Reagent for detecting disease marker by colloidal gold method as well as preparation method and application of reagent

Abstract

The invention provides a preparation method of a colloidal gold solution, a preparation method of colloidal gold labeled protein, and the colloidal gold solution and the colloidal gold labeled protein prepared by the method. The invention further discloses a kit containing the colloidal gold solution or the colloidal gold protein, a marking pad for the colloidal gold test strip, the colloidal gold test strip and application of the colloidal gold solution, the colloidal gold marking protein, the marking pad or the colloidal gold test strip in preparation of the kit for detecting disease markers in biological samples. For example, the kit provided by the invention can be used for detecting the C-reactive protein by a colloidal gold method.

Description

Technical Field

[0001] The present application belongs to the field of in vitro diagnosis, and mainly relates to a method for preparing colloidal gold, a prepared colloidal gold solution, a method for preparing a colloidal gold labeled protein, a prepared colloidal gold labeled protein, a kit containing the colloidal gold solution or the colloidal gold labeled protein, a labeling pad for a colloidal gold test strip, a colloidal gold test strip, and the 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 disease markers in biological samples. Background Art

[0002] C-reactive protein (CRP) refers to some proteins (acute proteins) in the plasma that rise sharply when the body is infected or the tissue is damaged. CRP can activate complement and strengthen the phagocytosis of phagocytes to play a conditioning role, thereby removing pathogenic microorganisms that invade the body and damaged, necrotic, and apoptotic tissue cells, and plays an important protective role in the body's natural immune process. CRP is not only a non-specific inflammatory marker, but it is also directly involved in cardiovascular diseases such as inflammation and atherosclerosis, and is the most powerful predictor and risk factor for cardiovascular disease. The interaction between CRP and complement C1q and FcTR enables it to exhibit many biological activities, including the host's defense response to infection, phagocytosis and regulation of inflammatory responses, etc. The combination with damaged cells, apoptotic cells and nuclear antigens makes it also play an important role in autoimmune diseases.

[0003] The determination of CRP has important clinical significance: (1) It rises rapidly within a few hours after the onset of various acute inflammations, tissue damage, myocardial infarction, surgical trauma, radiation damage and other diseases, and has a tendency to double, indicating possible concurrent 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 human body nonspecific resistance; when the patient's disease occurs, it may rise earlier than white blood cells and return to normal quickly, so it has extremely high sensitivity. (3) It can help identify the type of respiratory infection. CRP can be used for differential diagnosis of bacterial and viral infections: in bacterial infections, CRP levels increase; in viral infections, CRP does not increase or increases slightly. Therefore, CRP values ​​can help doctors identify the type of respiratory infection and give targeted drugs and treatments. (4) CRP is elevated in patients with malignant tumors. The combined detection of CRP and AFP can be used for differential diagnosis between liver cancer and benign liver diseases, and for judging the efficacy and prognosis of liver cancer. CRP rises before surgery and decreases after surgery, and its response is not affected by radiotherapy, chemotherapy, and corticosteroid treatment, which helps to evaluate the progression of the tumor. (5) To evaluate the severity of acute pancreatitis, when CRP is higher than 250 mg / L, it indicates extensive necrotizing pancreatitis. (6) CRP can also be used to predict the risk of coronary heart disease and myocardial infarction.

[0004] Although CRP qualitative detection reagents are widely used in rural hospitals, clinics and homes, the products currently on the market have many defects in such usage scenarios. The defects of these products are mainly concentrated in the following two aspects:

[0005] (1) Too high sensitivity. The main reason is that when inflammation occurs, the CRP content in the blood is high. The general threshold for inflammation diagnosis is 10 mg / L, so the sensitivity of the products developed by many manufacturers and institutions is generally lower than 10 mg / L, and then the colorimetric method is used to judge the positive and negative by matching color cards. Another method is to increase the sample dilution multiple. Some manufacturers need to dilute the sample 1000 times or even higher before testing. This requires more than 10 times the amount of diluent than ordinary products, which greatly increases the transportation cost and production cost; at the same time, it is necessary to cooperate with lower sample collection to achieve a higher dilution multiple. Lower sampling increases the sampling error rate, which has a greater impact on the accuracy of the test. The colorimetric method of judging the positive and negative and the severity of inflammation has high requirements for operators, and professional training is required to avoid incorrect use, especially in the home use scenario, which is highly subjective.

[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. In order to achieve lower sensitivity, many products use antibodies with lower sensitivity and larger sample dilution multiples. This method usually introduces the second problem, that is, 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 the inflammation.

[0007] Given the importance of CPR in clinical diagnosis, the development of effective reagents and methods for CPR detection will be of great significance. Summary of the invention

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

[0009] (1) preparing 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) heating the aqueous mixture of step (1) to boiling and maintaining the boiling temperature for 1-10 min, stopping the heating and cooling the mixture to room temperature;

[0011] (3) The solution obtained in step (2) is diluted with water to restore the volume of the aqueous mixture in step (1).

[0012] In some embodiments of the first aspect, the concentration of HAuCl4 in the aqueous mixed solution 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 mixed liquid is heated at a temperature of 100-500°C.

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

[0019] In a second aspect, the present application provides a colloidal gold solution prepared by the method described in the first aspect.

[0020] In a third aspect, the present application provides a method for preparing colloidal gold labeled protein, which comprises:

[0021] Adding the protein to be labeled to the colloidal gold solution of the second aspect to label the protein, thereby obtaining a labeled mixed solution;

[0022] Adding a labeled blocking solution to the labeled mixed solution for blocking;

[0023] After blocking, the supernatant was removed by centrifugation, and the renaturation solution was added to the precipitate for renaturation.

[0024] In some embodiments of the third aspect, the concentration of the protein to be labeled in the labeled mixed solution 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 is added to the precipitate in an amount equal to that of the precipitate.

[0027] In some embodiments of the third aspect, the labeling blocking solution 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 a pH of 8.0-9.5.

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

[0030] In some embodiments of the third aspect, the concentration of the protein to be labeled in the labeled mixed solution 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 a pH of 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 comprises centrifuging the renatured 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.

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

[0035] In some embodiments of the third aspect, the labeled diluent is a solution comprising 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% casein sodium salt, 1 w / v% polyvinyl pyrrolidone and 1 w / v% S9.

[0036] In some embodiments of the third aspect, the labeled diluent is a solution comprising 10-30 w / v% sucrose, 2.5-7.5 w / v% trehalose, 0.1-2 w / v% casein sodium salt, 0.1-2 w / v% polyvinyl pyrrolidone, 0.1-2 w / v% surfactant S9 and 10-100 mM Tris-HCl.

[0037] In some embodiments of the third aspect, the labeled diluent is a solution comprising 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% casein sodium salt, 1 w / v% polyvinyl pyrrolidone, 1 w / v% surfactant S9 and 50 mM Tris-HCl.

[0038] In a fourth aspect, the present application provides a colloidal gold-labeled protein prepared by the method described in the third aspect.

[0039] In a fifth aspect, the present application provides a kit, which includes the colloidal gold solution described in the second aspect or the colloidal gold protein described in the fourth aspect.

[0040] In a sixth aspect, the present application provides a labeling pad for a colloidal gold test strip, comprising at least two labeling lines separated from each other, wherein each of the at least two labeling lines is 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 marker 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, the present application provides a colloidal gold test strip comprising the marking pad described in the sixth aspect.

[0046] In some embodiments of the seventh aspect, the test strip further comprises 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] In an eighth aspect, the present application provides 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 detecting disease markers 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 marker is C-reactive protein, serum amyloid A, procalcitonin, glycosylated hemoglobin, cardiac troponin or N-terminal pro-B-type natriuretic peptide, new coronavirus surface antigen, influenza virus surface antigen or mycoplasma surface antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A picture of the prepared labeling pad is shown.

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

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

[0055] Figure 4 A picture of the prepared test card is shown.

[0056] Figure 5 A scatter plot of the test strips made using the prepared colloidal gold solution in the linear range is shown.

[0057] Figure 6The results of correlation analysis between 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 testing using conventional labeling method and annealing labeling method are shown in the figure. The upper figure is the result of conventional labeling method, and the lower figure is the result of annealing labeling method. DETAILED DESCRIPTION

[0059] The properties of colloidal gold mainly depend on the diameter of the gold particles, the absorption spectrum and its stability. The absorption spectrum of colloidal gold is an absorption peak, generally between 510 and 550 nm in the visible spectrum range. The absorption wavelength increases with the increase of the diameter of the gold particles, and at the same time produces a color change from light orange to blue-purple, and even brown. The size of colloidal gold particles is between 5 and 20 nm, with an absorption wavelength of 520 nm, and is wine red; between 20 and 40 nm, it mainly absorbs green light with a wavelength of 530 nm, and the solution is dark red; 60 nm colloidal gold solution mainly absorbs orange-yellow light with a wavelength of 600 nm.

[0060] The preparation of colloidal gold mainly adopts the chemical reduction method, that is, adding various reducing agents to the gold chloride aqueous solution to polymerize the gold ions into colloidal gold. The reduction method can be considered as a crystallization process, and the size of the particles depends on the nucleus formation rate and crystal growth rate caused 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 using the reduction method is that by changing the amount of reducing agent added, colloidal gold particles of different diameters (15-150nm) can be prepared. The basic rule is that the less the amount of reducing agent used, the larger the diameter of the colloidal gold particles.

[0061] Colloidal gold technology has the advantages of convenience, speed, specific sensitivity, strong stability, no need for special equipment and reagents, and intuitive results. It is therefore particularly suitable for grassroots inspection personnel, as well as for large-scale testing and large-area surveys. It has huge development potential and broad application prospects.

[0062] However, the conventional colloidal gold technology currently has some defects as shown below:

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

[0064] (1) The labeling efficiency is greatly affected by pH, and it is 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, resulting in differences in the labeling pH of each batch of labeled antibodies. It is difficult to achieve the optimal labeling pH for each batch, which leads to inconsistent labeling efficiency of each batch of gold-labeled antibodies and difficulty in accurately controlling batch differences.

[0066] 2. The conventional colloidal gold preparation method has certain risks in operation.

[0067] The conventional preparation method of colloidal gold is to prepare a colloidal solution of gold particles of a certain particle size by using chloroauric acid and a reducing agent. The conventional preparation method is to boil water and then add chloroauric acid and a reducing agent to react to obtain colloidal gold. This preparation method has great safety hazards during operation, especially in the process of adding chloroauric acid and a reducing agent when the water is boiling, which is prone to accidents such as scalding. At the same time, due to the influence of water vapor and high temperature, the operation process is difficult to finely control, and large batch differences are prone to occur. A safer, simpler and more stable method for preparing colloidal gold is increasingly in urgent need of production.

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

[0069] At present, the final concentration of chloroauric acid used in the colloidal gold solution prepared by most manufacturers is 0.01%, which is commonly known as one ten-thousandth of gold concentration. This concentration of colloidal gold requires extremely large containers and equipment when labeling in large quantities, which greatly affects production efficiency and is even a bottleneck process affecting production capacity in most cases. Especially in the process of centrifugal concentration, multiple large-capacity and high-speed centrifuges are required to meet the demand. Increasing the concentration of gold particles in colloidal gold has become particularly important to solve the bottleneck of mass production.

[0070] According to the characteristics of colloidal gold, the inventors of the present application have established a new method for preparing colloidal gold solutions by optimizing the final reaction concentration and reaction conditions of HAuCl4 solution and reducing agent, and have prepared high-quality and stable colloidal gold products. By adding the protein to be labeled (such as anti-CRP antibody or anti-DNP antibody) to the colloidal gold, the colloidal particles will agglomerate and form flocs, the aggregated precipitate and flocs are separated by centrifugation, and an alkaline colloidal gold refolding solution is added. The aggregated precipitate and flocs can be redispersed into a colloidal state by ultrasonic dispersion, and a pioneering method for preparing colloidal gold labeled proteins that first aggregate and then refold is developed. The colloidal gold solution or colloidal gold labeled protein prepared by the method of the present application can be used to prepare detection reagents, kits, detection cards, etc. for detecting targets (such as C-reactive protein) in biological samples, thereby achieving effective target detection.

[0071] The practice of this application employs, unless otherwise indicated, conventional techniques of chemistry, biochemistry, and analytical chemistry within the skill of the art.

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

[0073] As used herein, "internal reference antibody" refers to an antibody that determines whether the colloidal gold test strip of the present application is qualified and can be used for detection when the colloidal gold test strip is used for disease marker detection in biological samples.

[0074] As used in this article, the "C line" is also called the quality control line, and the "T line" is also called the test line. When conducting a test, you first need to see if the C line is displayed. If the C line shows a red line, it means that the test paper or test card is qualified, otherwise the test paper or test card is unqualified; even if the T line has a red line, it does not mean that the patient is positive, and the test paper or test card needs to be replaced and retested. If the C line shows a red line, the T line must also show a red line, indicating that the antigen test is positive and may be infected; if the T line does not show, it means the result is negative.

[0075] As used herein, "about" defines a range including plus or minus 10% of a reference value. For example, "about 30 nm" defines a range of 30 + / - 3 nm, ie, 27-33 nm.

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

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

[0078] (2) heating the aqueous mixture of step (1) to boiling and maintaining the boiling temperature for 1-10 min, stopping the heating and cooling the mixture to room temperature;

[0079] (3) The solution obtained in step (2) is diluted with water to restore the volume of the aqueous mixture 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 a range between any of the foregoing 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 a range between any of the foregoing 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 of 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 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 solution, reduction reaction, particle growth and stabilization, and preservation and storage. In some embodiments, the glassware used should be very clean. The properties of the glass surface play an important role in the start of the reduction process. If it is not clean, it will affect the start-up process, interfere with the generation of colloidal gold particles, and cause particles of different sizes or liquid turbidity. In some embodiments, the utensils used should be acid-washed and siliconized. In some embodiments, it is also possible to use special equipment without siliconization, that is, to directly prepare colloidal gold with cleaned utensils. In some embodiments, the colloidal gold prepared for the first time is used to stabilize the surface of the glassware and then discarded; or the prepared colloidal gold is used to coat the surface of the cleaned glassware, and then washed with double distilled deionized water, which is dedicated to the preparation of colloidal gold of the same diameter.

[0087] In some embodiments of the first aspect, the gold chloride plate is easy to absorb moisture and is inconvenient to weigh, so when preparing the gold chloride aqueous solution, the entire content (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 performed 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 waters used to prepare reagents should be double distilled deionized water or triple distilled deionized water, and attention should be paid to the cleanliness of the container, and any impurities and dust particles will interfere with the generation 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 embodiments, the reagents used, especially the reducing agent, must be weighed accurately because the diameter of the gold particles depends mainly 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 ranges between any of the foregoing values).

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

[0095] In a second aspect, the present application provides a colloidal gold solution prepared by the method described in the first aspect.

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

[0097] In some embodiments of the second aspect, the particle size of the colloidal gold in the prepared colloidal gold solution is 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] In a third aspect, the present application provides a method for preparing colloidal gold labeled protein, which comprises:

[0100] Adding the protein to be labeled to the colloidal gold solution of the second aspect to label the protein, thereby obtaining a labeled mixed solution;

[0101] Adding a labeled blocking solution to the labeled mixed solution for blocking;

[0102] After blocking, the supernatant was removed by centrifugation, and the renaturation solution was added to the precipitate for renaturation.

[0103] In some embodiments of the third aspect, the concentration of the protein to be labeled 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 mixed solution 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, the refolding solution is added to the precipitate in an amount equal to the volume of the precipitate. In some embodiments, the method further comprises a step of performing ultrasound after adding the refolding solution to allow the precipitate to be fully dispersed and re-melted.

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

[0108] In some embodiments of the third aspect, the refolding solution is a solution of PEG20000 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 aforementioned 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 aforementioned values) at 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 aforementioned 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 renaturation solution is in an alkaline pH environment, which can achieve a better renaturation effect.

[0109] In some embodiments, the colloidal gold particle size after refolding is increased by multiples of the original particle size, and a marker of the same size as the original particle size cannot be obtained. The smaller the initial particle size, the smaller the refolded particle size after agglutination; the larger the initial particle size, the larger the refolded particle size after agglutination. The sensitivity of the refolded marker is significantly improved compared with the normal marker that is labeled after adjusting the pH of the colloidal gold first.

[0110] In some embodiments of the third aspect, the protein to be labeled is an antibody, an antigen, a 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 mixed solution may 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 mixed solution may be 20 μg / mL.

[0114] In some embodiments of the third aspect, the method further comprises centrifuging the renatured 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 comprises a step of ultrasonicating after adding the labeled diluent to fully disperse and reconstitute the precipitate.

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

[0116] In some embodiments of the third aspect, the labeled diluent comprises 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 ranges between any of the foregoing values) sucrose.

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

[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 ranges between any of the foregoing values) of casein sodium salt.

[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 ranges between any of the foregoing values) of polyvinyl pyrrolidone.

[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 ranges between any of the foregoing values) of surfactant S9.

[0121] In some embodiments of the third aspect, the labeled diluent is a solution comprising 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% casein sodium salt, 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 comprising 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% casein sodium salt, 1 w / v% polyvinyl pyrrolidone, 1 w / v% surfactant S9 and 10-100 mM (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mM, or a range between any of the foregoing values) Tris-HCl.

[0123] In some embodiments of the third aspect, the labeled diluent is prepared by dissolving sucrose, trehalose, casein sodium salt, polyvinyl pyrrolidone, and surfactant S9 in a 50 mM Tris-HCl solution at pH 7.9-8.1, so that the concentration of sucrose is 20 w / v%, the concentration of trehalose is 5 w / v%, the concentration of casein sodium salt is 1 w / v%, and the concentration of surfactant S9 is 1 w / v%.

[0124] In some embodiments of the third aspect, the trade name of surfactant S9, pluracare1307, also known as Tetronic 1307, is a propylene oxide-ethylene oxide WAN-vinyl diamine copolymer, a typical zwitterionic surfactant with a hydrophilic-lipophilic balance (Hydrophilic Lipophilic Balance) greater than 24, and is a surfactant with good water solubility.

[0125] Colloidal gold particles adsorb surrounding ions and maintain the stability of the colloid by electrostatic repulsion. Once the various conditions that maintain the colloidal state are destroyed, the colloidal particles will aggregate and form flocs. The stability of colloidal gold mainly depends on the influence of electrolytes, the size and concentration of colloidal particles, etc. A small amount of electrolyte can cause the sol to precipitate. The application of stabilizers can enhance the stability of colloidal gold. Commonly used ones include bovine serum albumin (BSA), polyethylene glycol (molecular weight 20,000), ovalbumin, polyvinylamine pyrrolidone, gelatin, etc. Tween 20, starch, polydextrose, etc. can also improve the stability of colloidal gold.

[0126] The inventors of the present application have found that when colloidal gold is added to labeled proteins (such as antibodies) under acidic conditions, the colloidal particles will agglomerate and form flocs. The aggregated precipitates and flocs are separated by centrifugation, and an alkaline colloidal gold refolding solution is added. The aggregated precipitates and flocs can be redispersed into a colloidal state through ultrasonic dispersion.

[0127] The absorption peak was detected by a spectrophotometer, and the diameter of the renatured colloidal gold particles was about 60nm. The gold-labeled antibody normally labeled under alkaline conditions was prepared into a test strip (for example, for detecting CRP). The results showed that the sensitivity of the gold-labeled antibody renatured after labeling and aggregation under acidic conditions was 5 times that of the normally labeled gold-labeled antibody. That is, when measuring the same reference product, the color depth of the gold-labeled antibody renatured after labeling and aggregation under acidic conditions is 5 times that of the normally labeled gold-labeled antibody.

[0128] In some embodiments of the third aspect, when the colloidal gold solution described in the second aspect of the present application is used to prepare colloidal gold-labeled protein, the pH value does not need to be adjusted.

[0129] In a fourth aspect, the present application provides a colloidal gold-labeled protein prepared by the method described in the third aspect.

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

[0131] In a fifth aspect, the present application provides a kit, which includes 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 further include instructions for use to guide the user to use the kit for detection.

[0134] In a sixth aspect, the present application provides a labeling pad for a colloidal gold test strip, comprising at least two labeling lines separated from each other, wherein each of the at least two labeling lines is loaded with a first colloidal gold labeled protein and a second colloidal gold labeled protein.

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

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

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

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

[0139] In some embodiments of the sixth aspect, there are at least two marking lines in order to make the overall density of the colloidal gold uniform in the sample flow direction, so that when the test strip is tested, the colloidal gold intercepted by the T line has a uniform color and different shades to show the color change for 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 marker antibody.

[0141] In some embodiments of the sixth aspect, the second colloidal gold labeled protein and the protein to be labeled is an internal control antibody.

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

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

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

[0145] In some embodiments of the sixth aspect, the marking line is prepared by spraying a marking working liquid 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 spraying of the marking pad is performed 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 MP.

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

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

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

[0149] In some embodiments of the sixth aspect, the labeled diluent is a solution comprising 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% casein sodium salt, 1 w / v% polyvinyl pyrrolidone, 1 w / v% surfactant S9 and 50 mM Tris-HCl.

[0150] In some embodiments of the sixth aspect, the labeled diluent is prepared by dissolving sucrose, trehalose, casein sodium salt, polyvinyl pyrrolidone, and surfactant S9 in a 50 mM Tris-HCl solution 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 casein sodium salt 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 at 50±1° C. and a humidity of less than 10% for 24-72 hours (eg, 23-25 ​​hours).

[0152] In some embodiments of the sixth aspect, the marking pad prepared by the method is an integrated pad, which has obvious advantages in production efficiency and precision compared to the traditional "marking pad + sample pad" process. Since an intermediate product is reduced, the production steps are simpler and suitable for roll-type automated production instrument production. In addition, the cumbersome processes such as soaking and cutting in the traditional production process are reduced, reducing the risk of instability caused by human operation.

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

[0154] In some embodiments of the seventh aspect, the test strip further comprises a substrate (eg, 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 embodiments of the seventh aspect, the test strip may also be 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 the seventh aspect, the C line (quality control line) and the T line (detection line) are located on the 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] In an eighth aspect, the present application provides 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 detecting disease markers in biological samples.

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

[0161] In some embodiments of the eighth aspect, the disease marker is C-reactive protein, serum amyloid A, procalcitonin, glycosylated hemoglobin, cardiac troponin, N-terminal pro-B-type natriuretic peptide, new coronavirus surface antigen, influenza virus surface antigen or mycoplasma surface antigen.

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

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

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

[0165] In some embodiments, 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 embodiments, C-reactive protein is used to diagnose, assist in diagnosis, detect, assist in detection, monitor, assist in monitoring, predict, assist in predicting whether a subject suffers from acute inflammation, atherosclerosis, bacterial infection, viral infection, malignancy, liver cancer, benign liver disease, acute pancreatitis, coronary heart disease or myocardial infarction.

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

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

[0169] In some embodiments, serum amyloid A is a marker for viral infection, bacterial infection, malignant tumor metastasis, transplant rejection, rheumatoid arthritis, tuberculosis, leprosy, or sepsis.

[0170] In some embodiments, serum amyloid A is used to diagnose, assist in diagnosis, detect, assist in detection, monitor, assist in monitoring, predict, assist in predicting whether a subject has a viral infection, a bacterial infection, a malignant tumor metastasis, a transplant rejection reaction, rheumatoid arthritis, tuberculosis, leprosy or sepsis.

[0171] In some embodiments, serum amyloid A is used to predict, assist in predicting, assess, or assist in assessing whether a subject is at risk for viral infection, bacterial infection, malignant tumor metastasis, transplant rejection, rheumatoid arthritis, tuberculosis, leprosy, or sepsis.

[0172] In some embodiments, serum amyloid A is used to assess or assist in assessing the severity of a subject's viral infection, bacterial infection, malignant tumor metastasis, transplant rejection, rheumatoid arthritis, tuberculosis, leprosy, or sepsis.

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

[0174] In some embodiments, procalcitonin is used to diagnose, assist in diagnosis, detect, assist in detection, monitor, assist in monitoring, predict, assist in predicting whether a subject has a bacterial infection, a fungal infection, a parasitic infection, sepsis or multiple organ failure.

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

[0176] In some embodiments, procalcitonin is used to assess or assist in assessing the severity of a subject suffering from a bacterial infection, a fungal infection, a parasitic infection, sepsis, or multiple organ failure.

[0177] In some embodiments, glycated hemoglobin is a marker for diabetes.

[0178] In some embodiments, glycated hemoglobin is used to diagnose, assist in diagnosis, detect, assist in detection, monitor, assist in monitoring, predict, assist in predicting whether a subject has diabetes.

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

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

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

[0182] In some embodiments, cardiac troponin is used to diagnose, assist in diagnosis, detect, assist in detection, monitor, assist in monitoring, predict, assist in predicting whether a subject has acute myocardial infarction or acute coronary syndrome.

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

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

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

[0186] In some embodiments, N-terminal pro-B-type natriuretic peptide is used to diagnose, assist in diagnosis, detect, assist in detection, monitor, assist in monitoring, predict, assist in predicting whether a subject suffers from heart failure, hereditary heart disease, myocardial infarction or rheumatic heart disease.

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

[0188] In some embodiments, N-terminal pro-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 embodiments, the novel coronavirus surface antigen is a marker for COVID-19.

[0190] In some embodiments, the novel coronavirus surface antigen is used to diagnose, assist in diagnosis, detect, assist in detection, monitor, assist in monitoring, predict, and assist in predicting whether a subject has COVID-19.

[0191] In some embodiments, the novel coronavirus surface antigen is used to predict, assist in predicting, assess, or assist in assessing a subject's risk of contracting COVID-19.

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

[0193] In some embodiments, the influenza virus includes influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

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

[0195] In some embodiments, the influenza virus surface antigen is used to diagnose, assist in diagnosis, detect, assist in detection, monitor, assist in monitoring, predict, assist in predicting whether a subject has influenza A, influenza B, influenza C, or influenza D.

[0196] In some embodiments, influenza virus surface antigens are used to predict, assist in predicting, assess, or assist in assessing a subject's risk of contracting influenza A, influenza B, influenza C, or influenza D.

[0197] In some embodiments, influenza virus surface antigens are used to assess or assist in assessing the severity of influenza A, influenza B, influenza C, or influenza D in a subject.

[0198] In some embodiments, the mycoplasmas include Mycoplasma pneumoniae and Mycoplasma genitalium.

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

[0200] In some embodiments, the mycoplasma surface antigen is used to diagnose, assist in diagnosis, detect, assist in detection, monitor, assist in monitoring, predict, assist in predicting whether a subject has mycoplasma pneumonia or mycoplasma urogenital tract infection.

[0201] In some embodiments, the mycoplasma surface antigen is used to predict, assist in predicting, assess, or assist in assessing whether a subject is at risk of developing mycoplasma pneumonia or mycoplasma urogenital tract infection.

[0202] In some embodiments, the mycoplasma surface antigen is used to assess or assist in assessing the severity of mycoplasma pneumonia or mycoplasma urogenital tract infection in a subject.

[0203] In some embodiments, the method for preparing the colloidal gold solution provided in the present application is to first mix chloroauric acid and a reducing agent evenly at room temperature and then heat them for reaction. Compared with the traditional method of first heating water to boiling and then adding chloroauric acid and a reducing agent, the operational safety of the preparation process is greatly improved. At the same time, the precision of the operation at room temperature can be achieved to a higher level, thereby improving the preparation efficiency of colloidal gold and reducing batch differences.

[0204] In some embodiments, the preparation method of colloidal gold-labeled protein provided in the present application can greatly improve the labeling efficiency, while eliminating the step of adjusting the pH with potassium carbonate before labeling, and improving the labeling efficiency.

[0205] In some embodiments, the structural characteristics of CRP as a pentamer formed by the same structural subunits are combined with the characteristics of a gold-labeled antibody that binds multiple CRP monoclonal antibodies to a colloidal gold particle, and the "gold-labeled antibody-CRP antigen complex" of different complex sizes is formed under different CRP concentration conditions due to different intensities of the cascade reaction. That is, the higher the CRP content, the higher the intensity of the cascade reaction, the more cascades of the "gold-labeled antibody-CRP antigen complex" formed, and the larger the diameter of the cascade complex. On this basis, combined with the characteristics of the larger the lateral chromatography particle size, the slower the chromatography speed and the higher the sensitivity, by selecting a nitrocellulose membrane with a suitable pore size (e.g., 15 μm), the lower the CRP concentration, the lower the sensitivity and the lighter the color development. As the CRP concentration increases, the cascade reaction is strengthened, and the color development depth doubles with the increase in CRP concentration. That is, an ideal effect is achieved in which the color development is faint at a low concentration of 10 mg / L, and the color development depth of the detection line doubles with the increase in CRP concentration.

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

[0207] In some embodiments, the label pad prepared in the present application has a wider width (eg, 17 mm), and the precision of the product is improved by spraying the gold-labeled antibody multiple times.

[0208] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments.

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

[0210] Example

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

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

[0213] Raw material name Prepare 100mL <![CDATA[HAuCl4]]> 4g

[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] Raw material name Prepare 1000mL Disodium hydrogen phosphate dodecahydrate 58.01868g Sodium dihydrogen phosphate anhydrous 4.559g

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

[0218] Table 3. Formula of CRP sample diluent

[0219] Raw material name Prepare 1000mL Sodium chloride 16g Disodium hydrogen phosphate dodecahydrate 5.801868g Sodium dihydrogen phosphate dihydrate 0.4559g Twain 20 5mL Proclin300 0.5mL

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

[0221] Table 4.2 Formulation of w / v% sodium citrate solution

[0222] Raw material name Prepare 100mL Trisodium Citrate 2g

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

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

[0225] Raw material name Prepare 1000mL Tris 6.057g HCl Adjust pH to 8.0 ± 0.1

[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 make up to 1000 mL.

[0227] Table 6. Labeling blocking solution recipe

[0228] Raw material name Prepare 100mL BSA 10g

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

[0230] Table 7. Coating diluent formulation

[0231] Raw material name Prepare 100mL Sodium chloride 0.8g Trehalose 3g 0.2M PB 5mL Purified water 95mL

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

[0233] Table 8. Recipe for labeling diluent

[0234] Raw material name Prepare 100mL Sodium Caseinate 1g Trehalose 5g sucrose 20g PVP(K30) 1g S9 1g 50mM Tris-HCl Make up to 100mL

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

[0236] Table 9. Refolding solution formula

[0237] Raw material name Prepare 100mL dosage Boric acid 0.0124g PEG20000 0.1g

[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 make up to 100 mL.

[0239] Table 10. Recipe for Marking Pad Working Solution

[0240] Raw material name Prepare 100mL Colloidal gold labeled anti-CRP antibody 60mL Colloidal gold labeled anti-DNP antibody 8mL Marker diluent Make up to 100mL

[0241] Measure the above reagents and stir to make up to volume.

[0242] Table 11. Formula of CRP coating membrane C line coating working solution

[0243] Raw material name Prepare 100mL DNP-BSA 30mg Coating diluent Make up to 100mL

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

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

[0246] Raw material name Prepare 100mL CRP-0B 5mg Coating diluent Make up to 100mL

[0247] Take 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 In a container filled with purified water placed on an electric magnetic stirrer, add 20 mL of 4 w / v% HAuCl4 solution and 60 mL of 2% sodium citrate solution 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 and then turn off the heating. Continue stirring until it cools down.

[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 labeling verification.

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

[0254] 2. Preparation of Colloidal Gold Labeled Antibodies

[0255] The following process is carried out 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 marker

[0257] 2.1.1 Measure 1000mL of colloidal gold solution and place it in a clean beaker. Add a rotor and place it on a magnetic stirrer for rotation and stirring. Adjust the stirring speed so that 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 to react for 15 minutes.

[0259] 2.1.3 After the reaction is completed, add 10 mL of labeling blocking solution and stir the reaction for 15 minutes.

[0260] 2.1.4 After the blocking reaction is completed, the marker is transferred to a centrifuge tube and centrifuged at 10,000 rpm at 4°C for 5 minutes. After the centrifugation is completed, the supernatant is discarded, and an equal volume of the marker washing solution is added to the precipitate, and the precipitate is resuspended by ultrasonication with a cell disruptor (70% ultrasonication for 4 seconds, stop for 4 seconds, for a total of 3 minutes, with the ultrasonic rod extending about 1 / 3 into the liquid surface), and centrifuged at 10,000 rpm at 4°C for 30 minutes.

[0261] 2.1.5 After centrifugation, remove the supernatant and dilute the precipitate to 50 mL with labeled diluent. Mix by ultrasonication and label well. Store at 2-8℃ for later use.

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

[0263] 2.2.1 Measure 1000mL of colloidal gold solution and place it in a clean beaker. Add a rotor and place it on a magnetic stirrer for rotation and stirring. Adjust the stirring speed so that 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 to react for 15 minutes.

[0265] 2.2.3 After the reaction is completed, add 10 mL of labeling blocking solution and stir the reaction for 15 minutes.

[0266] 2.2.4 After the blocking reaction, transfer the marker to a centrifuge tube and centrifuge at 10,000 rpm at 4°C for 5 minutes. After the centrifugation, discard the supernatant, add an equal volume of marker washing solution, and resuspend with a cell disruptor (70% ultrasound for 4 seconds, stop for 4 seconds, for a total of 3 minutes, with the ultrasound rod extending about 1 / 3 of the liquid surface), and centrifuge at 10,000 rpm at 4°C for 30 minutes.

[0267] 2.2.5 After centrifugation, remove the supernatant and dilute the precipitate to 50 mL with labeled diluent. Mix by ultrasonication and label well. Store at 2-8℃ for later use.

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

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

[0270] 2.3.1 Measure 1000mL of colloidal gold solution and place it in a clean beaker. Add a rotor and place it on a magnetic stirrer for rotation and stirring. Adjust the stirring speed so that 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 minutes.

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

[0273] 2.3.4 After the reaction is completed, add 10 mL of labeling blocking solution and stir the reaction for 15 minutes.

[0274] 2.3.5 After the blocking is completed, centrifuge to remove the supernatant, and dilute the precipitate to 50 mL with the labeled diluent. After ultrasonic mixing, label it and store it at 2-8℃ for later use.

[0275] 2.4 Preparation of colloidal gold-labeled anti-DNP antibody markers by conventional methods

[0276] 2.3.1 Measure 1000mL of colloidal gold solution and place it in a clean beaker. Add a rotor and place it on a magnetic stirrer for rotation and stirring. Adjust the stirring speed so that 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 minutes.

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

[0279] 2.3.4 After the reaction is completed, add 10 mL of labeling blocking solution and stir the reaction for 15 minutes.

[0280] 2.3.5 After the blocking is completed, centrifuge to remove the supernatant, and dilute the precipitate to 50 mL with the labeled diluent. After ultrasonic mixing, label it and store it at 2-8℃ for later use.

[0281] 3. Preparation of CRP Labeling Pad

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

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

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

[0285] 3.4 After gold spraying, 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] Prepare the labeling pad as Figure 1 shown.

[0288] 4. Preparation of CRP Coating

[0289] 4.1 Take NC film and PVC board according to production volume, cut NC film into 30cm length. Peel off the protective film at the NC sticking position in the middle of the PVC bottom plate, and stick the smooth surface of NC film to the adhesive position with the rough surface of NC film facing up and the smooth surface facing down, and set aside.

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

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

[0292] 4.4 Clean the instrument according to the standard operation of the film scribing instrument. After cleaning, set the instrument's film scribing parameters, add the configured test line and quality control line solutions to the corresponding pumps, set the film scribing liquid volume to 1μL / cm, set the T scribing position to 31mm from the edge of the PVC board at the absorbent paper end, and the C scribing position to 26mm from the edge of the PVC board at the absorbent paper end, and perform the scribing operation.

[0293] 4.5 After the streaking is completed, the NC film is placed in a drying oven (temperature 50±1°C, humidity <10%) and dried for 47-49 hours.

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

[0295] Schematic diagram of CRP coating membrane Figure 2 shown.

[0296] 5. Preparation of the Detection Plate

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

[0298] 5.1 Prepare the dried marking pad, coating film and absorbent paper.

[0299] 5.2 Pasting of the marking pad: Peel off the protective film of the marking pad area of ​​the PVC board, align the marking pad with the edge of the PVC board marking pad pasting area and paste it, with the other end placed on the NC membrane, with the front side facing up and the gold-labeled antibody pasted close to the NC membrane.

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

[0301] 5.4 After pasting, package and seal according to the specified packaging specifications for storage.

[0302] The schematic diagram of the detection board is as follows Figure 3 shown.

[0303] 6. Preparation of test cards

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

[0305] 6.1 Cutting: Use a cutting machine to cut the test board into 3mm wide test strips.

[0306] 6.2 Card installation: Install the test strip into the card shell and press it tightly with a shell pressing machine.

[0307] 6.3 Packing: Put the jammed test strip into an aluminum foil bag and add 1g of bagged desiccant.

[0308] 6.4 Sealing: Use a sealing machine to seal the packed card shell so that the reagent card and desiccant are sealed in 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 shown.

[0311] Example 2. Performance Verification

[0312] 1. Test 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 gray value detected by the colloidal gold immunoassay analyzer for the linear reference test is shown in Table 13. The linear reference is the antigen with the product number RYZ004Ag1C-0001 produced by Shenzhen Ruimeng Innovation Biotechnology Co., Ltd., and is obtained after the C-reactive protein assay kit (immunoturbidimetry) produced by Siemens is tested and fixed.

[0316] The gray value was determined using a colloidal gold immunochromatographic analyzer model AGS1000 produced by Guangzhou Lanbo Biotechnology Co., Ltd.

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

[0318] Reference substance (mg / L) T C T / C 0 152 4735 0.032101 0.5 160 4420 0.036199 10 273 4565 0.059803 25 631 4539 0.139017 50 3702 4232 0.874764 100 12042 4533 2.656519 200 24425 4120 5.928398 400 40066 4945 8.102326

[0319] Figure 5 The results showed that within the range of 10 mg / L to 200 mg / L, the prepared colloidal gold test results had a high linear relationship.

[0320] 3. Precision determination

[0321] Determination method: Use the same batch number kit to repeat the determination of quality control samples of different concentrations 10 times, calculate the mean value (M) and standard deviation (SD) of the 10 determination results, and calculate the coefficient of variation (CV) according to the following formula.

[0322] CV=SD / M╳100%

[0323] in:

[0324] CV is the 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 yin and yang judgment value 10 mg / L

[0329]

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

[0331] Table 15. Precision verification results for median value 50 mg / L

[0332]

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

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

[0335]

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

[0337] 4. Clinical Validation

[0338] Take no less than 20 human serum samples of different concentrations reasonably distributed in the linear range and conduct a comparison test with the specified analytical system. Each sample is tested according to the requirements of the test reagent (kit) and the selected analytical system. Each sample is tested once, and the two groups of results are linearly fitted using the linear regression method to obtain the correlation coefficient (r) and slope of the linear regression equation. The correlation coefficient (r) ≥ 0.950, and the slope should be within [0.9, 1.1].

[0339] 4.1 The gray value results of clinical sample testing using colloidal gold immunoassay are shown in Table 17.

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

[0341]

[0342]

[0343] 4.2 Correlation analysis between test results and reference reagents

[0344] The results are as follows Figure 6 As shown, the test results of the present invention have a good correlation with the reference reagent, and R2 is 0.9979.

[0345] 5. Comparison between conventional labeling method and annealing labeling method

[0346] The test strips prepared by the conventional labeling method and the renaturing labeling method were adjusted to the same conditions for positive and negative judgment values, and the same batch of samples were tested. The darker the high value color and the higher the color gradient, the better the labeling effect.

[0347] Figure 7 The results showed that when the difference in adjustment sensitivity was not large, the reagent gradient prepared by the renaturation labeling method was significantly better than the reagent prepared by the conventional labeling method.

[0348] All patents, patent application disclosures, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. The exemplary embodiments of the inventions of this application are described above, but without departing from the essence and scope of this application, those skilled in the art can modify or improve the exemplary embodiments described in this application, and the resulting variants or equivalents also belong to the scope of this application.

Claims

1. A method for preparing a colloidal gold solution, comprising: (1) preparing an aqueous mixed solution of HAuCl4 and a reducing agent, wherein the concentration of HAuCl4 in the aqueous mixed solution is 0.03-0.05 w / v%, and the concentration of the reducing agent is 0.02-0.2 w / v%; (2) heating the aqueous mixture of step (1) to boiling and maintaining the boiling temperature for 1-10 minutes, stopping the heating and cooling the mixture to room temperature; (3) The solution obtained in step (2) is diluted with water to restore the volume of the aqueous mixture in step (1).

2. The method of claim 1, wherein: The concentration of HAuCl4 in the aqueous mixed solution is 0.04 w / v%; and / or The concentration of the reducing agent is 0.06 w / v%; and / or In step (2), the aqueous mixture is heated to boiling and maintained for 5 minutes.

3. The method of claim 1, wherein: The water used in the method is double distilled deionized water or triple distilled deionized water; and / or The reducing agent is trisodium citrate, white phosphorus, sodium ascorbate, tannic acid, sodium borohydride, hydrogen peroxide, ethanol or any combination thereof; and / or In step (2), the aqueous mixed solution is heated at a temperature of 100-500°C.

4. The method of claim 3, wherein: In step (2), the aqueous mixed liquid is heated at a temperature of 300°C.

5. A colloidal gold solution prepared by the method according to any one of claims 1 to 4.

6. A method for preparing colloidal gold labeled protein, comprising: Adding the protein to be labeled to the colloidal gold solution of claim 5 to label the protein, thereby obtaining a labeled mixed solution; Adding a labeled blocking solution to the labeled mixed solution for blocking; After blocking, the supernatant was removed by centrifugation, and the renaturation solution was added to the precipitate for renaturation.

7. The method of claim 6, wherein: The concentration of the labeled protein in the labeled mixed solution 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 Adding the refolding solution in an amount equal to the volume of the precipitate to the precipitate; and / or The labeling blocking solution is 10w / v% BSA; and / or The refolding solution is a solution containing 1-10 mM boric acid and 0.1-1 w / v % PEG20000 at a pH of 8.0-9.5; and / or The protein to be labeled is an antibody, an antigen, a recombinant protein or avidin.

8. The method of claim 6, wherein: The concentration of the labeled protein in the labeled mixed solution is 20-30 μg / mL; and / or The refolding solution is a solution containing 5 mM boric acid and 0.5 w / v% PEG20000 at pH 8.9-9.1; and / or The protein to be labeled is an anti-C-reactive protein (CRP) antibody or an anti-dinitrophenol (DNP) antibody.

9. The method according to claim 6, further comprising centrifuging the renatured 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.

10. The method of claim 9, wherein: The labeling diluent is a solution containing 10-30 w / v% sucrose, 2.5-7.5 w / v% trehalose, 0.1-2 w / v% casein sodium salt, 0.1-2 w / v% polyvinyl pyrrolidone and 0.1-2 w / v% S9.

11. The method of claim 9, wherein: The labeling diluent is a solution containing 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% casein sodium salt, 1 w / v% polyvinyl pyrrolidone and 1 w / v% S9.

12. The method of claim 9, wherein: The labeling diluent is a solution containing 10-30 w / v% sucrose, 2.5-7.5 w / v% trehalose, 0.1-2 w / v% casein sodium salt, 0.1-2 w / v% polyvinyl pyrrolidone, 0.1-2 w / v% surfactant S9 and 10-100 mM Tris-HCl.

13. The method of claim 9, wherein: The labeling diluent is a solution containing 20 w / v% sucrose, 5 w / v% trehalose, 1 w / v% casein sodium salt, 1 w / v% polyvinyl pyrrolidone, 1 w / v% surfactant S9 and 50 mM Tris-HCl.

14. Colloidal gold-labeled protein prepared by the method according to any one of claims 6 to 13.

15. A kit comprising the colloidal gold solution according to claim 5 or the colloidal gold-labeled protein according to claim 14.

16. A label pad for a colloidal gold test strip, comprising at least two separated label lines, each of the at least two label lines being loaded with a first colloidal gold labeled protein and a second colloidal gold labeled protein; Preferably, the first colloidal gold-labeled protein is prepared by the method according to any one of claims 6 to 13 and the protein to be labeled is an anti-disease marker antibody; and / or The second colloidal gold labeled protein is prepared by the method according to any one of claims 6 to 13, and the protein to be labeled is an internal reference antibody; More preferably, the anti-disease marker antibody is an anti-CRP antibody; and / or The internal reference antibody is an anti-DNP antibody.

17. A colloidal gold test strip comprising the label pad of claim 16; Preferably, the test strip further comprises a substrate, a nitrocellulose membrane and absorbent paper; More preferably, 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.

18. Use of the colloidal gold solution according to claim 5, the colloidal gold labeled protein according to claim 14, the labeling pad according to claim 16, or the colloidal gold test strip according to claim 17 in preparing a kit for detecting disease markers in biological samples; Preferably, the biological sample is blood or serum; and / or The disease marker is C-reactive protein, serum amyloid A, procalcitonin, glycosylated hemoglobin, cardiac troponin or N-terminal pro-B-type natriuretic peptide, new coronavirus surface antigen, influenza virus surface antigen or mycoplasma surface antigen.

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