High-sensitivity fecal occult blood detection kit and preparation method thereof

Abstract

The invention belongs to the technical field of fecal occult blood detection, and particularly relates to a high-sensitivity fecal occult blood detection kit and a preparation method thereof, and the preparation method of the kit comprises the following steps: synthesizing colloidal gold particles; carrying out surface modification on colloidal gold particles by adopting polyethylene glycol; covalently binding a hemoglobin specific antibody to the surface of the colloidal gold modified by polyethylene glycol by using a cross-linking agent; fixing the screened and optimized hemoglobin specific antibody on a detection line of a nitrocellulose membrane as a capture antibody, and forming a bispecific antibody system with the hemoglobin specific antibody in valence binding with the surface of colloidal gold; the method comprises the following steps: taking colloidal gold as a template, diluting and homogenizing a sample, adding antibody-coated magnetic beads, and then separating, cleaning and re-suspending the magnetic beads containing hemoglobin, thereby comprehensively upgrading the aspects of size control of colloidal gold particles, optimization of an antibody labeling strategy, innovation of a sample pretreatment technology and the like; the novel detection kit with excellent sensitivity and specificity is made, is more sensitive, specific and stable, and meets the requirements of clinical doctors and patients on rapid and accurate diagnosis of early gastrointestinal hemorrhage.

Description

Technical Field

[0001] The invention belongs to the technical field of fecal occult blood detection, and specifically relates to a high-sensitivity fecal occult blood detection kit and a preparation method thereof. Background Art

[0002] In the current healthcare field, early detection of digestive tract diseases, especially gastrointestinal bleeding, is the key to preventing the development of serious diseases. As an important and non-invasive screening tool, fecal occult blood testing has long been widely valued. Although traditional colloidal gold immunochromatography technology has been successfully applied to such tests, with the continuous development of science and technology and the improvement of public health awareness, some limitations of existing technologies have gradually emerged, and innovation and improvement are urgently needed.

[0003] Main defects and shortcomings of existing technology

[0004] 1. Limited detection sensitivity

[0005] Difficulty in detecting low-concentration hemoglobin: Conventional colloidal gold immunochromatography is not sensitive enough for detecting extremely low concentrations of hemoglobin, and may miss the diagnosis, especially in the case of early gastrointestinal lesions or mild bleeding.

[0006] Signal intensity constraint: The size of colloidal gold particles directly affects the signal intensity. Traditional technology is limited by the physical properties of colloidal gold particles and it is difficult to effectively amplify weak immune response signals, which limits the detection limit.

[0007] 2. Specificity needs to be improved

[0008] Cross-reaction risk: Existing test kits may produce false positive results due to non-specific antigen-antibody binding, such as interference from hemoglobin or other proteins from food sources, reducing the accuracy of diagnosis.

[0009] Influence of complex sample environment: Digestive tract samples usually contain a large amount of other substances, which may compete with the detection target or interfere with the specific binding of antibodies, increasing the complexity and uncertainty of the detection.

[0010] 3. Antibody labeling and stability

[0011] Antibody labeling efficiency: The surface properties of traditional colloidal gold particles limit the effective loading capacity of antibodies, affecting the sensitivity and reliability of detection.

[0012] Long-term stability issues: The labeled antibody-colloidal gold complex is prone to aggregation or inactivation during storage, shortening the effective use period of the kit. Summary of the invention

[0013] In order to solve the problems in the prior art, the present invention provides a high-sensitivity fecal occult blood detection kit and a preparation method thereof, which comprehensively upgrades the size control of colloidal gold particles, the optimization of antibody labeling strategy, the innovation of sample pretreatment technology, etc., to create a new detection kit with excellent sensitivity and specificity, so as to achieve greater sensitivity, specificity and stability, and meet the needs of clinicians and patients for rapid and accurate diagnosis of early gastrointestinal bleeding.

[0014] The present invention solves the technical problem by adopting the following technical solutions:

[0015] The present invention aims to provide a method for preparing a high-sensitivity fecal occult blood detection kit, comprising:

[0016] Synthesis of colloidal gold particles: Add chloroauric acid to deionized water, heat it, and then drop sodium citrate solution to generate colloidal gold particles;

[0017] Surface modification: The colloidal gold particles were surface modified with polyethylene glycol;

[0018] Antibody labeling: Hemoglobin-specific antibodies were covalently bound to polyethylene glycol-modified colloidal gold surfaces using N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide as cross-linking agents;

[0019] Construction of bispecific antibody system: The screened and optimized hemoglobin-specific antibody is fixed on the detection line of the nitrocellulose membrane as a capture antibody, and the hemoglobin-specific antibody bound to the colloidal gold surface constitutes a bispecific antibody system;

[0020] Sample pretreatment: dilute and homogenize the sample, add antibody-coated magnetic beads, and then separate the magnetic beads containing hemoglobin, wash and resuspend.

[0021] Furthermore, chloroauric acid was added to a flask filled with deionized water, and the flask was placed in a constant temperature water bath at 80°C and heated to boiling. After sodium citrate solution was added dropwise, the solution changed from yellow to dark red, and colloidal gold was generated. The solution was then kept warm at 80°C with constant stirring to promote the maturation and stability of the colloidal gold particles.

[0022] Furthermore, the average particle size of the synthesized colloidal gold particles is less than 5 nm.

[0023] Furthermore, the method for screening and optimizing hemoglobin-specific antibodies is to screen high-affinity hemoglobin-specific antibodies through phage display technology or hybridoma technology, and then ensure the high purity and consistency of the antibodies through affinity purification and in vitro amplification.

[0024] Furthermore, the method for preparing antibody-coated magnetic beads is to use magnetic beads based on polystyrene or silica, and to fix hemoglobin-specific antibodies on the surface thereof by covalent bonding.

[0025] Furthermore, during sample pretreatment, the magnetic beads containing hemoglobin are separated using an external magnet, excess liquid and unbound impurities are removed, and then the magnetic beads are washed and resuspended.

[0026] A high-sensitivity fecal occult blood detection kit is prepared by adopting the preparation method of the high-sensitivity fecal occult blood detection kit.

[0027] Aiming at the shortcomings of existing fecal occult blood detection kits in terms of sensitivity, specificity, repeatability and stability, the present invention proposes a new detection kit based on improved colloidal gold immunochromatography technology. Through a series of breakthroughs and optimizations in key technologies, a high-precision and high-reliability detection platform has been successfully constructed, including the synthesis of ultrafine colloidal gold particles, the selection and labeling of antibodies, the improvement of sample pretreatment procedures, and strict performance testing and clinical evaluation, as follows:

[0028] (I) Synthesis and labeling of ultrafine colloidal gold particles

[0029] Ultrafine colloidal gold synthesis: Using improved chemical synthesis methods, ultrafine colloidal gold with an average particle size of less than 5nm is prepared, which significantly enhances signal response. This particle has a larger surface area and can carry more antibody molecules, thereby improving detection sensitivity.

[0030] Surface modification and stability enhancement: Use polyethylene glycol (PEG) and other biocompatible materials to modify the surface of colloidal gold particles to prevent aggregation under different pH conditions, extend the shelf life of the kit, and ensure stability in various sample environments.

[0031] (II) Antibody selection and labeling optimization

[0032] Specific antibody screening: Through high-throughput screening technology, humanized monoclonal antibodies that bind highly specifically to human hemoglobin are selected to minimize nonspecific binding and improve detection specificity.

[0033] Efficient labeling strategy: Covalent bonding rather than physical adsorption is used to firmly bind antibodies to colloidal gold particles, preventing antibody shedding or inactivation, and ensuring stability and repeatability during the detection process.

[0034] 3. Sample pretreatment and testing process improvement

[0035] Sample purification and concentration: Introduce sample pretreatment technology based on magnetic bead separation. Through magnetic beads coated with specific antibodies, trace hemoglobin in fecal samples can be efficiently enriched to further enhance the detection signal and improve the detection sensitivity.

[0036] Rapid incubation and instant reading: Optimize immunochromatography conditions and shorten incubation time to a few minutes, facilitating instant test results, suitable for outpatient and home self-testing scenarios.

[0037] Compared with the prior art, the beneficial technical effects of the present invention are:

[0038] 1. Significantly improve detection sensitivity

[0039] Ultrafine colloidal gold particles: Ultrafine colloidal gold prepared by improved chemical synthesis technology has a larger surface area and higher labeling density, which enables more antibodies to be carried in each unit volume, greatly enhancing the signal response capability and detection sensitivity, and accurate detection results can be obtained even at very low hemoglobin concentrations.

[0040] 2. Enhance specificity and reduce false positives

[0041] Bispecific antibody system: Carefully selected high-affinity hemoglobin-specific antibodies constitute a bispecific antibody system, which significantly reduces the chance of nonspecific binding and ensures that only the real target can trigger the detection reaction, greatly improving the specificity and accuracy of the test and reducing the occurrence of false positives.

[0042] 3. Improve stability and durability

[0043] Surface functionalization: Surface modification of colloidal gold with biocompatible materials such as PEG not only enhances the stability between particles, but also improves the efficiency and durability of antibody labeling, ensuring that the kit maintains good performance during long-term storage or extreme environmental conditions.

[0044] 4. Simplify the operation process and improve the user experience

[0045] Efficient sample pretreatment technology: Through integrated enzymatic digestion, filtration and centrifugation steps, interfering substances in the sample are effectively removed, while efficient enrichment of trace hemoglobin is achieved, which greatly simplifies the sample pretreatment process, making the test faster and easier, and more suitable for outpatient and even home self-testing scenarios.

[0046] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0048] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0049] Example 1

[0050] A method for preparing a high-sensitivity fecal occult blood detection kit comprises the following steps:

[0051] 1. Synthesis and labeling of ultrafine colloidal gold particles

[0052] 1. Synthesis method

[0053] (1) Raw material preparation: Use high-quality chloroauric acid (HAuCl 4 ·3H 2 O) as a gold source, sodium citrate (C 6 H 5 Na 3 O 7 ·2H 2 O) as reducing agent and stabilizer, and deionized water as solvent.

[0054] (2) Synthesis steps:

[0055] Add an appropriate amount of chloroauric acid to a flask filled with deionized water and adjust to a predetermined concentration;

[0056] Place the flask in a constant temperature water bath at 80°C and heat to boiling;

[0057] Slowly add the pre-dissolved sodium citrate solution, and the solution will change from yellow to dark red, indicating the formation of colloidal gold;

[0058] Continue to keep the temperature at 80°C for several hours, stirring continuously to promote the maturation and stability of the colloidal gold particles;

[0059] The particle size distribution of the colloidal gold particles was confirmed using a transmission electron microscope (TEM) to ensure that the average particle size was less than 5 nm.

[0060] 2. Surface modification and antibody labeling

[0061] (1) Surface modification:

[0062] Polyethylene glycol (PEG) is used to modify the surface of colloidal gold particles to increase their biocompatibility and stability and prevent aggregation in physiological salinity and pH environments.

[0063] (2) Antibody labeling:

[0064] Hemoglobin-specific antibodies were covalently bound to the PEG-modified colloidal gold surface using N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) as cross-linking agents;

[0065] Optimize the ratio and conditions of antibody labeling to ensure that each colloidal gold particle surface is evenly covered with sufficient antibodies while maintaining the spatial conformation and biological activity of the antibodies.

[0066] 2. Construction of bispecific antibody system

[0067] 1. Antibody screening and optimization: First, high-affinity hemoglobin-specific antibodies are screened through phage display technology or hybridoma technology, and then affinity purification and in vitro amplification are performed to ensure the high purity and consistency of the antibodies.

[0068] 2. Build a system:

[0069] One of the antibodies is fixed on the detection line of the nitrocellulose membrane as the capture antibody;

[0070] Another antibody is labeled on colloidal gold particles and acts as a signal reporter antibody;

[0071] During construction, special attention is paid to the spatial arrangement and interaction between antibodies to avoid steric effects and ensure that the two can work synergistically on the same target without interfering with each other.

[0072] 3. Sample pretreatment and enrichment technology

[0073] 1. Magnetic bead separation technology

[0074] Preparation of magnetic beads: Use polystyrene or silica-based magnetic beads, and fix hemoglobin-specific antibodies on their surface by covalent bonding to make antibody-coated magnetic beads.

[0075] 2. Sample processing flow:

[0076] Fecal samples were initially diluted and homogenized to disperse solid components and release potential hemoglobin;

[0077] Add the pre-treated, antibody-coated magnetic bead mixture and gently shake it to allow the magnetic beads to fully contact and bind to the hemoglobin molecules in the sample;

[0078] The magnetic beads containing hemoglobin are separated using an external magnet to remove excess fluid and unbound impurities;

[0079] Wash and resuspend the beads to make them suitable for subsequent immunochromatographic detection.

[0080] Test Example 1: Sensitivity Test

[0081] Objective: To evaluate the detection capability of the kit for low concentration hemoglobin.

[0082] Methods: A series of gradient dilutions of hemoglobin standards were prepared, gradually decreasing from ultra-high concentration to near the expected detection limit, and the kit was used for continuous detection.

[0083] Evaluation criteria: Determine the minimum detection limit of the kit, that is, the lowest hemoglobin concentration that can stably give a positive signal.

[0084] Sample preparation: Prepare a series of hemoglobin reference substances with concentrations ranging from 1 ng / mL to 50 ng / mL, with a standard point configured at every 1 ng / mL interval.

[0085] Experimental steps: Three batches of the test reagents produced by our company were tested for three consecutive days, and the test was repeated 20 times each time. The lowest concentration with a positive detection rate of 95% was taken as the minimum detection limit. The samples with the detection limit concentration level were tested 40 times to verify the minimum detection limit.

[0086] Results: When the concentration is 5ng / mL, the positive detection rate is 95%, that is, C95. Therefore, this product uses the lowest concentration with a 95% positive detection rate, that is, 5ng / mL, as the minimum detection limit. The detection limit concentration level samples were tested 40 times to verify the minimum detection limit. The results showed that when the minimum detection limit was 5ng / mL, the positive detection rate also reached more than 95%.

[0087] Conclusion: This indicates that the kit has extremely high sensitivity and can capture extremely small changes in hemoglobin.

[0088] Test Example 2: Specificity Test

[0089] Purpose: To test the resistance of the test kit to non-blood related substances.

[0090] Method: Potential interfering substances, such as meat, vegetable extracts and other common digestive tract secretions, were added to the samples to compare their effects on the test results.

[0091] Evaluation criteria: Evaluate the ability of the test kit to correctly identify hemoglobin in the presence of multiple interferents, that is, the false positive rate.

[0092] Sample selection: Select positive samples containing human hemoglobin, as well as other interferents that may cause cross-reactions, such as animal hemoglobin, plant protein, antibiotics, etc.

[0093] Experimental steps: Use samples with and without interfering substances for testing and compare the test results.

[0094] Data processing: Compare the test results of positive samples and interferent samples to evaluate the anti-interference ability of the kit in the face of interferents.

[0095] Results: Among all the interferents tested, only the human hemoglobin sample showed an obvious positive signal, and the other samples were negative.

[0096] Conclusion: The results show that the kit has excellent specificity and almost no non-specific binding occurs

[0097] Test Example 3: Repeatability Test

[0098] Objective: To investigate the consistency of the test kit between batches and within batches.

[0099] Methods: Multiple batches of test kits were selected and standard samples of the same concentration were used to perform multiple independent tests.

[0100] Evaluation criteria: Evaluate the test results between all batches and within the same batch.

[0101] Sample selection: Use a hemoglobin reference with a concentration of 5 ng / mL as the test sample.

[0102] Experimental steps: Under the same conditions, the same sample was tested 20 times using the same batch of kits.

[0103] Results: All 40 test results were positive and the color was consistent

[0104] Conclusion: This shows that the kit has good repeatability and ensures the reliability of the test results.

[0105] Test Example 4: Stability Test

[0106] Purpose: To determine the performance retention of the kit under different storage conditions.

[0107] Method: The test kit was stored at the recommended temperature, high temperature and low temperature respectively, and the performance was tested at regular intervals until a performance degradation was observed.

[0108] Evaluation criteria: Record the shelf life of each set of test kits to ensure that they can maintain their effectiveness for at least one year under actual transportation and storage conditions.

[0109] Sample selection: Select a hemoglobin reference with a concentration of 5 ng / mL.

[0110] Experimental steps: Store the test kit at three temperature ranges: 2-8°C, 37°C, and -20°C, and perform testing using reference materials every other month for one year.

[0111] Data processing: Record each test result and evaluate the impact of different storage conditions on the performance of the kit.

[0112] Results: Under storage conditions of 2-8°C, the detection performance of the kit hardly decreased within one year; under 37°C conditions, the performance began to decline slightly, but remained above 95% before the 12th month; at -20°C, it showed better long-term memory stability.

[0113] Conclusion: This kit has excellent long-term stability under the recommended storage conditions and is adaptable to different logistics and storage conditions.

[0114] Test Example 5: Clinical Sample Testing

[0115] Sample collection: Stool samples from 200 patients with suspected GI bleeding were collected from the gastroenterology and gastrointestinal surgery departments of three large hospitals, including confirmed GI bleeding cases and healthy controls.

[0116] Testing method: All samples were tested using this kit, which was performed by an independent third-party laboratory and the results were compared with the gold standard (colonoscopy).

[0117] Data processing: Calculate the kit's accuracy and kappa value. Use SPSS and other statistical software to calculate the kit's total accuracy, positive accuracy, negative accuracy and Kappa consistency index to evaluate its clinical diagnostic efficacy.

[0118] result:

[0119] Test result matching four-grid table

[0120]

[0121] Summary table of positive coincidence rate, negative coincidence rate, total coincidence rate and kappa value

[0122] Positive / negative sample testing Positive coincidence rate 98.03％ Negative compliance rate 96.94％ Total compliance rate 97.50％ Kappa value 0.950

[0123] Conclusion: The clinical trial results show that the kit has high sensitivity and specificity, can effectively assist in the early diagnosis of gastrointestinal bleeding, and the kit is highly consistent with the gold standard, which helps to reduce unnecessary invasive examinations.

[0124] In summary, through the implementation of the technical solution of the present invention, the sensitivity and specificity of the fecal occult blood detection kit can be greatly improved, the possibility of nonspecific binding can be reduced, and at the same time, the magnetic bead separation technology can be used to achieve a high enrichment of trace hemoglobin in the sample, providing more reliable data support for clinical testing.

[0125] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0126] The embodiments of the present invention are described above, but the present invention is not limited to the above-mentioned specific implementation modes. The above-mentioned specific implementation modes are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which are all within the protection of the present invention.

Claims

1. A method for preparing a high-sensitivity fecal occult blood detection kit, characterized in that: include: Synthesis of colloidal gold particles: Add chloroauric acid to deionized water, then heat it, and then drop sodium citrate solution to generate colloidal gold particles; Surface modification: The colloidal gold particles were surface modified with polyethylene glycol; Antibody labeling: Hemoglobin-specific antibodies were covalently bound to polyethylene glycol-modified colloidal gold surfaces using N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide as cross-linking agents; Construction of bispecific antibody system: The screened and optimized hemoglobin-specific antibody is fixed on the detection line of the nitrocellulose membrane as a capture antibody, and the hemoglobin-specific antibody bound to the colloidal gold surface constitutes a bispecific antibody system; Sample pretreatment: dilute and homogenize the sample, add antibody-coated magnetic beads, and then separate the magnetic beads containing hemoglobin, wash and resuspend.

2. A method for preparing a high-sensitivity fecal occult blood detection kit as claimed in claim 1, characterized in that: Add chloroauric acid to a flask filled with deionized water, place the flask in a constant temperature water bath at 70-90°C, heat to boiling, and add sodium citrate solution. The solution changes from yellow to dark red, generating colloidal gold. Then continue to keep warm at 70-90°C, stirring constantly to promote the maturation and stability of the colloidal gold particles.

3. A method for preparing a high-sensitivity fecal occult blood detection kit as claimed in claim 2, characterized in that: The average particle size of the synthesized colloidal gold particles is less than 5nm.

4. The method for preparing a high-sensitivity fecal occult blood detection kit according to claim 1, characterized in that: The method for screening and optimizing hemoglobin-specific antibodies is to screen high-affinity hemoglobin-specific antibodies through phage display technology or hybridoma technology, and then ensure the high purity and consistency of the antibodies through affinity purification and in vitro amplification.

5. The method for preparing a high-sensitivity fecal occult blood detection kit according to claim 1, characterized in that: The preparation method of antibody-coated magnetic beads is to use polystyrene or silica as the basic material of magnetic beads, and fix the hemoglobin-specific antibody on the surface by covalent bonding.

6. The method for preparing a high-sensitivity fecal occult blood detection kit according to claim 1, characterized in that: During sample pretreatment, the magnetic beads containing hemoglobin are separated using an external magnet, excess liquid and unbound impurities are removed, and the beads are then washed and resuspended.

7. A high-sensitivity fecal occult blood detection kit, characterized in that: The method is adopted to prepare a high-sensitivity fecal occult blood detection kit as described in any one of claims 1 to 6.

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