A dry electrochemiluminescent lateral flow immunoassay strip and its application in immunoassay

Abstract

The present application discloses a dry electrochemiluminescent lateral flow immunoassay strip and its application in immunoassay, wherein the dry electrochemiluminescent lateral flow immunoassay strip comprises a lateral flow immunoassay strip, an electrode pad and a connection pad; the lateral flow immunoassay strip is composed of a sample pad, a binding pad, a detection pad with a backing NC membrane and an absorption pad stacked on a transparent adhesive plate; the electrode pad comprises an integrated closed bipolar electrode and a driving electrode; the integrated closed bipolar electrode comprises a closed bipolar electrode detection anode and a quality control anode and a shared cathode of the two anodes; the lateral flow immunoassay strip is inverted on the electrode pad; the T line and C line on the detection pad are in contact with the detection anode and the quality control anode on the electrode pad, respectively. The present invention adopts T / C for quantitative detection of biomarkers in ECL lateral flow immunoassay for the first time, solves the problem that the quality control signal is not fully utilized in the existing ECL lateral flow immunoassay, and improves the detection stability and storage stability.

Description

Technical Field

[0001] The invention relates to a dry electrochemiluminescent lateral flow immunoassay paper strip and application thereof in immunoassay. Background Art

[0002] The closed bipolar electrode electrochemiluminescence (ECL) cloth-based microfluidic chip is a combination of a closed bipolar electrode ECL and a cloth-based microfluidic chip. This type of chip has gradually developed into a means of detecting various biomarkers, but it still faces some problems. For example, the chip material is generally cloth material, and the material and source are unstable; the electrode is printed on rough cloth, and the resistance is large and unstable; it is necessary to build a hydrophobic dam on the cloth material to isolate the reporting channel and the supporting channel, and the chip needs to be pressed with a transparent plastic sheet, which easily causes the reaction solution to be connected, and the chip becomes an open bipolar electrode; the detection area and the quality control area are modified and recombined separately, and the role of the latter is not fully reflected. These have greatly limited the application scenarios of the closed bipolar electrode ECL cloth-based microfluidic chip.

[0003] The ECL lateral flow immunoassay strip is a combination of lateral flow immunoassay strip and ECL. Its preparation materials are nitrocellulose membrane (NC membrane), glass fiber and absorbent paper. It has the advantages of both ECL and lateral flow immunoassay strips and has been successfully applied to various immunoassays, but it still faces some problems. First, its detection pad is a backing-free NC membrane, which is relatively brittle and easy to break during use, which increases the difficulty of processing and experimental operation, affecting the accuracy and reliability of the test results. Secondly, during assembly, the electrodes need to be attached to the plastic base plate first, and then assembled in sequence, which increases the difficulty of processing and is difficult to mass produce. Thirdly, the detection line (T line) and the quality control line (C line) use different detection methods, and it is impossible to perform quantitative detection of biomarkers by T / C (the ratio of the signal value on the T line to the signal value on the C line). Finally, unstable tripropylamine needs to be added as a co-reactant during detection, and the operation process is complicated. These greatly limit the application of this type of test strip.

[0004] Dry chemical analysis technology is an analysis technology based on wet chemical analysis. More and more detection technologies are being applied to the field of dry chemical analysis. For example, lateral flow immunoassay strips are an analysis technology based on specific recognition of biomarkers and antibodies combined with certain markers. At present, lateral flow immunoassay strips on the market have the advantages of low price, easy use, rapid detection, and no need for expensive instruments. They are a conventional in vitro diagnostic method. However, these lateral flow immunoassay strip technologies have the disadvantages of high background, low sensitivity, long detection time, and difficulty in accurate quantitative detection. In addition, the detection of some biomarkers (such as luteinizing hormone (LH), cardiac troponin, etc.) still cannot meet the needs of family self-examination.

[0005] In summary, a new, fast, simple, accurate and easy to mass-produce technology suitable for hospital and home self-testing, which can effectively solve the shortcomings of the existing closed bipolar electrode ECL cloth-based microfluidic chip, ECL lateral flow immunoassay strip and lateral flow immunoassay strip has become a technical problem that needs to be urgently solved by technicians in this field. Summary of the invention

[0006] The purpose of the present invention is to provide a dry electrochemiluminescent lateral flow immunoassay test strip and its application in immunoassay. The test strip of the present invention integrates reaction reagent components on the test strip, and can realize dry quantitative detection of biomarkers. The detection method is fast, easy to operate and highly sensitive.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] Application of a dry electrochemiluminescent lateral flow immunoassay strip in immunoassay, wherein the dry electrochemiluminescent lateral flow immunoassay strip comprises a lateral flow immunoassay strip, an electrode pad and a connection pad;

[0009] The lateral flow immunoassay strip is composed of a sample pad, a conjugation pad, a detection pad with a backing NC membrane and an absorption pad stacked on a transparent adhesive plate; the detection pad is provided with a T line and a C line; the T line is coated with a T line capture antibody, and the C line is coated with a C line capture antibody; the conjugation pad is dried and fixed with an ECL signal antibody;

[0010] The electrode pad is formed of a conductive material on a hydrophobic bottom plate; the electrode pad includes an integrated closed bipolar electrode and a driving electrode; the integrated closed bipolar electrode includes a closed bipolar electrode detection anode and a quality control anode and a shared cathode of the two anodes; the driving electrode includes a negative driving electrode corresponding to the anode and a positive driving electrode corresponding to the shared cathode; the two anodes and the corresponding negative driving electrodes and the shared cathode and the corresponding positive driving electrode are naturally separated by the hydrophobic bottom plate;

[0011] The electrode pad is provided with double-sided tape between the detection anode, the shared cathode and the driving electrode for fixing part of the absorption pad and part of the detection pad, and double-sided tape is provided on one side of the quality control anode and the shared cathode for fixing part of the sample pad and part of the binding pad;

[0012] The lateral flow immunoassay test strip is inverted on the electrode pad; the NC membrane of the detection pad faces downward and the backing faces upward; the T line and C line on the detection pad are in contact with the detection anode and the quality control anode on the electrode pad respectively; the binding pad is close to one end of the quality control anode (away from the detection anode) and is in contact with the non-electrode area of ​​the electrode pad; the sample pad is close to the binding pad (away from the quality control anode) and is in contact with the non-electrode area of ​​the electrode pad; the absorption pad is close to one end of the detection anode and is in contact with the negative driving electrode corresponding to the detection anode;

[0013] The connection pad is located on the shared cathode, on the hydrophobic bottom plate between the shared cathode and the positive driving electrode, and is in contact with the positive driving electrode portion, so that the design can prevent the reaction solution from being connected to form an open bipolar electrode;

[0014] The sample pad and the binding pad are made of glass fiber, and the absorption pad and the connection pad are made of absorption paper; the electrode pad is constructed on a hydrophobic PET plastic plate by screen printing with conductive carbon ink; and the transparent adhesive plate is a transparent PET plastic plate;

[0015] The conjugate pad preparation process: the glass fiber is first treated with a conjugate pad treatment solution, then the ECL signal antibody mixed solution (6 μL) is evenly sprayed thereon, and then baked in a 37° C. oven for 0.5-2 h, thereby preparing a conjugate pad;

[0016] The sample pad preparation process: the glass fiber is first treated with a sample pad treatment solution, and then baked at 37° C. for 1-2 hours to obtain a sample pad;

[0017] The preparation process of the detection pad: the capture antibody solution of the T line and the capture antibody solution of the C line are modified and fixed on the same NC membrane by a gold spraying film streaking instrument, and then baked at 37° C. for 1-3 hours, thereby preparing the detection pad;

[0018] The absorption pad and the connection pad are both prepared by cutting absorbent paper with a laser cutting machine;

[0019] The transparent adhesive plate is prepared by cutting a transparent PET plastic plate with a laser cutting machine;

[0020] The binding pad treatment solution and the sample pad treatment solution are both phosphate buffered saline (PBS) containing 0.05% polyvinylpyrrolidone (PVP), 0.025% casein, and 0.1%-1% Tetronic1307 (abbreviated as S9), preferably 0.25% S9;

[0021] The T-line capture antibody solution comprises 15 mg / mL sucrose, 0.1% S9 and 0.1-1 mg / mL T-line capture antibody in PBS (pH 6.0-10.0), preferably with a pH of 8.0 and a T-line capture antibody concentration of 0.5 mg / mL;

[0022] The C-line capture antibody solution includes 5 mg / mL sucrose, 5 mg / mL trehalose, 0.1% S9, 0.25 mg / mL C-line capture antibody (such as mouse IgG) in PBS (pH 6.0-10.0), preferably pH 7.0.

[0023] The ECL signal antibody mixed solution is formed by mixing a solution of an intramolecular self-enhanced ECL signal antibody coupled to a C-line labeled antibody (i.e., a C-line ECL signal antibody) and a solution of an intramolecular self-enhanced ECL signal antibody coupled to a T-line labeled antibody (i.e., a T-line ECL signal antibody);

[0024] The preparation process of the T-line ECL signal antibody (or C-line ECL signal antibody) solution is as follows:

[0025] (1) The preparation process of the terpyridine ruthenium derivative (Ru(II)) is similar to the prior art (patent application number 202310708705.8).

[0026] (2) The preparation process of the complex of Ru(II) and L-lysine (L-Lys) (Ru(II)-L-Lys) is as follows:

[0027] Weigh 20 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 0.5 mg of N-hydroxysuccinimide (NHS) in 500 μL PBS; weigh 3.5 mg of Ru(Ⅱ) and add it to the above solution and shake to mix; then activate the carboxyl group of Ru(Ⅱ) in a constant temperature shaker at 25°C for 30 minutes to obtain an intermediate product (Ru(Ⅱ)-NHS);

[0028] Weigh 1 mg of L-Lys and dissolve it in 500 μL PBS; then add 500 μL of Ru(Ⅱ)-NHS synthesized in the above steps to the solution and shake to mix; then place it in a constant temperature shaker at 25°C for 8 hours to complete the amide connection; finally, use a Nanosep ultrafiltration centrifuge tube (1K MWCO, 8000 rpm, 5 min) to purify the solution after amide connection to obtain a purified product (Ru(Ⅱ)-L-Lys).

[0029] (3) Preparation process of T-line ECL signal antibody (or C-line ECL signal antibody) solution

[0030] First, 20 mg of EDC and 0.5 mg of NHS were weighed and added to 500 μL of Ru(Ⅱ)-L-Lys solution, and the carboxyl groups of Ru(Ⅱ)-L-Lys were activated in a constant temperature shaker at 25 °C for 30 min;

[0031] Next, add T-line labeled antibody (or C-line labeled antibody) to the activated Ru(Ⅱ)-L-Lys solution to make its concentration 0.2 mg / mL, and incubate in a constant temperature shaker at 25°C for 10 h to covalently couple Ru(Ⅱ)-L-Lys with T-line labeled antibody (or C-line labeled antibody);

[0032] Finally, the solution after the covalent coupling reaction was purified using a Nanosep ultrafiltration centrifuge tube (3K MWCO, 8000 rpm, 5 min) to obtain a T-line ECL signal antibody (or C-line ECL signal antibody) solution.

[0033] In order to achieve the above object, the present invention provides the use of the above dry ECL lateral flow immunoassay strip in immunoassay, comprising the following steps:

[0034] First, 20-40 μL of sample solution containing the biomarker to be tested is dripped onto the sample pad of the test strip, and the immune reaction time is waited for 1.5-9.5 min. The solution flows through the binding pad to form a complex with the T-line ECL signal antibody (T-line ECL signal antibody-biomarker), and then the immune complex continues to flow to the detection pad and combines with the T-line capture antibody on it to form an immune sandwich complex of "T-line ECL signal antibody-biomarker-T-line capture antibody". Preferably, 35 μL of sample solution containing the biomarker to be tested and the immune reaction time are 3.5 min.

[0035] Next, add 25-50 μL PBS to the sample pad to wash away excess residues for 3-7 min; then, add 15-40 μL PBS to the connection pad;

[0036] Finally, the dry electrochemiluminescent lateral flow immunoassay test strip is placed in an ECL instant detector (as disclosed in application number: 2024210642259) for detection, and an ECL trigger reaction is performed under a certain camera exposure time (50-450ms), camera white balance (2800-3200K) and driving voltage (9-13V). During the reaction, the ECL signals on the T line and the C line are automatically collected by the camera in the detector, and the ratio of the collected signal values ​​T / C is used to quantitatively detect the biomarker. The preferred camera exposure time is 166ms, the camera white balance is 2900K, and the driving voltage is 11V;

[0037] The biomarker is one of LH, cardiac troponin I or C-reactive protein.

[0038] Compared with the prior art, the present invention has the following advantages and effects:

[0039] 1. This patent invents a dry electrochemiluminescent lateral flow immunoassay test strip, which overcomes the shortcomings of the existing closed bipolar electrode ECL immunoassay chip and ECL lateral flow immunoassay test strip, which are complicated to prepare and operate, by integrating the reaction reagent components on the test strip.

[0040] 2. The test strip of the present invention uses NC membrane, glass fiber and absorbent paper as materials, which are materials with mature manufacturing processes on the market. The use of these materials further improves the practicality and universality of the test strip of the present invention.

[0041] 3. The present invention is the first to use a backing NC membrane to prepare an ECL lateral flow immunoassay strip, solving a series of problems encountered by existing ECL lateral flow immunoassay strips using a non-backing NC membrane (such as fragility, difficulty in processing, difficulty in experimental operation and mass production).

[0042] 4. The electrodes of the present invention are printed on a hydrophobic PET plastic plate, which not only avoids the disadvantages of unevenness of paper-based electrodes and cloth-based electrodes, but also simplifies the assembly of the ECL lateral flow immunoassay strip.

[0043] 5. The lateral flow immunoassay test strip of the present invention is inverted on the electrode pad, and no bottom plate is required to assemble the test strip, which simplifies the chelation of the test strip and the electrode pad, and solves the problems of weak pressing of PVC plastic plates in closed bipolar electrode ECL cloth-based microfluidic chips and easy cross-talk of reaction solutions during detection.

[0044] 6. The electrode pad of the present invention cleverly solves the problem that the reaction solutions in the reporter channel and the supporting channel of the existing closed bipolar electrode ECL cloth-based microfluidic chip are easily connected.

[0045] 7. The present invention uses intramolecular self-enhanced ECL signal antibodies. Compared with the existing ECL lateral flow immunoassay strips, there is no need to add co-reaction reagents again, thus avoiding the influence of the microenvironment, making ECL detection more accurate and easier to operate.

[0046] 8. The present invention is the first to apply the Ru(II)-L-Lys luminophore to the detection of dry electrochemiluminescent lateral flow immunoassay strips.

[0047] 9. The present invention adopts T / C for the first time in ECL lateral flow immunoassay to quantitatively detect biomarkers, which solves the problem of insufficient utilization of quality control signals in existing ECL lateral flow immunoassay and improves detection stability and storage stability.

[0048] 10. The present invention takes about 6.5 minutes from sample addition to completion of detection, which can achieve rapid quantitative detection.

[0049] 11. The test strip of the present invention is easy to operate. It only needs to drop the sample solution and buffer solution to be tested and then put it into the detector for analysis. No professional operation is required, and it is suitable for on-site instant detection.

[0050] 12. The test strips of the present invention are environmentally friendly, low-cost, and can be mass-produced using traditional test strip production processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is a schematic diagram of the overall structure of the dry electrochemiluminescent lateral flow immunoassay strip of the present invention;

[0052] Among them, 1-lateral flow immunoassay strip, 2-electrode pad, 3-connection pad.

[0053] Figure 2 It is a schematic diagram of the structure of the lateral flow immunoassay test strip of the present invention;

[0054] Among them, 1-1-sample pad, 1-2-binding pad, 1-3-detection pad, 1-3-1-NC membrane front side, 1-3-2-backing side, 1-3-3-T line, 1-3-4-C line, 1-4-absorption pad, 1-5-transparent adhesive plate.

[0055] Figure 3 Schematic diagram of the composition structure of the electrode pad of the present invention;

[0056] Among them, 2-1-hydrophobic bottom plate, 2-2-integrated closed bipolar electrode, 2-2-1-detection anode, 2-2-2-quality control anode, 2-2-3-shared cathode, 2-3-driving electrode, 2-3-1-negative driving electrode, 2-3-2-positive driving electrode.

[0057] Figure 4 It is a schematic diagram of the structural decomposition of the dry electrochemiluminescent lateral flow immunoassay test strip of the present invention;

[0058] Figure 5 This is a graph showing the relationship between T / C and different types of C-line antibody pairs.

[0059] Figure 6 This is the relationship diagram between T / C and C-line antibody pairs of the same type but different manufacturers.

[0060] Figure 7 Figure 2 is a graph showing the relationship between T / C and different types of surfactants in the bonding pad treatment solution.

[0061] Figure 8 The graph shows the relationship between T / C and different concentrations of surfactant in the conjugate pad treatment solution.

[0062] Fig. 9 This is the relationship diagram between T / C and the sample pad treatment solution buffer system.

[0063] Fig.10 This is a graph showing the relationship between T / C and different types of surfactants in the sample pad treatment solution.

[0064] Fig.11 This is a graph showing the relationship between T / C and different concentrations of surfactant in the sample pad treatment solution.

[0065] Fig.12This is the relationship diagram between T / C and NC film types.

[0066] Fig.13 The graph shows the relationship between T / C and the pH value of PBS in which the T-line capture antibody is present.

[0067] Fig.14 The graph shows the relationship between T / C and the pH value of PBS for capturing antibody in line C.

[0068] Fig.15 The graph is the relationship between T / C and the concentration of T-line capture antibody.

[0069] Fig.16 This is the relationship diagram between T / C and camera white balance.

[0070] Fig.17 This is a graph showing the relationship between T / C and camera exposure time.

[0071] Fig.18 This is the relationship diagram between T / C and driving voltage.

[0072] Fig.19 This is a graph showing the relationship between T / C and immune response time.

[0073] Fig. 20 This is the relationship between T / C and sample volume.

[0074] Fig.21 This is the relationship between T / C and sample dilution ratio.

[0075] Fig. 22 This is the analytical curve diagram for detecting different concentrations of LH (the inset is the linear fitting curve diagram of the data).

[0076] Fig.23 Evaluation of storage stability when detecting LH. DETAILED DESCRIPTION

[0077] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0078] Example 1

[0079] A dry electrochemiluminescent lateral flow immunoassay strip, the composition and structure of which are as follows Figure 1 to Figure 4 As shown, it includes a lateral flow immunoassay strip 1, an electrode pad 2 and a connection pad 3;

[0080] The lateral flow immunoassay strip 1 is composed of a sample pad 1-1, a conjugation pad 1-2, a detection pad 1-3 and an absorption pad 1-4 stacked on a transparent adhesive plate 1-5; the detection pad 1-3 is provided with a T line 1-3-3 and a C line 1-3-4; the T line 1-3-3 is coated with a T line capture antibody, and the C line 1-3-4 is coated with a C line capture antibody; the conjugation pad 1-2 is dried and fixed with an ECL signal antibody;

[0081] The electrode pad 2 is made of conductive material on a hydrophobic bottom plate 2-1; the electrode pad 2 includes an integrated closed bipolar electrode 2-2 and a driving electrode 2-3; the integrated closed bipolar electrode 2-2 includes a closed bipolar electrode detection anode 2-2-1 and a quality control anode 2-2-2 and a shared cathode 2-2-3 of the two anodes; the driving electrode 2-3 includes a negative driving electrode 2-3-1 corresponding to the anodes 2-2-1 and 2-2-2 and a positive driving electrode 2-3-2 corresponding to the shared cathode 2-2-3; the two anodes 2-2-1 and 2-2-2 and the corresponding negative driving electrode 2-3-1, and the shared cathode 2-2-3 and the corresponding positive driving electrode 2-3-2 are naturally separated by the hydrophobic bottom plate 2-1;

[0082] The electrode pad 2 is provided with a double-sided adhesive tape 2-4 between the detection anode 2-2-1, the shared cathode 2-2-3 and the driving electrode 2-3 for fixing part of the absorption pad 1-4 and part of the detection pad 1-3, and a double-sided adhesive tape 2-5 is provided on one side of the quality control anode 2-2-2 and the shared cathode 2-2-3 for fixing part of the sample pad 1-1 and part of the binding pad 1-2;

[0083] The lateral flow immunoassay test strip 1 is inverted on the electrode pad 2; the front side 1-3-1 of the NC film of the detection pad 1-3 faces downward, and the backing side 1-3-2 faces upward; the T line 1-3-3 and the C line 1-3-4 on the detection pad 1-3 are in contact with the detection anode 2-2-1 and the quality control anode 2-2-2 on the electrode pad 2 respectively; the conjugation pad 1-2 is close to the end of the quality control anode 2-2-2 (away from the detection anode 2-2-1) and is in contact with the non-electrode area of ​​the electrode pad 2; the sample pad 1-1 is close to the conjugation pad 1-2 (away from the quality control anode 2-2-2) and is in contact with the non-electrode area of ​​the electrode pad 2; the absorption pad 1-4 is close to the end of the detection anode 2-2-1 and is in contact with the negative drive electrode 2-3-1 corresponding to the detection anode 2-2-1;

[0084] The connection pad 3 is located on the shared cathode 2-2-3, on the hydrophobic bottom plate 2-1 between the shared cathode 2-2-3 and the positive drive electrode 2-3-2, and is in partial contact with the positive drive electrode 2-3-2. This design can prevent the reaction solution from being connected to form an open bipolar electrode.

[0085] The sample pad 1-1 and the binding pad 1-2 are made of glass fiber, the absorption pad 1-4 and the connection pad 3 are made of absorbent paper; the conductive material of the electrode pad 2 is conductive carbon ink, the hydrophobic bottom plate 2-1 for constructing the electrode is a hydrophobic PET plastic plate; the transparent adhesive plate 1-5 is a transparent PET plastic plate.

[0086] Example 2

[0087] The application of the dry electrochemiluminescent lateral flow immunoassay strip of the present invention in detecting LH, the detection process is as follows:

[0088] First, 30 μL of sample solution containing LH was dripped onto the sample pad 1-1 of the test strip, and the immune reaction time was waited for 3.5 minutes. The solution flowed through the binding pad 1-2 to form a complex with the T-line ECL signal antibody (T-line ECL signal antibody-LH). Then, the immune complex continued to flow to the detection pad 1-3 and combined with the T-line capture antibody on it to form an immune sandwich complex of "T-line ECL signal antibody-LH-T-line capture antibody".

[0089] Next, 30 μL of PBS was added to sample pad 1-1 to wash away excess residues for 3 min; then, 20 μL of PBS was added to connection pad 3;

[0090] Finally, the dry ECL lateral flow immunoassay strip was placed in a self-built ECL instant detector (application number: 2024210642259) for detection, and an ECL trigger reaction was performed under a certain camera exposure time (166ms), camera white balance (2800K) and driving voltage (11V). During the reaction, the ECL signals on the T line 1-3-3 and the C line 1-3-4 were automatically collected by the camera in the detector, and the ratio of the collected signal values ​​T / C was used to quantitatively detect LH;

[0091] The relationship between the type of C-line antibody pair (i.e., the C-line capture antibody and the corresponding C-line labeling antibody constitute the C-line antibody pair) and T / C in the dry electrochemiluminescent lateral flow immunoassay test strip is now tested using a sample solution containing 1mIU / mL LH and the test strip of the present invention as an example.

[0092] Set up the experimental group: select five C-line antibody pairs (goat anti-chicken IgY-chicken IgY (GC), goat anti-mouse IgG-mouse IgG (GM), goat anti-rabbit IgG-rabbit IgG (GR), rabbit anti-mouse IgG-mouse IgG (RM) and rabbit anti-goat IgG-goat IgG (RG)) for optimization experiment.

[0093] The test results are as follows Figure 5As shown in the figure, it can be seen that the ECL signal and T / C obtained by GC, GM and RG are not stable, which may be caused by the poor immunoactivity of these C-line antibody pairs in the ECL detection system. The detection results of RM and GR are good, but RM is better in stability, and its ECL signal and T / C on T line 1-3-3 are higher than those of GR. Therefore, RM is preferred in this study.

[0094] Example 3

[0095] The important factors affecting T / C in Example 2 (C-line antibody pairs of the same type but different manufacturers, surfactants of different types and concentrations in the treatment solution of the conjugate pad 1-2, buffer system of the treatment solution of the sample pad 1-1, surfactants of different types and concentrations in the treatment solution of the sample pad 1-1, type of NC membrane, pH value of PBS for T-line and C-line capture antibodies, concentration of T-line capture antibody, camera white balance, camera exposure time, driving voltage, immune response time, sample loading, sample dilution ratio) were optimized:

[0096] (1) Prefer the same type of C-line antibody from different manufacturers

[0097] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line and C-line capture antibody concentrations are both 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0098] 2. Set up the experimental group: select C-line antibody pairs (RM) from five manufacturers (Hangzhou Qitai Biotechnology Co., Ltd. (RM1), Beijing Solebow Technology Co., Ltd. (RM2), Luoyang Biotop Experimental Materials Center (RM3), Jiaozuo Yunzhiyu Biotechnology Co., Ltd. (RM4), Nanjing Xinfan Biotechnology Co., Ltd. (RM5)).

[0099] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Figure 6 shown.

[0100] It can be seen from the experimental results that the ECL signal and T / C stability of RM2, RM3 and RM5 are poor, which may be due to the poor compatibility of the process and storage conditions of the antibodies produced by these manufacturers with the ECL detection system. In addition, the T line and C line ECL signals of RM4 are relatively stable, but its T / C stability is poor. In contrast, the RM1 antibody pair has good stability in both ECL signal and T / C, and has a higher T / C. Therefore, the RM1 antibody pair is preferred.

[0101] (2) Preferred combination pad 1-2 treatment solution with different types of surfactants

[0102] 1. The LH concentration to be tested is 1mIU / mL and 5mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line and C-line capture antibody concentrations are both 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0103] 2. Set up the experimental group: select five surfactants (S9, S14, S16, S19 and S21) for optimization experiments.

[0104] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Figure 7 shown.

[0105] It can be seen from the experimental results that for the conjugate pads 1-2 treated with S9, S14 and S16 and the control group (Control), the ECL signal and T / C showed large standard deviations and poor stability. The possible reason for this phenomenon is that the conjugate pads 1-2 treated with these surfactants and those without any treatment cannot be well re-dissolved to release the ECL signal antibody, which affects the binding of the biomarker with the ECL signal antibody, and then affects the formation of "T line ECL signal antibody-LH-T line capture antibody" on the detection pad 1-3, resulting in unstable test results. In contrast, the test results shown by the conjugate pads 1-2 treated with S19 and S21 are more stable, especially when the LH concentration is 1mIU / mL, the T / C of S19 is higher than that of S21, while at 5mIU / mL, the difference between the two is not much. Therefore, S19 is preferred in this study.

[0106] (3) Optimum surfactant concentration in the treatment solution of the binding pad 1-2

[0107] 1. The LH concentration to be tested is 1mIU / mL and 5mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line and C-line capture antibody concentrations are both 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0108] 2. Set up experimental groups: Set different concentrations of S19 (0, 0.1%, 0.25%, 0.5%, 0.75% and 1%) for optimization experiments.

[0109] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Figure 8 shown.

[0110] It can be seen from the experimental results that when the S19 concentration in the treatment solution of the conjugate pad 1-2 reaches 0.5%, 0.75% and 1%, at LH concentrations of 1mIU / mL and 5mIU / mL, at least 1 of the 7 repeated tests will show almost no luminescence, and the ECL signal and T / C are both low, and the stability is extremely poor. The possible reason for this phenomenon is that the treatment of the conjugate pad 1-2 with too high a concentration of S19 will lead to obstruction of electrical conduction and hinder the normal generation of the ECL signal. In contrast, when the S19 concentration is 0, 0.1% and 0.25%, the ECL signal can be generated normally, but the S19 concentration is 0.25% and shows better stability. The possible reason for this phenomenon is that when the S19 concentration is 0 and 0.1%, its concentration is too low to effectively promote the re-dissolution and release of the ECL signal antibody and the binding of the biomarker to the ECL signal antibody, thereby affecting the stability. Therefore, the preferred concentration of S19 in the treatment solution of the conjugate pad 1-2 is 0.25%.

[0111] (4) Optimum sample pad 1-1 treatment solution buffer system

[0112] 1. The LH concentration to be tested is 1mIU / mL and 5mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line and C-line capture antibody concentrations are both 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0113] 2. Set up the experimental group: select five buffer systems (sodium hydrogen phosphate-citrate buffer (CASDP), PBS, Tris-HCl, glycine-NaOH buffer (GSH), borax-NaOH (BSH)) for optimization experiments.

[0114] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig. 9 shown.

[0115] It can be seen from the experimental results that the corresponding ECL signals and T / C obtained by CASDP, Tris-HCl, GSH and BSH buffers at LH concentrations of 1mIU / mL and 5mIU / mL are not stable, probably because the ion environment and pH value of these four buffers are not conducive to the immune reaction between biomarkers and antibodies or the generation of ECL signals. Relatively speaking, when the buffer system is PBS, the generated ECL signals and T / C show excellent stability. Therefore, PBS is preferred as the buffer system for the sample pad 1-1 treatment solution in this study.

[0116] (5) Optimize the type of surfactant in the sample pad 1-1 treatment solution

[0117] 1. The LH concentration to be tested is 1mIU / mL and 5mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line and C-line capture antibody concentrations are both 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0118] 2. Set up the experimental group: select five surfactants (S9, S14, S16, S19 and S21) for optimization experiments.

[0119] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.10 shown.

[0120] It can be seen from the experimental results that the ECL signal and T / C stability of the sample pad 1-1 treated with S14, S16 and S21 and the control group (Control) are poor. The possible reason is that the sample pad 1-1 treated with these surfactants cannot be well re-dissolved to release the ECL signal antibody, affecting the binding of the biomarker to the ECL signal antibody, and does not fully block the excess sites on the glass fiber and NC membrane, resulting in poor stability of the test results. In contrast, the sample pad 1-1 treated with S9 and S19 showed good stability, and when LH was 1mIU / mL and 5mIU / mL, the ECL signal was not much different, but the T / C of S19 was higher than that of S9. Therefore, S19 was preferred for treating the sample pad 1-1 in this study.

[0121] (6) Optimum surfactant concentration in sample pad 1-1 treatment solution

[0122] 1. The LH concentration to be tested is 1mIU / mL and 5mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line and C-line capture antibody concentrations are both 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0123] 2. Set up experimental groups: Set different concentrations of S19 (0, 0.1%, 0.25%, 0.5%, 0.75% and 1%) for optimization experiments.

[0124] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.11 shown.

[0125] It can be seen from the experimental results that when the S19 concentration reaches 0.5%, 0.75% and 1%, and the LH concentration is 1mIU / mL and 5mIU / mL, the ECL signal and T / C on the T line and C line are low and the stability is extremely poor. The results of 7 repeated tests show that there is a probability of inhibiting the generation of ECL signals, which leads to poor stability. The possible reason for this phenomenon is that too high a concentration of S19 is used to treat sample pad 1-1, which will lead to obstruction of electrical conduction and hinder the normal generation of ECL signals. In addition, when the S19 concentration is 0, the stability is also poor, which may be because the surfactant concentration is too low, resulting in poor sealing effect of excess sites on glass fiber and NC membrane or the immune binding of biomarkers to ECL signal antibodies is affected. In contrast, when the S19 concentration is 0.1% and 0.25%, the ECL signal and T / C stability are excellent, but the T / C of 0.25% is higher than 0.1%. Therefore, the S19 concentration in the sample pad 1-1 treatment solution is preferably 0.25%.

[0126] (7) Preferred detection pad 1-3 NC membrane type

[0127] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line and C-line capture antibody concentration is 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0128] 2. Set up the experimental group: select five NC membranes (HPM140 (Shenzhen Shenwei Industrial Co., Ltd.), CN95 (Sartorius), CN140 (Sartorius), 10547149 (Waterman), CN120 (Hangzhou Bluus Trading Co., Ltd.)) for optimization experiment.

[0129] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.12 shown.

[0130] It can be seen from the experimental results that the ECL signal and T / C stability of the NC membrane based on the CN95 model are very poor. The possible reason for this phenomenon is that the pore size of this type of NC membrane is large and the flow rate is too fast, resulting in insufficient time for the "T-line ECL signal antibody-biomarker-T-line capture antibody" to form, resulting in a low ECL signal and poor stability, which in turn makes the T / C stability very poor. The ECL signal and T / C of the NC membrane based on the HPM140 and CN120 models are relatively high, but the stability is poor. The possible reason for this phenomenon is that the sample solution flows slowly on the NC membrane, allowing the "T-line ECL signal antibody-biomarker-T-line capture antibody" to have sufficient time to react. However, it may be due to the unstable production process when the manufacturer produces the NC membrane, resulting in unstable quality of the produced NC membrane, which in turn makes the test results unstable. In contrast, the ECL signal and T / C based on the CN140 and 10547149 NC membranes are relatively high and extremely stable. The possible reason for this phenomenon is that the sample solution has a slow flow rate in the NC membrane, which allows sufficient time for the "T-line ECL signal antibody-biomarker-T-line capture antibody" to form, and the manufacturer produces the NC membrane with a stable production process, which makes the quality of the produced NC membrane stable. Given that the ECL signal and T / C of the CN140 NC membrane are higher than those of the 10547149, this study ultimately preferred the CN140 NC membrane purchased from Sartorius as the material for detection pads 1-3.

[0131] (8) Optimizing pH of PBS for T-line capture antibody

[0132] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line and C-line capture antibody concentration is 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0133] 2. Set up experimental groups: Set up PBS with different pH values ​​(6.0, 7.0, 8.0, 9.0 and 10.0) for optimization experiments.

[0134] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.13 shown.

[0135] It can be seen from the experimental results that when the pH value is 8.0, T / C is significantly higher than that of 6.0, 7.0, 9.0 and 10.0, and the standard deviation is the smallest, indicating that its stability is the best. This may be because when the pH value is 8.0, it is close to the isoelectric point of the T-line capture antibody, making the antibody in a state of easy precipitation, and the actual fixed amount, fixation firmness and activity on the NC membrane are relatively good, so the detection results are better. Therefore, the pH value of the T-line capture antibody PBS is preferably 8.0 in this study.

[0136] (9) Optimizing pH value of PBS for C-line capture antibody

[0137] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line and C-line capture antibody concentration is 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0138] 2. Set up experimental groups: set up PBS with different pH values ​​(6.0, 7.0, 8.0, 9.0 and 10.0).

[0139] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.14 shown.

[0140] It can be seen from the experimental results that when the pH value is 8.0, 9.0 and 10.0, the ECL signal and T / C stability are poor. This may be because when the pH value is greater than 8.0, the buffer environment is not conducive to the binding of the C-line capture antibody to the binding site on the NC membrane, which in turn affects the stability of the results. When the pH value is 6.0 and 7.0, the ECL signal and T / C stability are excellent, but the T / C at pH 7.0 is significantly higher. This may be because when the pH value is 7.0, it is close to the isoelectric point of the C-line capture antibody, making the antibody in a state of easy precipitation, and the actual fixed amount, fixation firmness and activity on the NC membrane are optimal. Therefore, in this study, the pH value of the C-line capture antibody PBS is preferably 7.0.

[0141] (10) Optimal T-line capture antibody concentration

[0142] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is 2800K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T-line capture antibody concentration is to be determined, the C-line capture antibody concentration is 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0143] 2. Set up experimental groups: Set different concentrations of T-line capture antibodies (0.1, 0.25, 0.5, 0.75 and 1 mg / mL) for optimization experiments.

[0144] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.15 shown.

[0145] It can be seen from the experimental results that when the concentration of T-line capture antibody is lower than 0.5 mg / mL, T / C gradually increases with the increase of T-line capture antibody concentration. However, when the concentration of T-line capture antibody is higher than 0.5 mg / mL, T / C gradually decreases with the increase of T-line capture antibody concentration. The possible reason for this phenomenon is that the excessive T-line capture antibody causes the ECL signal antibody to be retained in the C-line area, increasing the C-line ECL signal, which in turn causes T / C to decrease. Based on this study, the preferred T-line capture antibody concentration is 0.5 mg / mL.

[0146] (11) Optimize camera white balance

[0147] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is to be determined, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T line capture antibody concentration is 0.5mg / mL and the C line capture antibody concentration is 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0148] 2. Set up the experimental group: Set different camera white balances (2800, 2900, 3000, 3100 and 3200K) for optimization experiments.

[0149] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.16 shown.

[0150] It can be seen from the experimental results that when the camera white balance reaches 2900K, T / C reaches the highest. In the camera white balance range of 2800-3200K, the ratio of signal T / C to background T / C (SBR) gradually decreases. In order to obtain higher T / C and SBR, 2900K camera white balance is preferred.

[0151] (12) Optimize camera exposure time

[0152] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is 2900K, the camera exposure time is to be determined, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T line capture antibody concentration is 0.5mg / mL and the C line capture antibody concentration is 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0153] 2. Set up the experimental group: set different camera exposure times (50, 166, 250, 350 and 450ms) for optimization experiments.

[0154] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.17 shown.

[0155] It can be seen from the experimental results that when the exposure time is 50-166ms, T / C gradually increases; when it is 166-450ms, T / C shows a gradually decreasing trend. In the exposure time range of 50-450ms, SBR gradually decreases. In order to obtain higher T / C and SBR, the camera exposure time is preferably 166ms.

[0156] (13) Optimum driving voltage

[0157] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is 2900K, the camera exposure time is 166ms, the driving voltage is to be determined, the ECL signal antibody mixed solution is 6μL, the T line capture antibody concentration is 0.5mg / mL and the C line capture antibody concentration is 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is 30μL.

[0158] 2. Set up the experimental group: set different driving voltages (8, 9, 10, 11, 12 and 13 V) for optimization experiments.

[0159] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.18 shown.

[0160] It can be seen from the experimental results that when the driving voltage is lower than 11V, T / C increases with the increase of the driving voltage. This is because the driving voltage in this range has not yet fully caused the ECL reaction of the ECL signal antibody to occur, so the ECL signal and T / C increase with the increase of the driving voltage. However, when the driving voltage is higher than 11V, T / C decreases with the increase of the driving voltage. This may be because the driving voltage is too high, causing the ECL signal to be generated too quickly and quenched too quickly, resulting in the inability of the analysis equipment to fully capture the ECL signal, which in turn causes T / C to decrease. Therefore, the preferred driving voltage is 11V.

[0161] (14) Optimal immune response time

[0162] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is 2900K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T line capture antibody concentration is 0.5mg / mL and the C line capture antibody concentration is 0.25mg / mL, the immune reaction time is to be determined, and the sample volume is 30μL.

[0163] 2. Set up experimental groups: set different immune response times (1.5, 3.5, 5.5, 7.5 and 9.5 min) for optimization experiments.

[0164] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.19 shown.

[0165] It can be seen from the experimental results that when the immune response time is less than 3.5 minutes, the ECL signal and T / C increase with the increase of the immune response time. However, when the immune response time is higher than 3.5 minutes, the ECL signal and T / C basically reach a plateau. The possible reason for this phenomenon is that the formation of the "T-line ECL signal antibody-biomarker-T-line capture antibody" immune sandwich complex is basically saturated. Therefore, the preferred immune response time is 3.5 minutes.

[0166] (15) Optimal sample loading

[0167] 1. The LH concentration to be tested is 1mIU / mL, the camera white balance is 2900K, the camera exposure time is 166ms, the driving voltage is 11V, the ECL signal antibody mixed solution is 6μL, the T line capture antibody concentration is 0.5mg / mL and the C line capture antibody concentration is 0.25mg / mL, the immune reaction time is 3.5min, and the sample volume is to be determined.

[0168] 2. Set up experimental groups: Set different sample volumes (20, 25, 30, 35 and 40 μL) for optimization experiments.

[0169] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig. 20 shown.

[0170] It can be seen from the experimental results that when the sample volume is less than 35 μL, the ECL signal and T / C increase with the increase of the sample volume. However, when the sample volume is higher than 35 μL, the ECL signal and T / C basically reach a plateau. This phenomenon may be because the formation of the "T-line ECL signal antibody-biomarker-T-line capture antibody" immune sandwich complex has reached saturation. Therefore, the preferred sample volume for this study is 35 μL.

[0171] (16) Optimal sample dilution ratio

[0172] 1. The original concentrations of the LH samples to be tested were 1, 10 and 50 mIU / mL, the camera white balance was 2900K, the camera exposure time was 166 ms, the driving voltage was 11 V, the ECL signal antibody mixed solution was 6 μL, the T-line capture antibody concentration was 0.5 mg / mL and the C-line capture antibody concentration was 0.25 mg / mL, the immune reaction time was 3.5 min, and the sample volume was 35 μL.

[0173] 2. Set up the experimental group: set the volume ratio of sample to PBS to 1:6, 1:9 and 1:12 for optimization experiments.

[0174] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.21 shown.

[0175] It can be seen from the experimental results that when the volume ratio of the sample to PBS is 1:9, the slope of the fitting curve is the highest, reaching 2.145, and R 2 is 0.9987. In addition, the fitting curve when the volume ratio of the sample to PBS is 1:9 is better than that when the volume ratio of the sample to PBS is 1:6 and 1:12. The possible reason for this phenomenon is that at 1:6, the sample matrix affects the electrical conduction, thereby affecting the generation of the ECL signal, making T / C low when the LH sample is at a high concentration (10 and 50mIU / mL); at 1:12, the low concentration of biomarkers cannot make the total amount of the "T line ECL signal antibody-biomarker-T line capture antibody" complex reach a relative saturation state on the detection pad 1-3. Therefore, the preferred dilution ratio of the LH sample is 1:9.

[0176] Example 4

[0177] LH detection was performed using the dry electrochemiluminescent lateral flow immunoassay strip of Example 1 under the optimized conditions found in Example 3.

[0178] 1. The camera white balance was 2900K, the camera exposure time was 166ms, the driving voltage was 11V, the ECL signal antibody mixed solution was 6μL, the T-line capture antibody concentration was 0.5mg / mL and the C-line capture antibody concentration was 0.25mg / mL, the immune reaction time was 3.5min, and the sample volume was 35μL.

[0179] 2. Set up the experimental group: set several different LH concentrations (0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 11 and 22 mIU / mL), and use 0.01 mIU / mL of LH for blank control detection.

[0180] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig. 22 shown.

[0181] The experimental results show that when the LH concentration is in the range of 0.05-22mIU / mL, T / C increases with the increase of LH concentration. There is a good linear relationship between T / C (expressed by Y) and the logarithm of LH concentration (expressed by X), and the linear equation is Y=2.127X+2.832(R 2 =0.9985, n=5), and the detection limit was estimated to be 0.0496 mIU / mL.

[0182] The calculation method of detection limit is: L =Y b +3S b , where Y b Indicates the average T / C value of the blank control, S b represents the standard deviation of the blank control (5 repeated experiments), and the obtained Y L The corresponding LH concentration calculated from the value is the detection limit.

[0183] Example 5

[0184] The optimized conditions found in Example 3 were used to conduct actual sample detection experiments using dry electrochemiluminescent lateral flow immunoassay test strips.

[0185] 1. The camera white balance was 2900K, the camera exposure time was 166ms, the driving voltage was 11V, the ECL signal antibody mixed solution was 6μL, the T-line capture antibody concentration was 0.5mg / mL and the C-line capture antibody concentration was 0.25mg / mL, the immune reaction time was 3.5min, and the sample volume was 35μL.

[0186] 2. Set up the experimental group: dilute the collected urine sample 10 times to obtain three diluted samples with LH concentrations of 0.11, 1.21, and 6.96 mIU / mL for actual sample testing.

[0187] 3. The dry electrochemiluminescent lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are shown in Table 1.

[0188] Table 1

[0189]

[0190] As shown in Table 1, the test results of the test strips are 0.0104, 1.093 and 6.996 mIU / mL, respectively, the recovery rates are 93.69%, 91.08% and 100.22%, respectively, and the relative standard deviations are 8.51%, 9.78% and 10.42%, respectively. Therefore, the test strips of the present invention can achieve good detection of LH in actual urine samples.

[0191] Example 6

[0192] The storage stability experiment of the dry electrochemiluminescent lateral flow immunoassay test strip was carried out under the optimized conditions found in Example 3.

[0193] 1. The camera white balance was 2900K, the camera exposure time was 166ms, the driving voltage was 11V, the ECL signal antibody mixed solution was 6μL, the T-line capture antibody concentration was 0.5mg / mL and the C-line capture antibody concentration was 0.25mg / mL, the immune reaction time was 3.5min, and the sample volume was 35μL.

[0194] 2. Set up the experimental group: store the prepared dry electrochemiluminescent lateral flow immunoassay strips in a 37°C oven, and then test them at intervals of 0, 3, 3, 3, 5, 5, and 5 days.

[0195] 3. The dry electrochemical luminescence lateral flow immunoassay test strip detection process is the same as in Example 2. The test results are as follows: Fig.23 shown.

[0196] The experimental results show that the dry electrochemiluminescent lateral flow immunoassay strip can be stored for 19 days at 37°C. According to the Arrhenius equation, 19 days at 37°C is equivalent to about two and a half years at 4°C, which has good storage stability.

[0197] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of a dry electrochemiluminescent lateral flow immunoassay strip in the preparation of immunoassay products, characterized in that: The dry electrochemiluminescent lateral flow immunoassay paper strip comprises a lateral flow immunoassay paper strip, an electrode pad and a connection pad; The lateral flow immunoassay strip is composed of a sample pad, a conjugation pad, a detection pad with a backing NC membrane and an absorption pad stacked on a transparent adhesive plate; the detection pad is provided with a T line and a C line; the T line is coated with a T line capture antibody, and the C line is coated with a C line capture antibody; the conjugation pad is dried and fixed with an ECL signal antibody; The electrode pad includes an integrated closed bipolar electrode and a driving electrode; the integrated closed bipolar electrode includes a closed bipolar electrode detection anode and a quality control anode and a shared cathode of the two anodes; The lateral flow immunoassay strip is inverted on the electrode pad; the NC membrane of the detection pad faces downward and the backing faces upward; the T line and C line on the detection pad are in contact with the detection anode and the quality control anode on the electrode pad respectively; the conjugate pad is close to one end of the quality control anode and is in contact with the non-electrode area of ​​the electrode pad; the sample pad is close to the conjugate pad and is in contact with the non-electrode area of ​​the electrode pad; The absorption pad is close to one end of the detection anode and contacts the negative driving electrode corresponding to the detection anode; The preparation process of the detection pad: the capture antibody solution of the T line and the capture antibody solution of the C line are modified and fixed on the same NC membrane by a gold spraying film streaking instrument, and then baked at 37° C. for 1-3 hours, thereby preparing the detection pad; The T-line capture antibody solution includes 15 mg / mL sucrose, 0.1% S9 and 0.5 mg / mL T-line capture antibody in PBS, with a pH value of 8.0; The T-line capture antibody is rabbit anti-mouse IgG-mouse IgG or goat anti-rabbit IgG-rabbit IgG; The C-line capture antibody solution includes 5 mg / mL sucrose, 5 mg / mL trehalose, 0.1% S9, 0.25 mg / mL C-line capture antibody in PBS, with a pH value of 6.0-7.0; The preparation process of the conjugate pad is as follows: the glass fiber is first treated with a conjugate pad treatment solution, then the ECL signal antibody mixed solution is evenly sprayed on the glass fiber, and then baked in a 37° C. oven for 0.5-2 hours to prepare the conjugate pad; The conjugate pad treatment solution contains 0.25% S19 or S21; The sample pad preparation process: the glass fiber is first treated with a sample pad treatment solution, and then baked at 37° C. for 1-2 hours to obtain a sample pad; The sample pad treatment solution is a PBS buffer solution containing 0.1-0.25% S19 or S9; The sample pad and the combination pad are made of glass fiber, and the absorption pad and the connection pad are made of absorption paper; the electrode pad is constructed on a hydrophobic PET plastic plate by screen printing with conductive carbon ink; and the transparent adhesive plate is a transparent PET plastic plate.

2. The use according to claim 1, characterized in that: The connection pad is located on the shared cathode, on the hydrophobic bottom plate between the shared cathode and the positive driving electrode, and is in contact with a portion of the positive driving electrode.

3. The use according to claim 1, characterized in that: The ECL signal antibody mixed solution is formed by mixing an intramolecular self-enhanced ECL signal antibody solution coupled to a C-line labeled antibody and an intramolecular self-enhanced ECL signal antibody solution coupled to a T-line labeled antibody.

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