Solid-phase agglutination detection reagent card for expressing heterologous antigen by utilizing gene recombination and detection method of solid-phase agglutination detection reagent card

Abstract

The invention provides a solid-phase agglutination detection test paper card for expressing a heterologous antigen by utilizing gene recombination and a detection method thereof. A reaction area of the solid-phase agglutination detection test paper card comprises a reaction film, the reaction film is coated with captured protein and can enrich to-be-detected antibodies in a to-be-detected sample, and the captured protein is combined with the to-be-detected antibodies and then subjected to agglutination reaction with granular antigens in a detection reagent to form an agglutination compound. And the agglutination compound is intercepted in the reaction film for color development. The granular antigen is a cell carrying a specific antigen, and the cell expresses and displays the specific antigen on the surface of the cell in a gene recombination mode. The solid-phase agglutination detection test paper card provided by the invention has the advantages of simple detection steps, short detection time, high sensitivity, accurate detection result, strong specificity and the like, can be used for detecting various diseases and monitoring antibody level, and also has important significance in the fields of infectious disease prevention and control, medical diagnosis and the like.

Description

[0001] The present application claims priority to the Chinese prior application with the application number 2025106248916 and the filing date of 2025 / 05 / 15; the entire contents of which are incorporated herein as part of the present application. TECHNICAL FIELD

[0002] The present application belongs to the field of in vitro diagnosis, and in particular, relates to a solid-phase agglutination detection reagent card for expressing heterologous antigens by gene recombination and a detection method thereof. BACKGROUND

[0003] Immune agglutination detection is a classical immunology technique based on the specific binding reaction of antigen-antibody, and the detection purpose is achieved through the visible agglutination phenomenon. Immune agglutination detection can be applied in multiple scenarios, such as blood typing, infectious disease detection, autoimmune disease detection, pregnancy detection, etc. in clinical diagnosis. In addition, it also plays an important role in drug transfusion and transplantation, drug monitoring, forensic medicine and animal medicine, especially in rapid screening of animal epidemic diseases (such as brucellosis agglutination test). The advantages of immune agglutination detection are rapid (several minutes to several hours), simple operation, no need for expensive equipment, and suitable for basic medical treatment. Its limitations are lower sensitivity (microgram level) and susceptibility to prezone effect (antibody excess inhibits agglutination) or cross-reaction interference. In summary, immune agglutination detection forms visible agglutination through antigen-antibody cross-linking, although the sensitivity is limited, its rapidness and economy make it important in point-of-care testing (POCT).

[0004] The principle of immune agglutination is that antigen-antibody binding and cross-linking are required, the antigen needs to be multivalent (containing multiple epitopes), and the antibody needs to be bivalent or multivalent (such as IgM or IgG). After the combination of the two, a cross-linking network is formed, and visible agglutination particles are formed. The reaction environment requires certain electrolyte conditions, for example, the reaction needs to be carried out in an electrolyte environment such as physiological saline, which neutralizes the surface charge of the particles, reduces the repulsive force, and promotes agglutination. In order to be suitable for observation, sometimes the carrier amplification effect needs to be used, that is, the antigen is coupled with a solid-phase carrier, because a single solid-phase carrier (such as latex particles, nanometer microspheres) can simultaneously couple multiple antigens. Agglutination reaction includes direct agglutination and indirect agglutination. Direct agglutination: natural particulate antigens (such as bacteria, red blood cells) can directly combine with the antibody to be detected to occur agglutination reaction. Indirect agglutination: the antigen is coupled with a solid-phase carrier (such as latex microspheres, etc.) and then combined with the antibody to be detected to occur agglutination reaction.

[0005] According to the principle of direct agglutination described above, a solid-phase agglutination test paper card (small hole solid-phase agglutination) of natural particulate antigen can be prepared for detecting the antibodies produced after the body is infected with bacteria. The specific method is as follows: the capture protein is coated on the reaction film, after the sample is added, the capture protein captures the antibodies to be detected in the sample to form a capture protein-antibody to be detected complex, and then the dyed natural particulate antigen (such as Brucella) is added. The natural particulate antigen forms agglutination particles with the capture protein-antibody to be detected complex. Since the particle size of the agglutination particles is larger than the pore size of the reaction film, the agglutination particles are retained on the reaction film and develop color.

[0006] However, when detecting some antibodies, such as detecting viral antibodies in a sample, a natural particulate antigen with a surface containing viral antigens that matches the viral antibodies cannot be found, and therefore the detection of viral antibodies cannot be performed by preparing the solid-phase agglutination test paper card described above. The detection of viral antibodies in a sample is usually performed by ELISA detection or test strip detection. Although ELISA detection (such as indirect ELISA) has the advantages of high sensitivity, strong specificity, stable detection results, etc., it has a long detection time, usually 2-4 hours, and requires professional equipment such as an enzyme labeler, a thermostat, a plate washer, etc., and also depends on a laboratory environment and professional technicians, which is limited for primary or large-scale screening. Although the test strip detection method (such as a colloidal gold test strip) can quickly detect antibodies, usually within 15 minutes, and does not require incubation, washing, and other complicated steps, it requires the preparation of purified antigens, which is complex and time-consuming, and the coating concentration and the line concentration of the antigens on the test strip affect the sensitivity, specificity, and stability of the test strip, and therefore the coating concentration and the line concentration of the antigens need to be optimized through experiments. In addition, the test strip detection method highly depends on the specificity and purity of the antigens. If the specificity of the coated antigens or the labeled antigens is poor, the detection accuracy is low and positive results cannot be detected. If the purity of the coated antigens or the labeled antigens is insufficient (such as containing impurity proteins), these impurity proteins may cross-react with non-target antibodies (such as rheumatoid factor, heterophilic antibodies) in the sample, resulting in false positives.

[0007] Although the surface of the natural particulate antigen does not contain viral antigens, the viral antigens can be displayed on the surface of cells (including prokaryotic cells and eukaryotic cells) as particulate antigens through genetic recombination.

[0008] Therefore, there is an urgent need for a solid-phase agglutination test reagent card for expressing heterologous antigens through genetic recombination, which can broaden the detection approach of viral antibodies. SUMMARY

[0009] To address the challenges of existing technologies, this invention provides a solid-phase agglutination test strip and its detection method that utilize gene recombination to express heterologous antigens. The reaction zone of the solid-phase agglutination test strip includes a reaction membrane coated with a capture protein, which enriches the antibody to be tested in the sample. The capture protein binds to the antibody and then reacts with particulate antigens in the detection reagent to form an agglutination complex. This agglutination complex is retained in the reaction membrane and develops color. The particulate antigen is a cell carrying a specific antigen, which is expressed and displayed on its surface through gene recombination. The solid-phase agglutination test strip provided by this invention has advantages such as simple detection steps, short detection time, high sensitivity, accurate results, and strong specificity. It can be used not only for the detection of various diseases and the monitoring of antibody levels but also has significant implications for infectious disease control and medical diagnosis.

[0010] This invention provides a solid-phase agglutination test strip card for expressing heterologous antigens using gene recombination, comprising a capture protein and a detection reagent. The detection reagent contains particulate antigens, which are cells carrying specific antigens. The specific antigens specifically bind to the antibody to be tested. The cells carrying the specific antigens are cells that express and display the specific antigens through gene recombination.

[0011] When detecting certain antibodies, such as directly detecting viral antibodies in blood samples, it is impossible to find natural particulate antigens (referring to prokaryotic cells (bacteria, etc.) and eukaryotic cells (yeast, mammalian cells, etc.)) containing viral immunogenic proteins on their surface. Therefore, these antibodies cannot be detected by preparing solid-phase agglutination test strips. This invention utilizes gene recombination to express and display specific antigens on the cell surface, which serve as particulate antigens for use in solid-phase agglutination test strips to detect viral antibodies in samples. Commonly used methods for detecting viral antibodies in blood samples are ELISA and test strip methods (chromatographic or filtration methods).

[0012] Although this invention is less sensitive than ELISA (such as iELISA), it does not require professional technicians to operate with specialized equipment. It can be used for grassroots testing and large-scale screening without the need for a laboratory environment. Furthermore, the testing time is fast, with a single sample testing time of no more than ten minutes.

[0013] Compared to test strip detection methods, using colloidal gold test strips as an example, this invention offers several advantages. While both methods have similar sample detection times and can be used for basic-level testing and large-scale screening without laboratory intervention, this invention eliminates the need for antigen expression and purification. Instead, it involves displaying the antigen on the cell surface via gene recombination. In contrast, preparing colloidal gold test strips requires first expressing and purifying the antigen (which must contain at least two distinct epitopes), then coating the binding pad with the antigen as a label and applying it as a streak antigen to the detection line (T-line) (e.g., the double-antigen sandwich method). The antigen coating concentration and the antigen streak concentration (the coated antigen needs to be labeled with colloidal gold) affect the sensitivity, specificity, and stability of the test strip; therefore, these concentrations require further experimental optimization. On the other hand, the test strip method relies heavily on the specificity and purity of the antigen when detecting samples. If the specificity of the coated or labeled antigen is poor, the detection accuracy will be low, and no positive result will be detected. If the purity of the coated or labeled antigen is insufficient (e.g., containing impurity proteins), these impurity proteins may cross-react with non-target antibodies (e.g., rheumatoid factor, heterophilic antibodies) in the sample, leading to false positives. Furthermore, the solid-phase agglutination test strip card using gene recombination to express heterologous antigens provided by this invention adds washing solution to the reaction wells when detecting antibodies. The washing solution washes away non-specific binding impurities, improving specific binding. The washing solution can also adjust the reaction conditions such as ionic strength and pH value of the reaction wells to promote the agglutination reaction. In contrast, the test strip method does not have a washing solution step and relies on the natural migration of the sample on the membrane (chromatography). Non-specific binding impurities will migrate with the sample to the detection line (T line), and therefore residual non-specific binding impurities may bind with the labeled protein, producing background color and affecting the interpretation of the results.

[0014] The detection process of the solid-phase agglutination test strip card using gene recombination to express heterologous antigens provided by this invention can be summarized as follows: antibody enrichment - antibody agglutination with particulate antigen - washing - observation of agglutination. This invention eliminates the need for antigen purification and labeling when detecting viral antibodies. It indicates the presence of antibodies simply by displaying specific antigens on the cell surface and directly staining the cells as particulate antigens. Furthermore, the washing process addresses the non-specific binding issues in complex samples (such as blood samples), thus outperforming test strip methods in terms of specificity, sensitivity, and accuracy of result interpretation.

[0015] Furthermore, the cells carrying the specific antigens are prokaryotic cells or eukaryotic cells.

[0016] In some embodiments, the cell carrying the specific antigen is a prokaryotic cell.

[0017] Furthermore, the cells carrying the specific antigens include any one or more of bacteria, yeast, and animal cells.

[0018] In some embodiments, the cell carrying the specific antigen is Escherichia coli.

[0019] Furthermore, the capture protein is a ligand of the antibody to be tested or a monoclonal or polyclonal antibody targeting the antibody to be tested.

[0020] In some embodiments, the capture protein is streptococcal protein G.

[0021] Furthermore, the cells carrying the specific antigen may or may not be colorless. When they are not colorless, they need to be stained with dyes, or the chromogenic substance may be expressed inside the cells through transgenic methods, or a chromogenic substrate may be added to the cell culture medium and the substrate may be decomposed by enzymes produced by cell metabolism to release the chromogenic substance for staining.

[0022] In some methods, the cells carrying the specific antigen are not colorless themselves and are stained with red cinnabar reagent.

[0023] Furthermore, the solid-phase agglutination test strip card for expressing heterologous antigens using gene recombination also includes a reaction membrane, a permeate membrane, an absorbent pad, and a shell; the reaction membrane embeds the capture protein, and the shell is provided with sample application wells; the pore size of the reaction membrane and the permeate membrane is the same, which is 3-8 μm.

[0024] Furthermore, the solid-phase agglutination test strip card that utilizes gene recombination to express heterologous antigens also includes a washing solution.

[0025] Furthermore, the present invention provides a method for using the above-described solid-phase agglutination test strip card for expressing heterologous antigens through gene recombination, comprising the following steps:

[0026] (1) The captured protein package is embedded in the reaction membrane;

[0027] (2) Assemble the reaction membrane, permeate membrane, permeate membrane, absorbent pad and shell into a solid phase agglutination test paper card;

[0028] (3) Dilute or not dilute the sample to be tested;

[0029] (4) Add the sample to be tested and the test reagent to the sample well of the solid phase agglutination test strip in sequence to react. After the reaction, add washing solution; observe whether the sample well shows color.

[0030] Further, the sample to be tested in step (3) is any one or more of whole blood, serum, and plasma, with a dilution factor of 0-64 times.

[0031] Furthermore, in step (4), the amount of sample to be tested and the amount of test reagent added are the same, which is 5-20 μL; the reaction time is 10-180 seconds.

[0032] In some methods, a solid-phase agglutination test strip using a gene recombination expression heterologous antigen to detect rabies virus infection is used as an example. A solid-phase test strip for rabies virus is prepared, and the sample to be tested is rabies-immunized rabbit positive serum. The test results show that the positive serum can still be distinguished by the naked eye when diluted 512 times, indicating that the solid-phase agglutination test strip using gene recombination expression heterologous antigen provided by the present invention has high sensitivity.

[0033] In some methods, using a solid-phase agglutination test strip with recombinant gene expression of heterologous antigens to detect rabies virus infection is used as an example. A solid-phase test strip for rabies virus is prepared and clinical negative / positive serum samples from mice and rabbits are tested. The results show that the detection accuracy can reach 100%, indicating that the solid-phase agglutination test strip with recombinant gene expression of heterologous antigens provided by the present invention has high detection accuracy.

[0034] In some methods, the solid-phase agglutination detection reagent card for expressing heterologous antigens using gene recombination (taking the detection of rabies virus as an example) was optimized. The optimal expression system for rabies virus-G protein and the vector used therein were screened. The expression system was an Escherichia coli expression system and the vector was pET-28a(+). The nucleotide sequence of rabies virus-G protein was also optimized to improve the expression level, detection sensitivity, and specificity of rabies virus-G protein.

[0035] On the other hand, the present invention provides a solid-phase agglutination test strip, comprising a capture protein and a detection reagent, wherein the detection reagent contains a particulate antigen; the particulate antigen is a cell carrying a specific antigen, or a conjugate of a specific antigen and an inert carrier, wherein the specific antigen specifically binds to the antibody to be tested; the capture protein captures the antibody to be tested and reacts with the particulate antigen in the detection reagent to form an agglutination complex, which then develops color in the reaction area of ​​the test strip.

[0036] The solid-phase agglutination test strip provided by this invention changes the type of agglutination reaction to small-well solid-phase agglutination based on the principle of agglutination reaction. Because the reaction takes place in fixed small wells, the reaction is concentrated and rapid, resulting in a short reaction time and high color intensity. Washing with washing solution reduces the influence of other components and non-specific agglutination on the observation of true agglutination, and also eliminates interference from other colored substances in the sample, reducing non-specific interference. Whole blood, serum, and plasma can all be detected by this invention, and the results are clear and highly accurate. At the same time, the use of antibody enrichment method increases sensitivity; and it can also be equipped with detection and interpretation equipment to improve the objectivity of the detection.

[0037] The solid-phase agglutination test strip provided by this invention, compared to the plate agglutination test, firstly solves the problem of limited specificity and sensitivity of the plate agglutination test. By embedding the ligand of the antibody to be tested or monoclonal or polyclonal antibodies targeting the antibody to be tested on the reaction membrane, the antibody to be tested is captured and enriched, increasing the sensitivity of the detection. By adding washing solution, unreacted substances are removed, reducing the influence of interfering substances on the results and increasing the specificity of the detection. Secondly, it solves the problem of subjective interpretation of plate agglutination test results. The test reagents of the solid-phase agglutination test strip bind with the test substance and agglutinate, then develop color in the reaction wells. The results are not only fixed and concentrated, but also easy to interpret. Moreover, it can be equipped with detection and interpretation equipment to improve the objectivity of the detection and reduce the interference of human factors. The result interpretation is more objective and accurate than that of the plate agglutination test, which relies on visual observation of agglutination phenomena, and reduces the judgment differences between different operators. Furthermore, plate agglutination assays require a large amount of antigen and antibody materials and have a long detection time, while the solid-phase agglutination test strip of this invention requires only a minimum of 5 μL of sample, and the sample can be diluted, thus requiring even less sample and a shorter detection time of 0.5-5 minutes for detection and interpretation. In addition, whole blood, serum, and plasma can all be used for detection. Even if the sample has color or other interfering substances, washing with washing solution can reduce these interferences and improve interpretation accuracy. Compared to colloidal gold immunochromatography, the solid-phase agglutination test strip provided by this invention has the advantages of simple operation and rapid detection, but the principle or filtration of the solid-phase agglutination test strip of this invention differs from that of colloidal gold immunochromatography. When detecting antibodies using colloidal gold immunochromatography or immunofiltration, indirect methods or double-antigen sandwich methods are employed. This requires antigen purification before streaking the membrane as a detection line, and in indirect methods, the antibody is indirectly labeled or the antigen is labeled. In the solid-phase agglutination test strip of this invention, antibodies in the sample are first captured by the reaction membrane, and pre-stained antigen particles directly agglutinate with them. The capture protein in the solid-phase agglutination test strip can enrich the antibody in the sample beforehand, and the washing solution can eliminate non-specific interference. Colloidal gold immunochromatography or immunofiltration cannot achieve these two effects. Therefore, this invention can improve detection sensitivity, especially for low concentrations of the antibody in the sample, and also has good detection results. Furthermore, this invention is fundamentally different from common blood typing test strips. Blood typing test strips pre-embed monoclonal or polyclonal antibodies targeting blood type on the filtration membrane and fix them in place. When they encounter antigens on red blood cells in the blood sample, agglutination occurs, and after the addition of washing solution, the agglutinated antibodies remain on the filtration membrane, achieving the detection result. Therefore, blood typing test strips are essentially a direct antigen detection method. Solid-phase agglutination test strips, on the other hand, enrich the antibody to be tested and then add a known antigen, thus belonging to antibody detection.

[0038] Furthermore, the capture protein is a ligand of the antibody to be tested or a monoclonal or polyclonal antibody targeting the antibody to be tested.

[0039] The capture protein is used to enrich the antibody to be tested. When the capture protein is a ligand of the antibody to be tested, the ligand includes, but is not limited to, streptococcal protein G, Staphylococcus aureus protein A, galectin-3 which can enrich IgE binding protein, Sm22.6 and SmTAL1 of Schistosoma mansoni, ABA-1 produced by Ascaris suis, Anis1 and Anis2 produced by Anisakis, human FcεRI, FcεRII, etc.

[0040] When the capture protein acts as a ligand for the antibody to be tested, the antibodies in the sample enriched include both the antibody to be tested and antibodies of the same class. For example, streptococcal protein G can bind to the Fc fragment of IgG antibodies without affecting the function of the Fab fragment. If a specific bacterial antibody in serum is to be detected, and that antibody is IgG, streptococcal protein G can capture all IgG antibodies in the serum, including the antibody to be tested. This antibody will agglutinate with the corresponding particulate antigen in the detection reagent and be retained in the reaction wells for color development. The remaining non-specific antibodies are washed away by the washing solution and seep down the filter membrane.

[0041] In some embodiments, the present invention optimizes the sequence of streptococcal protein G to enhance its ability to bind IgG.

[0042] When the capture protein is a monoclonal or polyclonal antibody targeting the antibody to be tested, it can enrich the target antibody in the sample to be tested. The target antibody is not only IgG, but can also be IgM, IgE, or IgA or IgY antibodies.

[0043] The antibody to be tested is bivalent or multivalent, and the particulate antigen contains multiple epitopes, thus enabling the antibody to be tested and the particulate antigen to form an agglutination complex.

[0044] The solid-phase agglutination test strip of this invention is mainly used for the detection of animal infectious diseases, human infectious diseases, autoimmune diseases, pregnancy, drug transfusion and transplantation, drug monitoring, etc. Therefore, the antibodies to be detected are mainly located in the blood. The bivalent antibodies in the blood include IgG and IgE, and the multivalent antibodies include IgM.

[0045] The particulate antigens mentioned, such as those from bacteria and erythrocytes, are macromolecules with complex structures. Taking bacteria as an example, their cell walls, capsules, flagella, and other structures all have different antigenic determinants. Because there are a large number of different types of antigenic determinants on the surface of bacteria, they are multivalent.

[0046] Furthermore, the cell carrying the specific antigen is a naturally occurring cell carrying the specific antigen, or a cell that expresses and displays the specific antigen through gene recombination; the cell carrying the specific antigen is a prokaryotic cell or a eukaryotic cell.

[0047] Furthermore, the cells carrying the specific antigens include any one or more of bacteria, yeast, and animal cells.

[0048] Some cells carrying specific antigens are naturally occurring, primarily pathogenic bacteria such as Brucella and Staphylococcus aureus; others include common cells like erythrocytes, whose surface contains sialic acid receptors that can bind to viral hemagglutinin proteins, thus enabling antibody detection for various viruses (influenza, Newcastle disease, etc.). Other cells carrying specific antigens require gene recombination to express and display the specific antigens on their surface. Several expression systems can be used to prepare these cells: prokaryotic bacterial expression systems (e.g., E. coli), yeast expression systems (e.g., Pichia pastoris), insect cell-baculovirus systems (e.g., Sf9 cells), and mammalian cell expression systems (e.g., HEK293, CHO cells). Using the above expression systems also requires the construction of vectors. Vectors for prokaryotic expression systems include, but are not limited to, the pET series and pGEX series vectors, the pET-Surface vector based on the OmpA anchoring system, the pAIDA-I autotransporter vector that supports large protein display, and the pSDV (Surface Display Vector) that integrates the Lpp-OmpA anchoring system and is suitable for antigen display. Eukaryotic yeast expression vectors include, but are not limited to, pPICZ. Insect cell expression vectors include, but are not limited to, pFastBac. Mammalian cell expression vectors include, but are not limited to, pcDNA3.1 and lentiviral / adenoviral vectors.

[0049] In this invention, antigens that can stimulate the body to produce antibodies include protein antigens and non-protein antigens. The protein antigens include, but are not limited to, proteins on the surface of microorganisms (including bacteria, viruses, etc.), proteins on the surface of animal cells (including erythrocytes, tumor cells, etc.), and proteins on the surface of plant cells (including pollen, etc.). The non-protein antigens include, but are not limited to, chemical substances (including natural polypeptides, sugars, glycoproteins, toxins, hormones, etc.), drugs, and artificially synthesized polymers. The solid-phase detection agglutination test kit provided by this invention can detect whether the body has produced antibodies against the aforementioned biological and non-biological antigens.

[0050] Among the antigens mentioned above, some are proteins (such as proteins on the surface of bacteria and erythrocytes), and certain cells (such as bacteria and erythrocytes) naturally carry specific antigens. Therefore, these cells can be directly used as particulate antigens in the detection reagent. The size of these cells is smaller than the pore size of the reaction membrane and the filtration membrane. When the specific antigen carried on the cell reacts with the antibody to be tested, the resulting complex is larger than the pore size of the reaction membrane and the filtration membrane, and is thus trapped in the reaction membrane. Other antigens, although proteins, cannot be found to be naturally carried by cells (such as viruses). Therefore, it is necessary to express and display the specific antigen on the surface of prokaryotic or eukaryotic cells through gene recombination, and then use the cells carrying the specific antigen as particulate antigens in the detection reagent to react with the antibody to be tested in an agglutination reaction. Furthermore, for non-protein antigens (such as soluble antigens from Schistosoma eggs), it is impossible to express and display specific antigens on the surface of prokaryotic or eukaryotic cells through gene recombination. Therefore, additional physical (extraction, extraction, etc.) and chemical methods are required to obtain them. The obtained antigens are then coupled with an inert carrier to form a conjugate, which serves as the particulate antigen of the detection reagent and reacts with the antibody to be tested in an agglutination reaction.

[0051] Furthermore, the cells carrying the specific antigen may or may not be colorless. When they are colorless, they need to be stained with dyes, or the chromogenic substance may be expressed inside the cells through transgenic methods, or a chromogenic substrate may be added to the cell culture medium and the substrate may be decomposed by enzymes produced by cell metabolism to release the chromogenic substance for staining. The inert carrier may or may not be colorless. When it is colorless, it needs to be stained with dyes.

[0052] When the cells carrying the specific antigen are themselves colored, such as erythrocytes and Staphylococcus aureus, they will react with the test antibody and develop color on the reaction membrane. When the cells carrying the specific antigen are not colored, staining with dye is required to induce agglutination with the test antibody and develop color on the reaction membrane. Staining reagents and methods include, but are not limited to, red cinnabar, Congo red, Indian ink, phenol blue staining, Gram staining, acid-fast staining, crystal violet and brilliant green staining, etc. In addition to staining, adding a chromogenic substrate to the cell culture medium allows enzymes produced by the metabolism of the cells carrying the specific antigen to break down the substrate and release the chromogenic substance, achieving the staining purpose. For example, X-gal develops a blue color in Escherichia coli, CHROMagar develops a color in Candida albicans, and chromogenic medium produces a pink or green color in Staphylococcus aureus. In addition, specific colors can be formed by expressing color-producing substances inside cells carrying specific antigens through transgenic methods. For example, the green fluorescent protein gene can be transferred into E. coli to express EGFP, RFP, etc., which can form green fluorescence under excitation light, and red fluorescent protein genes, etc.

[0053] Inert carriers that are colored by nature include, but are not limited to, colloidal gold, latex microspheres, magnetic microspheres, and carbon powder. Inert carriers with luminescent properties include, but are not limited to, fluorescent microspheres. Inert carriers that are not colored by nature can be colored by dyeing.

[0054] Furthermore, the solid-phase agglutination test strip also includes a reaction membrane, a permeation membrane, an absorbent pad, and a shell; the reaction membrane encapsulates the captured protein, and the shell is provided with a sample application well; the pore size of the reaction membrane and the permeation membrane is the same, which is 3-8 μm.

[0055] The sample loading well can be of various forms, including single well, double well, or multiple wells.

[0056] Furthermore, the solid phase agglutination test strip also includes a washing solution.

[0057] The washing solution washes away non-agglomerated reactants and provides necessary ions to agglomerated reactants, reducing the interaction of charges between ions. It also contains active substances such as detergents to reduce non-specific adhesion on the reaction membrane.

[0058] On the other hand, the method of using the solid phase agglutination test strip as described above includes the following steps:

[0059] (1) The captured protein package is embedded in the reaction membrane;

[0060] (2) Assemble the reaction membrane, permeate membrane, permeate membrane, absorbent pad and shell into a solid phase agglutination test paper card;

[0061] (3) Dilute or not dilute the sample to be tested;

[0062] (4) Add the sample to be tested and the test reagent to the sample well of the solid phase agglutination test strip in sequence to react. After the reaction, add washing solution; observe whether the sample well shows color.

[0063] Further, the sample to be tested in step (3) is any one or more of whole blood, serum, and plasma, with a dilution factor of 0-64 times.

[0064] Furthermore, in step (4), the amount of sample to be tested and the amount of test reagent added are the same, which is 5-20 μL; the reaction time is 10-180 seconds.

[0065] In some methods, three representative solid-phase agglutination test strips were prepared: a cell solid-phase test strip naturally carrying specific antigens (for detecting brucellosis), a solid-phase agglutination test strip with direct antigen labeling (for detecting schistosomiasis egg infection), and a solid-phase agglutination test strip using gene recombination to express heterologous antigens (for detecting rabies virus infection).

[0066] In some methods, using naturally occurring cell-based solid-phase test strips carrying specific antigens to detect brucellosis as an example, a brucellosis rose benzene solid-phase test strip is prepared. The detection sensitivity of the test strip is compared with that of the rose benzene plate agglutination test. The sample to be tested is rabbit serum that is positive for brucellosis. The results show that the rabbit serum detected by the brucellosis rose benzene solid-phase test strip prepared in this invention can still be effectively identified as positive at a dilution of 2048 times, while the rose benzene plate agglutination test cannot effectively observe agglutination at a dilution of 128 times. Therefore, the naturally occurring cell-based solid-phase test strips carrying specific antigens provided by this invention can effectively improve sensitivity.

[0067] In some methods, taking the detection of Schistosoma egg infection as an example, the antigen-labeled solid-phase agglutination test strip is prepared using colloidal gold solid-phase test strips and latex solid-phase test strips containing soluble Schistosoma egg antigen. The sample to be tested is rabbit serum that is positive for soluble Schistosoma egg antigen. The rabbit serum is diluted to different multiples and its effectiveness in determining a positive result is tested. The results show that the rabbit serum can still effectively determine a positive result even when diluted 640 times. Therefore, the antigen-labeled solid-phase agglutination test strip provided by this invention can effectively improve sensitivity.

[0068] In some methods, taking the detection of brucellosis as an example, a brucellosis red rubella solid-phase test strip is prepared. The samples to be tested are rabbit serum positive for Brucella, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, Echinococcosis, Lawsonia intracellularis, and rabies virus. The test results show that the Brucella-positive serum test result is positive, and the others are negative. Therefore, the cell solid-phase test strip with naturally occurring specific antigens provided by the present invention has high specificity. Taking the detection of Schistosoma egg infection as an example, a solid-phase agglutination test strip containing soluble antigens of Schistosoma eggs was prepared. Rabbit serum positive for Schistosoma japonicum eggs, Brucella, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, Echinococcosis, and Lawsonia intracellularis was tested. The results showed that, except for the serum positive for Schistosoma japonicum eggs, all other sera were negative. Therefore, the antigen-labeled solid-phase agglutination test strip provided by this invention has high specificity. Taking the detection of rabies virus infection as an example, a solid-phase agglutination test strip for rabies virus was prepared using a gene recombination expression heterologous antigen solid-phase agglutination test strip. The test strip was used to detect rabies virus, Brucella, Schistosoma japonicum eggs, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, Echinococcosis, and Lawsonia intracellularis positive rabbit serum. The results showed that, except for rabies virus positive serum, all other serums were negative. Therefore, the solid-phase agglutination test strip for heterologous antigen expression using gene recombination provided by this invention has high specificity.

[0069] In some methods, cell-based solid-phase detection test strips naturally carrying specific antigens are used to detect brucellosis; antigen-labeled solid-phase agglutination test strips are used to detect schistosome egg infection; and solid-phase agglutination test strips using recombinantly expressed heterologous antigens are used to detect rabies virus infection. Serum samples from cattle, sheep, rabbits, and mice that were positive for brucellosis, schistosome eggs, and rabies virus at three different time points (one month apart) showed consistent results. Therefore, the solid-phase detection test strips provided by this invention exhibit good repeatability.

[0070] In some methods, using naturally occurring cell-based solid-phase test strips carrying specific antigens to detect brucellosis as an example, a comparison was made between the brucellosis rosacea solid-phase test strip prepared in this invention, the rosacea plate agglutination test, the commercially available iELISA kit (IDVET), and the cELISA kit (Keqian) on 10 positive bovine clinical serum samples. The results showed that the brucellosis rosacea solid-phase test strip prepared in this invention had a significantly higher detection accuracy than the rosacea plate agglutination test.

[0071] In some methods, the antigen-labeled solid-phase agglutination test strip is used to detect Schistosoma egg infection. Colloidal gold solid-phase test strips containing soluble antigens of Schistosoma eggs are prepared and tested on clinical negative / positive serum samples from cattle and sheep. The results show that the detection accuracy can reach 100%, indicating that the antigen-labeled solid-phase agglutination test strip provided by the present invention has high detection accuracy.

[0072] Furthermore, the present invention optimizes the sample dilution factor, sample volume, reaction time, and observation time of the solid phase test strip.

[0073] In some methods, using a cell solid-phase test strip naturally carrying specific antigens to detect brucellosis as an example, a brucellosis red erythrocyte sedimentation rate (RBR) solid-phase test strip is prepared. Blood is collected from rabbits, sheep, mice, and cattle infected with brucellosis, and their serum is separated. The serum is then diluted at a ratio of 1:(10-320) and tested through the test strip. The results show that when the serum is diluted at a ratio of 1:10, the test results for the above four animals are all positive. Therefore, when the sample to be tested is serum, the preferred dilution ratio of the serum is 10 times.

[0074] In some methods, cell solid-phase test strips naturally carrying specific antigens are used to detect brucellosis. For example, a brucellosis red erythrocyte sedimentation rate (RBR) solid-phase test strip is prepared. Blood is collected from rabbits infected with brucellosis, and their serum is separated. After dilution, 5 μL, 10 μL, and 20 μL of sample are added for detection. The amount of the test reagent added is the same as the amount of serum added. The results show that the amount added in all three systems can effectively determine the results. Therefore, from the perspective of saving samples and test reagents, the preferred amount of serum and test reagent added is 5 μL.

[0075] In some methods, cell-based solid-phase test strips naturally carrying specific antigens are used to detect brucellosis. For example, a brucellosis red rutin solid-phase test strip is prepared. Blood is collected from rabbits, sheep, mice, and cattle infected with brucellosis, and their serum is separated. After dilution, the serum is added to two reaction wells. In one well, the serum reacts with the test reagent for 10 seconds, followed by the addition of washing buffer. In the other well, the serum reacts with the test reagent for 3 minutes, followed by the addition of washing buffer. The results show that the detection results are consistent between the two reaction times. Therefore, considering time saving, the preferred reaction time is 10-20 seconds, and the observation time is set within 3 minutes.

[0076] In some methods, cell solid-phase test strips naturally carrying specific antigens are used to detect brucellosis. For example, a brucellosis rosé solid-phase test strip is prepared. Blood is collected from rabbits or mice infected or immunized with brucellosis. A portion of the blood is separated into serum, diluted, and added to the reaction wells. Another portion is directly added to the reaction wells with 10 μL of whole blood. After 10 seconds, rosé reagent is added, followed by washing buffer after another 10 seconds. The results show that the detection results of whole blood and serum are consistent. Therefore, this invention can use undiluted whole blood or serum for direct detection.

[0077] In some methods, using the detection of Schistosoma egg infection as an example, colloidal gold solid-phase test strips and latex solid-phase test strips containing soluble Schistosoma egg antigens are prepared. Rabbit positive serum is diluted and added to six reaction wells. The samples in these six wells react with the test reagent for 10s, 20s, 30s, 60s, 2min, and 3min, respectively, before adding washing buffer. Results show that both the colloidal gold solid-phase test strips and the latex solid-phase test strips can distinguish between positive and negative results at the above six reaction times. From a time-saving perspective, the serum sample should react with the test reagent for 10-60s before adding washing buffer; from the perspective of preventing sample drying and standardizing the test, the preferred reaction time is 10-20s, and the observation time is set at 1-10min.

[0078] Furthermore, the present invention screens the materials and number of layers of the reaction membrane and the permeation membrane.

[0079] Furthermore, the present invention also screens the formulation of the detergent.

[0080] Furthermore, the capture protein of the present invention is streptococcal protein G, which is optimized by constructing recombinant streptococcal protein G to enhance its ability to bind IgG.

[0081] Furthermore, in preparing a solid-phase agglutination detection reagent card that utilizes gene recombination to express heterologous antigens, the present invention optimizes the vector, cells, and sequence for expressing heterologous antigens.

[0082] Furthermore, in preparing the solid-phase agglutination detection reagent card using direct antigen labeling, the present invention screens the inert carriers conjugated to the antigen.

[0083] In some methods, optimization was carried out on solid-phase agglutination test kits that utilize direct antigen labeling (taking the detection of Schistosoma japonicum egg infection as an example), and different inert carriers were screened. The preferred inert carriers were colloidal gold and latex colored microspheres.

[0084] In another aspect, the present invention provides a solid-phase agglutination test strip card with direct antigen labeling, comprising a capture protein and a detection reagent, wherein the detection reagent contains a particulate antigen, the particulate antigen being a conjugate of a specific antigen and an inert carrier, and the specific antigen specifically binding to the antibody to be tested.

[0085] Furthermore, the specific antigen is a non-protein antigen.

[0086] Furthermore, the specific antigen is obtained through physical or chemical methods.

[0087] Furthermore, the capture protein is a ligand of the antibody to be tested or a monoclonal or polyclonal antibody targeting the antibody to be tested.

[0088] Furthermore, the inert carrier itself may have color, luminescent properties, or no color. When it is colorless, it needs to be dyed with dye.

[0089] Furthermore, the antigen-labeled solid-phase agglutination test strip also includes a reaction membrane, a permeation membrane, an absorbent pad, and a shell; the reaction membrane embeds the capture protein, and the shell is provided with a sample application well; the pore size of the reaction membrane and the permeation membrane is the same, which is 3-8 μm.

[0090] Furthermore, the antigen-labeled solid-phase agglutination test strip also includes a washing solution.

[0091] Furthermore, the present invention provides a method for using the above-described antigen-labeled solid-phase agglutination test strip, comprising the following steps:

[0092] (1) The captured protein package is embedded in the reaction membrane;

[0093] (2) Assemble the reaction membrane, permeate membrane, permeate membrane, absorbent pad and shell into a solid phase agglutination test paper card;

[0094] (3) Dilute or not dilute the sample to be tested;

[0095] (4) Add the sample to be tested and the test reagent to the sample well of the solid phase agglutination test strip in sequence to react. After the reaction, add washing solution; observe whether the sample well shows color.

[0096] Further, the sample to be tested in step (3) is any one or more of whole blood, serum, and plasma, with a dilution factor of 0-64 times.

[0097] Furthermore, in step (4), the amount of sample to be tested and the amount of test reagent added are the same, which is 5-20 μL; the reaction time is 10-180 seconds.

[0098] The present invention has the following beneficial effects:

[0099] 1. This invention provides a solid-phase agglutination test strip that utilizes gene recombination to express heterologous antibodies. The reaction area of ​​the solid-phase agglutination test strip includes a reaction membrane. The reaction membrane is coated with a capture protein, which can capture the antibody to be tested in the sample. After the capture protein binds to the antibody to be tested, it reacts with particulate antigens in the test reagent to form an agglutination complex. The agglutination complex is trapped in the reaction membrane because it exceeds the pore size of the reaction membrane and does not seep down.

[0100] 2. The particulate antigen is a cell carrying a specific antigen, which is expressed and displayed on the cell surface through gene recombination;

[0101] 3. The particulate antigen itself may be colored, or may be colored by staining, or may be colored by adding a chromogenic substrate to the cell culture medium and releasing the chromogenic substance after the enzyme produced by cell metabolism decomposes the substrate for staining, or may be colored by expressing the chromogenic substance in the cell through transgenic means.

[0102] 4. The solid-phase agglutination test strip card for expressing heterologous antibodies by gene recombination provided by this invention has the advantages of simple detection steps, short detection time, high sensitivity, accurate detection results, and strong specificity. It can not only be used for the detection of various diseases and the monitoring of antibody levels, but also has important significance for infectious disease prevention and control, medical diagnosis and other fields. Attached Figure Description

[0103] Figure 1 This is a physical image of the solid phase detection test strip card and a schematic diagram of the detection principle in Example 1;

[0104] Figure 2 The positive and negative determination criteria for the Rutin solid-phase test strip for brucellosis in Example 1 were established for samples diluted 10, 100, and 1000 times.

[0105] Figure 3 This is a schematic diagram of the insertion of the nucleotide sequence of the recombinant rabies virus-G protein into the pET-28a(+) vector in Example 1;

[0106] Figure 4 Example 1 shows the expression of recombinant rabies virus-G protein in Escherichia coli at different induction times;

[0107] Figure 5 The solubility of the recombinant rabies virus-G protein in Example 1;

[0108] Figure 6 The purification of recombinant rabies virus-G protein in Example 1;

[0109] Figure 7 The results of the Western Blot experiment on the recombinant rabies virus-G protein in Example 1 are shown.

[0110] Figure 8 The positive and negative determination criteria for solid-phase rabies virus test strips diluted 10, 100, and 1000 times are provided in Example 1.

[0111] Figure 9 The positive and negative criteria for solid-phase detection of soluble antigens from Schistosoma eggs in Example 1 were established for testing samples diluted 10, 100, and 1000 times.

[0112] Figure 10 The sensitivity test results of the Brassica naproxi solid phase agglutination test strip and the Brassica naproxi plate agglutination test for Brucella in Example 2 are shown.

[0113] Figure 11 The results of the red benzene plate agglutination test, red benzene solid phase agglutination test strip, and iELISA were used to detect the titer of Brucella rabbit polyclonal antibody serum in Example 2.

[0114] Figure 12 The results show the sensitivity of the solid-phase test strip for soluble antigens of Schistosoma eggs in Example 2.

[0115] Figure 13 The results of the solid-phase test strip sensitivity detection of rabies virus in Example 2 are shown.

[0116] Figure 14 This is the specific detection result of the Brucella virus red solid phase test strip card in Example 2;

[0117] Figure 15To implement the repeatability test results of the Brucella red erythrocyte sedimentation test strip;

[0118] Figure 16 Example 2: Brucella infection test strips were used to detect negative / positive whole blood results.

[0119] Figure 17 To implement the results of the two red brucellosis solid phase agglutination test strips and red brucellosis plate agglutination test for detecting negative / positive clinical samples of bovine brucellosis;

[0120] Figure 18 Example 3 illustrates the effect of different sample loading amounts on the test results of solid phase agglutination test strips.

[0121] Figure 19 Example 3 illustrates the effect of different reaction times on the test results of solid phase agglutination test strips;

[0122] Figure 20 This is a schematic diagram of the structure of the Streptococcus G protein in Example 6. Detailed Implementation

[0123] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0124] Example 1: Preparation of solid phase detection agglutination detection reagent card

[0125] Antigens that can stimulate the body to produce antibodies include protein antigens and non-protein antigens. Protein antigens include, but are not limited to, proteins on the surface of microorganisms (including bacteria, viruses, etc.), proteins on the surface of animal cells (including erythrocytes, tumor cells, etc.), and proteins on the surface of plant cells (including pollen, etc.). Non-protein antigens include, but are not limited to, chemical substances (including natural polypeptides, sugars, glycoproteins, toxins, hormones, etc.), drugs, and artificially synthesized polymers. The solid-phase detection agglutination test kit provided by this invention can detect whether the body has produced antibodies against the aforementioned biological and non-biological antigens.

[0126] Among the antigens mentioned above, some protein antigens (such as proteins on the surface of bacteria and erythrocytes) are naturally carried specific antigens by certain cells (such as bacteria and erythrocytes). Therefore, these cells can be directly used as particulate antigens in the detection reagent. The size of these cells is smaller than the pore size of the reaction membrane and the filtration membrane. However, when the specific antigens carried on the cell surface react with the antibody to be tested, the resulting complex is larger than the pore size of the reaction membrane and the filtration membrane, and is trapped in the reaction membrane. Other antigens, although proteins, cannot be found to be naturally carried by cells (such as viruses). Therefore, it is necessary to express and display the specific antigens on the surface of prokaryotic or eukaryotic cells through gene recombination, and then use the cells carrying the specific antigens as particulate antigens in the detection reagent to react with the antibody to be tested in an agglutination reaction. In addition, for non-protein antigens (such as soluble antigens from Schistosoma eggs), it is impossible to express and display specific antigens on the surface of prokaryotic or eukaryotic cells through gene recombination. Therefore, additional physical and chemical methods are required to obtain them. The obtained antigens are then coupled with an inert carrier to form a conjugate, which serves as the particulate antigen of the detection reagent and reacts with the antibody to be tested in an agglutination reaction.

[0127] Protein antigens are exemplified by those carried on the surface of bacteria and viruses, while non-protein antigens are exemplified by parasite eggs. Specifically, Brucella bacteria, rabies virus, and Schistosoma japonicum eggs are selected. Solid-phase test strips for detecting these three organisms are prepared accordingly. The physical image and schematic diagram of the solid-phase test strips are shown below. Figure 1 As shown, its agglutination principle is small-well solid-phase agglutination, which can increase the color intensity of the reaction. The solid-phase test strip contains three layers of glass fiber membranes. The top glass fiber membrane serves as the reaction membrane, embedding streptococcal G protein, while the bottom two glass fiber membranes serve as filtration membranes. The sample is added through the sample wells. The streptococcal G protein on the reaction membrane captures and enriches the target protein, improving detection sensitivity. Then, the detection reagent (containing particulate antigen) and washing solution are added sequentially. If the target protein is present, the target protein forms an agglutination complex with the particulate antigen and develops color in the sample wells. Washing with the washing solution reduces interference from other components and non-specific agglutination, improving the accuracy of the determination.

[0128] 1. Preparation of Rose Bengal Solid-Phase Test Strips for Brucellosis (Naturally Carrying Cell Solid-Phase Test Strips with Specific Antigens)

[0129] Brucella is a particulate antigen. The size of a single Brucella bacillus is smaller than the pore size of the reaction membrane and the filtration membrane. The size of the agglutination complex formed after the specific antigens carried on the surface of Brucella bind with the antibody to be tested is larger than the pore size of the reaction membrane and the filtration membrane. It can be trapped in the reaction membrane and develop color. Therefore, Brucella can be used directly as a detection reagent.

[0130] The specific steps are as follows:

[0131] (1) Embedding streptococcal G protein on the reaction membrane: Streptococcal G protein was sprayed onto the reaction membrane (Shanghai Jieyi Biotechnology Co., Ltd., GF2-S) using a colloidal gold spraying system at a concentration of 0.1 mg / mL and a spraying volume of 10 μL / cm. 2 After spraying, the reaction film is dried at 37°C.

[0132] (2) The reaction membrane 1 layer, the permeate membrane 2 layer (Shanghai Jieyi Biotechnology Co., Ltd., GL-B02), the absorbent pad (Shanghai Jieyi Biotechnology Co., Ltd., H5072) and the card shell are assembled to prepare a solid phase test paper card. The pore size of the reaction membrane and the permeate membrane is the same, which is 3μm-8μm.

[0133] (3) Prepare the detection reagent, which is Brucella stained with Rose Bengal reagent. The preparation method is as follows: 1) Preparation of bacterial suspension: Prepare a bacterial suspension of Brucella culture, heat it at 70-80℃ to kill it, and collect the bacterial cells by centrifugation at 2000rpm for 5min; 2) Preparation of buffer solution: Take 120g of sodium hydroxide, add it to 2000mL of 0.5% carbolic acid physiological saline, dissolve it, add 540mL of concentrated lactic acid, and then add 0.5% carbolic acid physiological saline to a total volume of 6000mL. Sterilize at 121℃ for 30min; 3) Preparation of Rose Bengal staining solution: Take Rose Bengal dye (Rose Bengal dye) 4g of Bengal (tetrachlorotetraiodofluorescein sodium salt) was added to 396mL of sterile distilled water and shaken thoroughly to dissolve. It was then stored at 4℃ for later use. 4) Preparation of particulate antigen solution: Weigh the precipitated bacterial cells and add 22.5mL of 0.5% carbolic acid saline per gram of bacterial cells to make a bacterial suspension. Stir with a magnetic stirrer for 30 minutes. Then add 1mL of red cinnamon dye per 35mL of bacterial suspension and stir with a magnetic stirrer for 30 minutes. Filter through gauze, centrifuge to precipitate and discard the supernatant. Weigh the wet weight of the stained bacterial cells that have settled. Add buffer solution at a ratio of 4-6mL per gram of stained bacterial cells and stir with a magnetic stirrer for 30 minutes. 5) Preparation of detection reagent (particulate antigen standardization): Standardize the antigen with 5 serum samples containing IgG antibodies. Prepare 5 IgG serum samples with negative serum to 25 IgA units / mL, 50 IgA units / mL, 100 IgA units / mL and 200 IgA units / mL respectively. The optimal concentration of the antigen dilution is the one that shows a "-" reaction at 25 units / mL serum; a "+" reaction at 50 units / mL serum; a "++" reaction at 100 units / mL serum; and a "+++" reaction at 200 units / mL serum. Then, dilute the particulate antigen solution according to this ratio.

[0134] (4) Prepare washing solution: 0.01 mol / L phosphate buffer + 1% Tween 20 + 1% Triton 100.

[0135] The criteria for determining positive or negative results when using the aforementioned Rose Bengal solid-phase test strip for brucellosis to test samples diluted 10, 100, and 1000 times are as follows: Figure 2 As shown.

[0136] 2. Preparation of solid-phase detection test strips for rabies virus (solid-phase agglutination detection reagent strips using gene recombination to express heterologous antigens)

[0137] Rabies virus is a particulate antigen, but the complex formed when it binds to the antibody to be tested is still smaller than the pore size of the reaction membrane and the filtration membrane, so it cannot be used as a detection reagent. However, the surface antigen of rabies virus is what stimulates the body to produce corresponding antibodies. Therefore, we chose to express and display the rabies virus surface antigen on the cell surface through gene recombination, and then use the cells carrying the rabies virus surface antigen as the particulate antigen in the detection reagent.

[0138] The specific steps are as follows:

[0139] (1) Embedding streptococcal G protein on the reaction membrane: The streptococcal G protein was sprayed onto the reaction membrane using a colloidal gold spraying system at a concentration of 0.1 mg / mL and a spraying volume of 10 μL / cm. 2 After spraying, the reaction film is dried at 37°C.

[0140] (2) The reaction membrane 1 layer, the permeate membrane 2 layer, the water-absorbing pad and the card shell are assembled to prepare a solid phase test paper card. The pore size of the reaction membrane and the permeate membrane is the same, which is 3μm-8μm.

[0141] (3) Equipped with detection reagents, the rabies virus-G protein is expressed and displayed on the surface of bacteria, yeast, or animal cells through gene recombination. Taking Escherichia coli as an example, the specific method is as follows: 1) Prepare Escherichia coli that can express and display the rabies virus-G protein: Insert the nucleotide sequence of the rabies virus-G protein (nucleotide sequence as shown in SEQ ID NO.2, and the corresponding protein sequence as shown in SEQ ID NO.1) into the pET-28a(+) vector (e.g. Figure 3 As shown), the recombinant plasmid was introduced into E. coli and expression was induced (as shown). Figures 4-7As shown, E. coli successfully expressed and displayed rabies virus surface antigen; 2) Preparation of antigen diluent: Prepare solution A (add NaCl to a mixture of 0.05M Na2CO3 and 0.5% carbolic acid to make the final concentration 2%) and solution B (add NaCl to a mixture of 0.1M NaHCO3 and 0.5% carbolic acid to make the final concentration 2%). Take 1.5 parts of solution A and 8.5 parts of solution B and mix them. The pH of this solution is 5.9; 3) Preparation of red benzene (RB) staining solution: Add 1g of red benzene to sterile physiological saline to 100mL, dissolve thoroughly, filter, seal, and store at 4℃; 4) Preparation of detection reagent (particulate antigen solution): Inoculate the E. coli prepared above that can express and display rabies virus surface antigen, culture at 37℃ for 2-24h, add IPTG to the bacteria to induce recombinant antigen expression, collect cells after 2-8 hours of induction; inactivate in a water bath at 70-80℃ for 30min, 500 Centrifuge at 0 rpm for 5-30 min, discard the supernatant, wash the cell sediment with sterile physiological saline, centrifuge 3 times, and finally discard the supernatant; add 10 mL of sterile physiological saline per gram of wet cells, shake thoroughly, stir at 4℃ for 30 min on a magnetic stirrer, centrifuge, discard the supernatant, add an equal volume of sterile physiological saline to the cell sediment, mix the cell suspension with 1% red erythrorhizon (RB) staining solution at a volume ratio of 30:1, shake thoroughly, incubate at 4℃ for 24 h, shaking several times during this period, centrifuge at 5000 rpm for 30 min, discard the supernatant; add 10 mL of antigen diluent per gram of wet cells, shake thoroughly, and the test reagent is ready.

[0142] (4) Prepare washing solution: 0.01 mol / L phosphate buffer + 1% Tween 20 + 1% Triton 100.

[0143] It should be noted that the above example uses bacteria (E. coli) to prepare bacteria capable of expressing and displaying rabies virus-G protein. Besides bacteria, cells can also be yeast (such as Pichia pastoris), animal cells (such as insect Sf9 cells, mammalian HEK239 cells, CHO, etc.). Different expression vectors are required for different cell types to express and display antigens.

[0144] The criteria for determining the positive or negative result of using the solid-phase rabies virus test strip to detect samples diluted 10, 100, and 1000 times are as follows: Figure 8 As shown.

[0145] 3. Preparation of solid-phase detection test strips for soluble antigens of Schistosoma eggs (antigen-labeled solid-phase agglutination detection reagent strips)

[0146] Although schistosome eggs are particulate antigens, the size of a single schistosome egg far exceeds the pore size of the reaction membrane and filtration membrane; therefore, schistosome eggs cannot be used as a detection reagent. Furthermore, what stimulates the body to produce corresponding antibodies are the soluble antigens carried by the schistosome eggs. These soluble antigens are not entirely proteins. Therefore, the soluble antigens carried by the schistosome eggs are coupled with an inert carrier to form the particulate antigen in the detection reagent, which then reacts with the antibody to be tested to induce an agglutination reaction.

[0147] The specific steps are as follows:

[0148] (1) Embedding streptococcal G protein on the reaction membrane: The streptococcal G protein was sprayed onto the reaction membrane using a colloidal gold spraying system at a concentration of 0.1 mg / mL and a spraying volume of 10 μL / cm. 2 After spraying, the reaction film is dried at 37°C.

[0149] (2) The reaction membrane 1 layer, the permeate membrane 2 layer, the water-absorbing pad and the card shell are assembled to prepare a solid phase test paper card. The pore size of the reaction membrane and the permeate membrane is the same, which is 3-8μm.

[0150] (3) Obtaining soluble antigens of Schistosoma japonicum eggs: 1) Infecting animals: Mice were infected with Schistosoma japonicum cercariae by tail vein injection; 2) Obtaining liver eggs: Mice were anesthetized and euthanized 6-8 weeks after infection, the liver was removed, the liver was cut into pieces, and put into a tissue grinder. An appropriate amount of physiological saline was added and the mixture was ground into a homogenate. Then, the homogenate is filtered through multiple layers of gauze to remove tissue fragments, resulting in a suspension containing schistosome eggs; 3) Egg purification: The egg suspension is carefully spread on a sucrose solution (30%-50% sucrose solution). After centrifugation, the eggs will concentrate in a specific sucrose density layer. This layer of liquid is collected, and the eggs are washed several times with physiological saline to obtain relatively pure Schistosoma japonicum eggs; 4) Egg disruption: The collected pure eggs are suspended in an appropriate amount of PBS and then disrupted using an ultrasonic disruptor; 5) Centrifugation: The disrupted egg suspension is centrifuged at 12000 r / min for 15-20 min, and the supernatant is collected, which is the crude Schistosoma japonicum egg antigen; 6) Antigen purification: To obtain a higher purity antigen, a Sephadex G-200 gel filtration chromatography column is used. The crude antigen is loaded onto the column and eluted with PBS. Different antigen components are separated according to molecular weight, and the elution peak containing the target antigen is collected to obtain the purified Schistosoma japonicum egg antigen.

[0151] (4) Provide testing reagents:

[0152] 1) Coupling soluble antigen from Schistosoma japonicum eggs with colloidal gold / latex colored microspheres to prepare a particulate antigen stock solution; 2) Preparing detection reagents (particulate antigen standardization): Standardizing the antigen with 5 serum samples containing specific IgG antibodies against soluble Schistosoma japonicum eggs, and diluting the 5 IgG serum samples with negative serum at 2, 4, 8, 16, 32, and 64 times respectively. The antigen dilution that shows a "++" reaction at 64 times dilution is the optimal concentration, and then diluting the particulate antigen stock solution according to this ratio;

[0153] (5) Prepare washing solution: 0.01 mol / L phosphate buffer + 1% Tween 20 + 1% Triton 100.

[0154] The positive and negative criteria for testing samples diluted 10, 100, and 1000 times using the solid-phase test strip of the soluble antigen of Schistosoma eggs are as follows: Figure 9 As shown.

[0155] The aforementioned streptococcal G protein can be replaced by Staphylococcus aureus protein A, galectin-3 which can enrich IgE binding protein, Sm22.6 and SmTAL1 of Schistosoma mansoni, ABA-1 produced by Ascaris suis, Anis1 and Anis2 produced by Anisakis, human FcεRI, FcεRII, etc., or can be replaced by monoclonal or polyclonal antibodies produced targeting the constant region of the antibody to be tested.

[0156] When the particulate antigen in the test reagent is a cell carrying a specific antigen, it reacts with the antibody to be tested to produce a colorimetric reaction. In addition to staining the cells carrying the specific antigen (staining methods include, but are not limited to, red cinnamon, Congo red, Indian ink, phenol blue staining, Gram staining, acid-fast staining, and crystal violet-brilliant green staining), such as using red cinnamon to stain Brucella and Escherichia coli that express and display rabies virus-G protein, the cells carrying the specific antigen can also be colored themselves, such as red blood cells and Staphylococcus aureus. In addition, a chromogenic substrate can be added to the cell culture medium, allowing enzymes produced by the metabolism of cells carrying the specific antigen to decompose the substrate and release chromogenic substances to achieve the staining purpose. For example, X-gal is used to show blue in Escherichia coli, CHROMagar is used to show color in Candida albicans, and Staphylococcus aureus shows pink or green in chromogenic medium. In addition, by transgenic means, chromogenic substances can be expressed inside cells carrying specific antigens to form specific colors. For example, the green fluorescent protein gene can be transferred into E. coli to express EGFP, RFP, etc. Therefore, when the cells carrying specific antigens bind to the antibody to be tested, they can form green fluorescence, red fluorescence, etc. under excitation light.

[0157] When the particulate antigen in the test reagent is a conjugate of a specific antigen and an inert carrier, the inert carrier, in addition to the colloidal gold and latex colored microspheres used above, also includes, but is not limited to, magnetic microspheres, fluorescent microspheres, carbon powder, etc. Inert carriers that are not colored themselves can be colored by dyeing.

[0158] This embodiment only provides specific preparation methods and procedures for solid-phase agglutination detection reagent cards for three pathogenic organisms. In addition to preparing solid-phase agglutination detection reagent cards for these three pathogenic organisms, this invention can also prepare solid-phase agglutination detection reagent cards for detecting, but not limited to, Class II diseases such as anthrax and toxoplasmosis; Class III diseases such as Escherichia coli infection, fowl cholera, coccidiosis, rabbit coccidiosis, pullorum disease, and bovine tuberculosis; plague; cholera; anthrax (pulmonary anthrax requires Class A management); tuberculosis; typhoid / paratyphoid fever; pertussis; diphtheria; neonatal tetanus; scarlet fever; brucellosis; gonorrhea; syphilis; malaria; leprosy; typhus; leishmaniasis; echinococcosis; and filariasis. Furthermore, the solid-phase agglutination detection reagent cards provided by this invention can be applied not only to the detection of the aforementioned animal and human infectious diseases, but also to the detection of, but not limited to, autoimmune diseases, pregnancy testing, drug transfusion and transplantation, drug monitoring, and forensic medicine.

[0159] The specific instructions for using the solid phase test strips are as follows:

[0160] 1. Dilute the sample to be tested by a ratio of 0-64 (preferably 10-fold). The sample to be tested is one of whole blood, serum, or plasma.

[0161] 2. Add 5-20 μL (preferably 5-10 μL) of the sample to be tested into the sample well of the solid phase test paper, and immediately add the same volume of test reagent, and wait for the reaction to proceed for 10-180 seconds.

[0162] 3. Add 3 drops of washing solution, wait 1 minute and observe whether the sample well shows color; color development indicates a positive result, no color development indicates a negative result.

[0163] It should be noted that when using whole blood for testing, fresh blood can be decolorized quickly, and positive and negative samples can be determined by the naked eye. However, negative samples will show a slightly reddish background, and positive samples will show dark red patches.

[0164] Example 2: Performance Testing of Solid Phase Detection Agglutination Detection Reagent Card

[0165] 1. Sensitivity detection

[0166] 1) Sensitivity of naturally occurring cell solid-phase detection test strips carrying specific antigens

[0167] Taking the detection of Brucella infection as an example, blood was collected from rabbits infected with Brucella and positive polyclonal antibody serum was isolated. The titer was detected using iELISA, and negative serum was used as a control.

[0168] The specific method for iELISA detection is as follows: Dilute the Brucella particulate antigen prepared in Example 1 to a suitable concentration with coating buffer and add 100 μL to each well of the ELISA plate. Incubate the ELISA plate overnight at 4°C or for 2 hours at 37°C to allow the antigen to adsorb onto the surface of the wells. Discard the liquid in the wells and wash the ELISA plate three times with washing buffer, soaking for 1 minute each time. Then pat dry on absorbent paper to remove unbound antigen and impurities. Add 5% BSA for blocking and incubate at 37°C for 1 hour. Dilute the serum to be tested appropriately with diluent, generally starting with a serial dilution of 1:100, and add 100 μL to each well of the antigen-coated ELISA plate. Also include a negative control well. Incubate the ELISA plate at 37°C for 1 hour to allow the specific antibodies in the serum to fully bind to the coating antigen. Discard the liquid in the wells. Wash the ELISA plate three times with washing buffer, soaking for 1 minute each time, then pat dry on absorbent paper to remove unbound serum proteins and other impurities. Add 100 μL of enzyme-labeled secondary antibody diluted with diluent according to the manufacturer's instructions. Incubate the ELISA plate at 37°C for 1 hour to allow the enzyme-labeled secondary antibody to bind to the specific antibody bound to the antigen. Wash the ELISA plate four times again with washing buffer, soaking for 1 minute each time, then pat dry on absorbent paper to remove unbound enzyme-labeled secondary antibody. Add 100 μL of substrate solution to each well, mix gently, and incubate the ELISA plate at 37°C in the dark for 30 minutes to allow the enzyme on the enzyme-labeled secondary antibody to catalyze the colorimetric reaction. When the color development reaches an appropriate level, add 100 μL of stop solution to each well to stop the enzymatic reaction. Measure the absorbance (OD value) of each well using a microplate reader at a wavelength of 450 nm.

[0169] The iELISA test results are shown in Table 1 below.

[0170] Table 1. Results of iELISA titer assay for Brucella rabbit-positive polyclonal antibody serum

[0171]

[0172] According to the data in Table 1, the iELISA titer of rabbit Brucella positive polyclonal antibody serum can reach 1:200000.

[0173] Furthermore, a Brucella solid-phase test strip was prepared according to the method in Example 1, and the detection sensitivity of the test strip was compared with that of the Brucella plate agglutination test.

[0174] The detection method for the Rose Bengal plate agglutination test should be performed according to the instructions of the Rose Bengal plate agglutination kit (Qingdao Lijian), as follows: Before the test, let the serum and antigen stand at room temperature for 30-60 minutes; mark the serum number to be tested on the glass plate, and set up negative serum and positive serum controls; then add 0.03 mL of the corresponding serum to be tested; add 0.03 mL of Rose Bengal plate agglutination antigen next to the serum to be tested; stir the serum and antigen with a sterile toothpick to mix them thoroughly, and observe the results within 5 minutes.

[0175] Test results as follows Figure 10 and Figure 11 As shown, the brucellosis rosé solid-phase test strip prepared according to this invention can still effectively detect positive rabbit serum at a dilution of 2048 times. In contrast, the rosé plate agglutination test shows agglutination at a dilution of 64 times, but agglutination is no longer effectively observed at a dilution of 128 times. Therefore, the cell solid-phase test strip naturally carrying specific antigens provided by this invention can effectively improve detection sensitivity, which is more than 20 times higher than that of the plate agglutination test.

[0176] 2) Sensitivity of antigen-labeled solid-phase agglutination detection kit

[0177] Taking the detection of Schistosoma egg infection as an example, solid-phase test strips for soluble Schistosoma egg antigen were prepared according to the method in Example 1, including colloidal gold solid-phase test strips and latex solid-phase test strips. Positive bovine serum was collected, diluted, and tested according to the method in Example 1, with negative serum as a control. The test results are shown in Table 2 below. Figure 12 As shown.

[0178] Table 2. Detection results of bovine schistosomiasis oocyte soluble antigen negative / positive serum at different dilution ratios.

[0179]

[0180] According to Table 2 and Figure 12 As a result, positive serum could still be distinguished by the naked eye even when diluted 512 times.

[0181] Furthermore, the positive sample was further diluted, and even at a dilution of 640 times, it was still visually distinguishable as positive or negative. Therefore, the antigen-labeled solid-phase agglutination detection reagent card provided by this invention has high sensitivity.

[0182] 3) Sensitivity of solid-phase agglutination detection kits using gene recombination to express heterologous antigens.

[0183] Taking the detection of rabies virus infection as an example, a solid-phase test strip for rabies virus was prepared according to the method in Example 1. Rabies-immunized rabbit positive serum was collected, diluted, and subjected to different dilutions. The results were then analyzed according to the method in Example 1 and compared using the iELISA method. Negative serum was used as a control. The results are shown in Table 3 below, where the results of the solid-phase test strip detecting positive serum are as follows: Figure 13 As shown.

[0184] Table 3. Results of rabies virus negative / positive serum from rabbits at different dilution ratios.

[0185]

[0186] According to Table 3 and Figure 13 As a result, the iELISA method demonstrated high sensitivity in detecting positive serum, accepting a positive result even when diluted 204,800 times. Although the sensitivity of the solid-phase agglutination test strip for detecting positive serum was lower than that of the iELISA method, it was still visually distinguishable between positive and negative results at a dilution of 512 times, indicating that the solid-phase agglutination test strip utilizing recombinant gene expression of heterologous antigens provided in this invention still maintains high sensitivity. Furthermore, compared to the iELISA method, the solid-phase agglutination test strip utilizing recombinant gene expression of heterologous antigens provided in this invention does not require specialized personnel or equipment for operation, enabling grassroots testing and large-scale screening without a laboratory environment. Moreover, the testing time is fast, with a single sample testing time not exceeding ten minutes.

[0187] 2. Specific detection

[0188] 1) Specificity of naturally occurring cell-based solid-phase test strips carrying specific antigens: Taking the detection of Brucella infection as an example, a Brucella infection Rose Bengal solid-phase test strip was prepared according to the method in Example 1. Using the test strip, Brucella, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, Echinococcosis, Lawsonia intracellularis, and rabies virus-positive rabbit serum were detected according to the detection method in Example 1. The test results are as follows: Figure 14 As shown, except for Brucella-positive serum which is positive, all other serums are negative. Therefore, the cell solid-phase test strip card with natural carrying specific antigens provided by this invention has high specificity.

[0189] 2) Specificity of the antigen-labeled solid-phase agglutination test strip: Taking the detection of Schistosoma egg infection as an example, solid-phase test strips containing soluble antigens of Schistosoma eggs were prepared according to the method in Example 1, including colloidal gold solid-phase test strips and latex solid-phase test strips. Using the test strips, rabbit serum positive for Schistosoma japonicum eggs, Brucella, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, Echinococcosis, and Lawsonia intracellularis were detected according to the detection method in Example 1. The test results showed that, except for the Schistosoma japonicum egg-positive serum, all other sera were negative. Therefore, the antigen-labeled solid-phase agglutination test strip provided by this invention has high specificity.

[0190] 3) Specificity of the solid-phase agglutination test strip using recombinant gene expression of heterologous antigens: Taking the detection of rabies virus infection as an example, a solid-phase test strip for rabies virus was prepared according to the method in Example 1. Using the test strip, rabbit serum positive for rabies virus, Brucella, Schistosoma japonicum eggs, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, Echinococcosis, and Lawsonia intracellularis was tested according to the detection method in Example 1. The test results showed that, except for rabies virus-positive serum, all other serums were negative. Therefore, the solid-phase agglutination test strip using recombinant gene expression of heterologous antigens provided by this invention has high specificity.

[0191] 3. Repeatability testing

[0192] 1) Reproducibility of naturally occurring cell-based solid-phase test strips carrying specific antigens: Taking the detection of Brucella infection as an example, Brucella infection Rutin solid-phase test strips were prepared according to the method in Example 1. Brucella-positive sera from cattle, sheep, rabbits, and mice were collected and tested three times at equal intervals, with a one-month interval, according to the method in Example 1. The test results are as follows: Figure 15 As shown, the positive results of three tests conducted on bovine and rabbit sera at different times were all positive, demonstrating that the cell solid-phase test strip card carrying the specific antigen provided by this invention has good repeatability.

[0193] 2) Repeatability of the antigen-labeled solid-phase agglutination test strip: Taking the detection of schistosome egg infection as an example, a solid-phase test strip containing soluble antigens of schistosome eggs was prepared according to the method in Example 1. Serum samples positive for schistosome eggs from cattle, sheep, rabbits, and mice were collected and tested using the same method as in Experiment 1). The test results showed that three tests conducted at different times on positive sera from different animals all yielded positive results, demonstrating that the antigen-labeled solid-phase agglutination test strip provided by this invention has good repeatability.

[0194] 3) Specificity of the solid-phase agglutination test strip using recombinant gene expression of heterologous antigens: Taking the detection of rabies virus infection as an example, a solid-phase test strip for rabies virus was prepared according to the method in Example 1. Rabies virus-positive sera from rabbits and mice were collected and tested using the same method as in Experiment 1). The test results showed that three tests conducted on positive sera from different animals at different times all yielded positive results, demonstrating that the solid-phase agglutination test strip using recombinant gene expression of heterologous antigens provided by this invention has good repeatability.

[0195] 4. Detection of whole blood samples

[0196] Taking the detection of brucellosis as an example, negative / positive whole blood and serum from mice and rabbits were used for comparative testing. After blood collection, 10 μL of undiluted whole blood was added for testing, and 10 μL of undiluted serum was also added for testing. Other operating procedures were the same as in Example 1. The test results are as follows. Figure 16 As shown in (B: whole blood; S: serum; N: negative; P: positive), the positive and negative results of whole blood and serum from mice and rabbits can be clearly distinguished, indicating that the Brucella infection solid-phase test strip provided by this invention can be used for direct detection with whole blood. Whole blood testing is an important way to improve the convenience of testing, and the Brucella infection solid-phase test strip can successfully detect and differentiate infection status in the blood of Brucella-positive animals. This result not only makes whole blood testing possible, but also provides a new approach for future Brucella screening in wild and free-range animals.

[0197] 5. Comparison of the accuracy of clinical sample testing

[0198] 1) Cell solid-phase test strips naturally carrying specific antigens for testing clinical samples.

[0199] Taking the detection of brucellosis as an example, a brucellosis rose benzene solid-phase test strip was prepared according to the method in Example 1. The results of the brucellosis rose benzene solid-phase test strip prepared in this invention, along with the results from the rose benzene plate agglutination test kit (Qingdao Lijian), the iELISA initial test (detecting the OD value of clinical samples), the commercial iELISA test kit (purchased from IDVET), and the cELISA test kit (purchased from Wuhan Keqian Biotechnology Co., Ltd.), were compared on 10 positive bovine clinical serum samples and 10 negative bovine serum samples. The detection method for the brucellosis rose benzene solid-phase test strip was as described in Example 1; the methods for the rose benzene plate agglutination test and the iELISA initial test were as described in Example 2; and the detection methods for the commercial iELISA test kit and the cELISA test kit were as described in their respective instructions. The criteria for judging positive / negative results for the initial iELISA test are as follows: Calculate the mean (X) and standard deviation (SD) of OD450 from multiple negative control samples, using X + 2SD as the cutoff value, with a cutoff value of 0.17; OD450 nm > 0.17 is positive; OD450 nm ≤ 0.17 is negative. The criteria for judging positive / negative results for IDVET-iELISA are as follows: S / P% ≤ 110%, negative; 110% < S / P% < 120%, questionable; S / P% ≥ 120%, positive. The criteria for judging positive / negative results for pre-cELISA are as follows: Inhibition rate (PI) = (Negative control OD450 nm - Detection sample OD450 nm) / Negative control OD450 nm × 100%; Inhibition rate (PI) ≥ 50%, positive; Inhibition rate (PI) < 50%, negative.

[0200] The test results of 10 bovine positive clinical serum samples and 10 bovine negative serum samples are shown in Table 4 below. The solid-phase assay card and red benzene plate test results for the 10 bovine positive clinical serum samples are as follows: Figure 17 As shown.

[0201] Table 4. Results of detecting positive bovine clinical serum using different methods

[0202]

[0203]

[0204] according to Figure 17The results in Table 4 show that the five methods yielded consistent results for detecting negative samples, with an accuracy rate of 100%. The results for detecting negative samples using iELISA and cELISA were also consistent, with an accuracy rate of 100%. The Rutaecarpa solid-phase test strip prepared in this invention detected 6 positive samples, while the Rutaecarpa plate agglutination kit detected only 1 positive sample. Although the accuracy rate of the Rutaecarpa solid-phase test strip is not as high as that of iELISA initial detection and commercial iELISA and cELISA, it significantly exceeds that of the Rutaecarpa plate agglutination test.

[0205] 2) Antigen-labeled solid-phase agglutination assay kit for detecting clinical samples

[0206] Taking the detection of Schistosoma egg infection as an example, a colloidal gold solid-phase test strip for soluble Schistosoma egg antigen was prepared according to the method in Example 1. Negative / positive sera were collected, with 30 positive cases (10 from cattle and 20 from sheep) and 40 negative cases (15 from cattle and 25 from sheep). The serum was tested according to the method described in Example 1, diluted 1:10, and the sample volume was 10 μL. The test results are shown in Tables 5-6 below.

[0207] Table 5. Negative / positive serum detection results of sheep schistosomiasis eggs

[0208]

[0209] Table 6. Negative / positive serum detection results of bovine schistosomiasis eggs

[0210]

[0211] According to the results in Table 5-6, the sensitivity and specificity of the negative / positive sera used to detect soluble antigens of Schistosoma eggs in cattle and sheep were both 100%, with a concordance rate of 100%.

[0212] Furthermore, the accuracy of using latex microsphere solid-phase test strips containing soluble antigens of Schistosoma eggs to detect the above-mentioned clinical samples can also reach 100%. Therefore, the antigen-labeled solid-phase agglutination test strips provided by this invention have high detection accuracy.

[0213] 3) Detection of clinical samples using a solid-phase agglutination assay kit for recombinant expression of heterologous antigens.

[0214] Taking the detection of rabies virus antibodies as an example, a solid-phase test strip for rabies virus was prepared according to the method in Example 1. Positive and negative sera were collected from rabies virus inactivated vaccine-immunized and non-immunized individuals. A total of 40 positive cases were collected (15 from mice and 25 from rabbits); 30 negative cases were collected (15 from mice and 15 from rabbits). The serum was tested according to the method described in Example 1, diluted 1:10, and the sample volume was 10 μL. The test results are shown in Tables 7-8 below.

[0215] Table 7. Negative / positive serum test results for rabies virus in mice

[0216]

[0217] Table 8. Results of negative / positive serological tests for rabbit rabies virus

[0218]

[0219] According to the results in Tables 7-8, the sensitivity and specificity of detecting rabies virus in mice and rabbits were both 100%, with a concordance rate of 100%. Therefore, the solid-phase agglutination detection reagent card using gene recombination to express heterologous antigens provided by this invention has high detection accuracy.

[0220] Example 3: Optimization of detection conditions for solid phase detection reagent cards

[0221] 1. Optimization of the dilution ratio of the test sample

[0222] Taking the detection of Brucella infection as an example, a Brucella virus red rubella solid-phase test strip was prepared according to the method in Example 1. Blood was collected from rabbits, sheep, mice, and cattle infected with Brucella, and positive serum was separated. The serum was then serially diluted at ratios of 1:10, 1:20, 1:40, 1:80, 1:160, and 1:320 before testing. The detection method was the same as in Example 1, and the test results are shown in Table 9 below.

[0223] Table 9. Detection results of Brucella positive serum from different animals after dilution at different ratios.

[0224]

[0225] According to the data in Table 9, the results show that when the serum was diluted at a ratio of 1:10, the test results of the above four animals were all positive. Therefore, when the sample to be tested is serum, the preferred dilution ratio of serum is 10 times.

[0226] Furthermore, whole blood and plasma from the four animals were diluted according to the stated dilution ratio and then tested. The results showed that the whole blood and plasma were diluted at a ratio of 1:10, and the test results for all four animals were positive.

[0227] 2. Optimization of sample loading and reagent dosage

[0228] Taking the detection of Brucella infection as an example, a Brucella infection red rutin solid-phase test strip was prepared according to the method in Example 1. Blood was collected from rabbits infected with Brucella, and positive serum was separated. After dilution 16-fold and 64-fold, 5 μL, 10 μL, and 20 μL of reagent were added for detection, respectively. The amount of reagent added was the same as that added to the serum. The detection method was the same as in Example 1, with negative serum used as a control. The detection results are as follows: Figure 18 As shown, the sample loading amounts for all three systems can effectively determine the results. Therefore, from the perspective of saving samples and test reagents, the preferred sample loading amount for serum and test reagents is 5 μL.

[0229] 3. Optimization of reaction time and observation time

[0230] 1) Optimization of reaction time and observation time for Brucella infection red rubella solid-phase test strips

[0231] Taking the detection of Brucella infection as an example, a Brucella virus red iodine solid-phase test strip was prepared according to the method in Example 1. Blood was collected from rabbits, sheep, mice, and cattle infected with Brucella, and positive serum was separated. After being diluted 16 times, the serum in one well was added to two reaction wells. The serum in one well reacted with the test reagent for 10 seconds, and then washing buffer was added. The serum in the other well reacted with the test reagent for 3 minutes, and then washing buffer was added. The remaining detection conditions were the same as in Example 1. The detection results are as follows: Figure 19 As shown, the detection results of the two reaction times are consistent, both being positive. Therefore, considering the need to save time, the reaction time is preferably 10-20 seconds, and the observation time is set within 3 minutes.

[0232] 2) Solid-phase test strips for soluble antigens of Schistosoma eggs

[0233] Taking the detection of Schistosoma egg infection as an example, colloidal gold solid-phase test strips and latex solid-phase test strips for soluble antigens of Schistosoma eggs were prepared according to the method in Example 1. Positive serum from rabbits and mice was collected, diluted 10-fold, and added to six reaction wells in 10 μL increments. The samples in the six reaction wells reacted with the detection reagent for 10 s, 20 s, 30 s, 60 s, 2 min, and 3 min, respectively, before adding washing buffer. Other detection conditions were the same as in Example 1. The results showed that both the colloidal gold solid-phase test strips and the latex solid-phase test strips could distinguish between positive and negative results at the above six reaction times. From a time-saving perspective, the sample reacts with the detection reagent for 10-60 s before adding washing buffer; from the perspective of preventing sample drying and standardizing the detection, the preferred reaction time between the sample and the detection reagent is 10-20 s, and the observation time is set at 1-10 min.

[0234] Example 4: Screening of Reaction Membranes and Permeation Membranes

[0235] 1. Screening of reaction membrane and permeation membrane materials

[0236] The types of reaction membranes and permeation membranes are shown in Table 10 below. Both reaction membranes and permeation membranes were purchased from Shanghai Jieyi Biotechnology Co., Ltd. Different types of reaction membranes and permeation membranes have different materials and textures, which will affect the non-specific binding and permeation rate during the detection process, and thus affect the detection accuracy. Therefore, it is necessary to screen out the optimal combination of reaction membranes and permeation membranes.

[0237] Table 10. Types of Reaction Membranes and Permeation Membranes

[0238]

[0239]

[0240] Taking the detection of Brucella infection as an example, Brucella virus red iodine solid-phase test strips were prepared by pairing different reaction membranes and permeation membranes listed in Table 10 above according to the method in Example 1. The permeability of different combinations of reaction membranes and permeation membranes for detecting positive and negative sera was then tested. Twenty positive rabbit sera and twenty negative rabbit sera were tested respectively, and the positive and negative accuracy rates were calculated. The test results are shown in Table 11 below.

[0241] Table 11. Positive and negative accuracy rates for different combinations of reaction membranes and permeate membranes used in detection.

[0242]

[0243] According to the data in Table 11, only combination 1 (reaction membrane 1 and permeate membrane 1) minimizes the false positive and false negative rates. Other combinations of reaction membranes and permeate membranes lead to higher false positive and false negative rates. The occurrence of false negatives and false positives in both negative and positive samples is a result of the combined effects of different reaction membranes and permeate membranes. If the reaction membrane material has a stronger non-specific adsorption capacity, the false positive rate will increase. If the pore size of the permeate membrane is too small, the sample permeation rate will be too slow, increasing the time the sample remains in the reaction membrane and thus increasing the risk of non-specific adsorption and the false positive rate. If both the reaction membrane and the permeate membrane have excessively large pore sizes, or if the permeate membrane has an excessively large pore size, the sample permeation rate will be too fast, resulting in insufficient reaction between the sample and the reaction membrane, weakening the positive signal. Therefore, when both the reaction membrane and the permeate membrane are combination 1, the sample detection effect is best, with the non-specific adsorption of the reaction membrane minimized and the permeation rate optimal.

[0244] 2. Screening of the number of reaction membrane and permeate membrane layers

[0245] Taking the detection of Brucella infection as an example, the number of layers of the reaction membrane and the permeate membrane in Example 1 were changed. The number of layers of the reaction membrane was 1-2 layers, and the number of layers of the permeate membrane was 1-3 layers. The reaction membrane and the permeate membrane with different numbers of layers were combined in pairs to prepare Brucella virus red sera solid-phase test strips according to the method in Example 1. The test strips were tested on 20 rabbit positive serum samples and 20 rabbit negative serum samples, and the positive accuracy rate and negative accuracy rate were calculated. The test results are shown in Table 12 below.

[0246] Table 12. Positive and negative accuracy rates of different combinations of reaction membrane and permeate membrane layers used for detection.

[0247]

[0248] Based on the data in Table 12, comparing the results of combinations 1-3, when the reaction membrane has only one layer, too few layers of the permeate membrane lead to an increased false negative rate because the permeate flow is too fast, and the antibodies in the sample do not have sufficient contact with the reaction membrane, weakening the positive signal. Conversely, too many layers of the permeate membrane lead to an increased false positive rate because the permeate flow is too slow, and sample residue may increase non-specific reactions, resulting in false positives. Therefore, when the reaction membrane has one layer and the permeate membrane has two layers, the false positive and false negative rates are lowest. Comparing the results of combinations 4-6, when the reaction membrane has two layers, both too many and too few layers of the permeate membrane result in severe false negatives and false positives.

[0249] Based on the above analysis, the reaction membrane is preferably a single layer, and the permeation membrane is preferably a double layer.

[0250] Example 5: Screening of Detergent Formula

[0251] The different detergent formulations are shown in Table 13 below.

[0252] Table 13. Combinations of different detergent formulations

[0253] Formulation Component Formulation 1 0.01 mol / L Phosphate buffer Formulation 2 0.01 mol / L Phosphate buffer + 1% Tween 20 + 1% Trion X100 Formulation 3 0.02 mol / L Tris-base Formulation 4 0.02 mol / L Tris-base + 0.5% PEG 20000

[0254] Taking the detection of Brucella infection as an example, the washing solution formula in Table 13 above was used to prepare Brucella virus red blood cell solid phase test strips according to the method in Example 1. The strips were tested on 20 rabbit positive serum samples and 20 rabbit negative serum samples, and the positive accuracy rate and negative accuracy rate were calculated. The test results are shown in Table 14 below.

[0255] Table 14. Positive and negative accuracy rates of different detergent formulations used for detection.

[0256]

[0257] According to the results in Table 14, Washing Solution Formula 2 showed the highest accuracy for detecting both positive and negative sera compared to the other formulas. This indicates that Washing Solution Formula 2 minimizes the probability of false negatives and false positives. If the elution power of the washing solution is too weak, non-specific substances cannot be fully eluted, remaining on the reaction membrane and interfering with the detection results, leading to false positives. If the elution power of the washing solution is too strong, on the one hand, it may alter the structure of non-specific proteins or other biomolecules in the sample, exposing more antigenic determinants or binding sites that bind to the capture proteins on the reaction membrane and particulate antigens in the detection reagent, thus producing false positives. On the other hand, excessively strong elution power may also elute the antibody already bound to the capture proteins and particulate antigens on the reaction membrane, resulting in false negatives. Therefore, the elution power of the washing solution should not be too weak or too strong, and Washing Solution Formula 2 has a moderate elution power, making it the preferred choice.

[0258] Example 6: Optimization of Streptococcal G Protein

[0259] Streptococcus protein G (SPG) can bind to the Fc terminus of human and various animal antibodies IgG. Similar to staphylococcus protein A (SPA), SPG exhibits stronger binding affinity and a broader binding spectrum to IgG compared to SPA. This embodiment will construct a recombinant streptococcus G protein to enhance its ability to bind IgG.

[0260] The structure of SPG is as follows: Figure 20As shown, starting from the N-terminus, there are three homologous structural regions A1, A2, and A3, each composed of 24 amino acids. These three homologous structures are separated by homologous regions B1 and B2, each composed of 51 amino acids. Next is a spacer region S, followed by homologous structural regions C1, C2, and C3, each composed of 55 amino acids, separated by regions D1 and D2. After region C3 is the hydrophilic region W, and finally region M. Studies have shown that the three homologous amino acid sequences C1, C2, and C3 of SPG are associated with binding to the Fc terminus of antibody IgG. Regions C1 and C2 differ by only two amino acids, while regions C1 and C3 differ by six amino acids. Furthermore, the binding affinity of region C3 to antibody IgG is seven times that of region C1. Therefore, in this embodiment, the IgG binding fragment of the SPG gene was reconstructed, retaining only the C region where protein G specifically binds to the Fc terminus of antibody IgG, and replacing the gene fragments in the C1 and C2 regions with the gene fragment in the C3 region, resulting in the C3-D1-C3-D2-C3 (rSPG) gene sequence. Simultaneously, the presence of rare E. coli codons (usage frequency <10%) in the rSPG sequence was detected. If rare codons were present, they were replaced with E. coli-preferred codons encoding the same amino acid. Finally, a TAA termination sequence was added to the 3' end of the sequence to construct the pET-28a(+)-rSGP recombinant plasmid. After induction of expression, the rSPG was purified by His column affinity chromatography, and the purified rSPG solution was dialyzed overnight in PBS before use.

[0261] Taking the detection of Brucella infection as an example, the purified rSPG was used to prepare Brucella virus red blood cell solid phase test strips according to the method in Example 1. Rabbit positive serum at different dilutions was tested and compared with Brucella virus red blood cell solid phase test strips prepared with unoptimized SPG. The test results are shown in Table 15 below.

[0262] Table 15. Results of using Streptococcal G protein before and after optimization for detecting rabbit positive serum.

[0263]

[0264]

[0265] According to the data in Table 15, the Brucella brucellosis solid-phase test strip made with the sequence-optimized rSPG could still detect positive results when the positive serum was diluted 2048 times, while the Brucella brucellosis solid-phase test strip made with the unoptimized SPG could not detect positive results when the positive serum was diluted 512 times. This indicates that optimizing the C region sequence can significantly improve the enrichment ability of SPG for IgG in the test sample. Since the target antibody is also IgG, this further improves the enrichment ability of the target antibody, thereby increasing the detection sensitivity.

[0266] Furthermore, the purified rSPG was diluted with PBS to different spray concentrations of 0, 0.05, 0.1, 0.2, and 0.5 mg / mL. Brucella virus red solid-phase test strips were prepared according to the method in Example 1, and rabbit positive serum at different dilutions was tested. The test results are shown in Table 16 below.

[0267] Table 16. Results of detecting rabbit positive serum at different concentrations of rSPG

[0268]

[0269] According to the results in Table 16, Brucella virus solid-phase test strips prepared with rSPG spray concentrations in the range of 0.05-0.5% can effectively detect positive results when positive serum is diluted 2048 times. However, considering the detection effect and cost savings, the optimal spray concentration is 0.1 mg / mL.

[0270] Example 7: Optimization of a solid-phase agglutination detection kit for expressing heterologous antigens using gene recombination.

[0271] Taking the detection of rabies virus as an example, the solid-phase test strip for rabies virus prepared in Example 1 was optimized.

[0272] 1. Optimization of cells carrying specific antigens

[0273] In Example 1, the rabies virus-G protein was expressed and displayed on the surface of E. coli using the E. coli prokaryotic expression system (vector pET-28a(+)). In this experiment, the rabies virus-G protein was expressed on the surface of other cells using other expression systems. The expression systems and vectors used are as follows: Pichia pastoris expression system, vector pPICZ; insect cell (sf9) expression system, vector pFsatBac; mammalian cell (HEK293) expression system, vector pcDNA3.1.

[0274] The rabies virus-G protein sequence from Example 1 was ligated into different vectors and introduced into corresponding expression systems for expression and display. The expression levels of the rabies virus-G protein prepared using the four different expression systems were detected by Western blot. The results showed that the rabies virus-G protein expressed and displayed using the *E. coli* prokaryotic expression system had the highest content.

[0275] Furthermore, the four types of cells prepared above were formulated into particulate antigen solutions according to the method in Example 1, and then diluted by the same factor to detect rabbit positive serum at different dilution factors. The detection results are shown in Table 17 below.

[0276] Table 17. Results of detecting rabbit positive serum at different dilutions using cells prepared with different expression systems

[0277] Cell 1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 Escherichia coli + + + + + + + + + + Pichia pastoris + + + + + + + + + - sf9 + + + + + + + + + - HEK293 + + + + + + + + - -

[0278] According to the data in Table 17, the cells carrying rabies virus-G protein prepared by the four different expression systems all have good sensitivity and specificity. However, compared with the Escherichia coli expression system, the cells prepared by the Escherichia coli expression system have higher sensitivity and specificity. Considering the production cost and culture conditions, Escherichia coli has low production cost and simple culture conditions, making it the preferred choice.

[0279] Therefore, the preferred expression system is the Escherichia coli expression system.

[0280] 2. Selection of carriers

[0281] This experiment screened vectors for E. coli expression systems. The vector used in Example 1 was pET-28a(+). This experiment compared the effects of different vectors on the expression and display of rabies virus-G protein in E. coli. The different vectors included pET-29a(+), pET-30a(+), pGEX-4T-1, and pAIDA-I. After ligating the rabies virus-G protein sequence into these four different vectors, expression and display were performed. The expression levels in the four cell types were then detected using Western blot. The results showed that the pET-28a(+) vector resulted in the highest expression and display level of rabies virus-G protein.

[0282] Furthermore, the four types of E. coli cells expressed using different vectors were prepared into particulate antigen solutions according to the method in Example 1, and then diluted by the same factor to detect rabbit positive serum at different dilution factors. The detection results are shown in Table 18 below.

[0283] Table 18. Results of detecting rabbit positive serum at different dilutions using reagents prepared with E. coli expressed by different vectors.

[0284] Vector 1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 pET-28a(+) + + + + + + + + + + pET-29a(+) + + + + + + + + + - pET-30a(+) + + + + + + + + - - pGEX-4T-1 + + + + + + + + + - pAIDA-I + + + + + + + + - -

[0285] Analysis of the data in Table 18 shows that the detection reagents prepared using E. coli expressed with different vectors all exhibit good sensitivity and specificity in detecting rabbit positive sera at different dilutions. However, compared to these, the rabies virus-G protein expressed using the pET-28a(+) vector demonstrates higher sensitivity and specificity. Therefore, the pET-28a(+) vector is the preferred vector for the E. coli expression system.

[0286] 3. Protein sequence optimization

[0287] The rabies virus-G protein nucleotide sequence used in Example 1 (as shown in SEQ ID NO. 2) is a nucleotide sequence optimized according to the codon preference of *E. coli*. This experiment compares the protein content expressed by *E. coli* before and after sequence optimization. The nucleotide sequence before optimization is shown in SEQ ID NO. 3. The two different sequences were ligated into the pET-28a(+) vector and introduced into *E. coli* for induced expression. The expression levels of four cell types were then detected by Western blot. The results showed that the pET-28a(+) vector ligated with the sequence of SEQ ID NO. 1 expressed and displayed the highest content of rabies virus-G protein.

[0288] Furthermore, E. coli cells expressing rabies virus-G protein using the two different nucleotide sequences mentioned above were prepared into particulate antigen solutions, which were then diluted by the same factor to detect rabbit positive sera at different dilutions. The detection results are shown in Table 19 below.

[0289] Table 19. Results of detecting rabbit positive serum at different dilutions using E. coli preparation reagents with different nucleotide sequences.

[0290] Nucleotide sequence 1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 SEQ ID NO. 2 + + + + + + + + + + SEQ ID NO. 3 + + + + + + + + - -

[0291] According to the data in Table 19, the optimized nucleotide sequence can improve the sensitivity and specificity of Escherichia coli expressing rabies virus-G protein for detection.

[0292] Therefore, optimizing codons can further improve the expression level of heterologous antigens and the detection sensitivity and specificity.

[0293] Example 8: Optimization of a solid-phase agglutination detection reagent card using direct antigen labeling

[0294] 1. Screening of specific non-protein antigens and inert vectors

[0295] Taking the detection of Schistosoma japonicum egg infection as an example, the solid-phase test strip for the soluble antigen of Schistosoma japonicum eggs prepared in Example 1 uses colloidal gold or latex colored microspheres as the inert carrier. The inert carrier is replaced with red blood cells, magnetic microspheres, and carbon powder. Rabbit positive serum is then tested. The serum is diluted 10 times and the sample volume is 10 μL. The other detection conditions are the same as in Example 1.

[0296] The specific preparation method of erythrocyte-labeled soluble antigen of Schistosoma eggs is as follows:

[0297] (1) Collection and processing of red blood cells: ① Collect blood from the jugular vein of healthy adult sheep and place it in an Erlenmeyer flask containing glass beads; ② Shake for 15-20 minutes; ③ Add an equal volume of Alderman's solution; ④ Place in a 4℃ refrigerator for 3 days to stabilize.

[0298] (2) Washing of red blood cells: ① Filter the stabilized defibrinated blood through a triangular funnel with a small amount of defatted cotton sandwiched between two layers of gauze to remove blood clots; ② Take an appropriate amount of red blood cell filtrate into a centrifuge tube and add 10 times the volume of 0.01M pH7.2 phosphate buffer; ③ Centrifuge at 2000 rpm for 10 minutes; ④ Aspirate the supernatant with a pipette with a rubber tube; ⑤ Resuspend the precipitate in an appropriate amount of PBS and centrifuge again; ⑥ Repeat steps ②-⑤ 5 times to suspend the red blood cells into a 5% suspension and store at 4℃ for later use.

[0299] (3) Red blood cell sensitization: Add 1% red blood cells to 200 mL of PBS solution (pH 6.4); add 0.6 mL of SEA antigen (1 mg / mL); shake in a water bath at 37°C for 45 min; centrifuge at 3500 rpm for 5 min and discard the supernatant; resuspend in 50 mL of PBS (pH 7.2); add 0.5 mL of rabbit negative serum for blocking (final serum concentration 1%); centrifuge at 3500 rpm for 1-5 min and discard the supernatant; add 50 mL of PBS (pH 7.2) and 0.5 mL of rabbit negative serum for blocking (final serum concentration 1%); test the effectiveness of the prepared reagent (red blood cell concentration is 4%); store at 4°C.

[0300] The test results showed that colloidal gold, latex colored microspheres, magnetic microspheres, and carbon-labeled soluble antigens of Schistosoma eggs could all differentiate between positive and negative results. However, the solid-phase test strips labeled with sensitive red blood cells could not differentiate between positive and negative results because the lyophilized sensitive red blood cell-labeled antigens could not be washed away with the washing solution, and negative serum showed the same color as positive serum. The reason for this is likely that the lyophilized red blood cell-labeled antigens exhibit severe non-specific adsorption to the reaction membrane.

[0301] Furthermore, colloidal gold, latex colored microspheres, magnetic microspheres, and carbon-labeled soluble antigens of Schistosoma eggs were used to detect rabbit negative serum. Twenty negative samples were tested for each method, and the false positive rate was calculated. The test results are shown in Table 20 below.

[0302] Table 20. Results of detection of soluble antigens from Schistosoma eggs labeled with different inert carriers in rabbit negative serum.

[0303]

[0304] According to the data in Table 20, the false positive rate of soluble Schistosoma egg antigen labeled with colloidal gold and latex colored microspheres for detecting rabbit negative serum was 0%, while false positives occurred with magnetic microspheres and charcoal, and the false positive rate of charcoal exceeded 50%, indicating serious non-specific adsorption. Therefore, colloidal gold and latex colored microspheres are preferred inert carriers.

[0305] 2. Screening of other non-protein antigens and inert carriers

[0306] In Experiment 1 above, the non-protein antigen was a soluble antigen of Schistosoma japonicum eggs, which included various substances such as proteins, polysaccharides, and glycoproteins. This experiment will prepare other types of solid-phase agglutination detection reagent cards using direct antigen labeling to detect antibodies produced by the body stimulated by carbohydrates, toxins, and drugs as non-protein antigens. The carbohydrates are capsular polysaccharides of Streptococcus pneumoniae and Haemophilus influenzae; the toxins are Clostridium difficile toxins A and B; and the drug is amoxicillin. The preparation method is the same as in Example 1 for preparing solid-phase agglutination detection reagent cards using direct antigen labeling. Inert antibodies can be selected from colloidal gold, latex colored microspheres, magnetic microspheres, and charcoal powder.

[0307] The positive sera for Streptococcus pneumoniae, Haemophilus influenzae, Clostridium difficile, and amoxicillin were tested using the corresponding solid-phase detection reagent cards according to the method in Example 1, and all results were positive.

[0308] Furthermore, solid-phase agglutination test kits labeled with different inert antibodies for detecting different antigens were used to test the corresponding negative sera. Twenty negative samples were tested for each, and the false positive rate was calculated. The test results are shown in Table 21 below.

[0309] Table 21. Solid-phase agglutination test kits for different antigens labeled with different inert antibodies used to detect false positives in negative samples.

[0310]

[0311] According to the data in Table 21, no false positives occurred when colloidal gold and latex colored microspheres were used to label polysaccharides, toxins, and small molecule drugs for detection. Therefore, colloidal gold and latex colored microspheres are preferred inert carriers.

[0312] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

[0313] sequence list

[0314] SEQ ID NO.1

[0315] Rabies virus amino acid sequence

[0316] LGPWSPIDIHHLSCPNNLVVEDEGCTNLSEFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVTDLDPYDKSLHSRVFPGGNCSGITVSSTYCSTNHDYTIWMPENLRLGTSCDIFTNSRGKRASKGGKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSDETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGSDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEVEDFVEVHLPDVHEQVSGVE

[0317] SEQ ID NO.2

[0318] Optimized nucleotide sequence of rabies virus

[0319]

[0320] SEQ ID NO.3

[0321] Unoptimized nucleotide sequence of rabies virus

[0322]

Claims

1. A solid-phase agglutination test strip card for expressing heterologous antigens using gene recombination, characterized in that, The device includes a capture protein and a detection reagent, wherein the detection reagent contains a particulate antigen, the particulate antigen being a cell carrying a specific antigen, and the specific antigen specifically binding to the antibody to be tested; the cell carrying the specific antigen is a cell that expresses and displays the specific antigen through gene recombination.

2. The solid-phase agglutination test strip card for expressing heterologous antigens using gene recombination as described in claim 1, characterized in that, The cells carrying the specific antigens are prokaryotic or eukaryotic cells.

3. The solid-phase agglutination test strip card for expressing heterologous antigens using gene recombination as described in claim 2, characterized in that, The cells carrying the specific antigens include any one or more of bacteria, yeast, and animal cells.

4. The solid-phase agglutination test strip card for expressing heterologous antigens using gene recombination as described in claim 3, characterized in that, The capture protein is a ligand of the antibody to be tested or a monoclonal or polyclonal antibody targeting the antibody to be tested.

5. The solid-phase agglutination test strip card for expressing heterologous antigens using gene recombination as described in claim 4, characterized in that, The cells carrying the specific antigen may or may not be colorless. When they are not colorless, they need to be stained with dyes, or the chromogenic substance may be expressed inside the cells through transgenic methods, or a chromogenic substrate may be added to the detection reagent.

6. The solid-phase agglutination test strip card for expressing heterologous antigens using gene recombination as described in claim 1, characterized in that, It also includes a reaction membrane, a permeation membrane, an absorbent pad, and a shell; the reaction membrane encapsulates the captured protein, and the shell is provided with a sample application well; the pore size of the reaction membrane and the permeation membrane is the same, which is 3-8 μm.

7. The solid-phase agglutination test strip card for expressing heterologous antigens using gene recombination as described in claim 1, characterized in that, It also includes detergent.

8. The method of using the solid-phase agglutination test strip card for expressing heterologous antigens by gene recombination as described in any one of claims 1-7, characterized in that, Includes the following steps: (1) The captured protein package is embedded in the reaction membrane; (2) Assemble the reaction membrane, permeate membrane, permeate membrane, absorbent pad and shell into a solid phase agglutination test paper card; (3) Dilute the sample to be tested; (4) Add the sample to be tested and the test reagent to the sample well of the solid phase agglutination test strip in sequence to react. After the reaction, add washing solution; observe whether the sample well shows color.

9. The method of use as described in claim 8, characterized in that, The sample to be tested in step (3) is any one or more of whole blood, serum, and plasma, with a dilution factor of 10-64 times.

10. The method of use as described in claim 9, characterized in that, In step (4), the amount of sample to be tested and the amount of test reagent added are the same, which is 5-20 μL; the reaction time is 10-180 seconds.

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