of gold standard solution by Jin Biaofu solutions, and the solution is sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and is dried at 37 ℃ overnight, so that the BZO/TCA/BAR gold bonding pad is prepared.
(4) MDMA/OPI/PCP gold conjugate pad preparation: adjusting the pH of a 40nm nano gold solution to 9.2, simultaneously adding MDMA, OPI, PCP antibody, keeping stand at 4 ℃ for 24 hours, adding BSA (BSA) to reach the final concentration of 10g/L, continuously stirring for 20 minutes, keeping stand at 4 ℃ for 4 hours, centrifuging for 30 minutes under the condition of 4 ℃ and 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare the gold standard stock solution which is concentrated by 20 times. Before use, each multi-antibody fusion tag Jin Biaoye is diluted into 6 times of gold standard solution by Jin Biaofu solutions, and the 6 times of gold standard solution is sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and is dried at 37 ℃ overnight to prepare the MDMA/OPI/PCP gold binding pad.
Preparation of solid antigen reaction membrane:
(1) The nitrocellulose membrane is coated with MET/COC/THC-BSA coupling antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; the concentration of the MET/COC/THC-BSA coupling antigen is 0.5 mg/mL, 0.6mg/mL and the concentration of the goat anti-mouse IgG antibody is 1.0mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
(2) The nitrocellulose membrane is coated with AMP/MOP/KET-BSA coupled antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; AMP/MOP/KET-BSA coupled antigen concentration is 0.8, 1.0mg/mL, goat anti-mouse IgG antibody concentration is 1.0mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
(3) BZO/TCA/BAR-BSA coupling antigen and goat anti-mouse IgG antibody are coated on the nitrocellulose membrane to form detection lines T1, T2 and T3 and a quality control line; BZO/TCA/BAR-BSA coupling antigen concentration is 0.5, 0.8mg/mL, sheep anti-mouse IgG antibody concentration is 1.15mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
(4) The nitrocellulose membrane is coated with MDMA/OPI/PCP-BSA coupling antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; MDMA/OPI/PCP-BSA coupling antigen concentration was 0.8, 1.0mg/mL, and goat anti-mouse IgG antibody concentration was 1.1mg/mL.
Example 4
A multiple-item combined detection kit for abuse of drugs in urine samples and saliva samples comprises a detection card, wherein the detection card comprises an upper plate block and a lower plate block, a sample adding hole and an observation port are formed in the upper plate block, 5 test strips are arranged between the upper plate block and the lower plate block, and each test strip comprises a sample pad, a combination pad, a reaction membrane, water absorbing paper and a pvc plate; the sample pad is formed by combining a filtering membrane and a glass fiber pad; the bonding pad is a multi-antibody fusion-labeled nano-gold bonding pad, and the multi-antibody fusion-labeled nano-gold bonding pad comprises a MET/COC/THC gold bonding pad, an AMP/MOP/KET gold bonding pad, a BZO/TCA/BAR gold bonding pad, an MDMA/OPI/PCP gold bonding pad and a BUP/MTD/TRA gold bonding pad.
The preparation method of the sample pad comprises the following steps:
(1) Treatment of the filtration membrane: soaking a filtering membrane (made of polyester fiber) with a molecular weight cut-off of 100-1000Da in a treatment solution containing 0.5% w/v of Tris-HCl, 1.0% w/v of N-acetylneuraminic acid, 0.5% w/v of Tween20, 0.5% w/v of anhydrous magnesium sulfate and 2% w/v of NaCl for 14 hours, taking out, and drying at 37 ℃ for 16 hours to obtain the filtering membrane for standby;
(2) Preparation of a glass fiber mat: the treatment solution containing 0.5% w/v Tris-HCl, 0.5% w/v TritonX-100 and 1.0% w/v S17 is coated on 0.59cm glass fiber, the coating thickness is 0.59cm, and the glass fiber pad is dried for 16 hours at 37 ℃ for standby;
(3) Combination of sample pads: and (3) flatly paving the filtering membrane in the step (1) on the glass fiber pad in the step (2) to obtain the sample pad.
The preparation of the multi-antibody fusion-labeled nano-gold binding pad comprises the following steps:
(1) MET/COC/THC gold conjugate pad preparation: regulating the pH of 30nm nano gold solution to 8.5, simultaneously adding MET, COC, THC antibody, standing at 4 ℃ for 24 hours, adding BSA (BSA) to the final concentration of 10g/L, continuously stirring for 20 minutes, standing at 4 ℃ for 4 hours, centrifuging for 30 minutes at 4 ℃ under 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare 20 times concentrated gold standard stock solution. Before use, each multi-antibody fusion mark Jin Biaoye is diluted into 6 times of gold standard liquid by Jin Biaofu solutions, and sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and dried overnight at 37 ℃ to prepare the MET/COC/THC gold binding pad.
(2) AMP/MOP/key gold conjugate pad preparation: adjusting the pH of a 20nm nano gold solution to 8.5, simultaneously adding AMP, MOP, KET antibody, keeping stand at 4 ℃ for 24 hours, adding BSA (BSA) to reach the final concentration of 10g/L, continuously stirring for 20 minutes, keeping stand at 4 ℃ for 4 hours, centrifuging for 30 minutes under the condition of 4 ℃ and 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare the gold standard stock solution which is concentrated by 20 times. Each multi-antibody fusion tag Jin Biaoye was diluted to a 5-fold gold standard solution with Jin Biaofu solutions before use, sprayed onto a polyester film with a gold spraying instrument at 2.5. Mu.L/cm, and dried overnight at 37℃to prepare an AMP/MOP/KET gold conjugate pad.
(3) BZO/TCA/BAR gold conjugate pad preparation: regulating the pH of 30nm nano gold solution to 9.0, simultaneously adding BZO, TCA, BAR antibody, the final concentration is 8 mug/mL, standing for 24 hours at 4 ℃, adding BSA to the final concentration of 10g/L, continuously stirring for 20 minutes, standing for 4 hours at 4 ℃, centrifuging for 30 minutes at 4 ℃ under 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare 20 times concentrated gold standard stock solution. Before use, each multi-antibody fusion mark Jin Biaoye is diluted into 8 times of gold standard solution by Jin Biaofu solutions, and the solution is sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and is dried at 37 ℃ overnight, so that the BZO/TCA/BAR gold bonding pad is prepared.
(4) MDMA/OPI/PCP gold conjugate pad preparation: adjusting the pH of a 40nm nano gold solution to 9.0, simultaneously adding MDMA, OPI, PCP antibody, keeping stand at 4 ℃ for 24 hours, adding BSA (BSA) to reach the final concentration of 10g/L, continuously stirring for 20 minutes, keeping stand at 4 ℃ for 4 hours, centrifuging for 30 minutes under the condition of 4 ℃ and 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare the gold standard stock solution which is concentrated by 20 times. Before use, each multi-antibody fusion tag Jin Biaoye is diluted into 6 times of gold standard solution by Jin Biaofu solutions, and the 6 times of gold standard solution is sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and is dried at 37 ℃ overnight to prepare the MDMA/OPI/PCP gold binding pad.
(5) BUP/MTD/TRA gold conjugate pad preparation: adjusting the pH of a 25nm nano gold solution to 9.5, simultaneously adding BUP, MTD, TRA antibody, keeping standing at 4 ℃ for 24 hours, adding BSA (BSA) to the final concentration of 10g/L, continuously stirring for 20 minutes, keeping standing at 4 ℃ for 4 hours, centrifuging for 30 minutes under the condition of 4 ℃ and 10000/min, and re-dissolving the obtained precipitate (namely nano gold particles) by adopting Jin Biaofu solution to prepare a gold standard stock solution which is concentrated by 20 times. Before use, each multi-antibody fusion tag Jin Biaoye is diluted into 10 times gold standard solution by Jin Biaofu solutions, sprayed on a polyester film by a metal spraying instrument according to 2.5 mu L/cm, and dried overnight at 37 ℃ to prepare BUP/MTD/TRA gold binding pads.
Preparation of solid antigen reaction membrane:
(1) The nitrocellulose membrane is coated with MET/COC/THC-BSA coupling antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; the concentration of the MET/COC/THC-BSA coupling antigen is 0.5 mg/mL, 0.6mg/mL and the concentration of the goat anti-mouse IgG antibody is 1.0mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
(2) The nitrocellulose membrane is coated with AMP/MOP/KET-BSA coupled antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; AMP/MOP/KET-BSA coupled antigen concentration is 0.8, 1.0mg/mL, goat anti-mouse IgG antibody concentration is 1.0mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
(3) BZO/TCA/BAR-BSA coupling antigen and goat anti-mouse IgG antibody are coated on the nitrocellulose membrane to form detection lines T1, T2 and T3 and a quality control line; BZO/TCA/BAR-BSA coupling antigen concentration is 0.5, 0.8mg/mL, sheep anti-mouse IgG antibody concentration is 1.2mg/mL; and after coating, drying at 37 ℃ for 4 hours to prepare the solid antigen reaction membrane.
(4) The nitrocellulose membrane is coated with MDMA/OPI/PCP-BSA coupling antigen and goat anti-mouse IgG antibody to form detection lines T1, T2 and T3 and a quality control line; MDMA/OPI/PCP-BSA coupling antigen concentration was 0.8, 1.0mg/mL, and goat anti-mouse IgG antibody concentration was 1.2mg/mL.
(5) BUP/MTD/TRA-BSA coupling antigen and goat anti-mouse IgG antibody are coated on the nitrocellulose membrane to form detection lines T1, T2 and T3 and a quality control line; BUP/MTD/TRA-BSA coupled antigen concentrations were 1.0, 1.2, 1.5mg/mL, and the goat anti-mouse IgG antibody concentration was 1.2mg/mL.
Example 5
The detection method of the kit comprises the following steps:
(1) The collected saliva sample or urine sample can be stored at 2-8deg.C for 3 days, and stored at-20deg.C for 3 days, and repeatedly frozen and thawed for no more than 3 times;
(2) 3 drops of the sample (80-100 mu L) are dripped into saliva or urine samples by a disposable plastic straw, the result is observed for 10 minutes, and the result is invalid after 15 minutes.
Analysis of detection results: the results corresponding to the detection lines T1, T2 and T3 are "+" respectively, and the results are positive; the results corresponding to the detection lines T1, T2 and T3 are respectively "-" which indicate that the detection lines are negative;
Example 6
The kit of example 5 was used to detect MET, COC, THC, AMP, MOP, KET, BZO, TCA, BAR, MDMA, OPI, PCP, BUP, MTD, TRA in saliva samples and urine samples, respectively.
1. Sample preparation
Positive samples: MET, COC, THC, AMP, MOP, KET, BZO, TCA, BAR, MDMA, OPI, PCP, BUP, MTD, TRA standard substances are respectively added into blank urine and saliva matrix samples to prepare different concentrations, and the concentrations are shown in the following tables 1 and 2;
Negative samples: fresh urine and saliva samples were randomly collected for 100 healthy non-drug groups.
2. Saliva matrix standard test results are shown in Table 3.
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3. Saliva negative sample detection results are shown in Table 4;
4. the detection results of the urine matrix standard are shown in Table 5;
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5. The urine negative sample test results are shown in Table 6.
In order to better prove that the kit has better technical effect, 9 comparative examples are given by taking example 4 as a reference, and the saliva sample and the urine sample are respectively detected by MET, COC, THC. The specific detection results of MET, COC, THC in saliva samples are shown in tables 7-9, and the specific detection results of MET, COC, THC in urine samples are shown in tables 10-12.
Comparative example 1
Unlike example 4, the sample pad was not laid with a filtration membrane, and the rest was the same.
Comparative example 2
Unlike example 4, the treatment liquid did not contain N-acetylneuraminic acid during the filtration membrane treatment, and the other operations were the same.
Comparative example 3
Unlike example 4, the treatment liquid did not contain anhydrous magnesium sulfate during the filtration membrane treatment, and the other operations were the same.
Comparative example 4
In the case of the filtration membrane treatment, the treatment solution contained 0.2% w/v N-acetylneuraminic acid, which was different from example 4, and the other operations were the same.
Comparative example 5
In the case of the filtration membrane treatment, 1.5% w/v N-acetylneuraminic acid was contained in the treatment solution, and the other steps were the same as those in example 4.
Comparative example 6
Unlike example 4, the glass fiber of step (2) was used to prepare the sample pad to a thickness of 0.4cm, and the rest of the procedure was the same.
Comparative example 7
Unlike example 4, three independent antibody gold conjugate pads were prepared using three antibodies separately labeled, respectively. The rest of the operations are the same.
Comparative example 8
Unlike example 4, the treatment liquid did not contain sodium chloride during the filtration membrane treatment, and the other operations were the same.
Comparative example 9
Unlike example 4, the filtration membrane was made of glass fiber.
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It should be noted that, the combination of the test strips of the test card in the present invention is not limited to the combination manner in the embodiment of the present invention. The specific introduction of the detection items is as follows: MET (methamphetamine), COC (cocaine), THC (tetrahydrocannabinol), AMP (amphetamine), MOP (morphine), KET (ketamine), BZO (benzodiazepine), TCA (tricyclic antidepressant), BAR (barbital), MDMA (methylenedioxymethamphetamine), OPI (opium), PCP (phencyclidine), BUP (buprenorphine), MTD (methadone), TRA (tramadol).
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (9)
1. The multi-item combined detection kit for drug abuse in urine samples and saliva samples is characterized by comprising a detection card, wherein the detection card comprises an upper plate block and a lower plate block, a sample adding hole and an observation port are formed in the upper plate block, a plurality of test strips are arranged between the upper plate block and the lower plate block, and each test strip comprises a sample pad, a binding pad, a reaction membrane, water absorbing paper and a pvc plate; the sample pad is formed by combining a filtering membrane and a glass fiber pad; the bonding pad is a multi-antibody fusion-labeled nano-gold bonding pad;
The preparation method of the sample pad comprises the following steps:
(1) Treatment of the filtration membrane: soaking a filtering membrane in a treatment solution containing 0.5% w/v of Tris-HCl, 0.5% -1.0% w/v of N-acetylneuraminic acid, 0.1-0.5% w/v of Tween20, 0.1-0.5% w/v of anhydrous magnesium sulfate and 1.5-2% w/v of NaCl for a period of time, and taking out and drying for later use;
(2) Preparation of a glass fiber mat: coating a treatment solution containing 0.5% w/v Tris-HCl, 0.1-0.5% w/v Triton X-100 and 0.5-1.0% w/v S17 on 0.5cm-0.59cm glass fiber, and drying to obtain a glass fiber mat for later use;
(3) Combination of sample pads: and (3) flatly paving the filtering membrane in the step (1) on the glass fiber pad in the step (2) to obtain the sample pad.
2. The kit for the multiple-unit detection of drug abuse in urine and saliva samples according to claim 1, wherein the filter membrane of step (1) is immersed in the above-mentioned treatment solution for 10-14 hours and dried at 37 ℃ for 12-16 hours.
3. The kit for the multiple-unit detection of drug abuse in urine and saliva samples according to claim 1, wherein the filter membrane in step (1) is made of polyester fiber and has a molecular weight cut-off of 100-1000Da.
4. The kit of claim 1, wherein the thickness of the treatment fluid applied to the glass fiber in step (2) is 0.5cm to 0.59cm, and the treatment fluid is dried at 37 ℃ for 12 to 16 hours.
5. The kit for the multiple joint detection of drug abuse in urine and saliva samples according to claim 1, wherein the number of the test strips is 2-5, and when the number of the test strips is 2, the multi-antibody fusion-labeled nano-gold binding pad comprises MET/COC/THC gold binding pad, AMP/MOP/key gold binding pad; when the number of the test strips is 3, the multi-antibody fusion labeling nano-gold binding pad comprises a MET/COC/THC gold binding pad, an AMP/MOP/KET gold binding pad and a BZO/TCA/BAR gold binding pad; when the number of the test strips is 4, the multi-antibody fusion labeling nano-gold binding pad comprises a MET/COC/THC gold binding pad, an AMP/MOP/KET gold binding pad, a BZO/TCA/BAR gold binding pad and an MDMA/OPI/PCP gold binding pad; when the number of the test strips is 5, the multi-antibody fusion labeling nano-gold binding pad comprises a MET/COC/THC gold binding pad, an AMP/MOP/KET gold binding pad, a BZO/TCA/BAR gold binding pad, an MDMA/OPI/PCP gold binding pad and a BUP/MTD/TRA gold binding pad.
6. The kit for multiple combined detection of drug abuse in urine and saliva samples according to claim 1, wherein the preparation of the multi-antibody fusion-labeled nanogold conjugate pad adopts multiple antibody fusion labeling modes, and specifically comprises the following steps:
S1, taking a 20-40nm nano gold solution, regulating pH to be alkaline, simultaneously adding 3 antibodies at the same speed, standing at 4 ℃ for 24 hours, adding BSA (BSA) to reach a final concentration of 10g/L, continuously stirring, standing at 4 ℃ for 4 hours, and centrifuging at 4 ℃ at 10000rpm/min for 30 minutes to obtain precipitates, namely the multi-antibody fusion nano gold particles;
S2, re-dissolving the multi-antibody fusion nano-gold particles in the step S1 by using Jin Biaofu solution to prepare gold-labeled stock solution which is 20 times concentrated, then diluting by using Jin Biaofu solution, spraying on a polyester film according to 2.5 mu L/cm, and drying overnight at 37 ℃ to prepare the multi-antibody fusion-labeled nano-gold binding pad.
7. The kit of claim 6, wherein the nanogold solution in step S1 is adjusted to a pH of 8.0-9.5.
8. The kit of claim 6, wherein the gold-labeled complex solution in step S2 has a pH of 8.0 and contains 1.0% w/v Tris-HCl, 0.5% w/v casein, 0.5% w/v tween 20 and 0.1% w/v Proclin 300.
9. The kit for the multiple combined detection of drug abuse in urine and saliva samples according to claim 1, wherein the reaction membrane is coated with three corresponding drug-BSA coupled antigens and goat anti-mouse IgG antibodies to form detection lines T1, T2, T3 and a quality control line C; the concentration of the BSA coupling antigen is 0.5-1.5mg/mL, the concentration of the goat anti-mouse IgG antibody is 1.0-1.2mg/mL, and the solid-phase antigen reaction membrane is prepared by drying the mixture for 4h at 37 ℃ after coating.
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