Preparation method of colloidal gold detection reagent, product and application thereof

Abstract

This application relates to the field of in vitro bioassay technology, and discloses a method for preparing a colloidal gold assay reagent, its product, and its application. The method for preparing the colloidal gold assay reagent comprises five steps: colloidal gold preparation, antibody labeling, double blocking, centrifugal purification, and dilution and reconstitution. In particular, the double blocking process involves a first blocking agent selected from one or more of bovine serum albumin, ovalbumin, and casein; and a second blocking agent selected from one or more of lysine, glycine, alanine, serine, threonine, valine, and asparagine. This allows the prepared colloidal gold assay reagent to achieve high sensitivity and excellent C / T line color development when applied to FSH detection, reducing false positives caused by nonspecific adsorption and improving the accuracy of test results. Its limit of detection (LOD) is as low as 10 mIU / mL, solving the problems of low sensitivity and uneven C / T line color development in existing FSH assays.

Description

Technical Field

[0001] This application relates to the field of in vitro biological detection technology, and in particular to a method for preparing a colloidal gold detection reagent, its product, and its application. Background Technology

[0002] Human follicle-stimulating hormone (FSH) is a glycoprotein hormone secreted by the anterior pituitary gland. Its concentration changes in blood and urine are closely related to the female menstrual cycle, follicle development and ovulation process, and are an important indicator for assessing female reproductive health.

[0003] Colloidal gold immunochromatography (CIM) has been widely used for the rapid detection of FSH due to its advantages such as simplicity, speed, and lack of complex instrumentation. Its basic principle is a double-antibody sandwich method: one specific antibody is labeled onto colloidal gold particles (gold-labeled antibodies), while another antibody is immobilized in the detection line region of a nitrocellulose membrane. When the sample contains the target antigen FSH, it binds to the gold-labeled antibody and is captured by the antibody on the detection line during chromatography, resulting in aggregation and color development. The preparation of colloidal gold immunochromatographic assay reagents typically includes steps such as the preparation of colloidal gold solution, antibody labeling and blocking, and test strip assembly. The blocking step aims to cover the unoccupied active sites on the surface of the colloidal gold particles with an inert substance to prevent non-specific adsorption, thus being crucial for improving the specificity and sensitivity of the detection.

[0004] The commonly used blocking method involves adding a certain concentration of a single macromolecular inert protein, such as bovine serum albumin (BSA) solution, to the reaction system after the antibody has been labeled and bound to the colloidal gold particles. After incubation, the BSA molecules adsorb and cover the remaining area on the surface of the colloidal gold particles. Then, the excess blocking agent is removed by centrifugation and other steps to obtain the blocked gold-labeled antibody complex.

[0005] Although this method is widely used and BSA can effectively shield most active sites as a blocking agent, the BSA molecule itself has a large three-dimensional volume and specific spatial structure. When it adsorbs onto the colloidal gold surface, it mainly covers relatively flat and open areas, making it difficult to effectively penetrate and tightly fill the gaps and depressions between antibody molecules and around the binding points between antibody molecules and the gold surface. These microscopic active sites that are not completely blocked are prone to non-specific adsorption of impurities, lipids, or other interfering substances in the sample matrix during subsequent detection, leading to increased detection background and decreased signal-to-noise ratio. Specifically, in practical applications, the product may exhibit problems such as insufficient detection sensitivity, uneven color development of the detection line, or prolonged interpretation time, making it difficult to meet the needs of accurate detection. Summary of the Invention

[0006] To overcome at least one of the problems existing in the prior art, one objective of this application is to provide a method for preparing colloidal gold detection reagents. This method involves five steps: colloidal gold preparation, antibody labeling, double blocking, centrifugal purification, and dilution and reconstitution. In particular, the double blocking process introduces two blocking agents of different sizes and properties, achieving synergistic coverage of the active sites on the surface of colloidal gold particles not occupied by antibodies. This reduces the non-specific adsorption of the colloidal gold detection reagent, thus solving the technical problems of incomplete blocking of the colloidal gold particle surface and high non-specific adsorption caused by the use of single BSA blocking, resulting in low detection sensitivity, uneven color development, and significant background interference. A second objective of this application is to provide colloidal gold detection reagents prepared by the aforementioned method. A third objective of this application is to provide colloidal gold detection products containing the aforementioned colloidal gold detection reagents. A fourth objective of this application is to provide applications of the aforementioned colloidal gold detection reagents or colloidal gold detection products.

[0007] Therefore, this application adopts the following technical solution: The first aspect of this application provides a method for preparing a colloidal gold detection reagent, comprising the following steps: S1. Preparation of colloidal gold solution: Colloidal gold solution was prepared by citrate reduction method; S2, Antibody labeling: Add antibody to the colloidal gold solution to carry out a labeling reaction and form a colloidal gold-antibody complex; S3, Double blocking: A first blocking agent is added to the colloidal gold-antibody complex for a first blocking treatment; then a second blocking agent is added for a second blocking treatment; S4. Centrifugal purification: After centrifugation and removal of the supernatant, the precipitate is collected after the sealed solution is removed. S5. Dilution and Reconstitution: Add dilution buffer to the precipitate and mix to prepare colloidal gold detection reagent; The first blocking agent is selected from one or more of bovine serum albumin, ovalbumin, and casein. The second blocking agent is selected from one or more of lysine, glycine, alanine, serine, threonine, valine, and asparagine.

[0008] The preparation method of the colloidal gold detection reagent in this application involves five major steps: colloidal gold preparation, antibody labeling, double blocking, centrifugal purification, and dilution and reconstitution. In particular, the double blocking process in step S3 limits the first blocking agent to one or more of bovine serum albumin, ovalbumin, and casein. The spatial structure of these large molecular proteins fully covers the main free sites of unbound antibodies on the surface of colloidal gold particles, blocking the non-specific binding of non-target substances to these sites. The second blocking agent is selected from one or more of lysine, glycine, alanine, serine, threonine, valine, and asparagine. These small molecular amino acids can penetrate into the steric hindrance region of the large molecular protein, filling the residual free sites not covered by the first blocking agent, forming a comprehensive and precisely filled double blocking protection. Step S1 prepares a colloidal gold solution using the citrate reduction method, providing a uniform and stable carrier for subsequent antibody labeling. Step S2 performs the labeling reaction to form a colloidal gold-antibody complex. Step S4, centrifugation purification, further avoids problems such as enhanced non-specific adsorption and increased detection background caused by impurities. Step S5, dilution and reconstitution, uses a dilution buffer to adjust the concentration and maintain the stability of the colloidal gold detection reagent, extending its shelf life. Therefore, the synergistic effect of each step effectively reduces non-specific binding of colloidal gold particles to non-target substances in the sample matrix during subsequent detection, improving the signal-to-noise ratio, sensitivity, and specificity of the detection, and laying the foundation for uniform color development of the detection line.

[0009] Preferably, in step S1, the process of preparing colloidal gold solution by citrate reduction involves adding chloroauric acid to water, heating to boiling, adding trisodium citrate, stirring continuously for 15-20 minutes until the solution turns purple-red, cooling, and obtaining colloidal gold solution.

[0010] Preferably, in step S2, the pH of the solution is adjusted to weakly alkaline before adding the antibody, and then the mixed antibody of FSH mAb2 and FSH mAb4 is added, followed by labeling for 25-40 minutes. More preferably, in step S2, the pH is adjusted using a potassium carbonate or sodium carbonate solution, and then the mixed antibody of FSH mAb2 and FSH mAb4 is added, followed by labeling for 30-40 minutes. Even more preferably, in step S2, the concentration of the potassium carbonate or sodium carbonate solution is 8%-15% (w / v).

[0011] In step S1, a colloidal gold solution with uniform particle size, stable dispersion, and a specific purple-red color is prepared using the citrate reduction method, providing a stable carrier for subsequent antibody labeling. In step S2, under weakly alkaline conditions, the electrostatic adsorption between the antibody and the colloidal gold particles is stronger, which is beneficial for improving the labeling efficiency and stability of the antibody and forming a more robust colloidal gold-antibody complex. The combined use of FSH mAb2 and FSH mAb4 can synergistically bind to different epitopes of the FSH antigen, enhancing the specific affinity between the antibody and the antigen and improving detection sensitivity.

[0012] Preferably, in step S3, the first sealing agent is selected from bovine serum albumin or ovalbumin. More preferably, in step S3, the first sealing agent is selected from bovine serum albumin.

[0013] Preferably, the second sealing agent is a mixture of lysine, glycine, and serine in a mass ratio of (2~5):(1~3):(1~2). Preferably, the second sealing agent is a mixture of lysine, glycine, and serine in a mass ratio of (2.8~5):(1.5~3):(1~2).

[0014] Preferably, the concentration of the first sealing agent is 8%~13% (w / v), and the concentration of the second sealing agent is 5%~10% (w / v). More preferably, the concentration of the first sealing agent is 10%~13% (w / v), and the concentration of the second sealing agent is 5%~10% (w / v).

[0015] Preferably, in step S3, the first sealing treatment takes 10-20 minutes, and the second sealing treatment takes 10-15 minutes. More preferably, in step S3, the first sealing treatment takes 15-20 minutes, and the second sealing treatment takes 12-15 minutes.

[0016] The first blocking agent selected is a large-molecule protein, which is cost-effective, biocompatible, and can effectively achieve steric hindrance blocking. The second blocking agent is preferably a mixture of lysine, glycine, and serine in a mass ratio of (2~5):(1~3):(1~2). Lysine, under near-neutral and weakly alkaline conditions, has a positively charged ε-amino group on its side chain, while colloidal gold particles typically have a negative charge. Therefore, lysine can preferentially and firmly adsorb onto the gold surface through strong electrostatic interactions, especially onto negatively charged active sites that BSA cannot reach due to steric hindrance. Glycine is the smallest amino acid in terms of molecular weight, has no side chains, and possesses strong permeability, enabling it to penetrate... The secondary sealing agent, which fills the tiny gaps or grooves created by steric hindrance that lysine and BSA molecules cannot enter, is a powerful polar compound that can form hydrogen bonds with oxygen atoms, hydration layers, or other polar molecules that may be present on the gold surface. This bond, with a strength between electrostatic and van der Waals forces, provides another stable binding mode. Therefore, controlling the secondary sealing agent, which is dominated by lysine and synergistically composed of glycine and serine, is beneficial for filling microscopic gaps with different properties, thereby achieving a denser secondary sealing.

[0017] Preferably, in step S4, the centrifugation speed is 9000~12000 rpm, and the centrifugation time is 25~30 min. More preferably, in step S4, the centrifugation speed is 10000~12000 rpm, and the centrifugation time is 25~30 min.

[0018] Preferably, in step S5, the dilution buffer comprises a buffer solution, a dispersant, a stabilizer, and a surfactant. More preferably, the concentration of the buffer solution is 0.01~0.1 mol / L, and the pH value of the buffer solution is 8~9.

[0019] Preferably, the buffer solution is selected from PBS buffer, Tris buffer, and Hepes buffer. More preferably, the buffer solution is selected from PBS buffer or Tris buffer.

[0020] Preferably, the dispersant is selected from one or more of polyvinylpyrrolidone, polyethylene glycol, and hydroxypropyl methylcellulose. More preferably, the dispersant is selected from one or more of polyvinylpyrrolidone and polyethylene glycol.

[0021] Preferably, the stabilizer is selected from one or more of sucrose, trehalose, mannitol, sorbitol, and bovine serum albumin. More preferably, the stabilizer is selected from one or more of sucrose, trehalose, mannitol, and bovine serum albumin. Even more preferably, the stabilizer is selected from sucrose and bovine serum albumin.

[0022] Preferably, the surfactant is selected from one or more of Tween-20, Tween-80, and Triton X-100. More preferably, the surfactant is selected from one or more of Tween-20 and Triton X-100.

[0023] Step S4 involves thorough precipitation under centrifugation, effectively removing unbound free antibodies, blocking agents, and other small molecule impurities. In step S5, the dilution buffer comprises a buffer solution, a dispersant, a stabilizer, and a surfactant. The buffer solution provides a physiologically compatible pH and ionic environment. The dispersant effectively prevents the aggregation of colloidal gold particles; the stabilizer maintains the activity of the antibody and colloidal gold; and the surfactant primarily improves the wettability and chromatographic flowability of each component. This system works together to ensure the final reagent exhibits good dispersion stability and chromatographic properties.

[0024] The second aspect of this application provides a colloidal gold detection reagent prepared according to the method for preparing colloidal gold detection reagents described in the first aspect of this application.

[0025] This colloidal gold detection reagent features thorough sealing, low non-specific adsorption, high sensitivity, and strong stability, resulting in accurate and reliable detection results.

[0026] A third aspect of this application provides a colloidal gold detection product, including a colloidal gold test strip, a colloidal gold test card, or a colloidal gold test pen coated with the colloidal gold detection reagent according to the second aspect of this application.

[0027] Preferably, in the colloidal gold detection product, the colloidal gold test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, wherein the conjugate pad is coated with the colloidal gold detection reagent according to the second aspect of this application.

[0028] In the colloidal gold test strip, the sample pad is used to receive the liquid sample to be tested, such as urine or serum, and can perform preliminary filtration or treatment of the sample; the conjugate pad is located downstream of the sample pad and is coated with the colloidal gold detection reagent according to the second aspect of this application. In use, when the sample to be tested flows through the conjugate pad, the gold-labeled antibody is reconstituted and carries the target antigen for chromatography; the nitrocellulose membrane is located downstream of the conjugate pad and is coated with a capture antibody that can bind to another epitope of the target antigen. This is the area where a specific immune reaction occurs and the results are displayed. The nitrocellulose membrane has a detection line and a control line for capturing the antigen-gold-labeled antibody complex, thereby aggregating and developing color; the absorbent pad is located at the end of the nitrocellulose membrane and provides chromatographic driving force by absorbing the waste liquid after flowing through the detection area and the control area, ensuring that the sample continues to flow forward.

[0029] The fourth aspect of this application provides the application of the colloidal gold detection reagent according to the second aspect of this application or the colloidal gold detection product according to the third aspect of this application in FSH detection.

[0030] Compared with the prior art, this application has at least the following beneficial effects: 1) The preparation method of the colloidal gold detection reagent in this application involves five major steps: colloidal gold preparation, antibody labeling, double blocking, centrifugal purification, and dilution and reconstitution. In particular, the double blocking process introduces two blocking agents of different sizes and properties, which reduces the non-specific adsorption of the colloidal gold detection reagent and the colloidal gold test paper, thereby improving its sensitivity and color uniformity when applied to FSH detection, reducing the interpretation time. Its limit of detection (LOD) is as low as 10 mIU / mL, color development can be achieved within 1 min, and the C / T line of the test paper can be basically interpreted within 10 min. The non-specific adsorption is low, and the detection results for interfering solutions of 200 mIU / mL LH, 100 μIU / mL TSH, and 1000 mIU / mL HCG are all negative.

[0031] 2) The double sealing ensures that the colloidal gold particles reduce local aggregation or non-specific retention during the chromatography process, thereby making the detection results develop faster and the bands more uniform, thus improving the accuracy and reproducibility of the detection results. Detailed Implementation

[0032] The following detailed description of the contents of this application is provided through specific embodiments, comparative examples, and tables, but is not limited to all the arguments and data.

[0033] It is particularly important to emphasize that, unless otherwise specified, the raw materials, reagents or devices used in this application can be obtained from conventional commercial sources.

[0034] Example of preparing dilution buffer: The steps for preparing a dilution buffer are as follows: Take 40 mL of 0.01 mol / L PBS buffer and add polyvinylpyrrolidone to a final concentration of 0.25% (w / v), sucrose to a final concentration of 3% (w / v), and BSA to a final concentration of 0.1% (w / v) in sequence. Stir thoroughly until dissolved, then add Tween-20 to a final concentration of 0.1% (v / v). Mix well and adjust the pH to 8.0 with dilute hydrochloric acid or sodium hydroxide solution. Finally, bring the volume to 50 mL with ultrapure water. After sterilization by filtration through a 0.22 μm filter membrane, store at 4 °C for later use.

[0035] Examples of colloidal gold detection reagents: The preparation method of the colloidal gold detection reagent of this application specifically includes the following steps: S1. Preparation of colloidal gold solution: Add 0.8~1g chloroauric acid to 100mL water, heat to boiling, add 2~2.4g trisodium citrate, stir continuously for 15~20min until the solution turns purple-red, cool, and obtain colloidal gold solution; S2, Antibody labeling: Add 2-5 mL of 8%-15% (w / v) potassium carbonate or sodium carbonate solution to the colloidal gold solution to adjust the pH, then add 1.5-2 mg of a mixed antibody of FSH mAb2 and FSH mAb4 in a mass ratio of (1-3):1, label for 30-40 min to form a colloidal gold-antibody complex. S3, Double blocking: Add the first blocking agent to the colloidal gold-antibody complex for a first blocking treatment of 10-20 min; then add the second blocking agent for a second blocking treatment of 10-15 min; S4. Centrifugal purification: Centrifuge the sealed solution at 9000~12000 rpm for 25~30 min, remove the supernatant and collect the precipitate; S5. Dilution and Reconstitution: Add 30-50 mL of dilution buffer to the precipitate, mix well, and prepare the colloidal gold detection reagent.

[0036] Regarding step S1, in some specific implementations, the amount of chloroauric acid can be 0.8g, 0.9g, or 1g, the amount of trisodium citrate can be 2, 2.1, 2.3, or 2.4g, and the stirring time can be 15min, 18min, or 20min.

[0037] Regarding step S2, in some specific embodiments, the concentration of potassium carbonate or sodium carbonate solution can be 8%, 10%, 12% or 15% (w / v), the mass ratio of the mixed antibody of FSH mAb2 and FSH mAb4 can be 1:1, 1.5:1, 2:1 or 3:1, and the labeling time can be 30 min, 35 min or 40 min.

[0038] Regarding step S3, in some specific embodiments, the first blocking agent may be selected from one or more of bovine serum albumin, ovalbumin, and casein, and the concentration of the first blocking agent may be 8%, 10%, or 13% (w / v); the second blocking agent may be selected from one or more of lysine, glycine, alanine, serine, threonine, valine, and asparagine, and the second blocking agent may be a mixture of lysine, glycine, and serine in a mass ratio of 2:1:1, 3:2:1, 4:1:2, 4:2:1, or 5:3:1, and the concentration of the second blocking agent may be 5%, 8%, or 10% (w / v); the first blocking treatment time may be 10 min, 15 min, or 20 min; and the second blocking treatment time may be 10 min, 12 min, or 15 min.

[0039] Regarding step S4, in some specific implementations, the centrifugation speed can be 9000 rpm, 10000 rpm, 11000 rpm or 12000 rpm, and the centrifugation time can be 25 min or 30 min.

[0040] Regarding step S5, in some specific embodiments, the dilution buffer includes a buffer, a dispersant, a stabilizer, and a surfactant. The concentration of the buffer is 0.01 mol / L, 0.05 mol / L, or 0.1 mol / L, and the pH value of the buffer is 8-9. The buffer may be selected from PBS buffer, Tris buffer, or Hepes buffer. The dispersant may be selected from one or more of polyvinylpyrrolidone, polyethylene glycol, and hydroxypropyl methylcellulose. The stabilizer may be selected from one or more of sucrose, trehalose, mannitol, sorbitol, and bovine serum albumin. The surfactant may be selected from one or more of Tween-20, Tween-80, and Triton X-100.

[0041] Based on the preparation method of the colloidal gold detection reagent of this application, the following examples and comparative examples are listed: Example 1

[0042] A method for preparing a colloidal gold detection reagent specifically includes the following steps: S1. Preparation of colloidal gold solution: Add 0.9g chloroauric acid to 100mL water, heat to boiling, add 2.3g trisodium citrate, stir continuously for 15~20min until the solution turns purple-red, cool, and obtain colloidal gold solution; S2, Antibody labeling: Add 2 mL of 10% (w / v) potassium carbonate solution to the colloidal gold solution to adjust the pH, then add 1.8 mg of a mixed antibody of FSH mAb2 and FSH mAb4 in a mass ratio of 2:1, label for 30 min to form a colloidal gold-antibody complex. S3, Double blocking: Add 0.5 mL of 10% (w / v) BSA solution prepared with 0.1 mol / L potassium carbonate solution as the first blocking agent to the colloidal gold-antibody complex, and stir for 15 min for the first blocking treatment; then add 0.4 mL of 5% (w / v) lysine aqueous solution as the second blocking agent, and continue stirring for 10 min for the second blocking treatment; S4. Centrifugal purification: Centrifuge the sealed solution at 10,000 rpm for 30 min, remove the supernatant, and collect the precipitate; S5. Dilution and Reconstitution: Add 40 mL of dilution buffer to the precipitate, mix well, and prepare the colloidal gold detection reagent. Example 2

[0043] The preparation method of a colloidal gold detection reagent is the same as in Example 1, except that in Example 2, the first blocking agent in step S3 is replaced with 0.5 mL of 10% (w / v) ovalbumin solution prepared with 0.1 mol / L potassium carbonate solution. Example 3

[0044] The preparation method of a colloidal gold detection reagent is the same as in Example 1, except that the second blocking agent in step S3 of Example 3 is replaced with 0.4 mL of an aqueous solution of a mixture of lysine, glycine and serine with a concentration of 5% (w / v) and a mass ratio of 4:2:1. Example 4

[0045] The preparation method of a colloidal gold detection reagent is the same as in Example 1, except that the second blocking agent in step S3 of Example 4 is replaced with 0.4 mL of an aqueous solution of a mixture of lysine, glycine and serine with a concentration of 5% (w / v) and a mass ratio of 2:1:1.

[0046] Comparative Example 1: The preparation method of a colloidal gold detection reagent is the same as in Example 1, except that the first blocking agent in step S3 of Comparative Example 1 is replaced with 0.5 mL of 16% (w / v) BSA solution prepared with 0.1 mol / L potassium carbonate solution. After stirring for 25 min for the first blocking treatment, the process proceeds directly to step S4.

[0047] Comparative Example 2: The preparation method of a colloidal gold detection reagent is the same as that in Example 1, except that the second blocking agent in step S3 of Comparative Example 2 is replaced with 0.4 mL of an aqueous solution of 5% (w / v) arginine.

[0048] Comparative Example 3: The preparation method of a colloidal gold detection reagent is the same as in Example 3, except that the second blocking agent in step S3 of Comparative Example 3 is replaced with 0.4 mL of an aqueous solution of a mixture of lysine, glycine and serine with a concentration of 5% (w / v) and a mass ratio of 3:4:3.

[0049] Application Examples 1-4, Comparative Examples 1-3: Preparation of colloidal gold test strips: Using a 60mm × 300mm white PVC substrate as a carrier, a glass fiber sample pad, after being impregnated and dried with a treatment solution containing 0.1 mol / L Tris-HCl and 2% (w / v) Triton X-100 at pH 8.0, was adhered to one end of the substrate. 1 mL each of the colloidal gold detection reagents prepared in Examples 1-4 and Comparative Examples 1-3 were linearly coated onto the glass fiber pad using a precision gold spraying spectrometer at a spraying rate of 1 μL / mm, forming a 6mm wide reagent strip. After drying at 37°C and <20% humidity for 20 hours, the strip was overlapped with the end of the sample pad by 1.5 mm. The nitrocellulose membrane, coated with FSH-capturing monoclonal antibody mAb1 (detection line T line) and goat anti-mouse IgG antibody (control line C line), is overlapped by 2.0 mm with the end of the conjugate pad and then overlapped by 2.0 mm with the end of the absorbent pad and the end of the nitrocellulose membrane. Finally, the assembled laminated plate is cut into test strips with a width of 4.0 mm to obtain the corresponding colloidal gold test strips for application examples 1-4 and application comparative examples 1-3.

[0050] Material performance testing: The colloidal gold test strips used in Examples 1-4 and Comparative Examples 1-3 were subjected to various performance tests, and the test methods are as follows: 1. Sensitivity Test: Take 100 mL of 0.01 mol / L pH 7.4 PBS buffer, add 0.1 g BSA, stir until dissolved, add FSH standard to prepare FSH samples with concentration gradients of 0, 10, 25, and 50 mIU / mL. Test 10 test strips in parallel for each concentration. Immerse the test strips in different concentrations of standard samples for 5-10 seconds, remove them and place them horizontally, observe the color development of the C / T lines, and record the time when the T line first appears as a clearly visible red line distinct from the background. Record this as the T line initial appearance time. According to the color chart, read the C line at 5 minutes, the T line at 10 minutes, and stop reading at 15 minutes. The lowest standard concentration that can clearly identify the T line is taken as the limit of detection (LOD). Finally, the sensitivity result of each test strip is the mode of the LOD values ​​of 10 parallel tests.

[0051] 2. Specificity Test: To evaluate the specificity of the test strips, high concentrations of potential interfering agent solutions were prepared: 200 mIU / mL of luteinizing hormone (LH), 100 μIU / mL of thyroid-stimulating hormone (TSH), and 1000 mIU / mL of human chorionic gonadotropin (HCG). Using these solutions, the colloidal gold test strips used in Examples 1-4 and Comparative Examples 1-3 were tested, with each test strip being tested 10 times in parallel under each interfering agent solution. The testing and interpretation methods were the same as the sensitivity test; a negative result was indicated by no T line and normal C line development.

[0052] 3. Color development performance test: FSH samples with concentrations of 10, 25, and 50 mIU / mL were used, and each test strip was tested 10 times in parallel. The test strips were immersed in the standard samples of different concentrations for 5-10 seconds, removed and placed horizontally to observe the color development of the C / T line. The T line was read according to the color card at 5, 10, and 15 minutes. The reading was stopped after 15 minutes. The color intensity of the T line was qualitatively rated according to the three-level standard of "strong, medium, and weak". The continuity, color uniformity, and edge clarity of the T line were qualitatively rated according to the three-level standard of "excellent, good, and poor" for color development uniformity.

[0053] The test performance of the colloidal gold test strips used in Examples 1-4 and Comparative Examples 1-3 is shown in Table 1 below:

[0054] In the colloidal gold test strips of Examples 1-4, the preparation method of the colloidal gold detection reagent involves five major steps: colloidal gold preparation, antibody labeling, double blocking, centrifugal purification, and dilution and reconstitution. In particular, the synergistic effect of the first and second blocking agents in the double blocking process results in high detection sensitivity of the colloidal gold test strips prepared using the method of this application. The limit of detection (LOD) is as low as 10 mIU / mL, the T line begins to develop color within 1 minute, the color uniformity reaches the excellent level, and the non-specific adsorption is low. The detection results for interfering solutions of 200 mIU / mL LH, 100 μIU / mL TSH, and 1000 mIU / mL HCG are all negative. This indicates that the colloidal gold detection reagent and test strip products obtained by the preparation method of this application have significantly optimized the blocking effect and detection performance, effectively solving the technical problems of low detection sensitivity, uneven color development, and large background interference in the prior art.

[0055] Compared to Application Example 1, Comparative Example 1 did not add a second blocking agent in step S3 of its colloidal gold detection reagent preparation method. Instead, it only performed single blocking by increasing the concentration of BSA and extending the blocking time. The results showed that the LOD of Comparative Example 1 was 15-20 mIU / mL, the initial T-line appearance time was longer than that of Application Example 1, and the color uniformity was poor. In the specificity test, the detection results for the interfering solution of 1000 mIU / mL HCG were all weakly positive. This may be because simply increasing the concentration of the macromolecular blocking agent and extending the time cannot overcome the limitations caused by its steric hindrance, and it still cannot penetrate to the tiny gaps to fill the residual sites, thus not fundamentally solving the problem of incomplete blocking. At the same time, excessively high BSA concentrations may cause protein molecules to aggregate, thereby interfering with the dispersibility of colloidal gold particles and the antigen-antibody binding efficiency, resulting in slow sample climbing speed and a still high level of non-specific adsorption.

[0056] Compared with Application Example 1, Comparative Example 2 replaced the second blocking agent in step S3 of the preparation method of the colloidal gold detection reagent with arginine. The results showed that the LOD of Comparative Example 2 was 25 mIU / mL, the colorimetric uniformity was good, and the specificity test showed weak positive results for the detection of interfering solution 200 mIU / mL LH. It can be seen that although arginine is also an amino acid, its molecular structure and charge characteristics differ from those of lysine, glycine, and serine preferred in this application. Its compatibility with residual sites on the colloidal gold surface is not as good as the range of second blocking agents specified in this application, and it cannot efficiently fill the residual sites. At the same time, its binding stability may be insufficient, and it is easy to detach from the colloidal gold surface, resulting in poor blocking effect and high non-specific adsorption, which in turn affects the detection sensitivity and colorimetric efficiency.

[0057] Compared to Application Example 3, Comparative Example 3 used a mixture of lysine, glycine, and serine in a mass ratio of 3:4:3 as the second blocking agent in step S3 of its colloidal gold detection reagent preparation method. This mixture is not within the preferred range of this application. The results showed that Comparative Example 3 had a LOD of 10-25 mIU / mL, a T-line initial appearance time of 40-50 s, and good color uniformity. This may be because insufficient lysine content led to inadequate coverage of its core active sites, while excessive glycine and serine content may have triggered competitive binding, thereby disrupting the synergistic blocking mechanism of the three amino acids. Simultaneously, the imbalance in proportions led to decreased stability of the blocking agent layer, thus affecting the detection sensitivity and color development efficiency.

[0058] Obviously, the above embodiments of this application are merely examples for clearly illustrating this application, and are not intended to limit the implementation of this application. For those skilled in the art, other variations or modifications can be made based on the above description. Any obvious variations or modifications derived from the technical solutions of this application are still within the protection scope of this application.

Claims

1. A method for preparing a colloidal gold detection reagent, characterized in that, Includes the following steps: S1. Preparation of colloidal gold solution: Colloidal gold solution was prepared by citrate reduction method; S2, Antibody labeling: Add antibody to the colloidal gold solution to carry out a labeling reaction and form a colloidal gold-antibody complex; S3, Double blocking: A first blocking agent is added to the colloidal gold-antibody complex for a first blocking treatment; Then add a second sealing agent for a second sealing treatment; S4. Centrifugal purification: After centrifugation and removal of the supernatant, the precipitate is collected after the sealed solution is removed. S5. Dilution and Reconstitution: Add dilution buffer to the precipitate and mix to prepare colloidal gold detection reagent; The first blocking agent is selected from one or more of bovine serum albumin, ovalbumin, and casein. The second blocking agent is selected from one or more of lysine, glycine, alanine, serine, threonine, valine, and asparagine.

2. The method for preparing the colloidal gold detection reagent according to claim 1, characterized in that, In step S1, the process of preparing colloidal gold solution by citrate reduction method is as follows: chloroauric acid is added to water, heated to boiling, trisodium citrate is added, and stirring is continued for 15-20 minutes until the solution turns purple-red. After cooling, colloidal gold solution is obtained.

3. The method for preparing the colloidal gold detection reagent according to claim 1, characterized in that, In step S2, the pH of the solution is adjusted to weakly alkaline before adding the antibody, and then a mixed antibody of FSH mAb2 and FSH mAb4 is added and labeled for 25-40 minutes.

4. The method for preparing the colloidal gold detection reagent according to claim 1, characterized in that, In step S3, the first sealing agent is selected from bovine serum albumin or ovalbumin; The second sealing agent is a mixture of lysine, glycine, and serine in a mass ratio of (2~5):(1~3):(1~2); The first sealing process takes 10-20 minutes; The second sealing process takes 10-15 minutes.

5. The method for preparing the colloidal gold detection reagent according to claim 1, characterized in that, In step S4, the centrifugation speed is 9000~12000 rpm, and the centrifugation time is 25~30 min.

6. The method for preparing the colloidal gold detection reagent according to claim 1, characterized in that, In step S5, the dilution buffer includes a buffer solution, a dispersant, a stabilizer, and a surfactant; The buffer solution is selected from one of PBS buffer, Tris buffer, and Hepes buffer. And / or, the dispersant is selected from one or more of polyvinylpyrrolidone, polyethylene glycol, and hydroxypropyl methylcellulose; And / or, the stabilizer is selected from one or more of sucrose, trehalose, mannitol, sorbitol, and bovine serum albumin; And / or, the surfactant is selected from one or more of Tween-20, Tween-80, and Triton X-100.

7. A colloidal gold detection reagent, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. A colloidal gold detection product, characterized in that, This includes colloidal gold test strips, colloidal gold test cards, or colloidal gold test pens coated with the colloidal gold test reagent as described in claim 7.

9. The colloidal gold testing product according to claim 8, characterized in that, The colloidal gold test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, wherein the conjugate pad is coated with the colloidal gold detection reagent of claim 7.

10. The application of a colloidal gold detection reagent as described in claim 7 or a colloidal gold detection product as described in claim 8 in FSH detection.

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