Colloidal gold immunochromatography semi-quantitative detection test strip and detection method

Abstract

The invention provides a colloidal gold immunochromatography semi-quantitative detection test strip and a detection method. The colloidal gold immunochromatography semi-quantitative detection test strip comprises a bottom plate, a sample loading pad, a colloidal gold pad, a nitrocellulose membrane and absorbent paper, and a sample to be detected sequentially flows through the sample loading pad, the colloidal gold pad, the nitrocellulose membrane and the absorbent paper; the nitrocellulose membrane is provided with a first detection line, a second detection line, a third detection line and a quality control line along the flowing direction of a sample, the first detection line is coated with a sheep monoclonal antibody with strong affinity with the sample to be detected, and the second detection line is coated with a rabbit monoclonal antibody with medium affinity with the sample to be detected; the third detection line is coated with a mouse monoclonal antibody with weak affinity with a to-be-detected sample, the colloidal gold pad is coated with a monoclonal antibody with high affinity with the to-be-detected sample, and the quality control line is coated with a mouse-derived, rabbit-derived and sheep-derived second antibody mixture. Compared with the method adopting the same antibody with different concentrations for coating, the method adopting the antibodies of different species has the advantages that the affinity difference of the antibodies can reach 10-100 times, and the sensitivity is higher.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnosis and immune detection, and particularly relates to a colloidal gold immunochromatography semi-quantitative detection test strip and a detection method. Background Art

[0002] Immunocolloidal gold labeling technology is a new type of immunolabeling technology that uses colloidal gold as a tracer marker and is applied to antigen-antibody reactions. Colloidal gold is polymerized into gold particles of a specific size by an aqueous solution of chloroauric acid under the action of a reducing agent. The particles form a stable colloidal state due to the electrostatic effect, also known as gold sol. Due to the characteristics of colloidal stability, strong protein adsorption and high electron density, colloidal gold can be seen as dark brown particles under a microscope at the gold-labeled protein binding site. When these markers are aggregated in large quantities at the corresponding ligands, red or pink spots can be seen with the naked eye. Therefore, it can be used in qualitative or semi-quantitative rapid immunoassay methods.

[0003] Colloidal gold immunochromatographic test paper is a strip-shaped fixed specific antibody or antigen in a certain area on the nitrocellulose (NC) membrane. The area has a control line and a test line that displays the results. When the sample is added to one end of the membrane, it moves forward with the help of the chromatographic force. After the sample moves along the membrane to reach the area, the corresponding antigen or antibody in the sample specifically binds to the antigen or antibody and is intercepted, making the area show a certain color. The visually observable colloidal gold marker obtains an intuitive color development result, while the free marker crosses this area to achieve the purpose of automatic separation. This method can interpret the test results with the naked eye and can achieve rapid detection of the target.

[0004] The double antibody sandwich method is usually used for detection. The detection reagent is usually a specific ligand (i.e., antibody Abl) of the antigen to be detected that is coupled to colloidal metal and is deposited on the conjugate pad. When the sample (urine, plasma, whole blood, etc.) is added to the sample pad, the sample quickly penetrates the conjugate pad due to the chromatographic force, the detection reagent is dissolved, and moves forward along the membrane strip with the sample flow. If the antigen to be detected is present in the sample, it will bind to the antibody in the detection reagent to form Ag-Abl. Subsequently, the sample passes through the test line area solidified with the capture reagent (generally another epitope-specific antibody Ab2 of the antigen to be detected), and the antigen to be detected-detection reagent complex is captured to form an Abl-Ag-Ab2 complex. The sample continues to move forward and passes through the quality control line area solidified with the anti-Abl antibody (secondary antibody Ab3) to form an Ab3-Abl-Ag-Ab2 complex. This method is usually used to detect analyses with multiple antigenic sites, such as human chorionic gonadotropin (HCG), human immunodeficiency virus (HIV), etc.

[0005] Semi-quantitative detection by colloidal gold immunochromatographic test strips can assess the risk and severity of certain high-risk special diseases in clinical practice and define the risk population. The main method is to set up multiple test lines and perform semi-quantitative detection through the color development results of different test lines, where different test lines are usually coated with different concentrations of the same antibody. However, the sensitivity of this method is not high enough, and some low-concentration test substances cannot be detected, resulting in inaccurate test results. For some test substances, the concentration is too high and the upper limit is too small to be detected. At the same time, the antibodies of the detection reagent cannot capture all the test antigens, causing the free antigens to occupy the sites of the capture reagents, resulting in false negative results (HOOK effect). Summary of the invention

[0006] Aiming at the shortcomings of the existing colloidal gold immunochromatographic semi-quantitative detection test paper, the present invention provides a colloidal gold immunochromatographic semi-quantitative detection test paper strip. The test paper strip can improve the detection sensitivity and expand the detection range.

[0007] To achieve the above object, the technical solution of the present invention is implemented as follows: a colloidal gold immunochromatographic semi-quantitative test strip comprises a bottom plate, a sample loading pad, a colloidal gold pad, a nitrocellulose membrane, and a blotting paper, wherein the sample loading pad, the colloidal gold pad, the nitrocellulose membrane, and the blotting paper are sequentially pasted on the top of the bottom plate, the colloidal gold pad and the blotting paper are respectively pressed on the top of the two ends of the nitrocellulose membrane in the length direction, the sample loading pad is pressed on the top of the end of the colloidal gold pad away from the nitrocellulose membrane, and the sample to be tested flows through the sample loading pad, the colloidal gold pad, and the blotting paper in sequence. , nitrocellulose membrane, absorbent paper, the nitrocellulose membrane is provided with a first detection line, a second detection line, a third detection line, and a quality control line along the sample flow direction, the first detection line is coated with a sheep monoclonal antibody with a strong affinity to the sample to be tested, the second detection line is coated with a rabbit monoclonal antibody with a medium affinity to the sample to be tested, the third detection line is coated with a mouse monoclonal antibody with a weak affinity to the sample to be tested, the colloidal gold pad is coated with a monoclonal antibody with a high affinity to the sample to be tested, and the quality control line is coated with a mixture of secondary antibodies from mouse, rabbit and sheep sources.

[0008] In one embodiment of the present invention, the colloidal gold pad is coated with a mixture of sheep monoclonal antibodies, rabbit monoclonal antibodies, and mouse monoclonal antibodies, and the antibody mixture coated with the colloidal gold pad has different binding sites with respect to the sample to be tested from the antibodies coated with the first detection line, the second detection line, and the third detection line.

[0009] In one embodiment of the present invention, the first detection line, the second detection line, the third detection line, and the quality control line are sequentially distributed along the sample flow direction.

[0010] In one embodiment of the present invention, the second detection line and the third detection line are both provided with an embedding strip at one end close to the colloidal gold pad, wherein the embedding strip is coated with plant-derived antibodies, the plant-derived antibodies coated with the embedding strip of the second detection line are sprayed with antibodies coated with the second detection line, and the plant-derived antibodies coated with the embedding strip of the third detection line are sprayed with antibodies coated with the third detection line.

[0011] In one embodiment of the present invention, the width of the embedded strip is 1-2 mm.

[0012] In one embodiment of the present invention, the secondary antibody mixture coated on the quality control line can be combined with the antibody coated on the colloidal gold pad.

[0013] On the other hand, the present invention also provides a method for detecting a colloidal gold immunochromatographic semi-quantitative test strip, using the colloidal gold immunochromatographic semi-quantitative test strip for detection, comprising at least the following steps: 1) Obtain the concentration range of the first detection line, the second detection line, and the third detection line; 2) Add the sample that may contain the target analyte to the sample pad, and wait for the sample to flow through the colloidal gold pad, nitrocellulose membrane, and finally into the absorbent paper; 3) Observe the colors of the first test line, the second test line, the third test line, and the quality control line, and determine the concentration range of the sample according to step 1.

[0014] The colloidal gold immunochromatographic semi-quantitative test strip obtained by the above technical solution has the following beneficial effects: 1. Differences in the immune systems of different species result in different abilities of the antibodies produced to bind to antigen epitopes. The affinity of sheep antibodies is greater than that of rabbit antibodies and greater than that of mouse antibodies. Compared with using the same antibody at different concentrations for coating, the affinity of the antibodies themselves can differ by 10-100 times when using antibodies of different species, and the sensitivity is higher.

[0015] 2. Pre-embed the plant-derived antibody at one end of the second and third test lines close to the colloidal gold pad and then cover the relevant test antigens, which can consume part of the test material in advance, greatly widen the detection range and avoid the HOOK effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the structure of the colloidal gold immunochromatographic semi-quantitative test strip of the present invention; Figure 2 It is a schematic diagram of the structure of a test strip for (C-reactive protein) in one embodiment of the present invention; Figure 3 The present invention is applied Figure 2 The testing process of the test strip; Figure 4 The present invention is applied Figure 2 Schematic diagram of the test strips for detecting samples of different concentrations; Figure 5 The present invention Figure 2 A schematic diagram of the structure after adding an embedded strip to the test strip; Figure 6 The present invention is applied Figure 5 The testing process of the test strip; Figure 7 The present invention is applied Figure 5 Schematic diagram of the test strips for detecting samples of different concentrations.

[0017] In the figure, 1, bottom plate; 2, sample loading pad; 3, colloidal gold pad; 4, nitrocellulose membrane; 5, absorbent paper; 41, first detection line; 42, second detection line; 43, third detection line; 44, quality control line; 45, embedding strip. DETAILED DESCRIPTION

[0018] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0019] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0020] The present invention generally relates to the field of immunoassay technology, and particularly to a colloidal gold immunochromatographic semi-quantitative test strip. The core of the present technical invention is based on immunocolloidal gold labeling technology to design a colloidal gold immunochromatographic semi-quantitative test strip to improve detection sensitivity, expand detection range, and avoid the HOOK effect.

[0021] The present invention is further explained below in conjunction with embodiments and drawings. It should be understood that the present invention is not limited to the specific embodiments described.

[0022] like Figure 1 , Figure 2As shown, the present invention proposes a colloidal gold immunochromatographic semi-quantitative test strip, comprising a bottom plate 1, a sample loading pad 2, a colloidal gold pad 3, a nitrocellulose membrane 4, and a wicking paper 5, wherein the sample loading pad 2, the colloidal gold pad 3, the nitrocellulose membrane 4, and the wicking paper 5 are sequentially pasted on the top of the bottom plate 1, the colloidal gold pad 3 and the wicking paper 5 are respectively pressed on the top of the two ends of the nitrocellulose membrane 4 in the length direction, the sample loading pad 2 is pressed on the top of the end of the colloidal gold pad 3 away from the nitrocellulose membrane 4, and the sample to be tested flows through the sample loading pad 2, the colloidal gold pad 3, the nitrocellulose membrane 4 in sequence. , absorbent paper 5, the nitrocellulose membrane 4 is provided with a first detection line 41, a second detection line 42, a third detection line 43, and a quality control line 44 along the sample flow direction, the first detection line 41 is coated with a sheep monoclonal antibody with a strong affinity to the sample to be tested, the second detection line 42 is coated with a rabbit monoclonal antibody with a medium affinity to the sample to be tested, the third detection line 43 is coated with a mouse monoclonal antibody with a weak affinity to the sample to be tested, the colloidal gold pad 3 is coated with a monoclonal antibody with a high affinity to the sample to be tested, and the quality control line 44 is coated with a mixture of secondary antibodies from mouse, rabbit and sheep sources.

[0023] like Figure 3-4 As shown, when the substance to be tested in the sample is at a relatively low concentration level, the antibody coated on the second detection line 42 and the third detection line 43 has a relatively weak affinity with the substance to be tested, so the substance to be tested will not be captured by the antibody, that is, no color will be developed at the positions of the second detection line 42 and the third detection line 43, and the antibody coated on the first detection line 41 has the strongest affinity with the substance to be tested, so the position of the first detection line 41 can be colored; When the substance to be tested in the sample is at a medium concentration level, since the antibody coated on the third detection line 43 has the weakest affinity with it, the substance to be tested will not be captured by it, that is, no color will be developed at the position of the third detection line 43, while the antibody coated on the first detection line 41 and the second detection line 42 has a stronger affinity with the substance to be tested, so it can effectively capture the substance to be tested, and color will be developed at the positions of the first detection line 41 and the second detection line 42; When the substance to be tested in the sample is at a higher concentration level, the antibodies coated on the first detection line 41, the second detection line 42 and the third detection line 43 can capture the substance to be tested, that is, color can be displayed at the positions of the first detection line 41, the second detection line 42 and the third detection line 43.

[0024] Specifically, the colloidal gold pad 3 is coated with a mixture of sheep monoclonal antibodies, rabbit monoclonal antibodies, and mouse monoclonal antibodies, and the antibody mixture coated with the colloidal gold pad 3 has different binding sites with respect to the sample to be tested from the antibodies coated with the first detection line 41, the second detection line 42, and the third detection line 43. The antigen of the sample to be tested is allowed to continue to flow after binding with the antibodies coated with the colloidal gold, and is respectively combined with the antibodies coated with the corresponding first detection line 41, the second detection line 42, and the third detection line 43 to form a double antibody sandwich structure.

[0025] Specifically, the first detection line 41, the second detection line 42, the third detection line 43, and the quality control line 44 are sequentially distributed along the sample flow direction.

[0026] like Figure 5-7 As shown, the second detection line 42 and the third detection line 43 are both provided with an embedding strip 45 at one end close to the colloidal gold pad 3, and the embedding strip 45 is coated with plant-derived antibodies, and the plant-derived antibodies coated with the embedding strip 45 of the second detection line 42 are sprayed with antibodies coated with the second detection line 42, and the plant-derived antibodies coated with the embedding strip 45 of the third detection line 43 are sprayed with antibodies coated with the third detection line 43. When the sample flows to the embedding strip 45, it will bind to the detection antibodies on the surface. Since the gaps in the membrane are occupied by the plant-derived antibodies, it will not stay on the nitrocellulose membrane 4, and will flow forward under the action of the chromatographic force, and finally flow to the absorbent paper 5. Since part of the sample to be tested is combined with the plant-derived antibodies, part of the sample is consumed, so that the detection range of the sample is increased when it subsequently flows into the second detection line 42 and the third detection line 43, and the HOOK effect is avoided.

[0027] Specifically, the embedding strip 45 has a width of 1-2 mm.

[0028] Specifically, the secondary antibody mixture coated on the quality control line 44 can be combined with the antibody coated on the colloidal gold pad 3. The quality control line 44 is used to determine whether the detection process is effective.

[0029] On the other hand, the present invention also provides a method for detecting a colloidal gold immunochromatographic semi-quantitative test strip, using the colloidal gold immunochromatographic semi-quantitative test strip for detection, comprising at least the following steps: 1) Obtain the concentration range of the first test line, the second test line, and the third test line: drip a series of concentration standard solutions containing the target analyte onto the sample pad. The sample first binds to the antibody on the colloidal gold pad, then binds to the antibodies on the first test line, the second test line, the third test line, and the quality control line on the nitrocellulose membrane, and finally flows onto the absorbent paper. The concentration range of the first test line, the second test line, the third test line, and the quality control line is determined by observing the color of the first test line, the second test line, the third test line, and the quality control line, and according to the concentration of the standard solution; 2) Add the sample that may contain the target analyte to the sample pad, and wait for the sample to flow through the colloidal gold pad, nitrocellulose membrane, and finally into the absorbent paper; 3) Observe the colors of the first test line, the second test line, the third test line, and the quality control line, and determine the concentration range of the sample according to step 1.

[0030] Example 1

[0031] Specific preparation method of colloidal gold test strips using C-reactive protein (CRP) as an example 1. Materials 1.1 CRP mouse monoclonal antibody, CRP rabbit monoclonal antibody, CRP goat monoclonal antibody, mouse secondary antibody, rabbit secondary antibody, goat secondary antibody were purchased from Shenzhen Feipeng Biological Co., Ltd.; plant-derived antibodies were purchased from Albertson Biotechnology Co., Ltd.; 1.2 Nitrocellulose membrane, purchased from Millipore; 1.3 Chloroauric acid, purchased from Sigma.

[0032] 2. Test strip composition The CRP colloidal gold (semi-quantitative) detection test strip of the present invention comprises four parts from bottom to top: a buffer pad, a colloidal gold pad, a nitrocellulose membrane and a water-absorbing paper.

[0033] 2.1 Labeling of colloidal gold solution Take a colloidal gold solution with 30-80 nm particles, adjust the pH to 7.2, 7.4 and 7.6 respectively, add CRP mouse monoclonal antibody A, CRP rabbit monoclonal antibody A and CRP goat monoclonal antibody A with a final concentration of 0.01 mg / mL respectively, add casein with a mass concentration of 1% for blocking after 1 hour, centrifuge to remove the supernatant, add 10% (v / v) 5% sugar solution to re-dissolve, and store for later use.

[0034] 2.2 Preparation of colloidal gold pad Take 1 mL of the labeled colloidal gold solution, add 10 mL of 0.1% Tween, 1% sugar in 0.1 mL PBS buffer, mix well, spread evenly on the glass fiber membrane, dry, and cut into 5 mm wide pieces for later use.

[0035] First colloidal gold pad: The selected colloidal gold solution contains CRP mouse monoclonal antibody A; Second colloidal gold pad: The selected colloidal gold solution contains CRP mouse monoclonal antibody A, CRP rabbit monoclonal antibody A and CRP sheep monoclonal antibody A at the same time.

[0036] 2.3 Preparation of nitrocellulose membrane First nitrocellulose membrane: Take CRP mouse monoclonal antibody B and dilute it to 1 mg / mL, 1.5 mg / mL and 3 mg / mL with 0.01M PB to form T1, T2 and T3 test line coating solutions respectively. Take mouse secondary antibody and dilute it to a final concentration of 2 mg / mL with 0.01M PB to form the quality control line coating solution. Spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm.

[0037] Second nitrocellulose membrane: CRP goat monoclonal antibody B, CRP rabbit monoclonal antibody B, and CRP mouse monoclonal antibody B were diluted to 1 mg / mL with 0.01M PB to form T1, T2, and T3 coating solutions, respectively. Mouse secondary antibody, rabbit secondary antibody, and goat secondary antibody were diluted with 0.01M PB to a final concentration of 2 mg / mL to form the quality control line coating solution. The test line coating solution and the quality control line coating solution were sprayed on the NC membrane at a speed of 1.2μL / cm.

[0038] Third nitrocellulose membrane: Take CRP mouse monoclonal antibody B and dilute it to 1 mg / mL, 1.5 mg / mL and 3 mg / mL with 0.01M PB to form T1, T2 and T3 test line coating solutions respectively. Take mouse secondary antibody and dilute it to a final concentration of 2 mg / mL with 0.01M PB to form the quality control line coating solution. Use 0.01M PB to dilute the plant-derived antibody to 2 mg / mL to form the embedding solution; spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm, and then spray the embedding solution at a speed of 1.2μL / cm at T2 and T3 1mm below the colloidal gold end. After the embedded strips are dried, spray them with T2 and T3 coating solutions to cover their surfaces.

[0039] Fourth nitrocellulose membrane: CRP sheep monoclonal antibody B, CRP rabbit monoclonal antibody B, and CRP mouse monoclonal antibody B were diluted to 1 mg / mL with 0.01M PB to form T1, T2, and T3 coating solutions respectively; mouse secondary antibody, rabbit secondary antibody, and sheep secondary antibody were diluted to a final concentration of 2 mg / mL with 0.01M PB to form a quality control line coating solution; plant-derived antibodies were diluted to 2 mg / mL with 0.01M PB to form an embedding solution; the detection line coating solution and the quality control line coating solution were sprayed on the NC membrane at a speed of 1.2μL / cm, and then the embedding solution was sprayed at a speed of 1.2μL / cm at T2 and T3 1mm below the colloidal gold end. After the embedded strips were dried, they were sprayed with T2 and T3 coating solutions to cover their surfaces.

[0040] 2.4 Test strip preparation The first process (control): Take a PVC plate and paste the sprayed first nitrocellulose membrane, press the first colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the first colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0041] The second process: take a PVC plate and paste the sprayed second nitrocellulose membrane, press the second colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the second colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0042] The third process: take a PVC plate and paste the sprayed third nitrocellulose membrane, press the first colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the first colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0043] The fourth process: Take a PVC plate and paste the sprayed fourth nitrocellulose membrane, press the second colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the second colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0044] 3. Test results After the test paper was made according to the method established by the present invention, CRP reference substances of different concentrations were taken for determination, and the results were as follows:

[0045] The results showed that compared with the first process, the second process used CRP monoclonal antibodies of different species. T1 of the second process used sheep monoclonal antibodies, which had a much higher affinity than mouse monoclonal antibodies, a sensitivity of up to 0.5 mg / mL, and a more obvious overall gradient. Compared with the first process, when detecting high-concentration samples, the third process sprayed a coating solution on the embedded color bands on the lower side of T2 and T3, which consumed part of the test material and caused the upper limit of detection to become wider. The sensitivity of the fourth process was 0.5 mg / mL, and the upper limit of detection could reach 150 mg / mL. The performance of the test strip far exceeded that of the first process.

[0046] Example 2

[0047] The specific preparation method of the colloidal gold test strip using lipoprotein-associated phospholipase A2 (LP-PLA2) as an example.

[0048] 1. Materials 1.1 LP-PLA2 mouse monoclonal antibody, LP-PLA2 rabbit monoclonal antibody, LP-PLA2 goat monoclonal antibody, mouse secondary antibody, rabbit secondary antibody, goat secondary antibody were purchased from Shenzhen Feipeng Biological Co., Ltd.; plant-derived antibodies were purchased from Albertson Biotechnology Co., Ltd.; 1.2 Nitrocellulose membrane, purchased from Millipore; 1.3 Chloroauric acid, purchased from Sigma.

[0049] 2. Test strip composition The LP-PLA2 colloidal gold (semi-quantitative) detection test strip of the present invention comprises four parts from bottom to top: a buffer pad, a colloidal gold pad, a nitrocellulose membrane and a water-absorbing paper.

[0050] 2.1 Labeling of colloidal gold solution Take the purple-red colloidal gold solution, adjust the pH to the optimal pH corresponding to LP-PLA2 mouse monoclonal antibody A, LP-PLA2 rabbit monoclonal antibody A, and LP-PLA2 sheep monoclonal antibody A, respectively, and then add each antibody to make the final concentration of 0.01 mg / mL. After 1 hour, add casein with a mass concentration of 1% for blocking, centrifuge to remove the supernatant, add 10% (v / v) 5% sugar solution to re-dissolve, and store for later use.

[0051] 2.2 Preparation of colloidal gold pad The labeled colloidal gold solution was mixed and diluted with 0.1 mL PBS buffer containing 0.1% Tween and 1% sugar at a ratio of 1:10 (v / v), spread evenly on the glass fiber membrane, dried, and cut into 5 mm wide pieces for later use.

[0052] First colloidal gold pad: The selected colloidal gold solution contains only LP-PLA2 mouse monoclonal antibody A; Second colloidal gold pad: The selected colloidal gold solution contains LP-PLA2 mouse monoclonal antibody A, LP-PLA2 rabbit monoclonal antibody A and LP-PLA2 sheep monoclonal antibody A at the same time.

[0053] 2.3 Preparation of nitrocellulose membrane First nitrocellulose membrane: Take LP-PLA2 mouse monoclonal antibody B and dilute it to 1 mg / mL, 1.5 mg / mL and 3 mg / mL with 0.01M PB (phosphate buffer) to form T1, T2 and T3 test line coating solutions, respectively. Dilute the mouse secondary antibody to a final concentration of 2 mg / mL with 0.01M PB to form the quality control line coating solution. Spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm.

[0054] Second nitrocellulose membrane: dilute LP-PLA2 sheep monoclonal antibody B, LP-PLA2 rabbit monoclonal antibody B, and LP-PLA2 mouse monoclonal antibody B to 1 mg / mL with 0.01M PB to form T1, T2, and T3 coating solutions, respectively. Dilute the mouse secondary antibody, rabbit secondary antibody, and sheep secondary antibody with 0.01M PB to a final concentration of 2 mg / mL to form the quality control line coating solution. Spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm.

[0055] Third nitrocellulose membrane: Take LP-PLA2 mouse monoclonal antibody B and dilute it to 1 mg / mL, 1.5 mg / mL and 3 mg / mL with 0.01M PB to form T1, T2 and T3 test line coating solutions respectively. Take mouse secondary antibody and dilute it to a final concentration of 2 mg / mL with 0.01M PB to form the quality control line coating solution. Use 0.01M PB to dilute the plant-derived antibody to 2 mg / mL to form the embedding solution; spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm, and then spray the embedding solution at a speed of 1.2μL / cm at T2 and T3 1mm below the colloidal gold end. After the embedded strips are dried, spray them with T2 and T3 coating solutions to cover their surfaces.

[0056] Fourth nitrocellulose membrane: Take LP-PLA2 sheep monoclonal antibody B, LP-PLA2 rabbit monoclonal antibody B, and LP-PLA2 mouse monoclonal antibody B and dilute them to 1 mg / mL with 0.01M PB to form T1, T2, and T3 coating solutions respectively; dilute the mouse secondary antibody, rabbit secondary antibody, and sheep secondary antibody with a final concentration of 2 mg / mL with 0.01M PB to form the quality control line coating solution; dilute the plant-derived antibody to 2 mg / mL with 0.01M PB to form the embedding solution; spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm, and then spray the embedding solution at a speed of 1.2μL / cm near T2 and T3 1mm below the colloidal gold end. After the embedded strips are dried, spray them with T2 and T3 coating solutions to cover their surfaces.

[0057] 2.4 Test strip preparation The first process (control): Take a PVC plate and paste the sprayed first nitrocellulose membrane, press the first colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the first colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0058] The second process: take a PVC plate and paste the sprayed second nitrocellulose membrane, press the second colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the second colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0059] The third process: take a PVC plate and paste the sprayed third nitrocellulose membrane, press the first colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the first colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0060] The fourth process: Take a PVC plate and paste the sprayed fourth nitrocellulose membrane, press the second colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the second colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0061] 3. Test results After the test paper was prepared according to the method established in the present invention, different concentrations of LP-PLA2 reference products were taken for determination, and the results were as follows:

[0062] The results showed that compared with the first process, the sensitivity of the second process using antibody products from different species was changed from 10ng / mL to 1ng / mL, and the sensitivity was significantly improved. The third process added embedded strips at T2 and T3, and the upper limit of LP-PLA2 detection could reach 900 ng / mL, far exceeding the control group of 300 ng / mL. The fourth process used the second and third processes at the same time, and its sensitivity and detection range were significantly higher than the control group.

[0063] Example 3

[0064] Specific preparation method of colloidal gold test strips using human chorionic gonadotropin (HCG) as an example 1. Materials 1.1 HCG mouse monoclonal antibody, HCG rabbit monoclonal antibody, HCG goat monoclonal antibody, mouse secondary antibody, rabbit secondary antibody, goat secondary antibody were purchased from Shenzhen Feipeng Biological Co., Ltd.; plant-derived antibodies were purchased from Albertson Biotechnology Co., Ltd.; 1.2 Nitrocellulose membrane, purchased from Millipore; 1.3 Chloroauric acid, purchased from Sigma.

[0065] 2. Test strip composition The HCG colloidal gold (semi-quantitative) detection test strip of the present invention comprises four parts from bottom to top: a buffer pad, a colloidal gold pad, a nitrocellulose membrane and a water-absorbing paper.

[0066] 2.1 Labeling of colloidal gold solution Take the purple-red colloidal gold solution, adjust the pH to the optimal pH value corresponding to HCG mouse monoclonal antibody A, HCG rabbit monoclonal antibody A, and HCG sheep monoclonal antibody A, respectively, and then add each antibody to make the final concentration of 0.01 mg / mL. After 1 hour, add casein with a mass concentration of 1% for blocking, centrifuge to remove the supernatant, add 10% (v / v) 5% sugar solution to re-dissolve, and store for later use.

[0067] 2.2 Preparation of colloidal gold pad Take 1 mL of the labeled colloidal gold solution, add 10 mL of 0.1% Tween, 1% sugar in 0.1 mL of PBS buffer (phosphate buffered saline), mix well, spread evenly on the glass fiber membrane, dry, and cut into 5 mm wide for later use.

[0068] First colloidal gold pad: The selected colloidal gold solution contains HCG mouse monoclonal antibody A; Second colloidal gold pad: The selected colloidal gold solution contains HCG mouse monoclonal antibody A, HCG rabbit monoclonal antibody A and HCG sheep monoclonal antibody A at the same time.

[0069] 2.3 Preparation of nitrocellulose membrane First nitrocellulose membrane: Take HCG mouse monoclonal antibody B and dilute it to 1 mg / mL, 1.5 mg / mL and 3 mg / mL with 0.01M PB (phosphate buffer) to form T1, T2 and T3 test line coating solutions respectively. Take mouse secondary antibody and dilute it to a final concentration of 2 mg / mL with 0.01M PB to form the quality control line coating solution. Spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm.

[0070] Second nitrocellulose membrane: dilute HCG sheep monoclonal antibody B, HCG rabbit monoclonal antibody B, and HCG mouse monoclonal antibody B to 1 mg / mL with 0.01M PB to form T1, T2, and T3 coating solutions, respectively. Dilute the mouse secondary antibody, rabbit secondary antibody, and sheep secondary antibody with 0.01M PB to a final concentration of 2 mg / mL to form the quality control line coating solution. Spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm.

[0071] Third nitrocellulose membrane: Take HCG mouse monoclonal antibody B and dilute it to 1 mg / mL, 1.5 mg / mL and 3 mg / mL with 0.01M PB to form T1, T2 and T3 test line coating solutions respectively. Take mouse secondary antibody and dilute it to a final concentration of 2 mg / mL with 0.01M PB to form the quality control line coating solution. Use 0.01M PB to dilute the plant-derived antibody to 2 mg / mL to form the embedding solution; spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm, and then spray the embedding solution at a speed of 1.2μL / cm at T2 and T3 1mm below the colloidal gold end. After the embedded strips are dried, spray them with T2 and T3 coating solutions to cover their surfaces.

[0072] Fourth nitrocellulose membrane: Take HCG sheep monoclonal antibody B, HCG rabbit monoclonal antibody B, and HCG mouse monoclonal antibody B and dilute them to 1 mg / mL with 0.01M PB to form T1, T2, and T3 coating solutions respectively; dilute the mouse secondary antibody, rabbit secondary antibody, and sheep secondary antibody with a final concentration of 2 mg / mL using 0.01M PB to form the quality control line coating solution; dilute the plant-derived antibody to 2 mg / mL with 0.01M PB to form the embedding solution; spray the test line coating solution and the quality control line coating solution on the NC membrane at a speed of 1.2μL / cm, and then spray the embedding solution at a speed of 1.2μL / cm near T2 and T3 1mm below the colloidal gold end. After the embedded strips are dried, spray them with T2 and T3 coating solutions to cover their surfaces.

[0073] 2.4 Test strip preparation The first process (control): Take a PVC plate and paste the sprayed first nitrocellulose membrane, press the first colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the first colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0074] The second process: take a PVC plate and paste the sprayed second nitrocellulose membrane, press the second colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the second colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0075] The third process: take a PVC plate and paste the sprayed third nitrocellulose membrane, press the first colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the first colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0076] The fourth process: Take a PVC plate and paste the sprayed fourth nitrocellulose membrane, press the second colloidal gold pad (cut into 5mm) to the NC membrane 1mm, press the buffer pad to the second colloidal gold pad 1mm, and then use absorbent paper to press the upper end of the NC membrane 1mm to make a test strip.

[0077] 3. Test results After the test paper was made according to the method established by the present invention, HCG reference substances of different concentrations were taken for determination, and the results were as follows:

[0078] The results showed that the second process used antibodies of different species, which significantly improved the sensitivity and had a clear gradient between different concentrations. The third process added embedded strips, which significantly delayed the HCG hook phenomenon and expanded the detection range. The fourth process used antibodies of different species and added embedded strips at the same time, which significantly improved the sensitivity and detection range.

[0079] The above technical solutions only reflect the preferred technical solutions of the technical solutions of the present invention. Some changes that may be made to certain parts thereof by technicians in this technical field all reflect the principles of the present invention and fall within the protection scope of the present invention.

[0080] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0081] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and the like are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defined as "first", "second", and the like may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0082] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "install", "connect", "connect", and "set" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood by specific circumstances.

Claims

1. A colloidal gold immunochromatographic semi-quantitative test strip, comprising a base plate (1), a sample loading pad (2), a colloidal gold pad (3), a nitrocellulose membrane (4), and a blotting paper (5), wherein the sample loading pad (2), the colloidal gold pad (3), the nitrocellulose membrane (4), and the blotting paper (5) are sequentially pasted on the top of the base plate (1), the colloidal gold pad (3) and the blotting paper (5) are respectively pressed on the top of both ends of the nitrocellulose membrane (4) in the length direction, the sample loading pad (2) is pressed on the top of the end of the colloidal gold pad (3) away from the nitrocellulose membrane (4), and the sample to be tested flows through the sample loading pad (2), the colloidal gold pad (3), and the nitrocellulose membrane (4) in sequence, and the nitrocellulose membrane (4) is provided with a first detection line (41), a second detection line (42), a third detection line (43), and a quality control line (44) along the sample flow direction, characterized in that: The first detection line (41) is coated with a sheep monoclonal antibody with a strong affinity to the sample to be tested, the second detection line (42) is coated with a rabbit monoclonal antibody with a medium affinity to the sample to be tested, the third detection line (43) is coated with a mouse monoclonal antibody with a weak affinity to the sample to be tested, the colloidal gold pad (3) is coated with a monoclonal antibody with a high affinity to the sample to be tested, and the quality control line (44) is coated with a mixture of mouse, rabbit and sheep secondary antibodies.

2. A colloidal gold immunochromatographic semi-quantitative test strip according to claim 1, characterized in that: The colloidal gold pad (3) is coated with a mixture of sheep monoclonal antibodies, rabbit monoclonal antibodies, and mouse monoclonal antibodies, and the antibody mixture coated on the colloidal gold pad (3) and the antibodies coated on the first detection line (41), the second detection line (42), and the third detection line (43) have different binding sites with respect to the sample to be tested.

3. A colloidal gold immunochromatographic semi-quantitative test strip according to claim 1, characterized in that: The first detection line (41), the second detection line (42), the third detection line (43), and the quality control line (44) are distributed in sequence along the sample flow direction.

4. A colloidal gold immunochromatographic semi-quantitative test strip according to claim 1, characterized in that: The second detection line (42) and the third detection line (43) are both provided with an embedding strip (45) at one end close to the colloidal gold pad (3), and the embedding strip (45) is coated with a plant-derived antibody. The plant-derived antibody coated by the embedding strip (45) of the second detection line (42) is sprayed with the antibody coated by the second detection line (42), and the plant-derived antibody coated by the embedding strip (45) of the third detection line (43) is sprayed with the antibody coated by the third detection line (43).

5. A colloidal gold immunochromatographic semi-quantitative test strip according to claim 4, characterized in that: The embedding strip (45) has a width of 1-2 mm.

6. A colloidal gold immunochromatographic semi-quantitative test strip according to claim 1, characterized in that: The secondary antibody mixture coated on the quality control line (44) can be combined with the antibody coated on the colloidal gold pad (3).

7. A method for detecting a colloidal gold immunochromatographic semi-quantitative test strip, using the colloidal gold immunochromatographic semi-quantitative test strip according to any one of claims 1 to 6 for detection, comprising at least the following steps: 1) Obtain the concentration range of the first detection line, the second detection line, and the third detection line; 2) Add the sample that may contain the target analyte to the sample pad, and wait for the sample to flow through the colloidal gold pad, nitrocellulose membrane, and finally into the absorbent paper; 3) Observe the colors of the first test line, the second test line, the third test line, and the quality control line, and determine the concentration range of the sample according to step 1.

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