[0028] In order to describe in detail the technical content, the achieved objectives and effects of the present invention, the following description will be given in conjunction with the embodiments and the accompanying drawings.
[0029] The first embodiment of the present invention is: a test paper suitable for the quantitative detection of peripheral blood immunochromatography, such as figure 1 As shown, it includes a bottom plate 1 and a top cover (not shown in the figure), the top cover is closed on the bottom plate 1, and the bottom plate 1 is sequentially provided with a sample pad 2, a closing pad 3, The reaction pad 4 and the absorbent pad 5; the sample pad 2 is coated with a first antibody, the first antibody has a tracer marker and can identify an epitope of the target object to be tested; the closed pad 3 RBC is coated thereon, and the closed pad 3 and the reaction pad 4 are partially laminated. The upper cover is provided with sample holes and observation windows in the corresponding areas above the sample pad 2 and the reaction pad 4, which is convenient for operation and for the analysis instrument to determine the result, which improves the convenience of the operation of the detection process and the accuracy of the method.
[0030] The length of the overlapping area of the closed pad 3 and the reaction pad 4 is 3 mm. The sealing pad 3 is prepared by the following method: the matrix material used to prepare the sealing pad 3 is soaked in a sealing solution, and then dried after being prepared, the sealing solution includes anti-erythrocyte antibodies, ethylenediammonium tetraacetic acid Disodium, BSA and Tris-HCl buffer. The concentration of the anti-erythrocyte antibody is 0.2 mg/ml; the concentration of the disodium edetate is 0.5 wt%; the concentration of the BSA is 5 wt%; the pH of the Tris-HCl buffer is 7.45 . Bovine serum albumin (BSA) is used to block non-specific protein binding sites to avoid false positive samples. Tris-HCl buffer is the pH value of the stable buffer, and disodium ethylenediaminetetraacetic acid is used as a chelate mixture. Adopting the sealing solution of the formula of the present invention for sealing can make the detection sensitivity higher. The first antibody is coated on the first antibody coating part 21 on the middle area of the sample pad 2, which is close to the sample hole of the upper cover, and the first antibody with the tracer marker is coated on the sample pad 2. The vicinity of the sample hole position can make the test object in the sample and the antibody have more sufficient contact time, and it is also more conducive to the release of the colloidal gold coupling substance carrying the test target more completely, which increases the overall detection sensitivity.
[0031] The sample pad 2 is also fixed with a quality control antibody, and the middle of the reaction pad 4 is also provided with a detection line 42 and a quality control line 41. The detection line 42 is located on the side close to the absorbent pad 5, and the quality control line 41 is located on the side close to the sealing pad 3, and the detection line 42 is close to the side of the absorbent pad 5, so that the antibodies on the detection line have more time to receive the colloidal gold conjugate from the sample pad carrying the target to be tested. It can detect a lower concentration of the target to be tested, thereby further improving the detection sensitivity of the system. The quality control line 41 is coated with an antigen capable of capturing quality control antibodies, and the detection line 42 is coated with a second antibody. The two antibodies can further reduce non-specific binding and improve detection sensitivity. Using a separate quality control area antibody, compared with the goat anti-mouse IgG used in the prior art, the separate quality control area antibody is not affected by the coating position, and can also reflect the total amount of antibody coating of the reagent card, reflecting the reaction of the reagent card The authenticity of the results.
[0032] The tracer marker is colloidal gold (other tracer markers can also be used), and colloidal gold is used as the marker, the marker has good stability and low price. The use of colloidal gold as a tracer marker, combined with the interpretation of the results of the colloidal gold test paper instrument, can easily realize automation, reduce the influence of subjective factors, and reduce interpretation errors, which not only makes the operation more convenient, but also can quickly obtain reliable diagnostic results. .
[0033] The target to be tested is PCT or NT-proBNP. The solution of the present invention is particularly suitable for PCT or NT-proBNP detection. The test paper prepared by the solution of the present invention is used for PCT or NT-proBNP detection with high sensitivity and only a detection time. 15min, which greatly improves the detection efficiency; in addition, the linear detection range of PCT can reach three orders of magnitude (0.50ng/ml~100.00ng/ml), the detection limit of NT-proBNP is as low as pg/ml, and the linear range is 200pg /ml~30000pg/ml.
[0034] The matrix materials of the sealing pad 3 and the sample pad 2 are both glass cellulose membranes, the matrix material of the reaction pad 4 is nitrocellulose membrane, and the anti-erythrocyte antibody is an anti-human erythrocyte antibody. The invention coats the antibody against human red blood cells on a separate glass cellulose membrane, which is convenient for process production, convenient for storage and transportation, and can effectively improve the stability of the reagent card.
[0035] The test paper of the present invention can be widely used in the preparation of immunochromatographic detection kits or in the quantitative detection of peripheral blood immunochromatographic detection.
[0036] The second embodiment of the present invention is: a PCT immunochromatographic quantitative detection test paper, comprising a bottom plate 1, on which a sample pad 2, a sealing pad 3 (matrix is glass fiber membrane), and a reaction pad 4 are sequentially arranged from left to right. The substrate is nitrocellulose membrane) and the absorbent pad 5. The first PCT monoclonal antibody labeled with colloidal gold (concentration range can be 0.3~1.5mg/mL) and the single C-band antibody labeled with colloidal gold are fixed on the sample pad 2; another one that can recognize PCT is fixed on the nitrocellulose membrane The second PCT monoclonal antibody of the epitope constitutes the detection line 42 (the concentration range can be 0.5-3 mg/mL) and the quality control line 41 (the concentration range can be) coated with a single C-band antibody antigen (DNP-BSA) It is 0.2~2.0mg/mL), and the quality control line 41 is used to test the validity of the test strip.
[0037] The test paper preparation procedure is as follows:
[0038] Step 1 Preparation of reaction pad 4:
[0039] The type of coating antibody is determined according to the target to be tested in the sample to be tested: the antibody of the present invention is a second PCT monoclonal antibody and a C-band antigen capable of capturing a single C-band antibody. The first procalcitonin monoclonal antibody at 6.92 mg/ml and the C-band antigen at 10.5 mg/ml were coated on a nitrocellulose membrane and dried for 24 hours at a temperature of 37° C. and a humidity of ≦40% RH.
[0040] Step 2 Preparation of sample pad 2
[0041] Take 40nm colloidal gold solution and add 0.2M K 2 CO 3 (Potassium carbonate) Adjust the pH to 7.5 (just adjust the pH to 7-8), take two pH-adjusted solutions, and add the first PCT monoclonal antibody and the C-band antibody (rabbit Anti-DNP antibody) to prepare an antibody composition with markers, stir at room temperature for 15 minutes, add BSA (bovine serum albumin) stabilizer at a ratio of 0.8%, and stir at room temperature for 30 minutes.
[0042] Centrifuge at 12000rpm for 15min, remove the supernatant, and take the concentrated precipitate; resuspend with 0.1% boric acid/PEG20000, repeat the centrifugation twice, and finally resuspend with a small amount (100~200μl can be used) 0.1% boric acid/PEG20000 to determine the optical density (optical density) , OD).
[0043] The recovered precipitation diluent was diluted with boric acid and coated on a 1.8cm×30cm glass cellulose membrane. The glass fiber was preliminarily used with a concentration of 5% casein, 0.2% trehalose, 2mM boric acid/PEG20000 buffer (2mM Soak in a working solution composed of boric acid/PEG20000) (pH 9.0). Dry the glass fiber at 37° C. for 24 hours, with a coating position near the sample inlet, add a liquid amount of 120 μl/sheet, and dry for 24 hours, at a temperature of 37° C., and a humidity of ≦40% RH.
[0044] Step 3 Preparation of closed pad 3:
[0045] Cut into 1cm×30cm glass fiber, use anti-human erythrocyte antibody with a concentration of 0.06mg/ml, 0.12% disodium edetate, 1% BSA (bovine serum albumin) and Tris-HCl buffer (three Soak in a sealing solution composed of hydroxymethylaminomethane-hydrochloric acid (pH7.4). It was dried at 37°C for 24 hours to obtain a closed pad 3.
[0046] Step 4 test paper assembly
[0047] After tearing off the double-sided tape on the bottom plate 1, stick the prepared reaction pad 4 on the bottom plate 1, and then press the absorbent pad 5 onto the 2mm right side of the reaction pad 4. The other end of the absorbent pad 5 is connected to the bottom plate 1. Aligned. Press the sealing pad 3 on the 3mm left side of the reaction pad 4 and paste it. Align one end of the sample pad with the bottom plate, and partially press the other end on the closed pad 3.
[0048] When the sample to be tested is added to the sample pad 2, it moves forward through chromatography, and the PCT in the sample reacts with the first PCT monoclonal antibody bound to the colloidal gold marker on the sample pad 2 to form a complex PCT-Mab-PCT. When the reaction complex continues to move forward through the second PCT monoclonal antibody coated on the nitrocellulose membrane (detection line 42) under the action of chromatography, the reaction complex is captured by the coated second PCT monoclonal antibody to form a complex (Mab-PCT-PCT-Mab-PCT) (detection line 42). At the same time, the individually labeled C-band antibody binds to the quality control line 41C antigen to show the band, and the colloidal gold test paper analyzer reads the detection line 42 The reaction signal is automatically converted into a quantitative value through signal conversion and a set standard curve, and the concentration of PCT in the sample is calculated to obtain the detection result of procalcitonin. The experiment found that the linear detection range is 0.50ng/ml~100.00ng/ml, and the linear relationship is good, which can achieve accurate quantification.
[0049] The third embodiment of the present invention is: a NT-proBNP immunochromatographic quantitative detection test paper, including a bottom plate 1, on which a sample pad 2, a sealing pad 3 (matrix is glass fiber membrane), and a reaction pad are sequentially arranged from left to right. 4 (Substrate is nitrocellulose membrane) and absorbent pad 5. The sample pad 2 is fixed with the first NT-proBNP monoclonal antibody labeled with colloidal gold (concentration range can be 0.3~1.5mg/mL) and the single C-band antibody labeled with colloidal gold; the nitrocellulose membrane is immobilized with NT-proBNP. The detection line formed by the monoclonal antibody of another epitope of proBNP (the second NT-proBNP monoclonal antibody (concentration range can be 0.5-3 mg/mL)) and the antigen concentration range for capturing the single C antibody can be 0.2-2.0 mg/ mL) constitutes a quality control line, which is used to test the validity of the test strip.
[0050] The test paper preparation procedure is as follows:
[0051] Step 1 Preparation of reaction pad 4:
[0052] Determine the type of coating antibody according to the target to be tested in the sample to be tested: the second N-terminal brain natriuretic peptide (NT-proBNP) monoclonal antibody and the C-band antigen (DNP-BSA) capable of capturing a single C-band antibody. The 15.42mg/ml second NT-proBNP monoclonal antibody and 8.02mg/ml C-band antigen were coated on the nitrocellulose membrane and dried for 24h at a temperature of 37°C and a humidity≦40%RH.
[0053] Step 2 Preparation of sample pad 2:
[0054] Take 40nm colloidal gold solution and add 0.2M K 2 CO 3 (Potassium carbonate) adjust the pH to 8.0, take two pH-adjusted solutions, add the first NT-proBNP monoclonal antibody and the C-band antibody (rabbit anti-DNP antibody) at the ratio of 15μg/ml to make the marker , Stir at room temperature for 15 minutes, add BSA as a stabilizer at a ratio of 0.8%, and stir at room temperature for 30 minutes.
[0055] Centrifuge at 12000 rpm for 15 minutes, remove the supernatant, and take the concentrated precipitate; resuspend with 0.1% boric acid/PEG20000, repeat the centrifugation twice, and finally resuspend with a small amount (100-200 microliters can be used) 0.1% boric acid/PEG20000 to determine the OD.
[0056] Dilute the recovered precipitation diluent with boric acid and coat it on a 1.8cm×30cm glass cellulose membrane. The coating position is near the sample inlet. The amount of liquid added is 120ul/sheet, dried for 24h, temperature 37℃, humidity≦ 40%RH.
[0057] Step 3 Preparation of closure pad 3
[0058] The glass fiber cut into 1cm×30cm contains anti-human erythrocyte antibody at a concentration of 0.06mg/ml, 0.12% disodium edetate, 1% BSA (bovine serum albumin) and Tris-HCl buffer (trihydroxymethyl) Hydrochloric acid) (pH7.4) working solution immersion. It was dried at 37°C for 24 hours to obtain a sample pad.
[0059] Step 4 test paper assembly
[0060] After tearing off the double-sided tape on the bottom plate 1, stick the prepared reaction pad 4 on the bottom plate 1, and then press the absorbent pad 5 onto the 2mm right side of the reaction pad 4. The other end of the absorbent pad 5 is connected to the bottom plate 1. Aligned. Press the sealing pad 3 on the 3mm left side of the reaction pad 4 and paste it. Align one end of the sample pad with the bottom plate, and partially press the other end on the closed pad 3.
[0061] When the sample to be tested is added to the sample pad 2, it moves forward by chromatography, and the NT-proBNP in the sample reacts with the NT-proBNP antibody bound to the colloidal gold marker on the sample pad 2 to form a complex NT-proBNP-Mab- NT-proBNP, when the reaction complex continues to move forward through the NT-proBNP antibody coated on the nitrocellulose membrane (detection line 42), the reaction complex is captured by the coated NT-proBNP antibody to form Mab- NT-proBNP-NT-proBNP-Mab-NT-proBNP (detection line), at the same time, the individually labeled C-band antibody binds to the quality control line C antigen to show the band, and the detection line 42 is read by the colloidal gold test paper analyzer The reaction signal is automatically converted into a quantitative value through signal conversion and a set standard curve, and the concentration of NT-proBNP in the sample is calculated to obtain the N-terminal brain natriuretic peptide (NT-proBNP) detection result. The experiment found that the detection line 42 of NT-proBNP ranges from 200pg/ml to 30000pg/ml.
[0062] The C-band antigen and the corresponding antibody in the present invention can also be combined with other individually matched antigen-antibody combinations, such as mouse anti-DNP antibodies.
[0063] The above are only the embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent transformations made using the content of the description and drawings of the present invention, or directly or indirectly applied in related technical fields, are included in the same reasoning The invention is within the scope of patent protection.
