Preparation method of test strip for quantitatively detecting tPA in nasal cavity

Abstract

The invention belongs to the field of immunodetection, and relates to a preparation method of a test strip for quantitatively detecting tPA in a nasal cavity, which comprises the following steps: 1) preparing a nitrocellulose membrane detection layer; one end of the nitrocellulose membrane detection layer is coated with an anti-human tPA monoclonal antibody Abtpa1 capable of recognizing human tPA, and the other end of the nitrocellulose membrane detection layer is coated with a goat anti-mouse IgG antibody; 2) preparing a sample pad; (3) preparing a combination pad, wherein the surface of the combination pad is sprayed with a colloidal gold labeled anti-human tPA monoclonal antibody Abtpa2; and (4) preparing a bottom plate, and assembling the nitrocellulose membrane detection layer prepared in the step (1), the sample pad prepared in the step (2), the combination pad prepared in the step (3) and absorbent paper on the bottom plate to obtain the quantitative detection nasal cavity tPA test strip. The invention provides the preparation method of the test strip for quantitatively detecting the tPA in the nasal cavity, which is accurate in detection, quick and convenient to use and simple to prepare.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassays and relates to a method for preparing a test strip for quantitatively detecting nasal tPA, and in particular to a method for preparing a test strip for quantitatively detecting nasal tPA based on colloidal gold immunochromatography and the test strip. Background Art

[0002] Tissue plasminogen activator (tPA) is a single-chain glycoprotein synthesized, secreted, and continuously released into the blood primarily by vascular endothelial cells. It is widely present in various tissues of the body, with the liver being the primary site of its inactivation. It has a high affinity for fibrin and converts tyrosine plasminogen into plasmin, which degrades fibrinogen and some coagulation factors, making it a key component of the fibrinolytic system. Its normal value is 0.3-0.5 u / mL (chromogenic substrate method), and both a decrease and an increase are clinically significant. A decrease indicates decreased fibrinolytic activity, which is seen in prethrombotic states and thrombotic disorders such as arterial thrombosis, deep venous thrombosis, and ischemic stroke. An increase indicates increased fibrinolytic activity, which is seen in primary and secondary fibrinolytic resistance, such as disseminated intravascular coagulation, acute promyelocytic leukemia, liver disease, coronary artery disease, hyperlipidemia, and stress reactions.

[0003] The invention application with application number CN202210107637 discloses the use of tPA in nasal secretions in the preparation of nasal polyps and their prognosis detection agents, and clearly discloses the use of tPA protein in nasal secretions in the prognosis detection of nasal polyps. Without directly traumating the nasal cavity, the expression level of tPA protein in nasal secretions can be monitored, and then it can be determined whether nasal polyps will recur, providing an important basis for early drug intervention.

[0004] Currently, a variety of tPA protein detection methods have been established, such as enzyme-linked immunosorbent assay, chemiluminescence, immunoturbidimetry, and time-resolved immunosorbent assay. However, these methods often require expensive instruments and reagents, or the test results cannot be obtained immediately and are not suitable for single-person testing.

[0005] Immunogold labeling is a unique immunoassay method that emerged in the early 1980s. It primarily utilizes the high electron density of gold particles. When these markers aggregate at their corresponding ligands, red or pink spots are visible to the naked eye. This technique is used in qualitative or semi-quantitative rapid immunoassays. Due to its rapidity, simplicity, accuracy, and lack of contamination, it has gained widespread adoption in detecting antigens and antibodies. While numerous immunogold labeling products are currently available in China, there are no products that utilize this method to quantitatively detect tPA protein concentrations in nasal secretions. Summary of the Invention

[0006] In order to solve the above technical problems existing in the background technology, the present invention provides a method for preparing a quantitative nasal tPA test strip that is accurate in detection, quick and convenient to use, and simple to prepare.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing a test strip for quantitatively detecting nasal tPA, characterized in that the method comprises the following steps:

[0009] 1) preparing a nitrocellulose membrane detection layer; one end of the nitrocellulose membrane detection layer is coated with an anti-human tPA monoclonal antibody Abtpa1 that can recognize human tPA, and the other end is coated with a goat anti-mouse IgG antibody;

[0010] 2) preparing a sample pad;

[0011] 3) preparing a conjugate pad, the surface of which is sprayed with colloidal gold-labeled anti-human tPA monoclonal antibody Abtpa2;

[0012] 4) preparing a base plate, assembling the nitrocellulose membrane detection layer prepared in step 1), the sample pad prepared in step 2), and the conjugate pad and absorbent paper prepared in step 3) on the base plate in sequence to obtain a quantitative nasal tPA test strip.

[0013] Preferably, the specific implementation method of step 1) adopted in the present invention is: the anti-human tPA monoclonal antibody Abtpa1 that can recognize human tPA is coated on the membrane surface using a film stripper to form a detection line T, and at the same time, the goat anti-mouse IgG is coated on the membrane surface using a film stripper to form a quality control line C.

[0014] Preferably, the anti-human tPA monoclonal antibody Abtpa1 in step 1) adopted in the present invention comprises three heavy chain complementary determining regions and three light chain complementary determining regions, the heavy chain complementary determining regions are CDR-VH1, CDR-VH2 and CDR-VH3; the light chain complementary determining regions are CDR-VL1, CDR-VL2 and CDR-VL3; wherein, the amino acid sequence of the heavy chain complementary determining region CDR-VH1 is GYTFSTYW; the amino acid sequence of the heavy chain complementary determining region CDR-VH2 is ILPGGGYT; the amino acid sequence of the heavy chain complementary determining region CDR-VH3 is ARGVDGHPAWFVY; the amino acid sequence of the light chain complementary determining region CDR-VL1 is QNVGTN; the amino acid sequence of the light chain complementary determining region CDR-VL2 is SAS; and the amino acid sequence of the light chain complementary determining region CDR-VL3 is QQYNTYPYT.

[0015] Preferably, the concentration of the anti-human tPA monoclonal antibody in step 1) of the present invention is 0.1-3.0 mg / ml, and the streak coating volume is 0.2-2.0 ul / cm.

[0016] Preferably, the concentration of goat anti-mouse IgG in step 1) used in the present invention is 0.1-3.0 mg / ml, and the streak coating amount is 0.2-2.0 ul / cm.

[0017] Preferably, the specific implementation of step 3) adopted by the present invention is:

[0018] 3.1) preparing colloidal gold solution by trisodium citrate reduction method;

[0019] 3.2) preparing colloidal gold-labeled anti-human tPA monoclonal antibody Abtpa2 based on the colloidal gold solution prepared in step 3.1);

[0020] 3.3) The colloidal gold-labeled anti-human tPA monoclonal antibody Abtpa2 prepared in step 3.2) was sprayed onto the surface of the pretreated glass fiber mat using a bioscratch gold sprayer to form a conjugate pad.

[0021] Preferably, the anti-human tPA monoclonal antibody Abtpa2 in step 3) adopted in the present invention comprises three heavy chain complementary determining regions and three light chain complementary determining regions, the heavy chain complementary determining regions are CDR-H1, CDR-H2 and CDR-H3; the light chain complementary determining regions are CDR-L1, CDR-L2 and CDR-L3; wherein, the amino acid sequence of the heavy chain complementary determining region CDR-H1 is GYTFTSYY; the amino acid sequence of the heavy chain complementary determining region CDR-H2 is IYPGNVNA; the amino acid sequence of the heavy chain complementary determining region CDR-H3 is ARWGLLYAMDY; the amino acid sequence of the light chain complementary determining region CDR-L1 is ESVDNYGTSF; the amino acid sequence of the light chain complementary determining region CDR-L2 is AAS; and the amino acid sequence of the light chain complementary determining region CDR-L3 is QQSKEVPHT.

[0022] Preferably, the specific implementation method of the pretreatment of the glass fiber mat used in step 3.3) of the present invention is:

[0023] a) preparing a conjugate pad treatment solution, wherein the content and composition of the conjugate pad treatment solution are 0.01 M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinylpyrrolidone, and the pH of the conjugate pad treatment solution is 8.7, where "%" means "g / 100 mL";

[0024] b) Soaking treatment: soak the glass fiber mat in the bonding pad treatment solution for 1-2 hours and then dry it in a drying room for 12-16 hours;

[0025] c) Collect the dried glass fiber mat, cut it, and store it in a dry and sealed environment for future use.

[0026] Preferably, the specific implementation of step 2) adopted by the present invention is:

[0027] 2.1) Prepare a sample pad treatment solution. The composition and content of the sample pad treatment solution are 0.01M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinylpyrrolidone. The pH of the sample pad treatment solution is 8.7, where "%" means "g / 100mL";

[0028] 2.2) Soaking treatment: soak a single sample pad in the conjugate pad treatment solution for 1-2 hours and then dry it in a drying room for 12-16 hours;

[0029] 2.3) Collect the dried sample pads, cut them, and store them in a dry and sealed environment for future use.

[0030] The quantitative nasal tPA test strip was prepared according to the preparation method of the quantitative nasal tPA test strip as described above.

[0031] The advantages of the present invention are:

[0032] The present invention provides a preparation method and test strip for quantitative detection of nasal tPA, the principle of which is based on the immunological principle of the double antibody sandwich method. When the sample is added to the sample absorption pad, the liquid in the sample first dissolves the colloidal gold-labeled mouse monoclonal antibody contained in the colloidal gold pad. Secondly, the antigen to be tested (tPA) in the sample combines with the mouse monoclonal antibody labeled with colloidal gold particles to form an antigen-antibody*colloidal gold complex, and moves toward the detection line by capillary action. Another mouse monoclonal antibody is fixed on the detection line of the nitrocellulose membrane. When the sample is chromatographed to the detection line, an antibody-antigen-antibody*colloidal gold double antibody sandwich complex can be further formed, and accumulates on the detection line (T line) to show a visible color reaction. The amount of colloidal gold reflects the amount of the antigen to be tested. Excess colloidal gold-labeled monoclonal antibodies that migrate from the sample pad can be captured by the quality control line (C line), forming a color reaction. This color reaction represents that the entire reaction system is correct. At the same time, the depth of the T line color reaction corresponds to the amount of the antigen being tested in the detection area. The absorption and scattering of light by colloidal gold particles at a specific wavelength is related to the amount of colloidal gold particles. The absorption and scattering of light by colloidal gold particles is detected by a photoelectric sensor, which is proportional to the content of tPA in the sample, thereby achieving the purpose of quantitative detection. In summary, the present invention is simple to manufacture, small in size, low in cost, and can be mass-produced; the reading time is short, and the reading can be read in 15 minutes to complete quantitative detection, which is suitable for clinical rapid diagnosis and on-site rapid testing occasions; it is easy to store and can be stored for a long time by simply placing it in an aluminum foil bag containing a desiccant, which is conducive to use and promotion in primary medical units. The quantitative analysis has high accuracy. The test strip of the present invention was used in a clinical comparison test of 58 random samples with the existing enzyme-linked immunosorbent assay. The clinical compliance rate of tpA concentration was 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic structural diagram of the reagent strip provided by the present invention;

[0034] in:

[0035] 1-sample pad; 2-conjugate pad; 3-nitrocellulose detection layer membrane; 4-T line; 5-C line; 6-absorbent paper; 7-base plate; DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0037] Example 1 Preparation of anti-human tPA monoclonal antibodies

[0038] 1.1) Animal immunization

[0039] Five 8-week-old BALB / c mice were immunized using the recombinant human tpA protein for injection (trade name: Aitongli) produced by Shanghai Boehringer Ingelheim Pharmaceuticals Co., Ltd. as the antigen, and two non-immunized mice were used as negative controls. After the initial immunization antigen was fully emulsified with an equal amount of Freund's complete adjuvant, the mice were immunized by multiple subcutaneous injections on the back, 100 μg / mouse. Three weeks later, the second immunization was performed with the same dose of antigen fully emulsified with Freund's incomplete adjuvant and then injected intraperitoneally. After that, the third immunization was performed with the same dose of antigen fully emulsified with Freund's incomplete adjuvant and then injected intraperitoneally 2 weeks later. Blood was collected from the tail vein 15 days after the third immunization to detect the antiserum titer.

[0040] 1.2) Antiserum titer detection

[0041] Antiserum titer was assessed using an indirect ELISA method: Antigen was diluted to a concentration of 3 μg / mL in PBS (8.5 g / L NaCl, 1.4 g / L Na2HPO4, 0.2 g / L NaH2PO4, pH = 7.4) and added to a 96-well plate at 100 μL / well. Incubate at 37°C for 2 h or at 4°C overnight. Wash the plate three times with PBST (8.5 g / L NaCl, 1.4 g / L Na2HPO4, 0.2 g / L NaH2PO4, 0.5% (v / v) Tween-20, pH = 7.4) and tap the plate. 1% bovine serum albumin (BSA) in PBS was added at 250 μL / well and blocked at 37°C for 2 h or at 4°C overnight. Wash the plate three times with PBST and tap the plate. The sera of 4 immune mice were diluted with PBS and added to the corresponding wells, 100 μL / well. The blank control was PBS solution, and the negative control was the serum of the mouse before immunization. The plate was coated at 37℃ for 1 hour and then washed. HRP-labeled goat anti-mouse IgG was diluted 1:5000 and added, 100 μL / well. The plate was coated at 37℃ for 1 hour and then washed. 100 μL of freshly prepared TMB colorimetric substrate solution was added to each well. After 10 minutes at 37℃, 100 μL of 1M hydrochloric acid was added to each well to stop the reaction. The OD was measured using an enzyme-linked detector. 450 nm value, read and observe the results. Select the one with the highest titer (titer 51200), and boost immunization one month after the third immunization. After 5 days, take the mouse spleen cells for cell fusion.

[0042] 1.3) Recovery and culture of SP2 / 0 myeloma cells

[0043] The frozen myeloma cells (SP2 / 0) were revived in advance. After the myeloma cells frozen in liquid nitrogen were quickly taken out, they were placed in a 37°C water bath and gently shaken to melt them quickly. Note that the mouth of the freezing tube cannot touch water to avoid contamination. Then the cells were transferred to a 24-well plate containing 2 ml of RPMI-1640 complete medium (RPMI-1640 medium containing 20% fetal bovine serum, fetal bovine serum purchased from Shanxi Runsheng Daye Biomaterial Co., Ltd.) and cultured in a 37°C, 5% CO2 incubator for half an hour. When all the cells grew attached to the wall, the medium was changed in time. After that, the cells were passaged every 3 days and the cells were adjusted to the optimal growth density. When the cells reached a certain activity, they were counted and prepared for fusion. Two days before cell fusion, the cells were passaged 1 to 4 and the cell concentration in each bottle was adjusted to 1×10 5 / ml.

[0044] 1.4) Preparation of feeder cells

[0045] 1.4.1) BALB / c mice were sacrificed by cervical distension after blood was drawn from their eyeballs. The mice were completely immersed in 75% alcohol for 5 minutes and then transferred to a dish on a laminar bench with the abdomen facing upward.

[0046] 1.4.2) Use forceps to lift the mouse chest and abdominal skin, make a small cut with scissors, and use two forceps to tear a larger cut in the skin. Lift the mouse peritoneum and cut it open. Find the mouse spleen and carefully remove the spleen with forceps and small scissors. Place it in a disposable dish. Carefully remove the fat, connective tissue, etc. attached to the spleen. Add 5 ml of RPMI-1640 medium (purchased from Hyclone, cat. no. SH30809.01). Use the needle of a syringe filled with 5 ml of RPMI-1640 medium to puncture the spleen. Carefully wash out the splenocytes, which are then sieved. Add the spleen cell suspension to a 10 ml centrifuge tube and centrifuge at 1100 rpm for 5 minutes. Discard the supernatant and wash twice with RPMI-1640 medium by centrifugation.

[0047] 1.4.3) Gently resuspend the cells in 5 ml of HAT medium and mix well. Count the cells and add more HAT medium until the cell concentration reaches 1×10 5 / ml.

[0048] 1.4.4) Pour the cell suspension into a 96-well cell culture plate at 130 μl / well and culture in a 37°C, 5% CO2 incubator.

[0049] 1.5) Preparation of immune spleen cell suspension

[0050] 1.5.1) Five days after the booster immunization, select BALB / c mice with the highest serum titer, remove their eyes, bleed, and collect and separate the serum as a positive control for antibody detection.

[0051] 1.5.2) Mice were sacrificed by cervical dislocation and immersed in 75% alcohol for 5 min. The mice were removed and placed in a petri dish on a sterile laminar bench with the abdomen facing upward.

[0052] 1.5.3) Use forceps to lift the mouse's chest and abdominal skin. Use scissors to make a small incision. Use two pairs of forceps to tear a larger incision in the skin. Then, use fresh forceps to lift the mouse's peritoneum and cut it open. Locate the mouse's spleen and carefully remove it. Place it in a disposable dish and carefully remove any fat and connective tissue.

[0053] 1.5.4) After rinsing with RPMI-1640 solution, add fresh RPMI-1640 solution. Use the needle of a syringe filled with 5 ml of RPMI-1640 medium to puncture the spleen and carefully wash out the splenocytes. Then, sieve the spleen to squeeze as many splenocytes as possible through the mesh and into the solution. Transfer the splenocyte suspension to a centrifuge tube and centrifuge at 1100 rpm for 5 minutes. Discard the supernatant and centrifuge twice.

[0054] 1.5.5) Gently resuspend the spleen cells in RPMI-1640 culture medium, count, and set aside.

[0055] 1.6) Preparation of SP2 / 0 Myeloma Cell Suspension

[0056] 1.6.1) Take two bottles of myeloma cells cultured in T75 culture flasks (change the medium one day before fusion, and the cells should be in the logarithmic growth phase at the time of fusion) and collect them into 50 ml centrifuge tubes.

[0057] 1.6.2) Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0058] 1.6.3) Add 30 ml of RPMI-1640 washing buffer to the pellet, gently resuspend, mix, and centrifuge again using the same method.

[0059] 1.6.4) Gently resuspend the spleen cells in 10 ml of RPMI-1640 culture medium, mix well, count, and set aside.

[0060] 1.7) Cell fusion

[0061] 1.7.1) will contain 1×10 8 A suspension of spleen cells containing 1×10 7 Mix the suspension of 100 SP2 / 0 myeloma cells in a 50ml centrifuge tube, add culture medium to 40ml, and mix thoroughly.

[0062] 1.7.2) Centrifuge at 1200 rpm for 5 minutes and discard the supernatant.

[0063] 1.7.3) Gently tap the bottom of the centrifuge tube to loosen and evenly distribute the cell clumps into a paste.

[0064] 1.7.4) Remove the prepared 50% PEG (MW 1450) and RPMI-1640 wash solution from the 4°C refrigerator and place it in a 37°C water bath to pre-warm.

[0065] 1.7.5) Use a 1 ml pipette to draw up 0.8 ml of 50% PEG (MW 1450) and slowly add it to the centrifuge tube while stirring for 60 seconds.

[0066] 1.7.6) Then gradually add 40 ml of preheated RPMI-1640 wash solution over 60 seconds to dilute the PEG and eliminate its fusogenic effect.

[0067] 1.7.7) Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0068] 1.7.8) Add 400 mL of HAT culture medium (purchased from Sigma, cat. no. H0262) and gently pipette to resuspend the pelleted cells.

[0069] 1.7.9) Add the fused cell suspension to 96-well culture plates containing feeder cells at 50 μl / well, covering 20 plates in total. Then, place the plates in a 37°C, 5% CO2 incubator for incubation.

[0070] 1.8) Screening and cloning of positive clones

[0071] Starting from the third day after fusion, observe the cell growth in each well every day. If contamination is found, treat it with sodium azide immediately. On the 7th day after fusion, replace the medium with HT culture medium (HT, 50×, purchased from Sigma, product number H0137). The next day after the medium change, aspirate the supernatant of the well where the clone appears for specific detection. The δ strain of the new coronavirus was used as the coating antigen and the indirect ELISA method was used for detection. The OD was measured using an enzyme-linked detector. 450, a P / N value > 2.1 is considered positive. Replace the medium in the positive wells with HT medium again, and test again with ELISA the next day for positive results. Select the cloned wells that are continuously positive for 2-3 subcloning cycles to screen for monoclonal hybridoma cells. Specific steps for subcloning: ① Blow away and mix the hybridoma cells in the positive fusion wells and determine the cell concentration; ② Prepare feeder cells in advance and suspend them in HT medium at 130 μl / well. Plate them into a 96-well plate, and then place the culture plate in a 37°C, 5% CO2 incubator until ready to use; ③ Take the hybridoma cells from the positive fusion wells and evenly distribute them into the 96-well plate prepared in step ②; ④ Place the culture plate in a 37°C, 5% CO2 incubator and culture for 7 days; ⑤ Screen the positive single colony wells using ELISA and subclone again; ⑥ After 2-3 consecutive subcloning cycles, if all subcloned cell colony wells are positive and the values are similar, then a monoclonal hybridoma cell is obtained; expand the culture of the monoclonal hybridoma cells to obtain the cell culture supernatant containing the monoclonal antibody. In this step, two qualified cell lines were screened and named Abtpa1 and Abtpa2.

[0072] 1.9) Ascites Preparation and Cell Preservation

[0073] The two positive monoclonal hybridoma cells selected were expanded and cultured. Monoclonal antibody ascites was prepared using conventional in vivo ascites induction methods. Antibodies were purified from the ascites using GE-HiTrap Protein A HP prepacked columns according to the manufacturer's instructions. The specific procedures were as follows:

[0074] a. Take 5 mL of ascites, add 0.5 mL of 1 M Tris (pH = 8.0) to adjust to pH = 8.0, and centrifuge at 20,000 g for 20 min to remove the precipitate.

[0075] b. After loading the column, wash with 10 column volumes of buffer A (100 mM Tris-Cl, pH = 8.0), and then wash with 10 column volumes of buffer B (10 mM Tris-Cl, pH = 8.0).

[0076] c. Elute the IgG using approximately three column volumes of IgG elution buffer (100 mM glycine, pH = 3.0). (Pre-load the collection tubes with 0.1 mL of IgG neutralization buffer (1 M Tris-Cl, pH = 8.0), with 0.9 mL of eluent per tube.)

[0077] d. Dialysis was performed with 50 volumes of PBS (8.5 g / L NaCl, 1.4 g / L Na2HPO4, 0.2 g / L NaH2PO4, pH = 7.4).

[0078] e. After ultrafiltration and concentration, adjust the concentration to 1 mg / ml with PBS and store at -70°C until use.

[0079] The purified antibodies were diluted serially and titered using an indirect ELISA assay. SDS-PAGE analysis revealed that both antibodies exhibited purity exceeding 95%, and ELISA titers exceeding 1:1,000,000. The purified antibodies were adjusted to a concentration of 1 mg / ml and stored at -70°C until use. These two monoclonal antibodies were designated Abtpa1 and Abtpa2, respectively.

[0080] 1.10) Sequence analysis of antibody variable regions

[0081] The hybridoma cells were sequenced and analyzed according to conventional methods to determine the gene sequence of the Abtpa1 monoclonal antibody, which contains three heavy chain complementary determining regions (CDR-VH1, CDR-VH2 and CDR-VH3) and three light chain complementary determining regions (CDR-VL1, CDR-VL2 and CDR-VL3). Among them, the amino acid sequence of CDR-VH1 is GYTFSTYW, the amino acid sequence of CDR-VH2 is ILPGGGYT, the amino acid sequence of CDR-VH3 is ARGVDGHPAWFVY, the amino acid sequence of CDR-VL1 is QNVGTN, the amino acid sequence of CDR-VL2 is SAS, and the amino acid sequence of CDR-VL3 is QQYNTYPYT.

[0082] At the same time, the gene sequence of the Abtpa2 monoclonal antibody was determined, which contains three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3). Among them, the amino acid sequence of CDR-H1 is GYTFTSYY, the amino acid sequence of CDR-H2 is IYPGNVNA, the amino acid sequence of CDR-H3 is ARWGLLYAMDY, the amino acid sequence of CDR-L1 is ESVDNYGTSF, the amino acid sequence of CDR-L2 is AAS, and the amino acid sequence of CDR-L3 is QQSKEVPHT.

[0083] Example 2 Preparation of a test strip for quantitative detection of nasal tpA using colloidal gold immunochromatography

[0084] 2.1 Nitrocellulose membrane coated with antibodies

[0085] a) Prepare coating buffer: To 80 ml of ultrapure water, add 0.15 g disodium hydrogen phosphate, 0.04 g sodium dihydrogen phosphate, 0.8 g sodium chloride, 0.02 g potassium chloride, and 5 g sucrose, sequentially. Stir well, adjust the pH to 7.5, and dilute to 100 ml.

[0086] b) Coating antibody dilution: Use the above coating buffer to dilute the anti-human tPA monoclonal antibody Abtpa1 and goat anti-mouse IgG antibody (product of Feipeng Biotechnology Co., Ltd.) to 2.0 mg / ml and 1.0 mg / ml respectively.

[0087] c) Streak Coating: Clean the streaking lines of a Gold Standard Bio-Stripper HM3035 20 times with cleaning solution and then with pure water. Aspirate the two diluted coating antibodies (anti-human tPA monoclonal antibody Abtpa1 and goat anti-mouse IgG antibody) into two separate streaking lines, and evacuate the air from the lines. Adjust the position of each streaking pen so that the N antigen detection line and the quality control line are 5 mm apart and the N antigen detection line is 4 mm from the bottom of the NC membrane. Streak the surface of nitrocellulose membrane CN140 with the two antibody solutions, using streaking parameters of 1 μl / cm and 100 mm / s.

[0088] 4) Drying: Dry the nitrocellulose membrane after streaking in a room at 30% relative humidity for 12 hours; collect the dried nitrocellulose membrane in a sealed aluminum foil bag and add a bag of desiccant to the bag;

[0089] 2.2 Sample Pad and Conjugate Pad Processing

[0090] a) Prepare treatment solutions: Sample pad treatment solution (pH = 8.7) composed of 0.01 M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinyl pyrrolidone; Conjugate pad treatment solution (pH = 8.7) composed of 0.01 M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinyl pyrrolidone. Prepare 1 L of each treatment solution according to the above recipe.

[0091] b) Soaking: Pour the treatment solution into an open container, place two glass fiber pads, and gently press to completely submerge the pads. Soak each for approximately 1-2 hours, then remove and place on a rack to air dry in a drying room for 12-16 hours. Treat the sample pad and conjugate pad separately using the above method.

[0092] c) Collect the dried sample pad and conjugate pad, and cut them into thin strips of 1.5 cm and 0.6 cm width respectively using a chopper.

[0093] d) Collect the sample pad and conjugate pad cut in the previous step and store them in a dry sealed bag.

[0094] 2.3 Colloidal gold labeling and spray pad

[0095] a) Preparation of colloidal gold solution:

[0096] The colloidal gold used in this product is prepared by the trisodium citrate reduction method. The specific method is as follows:

[0097] 1) Pour 99 ml of ultrapure water into a 250 ml round-bottom flask, then add 1 ml of 1% chloroauric acid (final concentration 0.01%). Place the round-bottom flask in an oil bath set to 120°C and heat to a full boil.

[0098] 2) Rapidly add 1.2 ml of 1% trisodium citrate and continue heating for 10 minutes. Observe the solution color change from yellow to blue, then to red, and finally to a stable red. Stop heating and cool in a water bath with stirring.

[0099] 3) Scan the cooled colloidal gold solution with a UV spectrophotometer at 400-600 nm to determine the maximum absorption peak wavelength and observe the peak shape and peak width of the maximum absorption peak. The colloidal gold has a single absorption peak at 520-530 nm.

[0100] b) Preparation, purification and concentration of colloidal gold-labeled anti-human tPA monoclonal antibodies:

[0101] 1) Place 10 ml of colloidal gold solution in a 50 ml round-bottom flask with a stirrer. Add 20 μl of 0.2 M potassium carbonate solution and stir on a magnetic stirrer for 5 minutes (speed should be sufficient to prevent bubbles). Slowly add the anti-human tPA monoclonal antibody Abtpa2 dropwise to a final concentration of 5 μg / ml (at a rate sufficient to allow 1 mg of protein to be added in 5 minutes). Stir at low speed for 45 minutes.

[0102] 3) Add 1000 μl of BSA (10% concentration BSA prepared in 40 mM Tris) at a constant speed, continue stirring for 15 minutes, and store at 4°C until use.

[0103] 4) Aliquot the labeled colloidal gold solution into 2 ml centrifuge tubes. Centrifuge at 12,000 g for 10 minutes, discard the supernatant, and resuspend the pellet in a volume of wash buffer equal to 1 / 5 of the original volume of the gold solution. Centrifuge again at 12,000 g for 10 minutes, discard the supernatant, and resuspend the pellet in a volume of wash buffer equal to 1 / 10 of the original volume of the gold solution. Store at 4°C until needed. (Wash buffer recipe: 0.01 M Tris, 1% BSA, 1% Tween-20, 5% sucrose, 0.3% polyvinylpyrrolidone, pH 8.7).

[0104] c) Loading of gold-labeled antibodies

[0105] The colloidal gold-labeled anti-human tPA monoclonal antibody Abtpa2 was sprayed onto the surface of the conjugate pad using a Shanghai Gold Standard Biofilm Sprayer HM3035 at a dosage of 8 ul / cm and a speed of 100 mm / s to form a conjugate pad. The conjugate pad was then transferred to a room with a relative humidity below 30% and placed for 1-2 hours before being collected.

[0106] 2.4 Assembly and cutting

[0107] 1) Assemble a large plate on a base plate and sequentially attach a sample pad, an antibody-coated nitrocellulose membrane, a conjugate pad, and an absorbent pad. The overlap between the sample pad and the conjugate pad is 1.2 mm, the overlap between the conjugate pad and the nitrocellulose membrane is 1.2 mm, and the overlap between the absorbent pad (Anhui Huaiyuan County Tongcheng Paper Products Co., Ltd., catalog number ABP-S370) and the nitrocellulose membrane is 2 mm.

[0108] 2) The assembled large plate is cut into 3 mm wide strips on a high-speed chopping machine to obtain a test strip for quantitative detection of nasal tpA using colloidal gold immunochromatography technology (see the detailed structure for details). Figure 1 ).

[0109] Example 3 Use of the colloidal gold test strips of the present invention and result determination

[0110] 1) Sample collection

[0111] Nasopharyngeal swab: Place the swab against the nostril and slowly penetrate along the bottom of the inferior nasal passage. When the swab reaches the posterior wall of the nasopharynx, gently rotate it once and slowly remove it. Place the collected swab into a centrifuge tube containing 1 ml of sample extraction solution (formula: 50 mM Tris, 1% Triton-100, 0.02% Proclin 300, pH = 8.2). Press the swab against the tube wall and rotate and squeeze it repeatedly about 10 times to maximize the release of the specimen.

[0112] 2) Use of test strips

[0113] Recombinant human tPA protein (recombinant human tPA protein for injection, produced by Shanghai Boehringer Ingelheim Pharmaceuticals Co., Ltd.) was diluted to 500 μg / L, 250 μg / L, 125 μg / L, 62.5 μg / L, 31.25 μg / L, 15.62 μg / L, and 7.815 μg / L solutions as standard solutions. A blank solution was also prepared. 100 μl of each standard and test sample was dripped onto the sample pad of the test strip. After 20 minutes, the test strips containing the developed standard and test sample were placed in an immunochromatographic reader (Beijing Meikang Biotechnology Research Center Co., Ltd.). The color value of the T line was read and recorded. The color value of the test sample was then applied to the standard curve to obtain the human tPA concentration result.

[0114] A clinical comparison experiment was conducted on 26 random samples using the colloidal gold immunochromatographic test strips of the present invention and an existing enzyme-linked immunosorbent assay (product of Wuhan Boster Bioengineering Co., Ltd., item number EK0897). The tPA concentrations measured by the two methods had a 100% clinical concordance rate, as shown in Table 1. This demonstrates that the present method can directly and quantitatively detect human tPA concentrations, requires no specialized training, is easy to operate, and is rapid, with results available in 20 minutes. It is therefore suitable for promotion and application in primary care hospitals and physical examination centers.

[0115] Table 1 Comparison results of random sample clinical testing

[0116]

[0117]

[0118] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a test strip for quantitatively detecting nasal tPA, characterized in that: The method for preparing the quantitative nasal tPA test strip comprises the following steps: 1) preparing a nitrocellulose membrane detection layer; one end of the nitrocellulose membrane detection layer is coated with an anti-human tPA monoclonal antibody Abtpa1 that can recognize human tPA, and the other end is coated with a goat anti-mouse IgG antibody; 2) Prepare sample pad; 3) preparing a conjugate pad, the surface of which is sprayed with colloidal gold-labeled anti-human tPA monoclonal antibody Abtpa2; 4) Preparing a base plate, assembling the nitrocellulose membrane detection layer prepared in step 1), the sample pad prepared in step 2), and the conjugate pad and absorbent paper prepared in step 3) on the base plate in sequence to obtain a quantitative nasal tPA test strip.

2. The method for preparing a test strip for quantitatively detecting nasal tPA according to claim 1, characterized in that: The specific implementation method of step 1) is: using a film stripper to coat the anti-human tPA monoclonal antibody Abtpa1 that can recognize human tPA on the membrane surface to form a detection line T, and at the same time using a film stripper to coat goat anti-mouse IgG on the membrane surface to form a quality control line C.

3. The method for preparing a test strip for quantitatively detecting nasal tPA according to claim 2, characterized in that: In the step 1), the anti-human tPA monoclonal antibody Abtpa1 comprises three heavy chain complementary determining regions and three light chain complementary determining regions, wherein the heavy chain complementary determining regions are CDR-VH1, CDR-VH2 and CDR-VH3; the light chain complementary determining regions are CDR-VL1, CDR-VL2 and CDR-VL3; wherein the amino acid sequence of the heavy chain complementary determining region CDR-VH1 is GYTFSTYW; the amino acid sequence of the heavy chain complementary determining region CDR-VH2 is ILPGGGYT; the amino acid sequence of the heavy chain complementary determining region CDR-VH3 is ARGVDGHPAWFVY; the amino acid sequence of the light chain complementary determining region CDR-VL1 is QNVGTN; the amino acid sequence of the light chain complementary determining region CDR-VL2 is SAS; and the amino acid sequence of the light chain complementary determining region CDR-VL3 is QQYNTYPYT.

4. The method for preparing a test strip for quantitatively detecting nasal tPA according to claim 3, characterized in that: In the step 1), the concentration of the anti-human tPA monoclonal antibody Abtpa1 is 0.1-3.0 mg / ml, and the streak coating volume is 0.2-2.0 ul / cm.

5. The method for preparing a test strip for quantitatively detecting nasal tPA according to claim 4, characterized in that: In step 1), the concentration of goat anti-mouse IgG is 0.1-3.0 mg / ml, and the streak coating volume is 0.2-2.0 ul / cm.

6. The method for preparing a test strip for quantitatively detecting nasal tPA according to claim 1, 2, 3, 4, or 5, characterized in that: The specific implementation of step 3) is: 3.1) Preparation of colloidal gold solution by trisodium citrate reduction method; 3.2) Preparing colloidal gold-labeled anti-human tPA monoclonal antibody Abtpa2 based on the colloidal gold solution prepared in step 3.1); 3.3) The colloidal gold-labeled anti-human tPA monoclonal antibody Abtpa2 prepared in step 3.2) was sprayed onto the surface of the pretreated glass fiber mat using a bioscratch gold sprayer to form a conjugate pad.

7. The method for preparing a test strip for quantitatively detecting nasal tPA according to claim 6, characterized in that: In the step 3), the anti-human tPA monoclonal antibody Abtpa2 comprises three heavy chain complementary determining regions and three light chain complementary determining regions, wherein the heavy chain complementary determining regions are CDR-H1, CDR-H2 and CDR-H3; the light chain complementary determining regions are CDR-L1, CDR-L2 and CDR-L3; wherein the amino acid sequence of the heavy chain complementary determining region CDR-H1 is GYTFTSYY; the amino acid sequence of the heavy chain complementary determining region CDR-H2 is IYPGNVNA; the amino acid sequence of the heavy chain complementary determining region CDR-H3 is ARWGLLYAMDY; the amino acid sequence of the light chain complementary determining region CDR-L1 is ESVDNYGTSF; the amino acid sequence of the light chain complementary determining region CDR-L2 is AAS; and the amino acid sequence of the light chain complementary determining region CDR-L3 is QQSKEVPHT.

8. The method for preparing a test strip for quantitatively detecting nasal tPA according to claim 7, characterized in that: The specific implementation method of the pretreatment of the glass fiber mat used in step 3.3) is: a) preparing a conjugate pad treatment solution, wherein the content and components of the conjugate pad treatment solution are 0.01 M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinyl pyrrolidone, and the pH of the conjugate pad treatment solution is 8.7; b) Soaking treatment: Soak the glass fiber mat in the bonding pad treatment solution for 1-2 hours and then dry it in a drying room for 12-16 hours; c) Collect the dried glass fiber mat, cut it and store it in a dry and sealed environment for future use.

9. The method for preparing a test strip for quantitatively detecting nasal tPA according to claim 8, characterized in that: The specific implementation of step 2) is: 2.1) Prepare a sample pad treatment solution, wherein the composition and content of the sample pad treatment solution are 0.01M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinylpyrrolidone, and the pH of the sample pad treatment solution is 8.7; 2.2) Soaking treatment: soak a single sample pad in the conjugate pad treatment solution for 1-2 hours and then dry it in a drying room for 12-16 hours; 2.3) Collect the dried sample pads, cut them, and store them in a dry and sealed environment for future use.

10. A test strip for quantitative detection of nasal tPA prepared according to the method for preparing a test strip for quantitative detection of nasal tPA according to any one of claims 1 to 9.

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