A gamma interferon electrochemiluminescence test strip and its application

Abstract

The present invention discloses an interferon-γ electrochemiluminescence test strip and its application. An interferon-γ electrochemiluminescence test strip of the present invention comprises a paper-based channel layer and an electrode layer; the paper-based channel layer comprises a sample loading pad, a conjugation pad, a detection pad and a water-absorbing pad which are overlapped in sequence; the sample loading pad is fixed with a sample buffer and a heterophilic antibody blocker; the conjugation pad is coated with a ruthenium-coupled labeled antibody; the detection pad is coated with a microsphere-coupled capture antibody and an electrochemiluminescence co-reactant buffer; the electrode layer is provided with a plurality of electrodes, one end of which is connected to the detection pad. The interferon-γ electrochemiluminescence test strip prepared by the present invention is used to detect interferon-γ, which has the advantages of good sensitivity and high detection efficiency, and provides an effective means for the rapid screening of interferon-γ.

Description

Technical Field

[0001] The invention relates to the technical field of medical detection, in particular to an interferon-γ electrochemiluminescence detection test strip and application thereof. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by infection with Mycobacterium tuberculosis. Mycobacterium tuberculosis can invade various organs of the human body, but mainly infects the lungs, which is called pulmonary tuberculosis. Tuberculosis is a chronic infectious disease, the number one cause of death from a single infectious source, and one of the 13th leading causes of death worldwide. Effective diagnosis is an important means to effectively reduce deaths from tuberculosis.

[0003] In the related technologies, the tuberculosis detection methods include tuberculin skin test, smear microscopy, bacterial culture, human interferon-γ induced protein 10 assay, enzyme-linked spot test, interferon-γ in vitro release enzyme-linked immunosorbent assay, fluorescence quantitative and probe PCR detection, and loop-mediated isothermal amplification technology, etc. However, there are many problems in these methods that make it impossible to accurately detect tuberculosis, such as interference by BCG and non-tuberculous mycobacteria, high subjectivity of judgment results, difficulty in sampling extrapulmonary tuberculosis, low positive rate, high requirements for laboratories and technicians, difficulty in sampling extrapulmonary tuberculosis and long time consumption, etc. Electrochemiluminescence detection of interferon-γ is the best method to make up for these defects, and there is no research report on the electrochemiluminescence interferon-γ detection method using paper substrate.

[0004] Therefore, the present invention proposes an interferon-γ electrochemiluminescence test strip, which has high detection accuracy and sensitivity and a simple detection method, and has far-reaching social significance and broad economic value for improving the diagnosis rate of tuberculosis. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides an electrochemiluminescent test strip for detecting interferon-γ and its application. The detection of interferon-γ by using the test strip has the advantages of high detection efficiency and sensitivity.

[0006] The present invention also provides an application of the interferon-γ electrochemiluminescence test strip in preparing a kit for detecting interferon-γ.

[0007] In a first aspect of the present invention, there is provided an interferon-γ electrochemiluminescence test strip, comprising a paper-based channel layer and an electrode layer;

[0008] The paper-based channel layer comprises a sample loading pad, a conjugation pad, a detection pad and a water-absorbing pad which are sequentially overlapped and arranged; the sample loading pad is fixed with a sample buffer and a heterophilic antibody blocker; the conjugation pad is coated with a ruthenium-coupled labeled antibody; the detection pad is coated with a microsphere-coupled capture antibody and an electrochemiluminescent co-reactant buffer;

[0009] The electrode layer is provided with a plurality of electrodes, one end of each electrode is connected to the detection pad.

[0010] The interferon-γ electrochemiluminescence test strip according to the embodiment of the present invention has at least the following beneficial effects: by adding sample buffer and heterophilic antibody blocker to the sample loading pad, the present invention can effectively increase the migration speed of the sample to be tested in the paper-based channel layer and reduce the interference of other heterophilic antibodies in the sample to be tested, which helps to improve the detection efficiency and detection sensitivity. The electrode layer of the present invention is used to provide the voltage required to generate a chemiluminescent signal on the detection pad.

[0011] The interferon-γ electrochemiluminescence test strip provided by the present invention has the advantages of strong detection specificity and high sensitivity, and the minimum detection limit of the interferon-γ standard positive product is as low as 4.4 pg / mL; secondly, the interferon-γ electrochemiluminescence test strip provided by the present invention has a wide range of applications and a short time, and each reaction only takes 5 to 10 minutes to produce a result.

[0012] In addition, the electrochemiluminescence detection test strip of the present invention is easy to carry and use, and is easy to expand production.

[0013] According to some embodiments of the present invention, the electrode layer is provided with three electrodes, namely a working electrode, a counter electrode and a reference electrode. The electrode layer is used to provide the voltage required for generating a chemiluminescent signal on the detection pad.

[0014] According to some embodiments of the present invention, the sample loading pad is obtained by the following preparation method: soaking the matrix material for preparing the sample loading pad with the sample buffer and the heterophilic antibody blocking agent, and then drying the matrix material;

[0015] Preferably, the matrix material of the sample loading pad is a glass cellulose membrane.

[0016] According to some embodiments of the present invention, the sample buffer comprises Tris-HCl, BSA, sodium caseinate, trehalose, sucrose, NaCl and PBS.

[0017] Preferably, the molar concentration of Tris-HCl is 3 mol / L to 6 mol / L;

[0018] Preferably, the pH value of the Tris-HCl is 7.0 to 7.6;

[0019] Preferably, the pH value of the PBS is 7 to 7.6.

[0020] According to some embodiments of the present invention, the sample buffer specifically consists of 0.05M Tris-HCl (pH 7.4), 1% BSA, 0.5% sodium caseinate, 0.5% trehalose, 2% sucrose, 0.9% NaCl and the remainder PBS buffer (pH 7.4).

[0021] According to some embodiments of the present invention, the method for preparing the PBS buffer comprises:

[0022] Dissolve 35.8g Na2HPO4·12H2O in 1000mL sterile water and 15.6g NaH2PO4·2H2O in 1000mL sterile water respectively, then mix 19mL NaH2PO4 and 81mL Na2HPO4, and dilute to 1000mL with water.

[0023] According to some embodiments of the present invention, the concentration of the heterophilic antibody blocker is 0.3 mg / mL to 0.8 mg / mL;

[0024] Preferably, the concentration of the heterophilic antibody blocker is 0.5 mg / mL.

[0025] According to some embodiments of the present invention, the conjugate pad is obtained by the following preparation method: terpyridine ruthenium and interferon-γ specific monoclonal antibody are mixed and reacted to obtain the ruthenium-coupled labeled antibody, and then the matrix material for preparing the conjugate pad is immersed in the ruthenium-coupled labeled antibody, and the conjugate pad is obtained after drying;

[0026] Preferably, the matrix material of the conjugate pad and the matrix material of the sample loading pad are the same material.

[0027] According to some embodiments of the present invention, the matrix material of the conjugate pad and the matrix material of the loading pad are both glass cellulose membranes.

[0028] According to some embodiments of the present invention, the molar ratio of the terpyridine ruthenium to the interferon-γ specific monoclonal antibody is 20 to 80:1;

[0029] Preferably, the molar ratio of the terpyridine ruthenium to the interferon-γ specific monoclonal antibody is 40:1.

[0030] According to some embodiments of the present invention, the mass concentration of the ruthenium-conjugated labeled antibody is 0.5 mg / mL to 1.5 mg / mL.

[0031] Preferably, the mass concentration of the ruthenium-coupled labeled antibody is 1 mg / mL.

[0032] According to some embodiments of the present invention, the detection pad is obtained by the following preparation method: soaking the matrix material for preparing the detection pad into the microsphere-coupled capture antibody and electrochemiluminescent co-reactant buffer, and drying the matrix material to obtain the detection pad.

[0033] According to some embodiments of the present invention, the matrix material of the detection pad is selected from at least one of filter paper and nylon membrane;

[0034] Preferably, the matrix material of the detection pad is a nylon membrane.

[0035] The present invention improves the matrix material of the paper-based channel layer and adopts nylon membrane as the matrix material of the detection pad of the present invention, which can further improve the detection rate of the luminescent signal and take into account the fluidity of the sample to be tested in the paper-based channel layer, which plays an important role in improving the detection efficiency of gamma interferon.

[0036] According to some embodiments of the present invention, the preparation method of the microsphere-coupled capture antibody includes: activating the plastic microspheres, adding the capture antibody in a ratio of plastic microspheres: capture antibody mass concentration of 1:1 to 2.5, and obtaining the product after reaction.

[0037] According to some embodiments of the present invention, the mass concentration ratio of the plastic microspheres: capture antibody is 1:2.

[0038] Preferably, the plastic microspheres are selected from any one of red plastic microspheres, yellow plastic microspheres and transparent plastic microspheres.

[0039] According to some embodiments of the present invention, the capture antibody is specifically the interferon-γ-specific monoclonal antibody B07.

[0040] According to some embodiments of the present invention, the electrochemiluminescent coreactant buffer comprises phosphate buffer, tripropylamine, a detergent and a preservative.

[0041] According to some embodiments of the present invention, the mass concentration ratio of the ruthenium-coupled labeled antibody to the microsphere-coupled capture antibody is 1:0.5-2.5;

[0042] Preferably, the mass concentration ratio of the ruthenium-coupled labeled antibody to the microsphere-coupled capture antibody is 1:1-2;

[0043] More preferably, the mass concentration ratio of the ruthenium-coupled labeled antibody to the microsphere-coupled capture antibody is 1:2.

[0044] According to some embodiments of the present invention, the test strip further comprises a sealing film layer, and the sealing film layer comprises a first sealing film layer and a second sealing film layer;

[0045] The first sealing film layer is provided with a sample adding hole corresponding to the position of the sample adding pad.

[0046] The first sealing film layer and the second sealing film layer are used to protect the reagent fixed in the test strip from evaporation, thereby improving the storage stability of the test strip. In addition, the sealing film layer can also effectively reduce the interference of environmental factors and has a certain promoting effect on improving the sensitivity of detection.

[0047] According to some embodiments of the present invention, the length of the sample adding pad is 20 mm to 30 mm.

[0048] Preferably, the length of the sample adding pad is 25 mm.

[0049] According to some embodiments of the present invention, the length of the conjugate pad is 5 mm to 15 mm;

[0050] Preferably, the length of the bonding pad is 10 mm.

[0051] According to some embodiments of the present invention, the length of the detection pad is 10 mm to 20 mm;

[0052] Preferably, the length of the detection pad is 15 mm.

[0053] According to some embodiments of the present invention, the length of the absorbent pad is 15 mm to 25 mm;

[0054] Preferably, the length of the water absorbent pad is 20 mm.

[0055] According to some embodiments of the present invention, the widths of the sample adding pad, the conjugation pad, the detection pad and the water absorption pad are 4 mm to 10 mm.

[0056] Preferably, the widths of the sample adding pad, the conjugation pad, the detection pad and the water absorbing pad are 4 mm to 8 mm.

[0057] More preferably, the width of the sample adding pad, the conjugation pad, the detection pad and the water absorption pad is 6 mm.

[0058] According to some embodiments of the present invention, the overlapping length of the sample adding pad, the conjugation pad, the detection pad and the water absorption pad is 1 mm to 3 mm;

[0059] Preferably, the overlapping length of the sample adding pad, the conjugation pad, the detection pad and the water absorption pad is 2 mm.

[0060] According to some embodiments of the present invention, the matrix material of the water absorbent pad is filter paper. The filter paper can effectively increase the migration speed of the sample to be tested in the paper-based channel layer, thereby improving the detection efficiency.

[0061] The second aspect of the present invention provides a use of the above-mentioned electrochemiluminescence test strip in the preparation of a kit for detecting interferon-γ.

[0062] The application of the embodiments of the present invention has at least the following beneficial effects:

[0063] (1) The detection of interferon-γ by the electrochemiluminescence test strip prepared by the present invention can effectively improve the detection rate of interferon-γ, and the detection method is simple and does not require training for operators.

[0064] (2) The test strip of the present invention has a unique electrochemical luminescent substance, which has a strong luminescent signal and little stray light interference, providing a solution for weak detection signals. In addition, since the reaction is carried out on a paper substrate, the detection cost is greatly reduced.

[0065] (3) The test strip of the present invention has a low detection limit, and the minimum detection limit for interferon-γ is as low as 4.4 pg / mL.

[0066] According to some embodiments of the present invention, the specific steps of detecting interferon-γ based on the electrochemiluminescence test strip include:

[0067] Step S1: collecting peripheral blood, obtaining peripheral blood containing interferon-γ after an interferon release test, and adding electrochemiluminescent co-reactant buffer and PBS buffer to obtain a sample to be tested;

[0068] Step S2: adding the sample to be tested into the sample addition hole of the electrochemiluminescence detection test strip, and after the reaction, placing the test strip in an electrochemiluminescence detector for detection to obtain a corresponding interferon-γ response curve;

[0069] Step S3: Calculate the concentration of interferon-γ in the sample to be tested according to the interferon-γ response curve and the pre-established interferon-γ standard curve.

[0070] According to some embodiments of the present invention, the volume ratio of the peripheral blood containing gamma interferon to the electrochemiluminescent co-reactant buffer is 2:1.

[0071] According to the application of the embodiment of the present invention, the detection of interferon-γ is used for non-diagnostic purposes.

[0072] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0074] Figure 1This is a schematic diagram of the decomposition of the layers of the interferon-γ electrochemiluminescence test strip according to an embodiment of the present invention.

[0075] Figure 2 This is a schematic diagram of an electrochemiluminescence test strip for detecting interferon-γ according to an embodiment of the present invention.

[0076] Figure 3 The electrochemiluminescence intensity measurement results of different dual antibody ratios in the examples of the present invention are shown.

[0077] Figure 4 This is a standard curve diagram of the interferon-γ paper-based electrochemiluminescence test strip according to an embodiment of the present invention.

[0078] Figure 5 This is a schematic diagram of detecting interferon-γ positive samples using an interferon-γ paper-based electrochemiluminescence test strip according to an embodiment of the present invention.

[0079] Reference numerals:

[0080] Paper-based channel layer 100, sample loading pad 110, conjugation pad 120, detection pad 130, water absorption pad 140;

[0081] Electrode layer 200;

[0082] A first sealing film layer 310;

[0083] The second sealing film layer 320 . DETAILED DESCRIPTION

[0084] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0085] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0086] In the description of the present invention, it should be understood that descriptions involving orientation, such as orientation or positional relationship indicated as up, down, etc., are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0087] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In the description of the embodiments of the present invention, several refers to more than two.

[0088] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0089] Example 1: An electrochemiluminescent test strip for interferon-γ

[0090] This embodiment is an electrochemiluminescent test strip for detecting interferon-γ. Figure 1 As shown, it includes: a sealing film layer, a paper-based channel layer 100 and an electrode layer 200, wherein the paper-based channel layer 100 is a "straight" paper-based microchannel composed of a sample pad 110, a conjugation pad 120, a detection pad 130 and a water-absorbing pad 140, as shown in FIG. Figure 2 As shown, the sample pad 110, the conjugation pad 120, the detection pad 130 and the absorbent pad 140 are overlapped in sequence, with an overlapping distance of about 2 mm, wherein the sample pad 110 is fixed with a sample buffer and a heterophilic antibody blocker (purchased from Jiangsu Fanbo Biological Products Co., Ltd., with a product number of HBR-5); the conjugation pad 120 is coated with a ruthenium-coupled labeled antibody, and the labeled antibody is specifically a gamma interferon-specific monoclonal antibody A20 (purchased from Reid Biotechnology Co., Ltd., with a batch number of 20200909), and the detection pad 130 is coated with a microsphere-coupled capture antibody and an electrochemiluminescent co-reactant buffer, and the capture antibody is specifically a gamma interferon-specific monoclonal antibody B07 (purchased from Reid Biotechnology Co., Ltd., with a batch number of 20201105). The electrode layer 200 is provided with three electrodes, namely a working electrode, a counter electrode and a reference electrode, and one end of the three electrodes is connected to the bottom of the detection pad 130 to provide the voltage required to generate a chemiluminescent signal on the detection pad 130.

[0091] In the interferon-γ electrochemiluminescence test strip of this embodiment, the sample pad 110 and the conjugate pad 120 are made of glass cellulose membrane, the detection pad 130 is made of nylon membrane, and the absorbent pad 140 is made of filter paper; wherein the length of the sample pad 110 is 25 mm, the length of the conjugate pad 120 is 10 mm, the length of the detection pad 130 is 15 mm, and the length of the absorbent pad 140 is 20 mm; the widths of the sample pad 110, the conjugate pad 120, the detection pad 130, and the absorbent pad 140 are all 6 mm. Using the above matrix material as the material of the paper-based channel layer 100 can further improve the detection rate of the luminescent signal, while taking into account the fluidity of the sample to be tested in the paper-based channel layer 100.

[0092] In the interferon-γ electrochemiluminescence test strip of the embodiment, the sealing film layer of the interferon-γ electrochemiluminescence test strip includes a first sealing film layer 310 and a second sealing film layer 320, wherein the first sealing film layer 310 is located on the side of the paper-based channel layer 100 away from the electrode layer 200, and the first sealing film layer 310 is provided with a sample loading hole corresponding to the position of the sample loading pad 110, for adding the sample to be tested into the sample loading pad 110; the second sealing film layer 320 is located on the side of the electrode layer 200 away from the paper-based channel layer 100, and after the first sealing film layer 310 and the second sealing film layer 320 are covered, they can be used to protect the paper-based channel layer 100 and the electrode layer 200 from external factors, which is conducive to improving the sensitivity of the test strip. In addition, the first sealing film layer 310 and the second sealing film layer 320 can also effectively prevent the evaporation of the reagent fixed in the paper-based channel layer 100, and improve the storage stability of the test strip.

[0093] The sample buffer and heterophilic antibody blocker are fixed in the sample addition area of ​​the interferon-γ electrochemiluminescence test strip of this embodiment, wherein the sample buffer is specifically composed of 0.05M Tris-HCl (pH value is 7.4), 1% BSA, 0.5% casein sodium, 0.5% trehalose, 2% sucrose, 0.9% NaCl and the balance PBS buffer (pH value is 7.4), and the sample buffer helps to dilute the sample to be tested and promote the rapid migration of the sample to be tested. The heterophilic antibody blocker can effectively eliminate the interference of heterophilic antibodies in the sample to be tested, thereby reducing false positives.

[0094] The conjugate pad 120 of the interferon-γ electrochemiluminescence test strip of this embodiment is coated with a ruthenium-coupled labeled antibody, wherein the ruthenium-coupled labeled antibody is specifically a terpyridine ruthenium-coupled labeled antibody. The detection pad 130 is coated with a microsphere-coupled capture antibody and an electrochemiluminescent co-reactant buffer. The microsphere-coupled capture antibody is a plastic microsphere-coupled capture antibody, and the electrochemiluminescent co-reactant buffer is composed of a phosphate buffer, tripropylamine, a detergent, and a preservative. The concentration ratio of the ruthenium-coupled labeled antibody to the microsphere-coupled capture antibody is 1:2, and the two antibodies can further reduce nonspecific binding and improve detection sensitivity.

[0095] Example 2: A method for preparing an electrochemiluminescent test strip for detecting interferon-γ

[0096] The specific method for making the interferon-γ electrochemiluminescence test strip in this embodiment is as follows:

[0097] 1. Preparation of the sample pad

[0098] In this embodiment, glass cellulose membrane is used as the matrix material of the sample loading pad 110. After the glass cellulose membrane is immersed in sample buffer and heterophilic antibody blocking agent (mass concentration of 0.5 mg / mL, purchased from Jiangsu Fanbo Biological Products Co., Ltd.), it is dried and cut into a size of 25 mm in length and 6 mm in width to obtain the sample loading pad 110.

[0099] The sample buffer is specifically composed of 0.05M Tris-HCl (pH 7.4), 1% BSA, 0.5% sodium caseinate, 0.5% trehalose, 2% sucrose, 0.9% NaCl and the remainder PBS buffer (pH 7.4). The sample buffer is used to dilute the sample to be tested and can promote the rapid migration of the sample to be tested.

[0100] 2. Preparation of conjugate pad

[0101] Under light-proof conditions, terpyridine ruthenium was balanced to room temperature (25±2°C), dissolved with DMSO, and mixed by oscillation after dissolution; the labeled antibody was taken out and dissolved, mixed by oscillation, diluted with PBS, and terpyridine ruthenium and interferon-γ specific monoclonal antibody A20 (purchased from Red Biotechnology Co., Ltd.) were mixed together at a molar ratio of 40:1, and then placed on a turntable at room temperature for 12 hours at 10 rpm. After the reaction was completed, a desalting column was placed to elute the unbound ruthenium to obtain the ruthenium-coupled labeled antibody, and the OD value was measured after the elution was completed. The results are shown in Table 1.

[0102] Table 1: Molar ratio of ruthenium-conjugated labeled antibodies

[0103] Terpyridine ruthenium: interferon-γ specific monoclonal antibody (molar ratio) 20:1 40:1 80:1 <![CDATA[γ interferon-specific monoclonal antibody (OD 277 )]]> 0.632 0.929 0.947 <![CDATA[Ruthenium (III) terpyridine (OD 455 )]]> 0.051 0.1044 0.1199

[0104] As can be seen from Table 1, according to the formula, the coupling efficiency is highest when the molar ratio of terpyridine ruthenium:γ-interferon-specific monoclonal antibody is 40:1 (ruthenium coupling labeling efficiency = molar concentration of ruthenium in ruthenium-labeled antibody solution / molar concentration of antibody), so this molar ratio is selected to prepare the ruthenium-coupled labeled antibody as the coating material of the conjugate pad 120.

[0105] The matrix material (glass cellulose membrane) for preparing the conjugate pad 120 is immersed in the ruthenium-coupled antibody obtained above, the mass concentration of the ruthenium-coupled antibody is 1 mg / mL, and after drying, it is cut into a size of 10 mm in length and 6 mm in width to obtain the conjugate pad 120.

[0106] 3. Preparation of test pad

[0107] The same amount of the ruthenium-coupled labeled antibody prepared above was dripped onto different detection papers, and the electrochemiluminescence intensity test was performed to select the suitable paper. The results are shown in Table 2.

[0108] Table 2: Test paper

[0109] Paper type filter paper Nylon membrane Nitrocellulose membrane Luminous intensity 16311 6374 none

[0110] The test results show that strong luminescent signals can be detected on both filter paper and nylon membrane. However, since filter paper cannot meet the antibody flow requirements of the experiment, nylon membrane is selected as the material of the detection pad 130 .

[0111] The plastic microspheres coupled with the capture antibody coated in the detection pad 130 are obtained by the following method:

[0112] Step S1: dilute the plastic microspheres with 0.05 mol / L MES with a pH value of 6.0, and then add 10 μL of 10 mg / mL NHS and EDC to activate the plastic microspheres. After mixing and reacting for 20 minutes, centrifuge at 20000×g for 20 minutes.

[0113] Step S2: After centrifugation, re-dissolve with MES, and then centrifuge again under the same conditions to wash the plastic microspheres.

[0114] Step S3: After the washing is completed, the capture antibody (specifically, interferon-γ-specific monoclonal antibody B07, purchased from Red Biotechnology Co., Ltd.) is added at a mass concentration ratio of 1:2 (plastic microspheres: capture antibody), and after addition, it is placed on a turntable for reaction for 1 hour. After the reaction is completed, centrifugation is performed under the same conditions as above;

[0115] Step S4: After centrifugation, discard the supernatant, add a blocking solution consisting of 0.05M Tris-HCl solution and 0.5% casein sodium, and then centrifuge at 20000×g for 20 minutes. After centrifugation, discard the supernatant, add a resuspension consisting of PBS (pH 7.4) and 1% BSA to obtain plastic microsphere-coupled capture antibodies.

[0116] In order to further obtain the optimal reaction concentration of ruthenium-coupled labeled antibody and plastic microsphere-coupled capture antibody, under the same conditions, the concentration of ruthenium-coupled labeled antibody was set to 1 mg / mL, and then 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL and 2 mg / mL of plastic microsphere-coupled capture antibody were reacted, and the electrochemiluminescence intensity was measured. The measurement results are shown in Figure 2. Figure 3As shown, the test results indicate that the optimal reaction concentration ratio of ruthenium-coupled labeled antibody to plastic microsphere-coupled capture antibody is 1:2.

[0117] The reaction concentration ratio of ruthenium-coupled labeled antibody to plastic microsphere-coupled capture antibody is 1:2 as a reference reaction concentration, the plastic microsphere-coupled capture antibody and electrochemiluminescent co-reactant buffer are fixed to the nylon membrane, and after drying, the membrane is cut into a size of 15 mm in length and 6 mm in width to obtain the detection pad 130.

[0118] The electrochemiluminescence co-reactant buffer consists of phosphate buffer, tripropylamine, detergent and preservative.

[0119] 4. Preparation of absorbent pad

[0120] The absorbent pad 140 of this embodiment is made of filter paper, and has a length of 20 mm and a width of 6 mm. The filter paper has good water absorption, which is conducive to the rapid migration of the sample to be tested and reduces the detection time.

[0121] 5. Assembly of test strips

[0122] The sample loading pad 110, the binding pad 120, the detection pad 130 and the absorbent pad 140 prepared above are overlapped in sequence to obtain the paper-based channel layer 100, and then the electrode layer 200 is assembled with the paper-based channel layer 100, and its electrode is connected to the detection pad 130 at a corresponding position to provide the voltage required to generate a chemiluminescent signal on the detection pad 130, and finally the first sealing film layer 310 and the second sealing film layer 320 are assembled, wherein the first sealing film layer 310 is provided with a sample loading hole at a position corresponding to the sample loading pad 110, and after the assembly is completed, the interferon-γ electrochemiluminescence detection test strip is obtained.

[0123] Test Example 1: Accuracy

[0124] 1. Experimental Materials

[0125] (1) The sample to be tested is peripheral blood of tuberculosis patients (the actual concentration of interferon-γ in the sample to be tested is 1000 pg / mL), and the negative sample is PBS.

[0126] (2) Interferon-γ standards, with concentrations set at 1 pg / mL, 8 pg / mL, 40 pg / mL, 200 pg / mL, 1000 pg / mL, and 5000 pg / mL, respectively.

[0127] (3) Electrochemiluminescence co-reactant buffer: phosphate buffer, tripropylamine, detergent and preservative.

[0128] (4) PBS: Dissolve 35.8 g of Na2HPO4·12H2O in 1000 mL of sterile water and 15.6 g of NaH2PO4·2H2O in 1000 mL of sterile water. Mix 19 mL of NaH2PO4 and 81 mL of Na2HPO4. After mixing, add water to dilute to 1000 mL for later use.

[0129] 2. Experimental methods

[0130] This detection example uses the interferon-γ electrochemiluminescence test strip prepared in Example 2 for detection. The detection process is as follows:

[0131] First, tuberculosis-specific antigens are used to stimulate the peripheral blood of the test subjects, causing activated T lymphocytes, NK cells, monocytes, dendritic cells and a small amount of hematopoietic stem cells to secrete interferon-γ. Then, interferon-γ is added to the sample wells of the test strip. Interferon-γ first binds to the ruthenium-coupled labeled antibody in the binding area, and then flows to the detection area to form a double antibody sandwich structure with the capture antibody labeled with plastic microspheres. The rest that is not bound to the capture antibody flows with the liquid to the water absorption area. Under the stimulation of the current in the detection area, ruthenium undergoes a color reaction, and the luminescence intensity is detected by an electrochemiluminescence analyzer to obtain the detection curve of interferon-γ. Finally, the sample concentration is calculated based on the standard curve.

[0132] The specific detection methods are as follows:

[0133] (1) Sampling: collect peripheral blood from tuberculosis patients, perform interferon release assays (IGRAs) to obtain peripheral blood containing γ interferon, mix the peripheral blood containing γ interferon with electrochemiluminescent co-reactant buffer in a volume ratio of 2:1, and then add PBS to make the volume to 150 μL to obtain the sample to be tested;

[0134] (2) Sample addition: Add 150 μL of the sample to be tested and standards of different solubility into the sample wells respectively;

[0135] (3) Reaction: The reaction lasts for 10 to 15 minutes, allowing the interferon-γ to bind to the ruthenium-coupled labeled antibody and the capture antibody coupled to the plastic microspheres on the test strip;

[0136] (4) Detection: Insert the test strip into the electrochemiluminescence analyzer for detection. The specific calculation process of the sample concentration is as follows: use the concentration of the standard as the horizontal axis (logarithmic coordinates) and the luminescence intensity as the vertical axis (normal coordinates). Draw a standard curve on semi-logarithmic coordinate paper. Find the corresponding concentration from the standard curve according to the luminescence intensity value of the sample to be tested, and then multiply it by the dilution factor; or use the concentration of the standard and the light intensity value to calculate the linear regression equation of the standard curve, substitute the light intensity value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0137] 3. Experimental results

[0138] The standard curve of the interferon-γ electrochemiluminescence test strip is as follows Figure 4 The linear regression equation of the standard curve is: y = 1322x + 187.3, R 2 =0.9905.

[0139] The test results of negative sample PBS and the sample to be tested are as follows Figure 5 As shown in the figure, it can be seen that the luminescence intensity value of the sample to be tested is 4156, and its detection concentration is 1004.6pg / mL calculated according to the standard curve; no obvious luminescence signal is detected in the negative sample. It can be seen that the interferon-γ electrochemiluminescence test strip of the present application scheme has good accuracy for trace detection.

[0140] Test Example 2 Repeatability and Minimum Detection Limit

[0141] This test example adopts the method of test example 1 to conduct a repeatability test on the interferon-γ electrochemiluminescence test strip prepared in Example 2 (the number of repetitions is 10 times, that is, 10 different interferon-γ electrochemiluminescence test strips are used to test samples of the same concentration), and the test results are shown in Table 3:

[0142] Table 3: Repeatability test of interferon-γ electrochemiluminescence test strips

[0143] Number of detections Luminous intensity 1st time 3723 2nd time 4156 3rd 4160 4th 3961 5th 3493 6th 4610 7th 3919 8th 3370 9th 4226 10th 3849 Average value(M) 3946.7 Standard Deviation (SD) 347.3 CV 8.7％

[0144] It can be seen from Table 3 that the CV value of the interferon-γ electrochemiluminescence test strip for testing the same sample is 8.7% (10% lower than the standard of the same batch), which shows that the interferon-γ electrochemiluminescence test strip of the present invention has the characteristic of good repeatability.

[0145] Furthermore, the sensitivity of the interferon-γ electrochemiluminescence test strip prepared in Example 2 was tested, and 10 different interferon-γ electrochemiluminescence test strips were used to test samples with an interferon-γ concentration of 0. The test results are shown in Table 4:

[0146] Table 4: Minimum detection limit of interferon-γ paper-based electrochemiluminescence test strips

[0147]

[0148]

[0149] It can be seen from Table 4 that the fitting concentration of the interferon-γ electrochemiluminescence test strip of the present invention is 4.4 pg / mL.

[0150] In summary, the test strip of the present invention constructs a paper-based electrochemiluminescence detection of interferon-γ, which uses a double antibody sandwich method to detect interferon-γ, and minimizes the production cost of the test strip, and has excellent detection efficiency. As a new detection method for interferon-γ, it provides an effective means for the rapid screening of interferon-γ.

[0151] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An interferon-γ electrochemiluminescence test strip, characterized in that: including a paper-based channel layer and an electrode layer; The paper-based channel layer comprises a sample loading pad, a conjugation pad, a detection pad and a water-absorbing pad which are sequentially overlapped and arranged; the sample loading pad is fixed with a sample buffer and a heterophilic antibody blocker; the conjugation pad is coated with a ruthenium-coupled labeled antibody; the detection pad is coated with a microsphere-coupled capture antibody and an electrochemiluminescent co-reactant buffer; The conjugate pad is prepared by the following preparation method: terpyridine ruthenium and interferon-γ specific monoclonal antibody are mixed and reacted to obtain the ruthenium-coupled labeled antibody, and then the matrix material for preparing the conjugate pad is immersed in the ruthenium-coupled labeled antibody, and the conjugate pad is obtained after drying; the molar ratio of the terpyridine ruthenium and the interferon-γ specific monoclonal antibody is 40-80:1; The detection pad is obtained by the following preparation method: soaking the matrix material for preparing the detection pad into the microsphere-coupled capture antibody and the electrochemiluminescent co-reactant buffer, and drying the matrix material of the detection pad is a nylon membrane, and the microspheres in the microsphere-coupled capture antibody are plastic microspheres; The electrode layer is provided with a plurality of electrodes, one end of each electrode is connected to the detection pad.

2. The interferon-γ electrochemiluminescence test strip according to claim 1, characterized in that: The sample loading pad is obtained by the following preparation method: soaking the matrix material for preparing the sample loading pad with the sample buffer and the heterophilic antibody blocking agent, and then drying the matrix material.

3. The interferon-γ electrochemiluminescence test strip according to claim 2, characterized in that: The matrix material of the sample loading pad is a glass cellulose membrane.

4. The interferon-γ electrochemiluminescence test strip according to claim 2, characterized in that: The sample buffer contained Tris-HCl, BSA, sodium caseinate, trehalose, sucrose, NaCl and PBS.

5. The interferon-γ electrochemiluminescence test strip according to claim 1, characterized in that: The matrix material of the conjugate pad and the matrix material of the sample loading pad are the same material.

6. The interferon-γ electrochemiluminescence test strip according to claim 1, characterized in that: The molar ratio of the terpyridine ruthenium to the interferon-γ specific monoclonal antibody is 40:

1.

7. The interferon-γ electrochemiluminescence test strip according to claim 1, characterized in that: The mass concentration of the ruthenium-coupled labeled antibody is 0.5 mg / mL to 1.5 mg / mL.

8. The interferon-γ electrochemiluminescence test strip according to claim 1, characterized in that: The preparation method of the microsphere-coupled capture antibody comprises: activating plastic microspheres, mixing the plastic microspheres and the capture antibody at a mass concentration ratio of 1:1 to 2.5, and reacting to obtain the microsphere-coupled capture antibody.

9. The interferon-γ electrochemiluminescence test strip according to claim 1, characterized in that: The electrochemiluminescent co-reactant buffer comprises phosphate buffer, tripropylamine, detergent and preservative.

10. The interferon-γ electrochemiluminescence test strip according to claim 1, characterized in that: The mass concentration ratio of the ruthenium-coupled labeled antibody to the microsphere-coupled capture antibody is 1:0.5-2.

5.

11. The interferon-γ electrochemiluminescence test strip according to any one of claims 1 to 10, characterized in that: The test paper strip further comprises a sealing film layer, wherein the sealing film layer comprises a first sealing film layer and a second sealing film layer; The first sealing film layer is provided with a sample adding hole corresponding to the position of the sample adding pad.

12. Use of the interferon-γ electrochemiluminescence test strip according to any one of claims 1 to 11 in preparing a kit for detecting interferon-γ.

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