Colloidal gold detection test paper with double window for detecting fecal occult blood and its use

Abstract

To provide a detection test paper for detecting occult blood in feces, which can delay the occurrence of the HOOK phenomenon and improve the accuracy of gold colloid detection. The reagent strip includes a PVC substrate, a sample pad, a marking pad, a cellulose nitrate membrane, and an absorbent pad. The cellulose nitrate membrane has a T1 detection line and a C quality control line on the left reagent strip from the side closest to the marking pad, and a T2 detection line and a C quality control line 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; The right-hand reagent strip also contains mouse anti-human hemoglobin monoclonal antibody treated with treatment solution, and is set on the sample pad or on the cellulose nitrate membrane between the T2 detection line and the marking pad. The gold colloid detection test strip with double window for detecting fecal occult blood.

Description

[Technical field]

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

[0002] Gastrointestinal bleeding is caused by multiple diseases and can be divided into dominant bleeding and recessive bleeding. The normal physiological blood loss in a 24-hour human gastrointestinal tract is 0.6 mL. If the amount of gastrointestinal bleeding is greater than 2 mL, it is considered pathological bleeding. The fecal occult blood test, which is commonly used in clinical practice, is a general screening indicator for detecting gastrointestinal bleeding, chronic gastrointestinal bleeding, and early diagnosis of malignant tumors. The fecal occult blood test is of great significance to the early detection, early diagnosis, and early treatment of gastrointestinal lesions and gastrointestinal malignant tumors, and to improving the survival rate and quality of life of patients.

[0003] The test and measurement of fecal occult blood using colloidal gold has the advantages of being fast and easy to operate, its sensitivity is relatively high, it can test and measure hemoglobin at a minimum of 100ng / mL, it can accurately test and measure asymptomatic, small amount, continuous, and invisible bleeding both to the naked eye and under a microscope, and it has the advantages of not being interfered with by drugs, iron supplements, animal blood, dietary restrictions, etc., and has high specificity. It has replaced the chemical method and has become the main method for testing fecal occult blood at present.

[0004] The test and measurement of fecal occult blood by the gold colloid method mainly uses the double antibody sandwich method as the test principle, and gold colloid marking is performed against human hemoglobin polyclonal and monoclonal antibodies, which specifically binds to human hemoglobin present in the feces, and the hemoglobin in human feces is tested and measured by the color development of the gold colloid marking. However, due to the theoretical limitations of the test method, the HOOK phenomenon is likely to occur, the results of the gold colloid method are false negative, clinically accurate diagnosis cannot be made, and the optimal timing of treatment is delayed. Next, in the case of clinically manifested black stools like asphalt, bright red blood or abnormal stools that are obviously brown or brown in appearance, if the test result is negative, it is necessary to repeat the test after diluting the sample many times. Clinically, the labor and time costs increase. In addition, hemoglobin is easily decomposed, so if the sample is stored at too high a temperature or takes too long, repeated measurements may give inaccurate results.

[0005] Most colloidal gold test kits currently on the market for detecting occult blood in feces are single-window test kits, and the result is negative or weakly positive when the hemoglobin content is >5mg / mL. Therefore, there are certain limitations to the use of colloidal gold for detecting occult blood in feces, which is likely to have a certain negative impact on the accuracy of colloidal gold for detecting occult blood in feces and the simple and fast characteristics of operation. 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 for detecting occult blood in stool with a double window, and use thereof, which 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 gold colloid detection test paper for detecting occult blood in feces with a double window, including a left reagent strip and a right reagent strip. The above-mentioned reagent strip includes a PVC substrate, a sample pad, a marking pad, a cellulose nitrate membrane, and a water-absorbing 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 on the left reagent strip, and a T2 detection line and a C quality control line on the right reagent strip, in that order, 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 reagent strip also contains mouse anti-human hemoglobin monoclonal antibody treated with a treatment solution, which is set on the sample pad or on the cellulose nitrate membrane between the T2 detection line and the marking pad as a neutralizing action just before the T2 detection line.

[0008] The present invention uses gold colloid immunochromatography, double anti-sandwich method as the basic principle. When the sample contains hemoglobin, after the sample is added, the hemoglobin will bind with the mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate in the marking pad to form 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)-gold colloid conjugate, which is red-purple in color, has clear color streaks, can be seen by the naked eye, does not require other auxiliary equipment, and is simple and convenient to operate.

[0009] Specifically, after a high concentration of test sample is added to the left reagent strip, when the test sample flows into the marking pad, hemoglobin binds to the mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate to form a complex, and this complex can specifically bind to the mouse anti-human hemoglobin monoclonal antibody (for coating) on ​​the T1 detection line. However, since there is excess hemoglobin in the sample, 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, and the complex cannot bind to the antibody on the T1 detection line, so the T1 detection line shows a weak positive or negative result.

[0010] After a high concentration of the test sample is added to the left reagent strip, when the test sample flows into the marking pad, hemoglobin and mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate bind to each other to form a complex hemoglobin-mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate, which continues to move forward by chromatography. When the excess hemoglobin passes through the closed line coated with mouse anti-human hemoglobin monoclonal antibody (for coating) treated with the treatment solution, it binds to it, and as it continues to move, the hemoglobin-mouse anti-human hemoglobin monoclonal antibody (for marking)-gold colloid conjugate specifically binds to the mouse anti-human hemoglobin monoclonal antibody (for coating) on ​​the T2 detection line, showing a reddish purple strip. In addition, after a high concentration of test sample is added to the right reagent strip, when the test sample flows through the sample pad, the hemoglobin first binds to the mouse anti-human hemoglobin monoclonal antibody (for coating) treated with the treatment liquid, and continues to move forward by chromatography. When 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, 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 the sample contains hemoglobin based on the color development of the detection line and the quality control line, and can also determine the concentration range of hemoglobin in the fecal sample based on the color development of the two detection lines, the T1 detection line coated on the left reaction area has high sensitivity and can detect hemoglobin at least 100ng / mL, and the T2 detection line coated on the right reaction area can detect hemoglobin at least 500ng / mL. The double window detection can delay the occurrence of the HOOK phenomenon and improve the accuracy of gold colloid 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 above 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, in the treatment liquid, 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. The chelating agent is one of aminopolycarboxylic acids and their sodium salts.

[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 can improve protein adsorption and the immune reaction rate by adjusting the ionic environment of the immune reaction system in combination with other components in the treatment solution. In addition, the synergistic effect between the components can be improved by adjusting the concentration of each component and the pH of the treatment solution, and in particular, the concentration of the chelating agent has a large effect on the treatment effect of the treatment solution, and if the concentration exceeds the limited range of the present invention, the detection rate of hemoglobin decreases.

[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. In addition, the 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 in front of the detection line, avoiding the excess hemoglobin competing to bind with the antibody on the detection line 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, and the other end of each of the marking pad and the water-absorbing pad is connected to 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 a use of the colloidal gold detection test paper in detecting hemoglobin, in which a sample to be measured is added to the sample pads of the left and right reagent strips, and the concentration range of hemoglobin 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 the sample contains hemoglobin. When both the detection line T and the quality control line C show red bands, it indicates that the sample contains hemoglobin. When only the quality control line C shows red bands, it indicates that the sample does not contain hemoglobin or the hemoglobin content in the sample is lower than the detection value. When the quality control line C does not show red bands, it indicates that the test strip is invalid. The T1 detection line coated on the left reaction area has high sensitivity and can detect hemoglobin at least 100ng / mL, and the T2 detection line coated on the right reaction area can detect hemoglobin at least 500ng / mL.

[0025] The colloidal gold detection test paper of the present invention can detect the range of hemoglobin concentrations in a sample. The sample is added to the sample pads of the left and right reagent strips, and the range of hemoglobin concentrations 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 < 100ng / mL. When the T1 detection line develops color and the T2 detection line does not develop color, it indicates that the hemoglobin concentration is 100ng / mL < 500ng / mL. When the T1 detection line develops color and the T2 detection line develops color, it indicates that the hemoglobin concentration is 500ng / mL < 5mg / mL. When the T1 detection line does not develop color and the T2 detection line develops color, it indicates that the hemoglobin concentration is ≥ 5mg / mL.

[0027] More preferably, when the T1 detection line does not develop color and the T2 detection line does not develop color, it is suggested that the hemoglobin concentration is < 100ng / mL. When the T1 detection line develops color and the T2 detection line does not develop color, it is suggested that the hemoglobin concentration is 100ng / mL ≦ hemoglobin concentration < 500ng / mL. When 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 is suggested that the hemoglobin concentration is 500ng / mL ≦ hemoglobin concentration < 10000ng / mL. When the T1 detection line develops strongly and the T2 detection line develops strongly, it is suggested that the hemoglobin concentration is 10000ng / mL. When 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 is suggested that the hemoglobin concentration is 10000ng / mL < hemoglobin concentration < 5mg / mL. If the T1 detection line does not develop color and the T2 detection line does develop color, it suggests a hemoglobin concentration range of ≧5 mg / mL.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention adds a detection window to the existing colloidal gold detection reagent strip, and the test reagent strip in the newly added window has added thereto 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 a processing solution can neutralize the excess hemoglobin, and then the colloidal gold hemoglobin antigen-antibody complex specifically binds to the hemoglobin antibody on the test line to show a positive result. The double window test can delay the appearance of the HOOK phenomenon and improve the accuracy of the colloidal gold test. (2) The present invention scientifically and objectively realizes rapid testing from qualitative to semi-quantitative, guarantees high sensitivity, and at the same time, can increase the upper limit of the linear testing range and improve the accuracy of the test reagent. In addition, the sample only needs to be processed once, eliminating the need for repeated dilution, saving time and labor costs. [Brief description of the drawings]

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

[0030] In the following, 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 colloidal gold 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 solution so that the sucrose content in the treatment solution is 8-15 mg / mL, the sodium chloride content is 5-10 g / L, the disodium hydrogen phosphate content is 0.5-3 g / L, the chelating agent content is 5-10 g / L, and the pH is adjusted 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 give 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 make the coating concentration 0.5-1.5 mg / mL. 1.1.4, Coating of left reagent strip: Spray the prepared C / T1 coating solution on the cellulose nitrate membrane at a rate of 1.0-1.2μL / cm to form the C quality control line and the T1 detection line, respectively. The coating size of the C quality control line is 34-35mm from the bottom edge of one end of the PVC substrate, and the coating size of the T1 detection line is 28-29mm from the bottom edge of the same end of the PVC substrate. Then dry it in a dryer at 37℃±2℃ for 12-24 hours to obtain the coated cellulose nitrate membrane. 1.1.5. Coating of the right side reagent strip: Spray the prepared C / T2 / closure line coating solution onto the cellulose nitrate membrane at a rate of 1.0-1.2 μL / cm to make the C quality control line, T2 detection line, and closure line, respectively. The coating size of the C quality control line is located 34-35 mm from the bottom edge of one end of the PVC substrate. The coating size of the T2 detection line is located 28-29 mm from the bottom edge of the same end of the PVC substrate. The coating size of the closure line is located 22-23 mm from the bottom edge of the same end of the PVC substrate. Then dry it in an electric heated air dryer at 37℃±2℃ for 12-24 hours to obtain a coated cellulose nitrate membrane.

[0032] 1.2 Marking 1.2.1. Preparation of colloidal gold: colloidal gold particles are mixed with mouse anti-human hemoglobin monoclonal antibody (for marking) and mouse IgG, respectively, to mark mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugate and mouse IgG-colloidal gold conjugate. 1.2.2. Preparation of marking solution: Dilute 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 3-6g / L casein, 4-7g / L trishydroxymethylaminomethane, 0.1-0.4mL / L Proclin300, 3-8g / L PVP-10, and 0.2-0.7mL / L Tween20 in aqueous solution, with a pH of 7.2-7.6. 1.3.2. Preparation of sample pad: The sample pad solution is spread on the glass fiber, placed in an electric heated air dryer at 37℃±2℃, and dried for 12-24 hours to obtain the sample pad.

[0034] 1.4 Assembly The method for assembling the above reagent strip is to provide a cellulose nitrate membrane in the middle of a PVC substrate. A marking pad and an absorbent pad are provided on both ends of the cellulose nitrate membrane, and one end of each is connected to the bottom side of the marking pad and the absorbent pad. The other end bottom sides of the marking pad and the absorbent pad are connected to the PVC substrate. A sample pad is provided on the upper side of the connection end between the marking pad and the PVC substrate. The sample pad has one end bottom side connected to the upper side of the marking pad and the other end bottom side connected to the PVC substrate. The left reagent strip and the right reagent strip are cut to the desired width and assembled in parallel to obtain a double-window detection gold colloid detection test paper.

[0035] (2) Preparation of colloidal gold detection test paper (on the right-hand 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 1.0-1.2μL / cm to form the C quality control line and the T2 detection line. The coating size of the C quality control line is 34-35mm from the bottom edge of one end of the PVC substrate. The coating size of the T2 detection line is 28-29mm from the bottom edge of the same end of the PVC substrate. Then dry it in an electric heated air dryer at 37℃±2℃ 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 Proclin300, 3-8 g / L PVP-10, 0.2-0.7 mL / L Tween20, 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, 0.05-0.1 mg / mL mouse anti-human hemoglobin monoclonal antibody (for coating), and a pH of 7.2-7.6. 1.3.2. Preparation of sample pad: Same as 1.3.2 in (1). 1.4 Assembly Same as 1.4 in (1).

[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 make the total volume 100 mL. 1.1.2. Preparation of solutions for coating C / T1 / T2 lines: Dilute sheep anti-mouse IgG and mouse anti-human hemoglobin monoclonal antibodies (for coating) in 10 mM PBS at pH 7.4 to prepare solutions at 1.0 mg / mL, 1.5 mg / mL, and 1.5 mg / mL, respectively. 1.1.3. Preparation of solution for coating of the closed line: Dilute mouse anti-human hemoglobin monoclonal antibody (for coating) with the closed line treatment solution to prepare a solution of 0.5 mg / mL. 1.1.4, Coating of the left reagent strip: Spray the prepared C / T1 coating solution on the cellulose nitrate membrane at a rate of 1.0 μL / cm to form the C quality control line and the T1 detection line, respectively. The coating size of the C quality control line is 34.5 mm from the bottom edge of one end of the PVC substrate, and the coating size of the T1 detection line is 28.5 mm from the bottom edge of the same end of the PVC substrate. Then dry it in a dryer at 37 ° C ± 2 ° C for 16 hours to obtain the coated cellulose nitrate membrane. 1.1.5. Coating of 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 make the C quality control line, T2 detection line, and closure line, respectively. The coating size of the C quality control line is 34.5 mm from the bottom edge of one end of the PVC substrate. The coating size of the T2 detection line is 28.5 mm from the bottom edge of the same end of the PVC substrate. The coating size of the closure line is 22.5 mm from the bottom edge of the same end of the PVC substrate. Then, dry it in an electric heated air dryer at 37℃±2℃ for 16 hours to obtain a 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 proportional amounts, and marked as mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugate and mouse IgG-colloidal gold conjugate. The absorbance measured by UV-Vis spectrophotometer was 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(hydroxymethylaminomethane), 0.016 mL of Proclin 300, 0.5 g of PVP-10, and 0.05 mL of Tween 20, in that order, and after completely dissolving, adjust the pH to 7.4, and finally dilute to 100 mL with purified water. 1.3.2. Preparation of sample pad: Then, 3.5mL of the sample pad solution is weighed out, laid on a 17mm*31mm glass fiber, and dried in an electric heated air dryer at 37℃±2℃ for 16 hours to obtain a 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, and then cut to the required width as necessary to obtain a double-window colloidal gold 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 preparation concentration is 1.5 mg / mL.

[0045] Example 4 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 (different chelating agent), the rest is the same as 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 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 make the total volume 100 mL.

[0046] Example 5 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 (different chelating agent), the rest is the same as 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 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 make the total volume 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 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 make the total volume 100 mL.

[0048] Example 7 On the right reagent strip, a mouse anti-human hemoglobin monoclonal antibody treated with a treatment solution is placed on the sample pad, i.e., preparation of a gold colloid detection test paper (i.e., the right reagent strip does not contain a closing line at this point). 1.1 Coating 1.1.1. Preparation of C / T1 / T2 line 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 prepare 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 solution 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 coating size of the C quality control line is 34.5 mm from the bottom edge of one end of the PVC substrate, and the coating size of the T1 detection line is 28.5 mm from the bottom edge of the same end of the PVC substrate. Then dry it in a dryer at 37°C ± 2°C for 12-24 hours to obtain the coated cellulose nitrate membrane. 1.1.3, Coating of the right reagent strip: Spray the prepared C / T2 coating solution on the cellulose nitrate membrane at a rate of 1.0 μL / cm to form the C quality control line and the T2 detection line. The coating size of the C quality control line is 34.5 mm from the bottom edge of one end of the PVC substrate, and the coating size of the T2 detection line is 28.5 mm from the bottom edge of the same end of the PVC substrate. Then dry it 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 proportional amounts, and marked as mouse anti-human hemoglobin monoclonal antibody (for marking)-colloidal gold conjugate and mouse IgG-colloidal gold conjugate. The absorbance measured by UV-Vis spectrophotometer was 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 trishydroxymethylaminomethane, 0.016 mL of Proclin300, 0.5 g of PVP-10, 0.05 mL of Tween20, 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), and after complete dissolution, adjust the pH to 7.4, and add purified water to make the volume 100 mL. 1.3.2. Preparation of sample pad: Then, 3.5mL of the solution is weighed out, spread on a 17mm*31mm glass fiber, and dried in an electric heated air dryer at 37℃±2℃ 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, and then cut to the required width as necessary to obtain a double-window colloidal gold detection test paper.

[0052] Comparative Example 1 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 0.1 mg / mL.

[0053] Comparative Example 2 Preparation of colloidal gold detection test paper (including a closing 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 2.0 mg / mL.

[0054] Comparative Example 3 Preparation of colloidal gold detection test paper (including a closing line on the right-hand reagent strip) Except for step 1.1.1 being different (no chelating agent added), 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, 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 make the total volume 100 mL.

[0055] Comparative Example 4 Preparation of colloidal gold detection test paper (including a closing line on the right-hand reagent strip) Except for step 1.1.1 being different (adding too much chelating agent), the rest is the same as 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 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 make the total volume 100 mL.

[0056] Comparative Example 5 Preparation of colloidal gold detection test paper (including a closing line on the right-hand reagent strip) Except for steps 1.1.1 and 1.1.3, the rest is the same as Example 1. Remove step 1.1.1. Preparation of closed line treatment solution from step 1.1.3: Dilute mouse anti-human hemoglobin monoclonal antibody (for coating) in 10 mM PBS buffer at pH 7.4 (i.e., similar to the solution for C / T1 / T2 line coating preparation) to 0.5 mg / mL.

[0057] Performance tests were carried out for the Examples and Comparative Examples, using the negative control, 50ng / mL hemoglobin, 100ng / mL hemoglobin, 200ng / mL hemoglobin, 500ng / mL hemoglobin, 1μg / mL hemoglobin, 10μg / mL hemoglobin, 100μg / mL hemoglobin, 5mg / mL hemoglobin, 10mg / mL hemoglobin, and 50mg / mL hemoglobin, with each concentration setting 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] JPEG0007660246000001.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] JPEG0007660246000002.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, in the immunochromatography reaction, Comparative Example 1 and Comparative Example 2 show that if the components or dosage of the closure line are not appropriate, it will affect the reaction environment of the antigen and antibody, affect the binding of the antigen and antibody, and ultimately affect the accuracy of the measurement 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 reagent strip can delay the appearance of the HOOK phenomenon and improve the accuracy of the gold colloid test, especially for high concentration samples, can accurately obtain positive results, and there is no need to dilute and re-measure multiple times, and at the same time, can combine the results of the double window to determine the concentration content range of hemoglobin.

[0059] In addition, by treating the closed line treatment solution, the results of Example 1 and Comparative Example 3 show that the detection rate of high-concentration hemoglobin can be improved by adding a chelating agent to the closed line treatment solution, and the chelating agent can adjust the ion environment in the immune reaction system and improve protein adsorption and immune reaction speed. However, Comparative Example 4 shows that a chelating agent with a high concentration affects the binding between hemoglobin and mouse anti-human monoclonal antibody, and the excess hemoglobin cannot be neutralized, and the HOOK effect cannot be delayed. In addition, Comparative Example 5 shows that if the closed line treatment solution is a normal PBS component, there will be a negative test result for a sample with a high concentration of hemoglobin.

[0060] Validation of clinical samples The present invention collected a total of 26 fecal occult blood positive samples, and confirmed the measurement values ​​by a third-party measurement reagent (Shanghai Transillumination Diagnostics Technology Co., Ltd. - Hemoglobin / Transferrin Measurement Kit (Flow Cytometry Fluorescence Method)), and the measurement results are shown in Table 3. Among them, 11 positive samples had a hemoglobin concentration of 100-500ng / mL, 9 positive samples had a hemoglobin concentration of 500ng / mL-5mg / mL, and 6 high-concentration positive samples had a hemoglobin concentration of >5mg / mL. [Table 3] JPEG0007660246000003.jpg199170*Note: "+" indicates a positive test result. "-" indicates a negative test result.

[0061] As shown in Table 3, the measurement results of clinical samples show that Example 1 has high sensitivity and accuracy, and that for samples with particularly high hemoglobin concentrations, the concentration range of hemoglobin in the sample can be determined simultaneously from the coloration results of the two T lines. In Comparative Examples 3 and 4, if no chelating agent is added to the treatment solution or if too much chelating agent is added, both of these affect 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 utilizing the specification of the present invention, or direct or indirect use in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A colloidal gold detection test paper for detecting occult blood in feces with a double window, comprising a left reagent strip and a right reagent strip, Each of the left and right reagent strips 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 is provided with a T1 detection line and a C quality control line in 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 in 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; The right-hand reagent strip also contains mouse anti-human hemoglobin monoclonal antibody treated with a treatment solution, and is placed on the sample pad or on the cellulose nitrate membrane between the T2 detection line and the marking pad. The treatment liquid is an aqueous solution obtained by mixing sucrose, sodium chloride, disodium hydrogen phosphate, and a chelating agent, In the treatment liquid, 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; A gold colloid detection test paper with double windows for detecting occult blood in feces, characterized in that the chelating agent is one of aminopolycarboxylic acids and their sodium salts.

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

5. 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; A gold colloid detection test paper with a double window for detecting occult blood in feces as described in any one of claims 1 to 4, characterized in that the antibody in the mouse anti-human hemoglobin monoclonal antibody-gold colloid conjugate, the antibody treated with the processing solution contained in the right-side reagent strip, and the antibody coated on the T1 detection line and the T2 detection line are different mouse anti-human hemoglobin monoclonal antibodies.

6. 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 connected to the bottom sides of one end, and a PVC substrate is connected to the bottom sides of the marking pad and the water-absorbing pad, respectively; A gold colloid detection test paper for detecting occult blood in feces with a double window as described in any one of claims 1 to 4, characterized in that a sample pad is provided on the upper side of the connection end between the marking pad and the PVC substrate, and one end bottom side of the sample pad is connected to the upper side of the marking pad and the other end bottom side is connected to the PVC substrate.

7. A use of the colloidal gold detection test paper according to any one of claims 1 to 4 in detecting hemoglobin, The use of the present invention is characterized in that 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.

8. 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 develops color and the T2 detection line develops color, it is suggested that the hemoglobin concentration is 500 ng / mL or less and less than 5 mg / mL. The use according to claim 7, 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.

Images