Fluorescent immunochromatographic test card for quantitative detection of isoniazid, preparation method and application thereof

Abstract

The present invention provides a fluorescent immunochromatographic test card for quantitative detection of isoniazid and a preparation method, a detection method and an application thereof, wherein the fluorescent microsphere pad of the fluorescent immunochromatographic test card is sprayed with isoniazid monoclonal antibodies labeled with fluorescent microspheres; the detection area on the nitrocellulose membrane of the fluorescent immunochromatographic test card is coated with isoniazid-carrier protein conjugate, and the quality control area is coated with goat anti-mouse IgG. In the present invention, the isoniazid monoclonal antibody is prepared using isoniazid-keyhole limpet hemocyanin conjugate as an immunogen, and the isoniazid-keyhole limpet hemocyanin conjugate has strong specificity and good immunogenicity, and the prepared isoniazid monoclonal antibody has strong specificity and high titer. The fluorescent immunochromatographic test card of the present invention is used to determine the isoniazid blood drug concentration in a sample, is simple to operate, and the result is immediately available, with a validity period of up to 18 months, no expensive instruments are required, and the maintenance cost is low, which is suitable for large-scale clinical promotion and application.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay detection, and in particular to a fluorescent immunochromatographic detection card for quantitatively detecting isoniazid, a preparation method and an application thereof. Background Art

[0002] Isoniazid (INH) is an important anti-tuberculosis drug currently used. It is used for various types and stages of pulmonary tuberculosis and various extrapulmonary tuberculosis. It is the first-line drug of choice for the prevention and treatment of tuberculosis and plays a vital role in the prevention of tuberculosis. However, the toxic and side effects of isoniazid cannot be ignored. Clinical data suggest that the use of isoniazid can cause liver function changes in about 20% of patients, about 1.6% of patients can develop symptomatic hepatitis, and a small number of patients can cause liver failure or even death. The individual differences in the efficacy and toxic side effects of isoniazid are mainly related to the patient's blood drug concentration. At present, multiple studies have found that when different patients use the standard dose of 5mg / kg of isoniazid, the blood drug concentration may differ by 3-7 times. The impact of low blood drug concentrations of anti-tuberculosis drugs on the treatment of anti-tuberculosis cannot be ignored. Many studies have pointed out that the failure or recurrence of anti-tuberculosis treatment and the occurrence of drug resistance are related to low blood drug concentrations. Failure to maintain effective drug concentrations and drug concentrations below the prescribed dose or below the minimum inhibitory concentration (MIC) are important factors that induce Mycobacterium tuberculosis to develop resistance to anti-tuberculosis drugs. The individual differences in isoniazid blood concentrations are large, which are related to individual absorption differences, age, weight, gender, pathophysiological status, and other comorbid medication conditions.

[0003] Therefore, a therapeutic concentration range of anti-tuberculosis drugs that takes into account both effectiveness and safety has been developed clinically, and drug concentration monitoring (Therapeutic Drug Monitoring, TDM) is carried out during treatment. Individualized dosing guided by drug exposure can reduce the occurrence of adverse drug reactions and increase the possibility of successful treatment, which is used to optimize the efficacy and safety of drugs.

[0004] At present, the main methods for monitoring the blood concentration of isoniazid include spectral analysis, high-performance liquid chromatography, and liquid chromatography-mass spectrometry. Liquid chromatography-mass spectrometry and high-performance liquid chromatography are also recommended technical methods based on drug specificity. Although both methods can accurately separate and determine the target substances in the sample and have good specificity and sensitivity, these two methods have high requirements for laboratory hardware facilities and the professional level of the testing personnel. The testing process is complicated, the instruments are expensive and require regular calibration and maintenance, and the cost of use is high, so there are certain limitations for clinical promotion. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a fluorescent immunochromatographic detection card for quantitative detection of isoniazid, a preparation method and an application thereof, so as to solve the problem of complex detection process in existing detection methods, realize low-cost and rapid detection, and facilitate clinical promotion.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A fluorescent immunochromatographic detection card for quantitative detection of isoniazid comprises a base plate and a sample pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped and pasted on the base plate; the fluorescent microsphere pad is sprayed with isoniazid monoclonal antibodies marked with fluorescent microspheres; the detection area on the nitrocellulose membrane is coated with isoniazid-carrier protein conjugate, and the quality control area is coated with sheep anti-mouse IgG.

[0008] Preferably, the isoniazid monoclonal antibody is secreted by hybridoma cell line 1H11. The hybridoma cell line 1H11 was deposited in the China Type Culture Collection CCTCC of Wuhan University, China on January 24, 2024, with a deposit number of CCTCC NO: C202427, and the deposit result is survival; the hybridoma cell line 1H11 is prepared using isoniazid-keyhole limpet hemocyanin (KLH) conjugate as an immunogen. The isoniazid-keyhole limpet hemocyanin (KLH) conjugate is a conjugate obtained by conjugating isoniazid and keyhole limpet hemocyanin (KLH).

[0009] Preferably, the isoniazid-carrier protein conjugate is a conjugate obtained by conjugating isoniazid with a carrier protein; the carrier protein is one of keyhole limpet hemocyanin, bovine serum albumin, and chicken yolk protein.

[0010] Preferably, the diameter of the fluorescent microsphere is 100-300 nm; and the surface of the fluorescent microsphere contains carboxyl groups.

[0011] The present invention also provides a method for preparing the fluorescent immunochromatographic detection card, comprising overlapping and pasting a sample pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water-absorbing pad in sequence and fixing them on a bottom plate, wherein the sample pad is treated with a bovine serum albumin solution containing sucrose before pasting, and the mass volume concentration of sucrose in the bovine serum albumin solution is 1-3%; the preparation method of the fluorescent microsphere pad comprises: spraying isoniazid monoclonal antibodies marked by the fluorescent microspheres onto a glass cellulose membrane, and obtaining the fluorescent microsphere pad after drying; the preparation method of the nitrocellulose membrane comprises: coating an isoniazid-carrier protein conjugate in a detection area, coating an anti-mouse IgG in a quality control area, and drying at 45-60° C. for 15-25 hours to obtain a nitrocellulose membrane coated with the isoniazid-carrier protein conjugate and the goat anti-mouse IgG.

[0012] Preferably, the method for preparing the fluorescent microsphere-labeled isoniazid monoclonal antibody comprises the following steps:

[0013] 1) mixing a fluorescent microsphere suspension, MES buffer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and performing an activation treatment to obtain activated fluorescent microspheres;

[0014] 2) Mixing the activated fluorescent microspheres in step 1) and isoniazid monoclonal antibodies for coupling to obtain isoniazid monoclonal antibodies labeled with fluorescent microspheres.

[0015] The detection method of the above fluorescent immunochromatographic detection card comprises the following steps:

[0016] The isoniazid pure product of different concentrations is added to a blank human serum matrix to prepare a calibrator; the concentration of isoniazid in the calibrator is 20 μg / ml, 10 μg / ml, 5 μg / ml, 2 μg / ml, 1 μg / ml, and 0.5 μg / ml, respectively; the calibrator is diluted 10-100 times and then added to the fluorescent immunochromatographic detection card, and immunochromatography is performed to measure the fluorescence intensity ratio T / C value of the detection area T line and the quality control area C line, and obtain the functional relationship between the concentration in the calibrator and the T / C value.

[0017] Take the sample to be tested, add it to the fluorescent immunochromatography test card, perform immunochromatography, measure the fluorescence intensity ratio T / C value of the detection area T line and the quality control area C line, and calculate the isoniazid content in the sample to be tested according to the function formula.

[0018] The present invention also provides the use of the fluorescent immunochromatographic detection card or the fluorescent immunochromatographic detection card prepared by the preparation method in detecting the isoniazid content in a sample.

[0019] The present invention also provides application of the detection method of the fluorescent immunochromatographic detection card in detecting the isoniazid content in serum, plasma or whole blood.

[0020] The present invention also provides a kit, comprising the fluorescent immunochromatographic detection card or the fluorescent immunochromatographic detection card prepared by the above preparation method.

[0021] The present invention provides a fluorescent immunochromatographic detection card for detecting isoniazid, a preparation method, a detection method and an application. The detection method is based on time-resolved fluorescent immunochromatographic technology and is implemented by the principle of competitive immune reaction. When the solution to be tested is added to the fluorescent immunochromatographic detection card, the isoniazid drug small molecules in the solution to be tested are combined with the isoniazid monoclonal antibody coated on the fluorescent microsphere pad and coupled to the fluorescent microsphere to form a complex of fluorescent microsphere-antibody-antigen, which is chromatographed along the nitrocellulose membrane to the quality control area (C) under the action of chromatography, and is captured by the coated sheep anti-mouse IgG to form a complex precipitated in the quality control area, while the fluorescent microsphere-isoniazid monoclonal antibody conjugate that is not combined with the isoniazid drug is captured by the antigen coated in the detection area (T), forming a complex precipitated in the detection area, and its fluorescence intensity is inversely proportional to the isoniazid content in the sample. Under the action of the excitation light source, the fluorescent substance emits a fluorescent signal of a specific wavelength, and the fluorescent immunochromatographic analyzer captures the fluorescent signal, which is automatically converted into a quantitative value through signal conversion and a set standard curve, and the isoniazid concentration in the solution to be tested is calculated.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a fluorescent immunochromatographic detection card for quantitative detection of isoniazid, as well as a preparation method and application. The preparation method uses isoniazid-keyhole limpet hemocyanin conjugate as an immunogen, has strong specificity and good immunogenicity; the prepared isoniazid monoclonal antibody has high titer and strong competitive effect, and the hybridoma cell line producing the antibody is numbered 1H11, and the hybridoma cell line 1H11 is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C202427. In addition, the fluorescent immunochromatographic detection card of the present invention is used to determine the isoniazid blood drug concentration in a sample, is simple to operate, and the results are available immediately. It is valid for up to 18 months, does not require expensive instruments, has low maintenance costs, and is suitable for large-scale clinical promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparative analysis chart of the titer of isoniazid monoclonal antibodies prepared using isoniazid-KLH and isoniazid-BSA as immunogens in Example 1 of the present invention.

[0025] Figure 2 This is a sensitivity (IC50) test analysis diagram of the isoniazid monoclonal antibody secreted by the hybridoma cell line 1H11 in Example 1 of the present invention.

[0026] Figure 3This is a sensitivity (IC50) test analysis diagram of the isoniazid monoclonal antibody secreted by the hybridoma cell line 4F3 in Example 1 of the present invention.

[0027] Figure 4 This is a correlation analysis diagram of the measurement results of the fluorescent immunochromatographic test card for quantitative detection of isoniazid in Example 4 of the present invention. DETAILED DESCRIPTION

[0028] The invention provides a fluorescent immunochromatographic detection card for quantitatively detecting isoniazid. The fluorescent immunochromatographic detection card comprises a base plate and a sample pad, a fluorescent microsphere pad, a nitrocellulose membrane coated with isoniazid-carrier protein conjugate and goat anti-mouse IgG, and a water-absorbing pad which are sequentially overlapped and pasted on the base plate; the fluorescent microsphere pad is sprayed with isoniazid monoclonal antibodies marked with fluorescent microspheres.

[0029] In the present invention, the fluorescent immunochromatographic detection card is preferably assembled according to the following method: a sample pad, a fluorescent microsphere pad, a nitrocellulose membrane coated with isoniazid-carrier protein conjugate and goat anti-mouse IgG, and a water-absorbing pad are overlapped and pasted and fixed on a bottom plate from left to right in sequence, the end of the sample pad is connected to the beginning of the fluorescent microsphere pad, the end of the fluorescent microsphere pad is connected to the beginning of the nitrocellulose membrane coated with isoniazid-carrier protein conjugate and goat anti-mouse IgG, the end of the nitrocellulose membrane coated with isoniazid-carrier protein conjugate and goat anti-mouse IgG is connected to the beginning of the water-absorbing pad, the beginning of the sample pad is aligned with the beginning of the bottom plate, the end of the water-absorbing pad is aligned with the end of the bottom plate, and then cut into test strips of appropriate width by a strip cutter, the test strips are mounted in a special plastic card shell, and assembled into a fluorescent immunochromatographic detection card; the width of the test strips is preferably 3.9-4.1 mm, more preferably 4.0 mm.

[0030] In the present invention, the sample pad is preferably treated by the following method, the steps are as follows: placing the sample pad in a bovine serum albumin solution containing sucrose, soaking it, and drying it to obtain the sample pad. The content of sucrose in the bovine serum albumin solution containing sucrose is preferably 1-3% (W / V), more preferably 2% (W / V); the solvent of the bovine serum albumin solution containing sucrose is preferably phosphate buffer; the concentration of the phosphate buffer is preferably 0.01-0.05 mol / L, more preferably 0.02 mol / L; the pH value of the phosphate buffer is preferably 7.1-7.5, more preferably 7.2; the soaking time is preferably 1.5-2.5h, more preferably 2h; the soaking temperature is preferably 15-25°C, more preferably 20°C; the drying temperature is preferably 45-55°C, more preferably 50°C; the drying time is preferably 5-8h, more preferably 6h; the drying method is preferably air drying and drying.

[0031] In the present invention, the fluorescent microsphere pad is sprayed with isoniazid monoclonal antibodies labeled with fluorescent microspheres; the fluorescent microsphere pad is preferably prepared by the following method: the isoniazid monoclonal antibodies labeled with fluorescent microspheres are loaded onto a Jining gold spraying film instrument, the gold spraying mode is selected to spray onto a glass cellulose membrane, and the fluorescent microsphere pad is obtained after drying; the spraying amount of the isoniazid monoclonal antibodies labeled with fluorescent microspheres is preferably 2.0-6.0 μl / cm, and most preferably 4 μl / cm; the loading device for the isoniazid monoclonal antibodies labeled with fluorescent microspheres is preferably a Jining gold spraying film instrument (model: XYZ3020); the drying time is preferably 18-24 hours, and more preferably 20 hours; the drying temperature is preferably 40-50° C., and more preferably 45° C.

[0032] In the present invention, the fluorescent microsphere-labeled isoniazid monoclonal antibody is preferably prepared by the following method:

[0033] 1) mixing a fluorescent microsphere suspension, MES buffer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and performing an activation treatment to obtain activated fluorescent microspheres;

[0034] 2) Mixing the activated fluorescent microspheres in step 1) and isoniazid monoclonal antibodies for coupling to obtain isoniazid monoclonal antibodies labeled with fluorescent microspheres.

[0035] In the above step 1), the diameter of the fluorescent microsphere is preferably 100-300 nm, and most preferably 200 nm; preferably, the surface of the fluorescent microsphere contains carboxyl groups; the fluorescent microsphere is preferably polystyrene encapsulated rare earth ion Eu + microspheres; the content of fluorescent microspheres in the fluorescent microsphere suspension is preferably 1% (W / V); the concentration of the MES buffer is preferably 40-60mM, more preferably 50mM; the pH value of the MES buffer is preferably 5.5-6.5, more preferably 6; the ratio of the fluorescent microsphere suspension, MES buffer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide is 40-60μL: 800-1200μL: 0.03-0.04mg: 0.3-0.4mg, preferably 50μL: 1000μL: 0.035mg: 0.35mg; the activation treatment time is preferably 10-30min, more preferably 15min; the activation treatment temperature is preferably 18-37°C, more preferably 26°C; the activation treatment is preferably carried out under shaking conditions.

[0036] In the above step 1), after obtaining the activated fluorescent microspheres, the activated fluorescent microspheres are centrifuged and the precipitate is resuspended; the centrifugal speed is preferably 12000-16000 rpm, more preferably 15000 rpm; the centrifugal temperature is preferably 10-20°C, more preferably 15°C; the centrifugal time is preferably 10-20min, more preferably 15min; the solvent for resuspending the precipitate is preferably MES buffer, and the volume is 1000 μL.

[0037] In the above step 2), the volume mass ratio of the activated fluorescent microspheres and the isoniazid monoclonal antibody is preferably 800-1200 μL: 40-60 μg, more preferably 1000 μL: 50 μg; the coupling time is preferably 100-150 min, more preferably 120 min; the coupling temperature is preferably 18-26° C., more preferably 20° C.; the coupling is preferably carried out under shaking conditions.

[0038] In the above step 2), after obtaining the isoniazid monoclonal antibody labeled with fluorescent microspheres, it is preferably further included to mix the isoniazid monoclonal antibody labeled with fluorescent microspheres and a bovine serum albumin aqueous solution, and perform a blocking treatment to obtain the blocked isoniazid monoclonal antibody labeled with fluorescent microspheres; the volume ratio of the isoniazid monoclonal antibody labeled with fluorescent microspheres to the bovine serum albumin aqueous solution is preferably 1-3:0.5-1.5, more preferably 2:1; the mass percentage of bovine serum albumin in the bovine serum albumin aqueous solution is preferably 2-4%, more preferably 3%; the blocking temperature is preferably 26-37°C, more preferably 30°C; the blocking time is preferably 1.5-3h, more preferably 2h.

[0039] In the present invention, the storage method of the blocked fluorescent microsphere-labeled isoniazid monoclonal antibody comprises the following steps: centrifuging the blocked fluorescent microsphere-labeled isoniazid monoclonal antibody, resuspending the precipitate, washing, and storing. Among them, the centrifugal speed is preferably 12000-16000rpm, more preferably 15000rpm, the centrifugal temperature is preferably 10-20℃, more preferably 15℃, and the centrifugal time is preferably 10-20min, more preferably 15min; the solvent for resuspending the precipitate is preferably a storage buffer, and the storage buffer is preferably a Tris buffer containing Proclin300 and bovine serum albumin, the mass percentage of Proclin300 in the Tris buffer is preferably 0.05%-0.1%, the mass percentage of bovine serum albumin in the Tris buffer is preferably 0.5%-1%, and the pH value of the Tris buffer is preferably 7.1-7.3, more preferably 7.2; the number of times the precipitate is washed is preferably 1-2 times, and the method for washing the precipitate is centrifugation again and resuspending the precipitate; the storage temperature is preferably 2-8℃, more preferably 4℃; and the storage is preferably kept away from light.

[0040] In the present invention, the isoniazid monoclonal antibody is preferably prepared using isoniazid-keyhole limpet hemocyanin (KLH) conjugate as an immunogen; the isoniazid-keyhole limpet hemocyanin (KLH) conjugate is a conjugate obtained by conjugating isoniazid with keyhole limpet hemocyanin (KLH); the preparation process of the isoniazid-keyhole limpet hemocyanin (KLH) conjugate is as follows: 40 mg of pure isoniazid (article number: I104690) purchased from Merck Biotechnology is weighed and dissolved in 2 mL of DMF to form a final concentration of 20 mg / mL. Then 90 μL of isoniazid with a concentration of 20 mg / mL is taken, mixed with 8 μL of EDC with a concentration of 100 mg / mL, and then 12 μL of NHS with a concentration of 50 mg / mL is added, and the mixture is evenly mixed and reacted for 15-60 minutes under stirring conditions at room temperature. After the reaction, the reaction mixture was centrifuged at 1600 r / min to discard the supernatant, 1 mL of 3 mg / mL keyhole limpet hemocyanin (KLH) solution was added and mixed, and the mixture was stirred at room temperature for 1-5 hours, and then dialyzed with phosphate buffer for 4 times, with the solution changed once every 12 hours. The dialysate was collected, and the concentration of the antigen was determined using the Quick Start Bradford Protein Assay Kit of BIO-RAD, USA, and the coupled complete antigen was identified by UV scanning. The isoniazid monoclonal antibody is prepared by the following method: a) using isoniazid-keyhole limpet hemocyanin conjugate as an immunogen to immunize Balb / c mice, and obtaining Balb / c mouse spleen cells capable of producing specific antibodies after boosting immunization; b) fusing the Balb / c mouse spleen cells capable of producing specific antibodies in step a) with myeloma cells SP20, measuring cell supernatants, and screening positive wells; c) cloning the fused cells in the positive wells in step b) to obtain a hybridoma cell line producing monoclonal antibodies, which is numbered as 1H11 and preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C202427; d) after sterile paraffin oil is injected into the peritoneal cavity of Balb / c mice, the hybridoma cell 1H11 in step c) is injected into the peritoneal cavity, and ascites is collected and purified to obtain isoniazid monoclonal antibodies with a purity of more than 90%.

[0041] The age of the Balb / c mice is preferably 7-9 weeks, more preferably 8 weeks. The present invention has no particular limitation on the amount of sterilized paraffin oil injected, and conventional doses in the art may be used. The time interval between the intraperitoneal injection of sterilized paraffin oil and the intraperitoneal injection of hybridoma cells into Balb / c mice is preferably 7-14 days, more preferably 10-12 days. The time interval between the intraperitoneal injection of hybridoma cells and the collection of ascites is preferably 7-10 days, more preferably 8-9 days. The method for purifying ascites is preferably affinity chromatography.

[0042] In the present invention, the nitrocellulose membrane coated with isoniazid-carrier protein conjugate preferably includes a detection area (T) and a quality control area (C); the detection area is preferably coated with isoniazid-bovine serum albumin conjugate, and the isoniazid-bovine serum albumin conjugate is purchased from Nanjing Fuxiao Biotechnology Co., Ltd. (Article No.: A163104L); the quality control area is preferably coated with goat anti-mouse IgG. The above-mentioned nitrocellulose membrane coated with isoniazid-carrier protein conjugate is preferably prepared by the following method: the detection area is coated with isoniazid-bovine serum albumin conjugate, and the quality control area is coated with goat anti-mouse IgG. After drying, a nitrocellulose membrane coated with isoniazid-bovine serum albumin conjugate and goat anti-mouse IgG is obtained; the drying temperature is preferably 45-60°C, more preferably 50°C; the drying time is preferably 15-25h, more preferably 20h.

[0043] In the present invention, the detection area is coated with isoniazid-bovine serum albumin conjugate preferably by the following method: spraying isoniazid-bovine serum albumin conjugate to the detection area on the nitrocellulose membrane to form a detection area. In the present invention, the concentration of the isoniazid-bovine serum albumin conjugate used for spraying is preferably 250-500 μg / mL, more preferably 400 μg / mL, and the spraying amount is preferably 0.75-1.5 μL / cm, more preferably 1.0 μL / cm; the spraying equipment is preferably a Jining gold spray film scratcher (model: XYZ3020).

[0044] In the present invention, before spraying the isoniazid-bovine serum albumin conjugate onto the detection area on the nitrocellulose membrane, the isoniazid-bovine serum albumin conjugate is preferably diluted; the dilution solution is preferably a phosphate buffer; the concentration of the phosphate buffer is preferably 0.005-0.02 mol / L, more preferably 0.01 mol / L; the pH value of the phosphate buffer is preferably 7.0-7.5, more preferably 7.2.

[0045] In the present invention, the quality control area is preferably coated with goat anti-mouse IgG by the following method: spray goat anti-mouse IgG onto the quality control area on the nitrocellulose membrane to form the quality control area. The concentration of the goat anti-mouse IgG used for spraying is preferably 150-250 μg / mL, more preferably 200 μg / mL, and the spraying amount is preferably 0.8-1.2 μL / cm, more preferably 1.0 μL / cm; the spraying equipment is preferably a Jining gold spray film scratcher (model: XYZ3020).

[0046] In the present invention, before spraying the goat anti-mouse IgG onto the quality control area on the nitrocellulose membrane, the goat anti-mouse IgG is preferably diluted; the dilution solution is preferably a phosphate buffer; the concentration of the phosphate buffer is preferably 0.005-0.02 mol / L, more preferably 0.01 mol / L; the pH value of the phosphate buffer is preferably 7.0-7.5, more preferably 7.2.

[0047] In the present invention, the method for using the fluorescent immunochromatographic detection card in detecting isoniazid comprises the following steps:

[0048] The method comprises adding pure isoniazid of different concentrations to a human serum matrix without isoniazid to prepare a calibrator; the concentration of isoniazid in the calibrator is 20 μg / ml, 10 μg / ml, 5 μg / ml, 2 μg / ml, 1 μg / ml and 0.5 μg / ml, respectively; the calibrator is diluted 10 to 100 times and then added to the fluorescent immunochromatographic detection card for immunochromatography, and the fluorescence intensity ratio T / C value of the detection area T line and the quality control area C line is measured to obtain the functional relationship between the concentration in the calibrator and the T / C value.

[0049] Take the sample to be tested, add it to the fluorescent immunochromatography test card for immunochromatography, measure the fluorescence intensity ratio T / C value of the detection area T line and the quality control area C line, and calculate the isoniazid content in the sample to be tested according to the function formula obtained above.

[0050] The following is a detailed description of a fluorescent immunochromatographic detection card for quantitative detection of isoniazid provided by the present invention, as well as its preparation method and application, in conjunction with the accompanying drawings and Examples 1-4. However, they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1: A fluorescent immunochromatographic test card for quantitative detection of isoniazid and its preparation method

[0052] The fluorescent immunochromatographic test card for quantitative detection of isoniazid in this embodiment comprises a base plate and a sample pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water-absorbing pad which are overlapped and pasted in sequence on the base plate; a detection area and a quality control area are arranged on the nitrocellulose membrane; the fluorescent microsphere pad is sprayed with isoniazid monoclonal antibodies labeled with fluorescent microspheres; the detection area on the nitrocellulose membrane is coated with isoniazid-bovine serum albumin conjugate, and the quality control area is coated with goat anti-mouse IgG.

[0053] The preparation method of the fluorescent immunochromatographic detection card comprises the following steps:

[0054] (1) Preparation of monoclonal antibodies against isoniazid

[0055] Preparation process of isoniazid-keyhole limpet hemocyanin conjugate (isoniazid-KLH): Weigh 40 mg of pure isoniazid (item number: I104690) purchased from Merck Biotechnology, dissolve in 2 mL of DMF to form a final concentration of 20 mg / mL, then take 90 μL of isoniazid with a concentration of 20 mg / mL, mix with 8 μL of EDC with a concentration of 100 mg / mL, and then add 12 μL of NHS with a concentration of 50 mg / mL, mix evenly and react for 15-60 minutes under stirring at room temperature. After the reaction is completed, the above reaction mixture is centrifuged at 1600 r / min to discard the supernatant, 1 mL of keyhole limpet hemocyanin (KLH) solution with a concentration of 3 mg / mL is added and mixed, react for 1-5 hours under stirring at room temperature, and dialyze with phosphate buffer 4 times, changing the solution once every 12 hours. The dialysate was collected and the concentration of the antigen was determined using the Quick Start Bradford Protein Assay Kit from BIO-RAD, USA. The coupled complete antigen was identified by UV scanning.

[0056] Preparation process of isoniazid monoclonal antibody: isoniazid-keyhole limpet hemocyanin (KLH) conjugate is used as an immunogen, diluted to 0.2 mg / ml with 10mM phosphate buffer, 500μl of the diluted immunogen is mixed with an equal volume of Freund's complete adjuvant, and emulsified completely. Balb / c mice are immunized by conventional methods. After 4 immunizations, mice are intraperitoneally injected with about 100μg of the immunogen for booster immunization. After 3 days, the spleen cells of Balb / c mice that produce specific antibodies are fused with myeloma cells SP20. The cell supernatant is measured by indirect competitive enzyme-linked immunosorbent assay to screen positive wells. The positive wells are cloned by limiting dilution method to obtain and establish a hybridoma cell line that produces isoniazid monoclonal antibody. The hybridoma cell line that produces isoniazid monoclonal antibody is named hybridoma cell line 1H11, which is deposited in China Center for Type Culture Collection CCTCC, and the deposit number is CCTCC NO: C202427.

[0057] Balb / c mice (8 weeks old) were intraperitoneally injected with sterile paraffin oil, and hybridoma cells were intraperitoneally injected 7-14 days later. Ascites was collected 7-10 days later. Ascites was purified by affinity chromatography, and the purity was determined to be above 90% by SDS-PAGE electrophoresis and stored at -20°C.

[0058] The isoniazid monoclonal antibody prepared by the method was subjected to an antibody titer comparison verification experiment and an antibody sensitivity comparison verification experiment with the isoniazid monoclonal antibody prepared by isoniazid-bovine serum albumin conjugate (isoniazid-BSA). From the results, it can be seen that the method of the present invention uses isoniazid-keyhole limpet hemocyanin conjugate as an immunogen, has strong specificity and good immunogenicity; the prepared isoniazid monoclonal antibody has a high titer and a strong competitive effect.

[0059] The method for preparing isoniazid monoclonal antibody with isoniazid-bovine serum albumin conjugate is as follows: in the step of preparing isoniazid monoclonal antibody with isoniazid-keyhole limpet hemocyanin conjugate, keyhole limpet hemocyanin is replaced with bovine serum albumin. The hybridoma cell line producing isoniazid monoclonal antibody prepared with isoniazid-bovine serum albumin conjugate as immunogen is named hybridoma cell line 4F3.

[0060] ① Antibody titer comparison and verification experiment:

[0061] The monoclonal antibodies prepared by immunizing mice with isoniazid-KLH and isoniazid-BSA were tested for titer using enzyme-linked immunosorbent assay (ELISA). At the same antibody concentration, the higher the OD value, the higher the antibody titer. The results are shown in Table 1 and Figure 1 As shown, the titer of the monoclonal antibody prepared by immunizing mice with isoniazid-KLH complete antigen was significantly higher than that of the monoclonal antibody prepared with isoniazid-BSA.

[0062] Table 1: Comparison of antibody titer results between 1H11 strain and 4F3 strain

[0063]

[0064] ② Antibody sensitivity comparison verification experiment (IC50)

[0065] The half inhibition rate (IC50) of the antibody was tested by indirect competitive enzyme-linked immunosorbent assay (ICELISA). In the ICELISA experiment, the OD value of the control well without isoniazid standard is B0, and the OD value of the well with isoniazid standard added is B. The binding rate is calculated as B / B0. When the binding rate is 50%, the corresponding concentration of isoniazid standard is IC50. The smaller the IC50 value, the higher the sensitivity and the stronger the specificity of the antibody. The results are shown in Table 2 and Figure 2 , Figure 3 As shown, the IC50 of the monoclonal antibody produced by immunizing mice with isoniazid-KLH complete antigen was 326.2 ng / ml, and the IC50 of the monoclonal antibody produced by immunizing mice with isoniazid-BSA complete antigen was 3210.8 ng / ml. Therefore, the sensitivity of the monoclonal antibody produced by the former was significantly higher than that of the latter by 10 times.

[0066] Table 2: Comparison of antibody sensitivity (IC50) between 1H11 strain and 4F3 strain

[0067]

[0068] (2) Preparation of isoniazid monoclonal antibody labeled with fluorescent microspheres

[0069] ① Activation: 50 μL of microsphere suspension with internally embedded fluorescent dye and surface modified with carboxyl functional groups purchased from Jiangsu Weidu Biotechnology Co., Ltd. was suspended in 1000 μL activation buffer (50 mM MES pH 6.0), 0.035 mg EDC and 0.35 mg NHS were added, mixed and activated at 26°C for 15 min to obtain suspension I;

[0070] ② Coupling: The suspension I activated in step ① was centrifuged at 15000 r / min for 15 min at 15°C, the supernatant was discarded, and the suspension was resuspended in 1000 μL activation buffer, 50 μg of isoniazid monoclonal antibody was added, and the mixture was mixed and shaken at 20°C for 120 min to obtain suspension II;

[0071] ③ Blocking: Add 500 μL of 3% bovine serum albumin solution to the suspension II obtained in step ②, mix well, and shake and block at 30° C. for 120 min to obtain suspension III;

[0072] ④ Storage: Centrifuge the suspension III in step ③ at 15000 r / min for 15 min at 15°C, discard the supernatant, resuspend in storage buffer (storage buffer is Tris buffer at pH 7.2 containing 0.07% Proclin 300 and 0.8% bovine serum albumin by mass), wash the microspheres twice with storage buffer, and finally resuspend in 500 μL storage buffer, mix well and store at 4°C in the dark.

[0073] (3) Preparation of fluorescent microsphere pad

[0074] The stored fluorescent microsphere-labeled isoniazid monoclonal antibody was diluted to 50 μg / mL with storage buffer, sprayed with gold using a gold sprayer at a spraying volume of 4.0 μl / cm, dried at 45°C for 20 h, and then taken out and sealed for storage.

[0075] (4) Preparation of nitrocellulose (NC) membrane coated with antibodies

[0076] Dilute isoniazid-bovine serum albumin conjugate to 400 μg / mL with 0.01 mol / L, pH 7.2 phosphate buffer, and spray it on the detection area (T) on the NC membrane using a gold spray film reader at a spray volume of 1.0 μL / cm; dilute goat anti-mouse IgG to 200 μg / mL with 0.01 mol / L, pH 7.2 phosphate buffer, and spray it on the quality control area (C) on the NC membrane using a gold spray film reader at a spray volume of 1.0 μL / cm; dry at 50°C for 20 h and set aside.

[0077] (5) Preparation of sample pad

[0078] The sample pad was soaked in a phosphate buffer solution containing 2% sucrose, 0.5% bovine serum albumin, pH 7.2, and 0.02 mol / L for 2 h, and dried at 50° C. for 6 h for later use.

[0079] (6) Assembly of test card

[0080] The sample pad, fluorescent microsphere pad, nitrocellulose membrane, and water-absorbing pad are overlapped and pasted on the bottom plate from left to right in sequence, the end of the sample pad is connected to the beginning of the fluorescent microsphere pad, the end of the fluorescent microsphere pad is connected to the beginning of the nitrocellulose membrane, the end of the nitrocellulose membrane is connected to the beginning of the water-absorbing pad, the beginning of the sample pad is aligned with the beginning of the bottom plate, the end of the water-absorbing pad is aligned with the end of the bottom plate, and then cut into 4.0 mm wide test strips with a strip cutter, installed in a special plastic card, and assembled into a fluorescent immunochromatographic test card. The plastic card is provided with a sample addition hole and a detection window.

[0081] Example 2: A method for quantitatively detecting isoniazid using a fluorescent immunochromatographic test card

[0082] (1) Sample pretreatment

[0083] Accurately pipette 20 μl of sample that has been freshly restored to room temperature into 180 μl of sample diluent and mix thoroughly.

[0084] (2) Detection using fluorescent immunochromatographic test card

[0085] Use a micropipette to accurately draw 90 μL of the sample solution to be tested after sample pretreatment into the sample well of the fluorescent immunochromatographic detection card prepared in Example 1, and react for 15 minutes at room temperature; insert the reaction-completed detection card into the card slot of the fluorescence detector (model: IGreat-IF-F10) produced by Igreat (Wuhan) Technology Co., Ltd., select the item to be tested through the touch screen, press the "Quick Test" button, and the fluorescence detector will automatically scan and test the fluorescent immunochromatographic detection card under the excitation / emission band of 365 / 610nm.

[0086] (3) Analysis of test results

[0087] After the test is completed, the fluorescence detector obtains the ratio of the fluorescence intensity of the detection area of ​​the fluorescence immunochromatography test card to the fluorescence intensity of the quality control area, and calculates the isoniazid content in the sample to be tested according to the following formula:

[0088] Y = D + (A - D) / [1 + (X / C) ^ B];

[0089] Among them, A = 0.282190, B = -1.745659, C = 1.720567, D = 2.035401;

[0090] Y is the ratio of the fluorescence intensity of the test area to the fluorescence intensity of the quality control area on the fluorescent immunochromatographic test card; X is the concentration of isoniazid in the sample.

[0091] Example 3: Anti-interference experiment of fluorescent immunochromatographic test card

[0092] Interfering substances were added to the isoniazid test sample, and the sample values ​​before and after the addition of the interfering substances were tested using the fluorescent immunochromatographic test card prepared in Example 1 to evaluate whether conventional clinical interfering substances had a significant effect on the test results. The experimental results are shown in Table 3. The interfering substances were a mixture of triglycerides, bilirubin and hemoglobin, and their concentrations in the test sample were 10 mg / ml, 1 mg / ml and 10 mg / ml, respectively.

[0093] Table 3: Effects of interfering substances on the determination results

[0094]

[0095] As shown in Table 3, the deviations of the test results of each sample before and after the addition of interfering substances were all within the range of ±10%, which is within the acceptable range.

[0096] Example 4: Correlation analysis of the measurement results of the fluorescent immunochromatographic test card

[0097] The fluorescent immunochromatographic test card prepared in the above Example 1 was compared with the high performance liquid chromatography method for determining isoniazid in human serum, wherein the human serum was a clinical serum sample. The high performance liquid chromatography method was performed according to the following scheme: Agilent 1260 Infinity II high performance liquid chromatograph, C18 column (150 mm × 4.6 mm, 5 μm), mobile phase: 25 mmol / L potassium dihydrogen phosphate buffer at pH 4.2-acetonitrile (85:15); detection wavelength: 240 nm; flow rate: 1 mL / min; 400 μl serum sample was added with 50 μl of 10% zinc sulfate precipitated protein, centrifuged at 5000 r / min, and 20 μl was taken for injection to determine the isoniazid peak height. The results are shown in Table 4. The correlation curve is shown in Figure 4 , it can be seen that the two fit well.

[0098] Table 4: Comparison of the isoniazid fluorescence immunochromatographic detection card of the present invention and the high performance liquid chromatography method for determining isoniazid in human serum

[0099] Serum sample number Fluorescence immunochromatography (μg / ml) HPLC (μg / ml) 1# 1.214 1.0 2# 2.732 1.3 3# 4.547 2.4 4# 3.470 1.8 5# 3.567 1.9 6# 5.903 4.6 7# 8.567 5.4 8# 7.191 4.0 9# 11.648 5.8 10# 7.263 4.7 11# 38.369 14.8 12# 17.384 7.2 13# 7.767 6.1 14# 11.105 8.2 15# 19.893 11.5 16# 9.875 6.7 17# 8.539 5.3 18# 12.542 9.6 19# 6.757 5.7 20# 15.782 11.6

[0100] From Table 4 and Figure 4 As shown in the figure, the two different methods detected serum samples of patients taking isoniazid, and the correlation was good. 2 =0.9125, which meets the requirements of clinical use.

[0101] It can be seen from the above embodiments that the present invention provides a fluorescent immunochromatographic detection card for quantitative detection of isoniazid, which has strong anti-interference ability and has the advantages of high detection accuracy and strong specificity.

Claims

1. A fluorescent immunochromatographic test card for quantitative detection of isoniazid, comprising a base plate and a sample pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped and pasted on the base plate, wherein a detection area and a quality control area are arranged on the nitrocellulose membrane; characterized in that: The fluorescent microsphere pad is sprayed with isoniazid monoclonal antibody marked with fluorescent microspheres; the detection area on the nitrocellulose membrane is coated with isoniazid-carrier protein conjugate, and the quality control area is coated with goat anti-mouse IgG; the isoniazid monoclonal antibody is produced by hybridoma cell line 1H11; the hybridoma cell line 1H11 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: C202427; the hybridoma cell line 1H11 is prepared by using isoniazid-keyhole limpet hemocyanin conjugate as an immunogen; the isoniazid-carrier protein conjugate is a conjugate obtained by conjugating isoniazid with a carrier protein; the carrier protein is one of keyhole limpet hemocyanin, bovine serum albumin, and chicken yolk protein; the fluorescent immunochromatographic detection card is used to detect the content of isoniazid in serum, plasma or whole blood.

2. The fluorescent immunochromatographic detection card according to claim 1, characterized in that: The diameter of the fluorescent microsphere is 100-300 nm; the surface of the fluorescent microsphere contains carboxyl groups.

3. A method for preparing the fluorescent immunochromatographic detection card according to any one of claims 1 to 2, comprising overlapping and pasting a sample pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water-absorbing pad in sequence on a bottom plate, characterized in that: The sample pad is treated with a bovine serum albumin solution containing sucrose before pasting, and the mass volume concentration of sucrose in the bovine serum albumin solution is 1-3%; the preparation method of the fluorescent microsphere pad comprises: spraying the isoniazid monoclonal antibody labeled by the fluorescent microsphere onto a glass cellulose membrane, and obtaining the fluorescent microsphere pad after drying; the preparation method of the nitrocellulose membrane comprises: coating the detection area with isoniazid-carrier protein conjugate, coating the quality control area with goat anti-mouse IgG, and drying at 45-60°C for 15-25h to obtain a nitrocellulose membrane coated with isoniazid-carrier protein conjugate and goat anti-mouse IgG.

4. The method for preparing a fluorescent immunochromatographic detection card according to claim 3, characterized in that: The method for preparing the fluorescent microsphere-labeled isoniazid monoclonal antibody comprises the following steps: 1) Mix the fluorescent microsphere suspension, MES buffer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and perform activation treatment to obtain activated fluorescent microspheres; 2) Mixing the activated fluorescent microspheres in step 1) and isoniazid monoclonal antibodies for coupling to obtain isoniazid monoclonal antibodies labeled with fluorescent microspheres.

5. The detection method of the fluorescent immunochromatographic detection card according to any one of claims 1 to 2, characterized in that: The following steps are involved: The calibrators of different concentrations are subjected to immunochromatography through the fluorescent immunochromatographic test card, and the fluorescence intensity ratio T / C value of the detection area T line and the quality control area C line is measured to obtain a function formula of the concentration and T / C value of the calibrator; Take the sample to be tested, perform immunochromatography through the fluorescent immunochromatography test card, measure the fluorescence intensity ratio T / C value of the detection area T line and the quality control area C line, and calculate the isoniazid content in the sample to be tested according to the function formula.

6. Application of fluorescent immunochromatographic detection card, characterized in that: The isoniazid content in the sample is detected by using the fluorescent immunochromatographic detection card prepared by the fluorescent immunochromatographic detection card described in any one of claims 1-2 or the method described in any one of claims 3-4.

7. Application of the detection method of the fluorescent immunochromatographic detection card, characterized in that: The isoniazid content in serum, plasma or whole blood is detected by the detection method described in claim 5.

8. A kit, characterized in that: The invention comprises the fluorescent immunochromatographic detection card according to any one of claims 1 to 2 or the fluorescent immunochromatographic detection card prepared by the method according to any one of claims 3 to 4.

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