Enhanced colloidal gold immunochromatography test strip pretreatment liquid and preparation method thereof

Abstract

The invention belongs to the technical field of colloidal gold immunochromatography test strips, and particularly relates to an enhanced colloidal gold immunochromatography test strip pretreatment solution and a preparation method thereof.The pretreatment solution comprises a sample pad pretreatment solution and a conjugate pad pretreatment solution, and the sample pad pretreatment solution comprises trehalose, Tween 20, BSA, a Proclin300 preservative and a borate buffer solution; the conjugate pad pretreatment solution comprises trehalose, Tween 20, BSA, PEG20000, a Proclin300 preservative, polyvinyl alcohol, chitosan and a borate buffer solution, and the enhanced colloidal gold immunochromatography test strip pretreatment solution can enhance the binding force between an antibody on the conjugate pad and colloidal gold while improving the permeability and diffusivity of a sample pad, and maximally maintain the activity of the antibody; and the detection sensitivity and accuracy are always kept at excellent levels, so that the overall performance of a colloidal gold immunochromatography detection product is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of colloidal gold immunochromatographic test strips, and in particular relates to an enhanced colloidal gold immunochromatographic test strip pretreatment liquid and a preparation method thereof. Background Art

[0002] Colloidal gold immunochromatography is a rapid detection method that combines colloidal gold labeling technology, immunoassay technology and chromatographic analysis technology. Its principle is to use colloidal gold particles as markers to detect target substances on the chromatographic medium through the specific binding reaction of antigen and antibody. This technology has the advantages of simple operation, rapid detection and intuitive results, and has been widely used in clinical diagnosis, food safety testing, environmental monitoring and other fields.

[0003] As the key carrier of this technology, the performance of colloidal gold immunochromatographic test strips directly determines the accuracy and reliability of the test results. Among them, the gold label conjugate pad and the sample pad, as important components of the test strip, respectively undertake the key tasks of carrying colloidal gold-labeled antibodies and pre-treating samples. The main function of the sample pad is to carry and pre-treat samples. When the sample is dripped onto the sample pad, the sample pad can quickly absorb the sample and properly treat the sample through the buffer system and additives therein, such as adjusting the pH value, removing impurities, etc., so that the target substance in the sample can fully combine with the colloidal gold-labeled antibody. At the same time, the sample pad can also control the flow rate of the sample to ensure that the sample can flow evenly on the test strip, thereby ensuring the accuracy and repeatability of the test results. The performance of the sample pad directly affects the pretreatment effect of the sample and the subsequent detection process. The gold label conjugate pad carries the colloidal gold-labeled antibody. During the detection process, the target substance in the sample and the colloidal gold-labeled antibody undergo a specific binding reaction on the gold label conjugate pad to form an antigen-antibody-colloidal gold complex. Then, the complex flows along the test strip under capillary action, reaches the detection line and quality control line on the nitrocellulose membrane, and further reacts with the antibody or antigen fixed on the membrane, thereby realizing the detection of the target substance. The performance of the gold label conjugate pad directly affects the stability, activity and binding efficiency of the colloidal gold labeled antibody with the target substance.

[0004] However, the existing gold label conjugate pad and sample pad pretreatment solution have many defects and shortcomings:

[0005] ① Poor stability: During storage and transportation, colloidal gold antibody conjugates are prone to aggregation and precipitation, which reduces their activity and affects the sensitivity and accuracy of detection. This stability problem is more prominent in some harsh environmental conditions, such as high temperature and high humidity.

[0006] ② Poor adaptability to complex samples: When faced with complex samples containing high concentrations of impurities and interfering substances, the existing pretreatment solution cannot effectively eliminate interference and is prone to produce false positive or false negative results. For example, some clinical samples may contain impurities such as cell fragments and proteins in the blood, which can affect the binding of colloidal gold-labeled antibodies to the target substance, resulting in inaccurate test results.

[0007] ③ Lack of effective anti-corrosion measures: The pretreatment liquid is easily contaminated by microorganisms during storage, causing it to deteriorate and affecting the service life and performance of the product. Especially in some environments with high humidity, the growth and reproduction of microorganisms are faster, making it easier to contaminate the pretreatment liquid.

[0008] ④ Limited environmental adaptability: Under different environmental conditions such as temperature and humidity, the performance of the existing pretreatment solution fluctuates greatly, making it difficult to ensure the consistency of the test results. For example, in a low-temperature environment, the fluidity of the pretreatment solution may deteriorate, affecting the flow rate of the sample on the test strip; in a high-temperature environment, the activity of the colloidal gold antibody conjugate may be affected, resulting in reduced detection sensitivity. Summary of the invention

[0009] In order to solve the problems in the prior art, the present invention provides an enhanced colloidal gold immunochromatography test strip pretreatment liquid and a preparation method thereof, so as to improve the permeability and diffusivity of the sample pad while enhancing the binding force between the antibody and the colloidal gold on the binding pad, thereby maintaining its activity to the greatest extent, ensuring that the detection sensitivity and accuracy are always at an excellent level, thereby improving the overall performance of the colloidal gold immunochromatography detection product.

[0010] The present invention solves the technical problem by adopting the following technical solutions:

[0011] The present invention aims to provide an enhanced colloidal gold immunochromatographic test strip pretreatment liquid, comprising a sample pad pretreatment liquid and a conjugate pad pretreatment liquid, wherein the sample pad pretreatment liquid comprises trehalose, Tween 20, BSA, Proclin 300 preservative and a borate buffer; and the conjugate pad pretreatment liquid comprises trehalose, Tween 20, BSA, PEG 20000, Proclin 300 preservative, polyvinyl alcohol, chitosan and a borate buffer.

[0012] Trehalose: As an excellent protective agent, it can maintain the spatial structure of colloidal gold antibody conjugates during drying and storage, prevent their denaturation and inactivation, and improve their stability.

[0013] Tween 20: Surfactant that reduces the surface tension of the liquid, promotes uniform diffusion of the sample on the sample pad, and ensures the accuracy and repeatability of the test.

[0014] BSA: Blocks nonspecific binding sites on the sample pad, reduces nonspecific adsorption, and improves detection specificity.

[0015] Proclin 300 preservative: effectively inhibits the growth and reproduction of microorganisms and extends the shelf life of the pretreatment liquid.

[0016] Borate buffer: provides a stable pH environment to ensure the activity of colloidal gold antibody conjugate.

[0017] PEG20000 and polyvinyl alcohol (PVA): enhance the dispersibility of colloidal gold, allowing it to be evenly distributed on the conjugate pad and improve detection sensitivity.

[0018] Chitosan (CS): Increases the stability of colloidal gold antibody conjugates and improves their ability to retain activity during storage and transportation.

[0019] Furthermore, the sample pad pretreatment solution includes 3% trehalose, 0.2% Tween 20, 1% BSA, 0.02% Proclin 300 preservative and 50 mM borate buffer.

[0020] Furthermore, the conjugate pad pretreatment solution includes 3% trehalose, 0.2% Tween 20, 1% BSA, 0.1% PEG20000, 0.02% Proclin 300 preservative, 0.5% polyvinyl alcohol, 0.3% chitosan and 2 mM borate buffer.

[0021] After a lot of experimental research and optimization, the best ratio between the ingredients was determined. For example, 3% trehalose can provide sufficient protection without adversely affecting the performance of other ingredients; 0.2% Tween 20 can ensure good surface activity without causing excessive foaming; 1% BSA can effectively block non-specific binding sites without causing excessive costs. 0.1% PEG20000, 0.02% Proclin 300 preservative, 0.5% polyvinyl alcohol (PVA) and 0.3% chitosan (CS) also achieved the best effect in their respective roles. These precise formula ratios are one of the key factors to achieve the excellent performance of the present invention.

[0022] A method for preparing an enhanced colloidal gold immunochromatographic test strip pretreatment solution comprises the following steps:

[0023] (1) Dissolving and mixing: adding the weighed sample pad pretreatment solution raw material and conjugate pad pretreatment solution raw material into respective containers and stirring to dissolve; then using borate buffer to make up the volume;

[0024] (2) Membrane filtration and ultraviolet disinfection: The sample pad pretreatment solution and the binding pad pretreatment solution after the fixed volume are filtered separately to remove tiny impurities and particles, and after ultraviolet disinfection, they are divided into sterile containers and then freeze-dried;

[0025] (3) Packaging: Use sealed bottles filled with inert gas and pack under nitrogen protection.

[0026] Furthermore, in step (1), after the sample pad pretreatment solution is completely dissolved, the solution is fixed to volume using 50 mM borate buffer and stirred evenly to ensure that the concentration of the solution is uniform; after the conjugate pad pretreatment solution is added with 2 mM borate buffer and stirred evenly, it is fixed to volume using purified water.

[0027] Furthermore, in step (2), freeze drying is performed using a -80°C freeze dryer.

[0028] Furthermore, in step (3), the pre-treated solution after packaging should be stored in an environment of 4°C.

[0029] Compared with the prior art, the beneficial technical effects of the present invention are:

[0030] (1) Significantly enhance stability and detection efficiency: By optimizing the formula and innovating the preparation process, the pretreatment solution can significantly improve the stability of the colloidal gold antibody conjugate, allowing it to maintain high activity under long-term storage and harsh environmental conditions. At the same time, it improves the sensitivity and accuracy of the detection, shortens the detection time, and improves the detection efficiency.

[0031] (2) Significantly improved adaptability and antiseptic properties for complex samples: It has excellent adaptability to various complex samples, can effectively eliminate interfering substances in the samples, and reduce the probability of false positive and false negative results. The added antiseptic ingredients and innovative preparation process can effectively inhibit the growth and reproduction of microorganisms, extend the shelf life of the pretreatment solution, and ensure the quality and performance of the product.

[0032] (3) Environmental adaptability optimization to ensure the reliability of test results under various conditions: After rigorous testing and verification, the pretreatment liquid can maintain stable performance under different environmental conditions such as temperature, humidity, and light, and is suitable for various application scenarios, providing users with reliable test results.

[0033] 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

[0034] 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.

[0035] 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.

[0036] Example 1

[0037] (1) Raw material preparation

[0038] According to the formula requirements, accurately weigh the required mass of 3% trehalose, 0.2% Tween 20, 1% BSA, 0.02% Proclin 300 preservative, 0.1% PEG20000, 0.5% polyvinyl alcohol (PVA) and 0.3% chitosan (CS) and the corresponding borate buffer.

[0039] Ensure that the quality of the raw materials used meets the relevant standards and is free of impurities and pollution.

[0040] (2) Dissolution and mixing

[0041] Add the weighed raw materials into appropriate containers.

[0042] For the sample pad pretreatment solution, 3% trehalose, 0.2% Tween 20, 1% BSA and 0.02% Proclin 300 preservative are first added to an appropriate amount of 50 mM borate buffer, and stirred and dissolved using a magnetic stirrer at a certain temperature (such as room temperature) until completely dissolved.

[0043] For the conjugate pad pretreatment solution, 3% trehalose, 0.2% Tween 20, 1% BSA, 0.02% Proclin 300 preservative, 0.1% PEG20000, 0.5% polyvinyl alcohol (PVA) and 0.3% chitosan (CS) were added to an appropriate amount of purified water and stirred to dissolve.

[0044] Then, slowly add 2 mM borate buffer to the conjugate pad pretreatment solution, stirring while adding to ensure uniform mixing.

[0045] (3) Volume determination and adjustment

[0046] After the sample pad pretreatment solution is completely dissolved, the solution is diluted to the required volume using 50 mM borate buffer while continuing to stir evenly to ensure that the concentration of the solution is uniform.

[0047] After adding 2 mM borate buffer to the conjugate pad pretreatment solution and stirring evenly, the solution was fixed to a predetermined volume using purified water.

[0048] The pH value of the solution can be tested using an instrument such as a pH meter. If necessary, the pH value can be adjusted to a suitable range by adding a small amount of acid or base (the optimal pH value is determined based on the characteristics of the colloidal gold antibody conjugate).

[0049] (4) Membrane filtration and ultraviolet disinfection

[0050] The pre-treatment liquid after the volume is fixed is filtered through a suitable filter device (such as a 0.2μm filter membrane) to effectively remove tiny impurities and particles to ensure the clarity of the pre-treatment liquid. Combined with ultraviolet disinfection, it further kills microorganisms to ensure sterility and no particle contamination.

[0051] The filtered pre-treated liquid is dispensed into sterile containers, such as sealed glass or plastic bottles.

[0052] (5) Low temperature freeze drying

[0053] Use -80℃ freeze dryer to improve the storage stability and long-term activity of pretreatment solution

[0054] (5) Packaging and transportation

[0055] Use sealed bottles filled with inert gas and package under nitrogen protection. The pretreatment solution should be stored in an environment of 4°C, avoiding direct sunlight and high temperature environment. During transportation, the temperature should also be controlled within the appropriate range to ensure that the performance of the pretreatment solution is not affected.

[0056] Comparative Example 1

[0057] The difference between this comparative example and Example 1 is that in the treatment solution, the sample pad pretreatment solution includes 3% trehalose, 0.2% Tween 20, 1% BSA, 0.02% Proclin 300 preservative and 50 mM borate buffer.

[0058] The conjugate pad pretreatment solution included 3% trehalose, 0.2% Tween 20, 1% BSA, 0.02% Proclin 300 preservative and 2 mM borate buffer.

[0059] Comparative Example 2

[0060] The difference between this comparative example and Example 1 is that in the treatment solution, the sample pad pretreatment solution includes 2% trehalose, 0.3% Tween 20, 2% BSA, 0.01% Proclin 300 preservative and 40 mM borate buffer.

[0061] The conjugate pad pretreatment solution includes 2% trehalose, 0.3% Tween 20, 2% BSA, 0.2% PEG20000, 0.01% Proclin 300 preservative, 0.4% polyvinyl alcohol, 0.4% chitosan and 3 mM borate buffer.

[0062] Comparative Example 3

[0063] The difference between this comparative example and Example 1 is that in the treatment solution, the sample pad pretreatment solution includes 4% trehalose, 0.1% Tween 20, 3% BSA, 0.03% Proclin 300 preservative and 60 mM borate buffer.

[0064] The conjugate pad pretreatment solution included 4% trehalose, 0.1% Tween 20, 3% BSA, 0.3% PEG20000, 0.03% Proclin 300 preservative, 0.6% polyvinyl alcohol, 0.2% chitosan and 1 mM borate buffer.

[0065] Test Example 1

[0066] 1. Material selection: Select materials with good adsorption performance and permeability as gold standard conjugate pad and sample pad

[0067] 2. Cutting: Cut the base material to the required size, ensuring accurate size and neat edges.

[0068] 3. Preprocessing:

[0069] Pretreatment group: a portion of the cut gold label conjugate pad and sample pad are immersed in the conjugate pad pretreatment solution and sample pad pretreatment solution of Example 1 of the present invention respectively. The immersion time is adjusted according to the properties of the material and the concentration of the pretreatment solution, generally 10-30 minutes.

[0070] Pretreatment control group 1: A portion of the cut gold label conjugate pad and sample pad are immersed in the conjugate pad pretreatment solution and sample pad pretreatment solution of comparative example 1 respectively. The immersion time is adjusted according to the properties of the material and the concentration of the pretreatment solution, as control group 1.

[0071] Pretreatment control group 2: A portion of the cut gold label conjugate pad and sample pad are immersed in the conjugate pad pretreatment solution and sample pad pretreatment solution of comparative example 2 respectively. The immersion time is adjusted according to the properties of the material and the concentration of the pretreatment solution, as control group 2.

[0072] Pretreatment control group 3: A portion of the cut gold label binding pad and sample pad are immersed in the binding pad pretreatment solution and sample pad pretreatment solution of comparative example 3 respectively. The immersion time is adjusted according to the properties of the material and the concentration of the pretreatment solution, as control group 3.

[0073] Unpretreated group: A portion of the cut gold-labeled conjugate pads and sample pads were used as the control group 4 without any pretreatment.

[0074] 4. Drying: Take out the soaked gold-labeled conjugate pad and sample pad as well as the untreated gold-labeled conjugate pad and sample pad, and dry them under appropriate conditions, such as in a well-ventilated environment or using a drying device, to ensure that the pads are completely dry without any moisture residue.

[0075] 5. Test strip assembly

[0076] Prepare components: In addition to the gold label conjugate pad and sample pad, you also need to prepare nitrocellulose membrane, absorbent paper, plastic base plate and other components.

[0077] Paste: Paste the nitrocellulose membrane in the middle of the plastic base plate, then paste the sample pad on one end of the nitrocellulose membrane (use the sample pads of the pretreatment group, pretreatment control group 1, pretreatment control group 2, pretreatment control group 3, and non-pretreatment group for comparative experiments), and paste absorbent paper on the other end. Paste the gold-labeled binding pad between the sample pad and the nitrocellulose membrane (also use the gold-labeled binding pads of the pretreatment group, pretreatment control group 1, pretreatment control group 2, pretreatment control group 3, and non-pretreatment group for comparative experiments), and ensure that the connection between the components is tight and seamless.

[0078] 6. Performance test:

[0079] After the test strips are assembled, multi-dimensional performance tests are carried out, including stability tests, detection efficiency tests, complex sample adaptability tests, etc. In the stability test, the test strips are placed under different temperature and humidity conditions, and the activity and stability of the colloidal gold antibody conjugate are regularly tested. In the detection efficiency test, the speed and accuracy of the test strips of the pretreatment group, pretreatment control group 1, pretreatment control group 2, pretreatment control group 3, and non-pretreatment group using the new pretreatment liquid of the present invention are compared when detecting the same samples. In the complex sample adaptability test, samples containing high concentrations of impurities and interfering substances are selected for testing to evaluate the control ability of the new pretreatment liquid of the present invention on false positive and false negative results. In the anti-corrosion and environmental adaptability test, the effect of the pretreatment liquid of the present invention on the activity of the colloidal gold antibody conjugate under different environmental conditions, as well as the stability of the overall performance of the test strips are evaluated.

[0080] 7. Test Results

[0081] The test results are shown in Table 1.

[0082] Table 1

[0083]

[0084]

[0085]

[0086] 8. Performance comparison test

[0087] The five groups of pads, namely the pre-treated group, the un-pre-treated group, the pre-treated control group 1, the pre-treated control group 2 and the pre-treated control group 3, were assembled into test strips for testing the same samples. The samples were stored at 4°C and 37°C for 1 week, 1 month and 6 months before testing.

[0088] During the detection process, the gold label release rate, the amount of gold label remaining on the gold label binding pad, the background clarity of the NC membrane and the positive color development were recorded. The detection results are shown in Table 2 below.

[0089] Table 2

[0090]

[0091]

[0092] By comparing the performance of the untreated group, pretreatment control group 1, pretreatment control group 2, pretreatment control group 3 and the pretreatment group using the pretreatment solution formula of the present invention after assembling the test strips, it can be clearly seen that the pretreatment solution formula of the present invention has significant advantages.

[0093] In the initial state, the pretreatment group had a fast gold label release rate, a small amount of gold label residue on the gold label binding pad, a clear and white NC membrane background, and obvious positive coloration with high clarity, while the non-pretreatment group, pretreatment control group 1, pretreatment control group 2, and pretreatment control group 3 all performed poorly in these aspects. After being stored at different temperatures (4°C and 37°C) for different time periods (one week, one month, and half a year), the performance gradually declined, the gold label release rate slowed down or even almost did not release, the amount of gold label residue on the gold label binding pad increased significantly, the NC membrane background color interfered seriously, and the positive coloration became weak or even no coloration. In contrast, the pretreatment group maintained good performance in all time periods, and even after half a year, it could still maintain a faster gold label release rate, less gold label residue on the gold label binding pad, a clear NC membrane background, and obvious positive coloration.

[0094] The present invention optimizes the formula of sample pad and conjugate pad pretreatment solution by introducing innovative ingredients such as polyvinyl alcohol (PVA) and chitosan (CS), significantly enhancing the stability, dispersibility and binding force of colloidal gold antibody conjugates. At the same time, innovative preparation processes such as deep filtration, ultraviolet disinfection, and low-temperature freeze-drying further improve the quality and performance of the pretreatment solution.

[0095] The present invention effectively solves the problems existing in the existing colloidal gold immunochromatography technology, such as poor stability, poor adaptability to complex samples, lack of effective antiseptic measures, and limited environmental adaptability. After multi-dimensional performance tests, including stability tests, detection efficiency tests, complex sample adaptability tests, etc., it is proved that the pretreatment liquid of the present invention can maintain stable performance under different temperature, humidity and light conditions, is suitable for the detection of various complex samples, and improves the accuracy and reliability of the detection. At the same time, the preparation process of the present invention is simple, the raw materials are easy to obtain, the cost is low, it is conducive to large-scale production and promotion and application, and provides more reliable and efficient technical support for clinical diagnosis, food safety testing, environmental monitoring and other fields, and has important practical application value and broad market prospects. The present invention has important significance and potential in the field of colloidal gold immunochromatography technology.

[0096] 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.

[0097] 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. An enhanced colloidal gold immunochromatographic test strip pretreatment solution, characterized in that: It includes a sample pad pretreatment liquid and a binding pad pretreatment liquid. The sample pad pretreatment liquid includes trehalose, Tween 20, BSA, Proclin 300 preservative and borate buffer; the binding pad pretreatment liquid includes trehalose, Tween 20, BSA, PEG 20000, Proclin 300 preservative, polyvinyl alcohol, chitosan and borate buffer.

2. The enhanced colloidal gold immunochromatographic test strip pretreatment solution according to claim 1, characterized in that: The sample pad pretreatment solution included 3% trehalose, 0.2% Tween 20, 1% BSA, 0.02% Proclin 300 preservative and 50 mM borate buffer.

3. The enhanced colloidal gold immunochromatographic test strip pretreatment solution according to claim 1, characterized in that: The conjugate pad pretreatment solution included 3% trehalose, 0.2% Tween 20, 1% BSA, 0.1% PEG20000, 0.02% Proclin 300 preservative, 0.5% polyvinyl alcohol, 0.3% chitosan and 2 mM borate buffer.

4. A method for preparing an enhanced colloidal gold immunochromatographic test strip pretreatment solution as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Dissolving and mixing: adding the weighed sample pad pretreatment solution raw material and conjugate pad pretreatment solution raw material into respective containers and stirring to dissolve; then using borate buffer to make up the volume; (2) Membrane filtration and ultraviolet disinfection: The sample pad pretreatment solution and the binding pad pretreatment solution after the fixed volume are filtered separately to remove tiny impurities and particles, and after ultraviolet disinfection, they are divided into sterile containers and then freeze-dried; (3) Packaging: Use sealed bottles filled with inert gas and pack under nitrogen protection.

5. The method for preparing a pretreatment solution for an enhanced colloidal gold immunochromatographic test strip according to claim 4, characterized in that: In step (1), after the sample pad pretreatment solution is completely dissolved, the solution is fixed to volume using 50 mM borate buffer and stirred evenly to ensure that the concentration of the solution is uniform; after adding 2 mM borate buffer to the conjugate pad pretreatment solution and stirring evenly, the solution is fixed to volume using purified water.

6. The method for preparing an enhanced colloidal gold immunochromatographic test strip pretreatment solution as claimed in claim 4, characterized in that: In step (2), freeze drying is performed using a -80°C freeze dryer.

7. The method for preparing an enhanced colloidal gold immunochromatographic test strip pretreatment solution as claimed in claim 4, characterized in that: In step (3), the pretreatment solution after packaging should be stored in an environment of 4°C.

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