A latex immunological detection kit based on dextran-carbon black gel microspheres and application thereof

Abstract

The application belongs to the technical field of medical immunity detection, and discloses a latex immunity detection kit based on dextran-carbon black gel microspheres and application thereof. The latex immunity detection kit comprises a dextran-carbon black gel card, and a preparation method of dextran-carbon black gel microspheres contained in the dextran-carbon black gel card is as follows: dextran, carbon black, an alkaline catalyst and a pore-forming agent are dissolved in water to prepare an aqueous phase; a dispersing agent and an oily solvent are mixed to prepare an oily phase; the aqueous phase is dropped into the oily phase to form a W / O type emulsion; finally, a crosslinking agent is added to generate a crosslinking reaction to prepare the dextran-carbon black gel microspheres. The dextran-carbon black gel microspheres are uniform, regular and high in structural strength, and are used to prepare the latex immunity detection kit. In the detection of anti-streptolysin O, the latex immunity detection kit can significantly improve visual contrast, improve the sensitivity and detection specificity of positive detection.

Description

Technical Field

[0001] This invention belongs to the field of medical immunoassay technology, specifically relating to a latex immunoassay kit based on dextran-carbon black gel microspheres and its application. Background Technology

[0002] Anti-streptolysin O (ASO) is a specific serological marker for group A hemolytic streptococcal (GAS) infection. Detecting elevated antibody titers can identify primary GAS infections such as acute pharyngitis, tonsillitis, and skin and soft tissue infections. It can also differentiate post-infectious immune complex-mediated complications such as rheumatic fever and acute glomerulonephritis, solving the diagnostic challenge of "occult infections" in clinical practice. This method provides crucial clues for investigating the etiology of unexplained fever, arthritis, and nephritis, and has an irreplaceable supplementary diagnostic role, especially for atypical GAS infections without obvious primary infection symptoms.

[0003] Currently, the main clinical methods for ASO detection include latex agglutination, immunoturbidimetry, and hemolysis inhibition. Each method has its own characteristics but also limitations. Latex agglutination is the most commonly used qualitative method. Its principle involves coating the surface of latex particles with streptococcal hemolysin O antigen. When mixed with a sample containing ASO antibodies, the antibodies bind to the antigen on the latex particles, causing agglutination. The result is determined by observing the agglutination phenomenon. This method is simple to operate and inexpensive, but it only provides qualitative or semi-quantitative results and cannot accurately determine the specific concentration of ASO. Furthermore, its sensitivity is relatively low, and false negatives are common. Immunoturbidimetry is currently the most widely used quantitative method in clinical practice, including scattering turbidimetry and transmission turbidimetry. Its principle utilizes the changes in light scattering or absorption produced by the immune complexes formed by antigen-antibody binding in solution to quantitatively determine the ASO concentration. Studies show that immunoturbidimetry has high accuracy and precision, with a coefficient of variation (CV) typically less than 2%. However, this method requires specialized testing instruments, resulting in high equipment costs and stringent sample pretreatment requirements, making it unsuitable for use in primary healthcare institutions. The hemolysis inhibition method was the earliest established ASO detection method. Its principle utilizes the property that ASO antibodies can neutralize the hemolytic activity of streptococcal hemolysin "O," and the ASO content is determined by observing the degree of hemolysis. This method has high sensitivity, but the operation is complex, requiring strict control of reaction conditions, and is affected by various factors such as temperature, pH, and reaction time, resulting in poor reproducibility. Furthermore, this method uses live red blood cells as an indicator system, posing biosafety risks, and the preservation and quality control of red blood cells also face challenges. Traditional methods also share a common problem: insufficient specificity. Actual clinical samples may contain interfering substances such as rheumatoid factor, autoantibodies, and high concentrations of nonspecific immunoglobulins. These substances may react nonspecifically with the test reagents, leading to false positive results, especially in patients with autoimmune diseases. Simultaneously, pathological factors such as hyperlipidemia, hemolysis, and jaundice can also affect the accuracy of the test results. Therefore, developing novel detection methods with higher sensitivity and specificity is of significant clinical importance.

[0004] Although the prior art discloses a latex immunoassay kit based on microcolumn gel cards, using cross-linked dextran microspheres as gel microspheres, the following problems still exist when using ordinary cross-linked dextran gel microspheres for detection: (1) Low visual contrast and difficulty in interpreting results: The color contrast between ordinary gel microspheres and white latex microspheres is not obvious, which leads to the blurring of the agglomeration band formed by the positive reaction, reducing the detection sensitivity and visual recognition, and may affect the accurate interpretation of the results; (2) "Positive drag" interference: Non-specific binding or other interference may occur during the detection process, causing abnormal drag or false positive signals in the gel column of samples that should be negative, i.e., the "positive drag" phenomenon, which reduces the specificity and accuracy of the detection; (3) Insufficient structural strength of gel microspheres: Ordinary cross-linked dextran gel microspheres have limited physical strength, and the structure may not be stable enough during operations such as centrifugation, affecting its separation effect as a molecular sieve and the overall stability of the detection system.

[0005] Therefore, it is urgent to solve the problems existing in the current technology, such as the difficulty in observing the detection results, susceptibility to non-specific interference (positive drag), and the need to improve the structural strength of the gel microspheres themselves. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a latex immunoassay kit based on dextran-carbon black gel microspheres and its application. This invention utilizes uniform, regular, and structurally strong dextran-carbon black gel microspheres to prepare a latex immunoassay kit, which can significantly improve visual contrast, enhance the sensitivity and recognition of positive detection, and effectively shield against positive carryover phenomena.

[0007] This invention provides a latex immunoassay kit based on dextran-carbon black gel microspheres.

[0008] Specifically, a latex immunoassay kit based on dextran-carbon black gel microspheres includes a dextran-carbon black gel card, wherein the dextran-carbon black gel card includes a card body and a micropillar gel tube, wherein the micropillar gel tube contains dextran-carbon black gel microspheres; The preparation method of the dextran-carbon black gel microspheres is as follows: Aqueous phase is prepared by dissolving dextran, carbon black, alkaline catalyst and pore-forming agent in water; An oil phase is prepared by mixing a dispersant and an oily solvent. The aqueous phase is added dropwise to the oil phase and stirred to form a W / O type emulsion; A crosslinking agent was added to the W / O type emulsion to induce a crosslinking reaction, thereby obtaining dextran-carbon black gel microspheres. The carbon black accounts for 0.05wt%-0.6wt% of the aqueous phase.

[0009] In some embodiments of the present invention, the carbon black accounts for 0.05 wt%-0.5 wt% of the aqueous phase, the dextran accounts for 25.0 wt%-38.0 wt% of the aqueous phase, the alkaline catalyst accounts for 3.0 wt%-10.0 wt% of the aqueous phase, the porogen accounts for 0.05 wt%-0.5 wt% of the aqueous phase, and the water accounts for 55.0 wt%-70.0 wt% of the aqueous phase. Preferably, the carbon black accounts for 0.05 wt%-0.4 wt% of the aqueous phase, the dextran accounts for 25.0 wt%-34.0 wt% of the aqueous phase, the alkaline catalyst accounts for 5.0 wt%-8.0 wt% of the aqueous phase, the porogen accounts for 0.1 wt%-0.3 wt% of the aqueous phase, and the water accounts for 60.0 wt%-68.0 wt% of the aqueous phase.

[0010] In some embodiments of the present invention, the dextran has a molecular weight of 20-40 kDa, and the hydroxyl groups on its molecular chain provide active sites for the cross-linking reaction. Preferably, the dextran has a molecular weight of 30-40 kDa.

[0011] In some embodiments of the present invention, the particle size of the carbon black is 1250-2500 mesh.

[0012] In some embodiments of the present invention, the alkaline catalyst includes one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, etc. These alkaline catalysts provide an alkaline environment in the reaction system, activating the hydroxyl groups of the dextran and promoting subsequent cross-linking reactions.

[0013] In some embodiments of the present invention, the porogen includes one or more of polyethylene glycol, n-heptane, and toluene, used to regulate the porosity and pore size distribution of the microspheres. Preferably, the porogen is polyethylene glycol. Polyethylene glycol has good hydrophilicity, and it can form specific interactions with the aqueous phase during the microsphere formation process, thereby forming a more uniform and regular pore structure inside the microspheres, which helps to optimize the detection effect. Moreover, n-heptane and toluene are highly toxic, while polyethylene glycol is non-toxic, odorless, and highly safe, making its use more environmentally friendly and safer.

[0014] In some embodiments of the present invention, the dispersant accounts for 2.0 wt%-8.0 wt% of the oil phase; preferably, the dispersant accounts for 3.0 wt%-4.0 wt% of the oil phase.

[0015] In some embodiments of the present invention, the oily solvent includes paraffin oil; the dispersant includes Span-80 and / or Tween-60, used to construct an oil-water dispersion system to control the morphology of microspheres.

[0016] In some embodiments of the present invention, the volume ratio of the aqueous phase to the oil phase is 1:(5-10); preferably, the volume ratio of the aqueous phase to the oil phase is 1:(6-9).

[0017] In some embodiments of the present invention, when preparing the W / O type emulsion, the stirring process is as follows: stirring at a rate of 300-500 r / min for 10-25 min.

[0018] In some embodiments of the present invention, the crosslinking agent includes one or more of epichlorohydrin, dichloroethyl ether, and glutaraldehyde; preferably, the crosslinking agent is epichlorohydrin. Microspheres prepared with epichlorohydrin have a high degree of crosslinking, while dichloroethyl ether and glutaraldehyde cannot provide sufficient crosslinking, resulting in the dextran-carbon black gel microspheres prepared with them exhibiting tendency for agglomerates to settle when detecting anti-streptolysin O, thus reducing detection sensitivity.

[0019] In some embodiments of the present invention, the mass ratio of the crosslinking agent to the dextran is 1:(1.0-1.5), such as 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5.

[0020] In some embodiments of the present invention, the crosslinking reaction process is as follows: stirring the reaction at a temperature of 15-35°C and a rotation speed of 200-500 r / min for 12-24 h, so that the hydroxyl groups of dextran undergo an etherification reaction with the crosslinking agent to form a three-dimensional crosslinked network.

[0021] In some embodiments of the present invention, after the crosslinking reaction is completed, a post-processing step is further included; the post-processing process is as follows: centrifuge at 2500-5000 rpm for 10-25 min, and collect microspheres with a mesh size of 120-270.

[0022] In some embodiments of the present invention, the dextran-carbon black gel card includes a card body and a micropillar gel tube, wherein the card body is used to fix and support the micropillar gel tube, and is, for example, a common flat plate shape, formed by one-time injection molding of transparent polypropylene plastic material. The micropillar gel tube contains at least one column, which is divided into three main functional areas from top to bottom: a sample application chamber, a reaction chamber, and a gel column. The sample application chamber is located at the top and can be a U-shaped or large column structure with an open top, used for adding serum samples. The reaction chamber connects the sample application chamber and the gel column, and is funnel-shaped, serving as the area where the corresponding antibody undergoes a specific immune response.

[0023] In some embodiments of the present invention, the gel column is located at the bottom and is filled with the dextran-carbon black gel microspheres, anti-human globulin and gel buffer for separating agglutinated and non-agglutinated serum samples by centrifugation.

[0024] In some embodiments of the present invention, the amount of the dextran-carbon black gel microspheres, the anti-human globulin, and the gel buffer added to each column is 20-40 μL, and the ratio of the three is (8-12):1:1. The concentration of the anti-human globulin is 9-20 mg / mL. The anti-human globulin can bind incomplete antibodies, promote agglutination, and improve sensitivity. This detection system based on dextran-carbon black gel microspheres significantly improves the clarity of result interpretation by creating a strong visual contrast between the black of the carbon black and the white of the latex particles. At the same time, the optimized microsphere structure and reaction conditions effectively reduce positive drag phenomenon, ensuring the accuracy and reliability of the detection.

[0025] In some embodiments of the present invention, the components of the gel buffer include Tris-HCl, sodium chloride, glucose, bovine serum albumin, sodium azide, and deionized water.

[0026] In some embodiments of the present invention, the latex immunoassay kit further includes serum diluent (dropper bottle), latex antigen suspension, positive control serum, and negative control serum.

[0027] The present invention also provides the application of the above-mentioned latex immunoassay kit in the detection of anti-streptolysin O.

[0028] The latex immunoassay kit based on dextran-carbon black gel microspheres provided by this invention can significantly improve the sensitivity and specificity of ASO detection.

[0029] Specifically, the molecular mechanism by which the dextran-carbon black gel microspheres enhance ASO detection sensitivity is multi-dimensional. First, carbon black possesses a high specific surface area and abundant surface functional groups, providing numerous adsorption sites for ASO antibodies and antigens, increasing the number of molecules that can effectively bind. Studies show that each gram of carbon black can adsorb hundreds of milligrams of protein; this strong adsorption capacity allows more ASO molecules to participate in the detection reaction, thereby enhancing the detection signal. Second, the addition of carbon black alters the pore structure of the cross-linked dextran gel, creating a microenvironment more conducive to antigen-antibody reactions. The carbon black particles in the composite material act as "spacers," increasing the porosity of the gel network, while their surface functional groups can regulate local pH and ionic strength, optimizing antigen-antibody reaction conditions. This improved microenvironment not only accelerates the reaction rate but also enhances the specificity of the reaction. Third, the optical properties of carbon black play a crucial role in signal amplification. In the microcolumn gel method, this optical enhancement effect can amplify the detection signal by several to tens of times, significantly improving detection sensitivity. Finally, the addition of carbon black can also affect detection performance by altering the rheological properties of the gel. The elastic modulus and viscosity of cross-linked dextran-carbon black composites differ from those of pure cross-linked dextran gels. This change in rheological properties affects the migration behavior of immune complexes in the gel, thereby optimizing the interpretation of detection results.

[0030] The latex immunoassay kit based on dextran-carbon black gel microspheres provided by this invention has a complex and important mechanism for improving the specificity of ASO detection. First, the addition of carbon black reduces non-specific binding through a competitive adsorption mechanism. The abundant functional groups on the carbon black surface preferentially adsorb non-specific proteins (such as albumin and globulins) in the sample, reducing the binding of these proteins to the detection site and lowering the background signal. This competitive adsorption effect is particularly crucial in clinical samples containing high concentrations of non-specific proteins. Second, certain functional groups on the carbon black surface can prevent non-specific binding through electrostatic repulsion. For example, when the carbon black surface is negatively charged, it can repel similarly negatively charged non-specific proteins, thereby improving detection specificity. This electrostatic repulsion effect can be optimized by adjusting the pH and ionic strength to achieve optimal specificity. Third, the hydrophobicity of the composite material helps improve specificity. Pure cross-linked dextran gels are generally highly hydrophilic and easily adsorb various hydrophilic proteins. The addition of carbon black can adjust the hydrophobicity of the composite material. By controlling the degree of surface modification of carbon black, the surface of the composite material can possess appropriate hydrophobicity, thereby selectively adsorbing target proteins and repelling non-specific proteins. Fourth, carbon black can act as a "molecular recognition enhancer," improving recognition specificity through specific interactions with antibodies or antigens. For example, some specially treated carbon black surfaces may contain specific recognition groups, which can form additional specific interactions with target molecules, thereby improving detection selectivity. Fifth, the porous structure of the composite material helps achieve size selectivity. The pore structure of cross-linked dextran-carbon black composites has a certain degree of size selectivity, which can prevent excessively large or small molecules from entering, thereby improving detection specificity. Finally, the antioxidant and anti-protein denaturation effects of carbon black may help maintain antibody activity and specificity. Carbon black has certain antioxidant properties, which can protect antibodies from oxidative damage and maintain their native conformation and binding activity. This protective effect is particularly important during long-term storage and use, and can improve the stability and reproducibility of detection reagents.

[0031] This invention utilizes dextran-carbon black gel microspheres to prepare a latex immunoassay kit. The cross-linked dextran and carbon black in the dextran-carbon black gel microspheres exhibit complementary functions. The cross-linked dextran provides a three-dimensional network structure and biocompatibility, while the carbon black imparts a high specific surface area and surface activity. The combination of these two components constructs a more comprehensive detection platform. This functional complementarity is manifested not only in enhanced adsorption capacity but also in synergistic signal amplification. Furthermore, a complex interfacial structure is formed between the cross-linked dextran and carbon black, and this interfacial region possesses unique physicochemical properties. Studies have shown that the density, polarity, and charge distribution of the interfacial region differ from the bulk phase; this interfacial effect can foster new functions such as selective adsorption and signal enhancement. Moreover, carbon black particles can act as "cross-linking points" or "reinforcing phases" within the cross-linked dextran network, altering the topology and mechanical properties of the gel network. An appropriate amount of carbon black can increase the cross-linking density of the network, improving the mechanical strength and stability of the gel; simultaneously, the presence of carbon black may also alter the relaxation behavior of the network, affecting molecular diffusion and reaction kinetics within the gel.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes dextran and carbon black to prepare dextran-carbon black gel microspheres using a reverse suspension crosslinking method. By controlling the amount of carbon black used and optimizing the reverse suspension crosslinking conditions, the prepared dextran-carbon black gel microspheres are made uniform and have high structural strength. The latex immunoassay kit prepared using this method can significantly improve visual contrast, enhance the sensitivity, identification, and detection specificity of positive detection in antigen detection coupled with white latex microsphere carriers, and effectively shield against positive drag phenomenon. Attached Figure Description

[0033] Figure 1 The graph shows the effect of carbon black dosage on the detection results in Experiment Example 1. Figure 2 The graph shows the effect of dextran molecular weight on the detection effect in Experiment Example 2; Figure 3 This is a graph showing the effect of carbon black particle size on the detection effect in Experiment Example 3; Figure 4 This is a graph showing the effect of the crosslinking agent on the detection effect in Experiment Example 4; Figure 5 This is a graph showing the effect of the dispersant on the detection effect in Experiment Example 5; Figure 6 This is a graph showing the effect of the porogen on the detection effect in Experiment Example 6; Figure 7 This is a diagram showing the results of the latex agglutination method for detecting sample P1 in Comparative Example 1. Figure 8 This is a diagram showing the effect of testing sample P1 in Example 1; Figure 9This is a graph showing the results of the latex agglutination method for detecting sample P2 in Comparative Example 1. Figure 10 This is a diagram showing the effect of testing sample P2 in Example 1; Figure 11 This is a diagram showing the results of the latex agglutination method for detecting sample P3 in Comparative Example 1. Figure 12 This is a diagram showing the effect of testing sample P3 in Example 1; Figure 13 This is a diagram showing the results of the latex agglutination method for detecting sample P4 in Comparative Example 1. Figure 14 This is a diagram showing the effect of testing sample P4 in Example 1; Figure 15 This is a diagram showing the results of the latex agglutination method for detecting sample P5 in Comparative Example 1. Figure 16 This is a diagram showing the effect of testing sample P5 in Example 1; Figure 17 This is a diagram showing the results of the latex agglutination method for detecting sample P6 in Comparative Example 1. Figure 18 This is a diagram showing the effect of testing sample P6 in Example 1; Figure 19 The image shows the appearance of the dextran-carbon black gel microspheres prepared in Example 2 under a microscope at 100x magnification. Figure 20 The image shows the appearance of the dextran gel microspheres prepared in Comparative Example 2 under a microscope at 100x magnification. Figure 21 This is a diagram showing the detection results in Example 2; Figure 22 This is a diagram showing the detection results in Comparative Example 2. Detailed Implementation

[0034] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0035] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0036] Experimental Example Experimental Example 1 The effect of carbon black dosage on the detection effect was studied. The dosage of each raw material component is shown in Table 1. The molecular weight of dextran is 40 (KD) and the particle size is 5-20 μm. The carbon black is 2000 mesh. The pore-forming agent is polyethylene glycol. The alkaline catalyst is sodium hydroxide. The dispersant is Span-80.

[0037] The preparation process of dextran-carbon black gel microspheres is as follows: (1) Preparation of aqueous phase: Weigh dextran, sodium hydroxide, carbon black, polyethylene glycol and purified water and place them in a 500ml reaction vessel. Stir with a stirrer. Stir until the solids are completely dissolved and the aqueous phase is orange and transparent.

[0038] (2) Preparation of oil phase: Weigh Span-80 and paraffin oil liquid and place them in a 2L reactor. Mix them evenly and set aside.

[0039] (3) Synthesis reaction Add the prepared aqueous phase to the oil phase and stir at a constant speed of 400 rpm for 20 minutes. After stirring, add epichlorohydrin to the reactor and control the reaction temperature to approximately 25°C. The total reaction time is 24 hours (starting from the addition of epichlorohydrin).

[0040] Table 1

[0041] Dextran-carbon black gel card: This consists of a card body and microcolumn gel tubes containing dextran-carbon black gel microspheres. The card body is flat and used to fix and support the microcolumn gel tubes, formed in a single injection molding process from transparent polypropylene plastic. The microcolumn gel tube contains eight columns, divided into three main functional areas from top to bottom: a sample loading chamber, a reaction chamber, and a gel column. The sample loading chamber, located at the top, can be a U-shaped structure with an open top, used for adding serum samples. The reaction chamber, connecting the sample loading chamber and the gel column, is funnel-shaped and is the area where the corresponding antibody undergoes a specific immune response. The gel column, located at the bottom, is filled with dextran-carbon black gel microspheres, anti-human globulin, and gel buffer, used for separating and detecting agglutinated positive serum samples and non-agglutinated negative serum samples by centrifugation. The amount of dextran-carbon black gel microspheres, anti-human globulin, and gel buffer added to each column is 30 μL, and the ratio of the three is 10:1:1. The concentration of anti-human globulin (anti-IgG) is 15 mg / mL. The formulation of the gel buffer is shown in Table 2.

[0042] Table 2 Gel Buffer

[0043] Test method: (1) Sampling and dilution: Take a test tube and add one drop of serum to be tested (about 25 microliters); mix the latex antigen suspension and add another drop of latex antigen suspension (about 25 microliters), mix and add to the sample addition cavity.

[0044] (2) Centrifugation: Place the dextran-carbon black gel card into a centrifuge and centrifuge at 800g for 10min.

[0045] (3) Interpretation of results: A white line appears on the reagent card, indicating a positive result. No line appears on the reagent card, indicating a negative result.

[0046] Test results as follows Figure 1 As shown, by Figure 1 It is known that the higher the amount of carbon black added, the darker the color of the test solution. Studies have shown that carbon black added in the aqueous phase at a ratio of 0.05%-0.5% can clearly distinguish the components, resulting in highly recognizable test results.

[0047] Experimental Example 2 The effect of dextran molecular weight on the detection effect was studied. The amounts of each raw material component are shown in Group 1 of Table 1, with dextran molecular weights of 30, 40, and 50 (KD). The detection method is as described in Experiment 1.

[0048] Low molecular weight dextran chains are short with dense cross-linking reaction sites, forming a dense network structure with small particle size and high degree of cross-linking; high molecular weight dextran chains are long and easily entangled, forming a loose and porous structure after cross-linking, with large particle size and high swelling ratio. Detection results show (e.g.) Figure 2 As shown in the figure, when the molecular weight of dextran is 30-40KD, its positive detection results are easy to observe and have high recognition. When the molecular weight of dextran is 50KD, due to the large particle size and high swelling rate of the formed microspheres, it is impossible to achieve good separation from the agglutinin (agglutinin formed by the specific binding of serum anti-streptolysin "O" factor with the coated latex antigen under the action of centrifugation) under the action of centrifugation.

[0049] Experimental Example 3 The effect of carbon black particle size on the detection effect was studied. The dosage of each raw material component is shown in Group 1 of Table 1, with carbon black sizes of 1250, 2000, and 2500 mesh. The detection method is as described in Experiment 1.

[0050] Transmittance depends on the scattering intensity of visible light (400-760nm) by the particles: when the particle size is <1 / 10 of the wavelength (i.e., <40nm, belonging to a colloidal dispersion system), Rayleigh scattering follows, and the scattering intensity is proportional to the fourth power of the particle size—the smaller the particle size, the weaker the scattering, and the stronger the light penetration. When the particle size is close to or larger than the wavelength (>100nm), Mie scattering follows, and the scattering intensity is proportional to the square of the particle size, and obvious directional scattering occurs, resulting in a significant decrease in transmittance. Test results show that carbon black with a particle size of 1250-2500 mesh can achieve good detection results (e.g., ...). Figure 3 (As shown).

[0051] Experiment Example 4 The effect of crosslinking agents on the detection results was studied. The amounts of each raw material component are shown in Group 1 of Table 1. The crosslinking agents selected were epichlorohydrin, dichloroethyl ether, and glutaraldehyde. The detection method is as described in Experiment 1. The detection results show (e.g.) Figure 4 As shown in the image, epichlorohydrin, dichloroethyl ether, and glutaraldehyde can all detect positive samples. However, because the microspheres made with epichlorohydrin have a higher degree of cross-linking, the aggregates formed during the detection process are less likely to fall off, resulting in higher detection sensitivity. In contrast, dichloroethyl ether and glutaraldehyde only increase the degree of cross-linking to a limited extent, and the aggregates fall off slightly, thus reducing the sensitivity.

[0052] Experimental Example 5 The effect of dispersants on the detection results was studied. The dosage of each raw material component is shown in Group 1 of Table 1. The dispersants selected were Span-80 and Tween-60. The detection method is as described in Experiment 1.

[0053] The results showed that Span-80 and Tween-60 were equally effective, both achieving good detection results (e.g., Figure 5 (As shown).

[0054] Experimental Example 6 The effect of porogens on the detection results was studied. The amounts of each raw material component are shown in Group 1 of Table 1. The porogens selected were n-heptane, toluene, and polyethylene glycol. The detection method is as described in Experiment 1.

[0055] The results showed that n-heptane, toluene, and polyethylene glycol yielded similar results, all achieving good detection performance (e.g., Figure 6 (As shown). However, due to the high toxicity of n-heptane and toluene, polyethylene glycol is the preferred choice.

[0056] Comparative Example 1 The latex agglutination method was used for testing, and the testing process is as follows: (1) Prepare 6 test tubes (or U-shaped plates); (2) Add 50 μL of positive serum to the first test tube (labeled as: 1 / 2 tube), add 80 μL of diluent to the second test tube (labeled as: 1 / 10 tube), add 50 μL of diluent to each of the subsequent test tubes (labeled as: 1 / 20 tube, 1 / 40 tube, 1 / 80 tube respectively), and add 50 μL of negative serum to the sixth test tube (labeled as: negative); (3) Add 20 μL of positive serum to 1 / 10 tube and mix well; take 50 μL of the mixture and add it to 1 / 20 tube and mix well; repeat this operation until 1 / 80 tube.

[0057] (4) Before using the reagents, allow them to reach room temperature; verify the negative and positive controls; add 1 drop of undiluted serum sample (50uL) to the black reaction slide; gently mix the latex reagent, place 1 drop of latex next to the sample, and stir the latex and sample together with a stirring rod. Spread the mixture in the black reaction area and gently shake the reaction slide to ensure thorough mixing of the sample and latex. Observe the results visually within 2 minutes. Under bright light, observe with the naked eye while shaking. The negative and positive controls are operated in the same manner as above.

[0058] Example 1 The raw material composition of the dextran-carbon black gel microspheres is shown in Table 3. The dextran has a molecular weight of 40 (KD) and a particle size of 5-20 μm. The carbon black is 2000 mesh. The pore-forming agent is polyethylene glycol. The alkaline catalyst is sodium hydroxide. The dispersant is Span-80.

[0059] Table 3. Raw material composition of dextran-carbon black gel microspheres in Example 1

[0060] The preparation process of dextran-carbon black gel microspheres is as follows: (1) Preparation of aqueous phase: Weigh dextran, sodium hydroxide, carbon black, polyethylene glycol and purified water and place them in a 500ml reaction vessel. Stir with a stirrer. Stir until the solids are completely dissolved and the aqueous phase is orange and transparent.

[0061] (2) Preparation of oil phase: Weigh Span-80 and paraffin oil liquid and place them in a 2L reactor. Mix them evenly and set aside.

[0062] (3) Synthesis reaction Add the prepared aqueous phase to the oil phase and stir at a constant speed of 400 rpm. Add epichlorohydrin to the reactor and control the reaction temperature to approximately 25°C. The total reaction time is 24 hours (starting from the addition of epichlorohydrin).

[0063] The testing process is as follows: (1) Sample preparation: Prepare 8 test tubes; Add 60 μL of diluent to the first test tube (labeled as: 1 / 5 tube), add 50 μL of diluent to each of the other 6 test tubes (labeled as: 1 / 10 tube, 1 / 20 tube, 1 / 40 tube, 1 / 80 tube, 1 / 160 tube, 1 / 320 tube respectively), and add 50 μL of negative serum to the 8th test tube (labeled as negative tube). Add 40 μL of positive serum to the 1 / 40 tube and mix well; take 50 μL of the mixture and add it to the 1 / 80 tube, mix well; repeat this operation, and finally take 50 μL from the 1 / 320 tube and discard it.

[0064] Add 50 μL of latex antigen suspension to each test tube and mix well.

[0065] (2) Numbering: Take out the test card (dextran-carbon black gel card) and mark it on the microtube of the test card (dextran-carbon black gel card). Tear the aluminum foil off the test card upright (tear the aluminum foil of the corresponding microtube as needed).

[0066] (3) Adding samples: Gently shake each test tube, take out 50 μL of antigen-serum mixture from each tube, and add it to the corresponding labeled microtube.

[0067] (4) Centrifugation: Place in a centrifuge and centrifuge at 3000 rpm (800×g) for 20 minutes.

[0068] Interpretation: After centrifugation, remove the test card, observe, determine the results, and record them.

[0069] The test samples used in Comparative Example 1 and Example 1 are shown in Table 4.

[0070] Table 4 Samples tested

[0071] Table 5. Results of Latex Aggregation Test in Comparative Example 1

[0072] Table 6. Test Results of Example 1

[0073] The results of the latex agglutination method in Comparative Example 1 are shown in Table 5. Figure 7 , 9 Figures 11, 13, 15, and 17 show the results of latex agglutination testing of samples P1, P2, P3, P4, P5, and P6 in Comparative Example 1. The test results of Example 1 are shown in Table 6. Figure 8 , 10 Figures 12, 14, 16, and 18 show the detection results for samples P1, P2, P3, P4, P5, and P6 in Example 1. The results show that the traditional latex agglutination method only detects positive results for samples P1-P6 at a dilution of 1 / 2, with a minimum detection limit of 158.5 IU / mL. In Example 1, samples P1 and P2 remained positive even at a dilution of 1 / 40, and P3-P6 remained positive even at a dilution of 1 / 80, with a minimum detection limit of 4.2 IU / mL. The detection sensitivity of this invention is higher than that of the traditional latex detection method, and the results are easier to distinguish.

[0074] Example 2 The raw material composition of the dextran-carbon black gel microspheres provided in Example 2 is shown in Table 7. The molecular weight of the dextran is 40 (KD) and the particle size is 5-20 μm. The carbon black is 2000 mesh.

[0075] Table 7. Raw material components of dextran-carbon black gel microspheres in Example 2

[0076] The preparation process of dextran-carbon black gel microspheres is as follows: (1) Preparation of aqueous phase: Weigh dextran, sodium hydroxide, carbon black, polyethylene glycol and purified water and place them in a 500ml reaction vessel. Stir with a stirrer. Stir until the solids are completely dissolved and the aqueous phase is orange and transparent.

[0077] (2) Preparation of oil phase: Weigh Span-80 and paraffin oil liquid and place them in a 2L reactor. Mix them evenly and set aside.

[0078] (3) Synthesis reaction Add the prepared aqueous phase to the oil phase and stir at a constant speed of 400 rpm. Add epichlorohydrin to the reactor and control the reaction temperature to approximately 25°C. The total reaction time is 24 hours (starting from the addition of epichlorohydrin).

[0079] Comparative Example 2 The raw material composition of the dextran gel microspheres provided in Comparative Example 2 is shown in Table 8. The molecular weight of the dextran is 40 (KD) and the particle size is 5-20 μm.

[0080] Table 8. Raw material composition of dextran gel microspheres in Comparative Example 2

[0081] The preparation process of dextran gel microspheres is as follows: (1) Preparation of aqueous phase: Weigh dextran, sodium hydroxide, polyethylene glycol and purified water and place them in a 500mL reactor. Stir with a stirrer until the solid is completely dissolved.

[0082] (2) Preparation of oil phase: Weigh Span-80 and paraffin oil liquid and place them in a 2L reactor. Mix them evenly and set aside.

[0083] (3) Synthesis reaction Add the prepared aqueous phase to the oil phase and stir at a constant speed of 400 rpm. Add epichlorohydrin to the reactor and control the reaction temperature to approximately 25°C. The total reaction time is 24 hours (starting from the addition of epichlorohydrin).

[0084] Figure 19 and Figure 20The images show the appearance of the dextran gels prepared in Example 2 and Comparative Example 2 under a microscope at 100x magnification. Figure 19 and Figure 20 It can be seen that the cross-linked dextran microspheres with added carbon black have a more uniform particle size.

[0085] Detection method process: (1) Sample preparation (serial dilution): Prepare 8 test tubes; Add 60 μL of sample diluent A to the first test tube (labeled as: 1 / 5 tube), and add 50 μL of sample diluent to each of the other seven test tubes (labeled as: 1 / 10 tube, 1 / 20 tube, 1 / 40 tube, 1 / 80 tube, 1 / 160 tube, 1 / 320 tube, and 1 / 640 tube, respectively). Add 40 μL of positive serum to 1 / 5 of the tube and mix well; take 50 μL of the mixture and add it to 1 / 10 of the tube and mix well; repeat this operation, and finally take 50 μL from 1 / 640 of the tube and discard it. Add 50 μL of polystyrene microsphere-antigen suspension to each test tube and mix well.

[0086] (2) Numbering: Take out the test card (carbon black cross-linked dextran gel card), mark the microtube on the test card, and tear the aluminum foil off the test card upright (tear the aluminum foil off the corresponding microtube as needed).

[0087] (3) Adding samples: Gently shake each test tube, take out 50 μL of antigen-serum mixture from each tube, and add it to the corresponding labeled microtube.

[0088] (4) Centrifugation: Place in a centrifuge and centrifuge at 3000 rpm (800×g) for 20 minutes.

[0089] Interpretation: After centrifugation, remove the test card, observe, determine the results, and record them.

[0090] Test results as follows Figure 21 and Figure 22 As shown, the results indicate that the dextran-carbon black gel card provided by this invention can significantly improve visual contrast, enhance the sensitivity and recognition of positive detection, and shield positive drag.

[0091] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A latex immunoassay kit based on dextran-carbon black gel microspheres, characterized in that, The invention includes a dextran-carbon black gel card, wherein the dextran-carbon black gel card comprises a card body and a micropillar gel tube, and the micropillar gel tube contains dextran-carbon black gel microspheres; The preparation method of the dextran-carbon black gel microspheres is as follows: Aqueous phase is prepared by dissolving dextran, carbon black, alkaline catalyst and pore-forming agent in water; An oil phase is prepared by mixing a dispersant and an oily solvent. The aqueous phase is added dropwise to the oil phase and stirred to form a W / O type emulsion; A crosslinking agent was added to the W / O type emulsion to induce a crosslinking reaction, thereby obtaining dextran-carbon black gel microspheres. The oily solvent is paraffin oil; The crosslinking agent includes one or more of epichlorohydrin, dichloroethyl ether, and glutaraldehyde; the mass ratio of the crosslinking agent to the dextran is 1:(1.0-1.5). The carbon black comprises 0.05 wt%-0.5 wt% of the aqueous phase, the dextran comprises 25.0 wt%-38.0 wt% of the aqueous phase, the alkaline catalyst comprises 3.0 wt%-10.0 wt% of the aqueous phase, the porogen comprises 0.05 wt%-0.5 wt% of the aqueous phase, and the water comprises 55.0 wt%-70.0 wt% of the aqueous phase. The dextran has a molecular weight of 30-40 kDa, and the carbon black has a particle size of 1250-2500 mesh.

2. The latex immunoassay kit according to claim 1, characterized in that, The alkaline catalyst includes one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate; the porogen includes one or more of polyethylene glycol, n-heptane, and toluene.

3. The latex immunoassay kit according to claim 1, characterized in that, The dispersant comprises 2.0wt%-8.0wt% of the oil phase; the dispersant includes Span-80 and / or Tween-60.

4. The latex immunoassay kit according to any one of claims 1-3, characterized in that, The volume ratio of the aqueous phase to the oil phase is 1:(5-10).

5. The latex immunoassay kit according to claim 4, characterized in that, The microcolumn gel tube comprises at least one column, which includes three functional areas from top to bottom: a sample application chamber, a reaction chamber, and a gel column; the gel column is filled with the dextran-carbon black gel microspheres, anti-human globulin, and gel buffer; the addition ratio of the dextran-carbon black gel microspheres, the anti-human globulin, and the gel buffer is (8-12):1:

1.

6. The latex immunoassay kit according to claim 5, wherein the concentration of the anti-human globulin is 9-20 mg / mL; and the components of the gel buffer include Tris-HCl, sodium chloride, glucose, bovine serum albumin, sodium azide, and deionized water.

7. The latex immunoassay kit according to claim 1, characterized in that, The latex immunoassay kit also includes serum diluent, latex antigen suspension, positive control serum, and negative control serum.

Images