A benzodiazepine BZO rapid detection kit

Abstract

The present invention discloses a benzodiazepine BZO rapid detection kit, including a bottom plate, a top cover and a reagent strip, wherein the bottom plate is provided with a card slot, and the reagent strip can be stably placed; the top cover is provided with a sample inlet and an observation port; the reagent strip is sequentially a sample pad, an immune colloidal gold sheet, an NC membrane and a water-absorbing filter paper from the sample application end; the immune colloidal gold sheet is a glass fiber pad coated with an anti-benzodiazepine monoclonal antibody labeled with colloidal gold; the NC membrane is a nitrocellulose membrane provided with a test line (T line) and a quality control line (C line), the test line is coated with a benzodiazepine-bovine serum albumin complex, and the quality control line is coated with goat anti-chicken IgY. The present invention further provides a method for preparing the test kit for the above-mentioned benzodiazepine drugs. The colloidal gold test kit for detecting benzodiazepine (BZO) drugs of the present invention is highly sensitive, highly specific, easy to operate and time-saving, and is very suitable for on-site and multi-person use.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a colloidal gold immunochromatography rapid detection kit related to benzodiazepine (BZO) drugs, which is used for drug rehabilitation centers, hospitals, special industries, high-risk population surveys, recruitment physical examination screening, food quarantine, etc. Background Art

[0002] Benzodiazepines have sedative, hypnotic, antianxiety, anticonvulsant, and muscle relaxant effects, and are used to treat a variety of physiological diseases, especially anxiety and insomnia. However, in the past few years, benzodiazepines have become a rapidly growing class of abused drugs in the new psychoactive substance market, and the non-medical use of this class of drugs has become one of the social issues of widespread concern. Long-term use of this drug can cause severe memory loss and cognitive impairment, physical and mental dependence, and strong withdrawal symptoms after discontinuation of the drug, including headaches, muscle pain, extreme anxiety and tension, irritability, excitement, and delirium. In severe cases, insomnia, impaired consciousness, loss of reason, hyperacusis, numbness, tingling in the limbs, hallucinations and epileptic seizures may occur, and even death may occur.

[0003] In view of the harmfulness of benzodiazepines, the state has classified them as psychotropic drugs for management. At present, there are many methods for detecting this type of drugs, most of which focus on chromatography, immunoassay, spectroscopy and electrochemistry. Each method has different focuses, but most of the instruments and equipment used are very expensive, which is not easy to carry out at the grassroots level.

[0004] The colloidal gold immunochromatography technique can directly detect benzodiazepines in blood, urine, and body fluids. This method has the advantages of high sensitivity, easy test strips, miniaturization, low cost, simple operation, and rapid response. In on-site testing, especially at the scene of drunk driving and drug driving, it is often used for the first step of screening. Summary of the invention

[0005] The purpose of the present invention is to overcome the existing technology and provide a rapid, convenient and sensitive benzodiazepine (BZO) rapid detection kit that does not rely on special equipment according to the needs of users.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A rapid detection kit for benzodiazepine (BZO) drugs comprises a reagent strip, which comprises a sample pad, an immunocolloidal gold sheet, an NC membrane and a water-absorbing filter paper in order from the sample application end; the immunocolloidal gold sheet is a glass fiber pad coated with an anti-benzodiazepine monoclonal antibody labeled with colloidal gold; the NC membrane is a nitrocellulose membrane provided with a detection line and a quality control line, the detection line is coated with a benzodiazepine-bovine serum albumin complex, and the quality control line is coated with goat anti-chicken IgY.

[0008] Furthermore, the test kit also includes a bottom plate and a top cover, the bottom plate is provided with a slot for stably placing the reagent strip; the top cover is provided with an injection port and an observation port; the injection port is located above the sample pad, and the observation port is located above the detection line and the quality control line.

[0009] In a preferred embodiment of the present invention, the width of the detection line (T line) and the quality control line (C line) is 0.8-0.5 cm.

[0010] In a preferred embodiment of the present invention, the sample addition port is in the shape of a funnel that is larger at the top and smaller at the bottom, which can effectively prevent the sample from overflowing due to the surface tension of the liquid.

[0011] In a preferred embodiment of the present invention, the observation window has a certain height, which can effectively isolate the reagent strip from being damaged by external forces and avoid dust contamination when it falls to the ground.

[0012] In a preferred embodiment of the present invention, 1 to 5 reagent strip placement slots are provided on the base, and different types of bases can be selected according to the frequency of use.

[0013] In a preferred embodiment of the present invention, the benzodiazepine antibody is chicken IgY, which can effectively reduce the interference of other metabolites in the human body due to the long phylogenetic distance between poultry and mammals. The concentration is OD30-60.

[0014] In a preferred embodiment of the present invention, one end of the sample pad overlaps with one end of the immunocolloidal gold sheet, the other end of the immunocolloidal gold sheet overlaps with one end of the NC membrane, and the other end of the NC membrane overlaps with the absorbent filter paper, so that the test kit can develop color even when placed vertically.

[0015] In a preferred embodiment of the present invention, the sample pad needs to be soaked in the treatment solution for 10-45 minutes, taken out and spun dry for 5-15 minutes, and placed in an oven for drying for 12-24 hours, wherein the soaking time is preferably 30 minutes.

[0016] In a preferred embodiment of the present invention, the treatment solution of the sample pad contains 0.02%-2% surfactant, 0.01%-1% polyvinyl pyrrolidone, 0.001%-0.1% casein sodium salt, 0.001%-0.1% EDTA.Na4, 0.05%-0.5% sodium cholate; the optimal pH is 8.0±0.1. By rationally formulating the sample pad soaked in the treatment solution, the release chromatography of the immune colloidal gold can be slowed down on the basis of complete release of the immune colloidal gold, which is conducive to the full reaction and combination of the antigen and antibody, and can effectively improve the reaction sensitivity of the test kit.

[0017] How to use the test kit:

[0018] Place the sample horizontally on the table, use a pipette to absorb the urine sample, and then add 3 to 5 drops of the sample into the square sample well. After 5 minutes of reaction, read the result. Note: Different pipettes need to be replaced for different samples.

[0019] Method for interpreting test kit results:

[0020] Negative: Two color bands appear in the observation port, that is, a red line appears at the position of the test line (T line) and the quality control line (C line), indicating that there is no benzodiazepine in the sample.

[0021] Positive: Only a red line appears at the quality control line (C line) in the observation port, and no line appears on the test line (T line), indicating that benzodiazepine is present in the sample.

[0022] Invalid: A red line appears at the position of the quality control line (C line), indicating that the sample addition and operation are correct. If no red line appears at the position of the quality control line, it means that the test result is uncertain and should be repeated.

[0023] How the test kit works:

[0024] If the urine sample contains benzodiazepines, the benzodiazepines in the urine sample will react with the benzodiazepine monoclonal antibodies in the anti-benzodiazepine monoclonal antibody colloidal gold layer coated on the NC membrane, competing for the antigen binding sites labeled on the anti-benzodiazepine monoclonal antibody colloidal gold layer. When the benzodiazepine concentration in the urine is higher than 300ng / ml, the benzodiazepines in the urine sample will first react with the benzodiazepine monoclonal antibodies and occupy all the antigen binding sites, thereby preventing the benzodiazepine monoclonal antibodies in the anti-benzodiazepine monoclonal antibody colloidal gold layer from binding to the benzodiazepine carrier in the benzodiazepine carrier complex layer on the test line. The test line cannot capture the colloidal gold particles and there is no red band, which is a positive result. If there is no benzodiazepine in the urine sample or its concentration is lower than the threshold concentration, the benzodiazepine monoclonal antibodies in the anti-benzodiazepine monoclonal antibody colloidal gold layer will combine with the benzodiazepine carrier in the benzodiazepine carrier complex layer on the test line, and the test line will capture the colloidal gold particles and present a red band, which is a negative result.

[0025] Beneficial effects of the present invention:

[0026] In the present invention, the drug antibody uses chicken IgY, and avian IgY can react with more epitopes on mammalian antigens to play a signal amplification role; in the preparation step of the immunocolloidal gold paper, the sample pad and the gold label pad are pretreated by using a pretreatment liquid with a reasonable formula, and a suitable gold spraying buffer is selected, which can slow down the release chromatography of the immunocolloidal gold during detection, which is beneficial to the full reaction and combination of the antigen and antibody, and improves the reaction sensitivity. At the same threshold, the amount of immunocolloidal gold can also be reduced to save costs.

[0027] The colloidal gold detection kit for detecting benzodiazepine drugs prepared by the present invention has the advantages of small batch difference, high sensitivity, strong specificity, simple and time-saving operation, naked eye judgment of detection results, no need for special instruments and professional technicians, low detection cost, convenient storage and transportation, etc., and is very suitable for on-site and multi-person use. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1-1 and 1-2 The top views of the single-person (left) and five-person (right) benzodiazepine (BZO) drug rapid detection kits of the present invention are shown in FIG. 1 and 4: top covers; 2 and 5: observation windows; 3 and 6: sample inlets.

[0029] Figure 2-1 and 2-2 Bottom views of the single-serving (left) and five-serving (right) benzodiazepine (BZO) drug rapid detection kits of the present invention.

[0030] Figure 3-1 and 3-2The top views of the bottom plates of the benzodiazepine (BZO) drug rapid detection kit for one person (left) and five people (right) of the present invention are shown in the figure: 9, 12: upper part of the card slot; 10, 13: bottom plate; 11, 14: lower part of the card slot.

[0031] Figure 4-1 and 4-2 The top views of the single-person (left) and five-person (right) benzodiazepine (BZO) drug rapid detection kits of the present invention without the top cover are shown in the figure: 15, 20: bottom plate; 16, 21: absorbent filter paper; 17, 22: NC membrane; 18, 23: immunocolloidal gold sheet; 19, 24: sample pad.

[0032] Figure 5-1 and 5-2 The top view and right view of the reagent strip of the benzodiazepine (BZO) drug rapid detection kit of the present invention are shown respectively. In the figure: 25, 31: absorbent filter paper; 26, 32: NC membrane; 27, 33: quality control line (C line); 28, 34: detection line (T line); 29, 35: immunocolloidal gold sheet; 30, 36: sample pad.

[0033] 6-1 and 6-2 are three-dimensional views of the single-person (left) and five-person (right) benzodiazepine (BZO) drug rapid detection kits of the present invention, respectively. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example

[0036] As shown in the figure, an embodiment of the rapid detection kit for benzodiazepine (BZO) drugs of the present invention includes a bottom plate, a top cover and a reagent strip. Figure 1-1 and 1-2 The top cover is for one person (left) and five people (right), with an injection port ( Figure 1-1 and 1-2 3, 6), observation port ( Figure 1-1 and 1-2 2, 5); the sample port is located above the sample pad, and the observation port is located above the test line and the quality control line. Figure 3-1 and 3-2 As shown, the bottom plate is provided with a slot to firmly place the reagent strip. Place the filter paper end of the reagent strip above the slot ( Figure 3-1 and 3-29, 12), the sample pad end is placed under the card slot ( Figure 3-1 and 3-2 11, 14), the placement diagrams of single serving (left) and five servings (right) are as follows Figure 4-1 and 4-2 As shown. The reagent strip is composed of the sample pad ( Figure 5-1 and 5-2 30, 36), immunocolloidal gold sheet ( Figure 5-1 and 5-2 29, 35), NC membrane ( Figure 5-1 and 5-2 26, 32) and absorbent filter paper ( Figure 5-1 and 5-2 25, 31); the immunocolloidal gold sheet is a glass fiber pad coated with anti-benzodiazepine monoclonal antibodies labeled with colloidal gold; the NC membrane is provided with a detection line ( Figure 5-1 and 5-2 28, 34) and quality control line ( Figure 5-1 and 5-2 The nitrocellulose membrane of 27, 33) is coated with a benzodiazepine-bovine serum albumin complex on the detection line, and the quality control line is coated with goat anti-chicken IgY.

[0037] Specifically, the production process in the embodiment of the present invention is as follows:

[0038] (1) Sample pad processing

[0039] Pour the EDTA sample pad treatment solution into a clean special container, put the glass fiber into the treatment solution and soak it for 30 minutes, take it out after soaking and spin dry, take it out and put it into a 45℃ oven to dry for 12-24 hours.

[0040] (2) Preparation of colloidal gold solution

[0041] Quickly pour 10% chloroauric acid solution into boiling ultrapure water and stir at high speed; add 10% trisodium citrate solution, stir for 5 minutes, then stop heating and continue stirring for 10 minutes; transfer the above solution to a heated stirrer and continue stirring at low speed for 20 minutes; place the above solution in a container in cooling water to below room temperature.

[0042] (3) Benzodiazepine (BZO) gold label solution labeling

[0043] Take the required gold water and place it on a stirrer for stirring. During the stirring process, add 0.2M potassium carbonate solution to adjust the pH and continue stirring for 5 minutes; quickly add benzodiazepine monoclonal antibody to the gold water solution and stir at room temperature for 30 minutes; add 10% BSA labeling blocking solution to the above solution and block for 30 minutes; centrifuge the labeled gold water solution at 2-8°C and 10000rpm for 45 minutes, remove the supernatant, add an appropriate amount of TCP gold label diluent to redissolve the gold label, so that the final labeling solution volume is about 1% of the gold water volume.

[0044] (4) Preparation of gold label for benzodiazepine (BZO)

[0045] Start the gold spraying machine and apply gold labels on the treated TB polyester film according to certain parameters. After completion, put the gold label strip into a 45℃ oven and dry it for 12-24 hours.

[0046] (5) Preparation of Benzodiazepine (BZO) Sheets

[0047] Tear off the adhesive tape at the NC film sticking place on the bottom plate, and stick the NC film to the corresponding position; start the film scribing instrument, set the parameters so that the C / T line spacing is 0.8cm; put the scribed sheet into a 45℃ oven to stabilize for 12-24h.

[0048] (6) Assembly

[0049] Cut the dried sheet into small strips with a width of 4 mm, place the cut reagent strips into the bottom plate of the mold, and close the top cover.

[0050] (7) Packaging

[0051] Place the assembled test strips in a bag containing desiccant and seal it for storage.

[0052] The present invention provides a benzodiazepine (BZO) rapid detection kit and a preparation process thereof, which combines a highly specific antibody-antigen reaction with an immunocolloidal gold chromatography technique, and improves upon the prior art by labeling a detection antibody with colloidal gold while labeling a control line antibody, and correspondingly performing a color development reaction on a second antibody spotted on a quality control area with the control line antibody labeled with colloidal gold, and achieving the color development of the detection line while ensuring that the color development of the quality control line is consistent.

[0053] Compared with the benzodiazepine detection kit with publication number CN102565409A, the benzodiazepine (BZO) rapid detection kit provided by the present invention has a more sensitive and rapid reaction and a lower cost.

[0054] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present application.

Claims

1. A benzodiazepine (BZO) rapid detection kit, comprising a reagent strip, which comprises a sample pad, an immune colloidal gold sheet, an NC membrane and a water-absorbing filter paper in order from the sample application end; the immune colloidal gold sheet is a glass fiber pad coated with an anti-benzodiazepine monoclonal antibody labeled with colloidal gold; the NC membrane is a nitrocellulose membrane provided with a detection line and a quality control line, the detection line is coated with a benzodiazepine-bovine serum albumin complex, the quality control line is coated with goat anti-chicken IgY, the anti-benzodiazepine monoclonal antibody is chicken IgY, and the concentration is an OD value of 30-60; The sample pad is prepared as follows: the glass fiber is placed in a treatment solution and soaked for 30 minutes, taken out after soaking and spun dry, taken out and placed in a 45°C oven, and dried for 12-24 hours; the treatment solution contains 0.02%-2% surfactant, 0.01%-1% polyvinyl pyrrolidone, 0.001%-0.1% casein sodium salt, 0.001%-0.1% EDTA.Na4, 0.05%-0.5% sodium cholate, and the pH value is 8.0±0.1; The reagent strip is composed of a sample pad, an immune colloidal gold sheet, and an NC film, which are overlapped and pasted on a PVC plastic sheet in sequence, cut into reagent strips with a width of 3-5 mm, and loaded into a box body template to form a test kit.

2. The kit according to claim 1, characterized in that It also includes a bottom plate and a top cover. The bottom plate is provided with a card slot to stably place the reagent strip; the top cover is provided with a sample addition port and an observation port; the sample addition port is located above the sample pad, and the observation port is located above the detection line and the quality control line.

3. The kit according to claim 2, characterized in that The bottom plate has various models, and is provided with 1 to 5 card slots according to the model, and the reagent strips are placed in the card slots.

4. The kit according to claim 2, characterized in that The sample adding port of the top cover is in the shape of a funnel which is wide at the top and narrow at the bottom.

5. The kit according to claim 1, characterized in that The preparation method of the immunocolloidal gold sheet and NC membrane is as follows: 1) Prepare gold labeling solution: take an appropriate amount of colloidal gold solution, the wavelength range corresponding to the maximum visible light absorbance value of the colloidal gold solution is 520-580nm, and add potassium carbonate with a concentration of 0.1-0.2 M to adjust the pH, and stir and mix for 5-10 min; add anti-benzodiazepine monoclonal antibody with a final concentration of 5-20 μg / mL to the mixed solution, and stir and mix for 30-45 min; then add an appropriate amount of 10% BSA as a blocking agent, and stir and mix for 30-45 min; centrifuge the labeled gold aqueous solution at a temperature of 2-8°C and 10000-12000 rpm for 30-45 min, remove the supernatant, and add an appropriate amount of gold label diluent to redissolve the gold label, so that the final labeling solution volume is about 1% of the gold water volume; 2) Preparation of coating solution: PBS buffer salt with a final concentration range of 0.001M to 0.05M and a pH value of 8.2; sucrose with a final concentration range of 0.1% to 1.2% by volume; trehalose with a final concentration range of 0.1% to 3% by volume; 3) Stripping and coating: Dilute the benzodiazepine-bovine serum albumin complex to an appropriate concentration with the stripping and coating solution, then stripe the diluted benzodiazepine-bovine serum albumin complex on the NC membrane, with one end adjacent to the immunocolloidal gold sheet as the detection line, and stripe goat anti-chicken IgY on the other end of the NC membrane as the quality control line; 4) Drying: Place the NC membrane after streaking in a dry environment at 37-50°C for 24-48 hours to obtain a NC membrane with a detection line and a quality control line.

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