Kit for animal epidemic disease detection
By using bovine serum albumin-polymer coupler diluent in ELISA kits, combined with inorganic salts and nonionic detergents, the problem of nonspecific IgG adsorption in ELISA kits was solved, improving the accuracy and reliability of detection.
Patent Information
- Application Number
- CN202511096916.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-06
- Publication Date
- 2025-11-18
AI Technical Summary
In animal disease detection, ELISA kits suffer from high background signals and false positive results due to non-specific IgG adsorption, affecting the accuracy and reliability of the detection.
A diluent containing bovine serum albumin-polymer coupler was used. Through steric hindrance and charge neutralization, combined with inorganic salts and nonionic detergents, the ionic strength was adjusted by steric hindrance and charge to reduce nonspecific binding, thereby reducing the IgG content in the sample.
It significantly reduces nonspecific IgG adsorption, improves detection accuracy, reduces false positive results, and provides a more reliable tool for detecting animal diseases.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of enzyme-linked immunoassay technology, and relates to a kit for animal disease detection. BACKGROUND
[0002] In the field of animal disease prevention and control, rapid and accurate detection methods are crucial for early diagnosis, epidemic control and animal health protection. Among them, enzyme-linked immunosorbent assay (ELISA) as a sensitive, rapid and simple detection technology has been widely used in animal disease detection. ELISA kit as the core tool of this technology, its performance and quality are directly related to the accuracy and reliability of the detection results. ELISA kit can detect pathogen antibodies or antigens in animal serum, plasma or other tissue samples through specific antigen-antibody reaction principle. In animal disease detection, ELISA kit has a wide range of applications, including but not limited to detection of avian influenza, Newcastle disease, porcine epidemic diarrhea, African swine fever and other important animal diseases. These kits usually contain necessary antigens, antibodies, enzyme-labeled secondary antibodies, substrates and other reagents, as well as solid-phase carriers (such as polystyrene plates) for fixing antigens or antibodies. The use method of ELISA kit is relatively simple, and the operator only needs to follow the instructions to operate, react the sample to be tested with the reagents in the kit, and then detect the reaction results by enzyme-labeled instrument. According to the color change or absorbance value of the results, it can be judged whether there is a specific pathogen antibody or antigen in the sample, so as to realize the rapid screening and diagnosis of animal diseases.
[0003] Despite the numerous advantages of ELISA kits in animal disease detection, several technical challenges remain in practical applications. One prominent issue is the high background signal or false-positive results caused by the adsorption of non-specific IgG onto the solid-phase carrier. Non-specific IgG refers to immunoglobulin G that does not bind to specific pathogens. During ELISA detection, these non-specific IgGs may adsorb onto the solid-phase carrier, non-specifically binding with subsequently added reagents and generating a background signal. Excessively high background signals can interfere with the detection of the target signal, reducing the accuracy of the results. Furthermore, the adsorption of non-specific IgG can also lead to false-positive results. When non-specific IgG cross-reacts with antigens or antibodies on the solid-phase carrier, it creates erroneous signals, causing negative samples to be misjudged as positive. This is particularly serious in animal disease detection, as false-positive results can mislead veterinarians or farmers in their diagnosis and treatment of animal diseases, adversely affecting animal health and farming efficiency. To address the technical problems caused by non-specific IgG adsorption, researchers are continuously exploring and improving the preparation methods and usage conditions of ELISA kits. For example, by optimizing the purity of antigens and antibodies, improving the surface treatment of solid-phase carriers, and strengthening the washing steps, the adsorption of nonspecific IgG and interference from background signals can be effectively reduced. Summary of the Invention
[0004] The purpose of this invention is to solve the technical problem of high background signal or false positives caused by non-specific IgG adsorption onto a solid-phase carrier in ELISA detection kits used for animal disease detection, particularly enzyme-linked immunosorbent assay (ELISA) kits. To address this need, this invention provides a kit for animal disease detection.
[0005] On one hand, the present invention relates to a kit for detecting animal diseases, the kit being an enzyme-linked immunosorbent assay (ELISA) kit, the ELISA kit containing a diluent for sample dilution, the diluent comprising 5-10 g / L bovine serum albumin and 10-30 g / L bovine serum albumin-polymer coupler.
[0006] Furthermore, in the kit for animal disease detection provided by the present invention, the bovine serum albumin-polymer coupler is prepared by cross-linking aminated bovine serum albumin and aldehyde-modified polysaccharide.
[0007] Furthermore, in the kit for animal disease detection provided by the present invention, the aldehyde-modified polysaccharide is selected from one of aldehyde-modified dextran, aldehyde-modified chitosan, or aldehyde-modified sodium alginate.
[0008] Furthermore, in the kit for animal disease detection provided by the present invention, the diluent further includes inorganic salts and nonionic detergents.
[0009] Furthermore, in the kit for detecting animal diseases provided by the present invention, the concentration of the inorganic salt is 0.1~15g / L.
[0010] Furthermore, in the kit for detecting animal diseases provided by the present invention, the inorganic salt is NaCl and / or KCl.
[0011] Furthermore, in the kit for animal disease detection provided by the present invention, the concentration of the nonionic detergent is 0.5~1g / L.
[0012] Furthermore, in the kit for animal disease detection provided by the present invention, the nonionic detergent is Tween-20.
[0013] On the other hand, the present invention relates to the application of the kit for detecting animal diseases in the preparation of enzyme-linked immunosorbent assay kits.
[0014] Furthermore, in the application provided by the present invention, the enzyme-linked immunosorbent assay kit includes a diluent for sample dilution, the diluent being used to reduce the IgG content in the sample.
[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention provides a kit for detecting animal diseases, particularly an enzyme-linked immunosorbent assay (ELISA) kit, which contains a carefully designed diluent comprising bovine serum albumin (BSA) and a BSA-polymer coupler. This diluent effectively addresses the technical problem of high background signal or false positives caused by nonspecific IgG adsorption onto a solid-phase carrier during ELISA testing. Furthermore, the BSA-polymer coupler is prepared by crosslinking aminated BSA with aldehyde-modified polysaccharides (such as aldehyde-modified dextran, aldehyde-modified chitosan, or aldehyde-modified sodium alginate). This structure significantly enhances the blocking effect and reduces nonspecific adsorption through steric hindrance and charge neutralization. In addition, the diluent also contains inorganic salts (such as NaCl and / or KCl) and nonionic detergents (such as Tween-20). The addition of these components further optimizes the blocking effect and reduces nonspecific binding by adjusting ionic strength and reducing hydrophobic interactions. Compared to conventional reagent kit diluents in existing technologies, the diluent provided by this invention can significantly reduce the IgG content in samples, thereby improving the accuracy of IgG detection and reducing false positive results. This technical solution not only improves the accuracy and reliability of ELISA detection but also provides a more effective and reliable detection tool for animal disease prevention and control, yielding significant social and economic benefits. Detailed Implementation
[0016] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.
[0017] In the following examples, bovine serum albumin (molecular weight 66.4 kDa), aminolated bovine serum albumin, aldehyde-modified dextran (molecular weight 3.4 kDa), aldehyde-modified chitosan (molecular weight 3.4 kDa), and aldehyde-modified sodium alginate (molecular weight 3.4 kDa) were all purchased from Xi'an Qiyue Biotechnology Co., Ltd.
[0018] Example 1 This embodiment provides the preparation of bovine serum albumin-polymer couplers.
[0019] Protein-polymer couplers are formed by cross-linking amino-coated proteins and aldehyde-coated polysaccharides under certain pH conditions.
[0020] This embodiment uses the general preparation steps, as follows: 250 mg / mL BSA-NH2 (aminated bovine serum albumin) and 300 mg / mL PS-CHO (aldehyde-modified polysaccharide) were thoroughly dispersed in deionized water for later use. A 0.05 M phosphate buffer solution with a pH of 7.4 was prepared to maintain pH stability. 100 μL of BSA-NH2 solution, 100 μL of PBS, and 80 μL of PS-CHO solution were added sequentially to centrifuge tubes. The tubes were shaken thoroughly for 30 seconds and then allowed to stand overnight. The mixed solution was filtered through a syringe using a 0.45 μm filter. Unreacted raw materials and salts were removed by dialysis in water using a dialysis bag. Finally, the solutions were freeze-dried to obtain bovine serum albumin-dextran couplers, bovine serum albumin-chitosan couplers, and bovine serum albumin-sodium alginate couplers, respectively.
[0021] Example 2 This embodiment provides diluents with different components, the composition of which is shown in Table 1.
[0022] Table 1: Components of the diluent
[0023] The preparation method involves sequentially adding the above-mentioned components to a solvent and then adjusting the volume accordingly.
[0024] Pig serum samples were used, and the IgG content in the samples was measured after dilution at a ratio of 1:40. The experimental results are shown in Table 2.
[0025] Table 2: IgG content in samples
[0026] Table 2 shows that the measurement results of IgG content in porcine serum varied significantly among different diluents, mainly due to the composition of the diluents and their inhibitory effect on non-specific binding. Diluents (1-3) containing BSA-polysaccharide couplers (such as dextran, chitosan, and sodium alginate) significantly reduced IgG detection values (0.398-0.478 g / L). The couplers enhanced the blocking effect and reduced non-specific adsorption through steric hindrance and charge neutralization. Diluents 4-6 (containing Tween-20 and salt): Further addition of Tween-20 (to reduce hydrophobic interaction) and NaCl / KCl (to adjust ionic strength) reduced IgG values to 0.205-0.227 g / L, indicating that the synergistic effect optimized the blocking effect. Diluent 7 (BSA only): Although containing 15 g / L BSA, it lacked couplers, and the IgG value (1.324 g / L) was much higher than that of the coupler diluents, indicating that the blocking efficiency of BSA alone was insufficient. Diluents 8-9 (uncoupled polysaccharides): Uncoupled chitosan / sodium alginate resulted in abnormally high IgG values (1.746-1.913 g / L), possibly due to the direct adsorption of IgG by charged polysaccharides or interference with antigen-antibody binding. Diluents 10 (pure PBS) and 11 (physiological saline): Without blocking agents, non-specific binding was maximized, resulting in the highest IgG values (2.511 g / L and 2.038 g / L, respectively). Diluent 12 (kit): Contains a high concentration of BSA (40 g / L), but the IgG value (1.135 g / L) is still higher than the optimized formulation, possibly due to sodium azide interference or the formulation not being optimized for porcine serum. The BSA-polysaccharide coupler forms a large molecular complex through covalent coupling, enhancing steric hindrance and covering more non-specific binding sites; different polysaccharides (neutral dextran, positively charged chitosan, negatively charged sodium alginate) neutralize the surface charge of the sample / carrier through charge complementarity, reducing electrostatic adsorption. In summary, the BSA-polysaccharide coupler combined with BSA dilution significantly reduces non-specific binding and improves the accuracy of IgG detection. Conventional dilutions (such as pure PBS or physiological saline) lack blocking agents, leading to higher false-positive signals.
[0027] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.
Claims
1. A kit for detecting animal diseases, said kit being an enzyme-linked immunosorbent assay (ELISA) kit, said ELISA kit comprising a diluent for sample dilution, characterized in that, The diluent comprises 5-10 g / L bovine serum albumin and 10-30 g / L bovine serum albumin-polymer coupler.
2. The reagent kit for detecting animal diseases according to claim 1, characterized in that, The bovine serum albumin-polymer coupler is prepared by cross-linking aminated bovine serum albumin with aldehyde-modified polysaccharide.
3. The reagent kit for detecting animal diseases according to claim 2, characterized in that, The aldehyde-modified polysaccharide is selected from one of aldehyde-modified dextran, aldehyde-modified chitosan, or aldehyde-modified sodium alginate.
4. The reagent kit for detecting animal diseases according to claim 1, characterized in that, The diluent also includes inorganic salts and nonionic detergents.
5. The reagent kit for detecting animal diseases according to claim 4, characterized in that, The concentration of the inorganic salt is 0.1~15 g / L.
6. The reagent kit for detecting animal diseases according to claim 5, characterized in that, The inorganic salt is NaCl and / or KCl.
7. The kit for detecting animal diseases according to claim 4, characterized in that, The concentration of the nonionic detergent is 0.5~1g / L.
8. The kit for detecting animal diseases according to claim 4, characterized in that, The nonionic detergent is Tween-20.
9. The use of the kit for detecting animal diseases according to any one of claims 1 to 8 in the preparation of an enzyme-linked immunosorbent assay (ELISA) kit.
10. The application according to claim 9, characterized in that, The enzyme-linked immunosorbent assay kit contains a diluent for sample dilution, which is used to reduce the IgG content in the sample.