Detection reagent strip for heart-type fatty acid binding protein and preparation method of detection reagent strip

Abstract

The invention belongs to the technical field of medical detection reagents, and particularly relates to a heart-type fatty acid binding protein detection reagent strip and a preparation method thereof. The detection reagent strip provided by the invention can effectively improve the detection sensitivity and accuracy of heart-type fatty acid binding protein in urine by comprehensively improving the composition of the reagent strip, the antibody type, the preparation process and parameters, the detection sensitivity is 10ng / mL, the accuracy reaches 100%, and the diagnosis sensitivity and accuracy of myocardial infarction are greatly improved. Moreover, the reagent strip is suitable for professional medical personnel to detect the human heart-type fatty acid binding protein in human urine in a medical institution, is very beneficial to early warning of myocardial infarction, and has a better application prospect in the fields of detection of hFABP and clinical diagnosis of acute myocardial infarction or acute myocardial injury.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection reagents, and in particular, relates to a detection reagent strip for cardiac fatty acid binding protein and a preparation method thereof. Background Art

[0002] Acute myocardial infarction (AMI) is a disease caused by acute blockage of the coronary arteries, which leads to insufficient blood supply to the corresponding myocardial area and myocardial necrosis. AMI seriously threatens human life and health and is one of the major causes of death and disability worldwide. Rapid diagnosis of early acute myocardial infarction and timely implementation of effective treatment are the key to reducing patient mortality.

[0003] Fatty acid binding protein (FABP) is a family of small molecule proteins with multiple functions. It is mainly found in the cytoplasm of cells that consume a lot of energy and plays a role in transporting free fatty acids in intracellular fat metabolism. Heart-type fatty acid binding protein (hFABP) was first discovered in the myocardium. It is a cytoplasmic protein of myocardial cells and is abundant in myocardial cells, accounting for about 4-8% of all soluble proteins in the heart. hFABP has a small molecular weight of about 15KD and is not decomposed in the blood. After acute myocardial infarction or acute myocardial injury occurs, hFABP is rapidly released into the blood and has specificity for the diagnosis of myocardial injury. It has important reference value in the early diagnosis of acute myocardial infarction and is one of the cardiac markers of early AMI. It is also very helpful for the early exclusion of AMI.

[0004] At present, there are many methods for detecting H-FABP. Among them, compared with many methods such as enzyme-linked immunosorbent assay, immunoturbidimetry, chemiluminescence, etc., the gold-label immunoassay is widely used in H-FABP detection because of its advantages of simplicity, speed, small amount of specimens required, and no restrictions on detection locations. However, the existing gold-label immunoassay often has problems with low detection sensitivity and specificity, and often leads to misjudgment or missed diagnosis. It cannot accurately reflect the early stage of myocardial infarction in patients, limiting its application in clinical diagnosis. In addition, the existing gold-label immunoassay mainly tests the patient's serum samples, which requires the patient to go to a medical institution for venous sampling and analysis. The detection time is relatively limited, which is not conducive to patient detection and timely identification, and the blood drawing process is easy to cause pain to the patient.

[0005] Therefore, establishing an H-FABP detection reagent with high sensitivity and accuracy and rapid detection of patients is of great significance for early warning of the occurrence of myocardial infarction and protecting the health of patients. Summary of the invention

[0006] In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing a detection reagent strip for cardiac fatty acid binding protein. The detection reagent strip prepared thereby is not only suitable for self-detection by physicians and patients, but also can improve the detection sensitivity (10 ng / mL) and accuracy (100%) of cardiac fatty acid binding protein, which is beneficial to improving the diagnostic sensitivity and accuracy of myocardial infarction and providing early warning of the occurrence of myocardial infarction.

[0007] At the same time, the purpose of the present invention is also to provide a detection reagent strip for heart-type fatty acid binding protein prepared by the above preparation method.

[0008] At the same time, the purpose of the present invention is also to provide a detection kit for heart-type fatty acid binding protein.

[0009] To achieve the above object, the technical solution adopted by the present invention is: A method for preparing a detection reagent strip for cardiac fatty acid binding protein comprises the following steps: (1) Preparation of binding pad: chloroauric acid, trisodium citrate, and proclin300 were mixed in water, and the pH of the solution was adjusted to 7.2-7.4 to obtain a colloidal gold solution; hFABP monoclonal antibody BF27104 was added to the colloidal gold solution and stirred, and then BSA solution was added and mixed to obtain a colloidal gold antibody labeling solution; The colloidal gold antibody labeling solution is centrifuged, and then the supernatant is discarded, the gold label precipitated at the bottom is retained, and the gold label is diluted with a colloidal gold diluent to obtain a colloidal gold antibody diluent; wherein the pH of the colloidal gold diluent is 9.2-9.4; the colloidal gold diluent comprises the following components in concentration: 2.2-2.6 mg / mL Tris-HCl, 1.8-2.2 mg / mL sodium caseinate, 150-250 mg / mL sucrose, and 0.18 wt%-0.22 wt% proclin300; Spraying the colloidal gold antibody dilution solution on the gold label pad and drying to obtain a conjugate pad; (2) Preparation of chromatographic membrane: sheep anti-mouse IgG polyclonal antibody was prepared into a solution with a concentration of 0.95-1.05 ug / uL using CT diluent to obtain a quality control line solution; hFABP monoclonal antibody BF27103 was prepared into a solution with a concentration of 1.15-1.25 ug / uL using CT diluent to obtain a test line solution; wherein the pH of the CT diluent was 8.2-8.4; the CT diluent included the following components in concentration: 0.4-0.5 mg / mL of sodium dihydrogen phosphate dihydrate, 2-3 mg / mL of sodium dihydrogen phosphate dodecahydrate, 0.3 wt%-0.5 wt% of Biolipidure-405, and 0.18 wt%-0.22 wt% of proclin300; The quality control line solution and the detection line solution are respectively drawn on the upper part and the lower part of the cellulose membrane at a dosage of 1.2-1.3 μL / cm to obtain a quality control line and a detection line, and then dried to obtain a chromatographic membrane; (3) Assembly of the reagent strip: The sample pad, the conjugate pad, the chromatography membrane and the absorbent pad are fixed on the bottom plate in sequence to obtain the detection reagent strip.

[0010] As a preferred solution, the detection object of the detection reagent strip is a human urine sample, and the detection target is cardiac fatty acid binding protein. hFABP in the patient's circulating blood is filtered out by the kidney glomerulus and excreted from the urine. Therefore, the reagent strip of the present invention can achieve rapid detection of hFABP by detecting urine. And it will not bring a detection burden to the patient.

[0011] As a preferred solution, the amount of hFABP monoclonal antibody BF27104 added to the colloidal gold solution is 4-6 μg / mL, more preferably 5 μg / mL.

[0012] As a preferred solution, the spraying amount of the colloidal gold antibody dilution solution on the gold label pad is 8-10 μL / cm, more preferably 9 μL / cm.

[0013] As a preferred solution, the material of the cellulose membrane is one of nitrocellulose membrane, cellulose acetate membrane, and nitric acid-cellulose acetate mixed membrane.

[0014] As a preferred solution, the absorbent pad is absorbent paper.

[0015] As a preferred solution, the model of the sample pad is NJ-Y8.

[0016] A detection reagent strip for heart-type fatty acid binding protein prepared by the above preparation method, the detection reagent strip comprises: a sample pad, a conjugation pad, a chromatographic membrane, a water absorbent pad and a bottom plate; the conjugation pad is a colloidal gold pad marked with hFABP monoclonal antibody BF27104; the chromatographic membrane is a cellulose membrane provided with a detection line and a quality control line; the detection line is coated with hFABP monoclonal antibody BF27103; the quality control line is coated with sheep anti-mouse IgG polyclonal antibody.

[0017] In order to facilitate the sample addition process and observe the test results, as a preferred solution, the test reagent strip also includes a protective shell; the shell is provided with a sample addition hole at a position relative to the sample pad, and an observation frame is provided at a position relative to the test line and the quality control line.

[0018] A detection kit for cardiac fatty acid binding protein mainly consists of the above detection reagent strip.

[0019] As a preferred solution, the detection kit includes a preset number of detection reagent strips. As a further preferred solution, the detection kit also includes a measuring cup for collecting fresh human urine samples.

[0020] The beneficial effects of the above technical solution of the present invention are: (i) The detection reagent strip for heart-type fatty acid binding protein provided by the present invention can realize the in vitro qualitative detection of human heart-type fatty acid binding protein (hFABP) in human urine, which can be used as auxiliary diagnosis in clinical applications for the detection of acute myocardial infarction or acute myocardial injury in humans. In addition, the reagent strip is suitable for professional medical personnel to detect human heart-type fatty acid binding protein in human urine in medical institutions, and is also suitable for patients to self-test.

[0021] (ii) The detection reagent strip provided by the present invention can effectively improve the detection sensitivity and accuracy of cardiac fatty acid binding protein in urine through comprehensive improvements in composition, antibody type, preparation process and parameters. The detection sensitivity is 10 ng / mL and the accuracy is 100%, which greatly improves the diagnostic sensitivity and accuracy of myocardial infarction, is conducive to early warning of the occurrence of myocardial infarction, and provides clinical evidence of the presence or absence of acute and chronic myocardial damage to facilitate accurate decision-making. It has good application prospects in the detection of hFABP and the clinical diagnosis of acute myocardial infarction or acute myocardial injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the structure of the heart-type fatty acid binding protein detection reagent strip provided in the present invention; in the figure, 1-sample pad, 2-binding pad, 3-chromatographic membrane, 4-water absorption pad. DETAILED DESCRIPTION

[0023] The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments and test examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical scheme of the present invention and should not be regarded as limiting the scope of protection of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the raw materials used are all commonly used in the art, available to the public or commercially available, unless otherwise specified.

[0024] In the technical solution of the present invention, the gold label pad can be made of materials familiar to technicians, such as glass fiber, polyester fiber, etc., which can be selected by technicians in a conventional manner. In the following embodiments of the present invention, the material of the gold label pad used is glass cellulose film.

[0025] Furthermore, the sample pad can be made of materials that are well known to technicians, and the technicians can routinely select. In the following embodiments of the present invention, the sample pad used is a NJ-Y8 sample pad.

[0026] Furthermore, the present invention does not specifically limit the size of the reagent strip, and technicians can make conventional settings based on the size of conventional reagent strips. Example

[0027] This embodiment provides a detection reagent strip for heart-type fatty acid binding protein, the detection object is a human urine sample, the detection target is heart-type fatty acid binding protein, and the detection reagent strip is mainly composed of a reagent strip; the structural schematic diagram of the reagent strip is as shown in Figure 1 As shown, it includes in sequence: a sample pad 1, a conjugate pad 2, a chromatography membrane 3 and a water absorbent pad 4; The conjugate pad is a colloidal gold pad marked with hFABP monoclonal antibody BF27104; the chromatographic membrane is a cellulose membrane with a detection line and a quality control line; the detection line is coated with hFABP monoclonal antibody BF27103; the quality control line is coated with goat anti-mouse IgG polyclonal antibody; the material of the cellulose membrane is nitrocellulose membrane. The absorbent pad is absorbent paper, which is pre-soaked in 0.9wt% sodium chloride solution and dried; the model of the sample pad is NJ-Y8.

[0028] The method for preparing the above-mentioned heart-type fatty acid binding protein detection reagent strip comprises the following steps: (1) Preparation of conjugate pad: 1 mL of chloroauric acid solution (concentration 1 wt%) and 1.5 mL of trisodium citrate solution (concentration 1 wt%) were added to 100 mL of ultrapure water, heated to boil, and then 0.2 wt% of proclin300 was added to adjust the solution pH to 7.3±0.1 to obtain a colloidal gold solution; Take the colloidal gold solution, add hFABP monoclonal antibody BF27104 at a dosage of 5 μg / mL and stir, then add BSA solution (50 mg / mL) accounting for 10% volume of the colloidal gold solution, continue stirring to obtain a colloidal gold antibody labeling solution; The colloidal gold antibody labeling solution is centrifuged, and then the supernatant is discarded, the gold label precipitated at the bottom is retained, and the gold label is diluted with a colloidal gold diluent to 10% of the volume of the original colloidal gold solution to obtain a colloidal gold antibody diluent; wherein the pH of the colloidal gold diluent is 9.3±0.1; the colloidal gold diluent includes the following components in concentration: 2.4 mg / mL Tris-HCl, 2 mg / mL sodium caseinate, 200 mg / mL sucrose, and 0.2 wt% proclin300; Spray the colloidal gold antibody dilution solution on the gold label pad at a dosage of 9 μL / cm and dry it to obtain a binding pad; (2) Preparation of chromatographic membrane: The goat anti-mouse IgG polyclonal antibody was prepared into a solution with a concentration of 1ug / uL using CT diluent to obtain a quality control line solution; The hFABP monoclonal antibody BF27103 was prepared into a solution with a concentration of 1.2 ug / uL using CT diluent to obtain a test line solution; The pH of the CT diluent is 8.3±0.1; the CT diluent includes the following components: 0.45 mg / mL sodium dihydrogen phosphate dihydrate, 2.6 mg / mL disodium hydrogen phosphate dodecahydrate, 0.4 wt% Biolipidure-405, and 0.2 wt% proclin300; The quality control line solution and the detection line solution are respectively drawn on the upper and lower parts of the cellulose membrane at a dosage of 1.25 μL / cm to obtain a quality control line and a detection line, and the lines are dried after drawing to obtain a chromatographic membrane; (3) Assembly of the reagent strip: Take the conjugate pad and chromatographic membrane prepared above, and also take the supporting bottom plate, sample pad and absorbent pad.

[0029] The sample pad, the binding pad, the chromatography membrane and the water absorption pad are sequentially bonded and fixed on the bottom plate to obtain a reagent strip. After obtaining the reagent strip, the reagent strip is assembled according to a preset number to obtain a detection reagent strip. The bottom plate is a plastic bottom plate.

[0030] The detection reagent strip for the above-mentioned heart-type fatty acid binding protein provided by the present invention has a detection principle of colloidal gold immunochromatography. The present invention encapsulates the heart-type fatty acid binding protein monoclonal antibody BF27104 on a colloidal gold pad. During the detection, a fresh urine sample is taken and dripped into the sample pad 1 area (5 drops), or the sample pad 1 is immersed in a measuring cup filled with urine for 5-8 seconds. The urine sample moves in the direction of the sample pad 1, the conjugation pad 2, the chromatographic membrane 3 and the absorbent pad 4 in sequence. When the urine sample contains human heart-type fatty acid binding protein, it first binds to the colloidal gold marker coated in the conjugation pad 2 to produce a reaction. Due to the chromatographic effect, the reaction complex will move forward along the binding pad 2 to the nitrocellulose membrane detection area in the chromatographic membrane 3. There are quality control line C (coated with goat anti-mouse antibody) and detection line T (coated with human heart-type fatty acid binding protein monoclonal antibody BF27103) on the nitrocellulose membrane. When the quality control line and the detection line appear at the same time, it can be judged as positive, indicating that the sample contains human heart-type fatty acid binding protein; when only the quality control line C appears, it is judged as negative, indicating that the sample does not contain human heart-type fatty acid binding protein or does not reach the detection threshold. The whole detection process takes 5-15 minutes to judge the result, and the result is invalid after 30 minutes.

[0031] In other embodiments, the reagent strip further includes a protective shell; the shell is provided with a sample addition hole at a position relative to the sample pad, and an observation frame is provided at a position relative to the detection line and the quality control line. The setting of the shell is prior art, and the present invention will not introduce it in detail. For example, the setting can be referred to the prior art such as CN101806804A.

[0032] This embodiment also provides a detection kit, which is mainly composed of the above-mentioned detection reagent strip.

[0033] The detection kit provided by the present invention may contain reagent strips of different preset numbers, and those skilled in the art can make routine selections based on the detection quantity requirements. For example, the reagent strips contained in the reagent strips may be 1 serving / box; 2 servings / box; 5 servings / box; 10 servings / box; 25 servings / box; 50 servings / box; or 100 servings / box.

[0034] In other embodiments, the detection kit further includes a measuring cup for collecting fresh human urine samples.

[0035] In other embodiments, the detection kit further includes a desiccant to maintain the effectiveness of the reagent strip.

[0036] Comparative Example 1 The comparative example provides a cardiac fatty acid binding protein detection reagent strip, and the preparation process and parameters thereof are substantially the same as those of Example 1, except that: the comparative example adopts bovine serum albumin (BSA) to replace the sodium caseinate in the colloidal gold diluent in Example 1, and the pH of the colloidal gold diluent is 8.3±0.1; the pH of the colloidal gold solution is 3.5±0.1; the sheep anti-mouse IgG polyclonal antibody is prepared into a solution with a concentration of 0.5ug / uL by using the CT diluent to obtain a quality control line solution; the hFABP monoclonal antibody BF27103 is prepared into a solution with a concentration of 1.7ug / uL by using the CT diluent to obtain a detection line solution; and other process conditions are the same as those of Example 1.

[0037] Comparative Example 2 The comparative example provides a cardiac fatty acid binding protein detection reagent strip, and its preparation process and parameters are basically the same as those in Example 1, except that: the pH of the colloidal gold diluent is 8.3±0.1; the pH of the colloidal gold solution is 3.5±0.1; the sheep anti-mouse IgG polyclonal antibody is prepared into a solution with a concentration of 0.5ug / uL by using the CT diluent to obtain a quality control line solution; the hFABP monoclonal antibody BF27103 is prepared into a solution with a concentration of 1.7ug / uL by using the CT diluent to obtain a detection line solution; and other process conditions are the same as those in Example 1.

[0038] Comparative Example 3 This comparative example provides a cardiac fatty acid binding protein detection reagent strip, and its preparation process and parameters are basically the same as those of Example 1, except that: the pH of the colloidal gold dilution in this comparative example is 8.3±0.1; the pH of the colloidal gold solution is 7.0±0.1, and other process conditions are the same as those of Example 1.

[0039] Comparative Example 4 This comparative example provides a cardiac fatty acid binding protein detection reagent strip, and its preparation process and parameters are basically the same as those of Example 1, except that the pH of the colloidal gold diluent used in this comparative example is 8.3±0.1, and other process conditions are the same as those of Example 1.

[0040] Comparative Example 5 This comparative example provides a cardiac fatty acid binding protein detection reagent strip, and its preparation process and parameters are basically the same as those of Example 1, except that: the CT diluent used in this comparative example does not contain Biolipidure-405.

[0041] Comparative Example 6 This comparative example provides a cardiac fatty acid binding protein detection reagent strip, and its preparation process and parameters are basically the same as those of Example 1, except that: the pH of the colloidal gold diluent used in this comparative example is 8.0±0.1, and other process conditions are the same as those of Example 1.

[0042] Comparative Example 7 The comparative example provides a cardiac fatty acid binding protein detection reagent strip, and its preparation process and parameters are basically the same as those of Example 1, except that: the pH of the CT diluent used in the comparative example is 9.3±0.1; the colloidal gold antibody diluent is sprayed on the gold label pad at a dosage of 7 μL / cm; the sheep anti-mouse IgG polyclonal antibody is prepared into a solution with a concentration of 0.5 ug / uL by using the CT diluent to obtain a quality control line solution; and other process conditions are the same as those of Example 1.

[0043] Comparative Example 8 This comparative example provides a cardiac fatty acid binding protein detection reagent strip, and its preparation process and parameters are basically the same as those of Example 1, except that: the pH of the colloidal gold solution used in this comparative example is 8.3±0.1; the pH of the CT diluent is 9.3±0.1, and other process conditions are the same as those of Example 1.

[0044] Comparative Example 9 This comparative example provides a cardiac fatty acid binding protein detection reagent strip, and its preparation process and parameters are basically the same as those of Example 1, except that: the pH of the colloidal gold solution used in this comparative example is 9.3±0.1, and other process conditions are the same as those of Example 1.

[0045] Test examples and evaluation of detection results (1) Detection effect of comparative example 1 The detection reagent strip provided in Comparative Example 1 was used to detect water, PBS, and samples containing hFABP antigens at different concentrations. The detection results are shown in Table 1.

[0046] Table 1. Test results of the reagent strip of Comparative Example 1 As shown in Table 1 and the color development results of the reagent strip, both water and PBS samples showed weak positive results. Therefore, the reagent strip of Comparative Example 1 will show a weak positive result and cannot be used for effective detection of samples.

[0047] (2) Detection effect of comparative example 2 The detection reagent strip provided in Comparative Example 2 was used to detect water, PBS, and samples containing hFABP antigens at different concentrations. The detection results are shown in Table 2.

[0048] Table 2. Test results of the reagent strip of Comparative Example 2 From Table 2 and the color development results of the reagent strip, it can be seen that compared with Comparative Example 1, the water and PBS samples tested this time are both negative. It can be seen that adding sodium caseinate to the colloidal gold diluent is beneficial to eliminate the weak positive results presented by the water and PBS samples in Comparative Example 1.

[0049] (3) Detection effect of comparative example 3 The test strip provided in Comparative Example 3 was used to test water, PBS, and samples containing hFABP antigens at different concentrations, and urine samples from four healthy subjects. The test results are shown in Table 3.

[0050] Table 3. Test results of the reagent strip of Comparative Example 3 Sample No. Sample name Comparative Example 3 Test Results Expected Results Sample 1 water Negative Negative Sample 2 PBS Negative Negative Sample 3 hFABP antigen sample (concentration 3200ng / mL) Positive Positive Sample 4 hFABP antigen sample (concentration 500ng / mL) Positive Positive Sample 5 hFABP antigen sample (concentration 200 ng / mL) Positive Positive Sample 6 hFABP antigen sample (concentration 100 ng / mL) Weak positive Positive Sample 7 Urine sample from a healthy person1 Negative Negative Sample 8 Urine sample from a healthy person2 Negative Negative Sample 9 Urine sample from a healthy person3 Negative Negative Sample 10 Urine samples from healthy people4 Negative Negative From Table 3 and the color development results of the reagent strip, it can be seen that the water and PBS samples tested in this test were negative, the lowest detection limit of the antigen was 200ng / mL, and the C and T lines were clearly colored. When the antigen was 100ng / mL, it was weakly positive and the color development was not obvious, indicating that the overall detection limit of the reagent strip was not high.

[0051] (4) Detection effect of comparative example 4 The detection reagent strip provided in Comparative Example 4 was used to detect water, PBS, and samples containing hFABP antigens at different concentrations, and 9 urine samples purchased from other institutions were also tested. The test results are shown in Table 4.

[0052] Table 4, test results of the reagent strip of comparative example 4 It can be seen from Table 4 and the color development results of the reagent strip that the 100 ng / mL antigen sample showed weak positivity and the color development was not obvious. The color development of the 9 urine samples was obviously poor and could not meet the detection requirements.

[0053] (5) Detection effect of comparative example 5 The detection reagent strip provided in Comparative Example 5 was used to detect water, PBS, and samples containing different concentrations of hFABP antigen, and 20 purchased urine samples were tested. The test results are shown in Table 5.

[0054] Table 5. Test results of the reagent strip of Comparative Example 5 From Table 5 and the color development results of the reagent strip, it can be seen that the hFABP antigen sample is 10 ng / mL and is weakly positive, and the color development is not obvious, but a new problem arises: the water test is positive, and the PBS test is negative. The positive accuracy of the purchased urine samples in Comparative Example 5 is improved compared with Comparative Example 4: Urine samples 3 and 8 of the purchased urine in Comparative Example 4 are negative; Urine samples 3 and 8 of the purchased urine in Comparative Example 5 are positive.

[0055] (6) Detection effect of comparative example 6 The detection reagent strip provided in Comparative Example 6 was used to detect water, PBS, and samples containing hFABP antigens at different concentrations. The detection results are shown in Table 6. Table 6. Test results of the reagent strip of Comparative Example 6 It can be seen from Table 6 and the color development results of the reagent strip that the 100 ng / mL antigen sample showed a weak positive result, but a border effect occurred during the test and could not meet the use requirements.

[0056] (7) Detection effect of comparative example 7 The test reagent strip provided in Comparative Example 7 was used to test water, PBS, and samples containing different concentrations of hFABP antigen, and 20 purchased urine samples. The test results are shown in Table 7.

[0057] Table 7. Test results of the reagent strip of Comparative Example 7 It can be seen from Table 7 and the color development results of the reagent strips that all 20 purchased urine samples were negative, which was inconsistent with the expected results.

[0058] (8) Detection effect of comparative example 8 The detection reagent strip provided in Comparative Example 8 was used to detect water, PBS, and samples containing different concentrations of hFABP antigen, and 20 purchased urine samples were also tested. The test results are shown in Table 8.

[0059] Table 8. Test results of the reagent strip of Comparative Example 8 It can be seen from Table 8 and the color development results of the reagent strip that the detection sensitivity and accuracy of the reagent strip of Comparative Example 8 are both poor.

[0060] (9) Detection effect of comparative example 9 The test reagent strip provided in Comparative Example 9 was used to test water, PBS, and samples containing different concentrations of hFABP antigen, and 20 purchased urine samples. The test results are shown in Table 9.

[0061] Table 9, Test results of the reagent strip of Comparative Example 9 From Table 9 and the color development results of the reagent strip, it can be seen that the sensitivity of the reagent strip of Comparative Example 9 is to develop antigen color to 25 ng / mL, with an accuracy of 100%, but the color development of the C line is not very obvious.

[0062] (10) Detection effect of Example 1 of the present invention The detection reagent strip provided in Example 1 of the present invention was used to detect 20 purchased urine samples. The detection results are shown in Table 10.

[0063] Table 10. Test results of the reagent strip of Example 10 As can be seen from Table 10, the detection reagent strip provided in the embodiment of the present invention has high sensitivity and good accuracy. The sensitivity: antigen color development reaches 10 ng / mL, the accuracy is 100%, and the C and T line color development is very obvious, which can effectively meet the detection needs.

[0064] In summary, the detection reagent strip provided by the present invention can effectively improve the detection sensitivity and accuracy of cardiac fatty acid binding protein in urine through comprehensive improvements in the reagent strip composition, antibody types, preparation process and parameters. The detection sensitivity is 10 ng / mL and the accuracy reaches 100%, which greatly improves the diagnostic sensitivity and accuracy of myocardial infarction, is conducive to early warning of the occurrence of myocardial infarction, and provides clinical evidence of the presence or absence of acute and chronic myocardial damage to facilitate accurate decision-making. It has good application prospects in the detection of hFABP and the clinical diagnosis of acute myocardial infarction or acute myocardial injury.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a test strip for detecting cardiac fatty acid binding protein, characterized in that: The following steps are involved: (1) Preparation of binding pad: chloroauric acid, trisodium citrate, and proclin300 were mixed in water, and the pH of the solution was adjusted to 7.2-7.4 to obtain a colloidal gold solution; hFABP monoclonal antibody BF27104 was added to the colloidal gold solution and stirred, and then BSA solution was added and mixed to obtain a colloidal gold antibody labeling solution; The colloidal gold antibody labeling solution is centrifuged, and then the supernatant is discarded, the gold label precipitated at the bottom is retained, and the gold label is diluted with a colloidal gold diluent to obtain a colloidal gold antibody diluent; wherein the pH of the colloidal gold diluent is 9.2-9.4; the colloidal gold diluent comprises the following components in concentration: 2.2-2.6 mg / mL Tris-HCl, 1.8-2.2 mg / mL sodium caseinate, 150-250 mg / mL sucrose, and 0.18 wt%-0.22 wt% proclin300; Spraying the colloidal gold antibody dilution solution on the gold label pad and drying to obtain a conjugate pad; (2) Preparation of chromatographic membrane: sheep anti-mouse IgG polyclonal antibody was prepared into a solution with a concentration of 0.95-1.05 ug / uL using CT diluent to obtain a quality control line solution; hFABP monoclonal antibody BF27103 was prepared into a solution with a concentration of 1.15-1.25 ug / uL using CT diluent to obtain a test line solution; wherein the pH of the CT diluent was 8.2-8.4; the CT diluent included the following components in concentration: 0.4-0.5 mg / mL of sodium dihydrogen phosphate dihydrate, 2-3 mg / mL of sodium dihydrogen phosphate dodecahydrate, 0.3 wt%-0.5 wt% of Biolipidure-405, and 0.18 wt%-0.22 wt% of proclin300; The quality control line solution and the detection line solution are respectively drawn on the upper part and the lower part of the cellulose membrane at a dosage of 1.2-1.3 μL / cm to obtain a quality control line and a detection line, and then dried to obtain a chromatographic membrane; (3) Assembly of the reagent strip: The sample pad, the conjugate pad, the chromatography membrane and the absorbent pad are fixed on the bottom plate in sequence to obtain the detection reagent strip.

2. The method for preparing a detection reagent strip for heart-type fatty acid binding protein according to claim 1, characterized in that: The detection object of the detection reagent strip is a human urine sample, and the detection target is heart-type fatty acid binding protein.

3. The method for preparing a detection reagent strip for heart-type fatty acid binding protein according to claim 1, characterized in that: The amount of hFABP monoclonal antibody BF27104 added to the colloidal gold solution was 4-6 μg / mL.

4. The method for preparing a test strip for detecting heart-type fatty acid binding protein according to claim 1, characterized in that: The spraying amount of colloidal gold antibody dilution on the gold label pad is 8~10μL / cm.

5. The method for preparing a detection reagent strip for heart-type fatty acid binding protein according to claim 1, characterized in that: The material of the cellulose membrane is one of nitrocellulose membrane, cellulose acetate membrane and nitric acid-cellulose acetate mixed membrane.

6. The method for preparing a detection reagent strip for heart-type fatty acid binding protein according to claim 1, characterized in that: The absorbent pad is absorbent paper.

7. The method for preparing a detection reagent strip for heart-type fatty acid binding protein according to claim 1, characterized in that: The model of the sample pad is NJ-Y8.

8. A test strip for detecting heart-type fatty acid binding protein prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The detection reagent strip comprises: a sample pad, a conjugation pad, a chromatographic membrane, a water-absorbing pad and a bottom plate; the conjugation pad is a colloidal gold pad marked with hFABP monoclonal antibody BF27104; the chromatographic membrane is a cellulose membrane provided with a detection line and a quality control line; the detection line is coated with hFABP monoclonal antibody BF27103; the quality control line is coated with sheep anti-mouse IgG polyclonal antibody.

9. The heart-type fatty acid binding protein detection reagent strip according to claim 8, characterized in that: The detection reagent strip also includes a protective shell; the shell is provided with a sample addition hole at a position relative to the sample pad, and an observation frame at a position relative to the detection line and the quality control line.

10. A detection kit for heart-type fatty acid binding protein, characterized in that: The detection kit is mainly composed of the detection reagent strip according to claim 8 or 9.

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