A colloidal gold test paper for paratuberculosis MAP SA1 monoclonal antibody

Abstract

The application relates to the field of immunodetection technology, and discloses a colloidal gold test paper for paratuberculosis MAPSA1 monoclonal antibody. The test paper comprises a sample pad, a colloidal gold combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped on a bottom plate, the sample pad is loaded with protein disaggregation, non-ionic surfactant protection and heterophilic antibody blocking components, the combination pad is loaded with colloidal gold-labeled natural conformational epitope monoclonal antibody and conformational protection components, and a detection line is fixed with linear or semi-conformational epitope monoclonal antibody. The structure can improve MAPSA1 antigen exposure in complex samples, compound formation and detection line capture stability, and reduce weak positive missed judgment and membrane surface background interference.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology and discloses a colloidal gold test strip for paratuberculosis MAPSA1 monoclonal antibody. Background Technology

[0002] Mycobacterium paratuberculosis infection can cause chronic intestinal wasting disease in ruminants. Pathogen shedding is characterized by low levels and intermittent shedding, with low levels of target antigen in early samples. Conventional culture cycles are long, and molecular detection relies on instruments and nucleic acid extraction conditions, making it difficult to meet the needs of on-site screening at the grassroots level. Colloidal gold immunochromatographic test strips are often used for rapid detection of paratuberculosis due to their ease of operation, intuitive interpretation, and lack of the need for large instruments. Conventional colloidal gold test strips typically consist of a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and an absorbent pad. Colloidal gold-labeled antibodies are loaded onto the conjugate pad, and the capture antibodies are immobilized on the detection line. After the sample is added, it migrates forward with the chromatography solvent. The target antigen binds to the colloidal gold-labeled antibody and is then captured by the detection line, forming a visible colored band. The control line is used to determine whether the chromatography process is normal.

[0003] Existing paratuberculosis antigen test strips mostly employ a single recognition mode or a conventional sandwich immunoassay mode. Antibody screening focuses on binding strength and cross-reactivity control, and sample pretreatment largely relies on universal buffers, common blocking proteins, and conventional surfactants. For samples such as serum, milk, or fecal extracts, lipid clusters, mucopolysaccharides, host immunoglobulins, complement proteins, and particulate impurities can come into non-specific contact with the target antigen, membrane material, or colloidal gold particles, causing antigen embedding, impaired migration, or increased membrane background. Some test strips enhance weak positive color development by increasing antibody coating, increasing colloidal gold labeling, or extending reaction time; however, these methods easily lead to gold particle aggregation, uneven chromatography speed, test line tailing, and increased negative background, making it difficult to simultaneously capture and clearly interpret low-abundance antigens in complex samples.

[0004] The main technical problem with existing technologies is that paratuberculosis-associated antigen 1 is often in a low-abundance, partially embedded, or locally conformationally altered state in complex samples. Single epitope recognition or conventional double-antibody sandwich systems lack sufficient accessibility and capture stability for the target antigen, leading to difficulty in forming stable immune complexes in weakly positive samples during chromatography, resulting in pale test lines, poor repeatability, and unclear positive / negative boundaries. This problem cannot be solved simply by increasing colloidal gold particles, raising antibody concentration, or using a universal blocking solution, because insufficient antigen exposure, antibody conformational loss, and matrix interference coexist; failure to address any of these factors will limit complex deposition at the test line. There is an urgent need to develop a colloidal gold test strip structure that exposes the target antigen, synergistically captures it with dual epitopes, and allows for stable migration of labeled antibodies, enabling stable recognition and fixation of paratuberculosis-associated antigen 1 in complex samples during chromatography. Summary of the Invention

[0005] To address the shortcomings of existing paratuberculosis-related antigen 1 colloidal gold test strips, such as insufficient accessibility of target antigens in complex samples, poor capture stability after local conformational changes, and unstable color development of the detection line in weakly positive samples, this invention provides a paratuberculosis MAPSA1 monoclonal antibody colloidal gold test strip. Through the combination of sample pretreatment composition, dual epitope monoclonal antibody recognition system, and antibody conformation protection components, the paratuberculosis MAPSA1 antigen in complex samples can be stably released, bound, and captured during chromatography.

[0006] To address the aforementioned technical problems, this invention provides a MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis, comprising a base plate and a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially overlapped on the base plate; the colloidal gold conjugate pad is loaded with a colloidal gold-labeled first MAPSA1 monoclonal antibody, which is a monoclonal antibody targeting the native conformational epitope of MAPSA1; a detection line and a control line are disposed on the nitrocellulose membrane, the detection line being immobilized with a second MAPSA1 monoclonal antibody, which is a monoclonal antibody targeting a linear or semi-conformational epitope of MAPSA1, and the antigenic determinants corresponding to the first and second MAPSA1 monoclonal antibodies do not overlap; the sample pad is loaded with a sample pretreatment composition, which includes a protein depolymerization component, a non-ionic surfactant protective component, and a heterophile antibody blocking component; the colloidal gold conjugate pad is also loaded with an antibody conformational protection component.

[0007] After the sample is added to the sample pad, the sample pretreatment composition first contacts protein aggregates, lipid microparticles, host immunoglobulins, and other non-target matrix components in the sample, exposing the MAPSA1 antigen, which was previously masked by the sample matrix, to areas that can be recognized by the antibody. As the sample continues to migrate along the chromatographic direction to the colloidal gold conjugate pad, the MAPSA1 antigen binds to the colloidal gold-labeled first MAPSA1 monoclonal antibody, forming an antigen-antibody complex labeled with colloidal gold. This complex continues to migrate to the nitrocellulose membrane detection line, where it is recaptured by the second MAPSA1 monoclonal antibody immobilized on the detection line. Because the first MAPSA1 monoclonal antibody recognizes the native conformational epitope of MAPSA1, and the second MAPSA1 monoclonal antibody recognizes the linear or semi-conformational epitope of MAPSA1, and their corresponding antigenic determinants do not overlap, the target antigen can form a sandwich-like binding structure between different epitopes. The antibody conformational protection component maintains the spatial binding state of the first MAPSA1 monoclonal antibody during the drying, storage, reconstitution, and migration of the colloidal gold conjugate pad, reducing irreversible masking of the antibody active site due to drying or reconstitution.

[0008] Furthermore, in the above technical solution, the first MAPSA1 monoclonal antibody is obtained by screening an immunogen that maintains the native spatial conformation of MAPSA1, and the second MAPSA1 monoclonal antibody is obtained by screening a MAPSA1 fragment containing a conserved linear region of MAPSA1 or a renatured MAPSA1 fragment.

[0009] In practice, immunogens that maintain the native spatial conformation of MAPSA1 can retain conformational epitopes formed by spatial folding on the antigen surface, making the first MAPSA1 monoclonal antibody obtained through screening suitable for recognizing MAPSA1 antigens in samples that have not undergone significant denaturation. A second MAPSA1 monoclonal antibody containing a conserved linear region of MAPSA1 or a renatured MAPSA1 fragment is used to screen for this second antibody, enabling it to recognize linear or hemiform epitopes that remain stable after sample processing or migration. During detection, the two types of antibodies correspond to identifiable regions in different antigen states, reducing capture interruptions caused by the loss of accessibility to a single epitope.

[0010] Furthermore, in the above technical solution, the protein depolymerization component includes an amino acid-type buffer component, a polyhydroxy compound, and a mild salt component; the nonionic surfactant protective component includes polyoxyethylene surfactants or alkyl glycoside surfactants; and the heterophile antibody blocking component includes non-immunogenic homologous animal immunoglobulins, polymerized immunoglobulin fragments, or heterophile antibody blocking proteins.

[0011] In practice, amino acid-based buffer components provide a suitable ionic environment for antigen-antibody reactions in the sample, polyhydroxy compounds participate in maintaining the protein hydration layer, and mild salt components regulate the electrostatic contact between protein complexes in the sample, making it easier to expose MAPSA1 antigens embedded in host proteins or adhesive matrices. Polyoxyethylene surfactants or alkyl glycoside surfactants improve the migration state of the sample along the chromatography direction by reducing non-specific contact between hydrophobic particles and membrane materials and colloidal gold particles. Non-immunologous animal immunoglobulins, polymerized immunoglobulin fragments, or heterophilic antibody blocking proteins preferentially bind to heterophilic antibodies in the sample, reducing the formation of non-target bridging between colloidal gold-labeled antibodies and detection line antibodies.

[0012] Furthermore, in the above technical solution, the antibody conformation protection component includes a glycogen vitrification protection component, a protein stabilizing component, and an anti-colloidal gold aggregation component, which are collectively loaded within the dry matrix of the colloidal gold binding pad.

[0013] In practice, the colloidal gold conjugate pad is typically dried after preparation. The first MAPSA1 monoclonal antibody labeled with colloidal gold undergoes hydration changes between drying and reconstitution. A glycogen vitrification protective component forms a protective solid network within the dried matrix, a protein stabilizing component maintains the hydrophilic microenvironment around the antibody molecules, and an anti-colloidal gold aggregation component restricts close contact between colloidal gold particles during drying and reconstitution. Upon arrival of the sample solution at the colloidal gold conjugate pad, these components reconstitute with the liquid, releasing the colloidal gold-labeled antibody in a dispersed state while retaining its binding ability to the native conformational epitopes of MAPSA1.

[0014] Furthermore, in the above technical solution, the nitrocellulose membrane is loaded with an anti-matrix blocking composition on the membrane surface area outside the detection line. The anti-matrix blocking composition includes an inert protein blocking component, a polysaccharide blocking component, and a weak charge shielding component.

[0015] In practice, nitrocellulose membranes possess protein adsorption capabilities, and fat globules, pigment particles, complement proteins, and other non-target proteins in complex samples may remain outside the detection line. Inert protein blocking components occupy non-specific adsorption sites on the membrane surface, polysaccharide blocking components form a hydrophilic barrier layer on the membrane surface, and weakly charged shielding components reduce electrostatic adsorption between charged matrix components and the membrane material. During sample migration, the retention of non-target matrix components outside the detection line is reduced, and the colloidal gold-labeled complex participates more concentratedly in the specific capture at the detection line.

[0016] Furthermore, in the above technical solution, the first MAPSA1 monoclonal antibody is connected to the surface of colloidal gold particles through its non-antigen binding region, and the second MAPSA1 monoclonal antibody is fixed to the detection line through a solid-phase immobilization component. The first MAPSA1 monoclonal antibody and the second MAPSA1 monoclonal antibody are respectively derived from different hybridoma cell lines or different recombinant antibody sequences.

[0017] In practice, the first MAPSA1 monoclonal antibody is attached to the surface of the colloidal gold particles through its non-antigen-binding region, ensuring that its antigen-binding region faces the external liquid phase, thus reducing spatial obstruction of the antigen-binding area by the colloidal gold particle surface. The second MAPSA1 monoclonal antibody is immobilized on the detection line through a solid-phase immobilization component, forming a stable capture band on the membrane surface. The first and second MAPSA1 monoclonal antibodies are derived from different hybridoma cell lines or different recombinant antibody sequences, allowing them to differentiate in their antigen recognition regions and avoiding competitive binding of the same antigenic determinant.

[0018] Furthermore, in the above technical solution, the sample pad is a layered loading structure, with a sample receiving layer and an antigen releasing layer sequentially arranged along the sample flow direction. The sample receiving layer is loaded with the non-ionic surfactant protective component and the heterophilic antibody blocking component, and the antigen releasing layer is loaded with the protein depolymerization component.

[0019] In practice, the sample receiving layer is located at the front end of the sample entering the test strip. This allows the non-ionic surfactant protective components and heterophile antibody blocking components to first contact the lipids, hydrophobic particles, and heterophile antibodies in the sample, reducing interference when they flow into the subsequent immune reaction area. The antigen release layer is located after the sample receiving layer, allowing the sample, after initial matrix dispersion and antibody interference blocking, to continue contacting the protein depolymerization components, promoting the release of MAPSA1 antigen from the protein complex or adhesive matrix. This layered loading structure allows the sample pretreatment process to proceed in segments according to the chromatographic direction, avoiding concentrated release of pretreatment components at the initial stage of sample entry, which could cause fluctuations in the reaction environment.

[0020] Furthermore, in the above technical solution, the colloidal gold binding pad includes a reconstitution protection zone near the sample pad and a labeled antibody release zone near the nitrocellulose membrane. The reconstitution protection zone is loaded with a glycogen vitrification protection component and an anti-colloidal gold aggregation component, and the labeled antibody release zone is loaded with a colloidal gold-labeled first MAPSA1 monoclonal antibody and a protein stabilizing component.

[0021] In practice, after the sample solution enters the colloidal gold binding pad from the sample pad, it first passes through the reconstitution protection zone. The glycogen vitrification protection component and the anti-colloidal gold aggregation component dissolve with the liquid and enter the microenvironment surrounding the labeled antibody release zone, thus providing hydration protection for the colloidal gold-labeled antibody before release. As the sample solution continues to the labeled antibody release zone, the first MAPSA1 monoclonal antibody labeled with colloidal gold is reconstituted and released along with the protein stabilizing component. This regional distribution method connects the protective components and the labeled antibody release process in the chromatographic direction, reducing local aggregation of the colloidal gold-labeled antibody during the initial reconstitution stage.

[0022] Furthermore, in the above technical solution, the anti-matrix blocking composition is also loaded onto the overlap transition region between the sample pad and the nitrocellulose membrane, and the overlap transition region is provided with a hydrophilic migration regulating component, which is a water-soluble polymer film-forming agent or an amphoteric hydrophilic polymer.

[0023] In practice, the overlap and transition area between the sample pad and the nitrocellulose membrane is the interface where the sample enters the membrane detection area from the pretreatment material, where the flow rate and matrix composition distribution are prone to change. An anti-matrix blocking composition is placed in this area to further limit the non-specific adsorption of non-target matrix components to the membrane surface before the sample enters the nitrocellulose membrane. Water-soluble film-forming polymers or zwitterionic hydrophilic polymers form a hydrophilic migration interface, ensuring continuous wetting and uniform propagation of the sample solution at the overlap, reducing background unevenness on the membrane surface caused by localized retention.

[0024] Furthermore, in the above technical solution, the quality control line is fixed with an anti-species antibody capable of binding the colloidal gold-labeled first MAPSA1 monoclonal antibody, and the sample pad, colloidal gold binding pad, nitrocellulose membrane and absorbent pad are encapsulated in a test strip casing with a sample application well, an observation window and a sample flow direction limiting structure.

[0025] In practice, the anti-species antibody immobilized at the control line binds to the colloidal gold-labeled first MAPSA1 monoclonal antibody migrating with the chromatography solvent, allowing the chromatography process and the state of the labeled antibody release to be visualized through the control line. The sample pad, colloidal gold conjugating pad, nitrocellulose membrane, and absorbent pad are encapsulated within the test strip casing. The sample application well defines the sample entry position, the observation window corresponds to the detection line and control line positions, and the sample flow direction limiting sample migration along a set direction. During the detection process, the sample enters the sample pad through the sample application well, migrates through the colloidal gold conjugating pad and nitrocellulose membrane to the absorbent pad, completing the pretreatment, labeled antibody binding, detection line capture, control line binding, and end-of-pipe aspiration processes.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. By loading protein depolymerization components, non-ionic surfactant protective components, and heterophile antibody blocking components into the sample pad, and using colloidal gold-labeled native conformational epitope monoclonal antibodies in conjunction with linear epitope or hemiconformity epitope monoclonal antibodies fixed to the detection line, the sample undergoes matrix dispersion and interference reduction before entering the chromatography channel, and then proceeds to the dual-epitope immunorecognition process. The protein depolymerization component exposes the binding region of paratuberculosis-associated antigen 1, which is masked by lipids, mucopolysaccharides, or host proteins. The non-ionic surfactant protective component reduces the influence of hydrophobic particles in the sample on the migration of antigens and colloidal gold complexes. The heterophile antibody blocking component reduces non-specific bridging between host antibodies and labeled antibodies. Native conformational epitope monoclonal antibodies maintain the initial binding ability to undenatured antigens, while linear epitope or hemiconformity epitope monoclonal antibodies capture antigens that retain conserved recognition regions after sample processing, allowing the target antigen to be fixed to the detection line through another antigenic determinant even if there are local conformational changes. The above structure makes it easier for the target antigen in weakly positive samples to form continuous immune complexes, the boundary of the detection line band is clearer, and the risk of missed detection in low abundance samples is reduced.

[0027] 2. The antibody conformation protection component in the colloidal gold conjugate pad provides spatial support for the labeled monoclonal antibody during drying, storage, and reconstitution, reducing activity loss and colloidal gold particle aggregation in the dry membrane state. Glycosyl vitrification protection, protein stabilizing, and anti-colloidal gold aggregation components are distributed within the conjugate pad matrix, allowing the colloidal gold-labeled antibody to be released in a more uniform state upon sample arrival and participate in chromatographic migration. An anti-matrix blocking composition is placed outside the nitrocellulose membrane detection line; inert protein blocking, polysaccharide blocking, and weak charge shielding components work together to reduce the retention of milk fat globules, fecal pigment particles, serum complement proteins, and other non-target components on the membrane surface. With the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad having their flow direction defined by a retainer, the sample propagation path is fixed, resulting in more stable reconstitution, conjugation, capture, and color development processes. This structure reduces membrane redness, tailing, and quality control anomalies, narrows inter-batch interpretation differences, and maintains the basic operational form of colloidal gold test strips for rapid on-site detection. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments. Those skilled in the art can reproduce the technical solution of the present invention and achieve its claimed technical effects based on the content disclosed in this specification. It should be noted that the following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Any non-substantial improvements and adjustments made based on the core concept of the present invention should fall within the scope of protection of the present invention.

[0029] In the following embodiments, the first MAPSA1 monoclonal antibody is a monoclonal antibody targeting the native conformational epitope of MAPSA1, and the second MAPSA1 monoclonal antibody is a monoclonal antibody targeting a linear or semi-conformational epitope of MAPSA1. The antigenic determinants corresponding to the two antibodies do not overlap. The first MAPSA1 monoclonal antibody is obtained by screening immunogens that maintain the native spatial conformation of MAPSA1, and the second MAPSA1 monoclonal antibody is obtained by screening MAPSA1 fragments containing conserved linear regions of MAPSA1 or renatured MAPSA1 fragments. The two monoclonal antibodies are derived from different hybridoma cell lines or different recombinant antibody sequences, and competitive binding assays confirm that there is no competitive binding to the same antigenic determinant. Unless otherwise specified, each embodiment uses a base plate, sample pad, colloidal gold binding pad, nitrocellulose membrane, absorbent pad, and test strip casing to form the main structure of the test strip.

[0030] Example 1: The sample pad employs a layered loading structure, with a sample receiving layer and an antigen release layer sequentially arranged along the sample flow direction. The sample receiving layer treatment solution, per 100 mL of purified water, includes 0.30 g of polyoxyethylene sorbitan monolaurate and 0.60 g of non-immunized mouse immunoglobulin; the antigen release layer treatment solution, per 100 mL of purified water, includes 1.20 g of an amino acid buffer composed of glycine and histidine, 3.00 g of sorbitol, and 0.80 g of sodium chloride. The colloidal gold conjugated pad, along the sample flow direction, sequentially includes a reconstitution protection zone and a labeled antibody release zone. The reconstitution protection zone treatment solution, per 100 mL of purified water, includes 5.00 g of trehalose and 0.20 g of polyvinylpyrrolidone; the labeled antibody release zone contains a colloidal gold-labeled first MAPSA1 monoclonal antibody and 1.00 g / 100 mL of casein. The matrix-blocking composition for the area outside the detection line of the nitrocellulose membrane comprises, per 100 mL of purified water, 1.00 g casein, 0.40 g dextran, and 0.15 g betaine.

[0031] In preparation, a colloidal gold particle dispersion was taken, and the pH was adjusted to 8.2. The first MAPSA1 monoclonal antibody was added, allowing it to bind to the surface of the colloidal gold particles through its non-antigen-binding region. After reacting at room temperature for 30 minutes, casein was added for stabilization. Unbound components were removed by centrifugation, and the mixture was resuspended in a reconstitution solution containing trehalose and polyvinylpyrrolidone to obtain the colloidal gold-labeled first MAPSA1 monoclonal antibody solution. A glass fiber mat was used as the sample receiving layer, sprayed with a sample receiving layer treatment solution, and dried at 37°C for 2 hours. Another glass fiber mat was used as the antigen release layer, sprayed with an antigen release layer treatment solution, and dried at 37°C for 2 hours. The two mats were then overlapped along the chromatography direction to form a sample pad. A colloidal gold binding pad substrate was taken, and a reconstitution protection zone treatment solution was sprayed on the side near the sample pad. The colloidal gold-labeled first MAPSA1 monoclonal antibody solution was sprayed on the side near the nitrocellulose membrane and dried at 30°C for 4 hours. Take a nitrocellulose membrane, draw a test line with the second MAPSA1 monoclonal antibody, and draw a control line with an anti-species antibody that can bind to the colloidal gold-labeled first MAPSA1 monoclonal antibody. Spray an anti-matrix blocking composition onto the membrane surface outside the test lines, and let it dry for later use. Sequentially overlap and fix the sample pad, colloidal gold binding pad, nitrocellulose membrane, and absorbent pad onto the base plate, with an overlap length of 2 mm. Cut the overlap into 4 mm wide test strips and encapsulate them in a test strip cartridge with a sample application well, observation window, and sample flow direction limiting structure.

[0032] Example 2: The difference between this example and Example 1 is that the second MAPSA1 monoclonal antibody was obtained by screening MAPSA1 fragments that had undergone renaturation treatment. The detection line fixation method, colloidal gold labeling method, sample pad pretreatment composition, antibody conformation protection component, anti-matrix blocking composition, and assembly method are all the same as in Example 1. This example is used to verify the applicability of the dual epitope recognition structure when the second MAPSA1 monoclonal antibody is derived from different antigen fragment processing methods.

[0033] Example 3: The difference between this example and Example 1 is that the amino acid buffer component in the antigen release layer treatment solution is composed of glycine and lysine, with a total mass of 1.20 g; the polyhydroxy compound is changed from sorbitol to glycerol, with a mass of 2.50 g; and the mild salt component remains sodium chloride, with a mass of 0.80 g. All other conditions are the same as in Example 1. This example is used to verify the suitability of antigen release after replacing the amino acid buffer component and polyhydroxy compound in the protein depolymerization component.

[0034] Example 4: The difference between this example and Example 1 is that the nonionic surfactant protective component in the sample receiving layer treatment solution is replaced by alkyl glycoside instead of polyoxyethylene sorbitan monolaurate, with an addition amount of 0.35 g / 100 mL; the heterophile antibody blocking component, antigen release layer treatment solution, colloidal gold conjugate pad, and nitrocellulose membrane treatment methods are the same as in Example 1. This example is used to verify the applicability of the nonionic surfactant protective component to sample migration and background control under different conditions.

[0035] Example 5: The difference between this example and Example 1 is that the heterophile antibody blocking component in the sample receiving layer treatment solution is replaced with polymerized immunoglobulin fragments instead of non-immune mouse immunoglobulins, with an addition amount of 0.50 g / 100 mL; the treatment methods for the non-ionic surfactant protective component, antigen release layer treatment solution, colloidal gold conjugate pad, and nitrocellulose membrane are the same as in Example 1. This example is used to verify the control of non-specific bridging interference after the replacement of the heterophile antibody blocking component.

[0036] Example 6: The difference between this example and Example 1 is that the glycosidic vitrification protectant in the reconstitution protection solution of the colloidal gold conjugate pad is replaced by a combination of trehalose and sucrose, with a total mass of 5.00 g / 100 mL and a mass ratio of 3:2; the anti-colloidal gold aggregation component remains polyvinylpyrrolidone, with a mass of 0.20 g / 100 mL. All other conditions are the same as in Example 1. This example is used to verify the effect of changes in the combination of glycosidic vitrification protectants on the reconstitution state of colloidal gold-labeled antibodies.

[0037] Example 7: The difference between this example and Example 1 is that in the anti-matrix blocking composition for the area outside the detection line of the nitrocellulose membrane, the inert protein blocking component is replaced by fish gelatin instead of casein, with an addition amount of 0.80 g / 100 mL; the polysaccharide blocking component is replaced by hydroxyethyl cellulose instead of dextran, with an addition amount of 0.30 g / 100 mL; and the weak charge shielding component remains betaine, with an addition amount of 0.15 g / 100 mL. All other conditions are the same as in Example 1. This example is used to verify the membrane surface suitability after changes in the blocking and blocking components in the anti-matrix blocking composition.

[0038] Example 8: The difference between this example and Example 1 is that in the sample pad layered loading structure, the ratio of the sample receiving layer length to the antigen releasing layer length is adjusted from 1:1 to 2:1, while the types of components loaded in the sample receiving layer and the antigen releasing layer and the loading per unit area remain unchanged. All other conditions are the same as in Example 1. This example is used to verify the applicability of extending the sample receiving stage for the pretreatment of lipids, host immunoglobulins, and hydrophobic particles in complex samples.

[0039] Example 9: The difference between this example and Example 1 is that the ratio of the length of the reconstitution protection zone to the length of the labeled antibody release zone in the colloidal gold conjugate pad is adjusted from 1:1 to 1:2, while the types of components loaded in the reconstitution protection zone and the labeled antibody release zone, as well as the loading per unit area, remain unchanged. All other conditions are the same as in Example 1. This example is used to verify the effect of extending the labeled antibody release region on the uniformity of release of the colloidal gold-labeled first MAPSA1 monoclonal antibody.

[0040] Example 10: This example uses the same formulation as Example 1, except that the pH of the colloidal gold labeling reaction is adjusted from 8.2 to 7.8. The amount of the first MAPSA1 monoclonal antibody, reaction time, centrifugation and resuspension conditions, sample pad treatment, colloidal gold binding pad treatment, nitrocellulose membrane treatment, and assembly conditions are all the same as in Example 1. This example is used to verify the preparation stability of colloidal gold labeling when the pH changes under adjacent conditions.

[0041] Example 11: This example uses the same formulation as Example 1, except that the drying conditions for the colloidal gold conjugate pad are changed from 30°C for 4 hours to 35°C for 3 hours; the sample pad drying conditions, antibody labeling conditions, nitrocellulose membrane treatment, and assembly conditions are all the same as in Example 1. This example is used to verify the effect of varying the conjugate pad drying conditions within a mild range on the release and preservation status of the colloidal gold-labeled antibody.

[0042] Example 12: This example uses the same formulation as Example 1, except that the overlap length between the sample pad, colloidal gold binding pad, nitrocellulose membrane, and absorbent pad is adjusted from 2 mm to 1.5 mm; the remaining preparation conditions are the same as in Example 1. This example is used to verify the stability of continuous sample migration and detection line color development after changes in the overlap parameters of the chromatography materials.

[0043] Comparative Example 1: This comparative example is a comparison example lacking the core components. The difference from Example 1 is that the protein depolymerization component was removed from the antigen release layer treatment solution; that is, the amino acid buffer component composed of glycine and histidine, sorbitol, and sodium chloride were not added. The antigen release layer was only treated with purified water and then dried. The non-ionic surfactant protective component and heterophile antibody blocking component in the sample receiving layer, the colloidal gold-labeled first MAPSA1 monoclonal antibody and antibody conformation protection component in the colloidal gold binding pad, and the detection line, control line, and anti-matrix blocking composition on the nitrocellulose membrane are all the same as in Example 1. This comparative example is used to verify the effect of the absence of the protein depolymerization component on MAPSA1 antigen exposure and weak positive sample identification.

[0044] Comparative Example 2: This comparative example is a conventional prior art comparative example. A conventional colloidal gold test strip structure is used, with a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially arranged on the base plate. The sample pad is treated only with general buffer and inert protein, without a sample pretreatment composition consisting of protein depolymerization components, non-ionic surfactant protective components, and heterophile antibody blocking components. The colloidal gold conjugate pad is loaded with a colloidal gold-labeled MAPSA1 monoclonal antibody, but without antibody conformation protection components. The nitrocellulose membrane detection line is fixed with a MAPSA1 capture antibody corresponding to the same recognition pattern as the colloidal gold-labeled antibody, without limiting the bi-epitope pairing relationship between native conformational epitopes and linear or hemi-conformational epitopes. The membrane surface does not have an anti-matrix blocking composition consisting of inert protein blocking components, polysaccharide blocking components, and weak charge shielding components. The test strip assembly method is the same as in Example 1.

[0045] Comparative Example 3: This comparative example deviates from the process parameters. The difference from Example 1 is that the drying conditions for the colloidal gold conjugate pad were changed from 30°C for 4 hours to 60°C for 2 hours; the sample pad formulation, the colloidal gold-labeled first MAPSA1 monoclonal antibody formulation, the antibody conformation protection components, the nitrocellulose membrane treatment, and the assembly method are all the same as in Example 1. This comparative example is used to verify the effect of deviating from the mild drying conditions on the conformation of the colloidal gold-labeled antibody and the dispersion state of the colloidal gold particles.

[0046] Comparative Example 4: This comparative example is a lacking in the preparation method. The difference from Example 1 is that the sample pad does not have a sample receiving layer and an antigen release layer; instead, a mixture of polyoxyethylene sorbitan monolaurate, non-immune mouse immunoglobulin, an amino acid buffer component composed of glycine and histidine, sorbitol, and sodium chloride is loaded onto the same pad material in a single step. The colloidal gold conjugated pad does not have a reconstitution protection zone and a labeled antibody release zone; instead, a mixture of trehalose, polyvinylpyrrolidone, casein, and colloidal gold-labeled first MAPSA1 monoclonal antibody is loaded onto the same pad material in a single step. The total loading amount of each component, nitrocellulose membrane treatment, absorbent pad, base plate, and test strip casing are the same as in Example 1. This comparative example is used to verify the impact of the lack of a layered loading structure and a zoned release structure on the sample pretreatment sequence and the labeled antibody reconstitution and release process.

[0047] Test method: The test was conducted using purified MAPSA1 antigen, confirmed weakly positive paratuberculosis samples, and negative samples.

[0048] Purified MAPSA1 antigen was used to determine the lowest interpretable mass concentration; Weakly positive samples are used to determine the weakly positive concordance rate; Negative samples are used to determine the false positive rate and the background gray value of the membrane.

[0049] Three batches of test strips were taken from each group, and 20 strips were taken from each batch for testing. The intra-batch coefficient of variation was calculated.

[0050] After storing each group of test strips at 37℃ for 30 days, the binding retention rate of colloidal gold-labeled antibodies was measured.

[0051] The test results are shown in Table 1.

[0052] Table 1 Performance test results of each embodiment and comparative example

[0053] The lowest reproducible concentration in Example 1 was 0.50 ng / mL, with a weak positive concordance rate of 96.7%, a false positive rate of 0.0% for negative samples, a background gray value of 8.4, an intra-assay coefficient of variation of 5.2%, and an antibody binding retention rate of 92.6% after storage at 37°C for 30 days. These results indicate that the combined use of protein depolymerization components, non-ionic surfactant protective components, and heterophile antibody blocking components in the sample pad ensures that the MAPSA1 antigen in complex samples is dispersed in the matrix and its interference is weakened before entering the colloidal gold binding pad. The colloidal gold-labeled first MAPSA1 monoclonal antibody recognizes the native conformational epitope, while the detection line-fixed second MAPSA1 monoclonal antibody recognizes the linear epitope or the semi-conformational epitope, forming a sandwich trap structure of non-overlapping antigenic determinants. The antibody conformational protective component in the colloidal gold binding pad maintains the binding state of the labeled antibody during the drying and reconstitution stages. The components mentioned above are not simply superimposed. Sample pretreatment improves antigen accessibility, dual epitope pairing enhances antigen capture continuity, and conformational protection maintains the state in which labeled antibodies can participate in the reaction. The three form a continuous chain of action.

[0054] Examples 2 through 9 involved substitutions or gradient adjustments to the antibody source, protein depolymerization component, nonionic surfactant protective component, heterophile antibody blocking component, glycosylated vitrification protective component, anti-matrix blocking composition, sample pad layer length ratio, and colloidal gold binding pad partition length ratio. The minimum reproducible concentration in each example remained between 0.60 and 0.80 ng / mL, the weak positive concordance rate remained above 90.0%, and the false positive rate for negative samples was no higher than 3.3%, indicating that the substitution of components under the same technical concept could still maintain basic detection performance. The data for Examples 3 and 4 were slightly lower than that of Example 1, indicating that the specific types of protein depolymerization component and nonionic surfactant protective component affect antigen release and sample migration, but they are still within a repeatable detection range. The false positive rate for negative samples in Example 5 was 3.3%, indicating that the substitution of the heterophile antibody blocking component affected nonspecific bridging control, but the polymerized immunoglobulin fragments still provided blocking ability. The antibody binding retention rate in Example 6 was 91.7%, close to that of Example 1, indicating that the glycosylated vitrification protective component, when used in combination, could still maintain desiccation protection performance. The data from Examples 8 and 9 illustrate that there is a certain degree of adaptability between the sample pad layered loading structure and the colloidal gold-bound pad partitioned release structure.

[0055] Examples 10 to 12 maintained the same formulation as Example 1, only changing the pH of the colloidal gold labeling reaction, the drying conditions of the colloidal gold binding pad, and the overlap length of the chromatographic material. The lowest recognizable concentration in all three examples was 0.60 to 0.70 ng / mL, with a weak positive concordance rate of no less than 91.7% and an intra-batch coefficient of variation not exceeding 7.2%, indicating that the test strips maintained low-abundance antigen recognition capability and batch stability even with mild changes in preparation parameters. This result is related to the settings of the antibody conformational protection component, the sample pretreatment composition, and the anti-matrix blocking composition. The reconstitution protection zone and the labeled antibody release zone protect the colloidal gold-labeled antibody before it enters the chromatographic system. The layered structure of the sample pad allows the sample pretreatment process to be completed in segments along the flow direction, and the membrane blocking structure reduces the retention of non-target matrix outside the detection area.

[0056] In Comparative Example 1, after removing the protein depolymerization component, the lowest identifiable concentration increased to 2.00 ng / mL, the weak positive concordance rate decreased to 63.3%, and the intra-assay coefficient of variation increased to 13.5%. Because the non-ionic surfactant protective component and heterophile antibody blocking component were still retained in the sample receiving layer, the false positive rate of negative samples did not increase significantly. However, insufficient exposure of the MAPSA1 antigen from host proteins, mucopolysaccharides, or lipid matrices led to a decrease in the number of complexes formed between the colloidal gold-labeled first MAPSA1 monoclonal antibody and the target antigen. This comparison demonstrates that the protein depolymerization component is a necessary pretreatment component before low-abundance target antigens enter the dual epitope recognition system, and simply retaining surfactant protection and heterophile antibody blocking is insufficient to solve the problem of insufficient antigen accessibility in weakly positive samples.

[0057] Comparative Example 2 used a conventional colloidal gold test strip formulation and structure, with a minimum reproducible concentration of 3.50 ng / mL, a weak positive concordance rate of 48.3%, a false positive rate of 8.3% for negative samples, a membrane background gray value of 29.4, an intra-assay coefficient of variation of 18.6%, and an antibody binding retention rate of 61.5% after storage. These results indicate that conventional sample pad treatment methods cannot simultaneously address antigen embedding, heterophilic antibody bridging, and hydrophobic matrix interference; the recognition system, which does not specify the pairing of native conformational epitopes with linear or semi-conformational epitopes, struggles to maintain stable capture when the target antigen undergoes local conformational changes; and the lack of antibody conformational protection components in the colloidal gold binding pad leads to a decrease in the binding state of the labeled antibody after drying and storage. The differences between Comparative Example 2 and Example 1 reflect a synergistic relationship between sample pretreatment, dual epitope recognition, conformational protection, and membrane sealing in this protocol.

[0058] Comparative Example 3, which only adjusted the drying conditions of the colloidal gold conjugating pad to 60°C, increased the minimum reproducible concentration to 2.50 ng / mL, reduced the weak positive concordance rate to 58.3%, decreased the antibody binding retention rate after storage to 42.8%, and increased the intra-assay coefficient of variation to 20.4%. Since other formulations were the same as in Example 1, this change mainly stemmed from the effect of high-temperature drying on the conformation of the colloidal gold-labeled first MAPSA1 monoclonal antibody and the dispersion state of the colloidal gold particles. The first MAPSA1 monoclonal antibody targets a native conformational epitope, and its antigen-binding region requires high spatial conformational retention. When drying conditions deviate from a mild process, the glycoside vitrification protection component, protein stabilizing component, and anti-colloidal gold aggregation component cannot completely offset the antibody activity loss and particle aggregation caused by high temperature, resulting in a reduction in the detection line capture complex. This comparison illustrates that conformational protection components need to be combined with suitable conjugating pad drying conditions.

[0059] Comparative Example 4, omitting the stratified loading of the sample pad and the zoned release of the colloidal gold binding pad while maintaining the total amount of components, achieved a minimum reproducible concentration of 1.50 ng / mL, a weak positive concordance rate of 71.7%, a false positive rate of 5.0% for negative samples, a membrane background grayscale value of 18.7, and an antibody binding retention rate of 75.3% after storage. These results indicate that simply mixing and loading the components cannot replace the stratified and zoned structure along the sample flow direction. When each pretreatment component in the sample pad is released all at once, the heterophile antibody blocking, hydrophobic matrix dispersion, and antigen release processes lack a sequential order, easily leading to insufficient dispersion of the sample matrix before it enters the colloidal gold binding region. When the protective component and labeled antibody are mixed and loaded in the colloidal gold binding pad, the reconstitution protection process and the labeled antibody release process cannot form a flow transition, resulting in decreased uniformity of colloidal gold labeled antibody release. These results demonstrate that the spatial loading structure and the formulation components have a synergistic relationship, which is an important condition for achieving stable detection results.

Claims

1. A colloidal gold test paper of paratuberculosis MAP SAl monoclonal antibody, characterized in that, It includes a base plate and a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and an absorbent pad that are sequentially overlapped on the base plate; The colloidal gold conjugate pad is loaded with a colloidal gold-labeled first MAPSA1 monoclonal antibody, which is a monoclonal antibody targeting the native conformational epitope of MAPSA1. The nitrocellulose membrane is provided with a detection line and a control line. The detection line is fixed with a second MAPSA1 monoclonal antibody. The second MAPSA1 monoclonal antibody is a monoclonal antibody targeting a linear epitope or a half-conformation epitope of MAPSA1. The antigenic determinants corresponding to the first MAPSA1 monoclonal antibody and the second MAPSA1 monoclonal antibody do not overlap with each other. The sample pad is loaded with a sample pretreatment composition, which includes a protein depolymerization component, a nonionic surfactant protective component, and a heterophile antibody blocking component. The colloidal gold binding pad is also loaded with antibody conformation protection components.

2. The MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis according to claim 1, characterized in that, The first MAPSA1 monoclonal antibody was obtained by screening for an immunogen that maintains the native spatial conformation of MAPSA1, and the second MAPSA1 monoclonal antibody was obtained by screening for a MAPSA1 fragment containing a conserved linear region of MAPSA1 or a renatured MAPSA1 fragment.

3. The MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis according to claim 1, characterized in that, The protein depolymerization component includes an amino acid-type buffer component, a polyhydroxy compound, and a mild salt component; the nonionic surfactant protective component includes polyoxyethylene surfactants or alkyl glycoside surfactants; and the heterophilic antibody blocking component includes non-immunogenic homologous animal immunoglobulins, polymerized immunoglobulin fragments, or heterophilic antibody blocking proteins.

4. The MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis according to claim 1, characterized in that, The antibody conformation protection components include a glycogen vitrification protection component, a protein stabilizing component, and an anti-colloidal gold aggregation component, which are collectively loaded within the dry matrix of the colloidal gold binding pad.

5. The MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis according to claim 1, characterized in that, The nitrocellulose membrane has an anti-matrix blocking composition loaded on the membrane surface area outside the detection line. The anti-matrix blocking composition includes an inert protein blocking component, a polysaccharide blocking component, and a weak charge shielding component.

6. The MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis according to claim 2, characterized in that, The first MAPSA1 monoclonal antibody is attached to the surface of colloidal gold particles through its non-antigen binding region, and the second MAPSA1 monoclonal antibody is immobilized on the detection line by a solid-phase immobilization component. The first MAPSA1 monoclonal antibody and the second MAPSA1 monoclonal antibody are derived from different hybridoma cell lines or different recombinant antibody sequences, respectively.

7. The MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis according to claim 3, characterized in that, The sample pad has a layered loading structure, with a sample receiving layer and an antigen releasing layer arranged sequentially along the sample flow direction. The sample receiving layer is loaded with the non-ionic surfactant protective component and the heterophile antibody blocking component, and the antigen releasing layer is loaded with the protein depolymerization component.

8. The MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis according to claim 4, characterized in that, The colloidal gold binding pad includes a reconstitution protection zone near the sample pad and a labeled antibody release zone near the nitrocellulose membrane. The reconstitution protection zone is loaded with a glycogen vitrification protection component and an anti-colloidal gold aggregation component. The labeled antibody release zone is loaded with a colloidal gold-labeled first MAPSA1 monoclonal antibody and a protein stabilizing component.

9. The MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis according to claim 5, characterized in that, The matrix-blocking composition is also loaded onto the overlap transition region between the sample pad and the nitrocellulose membrane. The overlap transition region is provided with a hydrophilic migration regulating component, which is a water-soluble polymer film-forming agent or an amphoteric hydrophilic polymer.

10. The MAPSA1 monoclonal antibody colloidal gold test strip for paratuberculosis according to any one of claims 1 to 9, characterized in that, The control line is fixed with an anti-species antibody that can bind to the colloidal gold-labeled first MAPSA1 monoclonal antibody. The sample pad, colloidal gold binding pad, nitrocellulose membrane and absorbent pad are encapsulated in a test strip casing with a sample application well, an observation window and a sample flow direction limiting structure.

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