Reagent for detecting disease marker by colloidal gold method and preparation method and use thereof

Abstract

The application provides a preparation method of a colloidal gold solution, a preparation method of a colloidal gold labeled protein, a colloidal gold solution and a colloidal gold labeled protein prepared by the method, a kit comprising the colloidal gold solution or the colloidal gold protein, a labeling pad for a colloidal gold test strip, a colloidal gold test strip, and use of the colloidal gold solution, the colloidal gold labeled protein, the labeling pad or the colloidal gold test strip in preparing a kit for detecting a disease marker in a biological sample. For example, the kit of the application can be used for detecting C-reactive protein by a colloidal gold method.

Description

Technical Field

[0001] This application belongs to the field of in vitro diagnostics and mainly relates to methods for preparing colloidal gold, prepared colloidal gold solutions, methods for preparing colloidal gold-labeled proteins, prepared colloidal gold-labeled proteins, kits containing colloidal gold solutions or colloidal gold-labeled proteins, labeling pads for colloidal gold test strips, colloidal gold test strips, and the use of said colloidal gold solutions, said colloidal gold-labeled proteins, said labeling pads, or said colloidal gold test strips in the preparation of kits for the detection of disease biomarkers in biological samples. Background Technology

[0002] C-reactive protein (CRP) refers to proteins that rapidly increase in plasma levels (acute proteins) when the body is infected or tissues are damaged. CRP plays an opsonizing role by activating complement and enhancing phagocytosis, thereby clearing invading pathogens and damaged, necrotic, and apoptotic tissues and cells, playing a crucial protective role in the body's innate immune process. CRP is not only a non-specific inflammatory marker, but it also directly participates in inflammation and cardiovascular diseases such as atherosclerosis, and is one of the most potent predictors and risk factors for cardiovascular diseases. The interaction of CRP with complement C1q and FcTR gives it many biological activities, including host defense responses to infection, phagocytosis in inflammatory responses, and regulatory functions. Its binding to damaged cells, apoptotic cells, and nuclear antigens also makes it important in autoimmune diseases.

[0003] The measurement of CRP has important clinical significance: (1) It rises rapidly within hours after the onset of various acute inflammations, tissue damage, myocardial infarction, surgical trauma, radiation damage, and other diseases, and tends to increase exponentially, indicating possible infection or thromboembolism. (2) CRP is closely related to other inflammatory factors, such as total white blood cell count, erythrocyte sedimentation rate, and polymorphonuclear leukocytes, and plays an active role in the inflammatory response, giving the body non-specific resistance; it can rise earlier than white blood cells when the patient's disease occurs, and it recovers to normal very quickly, so it has extremely high sensitivity. (3) It can help identify the type of respiratory infection. CRP can be used for the differential diagnosis of bacterial and viral infections: CRP levels rise in bacterial infections; while CRP does not rise or rises slightly in viral infections. Therefore, CRP values ​​can help doctors identify the type of respiratory infection and give targeted drugs and treatments. (4) CRP levels are elevated in patients with malignant tumors. Combined detection of CRP and AFP can be used to differentiate liver cancer from benign liver diseases and to assess the efficacy and prognosis of liver cancer. CRP levels rise before surgery and decrease after surgery, and its response is not affected by radiotherapy, chemotherapy, or corticosteroid therapy, which helps in assessing tumor progression. (5) CRP can be used to assess the severity of acute pancreatitis. When CRP is higher than 250 mg / L, it suggests extensive necrotizing pancreatitis. (6) CRP can also be used to predict the risk of coronary heart disease and myocardial infarction.

[0004] Although CRP qualitative testing reagents are widely used in rural hospitals, clinics, and homes, current market products have several shortcomings in these applications. These shortcomings mainly focus on the following two aspects:

[0005] (1) Excessive sensitivity. The main reason is that CRP levels are high in the blood during inflammation. The general diagnostic threshold for inflammation is 10 mg / L, so many manufacturers and institutions develop products with sensitivity below 10 mg / L, and then use colorimetric methods to determine positive or negative results by matching color charts. Another method is to increase the sample dilution factor. Some manufacturers require the sample to be diluted 1000 times or even higher before testing. This requires more than 10 times the amount of diluent used as in ordinary products, which greatly increases transportation and production costs. At the same time, lower sample sizes are required to achieve higher dilution factors, which increases the sampling error rate and has a significant impact on the accuracy of the test. The colorimetric method for determining positive or negative results and the severity of inflammation requires high skill from the operator and professional training to avoid incorrect use, especially in home use scenarios, where subjectivity is strong.

[0006] (2) There is no obvious gradient in the color development of low, medium, and high values, or there is no obvious difference in the depth of color development. Many products use antibodies with lower sensitivity and larger sample dilution factors in order to achieve lower sensitivity. This approach usually introduces a second problem, which is that there is no obvious gradient in the low, medium, and high values. Especially when testing samples with medium and high values, there is usually no obvious difference in color development, and doctors cannot make a preliminary judgment on the severity of inflammation.

[0007] Given the importance of CPR in clinical diagnosis, it is of great significance to develop effective reagents and methods for CPR detection. Summary of the Invention

[0008] In a first aspect, this application provides a method for preparing a colloidal gold solution, comprising:

[0009] (1) Prepare an aqueous mixture of HAuCl4 and a reducing agent, wherein the concentration of HAuCl4 in the aqueous mixture is 0.03-0.05 w / v%, and the concentration of the reducing agent is 0.02-0.2 w / v%.

[0010] (2) Heat the aqueous mixture described in step (1) to boiling and maintain for 1-10 min, then stop heating and cool to room temperature;

[0011] (3) Use water to bring the solution obtained in step (2) to a volume equal to that of the aqueous mixture described in step (1).

[0012] In some embodiments of the first aspect, the concentration of HAuCl4 in the aqueous mixture is 0.04 w / v.

[0013] In some embodiments of the first aspect, the concentration of the reducing agent is 0.06 w / v.

[0014] In some embodiments of the first aspect, in step (2), the aqueous mixture is heated to boiling and maintained for 5 minutes.

[0015] In some embodiments of the first aspect, the water used in the method is double-distilled deionized water or triple-distilled deionized water.

[0016] In some embodiments of the first aspect, the reducing agent is trisodium citrate, white phosphorus, sodium ascorbate, tannic acid, sodium borohydride, hydrogen peroxide, ethanol, or any combination thereof.

[0017] In some embodiments of the first aspect, in step (2), the aqueous mixture is heated at a temperature of 100-500°C.

[0018] In some embodiments of the first aspect, in step (2), the aqueous mixture is heated at a temperature of 300°C.

[0019] Secondly, this application provides a colloidal gold solution prepared by the method described in the first aspect.

[0020] Thirdly, this application provides a method for preparing colloidal gold-labeled proteins, comprising:

[0021] The protein to be labeled is added to the colloidal gold solution described in the second aspect for labeling, thereby obtaining a labeled mixture;

[0022] Add a sealing agent to the labeled mixture for sealing;

[0023] After sealing, centrifuge to remove the supernatant, and add refolding solution to the precipitate for refolding.

[0024] In some embodiments of the third aspect, the concentration of the labeled protein in the labeled mixture is 10-30 μg / mL.

[0025] In some embodiments of the third aspect, the volume ratio of the added labeling blocking solution to the colloidal gold solution is 1:100.

[0026] In some embodiments of the third aspect, the refolding solution of equal volume to the precipitate is added to the precipitate.

[0027] In some embodiments of the third aspect, the labeling blocking fluid is 10 w / v% BSA.

[0028] In some embodiments of the third aspect, the refolding solution is a solution containing 1-10 mM boric acid and 0.1-1 w / v% PEG20000 at pH 8.0-9.5.

[0029] In some embodiments of the third aspect, the protein to be labeled is an antibody, antigen, recombinant protein, or avidin.

[0030] In some embodiments of the third aspect, the concentration of the protein to be labeled in the labeled mixture is 20-30 μg / mL.

[0031] In some embodiments of the third aspect, the refolding solution is a solution containing 5 mM boric acid and 0.5 w / v% PEG20000 at pH 8.9-9.1.

[0032] In some embodiments of the third aspect, the protein to be labeled is an anti-C-reactive protein (CRP) antibody or an anti-dinitrophenol (DNP) antibody.

[0033] In some embodiments of the third aspect, the method further includes centrifuging the refolded solution to remove the supernatant and adding a label diluent to the precipitate, wherein the volume ratio of the precipitate to the label diluent is 1:50.

[0034] In some embodiments of the third aspect, the labeling diluent is a solution comprising 10-30 w / v% sucrose, 2.5-7.5 w / v% trehalose, 0.1-2 w / v% sodium caseinate, 0.1-2 w / v% polyvinylpyrrolidone and 0.1-2 w / v% surfactant S9.

[0035] In some embodiments of the third aspect, the labeling diluent is a solution containing 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% sodium caseinate, 1 w / v% polyvinylpyrrolidone and 1 w / v% S9.

[0036] In some embodiments of the third aspect, the labeling diluent is a solution containing 10-30 w / v% sucrose, 2.5-7.5 w / v% trehalose, 0.1-2 w / v% sodium caseinate, 0.1-2 w / v% polyvinylpyrrolidone, 0.1-2 w / v% surfactant S9, and 10-100 mM Tris-HCl.

[0037] In some embodiments of the third aspect, the labeling diluent is a solution containing 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% sodium caseinate, 1 w / v% polyvinylpyrrolidone, 1 w / v% surfactant S9 and 50 mM Tris-HCl.

[0038] Fourthly, this application provides colloidal gold-labeled proteins prepared by the method described in the third aspect.

[0039] Fifthly, this application provides a kit comprising the colloidal gold solution described in the second aspect or the colloidal gold protein described in the fourth aspect.

[0040] In a sixth aspect, this application provides a labeling pad for colloidal gold test strips, comprising at least two spaced-apart labeling lines, each of the at least two labeling lines being loaded with a first colloidal gold labeled protein and a second colloidal gold labeled protein.

[0041] In some embodiments of the sixth aspect, the first colloidal gold-labeled protein is prepared by the method described in the third aspect, and the protein to be labeled is an anti-disease biomarker antibody.

[0042] In some embodiments of the sixth aspect, the second colloidal gold-labeled protein is prepared by the method described in the third aspect, and the protein to be labeled is an internal control antibody.

[0043] In some embodiments of the sixth aspect, the anti-disease marker antibody is an anti-CRP antibody.

[0044] In some embodiments of the sixth aspect, the internal control antibody is an anti-DNP antibody.

[0045] In a seventh aspect, this application provides a colloidal gold test strip that includes the marking pad described in the sixth aspect.

[0046] In some embodiments of the seventh aspect, the test strip further includes a substrate, a nitrocellulose membrane, and absorbent paper.

[0047] In some embodiments of the seventh aspect, the substrate is a PVC board.

[0048] In some embodiments of the seventh aspect, the marking pad, the nitrocellulose membrane, and the absorbent paper are sequentially attached to the substrate, and the marking pad and the absorbent paper are each partially placed on the nitrocellulose membrane.

[0049] Eighthly, this application provides the use of the colloidal gold solution described in the second aspect, the colloidal gold labeled protein described in the fourth aspect, the labeling pad described in the sixth aspect, or the colloidal gold test strip described in the seventh aspect in the preparation of a kit for the detection of disease biomarkers in biological samples.

[0050] In some embodiments of the eighth aspect, the biological sample is blood or serum.

[0051] In some embodiments of the eighth aspect, the disease markers are C-reactive protein, serum amyloid A, procalcitonin, glycated hemoglobin, cardiac troponin or N-terminal pro-B-type natriuretic peptide, novel coronavirus surface antigen, influenza virus surface antigen or mycoplasma surface antigen. Attached Figure Description

[0052] Figure 1 An image of the prepared marking pad is shown.

[0053] Figure 2 A schematic diagram of the CRP-coated membrane is shown.

[0054] Figure 3 A schematic diagram of the detection board is shown.

[0055] Figure 4 An image of the prepared test card is shown.

[0056] Figure 5 The diagram shows a scatter plot of the test strips prepared using the colloidal gold solution within the linear range.

[0057] Figure 6The correlation analysis results of the test results using the prepared colloidal gold solution and the test results using the reference reagent are shown.

[0058] Figure 7 The results of sensitivity tests using the conventional labeling method and the reversible labeling method are shown in the graph. The top graph shows the results using the conventional labeling method, and the bottom graph shows the results using the reversible labeling method. Detailed Implementation

[0059] The properties of colloidal gold mainly depend on the diameter of the gold particles, their absorption spectrum, and their stability. The absorption spectrum of colloidal gold consists of a single absorption peak, generally within the visible spectrum range of 510–550 nm. The absorption wavelength increases with increasing particle diameter, resulting in color variations ranging from pale orange-yellow to bluish-violet and even brownish-red. Colloidal gold particles with a size between 5 and 20 nm absorb light at 520 nm, appearing wine-red; those between 20 and 40 nm primarily absorb green light at 530 nm, resulting in a deep red solution; and colloidal gold solutions with a particle size of 60 nm primarily absorb orange-yellow light at 600 nm.

[0060] Colloidal gold is primarily prepared using a chemical reduction method, which involves adding various reducing agents to an aqueous solution of gold chloride, causing gold ions to polymerize into colloidal gold. The reduction method can be considered a crystallization process; the particle size depends on the rate of crystal nucleation and growth induced by the reducing agent. Commonly used reducing agents include white phosphorus, sodium ascorbate, trisodium citrate, tannic acid, sodium borohydride, hydrogen peroxide, and ethanol. The advantage of the reduction method is that colloidal gold particles of different diameters (15–150 nm) can be prepared by varying the amount of reducing agent added. A basic rule is that the smaller the amount of reducing agent, the larger the diameter of the colloidal gold particles.

[0061] Colloidal gold technology has advantages such as convenience, speed, specificity, high stability, no need for special equipment and reagents, and intuitive result interpretation. Therefore, it is particularly suitable for grassroots testing personnel, as well as for large-scale testing and large-scale surveys, and has great development potential and broad application prospects.

[0062] However, conventional colloidal gold technology currently has some drawbacks as follows:

[0063] 1. Conventional colloidal gold labeling methods have defects in operation and labeling performance.

[0064] (1) The labeling efficiency is greatly affected by pH, making it difficult to achieve the optimal labeling efficiency.

[0065] (2) The pH of each batch of colloidal gold and the concentration of K2CO3 used to adjust the pH are different, which leads to the difference in the labeling pH of each batch of labeled antibody. It is difficult to achieve the optimal labeling pH for each batch, resulting in inconsistent labeling efficiency of each batch of gold-labeled antibody and difficulty in accurately controlling the batch-to-batch difference.

[0066] 2. Conventional methods for preparing colloidal gold involve certain operational risks.

[0067] The conventional method for preparing colloidal gold involves using chloroauric acid as a reducing agent to create a colloidal solution of gold particles of a specific size. The conventional process involves boiling water before adding chloroauric acid and the reducing agent, leading to the reaction and the formation of colloidal gold. This method presents significant safety risks, particularly the addition of chloroauric acid and the reducing agent while the water is boiling, which can easily result in burns. Furthermore, the effects of steam and high temperatures make precise control of the process difficult, leading to significant batch-to-batch variations. Therefore, a safer, simpler, and more stable method for preparing colloidal gold is increasingly needed in production.

[0068] 3. The concentration of colloidal gold prepared by conventional methods is relatively low, which has a certain impact on subsequent production efficiency and labeling efficiency.

[0069] Currently, most manufacturers use chloroauric acid with a final concentration of 0.01% (commonly known as 0.01% gold concentration) in their colloidal gold solutions. This concentration requires extremely large containers and equipment for large-scale labeling, significantly impacting production efficiency and often becoming a bottleneck affecting capacity. Especially in processes like centrifugation and concentration, multiple large-capacity, high-speed centrifuges are needed to meet the requirements. Therefore, increasing the gold particle concentration in colloidal gold solutions has become crucial for overcoming this bottleneck in large-scale production.

[0070] Based on the properties of colloidal gold, the inventors of this application established a novel method for preparing colloidal gold solutions by optimizing the final concentration and reaction conditions of the HAuCl4 solution and reducing agent, resulting in high-quality and stable colloidal gold products. By adding a protein to be labeled (e.g., anti-CRP antibody or anti-DNP antibody) to the colloidal gold, the colloidal particles aggregate and form flocs. Centrifugation separates the aggregated precipitate and flocs. Adding an alkaline colloidal gold refolding solution and using ultrasonic dispersion allows the aggregated precipitate and flocs to redisperse into a colloidal state, thus developing a pioneering method for preparing colloidal gold-labeled proteins through a process of aggregation followed by refolding. The colloidal gold solutions or colloidal gold-labeled proteins prepared using this method can be used to prepare diagnostic reagents, kits, and test cards for detecting targets (e.g., C-reactive protein) in biological samples, thereby achieving effective target detection.

[0071] Unless otherwise specified, this application is implemented using conventional chemical, biochemical and analytical chemical techniques in the art.

[0072] To facilitate understanding of this application, some terms used herein are first defined.

[0073] As used herein, "internal reference antibody" refers to the antibody used to determine whether the colloidal gold test strip is qualified and suitable for detection when the colloidal gold test strip of this application is used to detect disease biomarkers in biological samples.

[0074] As used in this article, the "C-line" is also known as the control line, and the "T-line" is also known as the test line. When conducting a test, first check if the C-line is visible. If the C-line is red, the test strip or test card is qualified; otherwise, it is unqualified. Even if the T-line is red, it does not necessarily mean the patient is positive; the test strip or test card needs to be replaced and the test repeated. If both the C-line and T-line are red, the antigen test is positive, possibly indicating an infection. If the T-line is not visible, the result is negative.

[0075] As used in this article, “about” defines a range including a reference value plus or minus 10%. For example, “about 30nm” defines a range of 30+ / -3nm, that is, 27-33nm.

[0076] In a first aspect, this application provides a method for preparing a colloidal gold solution, comprising:

[0077] (1) Prepare an aqueous mixture of HAuCl4 and a reducing agent, wherein the concentration of HAuCl4 in the aqueous mixture is 0.03-0.05 w / v% and the concentration of the reducing agent is 0.02-0.2 w / v%.

[0078] (2) Heat the aqueous mixture described in step (1) to boiling and maintain for 1-10 minutes, then stop heating and cool to room temperature;

[0079] (3) Use water to bring the solution obtained in step (2) to a volume equal to that of the aqueous mixture described in step (1).

[0080] In some embodiments of the first aspect, the concentration of HAuCl4 in the aqueous mixture is 0.03, 0.035, 0.04, 0.045 or 0.05 w / v%, or any range between the aforementioned values.

[0081] In some embodiments of the first aspect, the concentration of HAuCl4 in the aqueous mixture is 0.04 w / v.

[0082] In some embodiments of the first aspect, the concentration of the reducing agent is 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.2 w / v%, or any range between the aforementioned values.

[0083] In some embodiments of the first aspect, the concentration of the reducing agent is 0.06 w / v.

[0084] In some embodiments of the first aspect, the aqueous mixture described in step (1) is heated to boiling and maintained for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes, or any range between the aforementioned values.

[0085] In some embodiments of the first aspect, the aqueous mixture described in step (1) is heated to boiling and maintained for 5 minutes.

[0086] In some embodiments of the first aspect, the preparation of colloidal gold involves multiple steps, including material preparation, preparation of gold salt solutions, reduction reaction, particle growth and stabilization, and preservation and storage. In some embodiments, the glassware used should be very clean, as the properties of the glass surface play a crucial role in initiating the reduction process; uncleanliness will affect the initiation process, interfere with the formation of colloidal gold particles, and result in inconsistent particle sizes or liquid turbidity. In some embodiments, the glassware used should be acid-washed and silanized. In some embodiments, silanization can be omitted, and specialized glassware can be used, i.e., the cleaned glassware is used directly for colloidal gold preparation. In some embodiments, the surface of the glassware is stabilized with the colloidal gold prepared in the first step and then discarded; or the prepared colloidal gold is coated onto the cleaned glassware surface, followed by washing with double-distilled deionized water, and used exclusively for preparing colloidal gold of the same diameter.

[0087] In some embodiments of the first aspect, gold chloride is highly hygroscopic and inconvenient to weigh; therefore, when preparing an aqueous solution of gold chloride, the entire contents (1g) in the bottle can be dissolved at once to prepare a 4w / v% aqueous solution. It can be stored at 4°C for several months.

[0088] In some embodiments of the first aspect, the method is carried out under stirring conditions.

[0089] In some embodiments of the first aspect, the water used in the method is double-distilled deionized water or triple-distilled deionized water. In some embodiments, all water used to prepare the reagents should be double-distilled deionized water or triple-distilled deionized water, and care should be taken to ensure the cleanliness of the containers, as any impurities and dust particles can interfere with the formation of colloidal gold.

[0090] In some embodiments of the first aspect, the reducing agent is trisodium citrate, white phosphorus, sodium ascorbate, tannic acid, sodium borohydride, hydrogen peroxide, ethanol, or any combination thereof.

[0091] In some specific embodiments of the first aspect, the reducing agent is trisodium citrate.

[0092] In some implementation schemes, the reagents used, especially the reducing agent, must be weighed precisely, since the diameter of the gold particles depends primarily on the concentration of the reducing agent.

[0093] In some embodiments of the first aspect, in step (2), the aqueous mixture is heated at a temperature of 100, 150, 200, 250, 300, 350, 400, 450 or 500°C (or any range between the aforementioned values).

[0094] In some embodiments of the first aspect, in step (2), the aqueous mixture is heated at a temperature of 300°C.

[0095] Secondly, this application provides a colloidal gold solution prepared by the method described in the first aspect.

[0096] In some implementations of the second aspect, an ultraviolet spectrophotometer can be used to detect the absorption peaks of colloidal gold particles.

[0097] In some embodiments of the second aspect, the colloidal gold in the prepared colloidal gold solution has a particle size of about 30 nm.

[0098] In some embodiments of the second aspect, the prepared colloidal gold solution can be stored at 4°C. In some embodiments, the colloidal gold solution can be stored in a sterile container. In some embodiments, the colloidal gold solution can be stored in a sterile container at 4°C for 6 months.

[0099] Thirdly, this application provides a method for preparing colloidal gold-labeled proteins, comprising:

[0100] The protein to be labeled is added to the colloidal gold solution described in the second aspect for labeling, thereby obtaining a labeled mixture;

[0101] Add a sealing agent to the labeled mixture for sealing;

[0102] After sealing, centrifuge to remove the supernatant, and add refolding solution to the precipitate for refolding.

[0103] In some embodiments of the third aspect, the concentration of the labeled protein in the labeled mixture is 10-30 μg / mL, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 μg / mL, or a range or value between any two of the above values.

[0104] In some specific embodiments of the third aspect, the concentration of the protein to be labeled in the labeled mixture is 20 or 30 μg / mL.

[0105] In some embodiments of the third aspect, the volume ratio of the added labeling blocking solution to the colloidal gold solution is 1:100.

[0106] In some embodiments of the third aspect, an equal volume of the refolding solution is added to the precipitate. In some embodiments, the method further includes a step of sonication after adding the refolding solution to ensure thorough dispersion and remelting of the precipitate.

[0107] In some embodiments of the third aspect, the labeling blocking fluid is 10 w / v% BSA.

[0108] In some embodiments of the third aspect, the refolding solution is a solution of PEG20000 with pH 8.0-9.5 (e.g., 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, or 9.5, or any range between the foregoing values) containing 1-10 mM boric acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM, or any range between the foregoing values) and 0.1-1 w / v% (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 w / v%, or any range between the foregoing values). In some embodiments, the boric acid in the refolding solution acts as a lubricant. In some embodiments, the PEG in the refolding solution acts as a stabilizer. In some embodiments, the refolding solution is in a slightly alkaline pH environment, which can improve the refolding effect.

[0109] In some implementations, the refolded colloidal gold particle size is several times larger than the original particle size, making it impossible to obtain a marker with a particle size close to the original size. Smaller initial particle sizes result in smaller refolded particle sizes after aggregation; conversely, larger initial particle sizes result in larger refolded particle sizes after aggregation. The refolded markers show a significant improvement in sensitivity compared to normal markers labeled after adjusting the pH of the colloidal gold.

[0110] In some embodiments of the third aspect, the protein to be labeled is an antibody, antigen, recombinant protein, or avidin.

[0111] In some embodiments of the third aspect, the protein to be labeled is an anti-C-reactive protein (CRP) antibody or an anti-dinitrophenol (DNP) antibody.

[0112] In some embodiments of the third aspect, when the protein to be labeled is an anti-CRP antibody, the concentration of the anti-CRP antibody in the labeled mixture can be 30 μg / mL.

[0113] In some embodiments of the third aspect, when the protein to be labeled is an anti-DNP antibody, the concentration of the anti-DNP antibody in the labeled mixture can be 20 μg / mL.

[0114] In some embodiments of the third aspect, the method further includes centrifuging the refolded solution to remove the supernatant and adding a labeled diluent to the precipitate, wherein the volume ratio of the precipitate to the labeled diluent is 1:50. In some embodiments, the method further includes a step of sonication after adding the labeled diluent to ensure thorough dispersion and remelting of the precipitate.

[0115] In some embodiments of the third aspect, the labeling diluent is a solution comprising 10-30 w / v% sucrose, 2.5-7.5 w / v% trehalose, 0.1-2 w / v% sodium caseinate, 0.1-2 w / v% polyvinylpyrrolidone and 0.1-2 w / v% surfactant S9.

[0116] In some embodiments of the third aspect, the labeled diluent contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 w / v% (or any range between the aforementioned values) of sucrose.

[0117] In some embodiments of the third aspect, the labeled diluent contains 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 w / v% (or any range between the aforementioned values).

[0118] In some embodiments of the third aspect, the labeled diluent comprises 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 w / v% (or any range between the foregoing values).

[0119] In some embodiments of the third aspect, the labeled diluent comprises 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 w / v% (or any range between the foregoing values) of polyvinylpyrrolidone.

[0120] In some embodiments of the third aspect, the labeled diluent comprises 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 w / v% (or any range between the aforementioned values) of surfactant S9.

[0121] In some embodiments of the third aspect, the labeling diluent is a solution containing 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% sodium caseinate, 1 w / v% polyvinylpyrrolidone (PVP) and 1 w / v% surfactant S9.

[0122] In some embodiments of the third aspect, the labeled diluent is a solution containing 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% sodium caseinate, 1 w / v% polyvinylpyrrolidone, 1 w / v% surfactant S9, and 10-100 mM (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM, or any range between the foregoing values) Tris-HCl.

[0123] In some embodiments of the third aspect, the labeling diluent is a solution of sucrose, trehalose, sodium caseinate, polyvinylpyrrolidone, and surfactant S9 dissolved in 50 mM Tris-HCl at pH 7.9-8.1, such that the concentration of sucrose is 20 w / v, the concentration of trehalose is 5 w / v, the concentration of sodium caseinate is 1 w / v, and the concentration of surfactant S9 is 1 w / v.

[0124] In some embodiments of the third aspect, surfactant S9, trade name pluracare 1307, also known as Tetronic 1307, is a copolymer of propylene oxide, ethylene oxide, and vinyl diamine. It is a typical amphoteric surfactant with a hydrophilic-lipophilic balance greater than 24, making it a highly water-soluble surfactant.

[0125] Colloidal gold particles maintain their stability through electrostatic repulsion by adsorbing surrounding ions. Once the conditions maintaining the colloidal state are disrupted, the colloidal particles aggregate and flocculents form. The stability of colloidal gold mainly depends on the influence of electrolytes, particle size, and concentration. Even small amounts of electrolytes can cause sol aggregation. Stabilizers can enhance the stability of colloidal gold. Commonly used stabilizers include bovine serum albumin (BSA), polyethylene glycol (molecular weight 20,000), ovalbumin, polyvinylpyrrolidone, and gelatin. Tween 20, starch, and glucan can also improve the stability of colloidal gold.

[0126] The inventors of this application have discovered that when colloidal gold is added to labeled proteins (such as antibodies) under acidic conditions, the colloidal particles will aggregate and form flocs. Centrifugation separates the aggregated precipitates and flocs. Adding an alkaline colloidal gold refolding solution and ultrasonic dispersion can redisperse the aggregated precipitates and flocs into a colloidal state.

[0127] The absorption peak was detected using a spectrophotometer, and the diameter of the refolded colloidal gold particles was approximately 60 nm. Test strips were prepared simultaneously with normally labeled gold-labeled antibodies under alkaline conditions (e.g., for CRP detection). Results showed that the sensitivity of the gold-labeled antibody, after aggregation and refolding under acidic conditions, was five times that of the normally labeled gold-labeled antibody. That is, when testing the same reference sample, the colorimetric depth of the gold-labeled antibody, after aggregation and refolding under acidic conditions, was five times that of the normally labeled gold-labeled antibody.

[0128] In some embodiments of the third aspect, pH adjustment is not required when preparing colloidal gold-labeled proteins using the colloidal gold solution described in the second aspect of this application.

[0129] Fourthly, this application provides colloidal gold-labeled proteins prepared by the method described in the third aspect.

[0130] In some embodiments of the fourth aspect, the prepared colloidal gold-labeled protein (e.g., colloidal gold-labeled protein solution) can be stored at 2-8°C for 3-6 months to prevent it from deteriorating or agglomerating.

[0131] Fifthly, this application provides a kit comprising the colloidal gold solution described in the second aspect or the colloidal gold protein described in the fourth aspect.

[0132] In some embodiments of the fifth aspect, the kit may further include diluents, washing solutions, blocking solutions, etc., required for detection.

[0133] In some embodiments of the fifth aspect, the kit may also include instructions for use to guide users in using the kit for testing.

[0134] In a sixth aspect, this application provides a labeling pad for colloidal gold test strips, comprising at least two spaced-apart labeling lines, each of the at least two labeling lines being loaded with a first colloidal gold labeled protein and a second colloidal gold labeled protein.

[0135] In some embodiments of the sixth aspect, the marking lines are at least two, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0136] In some embodiments of the sixth aspect, the marking lines are two.

[0137] In some embodiments of the sixth aspect, the interval between the marking lines is 3-10 mm, for example 3, 4, 5, 6, 7, 8, 9, 10 mm, or any value or range between any two of the above values.

[0138] In some specific embodiments of the sixth aspect, the spacing between the marking lines is 5 mm.

[0139] In some embodiments of the sixth aspect, the marking lines are at least two in order to make the overall density of colloidal gold uniform in the sample flow direction, so that when the test strip is detected, the colloidal gold trapped by the T line has a uniform color with different shades, so as to show the color change with the target concentration.

[0140] In some embodiments of the sixth aspect, the first colloidal gold-labeled protein is prepared by the method described in the third aspect, and the protein to be labeled is an anti-disease biomarker antibody.

[0141] In some embodiments of the sixth aspect, the second colloidal gold-labeled protein and the protein to be labeled are internal control antibodies.

[0142] In some specific implementations of the sixth aspect, the anti-disease marker antibody is an anti-CRP antibody.

[0143] In some specific implementations of the sixth aspect, the internal reference antibody is an anti-DNP antibody.

[0144] In some embodiments of the sixth aspect, the padding material of the marking pad is a glass cellulose film, a polyester film, or a plant fiber film, etc.

[0145] In some embodiments of the sixth aspect, the marking line is prepared by spraying a marking working fluid onto the pad material of the marking pad. In some embodiments of the sixth aspect, the spraying is performed 1-4 times, for example, 1, 2, 3, or 4 times. In some specific embodiments of the sixth aspect, the marking pad is sprayed 2 times. In some embodiments of the sixth aspect, the spraying parameters are generally a spray volume of 4 μL / cm and an air pressure of 0.2 MPa.

[0146] In some embodiments of the sixth aspect, the labeling pad working solution comprises 60 v / v% of a first colloidal gold-labeled protein and 8 v / v% of a second colloidal gold-labeled protein.

[0147] In some specific embodiments of the sixth aspect, the labeling pad working solution comprises 60 v / v% of a first colloidal gold-labeled protein, 8 v / v% of a second colloidal gold-labeled protein, and 32 v / v% of a labeling diluent.

[0148] In some embodiments of the sixth aspect, the labeling diluent is a solution containing 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% sodium caseinate, 1 w / v% polyvinylpyrrolidone (PVP) and 1 w / v% surfactant S9.

[0149] In some embodiments of the sixth aspect, the labeling diluent is a solution containing 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% sodium caseinate, 1 w / v% polyvinylpyrrolidone, 1 w / v% surfactant S9 and 50 mM Tris-HCl.

[0150] In some embodiments of the sixth aspect, the labeling diluent is a solution of sucrose, trehalose, sodium caseinate, polyvinylpyrrolidone, and surfactant S9 dissolved in 50 mM Tris-HCl at pH 7.9-8.1, such that the concentration of sucrose is 20 w / v, the concentration of trehalose is 5 w / v, the concentration of sodium caseinate is 1 w / v, and the concentration of surfactant S9 is 1 w / v.

[0151] In some embodiments of the sixth aspect, the marking pad is dried in an environment of 50±1°C and less than 10% humidity for 24-72 hours (e.g., 23-25 ​​hours).

[0152] In some implementations of the sixth aspect, the marking pad prepared by the method is an integral pad, which has significant advantages in production efficiency and precision compared to the traditional "marking pad + sample pad" process. Because it eliminates one intermediate step, the production process is simpler and suitable for automated roll-to-roll production. Furthermore, it reduces the cumbersome processes of soaking and cutting required in traditional production processes, lowering the risk of instability due to human error.

[0153] In a seventh aspect, this application provides a colloidal gold test strip that includes the marking pad described in the sixth aspect.

[0154] In some embodiments of the seventh aspect, the test strip further includes a substrate (e.g., a PVC board), a nitrocellulose membrane, and absorbent paper.

[0155] In some embodiments of the seventh aspect, the marking pad, the nitrocellulose membrane, and the absorbent paper are sequentially attached to the substrate, and the marking pad and the absorbent paper are each partially placed on the nitrocellulose membrane.

[0156] In some implementation schemes of the seventh aspect, the test strip may also be presented in the form of a test card.

[0157] In some embodiments of the seventh aspect, the method for preparing the test strip is well known to those skilled in the art.

[0158] In some embodiments of aspect seven, the C-line (control line) and T-line (detection line) are located on a nitrocellulose membrane. In some embodiments, the T-line is coated with anti-CRP antibody at a concentration of 0.05 mg / mL. In some embodiments, the C-line is coated with DNP-BSA at a concentration of 0.3 mg / mL.

[0159] Eighthly, this application provides the use of the colloidal gold solution described in the second aspect, the colloidal gold labeled protein described in the fourth aspect, the labeling pad described in the sixth aspect, or the colloidal gold test strip described in the seventh aspect in the preparation of a kit for the detection of disease biomarkers in biological samples.

[0160] In some embodiments of the eighth aspect, the biological sample is blood or serum.

[0161] In some implementations of the eighth aspect, the disease markers are C-reactive protein, serum amyloid A, procalcitonin, glycated hemoglobin, cardiac troponin, N-terminal pro-B-type natriuretic peptide, novel coronavirus surface antigen, influenza virus surface antigen, or mycoplasma surface antigen.

[0162] In some implementations, the novel coronavirus surface antigen is the nucleocapsid protein (N protein) or the spike protein (S protein).

[0163] In some implementations, the influenza virus surface antigen is hemagglutinin (HA) or neuraminidase (NA).

[0164] In some implementations, the mycoplasma surface antigen is the P1 outer membrane protein.

[0165] In some implementations, C-reactive protein is a marker for acute inflammation, atherosclerosis, bacterial infection, viral infection, malignancy, liver cancer, benign liver disease, acute pancreatitis, coronary heart disease, or myocardial infarction.

[0166] In some implementations, C-reactive protein is used for diagnosis, auxiliary diagnosis, detection, auxiliary detection, monitoring, auxiliary monitoring, prediction, and auxiliary prediction of whether a subject has acute inflammation, atherosclerosis, bacterial infection, viral infection, malignancy, liver cancer, benign liver disease, acute pancreatitis, coronary heart disease, or myocardial infarction.

[0167] In some implementations, C-reactive protein is used to predict, assist in predicting, assess, or assist in assessing the risk of a subject having acute inflammation, atherosclerosis, bacterial infection, viral infection, malignancy, liver cancer, benign liver disease, acute pancreatitis, coronary heart disease, or myocardial infarction.

[0168] In some implementations, C-reactive protein is used to assess or assist in assessing the severity of acute inflammation, atherosclerosis, bacterial infection, viral infection, malignancy, liver cancer, benign liver disease, acute pancreatitis, coronary heart disease, or myocardial infarction in a subject.

[0169] In some implementations, serum amyloid A is a marker of viral infection, bacterial infection, metastatic malignancy, transplant rejection, rheumatoid arthritis, tuberculosis, leprosy, or sepsis.

[0170] In some implementations, serum amyloid A is used for diagnosis, auxiliary diagnosis, detection, auxiliary detection, monitoring, auxiliary monitoring, prediction, and auxiliary prediction of whether a subject has a viral infection, bacterial infection, metastatic malignant tumor, transplant rejection, rheumatoid arthritis, tuberculosis, leprosy, or sepsis.

[0171] In some implementations, serum amyloid A is used to predict, assist in predicting, assess, or assist in assessing the risk of a subject having a viral infection, bacterial infection, metastatic malignancy, transplant rejection, rheumatoid arthritis, tuberculosis, leprosy, or sepsis.

[0172] In some implementations, serum amyloid A is used to assess or assist in assessing the severity of viral infection, bacterial infection, metastatic malignancy, transplant rejection, rheumatoid arthritis, tuberculosis, leprosy, or sepsis in a subject.

[0173] In some implementations, procalcitonin is a marker of bacterial infection, fungal infection, parasitic infection, sepsis, or multiple organ failure.

[0174] In some implementation schemes, procalcitonin is used for diagnosis, auxiliary diagnosis, detection, auxiliary testing, monitoring, auxiliary surveillance, prediction, and auxiliary prediction of whether a subject has a bacterial infection, fungal infection, parasitic infection, sepsis, or multiple organ failure.

[0175] In some implementations, procalcitonin is used to predict, assist in predicting, assess, or assist in assessing the risk of a subject having a bacterial infection, fungal infection, parasitic infection, sepsis, or multiple organ failure.

[0176] In some implementations, procalcitonin is used to assess or assist in the assessment of the severity of a subject’s bacterial infection, fungal infection, parasitic infection, sepsis, or multiple organ failure.

[0177] In some implementations, glycated hemoglobin is a biomarker for diabetes.

[0178] In some implementations, glycated hemoglobin is used for diagnosis, auxiliary diagnosis, detection, auxiliary detection, monitoring, auxiliary monitoring, prediction, and auxiliary prediction of whether a subject has diabetes.

[0179] In some implementations, glycated hemoglobin is used to predict, assist in predicting, assess, or assist in assessing the risk of a subject having diabetes.

[0180] In some implementations, glycated hemoglobin is used to assess or assist in assessing the severity of diabetes in a subject.

[0181] In some implementations, cardiac troponin is a biomarker for acute myocardial infarction or acute coronary syndrome.

[0182] In some implementations, cardiac troponin is used for diagnosis, auxiliary diagnosis, detection, auxiliary detection, monitoring, auxiliary monitoring, prediction, and auxiliary prediction of whether a subject has acute myocardial infarction or acute coronary syndrome.

[0183] In some implementations, cardiac troponin is used to predict, assist in predicting, assess, or assist in assessing the risk of a subject having an acute myocardial infarction or acute coronary syndrome.

[0184] In some implementations, cardiac troponin is used to assess or assist in assessing the severity of a subject's acute myocardial infarction or acute coronary syndrome.

[0185] In some implementations, N-terminal pro-B-type natriuretic peptide is a marker of heart failure, hereditary heart disease, myocardial infarction, or rheumatic heart disease.

[0186] In some implementations, N-terminal B-type natriuretic peptide is used for diagnosis, auxiliary diagnosis, detection, auxiliary detection, monitoring, auxiliary monitoring, prediction, and auxiliary prediction of whether a subject has heart failure, hereditary heart disease, myocardial infarction, or rheumatic heart disease.

[0187] In some implementations, N-terminal B-type natriuretic peptide is used to predict, assist in predicting, assess, or assist in assessing the risk of a subject having heart failure, hereditary heart disease, myocardial infarction, or rheumatic heart disease.

[0188] In some implementations, N-terminal B-type natriuretic peptide is used to assess or assist in assessing the severity of heart failure, hereditary heart disease, myocardial infarction, or rheumatic heart disease in a subject.

[0189] In some implementations, the novel coronavirus surface antigen is a biomarker for COVID-19.

[0190] In some implementation schemes, the novel coronavirus surface antigen is used for diagnosis, auxiliary diagnosis, detection, auxiliary testing, monitoring, auxiliary surveillance, prediction, and auxiliary prediction of whether a subject has COVID-19.

[0191] In some implementations, the novel coronavirus surface antigen is used to predict, assist in predicting, assess, or assist in assessing whether a subject is at risk of having COVID-19.

[0192] In some implementations, the novel coronavirus surface antigen is used to assess or assist in assessing the severity of a subject's COVID-19 infection.

[0193] In some implementation schemes, influenza viruses include influenza A, influenza B, influenza C, and influenza D viruses.

[0194] In some implementation schemes, influenza virus surface antigen is a marker for influenza A, influenza B, influenza C, or influenza D.

[0195] In some implementation schemes, influenza virus surface antigen is used for diagnosis, auxiliary diagnosis, detection, auxiliary testing, monitoring, auxiliary surveillance, prediction, and auxiliary prediction of whether an individual has influenza A, influenza B, influenza C, or influenza D.

[0196] In some implementation schemes, influenza virus surface antigen is used to predict, assist in predicting, assess, or assist in assessing whether an individual is at risk of having influenza A, influenza B, influenza C, or influenza D.

[0197] In some implementation schemes, influenza surface antigen is used to assess or assist in assessing the severity of a subject’s influenza A, influenza B, influenza C, or influenza D infection.

[0198] In some implementations, mycoplasma includes Mycoplasma pneumoniae and Mycoplasma genitalium.

[0199] In some implementations, mycoplasma surface antigen is a marker for mycoplasma pneumonia or mycoplasma urogenital infection.

[0200] In some implementation schemes, mycoplasma surface antigen is used for diagnosis, auxiliary diagnosis, detection, auxiliary detection, monitoring, auxiliary monitoring, prediction, and auxiliary prediction of whether an individual has mycoplasma pneumonia or mycoplasma urogenital infection.

[0201] In some implementations, mycoplasma surface antigen is used to predict, assist in predicting, assess, or assist in assessing the risk of a subject having mycoplasma pneumonia or mycoplasma genitourinary infection.

[0202] In some implementations, mycoplasma surface antigen is used to assess or assist in assessing the severity of a subject's mycoplasma pneumonia or mycoplasma genitourinary infection.

[0203] In some embodiments, the method for preparing colloidal gold solution provided in this application involves mixing chloroauric acid and a reducing agent uniformly at room temperature before heating and reacting. Compared with the traditional method of heating water to boiling before adding chloroauric acid and a reducing agent, this method significantly improves the operational safety of the preparation process. At the same time, it allows for more precise operation at room temperature, thereby improving the preparation efficiency of colloidal gold and reducing batch-to-batch variation.

[0204] In some embodiments, the method for preparing colloidal gold-labeled proteins provided in this application can significantly improve labeling efficiency, while eliminating the step of adjusting the pH with potassium carbonate before labeling, and further improving labeling efficiency.

[0205] In some implementations, the structural characteristic of CRP forming pentamers with identical subunits is combined with the property of gold-labeled antibodies binding multiple CRP monoclonal antibodies onto a single colloidal gold particle. This results in "gold-labeled antibody-CRP antigen complexes" of varying sizes due to the different intensities of the cascade reaction under different CRP concentrations. Specifically, higher CRP content leads to a stronger cascade reaction, resulting in more cascaded "gold-labeled antibody-CRP antigen complexes" and larger complex diameters. Building upon this, and considering the characteristics of larger particle size in lateral chromatography resulting in slower chromatography speed and higher sensitivity, by selecting a nitrocellulose membrane with an appropriate pore size (e.g., 15 μm), the desired effect is achieved: lower CRP concentrations result in lower sensitivity and shallower color development; as CRP concentration increases, the cascade reaction intensifies, and the color depth doubles with increasing CRP concentration. This achieves the ideal effect of barely visible color development at a low CRP concentration of 10 mg / L, with the color depth of the detection line doubling as the CRP concentration increases.

[0206] In some embodiments, the marking pad prepared in this application simplifies the manufacturing process by combining the sample pad and the marking pad into one, thereby making the product more suitable for automated production of roll materials.

[0207] In some implementations, the label pad prepared in this application is wider (e.g., 17 mm), which improves the precision of the product by spraying the gold-labeled antibody multiple times.

[0208] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0209] The following examples are for illustrative purposes only and are not intended to limit the scope of this application.

[0210] Example

[0211] The solution preparation details used in this application are shown in Tables 1-12 below:

[0212] Table 1. Formulation of 4 w / v% HAuCl4 solution

[0213]

[0214] Weigh the above reagents, add 100 mL of purified water, and stir to dissolve.

[0215] Table 2. Formulation of 0.2M PB solution

[0216]

[0217] Weigh the above reagents, add 1000mL of purified water, stir to dissolve, and the pH value should be between 7.2 and 7.6.

[0218] Table 3. Formulation of CRP sample dilution solution

[0219]

[0220] Weigh the above reagents, add 800 mL of purified water, stir to dissolve, and then bring the volume to 1000 mL. The pH value should be between 7.2 and 7.6.

[0221] Table 4. Formulation of 2w / v% sodium citrate solution

[0222]

[0223] Weigh the above reagents, add 100 mL of purified water, and stir to dissolve.

[0224] Table 5. Formulation of 0.05M Tris-HCl solution

[0225]

[0226] Weigh the above reagents, add 800 mL of purified water, stir to dissolve, adjust the pH to 8.0 ± 0.1 with HCl, and bring the volume to 1000 mL.

[0227] Table 6. Formulation of the labeling blocking solution

[0228]

[0229] Weigh the above reagents, add 100 mL of purified water, and stir to dissolve.

[0230] Table 7. Formulation of coating diluent

[0231]

[0232] Weigh the above reagents and stir to dissolve.

[0233] Table 8. Formulation of Labeled Diluent

[0234]

[0235] Weigh the above reagents and stir to dissolve.

[0236] Table 9. Formulation of refolding solution

[0237]

[0238] Weigh the above reagents, add 80 mL of purified water, stir to dissolve, adjust the pH to 9.0 ± 0.1 with NaOH, and bring the volume to 100 mL.

[0239] Table 10. Formulation of working solution for marker pads

[0240]

[0241] Measure the above reagents and stir to a final volume.

[0242] Table 11. Formulation of CRP-coated membrane C-line coating working solution

[0243]

[0244] Measure the above reagents, make up to volume, and mix well.

[0245] Table 12. Formulation of CRP-coated membrane T-line coating working solution

[0246]

[0247] Measure the above reagents, make up to volume, and mix well.

[0248] Example 1. Preparation of CRP test card

[0249] 1. Preparation of colloidal gold solution

[0250] 1.1 Add 20 mL of 4 w / v HAuCl4 solution and 60 mL of 2% sodium citrate solution to a container of purified water placed on an electric heating magnetic stirrer while stirring, so that the final concentration of HAuCl4 is 0.04 w / v and the final concentration of sodium citrate is 0.06 w / v.

[0251] 1.2 Turn on the heating and heat at 300℃ until the solution boils. Continue heating for 5 minutes, then turn off the heating and continue stirring until cooled.

[0252] 1.3 After cooling, dilute to 2L with purified water, and take 5mL of colloidal gold solution for quality verification such as pH measurement, UV spectrophotometer detection, and label verification.

[0253] 1.4 After passing the inspection, affix a label and store at room temperature for later use.

[0254] 2. Preparation of colloidal gold-labeled antibodies

[0255] The following process is performed according to the amount used to prepare 1000 mL of colloidal gold-labeled antibody.

[0256] 2.1 Preparation of colloidal gold-labeled anti-CRP antibody markers

[0257] 2.1.1 Measure 1000 mL of colloidal gold solution into a clean beaker, add the rotor and place it on a magnetic stirrer to rotate and stir. Adjust the stirring speed so that the height of the stirring vortex is about 1 / 3 of the height from the highest point of the liquid surface to the bottom.

[0258] 2.1.2 Quickly add 30 mg of anti-CRP antibody and stir for 15 min.

[0259] 2.1.3 After the reaction is complete, add 10 mL of labeled blocking solution and stir for 15 min.

[0260] 2.1.4 After the blocking reaction was completed, the labeled substance was transferred to a centrifuge tube and centrifuged at 10,000 rpm and 4°C for 5 min. After centrifugation, the supernatant was discarded, and an equal volume of refolding solution was added to the precipitate. The precipitate was then resuspended by sonication using a cell disruptor (sonication at 70% for 4 seconds, pause for 4 seconds, for a total of 3 min, with the sonicator inserted to about 1 / 3 of the liquid surface). The precipitate was then centrifuged at 10,000 rpm for 30 min at 4°C.

[0261] 2.1.5 After centrifugation, remove the supernatant, and dilute the precipitate to 50 mL with labeled diluent. Sonicate thoroughly, label, and store at 2-8℃.

[0262] 2.2 Preparation of colloidal gold-labeled anti-DNP antibody markers

[0263] 2.2.1 Measure 1000 mL of colloidal gold solution into a clean beaker, add the rotor, and place it on a magnetic stirrer to rotate and stir. Adjust the stirring speed so that the height of the stirring vortex is about 1 / 3 of the height from the highest point of the liquid surface to the bottom.

[0264] 2.2.2 Quickly add 20 mg of anti-DNP antibody and stir for 15 min.

[0265] 2.2.3 After the reaction is complete, add 10 mL of labeled blocking solution and stir for 15 min.

[0266] 2.2.4 After the blocking reaction was completed, the labeled material was transferred to a centrifuge tube and centrifuged at 10,000 rpm and 4°C for 5 min. After centrifugation, the supernatant was discarded, an equal volume of refolding solution was added, and the cells were resuspended by sonication using a cell disruptor (sonication at 70% for 4 seconds, pause for 4 seconds, for a total of 3 min, with the sonicator inserted to about 1 / 3 of the liquid surface). The cells were then centrifuged at 10,000 rpm for 30 min at 4°C.

[0267] 2.2.5 After centrifugation, remove the supernatant, and dilute the precipitate to 50 mL with labeled diluent. Sonicate thoroughly, label, and store at 2-8℃.

[0268] 2.3 Preparation of colloidal gold-labeled anti-CRP antibody markers using conventional methods

[0269] The following process is performed according to the amount used to prepare 1000 mL of colloidal gold-labeled antibody.

[0270] 2.3.1 Measure 1000 mL of colloidal gold solution into a clean beaker, add the rotor and place it on a magnetic stirrer to rotate and stir. Adjust the stirring speed so that the height of the stirring vortex is about 1 / 3 of the height from the highest point of the liquid surface to the bottom.

[0271] 2.3.2 Add 20 mL of 0.2 M potassium carbonate solution and stir for 15 min.

[0272] 2.3.3 Quickly add 30 mg of anti-CRP antibody and stir for 15 min.

[0273] 2.3.4 After the reaction is complete, add 10 mL of labeled blocking solution and stir for 15 min.

[0274] 2.3.5 After sealing, centrifuge to remove the supernatant, and dilute the precipitate to 50 mL with labeled diluent. Sonicate to mix thoroughly, label, and store at 2-8℃ for later use.

[0275] 2.4 Preparation of Colloidal Gold-Labeled Anti-DNP Antibody Markers Using Conventional Methods

[0276] 2.3.1 Measure 1000 mL of colloidal gold solution into a clean beaker, add the rotor and place it on a magnetic stirrer to rotate and stir. Adjust the stirring speed so that the height of the stirring vortex is about 1 / 3 of the height from the highest point of the liquid surface to the bottom.

[0277] 2.3.2 Add 20 mL of 0.2 M potassium carbonate solution and stir for 15 min.

[0278] 2.3.3 Quickly add 30 mg of anti-DNP antibody and stir for 15 min.

[0279] 2.3.4 After the reaction is complete, add 10 mL of labeled blocking solution and stir for 15 min.

[0280] 2.3.5 After sealing, centrifuge to remove the supernatant, and dilute the precipitate to 50 mL with labeled diluent. Sonicate to mix thoroughly, label, and store at 2-8℃ for later use.

[0281] 3. Preparation of CRP Marking Pads

[0282] 3.1 Obtain the corresponding quantity of glass cellulose membrane (17mm wide) and gold-labeled antibody according to the production volume.

[0283] 3.2 Prepare the corresponding volume of labeling pad working solution according to the labeling working solution formula shown in Table 10.

[0284] 3.3 Clean the instrument according to the standard operating requirements of the spray pad machine. After cleaning, set the parameters, add the prepared gold-labeled antibody to the corresponding pump, set the spray volume to 4 μL / cm, the air pressure to 0.2 MPa, and the spray pad position to be 3 mm away from the edge of the labeling pad with an interval of 5 mm. Repeat the spray pad operation twice.

[0285] 3.4 After the gold spraying is completed, place the gold label pad in a drying oven (50±1℃, humidity less than 10%) and dry for 23-25 ​​hours.

[0286] 3.5 After drying, proceed to the next process or dry and seal for storage.

[0287] The prepared marking pads are as follows Figure 1 As shown.

[0288] 4. Preparation of CRP-coated membranes

[0289] 4.1 Requisition NC film and PVC board according to production volume, and cut the NC film into 30cm lengths. Peel off the protective film from the NC adhesive area in the middle of the PVC base board, and attach the smooth side of the NC film to the adhesive area with the rough side facing up and the smooth side facing down, for later use.

[0290] 4.2 Preparation of CRP T-line coating working solution: Measure the prepared coating diluent, add CRP coating antibody to a final concentration of 0.05 mg / mL, mix thoroughly, label, and set aside.

[0291] 4.3 CRP C-line coating working solution: Measure the prepared coating diluent, add DNP-BSA to a final concentration of 0.3 mg / mL, mix thoroughly, label, and set aside.

[0292] 4.4 Clean the instrument according to the standard operation of the scribing machine. After cleaning, set the scribing parameters of the instrument, add the prepared detection line and quality control line solutions to the corresponding pumps, set the scribing liquid volume to 1μL / cm, the T scribing position to be 31mm away from the edge of the PVC board at the end of the absorbent paper, and the C scribing position to be 26mm away from the edge of the PVC board at the end of the absorbent paper, and then perform the scribing operation.

[0293] 4.5 After scribing, place the NC film in a drying oven (temperature 50±1℃, humidity <10%) and dry for 47-49 hours.

[0294] 4.6 After drying, proceed to the next process or dry and seal for storage.

[0295] A schematic diagram of the CRP-coated membrane is shown below. Figure 2 As shown.

[0296] 5. Preparation of large test plates

[0297] The preparation was carried out in a drying room at a temperature of 18-28℃ and a humidity of 10%-30%.

[0298] 5.1 Prepare dried marking pads, wrapping film, and absorbent paper.

[0299] 5.2 Adhesion of the labeling pad: Peel off the protective film of the PVC board labeling pad area, align the edge of the labeling pad with the edge of the PVC board labeling pad adhesive area and adhere it, with the other end resting on the NC film, making sure the front is facing up and the gold-labeled antibody is adhered close to the NC film.

[0300] 5.3 Applying absorbent paper: Peel off the protective film from the absorbent paper area of ​​the PVC board, align one end of the cut absorbent paper with the edge of the PVC board and apply it, and place the other end on the NC film.

[0301] 5.4 After the adhesive is applied, package it according to the specified packaging specifications, dry it, seal it, and store it.

[0302] A schematic diagram of the large board testing is shown below. Figure 3 As shown.

[0303] 6. Preparation of the test card

[0304] The preparation is carried out in a drying room at a temperature of 18-28℃ and a humidity of 10%-30%.

[0305] 6.1 Cutting strips: Cut the large test plate into test strips with a width of 3mm using a strip cutter.

[0306] 6.2 Mounting: Mount the test strip into the housing and press it tightly with a housing press.

[0307] 6.3 Packaging: Place the test strip with the casing into an aluminum foil bag and add 1g of bagged desiccant.

[0308] 6.4 Sealing: The card holders in the bag are sealed with a sealing machine to keep the reagent cards and desiccant sealed inside the aluminum foil bag.

[0309] 6.5 Storage: Store the prepared test cards in a dry place at room temperature.

[0310] Prepared test cards such as Figure 4 As shown.

[0311] Example 2. Performance Verification

[0312] 1. Testing Method

[0313] The sample was diluted with CRP sample diluent at a ratio of 100:1. The sample volume was 50 μL, and the reaction time was 10 min.

[0314] 2. Linearity determination

[0315] The grayscale values ​​detected by the colloidal gold immunoassay analyzer for the linear reference test are shown in Table 13 below. The linear reference is antigen with catalog number RYZ004Ag1C-0001 produced by Shenzhen Ruimeng Innovation Biotechnology Co., Ltd., and the values ​​were obtained after testing and determination using a C-reactive protein assay kit (immunoturbidimetric method) produced by Siemens.

[0316] The grayscale value was determined using an AGS1000 colloidal gold immunochromatographic analyzer manufactured by Guangzhou Lanbo Biotechnology Co., Ltd.

[0317] Table 13. Gray values ​​detected by colloidal gold immunoassay analyzer for linear reference test

[0318]

[0319] Figure 5 The results showed that the prepared colloidal gold exhibited a high linearity in the concentration range of 10 mg / L to 200 mg / L.

[0320] 3. Precision determination

[0321] Assay method: Using the same batch of reagent kits, the quality control samples of different concentrations were measured 10 times each. The mean (M) and standard deviation (SD) of the 10 measurements were calculated, and the coefficient of variation (CV) was obtained according to the following formula.

[0322] CV = SD / M × 100%

[0323] in:

[0324] CV stands for coefficient of variation.

[0325] SD is the standard deviation of 10 measurements;

[0326] M is the average value of the 10-ohm measurement results.

[0327] 3.1 The precision verification results of the yin-yang judgment value of 10 mg / L are shown in Table 14.

[0328] Table 14. Precision verification results of the yin-yang judgment value of 10 mg / L

[0329]

[0330] 3.2 The precision verification results at a median of 50 mg / L are shown in Table 15.

[0331] Table 15. Precision verification results at a median of 50 mg / L

[0332]

[0333] 3.3 The precision verification results for the high value of 200 mg / L are shown in Table 16.

[0334] Table 16. Precision verification results for high value of 200 mg / L

[0335]

[0336] The results in Tables 14-16 show that the precision verification results (CV) for low, medium, and high values ​​are all within 10%, which meets the performance requirements of the product.

[0337] 4. Clinical validation

[0338] Take no fewer than 20 human serum samples with different concentrations reasonably distributed within the linear range and conduct a comparative experiment with the specified analytical system. Each sample should be tested according to the requirements of the test reagent (kit) and the selected analytical system. Each sample should be tested once. Use the linear regression method to perform linear fitting on the two sets of results, and obtain the correlation coefficient (r) and slope of the linear regression equation. The correlation coefficient (r) should be ≥0.950, and the slope should be within [0.9, 1.1].

[0339] 4.1 The grayscale values ​​of the clinical samples tested using a colloidal gold immunoassay analyzer are shown in Table 17.

[0340] Table 17. Gray values ​​detected by colloidal gold immunoassay analyzer for clinical sample testing

[0341]

[0342] 4.2 Correlation analysis between test results and reference reagent

[0343] The results are as follows Figure 6 As shown, the test results of this invention have a good correlation with the reference reagent, with R² being 0.9979.

[0344] 5. Comparison of conventional labeling methods and complex labeling methods

[0345] When test strips prepared by conventional labeling methods and renaturation labeling methods are tested on the same batch of samples under the condition that the positive and negative judgment values ​​are adjusted to be consistent, the deeper the color development of the high value and the higher the color gradient, the better the labeling effect.

[0346] Figure 7 The results show that, when the sensitivity differences are not significant, the reagent gradient prepared by the refolding labeling method is significantly better than that prepared by the conventional labeling method.

[0347] All patents, patent application publications, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. Exemplary embodiments of the inventions described above have been described; however, those skilled in the art can modify or improve the exemplary embodiments described herein without departing from the spirit and scope of this application, and such variations or equivalents also fall within the scope of this application.

Claims

1. A method for preparing colloidal gold-labeled antibodies, comprising: (1) Prepare an aqueous mixture of HAuCl4 and trisodium citrate, wherein the concentration of HAuCl4 in the aqueous mixture is 0.04 w / v% and the concentration of trisodium citrate is 0.06 w / v%. (2) Heat the aqueous mixture described in step (1) to boiling at a temperature of 100-500℃ and maintain it for 1-10 minutes, then stop heating and cool to room temperature; (3) Use water to adjust the volume of the solution obtained in step (2) back to the volume of the aqueous mixture described in step (1) to obtain a colloidal gold solution; (4) Add the antibody to be labeled to the colloidal gold solution obtained in step (3), stir and react for 15 minutes to obtain the labeled mixture, wherein the antibody to be labeled is an anti-C-reactive protein antibody or an anti-dinitrophenol antibody; (5) Add a marking and sealing solution to the marked mixture for sealing; and (6) After sealing, centrifuge to remove the supernatant, add an equal volume of refolding solution to the precipitate, resuspend the precipitate by sonication using a cell disruptor, centrifuge to remove the supernatant, and add a label diluent to the precipitate. The refolding solution is a solution containing 2 mM boric acid and 0.5 w / v% PEG20000 with a pH of 8.9-9.1; and The labeling diluent is a solution containing 10-30 w / v% sucrose, 2.5-7.5 w / v% trehalose, 0.1-2 w / v% sodium caseinate, 0.1-2 w / v% polyvinylpyrrolidone, 0.1-2 w / v% surfactant S9, and 10-100 mM Tris-HCl.

2. The method as described in claim 1, wherein, The concentration of the antibody to be labeled in the labeled mixture is 10-30 μg / mL; and / or The volume ratio of the added labeling blocking solution to the colloidal gold solution is 1:100; and / or The labeling and sealing solution is 10 w / v% BSA.

3. The method as described in claim 1, wherein, The concentration of the antibody to be labeled in the labeled mixture is 20-30 μg / mL.

4. The method of claim 1, wherein, The volume ratio of the precipitate to the labeled diluent is 1:

50.

5. The method of claim 1, wherein, The labeling diluent is a solution containing 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% sodium caseinate, 1 w / v% polyvinylpyrrolidone, 1 w / v% surfactant S9 and 50 mM Tris-HCl.

6. The method of claim 1, wherein, In step (2), the aqueous mixture is heated to boiling and maintained for 5 minutes.

7. The method of claim 1, wherein, The water used in step (3) is double-distilled deionized water or triple-distilled deionized water.

8. The method of claim 1, wherein, In step (2), the aqueous mixture is heated at a temperature of 300°C.

9. A colloidal gold-labeled antibody prepared by the method of any one of claims 1 to 8.

10. A kit comprising the colloidal gold-labeled antibody of claim 9.

11. A labeling pad for a colloidal gold test strip, comprising at least two spaced-apart labeling lines, each of the at least two labeling lines being loaded with a first colloidal gold-labeled antibody and a second colloidal gold-labeled antibody; in, The first colloidal gold-labeled antibody is prepared by the method according to any one of claims 1 to 8, and the antibody to be labeled is an anti-C-reactive protein antibody; and / or The second colloidal gold-labeled antibody is prepared by the method described in any one of claims 1 to 8, and the antibody to be labeled is an anti-dinitrophenol antibody.

12. A colloidal gold test strip comprising the marking pad as described in claim 11; The test strip also includes a substrate, a nitrocellulose membrane, and absorbent paper; and The marking pad, the nitrocellulose membrane, and the absorbent paper are sequentially attached to the substrate, and the marking pad and the absorbent paper are each partially placed on the nitrocellulose membrane.

13. Use of the colloidal gold-labeled antibody of claim 9, the labeling pad of claim 11, or the colloidal gold test strip of claim 12 in the preparation of a kit for the detection of disease biomarkers in biological samples; wherein the disease biomarker is C-reactive protein.

14. The use as described in claim 13, wherein the biological sample is blood or serum.

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