Colloidal gold test paper for double-window detection of fecal occult blood and application thereof

Abstract

To provide a detection test paper for detecting fecal occult blood, which can delay occurrence of a HOOK phenomenon and improve accuracy of colloidal gold detection.SOLUTION: A reagent strip includes a PVC substrate, a sample pad, a marking pad, a nitrocellulose membrane and a water absorption pad. The nitrocellulose membrane is arranged with a T1 detection line and a C quality control line on a reagent strip on a left side of the marking pad sequentially from a side closer to the marking pad, and arranged with a T2 detection line and the C quality control line on a reagent strip on a right side sequentially. The T1 detection line and T2 detection line are coated with a mouse anti-human hemoglobin monoclonal antibody, and the marking pad contains a mouse anti-human hemoglobin monoclonal antibody- colloidal gold conjugate. The right reagent strip also contains a mouse anti-human hemoglobin monoclonal antibody treated with a treatment solution. A double-window colloidal gold detection test paper for detecting fecal occult blood is set on the sample pad or on the nitrocellulose membrane between the T2 detection line and the marking pad.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the technical field of immunodetection, and in particular to a double-window colloidal gold detection test paper for detecting fecal occult blood and its use. [Background technology]

[0002] Gastrointestinal bleeding can be caused by multiple diseases and can be divided into dominant and recessive bleeding. The normal 24-hour gastrointestinal bleeding volume in a normal human is 0.6 mL. Gastrointestinal bleeding volume greater than 2 mL is considered pathological bleeding. Fecal occult blood testing, commonly used in clinical practice, is a common screening indicator for detecting gastrointestinal bleeding, chronic gastrointestinal bleeding, and early diagnosis of malignant tumors. Fecal occult blood testing is important for the early detection, diagnosis, and treatment of gastrointestinal lesions and gastrointestinal malignant tumors, as well as improving patients' survival rates and quality of life.

[0003] Colloidal gold testing for fecal occult blood has the advantages of being fast and easy to operate, and is relatively sensitive, being able to test for hemoglobin levels as low as 100ng / mL, and being able to accurately test for asymptomatic, small amounts of bleeding that is persistent and cannot be seen with the naked eye or under a microscope. It is not affected by drugs, iron supplements, animal blood, or dietary restrictions, and has high specificity. It has replaced chemical methods as the primary method for testing fecal occult blood.

[0004] The colloidal gold method primarily uses the double-antibody sandwich technique to test for fecal occult blood. Gold colloids are then specifically labeled with anti-human hemoglobin polyclonal and monoclonal antibodies, which then specifically bind to the hemoglobin present in the feces. The color development of the colloidal gold marking allows for the detection and measurement of hemoglobin in human feces. However, due to limitations in the testing method, the hook phenomenon is prone to occur, resulting in false-negative results from the colloidal gold method, which can lead to clinically inaccurate diagnoses and delays in optimal treatment timing. Furthermore, in cases where the clinical manifestation is asphalt-like black stool, bright red blood, or abnormal stools that appear clearly brown or brown, a negative test result requires repeated dilution of the sample. This increases clinical labor and time costs. Furthermore, because hemoglobin is easily degraded, repeated testing can produce inaccurate results if the sample is stored at too high a temperature or for too long.

[0005] Most colloidal gold test kits currently on the market for detecting fecal occult blood are single-window test kits that produce negative or weakly positive results when the hemoglobin content is >5 mg / mL. Therefore, there are certain limitations to using colloidal gold to detect fecal occult blood, which can have a negative impact on the accuracy and ease of operation of colloidal gold for detecting fecal occult blood. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention aims to provide a colloidal gold detection test paper with a double window for detecting occult blood in feces, and its use. This test paper adds a detection window to the conventional colloidal gold detection reagent strip, and adds a mouse anti-human hemoglobin monoclonal antibody treated with a processing solution to the detection reagent strip with the additional window, thereby exerting a neutralizing effect just before the T2 detection line, delaying the occurrence of the HOOK phenomenon, and improving the accuracy of colloidal gold detection.

[0007] The object of the present invention is achieved by the following technical methods. In a first aspect, the present invention provides a double-window gold colloid detection test paper for detecting fecal occult blood, including a left-hand reagent strip and a right-hand reagent strip. The reagent strips include a PVC substrate and a sample pad, a marking pad, a cellulose nitrate membrane, and an absorbent pad, which are connected in sequence on the PVC substrate. The cellulose nitrate membrane is provided with a T1 detection line and a C quality control line, in this order, on the left-hand reagent strip, and a T2 detection line and a C quality control line on the right-hand reagent strip, from the side closest to the marking pad. The T1 detection line and the T2 detection line are coated with mouse anti-human hemoglobin monoclonal antibody, and the marking pad contains a mouse anti-human hemoglobin monoclonal antibody-gold colloid conjugate. The right-hand reagent strip also contains mouse anti-human hemoglobin monoclonal antibody treated with a treatment solution, which is placed on the sample pad or on the cellulose nitrate membrane between the T2 detection line and the marking pad for neutralization just before the T2 detection line.

[0008] This invention is based on the double anti-sandwich method of colloidal gold immunochromatography. When hemoglobin is present in a sample, it binds to the mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugate in the marking pad, forming an antigen-antibody complex. As the chromatography proceeds, this complex is captured by the mouse anti-human hemoglobin monoclonal antibody (for coating) coated on the cellulose nitrate membrane, forming a mouse anti-human hemoglobin monoclonal antibody (for coating)-hemoglobin-mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugate, which is reddish-purple in color with clear color streaks visible to the naked eye. No additional auxiliary equipment is required, making the operation simple and convenient.

[0009] Specifically, after a high concentration of test sample is added to the left reagent strip, when the test sample flows onto the marking pad, hemoglobin binds to the mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate to form a complex, which can specifically bind to the mouse anti-human hemoglobin monoclonal antibody (for coating) on the T1 detection line. However, because the sample contains excess hemoglobin, it competes with the complex hemoglobin-mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate to bind to the antibody on the T1 detection line, preventing the complex from binding to the antibody on the T1 detection line, resulting in a weak positive or negative result on the T1 detection line.

[0010] After a high concentration of test sample is added to the left reagent strip, the test sample flows through the marking pad, where hemoglobin binds to the mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugate to form a complex hemoglobin-mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugate, which continues to move forward by chromatography. Excess hemoglobin binds to the closed line coated with mouse anti-human hemoglobin monoclonal antibody (for coating) treated with a treatment solution, and as it continues to move, the hemoglobin-mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugate specifically binds to the mouse anti-human hemoglobin monoclonal antibody (for coating) on the T2 detection line, forming a reddish-purple strip. Also, after a high concentration of test sample is added to the reagent strip on the right, as the test sample flows through the sample pad, hemoglobin first binds to the mouse anti-human hemoglobin monoclonal antibody (for coating) treated with the treatment solution and continues to move forward by chromatography. As it flows through the marking pad, the remaining hemoglobin binds to the mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate to form a complex hemoglobin-mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate, which continues to move and specifically binds to the mouse anti-human hemoglobin monoclonal antibody (for coating) on the T2 detection line, showing a reddish-purple strip.

[0011] The present invention can determine whether a sample contains hemoglobin based on the color development of the detection line and quality control line, and can also determine the concentration range of hemoglobin in a fecal sample based on the color development of the two detection lines. The T1 detection line coated on the left reaction area is highly sensitive and can detect hemoglobin at a minimum of 100 ng / mL, while the T2 detection line coated on the right reaction area can detect hemoglobin at a minimum of 500 ng / mL. The double-window detection delays the occurrence of the HOOK phenomenon and improves the accuracy of colloidal gold detection.

[0012] Preferably, the coating concentration of the mouse anti-human hemoglobin monoclonal antibody coated on the T1 detection line is 1.0 to 2.0 mg / mL. The coating concentration of the mouse anti-human hemoglobin monoclonal antibody coated on the T2 detection line is 1.0 to 2.0 mg / mL.

[0013] Preferably, the mouse anti-human hemoglobin monoclonal antibody treated with the treatment solution has an antibody concentration of 0.5 to 1.5 mg / mL on the cellulose nitrate membrane between the T2 detection line and the marking pad, and an antibody concentration of 0.05 to 0.1 mg / mL on the sample pad.

[0014] Specifically, on the cellulose nitrate membrane between the T2 detection line and the marking pad, the antibody concentration in the treatment solution of the mouse anti-human hemoglobin monoclonal antibody treated with the treatment solution is 0.5 to 1.5 mg / mL.On the sample pad, the antibody concentration in the treatment solution-containing sample pad solution of the mouse anti-human hemoglobin monoclonal antibody treated with the treatment solution is 0.05 to 0.1 mg / mL.

[0015] Preferably, the treatment liquid is an aqueous solution obtained by mixing sucrose, sodium chloride, disodium hydrogen phosphate, and a chelating agent, and the pH is adjusted to 7.2 to 7.6.

[0016] Preferably, the treatment solution contains 8 to 15 mg / mL of sucrose, 5 to 10 g / L of sodium chloride, 0.5 to 3 g / L of disodium hydrogen phosphate, and 5 to 10 g / L of a chelating agent, which is an aminopolycarboxylic acid or a sodium salt thereof.

[0017] Preferably, the chelating agent is one of ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetic acid, hydroxyethylethylenediaminetriacetic acid (HEDTA), hydroxyethylethylenediaminetriacetic acid sodium salt, aminotriacetic acid (NTA), and aminotriacetic acid sodium salt.

[0018] The addition of a chelating agent can improve the detection rate of high-concentration hemoglobin, and the chelating agent, in combination with other components in the treatment solution, can improve protein adsorption and the immune reaction rate by adjusting the ionic environment of the immune reaction system. Furthermore, adjusting the concentrations of each component and the pH of the treatment solution can improve the synergistic effects between the components. In particular, the concentration of the chelating agent has a significant impact on the treatment effect of the treatment solution, and if the concentration exceeds the limited range of the present invention, the hemoglobin detection rate will decrease.

[0019] Preferably, the C quality control line is coated with sheep anti-mouse IgG, and the coating concentration is 0.5 to 2.0 mg / mL.

[0020] Preferably, the marking pad contains a mouse anti-human hemoglobin monoclonal antibody-gold colloid conjugate and a mouse IgG-gold colloid conjugate. The concentration of the mouse anti-human hemoglobin monoclonal antibody-gold colloid conjugate is OD20-OD60. The mouse anti-human hemoglobin monoclonal antibody (for coating) coated on the T1 detection line and the T2 detection line and the mouse anti-human hemoglobin monoclonal antibody (for coating) treated with a treatment solution are antibodies against the same antigen epitope. Furthermore, these antibodies and the mouse anti-human hemoglobin monoclonal antibody (for marking) contained in the marking pad are antibodies against different antigen epitopes.

[0021] The mouse anti-human hemoglobin monoclonal antibody treated with the treatment solution captures excess hemoglobin before the detection line, preventing excess hemoglobin from competing with the antibody on the detection line to bind and resulting in weak positive or false negative results, increasing the upper limit of the detection range, and delaying the HOOK phenomenon.

[0022] Preferably, a cellulose nitrate film is provided in the middle of the PVC substrate. A marking pad and a water-absorbing pad are provided at both ends of the cellulose nitrate film, and one end of each of the marking pad and the water-absorbing pad is connected to the bottom side, while the other end of each of the marking pad and the water-absorbing pad is connected to the bottom side of the PVC substrate. A sample pad is provided above the connection end between the marking pad and the PVC substrate. One end of the bottom side of the sample pad is connected to the top side of the marking pad, and the other end of the bottom side is connected to the PVC substrate.

[0023] In a second aspect, the present invention further provides the use of colloidal gold detection test paper in hemoglobin detection, in which a sample to be measured is added to the sample pads of the left and right reagent strips, and the hemoglobin concentration range is determined based on the color development of the T1 detection line and the T2 detection line.

[0024] The colloidal gold detection test strip of the present invention can detect whether a sample contains hemoglobin. When both the detection line T and the quality control line C show a red band, it indicates that the sample contains hemoglobin. When only the quality control line C shows a red band, it indicates that the sample does not contain hemoglobin or that the hemoglobin content in the sample is lower than the detection value. When the quality control line C does not show a red band, it indicates that the test strip is invalid. The T1 detection line coated on the left reaction area is highly sensitive and can detect hemoglobin at a minimum of 100 ng / mL, and the T2 detection line coated on the right reaction area can detect hemoglobin at a minimum of 500 ng / mL.

[0025] The colloidal gold detection test paper of the present invention can detect a range of hemoglobin concentrations in a sample. The sample is applied to the sample pads on the left and right reagent strips, and the hemoglobin concentration range can be determined based on the color development of the two detection lines.

[0026] Preferably, when the T1 detection line does not develop color and the T2 detection line does not develop color, it indicates that the hemoglobin concentration is <100 ng / mL. When the T1 detection line develops color and the T2 detection line does not develop color, it indicates that the hemoglobin concentration is 100 ng / mL or less and <500 ng / mL. When the T1 detection line develops color and the T2 detection line develops color, it indicates that the hemoglobin concentration is 500 ng / mL or less and <5 mg / mL. When the T1 detection line does not develop color and the T2 detection line develops color, it indicates that the hemoglobin concentration is ≥5 mg / mL.

[0027] More preferably, if the T1 detection line does not develop color and the T2 detection line does not develop color, it indicates a hemoglobin concentration < 100 ng / mL. If the T1 detection line develops color and the T2 detection line does not develop color, it indicates a hemoglobin concentration of 100 ng / mL or less < 500 ng / mL. If the T1 detection line develops strongly (stronger than the T2 detection line) and the T2 detection line develops weakly (weaker than the T1 detection line), it indicates a hemoglobin concentration of 500 ng / mL or less < 10,000 ng / mL. If the T1 detection line develops strongly and the T2 detection line develops strongly, it indicates a hemoglobin concentration of 10,000 ng / mL. If the T1 detection line develops weakly (weaker than the T2 detection line) and the T2 detection line develops strongly (stronger than the T1 detection line), it indicates a hemoglobin concentration of 10,000 ng / mL < 5 mg / mL. If the T1 detection line is not colored and the T2 detection line is colored, it indicates a hemoglobin concentration range of ≧5 mg / mL.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention adds a detection window to the existing colloidal gold detection reagent strip. The test reagent strip in the newly added window contains a mouse anti-human hemoglobin monoclonal antibody treated with a processing solution. When a sample contains a high concentration of hemoglobin, the mouse anti-human hemoglobin monoclonal antibody treated with the processing solution can neutralize the excess hemoglobin. The colloidal gold hemoglobin antigen-antibody complex then specifically binds to the hemoglobin antibody on the test line, indicating a positive result. This double-window test can delay the appearance of the HOOK phenomenon and improve the accuracy of colloidal gold tests. (2) This invention scientifically and objectively realizes rapid testing from qualitative to semi-quantitative, guarantees high sensitivity, and at the same time, increases the upper limit of the linear test range, improves the accuracy of the test reagent, and only requires one sample treatment, eliminating the need for repeated dilution, saving time and labor costs. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 10 is a diagram showing the detection results of the test paper of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] Hereinafter, technical aspects of the present invention will be described using specific examples, but the scope of protection of the present invention is not limited thereto. (1) Preparation of gold colloid detection test paper (on the right-hand reagent strip, a mouse anti-human hemoglobin monoclonal antibody treated with a treatment solution is placed on the cellulose nitrate membrane between the T2 detection line and the marking pad to form a closed line).

[0031] 1.1 Coating 1.1.1 Preparation of closed line treatment solution: Mix sucrose, sodium chloride, disodium hydrogen phosphate, and a chelating agent, then dilute the mixture to adjust the sucrose content in the treatment solution to 8-15 mg / mL, the sodium chloride content to 5-10 g / L, the disodium hydrogen phosphate content to 0.5-3 g / L, and the chelating agent content to 5-10 g / L, and adjust the pH to 7.2-7.6. 1.1.2. Preparation of solutions for C / T1 / T2 line coating: Dilute sheep anti-mouse IgG and mouse anti-human hemoglobin monoclonal antibodies (for coating) with 10 mM PBS at pH 7.2-7.6 to achieve coating concentrations of 0.5-2.0 mg / mL, 1.0-2.0 mg / mL, and 1.0-2.0 mg / mL, respectively. 1.1.3. Preparation of solution for closed line coating: Dilute mouse anti-human hemoglobin monoclonal antibody (for coating) with closed line treatment solution to a coating concentration of 0.5 to 1.5 mg / mL. 1.1.4. Coating the left-side reagent strip: Spray the prepared C / T1 coating solutions onto the cellulose nitrate membrane at a rate of 1.0-1.2 μL / cm to form the C quality control line and the T1 detection line, respectively. The C quality control line should be coated 34-35 mm from the bottom edge of one end of the PVC substrate, and the T1 detection line should be coated 28-29 mm from the bottom edge of the same end of the PVC substrate. Then, dry in a dryer at 37°C ± 2°C for 12-24 hours to obtain the coated cellulose nitrate membrane. 1.1.5. Coating the right-side reagent strip: Spray the prepared C / T2 / closure line coating solutions onto the cellulose nitrate membrane at a rate of 1.0-1.2 μL / cm to create the C quality control line, T2 detection line, and closure line, respectively. The C quality control line should be coated 34-35 mm from the bottom edge of one end of the PVC substrate. The T2 detection line should be coated 28-29 mm from the bottom edge of the same end of the PVC substrate. The closure line should be coated 22-23 mm from the bottom edge of the same end of the PVC substrate. Then, dry the strips in an electrically heated air dryer at 37°C ± 2°C for 12-24 hours to obtain the coated cellulose nitrate membrane.

[0032] 1.2 Marking 1.2.1. Preparation of colloidal gold: Colloidal gold particles were mixed with mouse anti-human hemoglobin monoclonal antibody (for marking) and mouse IgG, respectively, to produce mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugates and mouse IgG-colloidal gold conjugates. 1.2.2. Preparation of marking solution: Dilute the mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate and mouse IgG-gold colloid conjugate with a diluent containing 50-55 g / L sucrose, 8-15 g / L BSA, and 6-9 g / L disodium hydrogen phosphate to obtain marking solutions with OD20-OD60 and OD10-OD60. 1.2.3. Apply the prepared marking solution to the polyester fiber film at a rate of 1.8-2.0 μL / cm, and dry it in an electric heated air dryer at 37°C ± 2°C for 12-24 hours to obtain a marking pad.

[0033] 1.3 Sample Pad 1.3.1. Preparation of sample pad solution: Mix an aqueous solution of 3-6 g / L casein, 4-7 g / L trishydroxymethylaminomethane, 0.1-0.4 mL / L Proclin 300, 3-8 g / L PVP-10, and 0.2-0.7 mL / L Tween 20, with a pH of 7.2-7.6. 1.3.2. Preparation of sample pad: The sample pad solution is spread on a glass fiber and placed in an electric heated air dryer at 37°C ± 2°C for 12 to 24 hours to obtain a sample pad.

[0034] 1.4 Assembly The above-mentioned reagent strip is assembled by placing a cellulose nitrate membrane in the middle of a PVC substrate. A marking pad and an absorbent pad are placed on both ends of the cellulose nitrate membrane, respectively, and are connected to the bottom of one end of the marking pad and the absorbent pad, respectively. The bottom of the other ends of the marking pad and the absorbent pad are each connected to the PVC substrate. A sample pad is placed above the connected end between the marking pad and the PVC substrate. One bottom end of the sample pad is connected to the top of the marking pad, and the other bottom end is connected to the PVC substrate. The left and right reagent strips are cut to the desired width and assembled in parallel to obtain a double-window gold colloid detection test paper.

[0035] (2) Preparation of colloidal gold detection test paper (on the right reagent strip, mouse anti-human hemoglobin monoclonal antibody treated with treatment solution is placed on the sample pad)

[0036] 1.1 Coating 1.1.1 Preparation of coating solution for C / T1 / T2 wire: Same as 1.1.2 in (1). 1.1.2. Coating of left reagent strip: Same as 1.1.4 in (1). 1.1.3. Coating the right-side reagent strip: Spray the prepared C / T2 coating solution onto the cellulose nitrate membrane at a rate of 1.0-1.2 μL / cm to form the C quality control line and the T2 detection line, respectively. The C quality control line should be coated 34-35 mm from the bottom edge of one end of the PVC substrate. The T2 detection line should be coated 28-29 mm from the bottom edge of the same end of the PVC substrate. Then, dry in an electric-heated fan dryer at 37°C ± 2°C for 12-24 hours to obtain the coated cellulose nitrate membrane.

[0037] 1.2 Marking 1.2.1 Preparation of gold colloid: Same as 1.2.1 in (1). 1.2.2. Preparation of marking solution: Same as 1.2.2 in (1). 1.2.3. Obtain the marking pad: Same as 1.2.3 in (1).

[0038] 1.3 Sample Pad 1.3.1. Preparation of sample pad solution: Prepare an aqueous solution containing 3-6 g / L casein, 4-7 g / L trishydroxymethylaminomethane, 0.1-0.4 mL / L Proclin 300, 3-8 g / L PVP-10, 0.2-0.7 mL / L Tween 20, 8-15 mg / mL sucrose, 5-10 g / L sodium chloride, 0.5-3 g / L disodium hydrogen phosphate, 5-10 g / L chelating agent, and 0.05-0.1 mg / mL mouse anti-human hemoglobin monoclonal antibody (for coating), with a pH of 7.2-7.6. 1.3.2. Preparation of sample pad: Same as 1.3.2 in (1). 1.4 Assembly (1) Same as 1.4.

[0039] Example 1 Preparation of colloidal gold detection test paper (right reagent strip contains a closure line) 1.1 Coating 1.1.1 Preparation of closed line treatment solution: First, measure out 80 mL of purified water, then add 1 g of sucrose, 0.8 g of sodium chloride, 0.2 g of disodium hydrogen phosphate, and 1 g of ethylenediaminetetraacetic acid in that order, and stir until completely dissolved. Then, adjust the pH to 7.4 and add purified water to bring the total volume to 100 mL. 1.1.2. Preparation of coating solution for C / T1 / T2 wires: Dilute sheep anti-mouse IgG and mouse anti-human hemoglobin monoclonal antibodies (for coating) in 10 mM PBS at pH 7.4 to 1.0 mg / mL, 1.5 mg / mL, and 1.5 mg / mL, respectively. 1.1.3. Preparation of coating solution for the occluding line: Dilute mouse anti-human hemoglobin monoclonal antibody (for coating) with the occluding line treatment solution to a concentration of 0.5 mg / mL. 1.1.4. Coating the left-side reagent strip: Spray the prepared C / T1 coating solutions onto the cellulose nitrate membrane at a rate of 1.0 μL / cm to coat the C quality control line and the T1 detection line, respectively. The C quality control line should be coated 34.5 mm from the bottom edge of one end of the PVC substrate, and the T1 detection line should be coated 28.5 mm from the bottom edge of the same end of the PVC substrate. Then, dry the membrane in a dryer at 37°C ± 2°C for 16 hours to obtain the coated cellulose nitrate membrane. 1.1.5. Coating the right-side reagent strip: Spray the prepared C / T2 / closure line coating solution onto the cellulose nitrate membrane at a rate of 1.0 μL / cm to create the C quality control line, T2 detection line, and closure line, respectively. The C quality control line should be coated 34.5 mm from the bottom edge of one end of the PVC substrate. The T2 detection line should be coated 28.5 mm from the bottom edge of the same end of the PVC substrate. The closure line should be coated 22.5 mm from the bottom edge of the same end of the PVC substrate. Then, dry the strip in an electrically heated air dryer at 37°C ± 2°C for 16 hours to obtain the coated cellulose nitrate membrane.

[0040] 1.2 Marking 1.2.1. Preparation of colloidal gold: Colloidal gold particles were mixed with mouse anti-human hemoglobin monoclonal antibody (for marking) and mouse IgG in proportions to produce mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugates and mouse IgG-colloidal gold conjugates. The absorbances measured using a UV-visible spectrophotometer were OD129.5 and OD130.1, respectively. 1.2.2. Preparation of marking solution: Mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate and mouse IgG-gold colloid conjugate are diluted with a diluent containing 50 g / L sucrose, 10 g / L BSA, and 7 g / L disodium hydrogen phosphate to obtain marking solutions with OD20 and OD10. 1.2.3. Apply the prepared marking solution to a polyester cellulose film at a rate of 2.0 μL / cm, and dry it in an electric-heated air dryer at 37°C ± 2°C for 16 hours to obtain a marking pad.

[0041] 1.3 Sample Pad 1.3.1. Preparation of sample pad solution: First, measure out 80 mL of purified water, then add 0.4 g of casein, 0.48 g of tris(hydroxymethyl)aminomethane, 0.016 mL of Proclin 300, 0.5 g of PVP-10, and 0.05 mL of Tween 20 in that order. After dissolving completely, adjust the pH to 7.4, and finally add purified water to make the total volume 100 mL. 1.3.2. Preparation of sample pad: Then, 3.5 mL of the sample pad solution is weighed out and spread on a 17 mm * 31 mm glass fiber, and dried in an electric heated air dryer at 37 ° C ± 2 ° C for 16 hours to obtain the sample pad.

[0042] 1.4 Assembly The absorbent pad, sample pad, marking pad, coated cellulose nitrate membrane, and PVC substrate are connected in order to assemble the test paper, which is then cut to the required width as needed to obtain a double-window gold colloid detection test paper.

[0043] Example 2 Preparation of colloidal gold detection test paper (including a closure line on the right-hand reagent strip) Except for step 1.1.3, the rest is the same as Example 1. 1.1.3. Preparation of occlusion line coating solution: The preparation concentration is 1 mg / mL.

[0044] Example 3 Preparation of colloidal gold detection test paper (including a closure line on the right-hand reagent strip) Except for step 1.1.3, the rest is the same as Example 1. 1.1.3. Preparation of occlusion line coating solution: The concentration should be 1.5 mg / mL.

[0045] Example 4 Preparation of Colloidal Gold Detection Test Paper (Containing a Closure Line on the Right Reagent Strip) The procedure is the same as in Example 1 except that step 1.1.1 is different (the chelating agent is different). 1.1.1 Preparation of closed line treatment solution: First, measure out 80 mL of purified water, then add 1 g of sucrose, 0.8 g of sodium chloride, 0.2 g of disodium hydrogen phosphate, and 1 g of hydroxyethylethylenediaminetriacetic acid in that order, and stir until completely dissolved. Then, adjust the pH to 7.4 and add purified water to bring the total volume to 100 mL.

[0046] Example 5 Preparation of colloidal gold detection test paper (including a closure line on the right-hand reagent strip) The procedure is the same as in Example 1 except that step 1.1.1 is different (different chelating agent). 1.1.1 Preparation of closed line treatment solution: First, measure out 80 mL of purified water, then add 1 g of sucrose, 0.8 g of sodium chloride, 0.2 g of disodium hydrogen phosphate, and 1 g of aminotriacetic acid in that order, and stir until completely dissolved. Then, adjust the pH to 7.4 and add purified water to bring the total volume to 100 mL.

[0047] Example 6 Preparation of colloidal gold detection test paper (including a closure line on the right-hand reagent strip) Except for step 1.1.1 being different (the amount of chelating agent added is different), the rest is the same as in Example 1. 1.1.1 Preparation of closed line treatment solution: First, measure out 80 mL of purified water, then add 1 g of sucrose, 0.8 g of sodium chloride, 0.2 g of disodium hydrogen phosphate, and 0.8 g of ethylenediaminetetraacetic acid in that order, and stir until completely dissolved. Then, adjust the pH to 7.4 and add purified water to bring the total volume to 100 mL.

[0048] Example 7: Preparation of a gold colloid detection test paper (i.e., the right-side reagent strip does not contain a closure line at this point) by placing a mouse anti-human hemoglobin monoclonal antibody treated with a treatment solution on the sample pad on the right-side reagent strip. 1.1 Coating 1.1.1 Preparation of C / T1 / T2 wire coating solution: Dilute sheep anti-mouse IgG and mouse anti-human hemoglobin monoclonal antibodies (for coating) in 10 mM PBS at pH 7.4 to 1.0 mg / mL, 1.5 mg / mL, and 1.5 mg / mL, respectively. 1.1.2. Coating the left-side reagent strip: Spray the prepared C / T1 coating solutions onto the cellulose nitrate membrane at a rate of 1.0 μL / cm to form the C quality control line and the T1 detection line. The C quality control line should be coated 34.5 mm from the bottom edge of one end of the PVC substrate, and the T1 detection line should be coated 28.5 mm from the bottom edge of the same end of the PVC substrate. Then, dry the membrane in a dryer at 37°C ± 2°C for 12-24 hours to obtain the coated cellulose nitrate membrane. 1.1.3. Coating the right-side reagent strip: Spray the prepared C / T2 coating solution onto the cellulose nitrate membrane at a rate of 1.0 μL / cm to create the C quality control line and the T2 detection line. The C quality control line should be coated 34.5 mm from the bottom edge of one end of the PVC substrate, and the T2 detection line should be coated 28.5 mm from the bottom edge of the same end of the PVC substrate. Then, dry the membrane in a dryer at 37°C ± 2°C for 16 hours to obtain the coated cellulose nitrate membrane.

[0049] 1.2 Marking 1.2.1. Preparation of colloidal gold: Colloidal gold particles were mixed with mouse anti-human hemoglobin monoclonal antibody (for marking) and mouse IgG in proportions to produce mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugates and mouse IgG-colloidal gold conjugates. The absorbances measured using a UV-visible spectrophotometer were OD129.5 and OD130.1, respectively. 1.2.2. Preparation of marking solution: Mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate and mouse IgG-gold colloid conjugate are diluted with a diluent containing 50 g / L sucrose, 10 g / L BSA, and 7 g / L disodium hydrogen phosphate to obtain marking solutions with OD20 and OD10. 1.2.3. Apply the prepared marking solution to a polyester cellulose film at a rate of 2.0 μL / cm, and dry it in an electric-heated air dryer at 37°C ± 2°C for 16 hours to obtain a marking pad.

[0050] 1.3 Sample Pad 1.3.1. Preparation of sample pad solution: First, measure out 80 mL of purified water, then add 0.4 g of casein, 0.48 g of tris(hydroxymethyl)aminomethane, 0.016 mL of Proclin 300, 0.5 g of PVP-10, 0.05 mL of Tween 20, 1 g of sucrose, 0.8 g of sodium chloride, 0.2 g of disodium hydrogen phosphate, 1 g of ethylenediaminetetraacetic acid, and 1.02 mL of mouse anti-human hemoglobin antibody (for coating) (original concentration 4.9 mg / mL, concentration content in sample pad solution 0.05 mg / mL). After complete dissolution, adjust the pH to 7.4, and add purified water to make the total volume 100 mL. 1.3.2. Preparation of sample pad: Then, 3.5 mL of the solution is weighed and spread on a 17 mm x 31 mm glass fiber, and dried in an electric heated air dryer at 37°C ± 2°C for 16 hours to obtain a sample pad.

[0051] 1.4 Assembly The absorbent pad, sample pad, marking pad, coated cellulose nitrate membrane, and PVC substrate are connected in order to assemble the test paper, which is then cut to the required width as needed to obtain a double-window gold colloid detection test paper.

[0052] Comparative Example 1 Preparation of Colloidal Gold Detection Test Paper (Containing a Closure Line on the Right Reagent Strip) Except for step 1.1.3, the rest is the same as Example 1. 1.1.3. Preparation of occlusion line coating solution: The concentration of the solution should be 0.1 mg / mL.

[0053] Comparative Example 2 Preparation of colloidal gold detection test paper (including a closure line on the right-hand reagent strip) Except for step 1.1.3, the rest is the same as Example 1. 1.1.3. Preparation of occlusion line coating solution: The concentration should be 2.0 mg / mL.

[0054] Comparative Example 3 Preparation of colloidal gold detection test paper (including a closure line on the right-hand reagent strip) The procedure was the same as in Example 1 except that step 1.1.1 was different (no chelating agent was added). 1.1.1 Preparation of closed line treatment solution: First, measure out 80 mL of purified water, then add 1 g of sucrose, 0.8 g of sodium chloride, and 0.2 g of disodium hydrogen phosphate in that order, and stir until completely dissolved. Then, adjust the pH to 7.4 and add purified water to bring the total volume to 100 mL.

[0055] Comparative Example 4 Preparation of Colloidal Gold Detection Test Paper (Right Reagent Strip Includes Closure Line) The procedure was the same as in Example 1 except that step 1.1.1 was different (addition of too much chelating agent). 1.1.1 Preparation of closed line treatment solution: First, measure out 80 mL of purified water, then add 1 g of sucrose, 0.8 g of sodium chloride, 0.2 g of disodium hydrogen phosphate, and 1.2 g of ethylenediaminetetraacetic acid in that order, and stir until completely dissolved. Then, adjust the pH to 7.4 and add purified water to bring the total volume to 100 mL.

[0056] Comparative Example 5 Preparation of Colloidal Gold Detection Test Paper (Right Reagent Strip Includes Closure Line) The rest is the same as Example 1, except that Step 1.1.1 and Step 1.1.3 are different. Remove step 1.1.1. Preparation of closed line treatment solution in step 1.1.3: Dilute mouse anti-human hemoglobin monoclonal antibody (for coating) with 10 mM PBS buffer at pH 7.4 (i.e., the same as the solution for C / T1 / T2 line coating preparation) to 0.5 mg / mL.

[0057] Performance tests were conducted on the Examples and Comparative Examples using the negative control, 50 ng / mL hemoglobin, 100 ng / mL hemoglobin, 200 ng / mL hemoglobin, 500 ng / mL hemoglobin, 1 μg / mL hemoglobin, 10 μg / mL hemoglobin, 100 μg / mL hemoglobin, 5 mg / mL hemoglobin, 10 mg / mL hemoglobin, and 50 mg / mL hemoglobin. Each concentration setting was repeated 10 times, and the results are shown in Tables 1 and 2. The intensity of the color development results is shown in Figure 1. [Table 1] JPEG2025117497000001.jpg168170*Note: "+" indicates a positive test result. "-" indicates a negative test result. 10(+) means all 10 test results are positive. 10(-) means all 10 test results are negative. [Table 2] JPEG2025117497000002.jpg170170*Note: "+" indicates a positive test result. "-" indicates a negative test result. 10(+) means all 10 test results are positive. 10(-) means all 10 test results are negative.

[0058] Results Analysis: As shown in Tables 1 and 2, Comparative Examples 1 and 2 show that in the immunochromatographic reaction, inappropriate components or dosage of the closure line can affect the reaction environment between the antigen and antibody, affect the binding of the antigen and antibody, and ultimately affect the accuracy of the test results. Within the limited concentration range of the present invention, the mouse anti-human hemoglobin monoclonal antibody treated with the treatment solution contained in the right-hand reagent strip can delay the occurrence of the HOOK phenomenon and improve the accuracy of the gold colloid test, especially for high-concentration samples, to obtain accurate positive results without the need for repeated dilution and re-test. At the same time, the double-window results can be combined to determine the hemoglobin concentration range.

[0059] Furthermore, the results of Example 1 and Comparative Example 3 indicate that adding a chelating agent to the closed line treatment solution can improve the detection rate of high-concentration hemoglobin, adjusting the ionic environment in the immune reaction system and improving protein adsorption and immune reaction rates. However, Comparative Example 4 demonstrates that excessively high concentrations of chelating agent affect the binding between hemoglobin and mouse anti-human monoclonal antibodies, making it impossible to neutralize excess hemoglobin and delay the HOOK effect. Furthermore, Comparative Example 5 demonstrates that if the closed line treatment solution is made of standard PBS components, the test results for samples with high hemoglobin concentrations are negative.

[0060] Clinical sample validation In this study, a total of 26 fecal occult blood-positive samples were collected and the measurements were confirmed using a third-party test reagent (Shanghai Tongjing Diagnostic Technology Co., Ltd. - Hemoglobin / Transferrin Test Kit (Flow Cytometry Fluorescence Method)). The test results are shown in Table 3. Among them, 11 positive samples had hemoglobin concentrations of 100-500 ng / mL, 9 positive samples had hemoglobin concentrations of 500 ng / mL-5 mg / mL, and 6 positive samples had high hemoglobin concentrations of >5 mg / mL. [Table 3] JPEG2025117497000003.jpg199170*Note: "+" indicates a positive test result. "-" indicates a negative test result.

[0061] As shown in Table 3, the measurement results of clinical samples showed that Example 1 had high sensitivity and accuracy, and that for samples with particularly high hemoglobin concentrations, the concentration range of hemoglobin in the sample could be simultaneously determined from the color reaction results of the two T lines. In Comparative Examples 3 and 4, adding no chelating agent to the treatment solution or adding too much chelating agent affected the detection accuracy of the colloidal gold test paper.

[0062] The above are merely preferred embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structure or equivalent flow conversion made by using the specification of the present invention, or any direct or indirect use in other related technical fields, also falls within the patent protection scope of the present invention.

Claims

1. A colloidal gold detection test paper for detecting fecal occult blood with a double window, including a left reagent strip and a right reagent strip, The reagent strip includes a PVC substrate and a sample pad, a marking pad, a cellulose nitrate membrane, and an absorbent pad, which are connected in series on the PVC substrate; The cellulose nitrate membrane has a T1 detection line and a C quality control line provided in this order on the left reagent strip from the side closest to the marking pad, and a T2 detection line and a C quality control line provided in this order on the right reagent strip; The T1 and T2 detection lines are coated with mouse anti-human hemoglobin monoclonal antibody, and the marking pad contains mouse anti-human hemoglobin monoclonal antibody-gold colloid conjugate. A gold colloid detection test paper with a double window for detecting fecal occult blood, characterized in that the reagent strip on the right also contains a mouse anti-human hemoglobin monoclonal antibody treated with a processing solution, and is set on the sample pad or on the cellulose nitrate membrane between the T2 detection line and the marking pad.

2. 2. The double-window gold colloid detection test paper for detecting fecal occult blood according to claim 1, characterized in that the coating concentration of the mouse anti-human hemoglobin monoclonal antibody coated on the T1 detection line is 1.0 to 2.0 mg / mL, and the coating concentration of the mouse anti-human hemoglobin monoclonal antibody coated on the T2 detection line is 1.0 to 2.0 mg / mL.

3. The colloidal gold detection test paper for detecting fecal occult blood with a double window according to claim 1, characterized in that the mouse anti-human hemoglobin monoclonal antibody treated with the treatment solution has an antibody concentration of 0.5 to 1.5 mg / mL on the cellulose nitrate membrane between the T2 detection line and the marking pad, and an antibody concentration of 0.05 to 0.1 mg / mL on the sample pad.

4. 2. The double-window colloidal gold detection test paper for detecting fecal occult blood according to claim 1, wherein the treatment liquid is an aqueous solution obtained by mixing sucrose, sodium chloride, disodium hydrogen phosphate, and a chelating agent.

5. In the treatment solution, the sucrose content is 8 to 15 mg / mL, the sodium chloride content is 5 to 10 g / L, the disodium hydrogen phosphate content is 0.5 to 3 g / L, and the chelating agent content is 5 to 10 g / L; 5. The double-window colloidal gold detection test paper for detecting fecal occult blood according to claim 4, wherein the chelating agent is one of aminopolycarboxylic acids and their sodium salts.

6. The double-window gold colloid detection test paper for detecting fecal occult blood according to claim 1, characterized in that the C quality control line is coated with sheep anti-mouse IgG at a coating concentration of 0.5 to 2.0 mg / mL.

7. The marking pad contains a mouse anti-human hemoglobin monoclonal antibody-gold colloid conjugate and a mouse IgG-gold colloid conjugate, and the concentration of the mouse anti-human hemoglobin monoclonal antibody-gold colloid conjugate is OD20-OD60; The double-window colloidal gold detection test paper for detecting fecal occult blood according to any one of claims 1 to 6, characterized in that the antibody in the mouse anti-human hemoglobin monoclonal antibody-colloidal gold conjugate, the antibody treated with the treatment solution contained in the right-hand reagent strip, and the antibody coated on the T1 detection line and the T2 detection line are different mouse anti-human hemoglobin monoclonal antibodies.

8. A cellulose nitrate film is provided in the middle of the PVC substrate, A marking pad and a water-absorbing pad are provided at both ends of the cellulose nitrate membrane, and the marking pad and the water-absorbing pad are respectively connected to the bottom side of one end thereof, and a PVC substrate is respectively connected to the bottom side of the other end thereof; 7. A double-window gold colloid detection test paper for detecting occult blood in feces according to any one of claims 1 to 6, characterized in that a sample pad is provided above the connection end between the marking pad and the PVC substrate, and the bottom side of one end of the sample pad is connected to the top side of the marking pad and the bottom side of the other end is connected to the PVC substrate.

9. Use of the colloidal gold detection test paper according to any one of claims 1 to 8 in hemoglobin detection, The use of the present invention is characterized in that the sample to be measured is applied to the sample pads of the left and right reagent strips, and the hemoglobin concentration range is determined based on the color development of the T1 detection line and the T2 detection line.

10. If the T1 detection line does not develop color and the T2 detection line does not develop color, it indicates that the hemoglobin concentration is <100 ng / mL. If the T1 detection line develops color and the T2 detection line does not develop color, it is suggested that the hemoglobin concentration is 100 ng / mL or less and less than 500 ng / mL. When the T1 detection line and the T2 detection line are colored, it is suggested that the hemoglobin concentration is 500 ng / mL or less and less than 5 mg / mL. The use according to claim 9, characterized in that if the T1 detection line does not develop color and the T2 detection line develops color, it indicates that the hemoglobin concentration is ≧5 mg / mL.

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