Solid-phase agglutination detection test paper card directly marked by antigen and detection method of solid-phase agglutination detection test paper card

Abstract

The invention provides a solid-phase agglutination detection test paper card directly marked by antigens 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 conjugate formed by coupling a specific antigen and an inert carrier, the specific antigen is not a protein, and the specific antigen is specifically combined with an antibody to be detected. 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 test paper card directly labeled with antigens and a detection method thereof. BACKGROUND

[0003] Immune agglutination detection is a classic immunology technology based on the specific binding reaction of antigen-antibody, and the detection purpose is achieved through the visible agglutination phenomenon. The principle is that immune agglutination requires antigen-antibody binding and cross-linking, 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, neutralizing the surface charge of the particles, reducing the repulsive force, and promoting agglutination. In order to be suitable for observation, sometimes the carrier amplification effect needs to be used. Agglutination reaction includes direct agglutination and passive agglutination. If the surface of the cell (such as bacteria, red blood cells) naturally contains the antigen corresponding to the antibody to be detected, the cell is a natural particulate antigen and can be directly used for direct agglutination, which directly combines with the antibody. If the surface of the cell does not contain the antigen corresponding to the antibody to be detected, and the antigen (non-protein antigen) cannot be expressed on the surface of the cell by gene recombination, the antigen needs to be adsorbed to the surface of an inert carrier (such as latex microspheres, red blood cells, carbon powder), and the coupling of the antigen and the inert carrier is used as a particulate antigen. The particulate antigen and the antibody undergo passive agglutination.

[0004] In the existing immune agglutination detection (including direct agglutination detection and indirect agglutination detection), the pretreated sample to be detected is usually mixed directly with the particulate antigen on a reaction plate or a glass slide. If the sample to be detected contains the target antibody, the target antibody and the particulate antigen will undergo an agglutination reaction, otherwise no agglutination reaction will occur. However, a large amount of non-specific substances in the pretreated sample to be detected will combine with the particulate antigen, resulting in low detection specificity and low sensitivity.

[0005] The immune agglutination detection can be applied in multiple scenes, such as blood type identification, infectious disease detection, autoimmune disease detection, pregnancy detection and the like in clinical diagnosis. In addition, the immune agglutination detection has important roles in drug transfusion and transplantation, drug monitoring, forensic medicine and animal medicine, and the like, especially in rapid screening of animal epidemic diseases (such as brucellosis agglutination test). The immune agglutination detection has the advantages of rapidity (several minutes to several hours), simple operation, no need of expensive equipment, and suitability for primary medical treatment. However, the immune agglutination detection has the limitations of lower sensitivity (microgram level), and is easily interfered by the prozone effect (antibody excess inhibits agglutination) or cross reaction. In summary, the immune agglutination detection is widely applied in the fields of infectious disease diagnosis, blood type matching and pregnancy detection, and the like, by forming visible agglutination through antigen-antibody cross-linking. Although the sensitivity is limited, the rapidity and economy make the immune agglutination detection still have important values in point-of-care testing (POCT).

[0006] The flat plate agglutination test is often used for antibody screening or detection of pathogenic factors, including detection of infectious diseases (animal or human), detection of autoimmune diseases, detection of pregnancy, detection of drug transfusion and transplantation, detection of drug monitoring, detection of forensic medicine and animal medicine, and the like. However, the flat plate agglutination test has the following problems: (1) blood needs to be collected to separate serum; (2) more antigens and antibodies are needed for testing (30 μL for each); (3) human judgment is needed, and therefore the subjectivity is high; (4) the detection effect is poor for weak positive, that is, the sensitivity is not high; and (5) the reaction needs a certain time length and the like.

[0007] Therefore, it is urgent to find a new detection method to replace the above-mentioned immune agglutination test and solve the above-mentioned problems. SUMMARY

[0008] In order to solve the problems in the prior art, the present application provides an antigen directly labeled solid phase agglutination detection test paper card and a detection method thereof. The reaction area of the solid phase agglutination detection test paper card comprises a reaction film, and the reaction film is coated with a capture protein. The capture protein can enrich the antibody to be detected in the sample to be detected. After the capture protein is combined with the antibody to be detected, an agglutination reaction occurs with the particulate antigen in the detection reagent to form an agglutination complex. The agglutination complex is trapped in the reaction film to develop color. The particulate antigen is a conjugate of a specific antigen and an inert carrier. The specific antigen is not a protein. The specific antigen specifically binds to the antibody to be detected. The solid phase agglutination detection test paper card provided by the present application has the advantages of simple detection steps, short detection time, high sensitivity, accurate detection results, strong specificity and the like. The solid phase agglutination detection test paper card can be used not only for detection of various diseases and monitoring of antibody levels, but also has important significance in the fields of infectious disease prevention and control, medical diagnosis and the like.

[0009] The present application provides an antigen directly labeled solid phase agglutination detection test paper card, which comprises a capture protein and a detection reagent. The detection reagent comprises a particulate antigen. The particulate antigen is a conjugate of a specific antigen and an inert carrier. The specific antigen specifically binds to the antibody to be detected.

[0010] In some embodiments, the capture protein is streptococcal protein G.

[0011] Further, the specific antigen is a non-protein antigen.

[0012] Further, the specific antigen is obtained by physical or chemical methods.

[0013] Further, the capture protein is a ligand of the antibody to be detected or a monoclonal antibody targeting the antibody to be detected.

[0014] Further, the inert carrier itself has a color or has a luminescent property or itself does not have a color, and when it itself does not have a color, it needs to be dyed by a dye.

[0015] In some embodiments, the antigen directly labeled solid phase agglutination test reagent card (for detecting Schistosoma japonicum egg infection) is optimized, and different inert carriers are screened, and the inert carrier is preferably colloidal gold and latex color microspheres. Further, the antigen directly labeled solid phase agglutination test reagent card further comprises a reaction membrane, a filtration membrane, a water absorption pad, and a shell; the reaction membrane is embedded with a capture protein, and the shell is provided with a sample addition hole; the pore size of the reaction membrane and the filtration membrane is the same, and is 3-8 μm.

[0016] Further, the antigen directly labeled solid phase agglutination test reagent card further comprises a washing solution.

[0017] In another aspect, the present application provides a use method of the above-mentioned antigen directly labeled solid phase agglutination test reagent card, comprising the following steps:

[0018] (1) embedding the capture protein into the reaction membrane;

[0019] (2) assembling the reaction membrane, the filtration membrane, the filtration membrane, the water absorption pad, and the shell into a solid phase agglutination test reagent card;

[0020] (3) diluting or not diluting the sample to be detected;

[0021] (4) sequentially adding the sample to be detected and the detection reagent into the sample addition hole of the solid phase agglutination test reagent card for reaction, adding a washing solution after the reaction; and observing whether the sample addition hole develops color.

[0022] Further, the sample to be detected in step (3) is any one or more of whole blood, serum, and plasma, and the dilution multiple is 0-64.

[0023] Further, the sample to be detected and the detection reagent in step (4) are added in the same amount, which is 5-20 μL; and the reaction time is 10-180 seconds.

[0024] In some modes, the antigen directly labeled solid phase agglutination test reagent card is taken as an example for detecting schistosome egg infection, and the colloidal gold solid phase test paper card and the latex solid phase test paper card of schistosome egg soluble antigen are prepared. The rabbit serum to be detected is positive for schistosome egg soluble antigen, and the rabbit serum is diluted to different multiples and detected whether it can be effectively judged as positive. The results show that when the rabbit serum is diluted by 640 times, it can still be effectively judged as positive. Therefore, the antigen directly labeled solid phase agglutination test reagent card can effectively improve the sensitivity.

[0025] In some modes, the antigen directly labeled solid phase agglutination test reagent card is taken as an example for detecting schistosome egg infection, and the colloidal gold solid phase test paper card of schistosome egg soluble antigen is prepared. The clinical negative / positive serum samples of cattle and sheep are detected, and the results show that the detection accuracy can reach 100%, indicating that the antigen directly labeled solid phase agglutination test reagent card has high detection accuracy.

[0026] In some modes, the antigen directly labeled solid phase agglutination test reagent card is taken as an example for detecting schistosome egg infection, and the colloidal gold solid phase test paper card and the latex solid phase test paper card of schistosome egg soluble antigen are prepared. The rabbit positive serum is diluted and added to 6 reaction holes respectively. The samples in the 6 reaction holes are reacted with the test reagent for 10s, 20s, 30s, 60s, 2min and 3min respectively, and then the washing liquid is added. The results show that the colloidal gold solid phase test paper card and the latex solid phase test paper card can distinguish negative and positive under the above 6 reaction times. From the perspective of saving time, the washing liquid is added after the serum sample is reacted with the test reagent for 10-60s; from the perspective of preventing sample drying and standardizing detection, the reaction time of the sample and the test reagent is preferably 10-20s, and the observation time is set to 1-10min.

[0027] On the other hand, the application provides a solid phase agglutination test paper card, which comprises a capture protein and a test reagent, wherein the test reagent comprises a particulate antigen; the particulate antigen is a cell carrying a specific antigen, or a conjugate of the specific antigen coupled with an inert carrier, and the specific antigen specifically binds to the antibody to be detected; the capture protein captures the antibody to be detected and reacts with the particulate antigen in the test reagent to form an agglutination complex, and develops color in the reaction area of the test paper card.

[0028] The solid-phase agglutination test paper card provided by the application changes the type of agglutination reaction to small-hole solid-phase agglutination according to the principle of agglutination reaction. Since the reaction is carried out in fixed small holes, the reaction is concentrated and rapid, the reaction time is short, and the color development intensity is high. By washing with a washing liquid, the observation of real agglutination is reduced, the interference of other colored substances in the sample to be detected is excluded, non-specific interference is reduced, whole blood, serum and plasma can be detected by the application, the determination result is clear, and the accuracy of interpretation is high. Meanwhile, the method of antibody enrichment is used to increase the sensitivity. In addition, a detection and interpretation equipment can be matched to improve the objectivity of detection.

[0029] The solid phase agglutination test paper card provided by the application firstly solves the problem of limited specificity and sensitivity of the flat plate agglutination test, and increases the sensitivity of the detection by embedding the ligand of the antibody to be detected or the monoclonal antibody or polyclonal antibody with the antibody to be detected as a target on the reaction film to capture the antibody to be detected to realize the enrichment of the antibody to be detected. The influence of interfering substances on the result is reduced by adding the washing liquid to remove the unreacted substances, and the specificity of the detection is increased. Secondly, the subjective problem of the result determination of the flat plate agglutination test is solved. The detection reagent matched with the solid phase agglutination test paper card is combined with the substance to be detected to agglutinate, and the color is developed in the reaction hole. The result is not only fixed in position but also concentrated, and is easy to judge. Moreover, the detection and judgment equipment can be matched to improve the objectivity of the detection and reduce the interference of human factors. Compared with the result determination of the flat plate agglutination test which depends on the naked eye observation of the agglutination phenomenon, the result determination is more objective and accurate, and the judgment difference between different operators is reduced. In addition, the flat plate agglutination test needs a large amount of antigen and antibody materials, and the detection time is long. The solid phase agglutination test paper card provided by the application only needs 5 muL of the sample to be detected, and the sample to be detected can be diluted, so the actual sample amount is smaller, and the detection time is shorter, which can be completed in 0.5-5 minutes. In addition, whole blood, serum and plasma can be used for detection. Even if the sample to be detected has color or other interference, the interference can be reduced after washing with the washing liquid, and the judgment accuracy is improved. Compared with the colloidal gold immunochromatography, the solid phase agglutination test paper card provided by the application has the advantages of simple operation and rapid detection, but the principle or filtration of the solid phase agglutination test paper card and the colloidal gold immunochromatography is different. The colloidal gold immunochromatography or the filtration detection of the antibody adopts an indirect method or a double antigen sandwich method, needs to purify the antigen to be coated as a detection line, and in the indirect method, the antibody is indirectly labeled or the antigen is labeled. The antibody is captured by the reaction film first, and the antigen particles with pre-stained color are directly used to form agglutination with the antibody. The capture protein in the solid phase agglutination test paper card can first enrich the antibody to be detected in the sample to be detected, and the washing liquid can exclude non-specific interference. The colloidal gold immunochromatography or the immunofiltration method cannot achieve these two effects, so the application can improve the detection sensitivity, especially for the antibody to be detected with low content in the sample to be detected, and also has good detection effect. In addition, the application has essential difference from the common blood type detection card. The blood type detection card embeds the monoclonal antibody or polyclonal antibody for blood type on the filtration membrane and fixes it. When encountering the antigen on the red blood cells in the sample to be detected, agglutination is formed, and after the washing liquid is added, the agglutination is retained on the filtration membrane to achieve the detection result. Therefore, the reaction essence of the blood type detection card is a direct antigen detection. The solid phase agglutination test paper card is to enrich the antibody to be detected and then add the known detection antigen, which belongs to antibody detection.

[0030] Further, the capture protein is the ligand of the antibody to be detected or the monoclonal antibody or polyclonal antibody with the antibody to be detected as a target.

[0031] The capture protein is used to enrich the antibody to be detected, and when the capture protein is a ligand of the antibody to be detected, the ligand includes but is not limited to streptococcal protein G, protein A of Staphylococcus aureus, a binding protein galectin-3 that can enrich IgE, Sm22.6 and SmTAL1 of Schistosoma mansoni, ABA-1 produced by Ascaris suum, Anis1 and Anis2 produced by Heterakis sp., human FcεRI, FcεRII, etc.

[0032] When the capture protein is a ligand of the antibody to be detected, the antibody in the sample to be detected that is enriched by the capture protein includes the antibody to be detected and an antibody belonging to the same class as the antibody to be detected. Taking streptococcal protein G as an example, it can bind to the Fc segment of an IgG antibody without affecting the function of the Fab segment of the IgG antibody. If a certain bacterial antibody in serum is to be detected, the antibody is IgG, and streptococcal protein G can capture all IgG antibodies in the serum, and the certain bacterial antibody to be detected is also included. The bacterial antibody will have an agglutination reaction with the corresponding particulate antigen in the detection reagent and be trapped in the reaction well to develop color, and the remaining non-specific antibodies are washed away along with the filter membrane after being washed with a washing solution.

[0033] In some modes, the sequence of streptococcal protein G is optimized to make it stronger in binding to IgG.

[0034] When the capture protein is a monoclonal antibody or a polyclonal antibody that takes the antibody to be detected as a target, it can enrich the target antibody in the sample to be detected. The form of the target antibody is not only IgG, but also IgM, IgE or IgA, IgY antibody.

[0035] The antibody to be detected is bivalent or multivalent, and the particulate antigen contains multiple epitopes, so that the antibody to be detected and the particulate antigen can form an agglutination complex.

[0036] The solid-phase agglutination test paper card of the application is mainly used for detection of animal infectious diseases, human infectious diseases, autoimmune diseases, pregnancy detection, drug transfusion and transplantation, drug monitoring, etc., so the antibody to be detected is mainly in blood, and the bivalent antibody in blood includes IgG and IgE, and the multivalent antibody includes IgM.

[0037] The particulate antigen such as bacteria, red blood cells, etc. is a macromolecule with a complex structure. Taking bacteria as an example, different antigenic determinants exist on the cell wall, capsule, flagellum, etc. of the bacteria, and because a large number of different types of antigenic determinants exist on the surface of the bacteria, the bacteria are multivalent.

[0038] Further, the cell carrying the specific antigen is a cell naturally carrying the specific antigen, or a cell expressing and displaying the specific antigen by genetic recombination; and the cell carrying the specific antigen is a prokaryotic cell or a eukaryotic cell.

[0039] Further, the cells carrying specific antigens include any one or more of bacteria, yeast, and animal cells.

[0040] Part of the cells carrying specific antigens are cells naturally carrying specific antigens, mainly pathogenic bacteria such as Brucella, Staphylococcus aureus, and the like; and also include common other cells such as red blood cells, which contain sialic acid receptors on the surface and can bind to the hemagglutinin protein of viruses, and thus can be used for antibody detection of various viruses (influenza, Newcastle disease, etc.). Another part of the cells carrying specific antigens need to express and display specific antigens on the cell surface by genetic recombination, which can be prepared by using the following expression systems: prokaryotic expression system bacteria (such as E. coli), yeast expression system (such as Pichia pastoris), insect cell-baculovirus system (such as Sf9 cells), and mammalian cell expression system (such as HEK293, CHO cells). The above expression systems also need to construct vectors, the prokaryotic expression system vectors include but are not limited to pET series, pGEX series vectors, pET-Surface vector based on OmpA anchoring system, autotransporter vector pAIDA-I supporting large protein display, pSDV (Surface Display Vector) suitable for antigen display integrating Lpp-OmpA anchoring system; 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, lentivirus / adenovirus vectors.

[0041] In the present application, the 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 red blood cells, tumor cells, etc.), and proteins on the surface of plant cells (including pollen, etc.), and 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 reagent card provided by the present application can detect whether the body has produced antibodies to the above biological antigens and non-biological antigens.

[0042] Among the antigens mentioned above, some are proteins (such as proteins on the surface of bacteria, red blood cells, etc.), and some cells (such as bacteria, red blood cells, etc.) naturally carry specific antigens, so these cells can be directly used as particulate antigens in the detection reagent. The size of the cells is smaller than the pore size of the reaction membrane and the filtration membrane, and when the specific antigens carried on the cells have agglutination reaction with the antibodies to be detected, the complex formed has a size larger than the pore size of the reaction membrane and the filtration membrane and is trapped in the reaction membrane. Although some antigens are proteins, no cells naturally carrying specific antigens can be found (such as viruses), so it is necessary to express and display the specific antigens on the surface of prokaryotic or eukaryotic cells by genetic recombination, and then use the cells carrying specific antigens as particulate antigens in the detection reagent to have agglutination reaction with the antibodies to be detected. In addition, for non-protein antigens (such as soluble antigens of schistosome eggs), it is not possible to express and display the specific antigens on the surface of prokaryotic or eukaryotic cells by genetic recombination, so it is necessary to obtain the antigens by additional physical (extraction, extraction, etc.) or chemical methods, couple the obtained antigens with inert carriers to form conjugates as particulate antigens of the detection reagent, and have agglutination reaction with the antibodies to be detected.

[0043] Further, the cells carrying specific antigens have color or do not have color by themselves, and when they do not have color by themselves, it is necessary to be dyed by dye, or to express color-developing substances in the cells by transgenic method, or to add color-developing substrates to the culture medium of the cells to release color-developing substances after the substrates are decomposed by enzymes metabolized by the cells to achieve dyeing; the inert carriers have color or luminescent performance or do not have color by themselves, and when they do not have color by themselves, it is necessary to be dyed by dye.

[0044] When the cells carrying specific antigens have color by themselves, such as red blood cells, Staphylococcus aureus, etc., they can develop color on the reaction membrane after agglutination reaction with the antibodies to be detected. When the cells carrying specific antigens do not have color by themselves, it is necessary to be dyed by dye to develop color on the reaction membrane after agglutination reaction with the antibodies to be detected. The dyeing reagents and methods include but are not limited to tiger red reagent, congo red, Indian ink, trypan blue staining, gram staining, acid-fast staining, crystal violet and malachite green staining, etc. In addition to dyeing, color-developing substrates can be added to the culture medium of the cells to release color-developing substances after the substrates are decomposed by enzymes metabolized by the cells to achieve the purpose of dyeing, for example, X-gal is used to develop blue color in E. coli, CHROMagar is used to develop color in Candida, and Staphylococcus aureus is cultured in color-developing medium to develop pink or green color. In addition, color-developing substances can be expressed in the cells carrying specific antigens by transgenic method to form specific colors, for example, green fluorescent protein gene is introduced into E. coli to express EGFP, RFP, etc., which can form green fluorescence under excitation light, and red fluorescent protein gene, etc.

[0045] The inert carriers with color include, but are not limited to, colloidal gold, latex microspheres, magnetic microspheres, carbon powder, etc., the inert carriers with luminescence performance include, but are not limited to, fluorescent microspheres, and the inert carriers without color can be dyed with dyes to have color.

[0046] Further, the solid phase agglutination test paper card further comprises a reaction membrane, a filtration membrane, a water absorption pad and a shell; the reaction membrane is embedded with capture proteins, and the shell is provided with a sample adding hole; the reaction membrane and the filtration membrane have the same pore size of 3-8 μm.

[0047] The sample adding hole has various forms, which can be a single hole, a double hole or a multi-hole.

[0048] Further, the solid phase agglutination test paper card further comprises a washing liquid.

[0049] The washing liquid washes non-agglutination reactants and provides necessary ions for agglutination reactants, reduces the interaction of ionic charges, and contains active substances such as detergents to reduce non-specific adhesion on the reaction membrane.

[0050] On the other hand, the use method of the solid phase agglutination test paper card as described above comprises the following steps:

[0051] (1) embedding capture proteins into a reaction membrane;

[0052] (2) assembling the reaction membrane, the filtration membrane, the filtration membrane, the water absorption pad and the shell into a solid phase agglutination test paper card;

[0053] (3) diluting or not diluting the sample to be tested;

[0054] (4) sequentially adding the sample to be tested and the detection reagent into the sample adding hole of the solid phase agglutination test paper card for reaction, adding a washing liquid after the reaction, and observing whether the sample adding hole shows color.

[0055] Further, the sample to be tested in step (3) is any one or more of whole blood, serum and plasma, and the dilution multiple is 0-64.

[0056] Further, the sample to be tested and the detection reagent in step (4) have the same sample adding amount of 5-20 μL; and the reaction time is 10-180 seconds.

[0057] In some modes, three representative solid phase agglutination test paper cards are prepared respectively, which are a cell solid phase test paper card naturally carrying specific antigens (for detecting brucellosis), an antigen directly labeled solid phase agglutination test reagent card (for detecting schistosome egg infection) and a solid phase agglutination test reagent card using gene recombinant expression of heterologous antigens (for detecting rabies virus infection).

[0058] In some modes, the natural specific antigen carrying cell solid phase test paper card is used to detect brucellosis. The brucellosis rose solid phase test paper card is prepared, and the detection sensitivity of the test paper card and the rose plate agglutination test is compared. The sample to be detected is rabbit serum positive for brucellosis. The results show that the rabbit serum detected by the brucellosis rose solid phase test paper card prepared by the application can still be effectively determined as positive at a dilution of 2048 times, while the rose plate agglutination test cannot effectively observe the agglutination at a dilution of 128 times. Therefore, the natural specific antigen carrying cell solid phase test paper card provided by the application can effectively improve the sensitivity.

[0059] In some modes, the solid phase agglutination test reagent card for expressing heterologous antigen by gene recombination is used to detect rabies virus infection. The solid phase test paper card for rabies virus is prepared, and the sample to be detected is positive serum of a rabbit immunized with rabies. The detection results show that the positive serum can still be distinguished by naked eye at a dilution of 512 times, indicating that the solid phase agglutination test reagent card for expressing heterologous antigen by gene recombination provided by the application has high sensitivity.

[0060] In some modes, the natural specific antigen carrying cell solid phase test paper card is used to detect brucellosis. The brucellosis rose solid phase test paper card is prepared, and the sample to be detected is rabbit serum positive for brucellosis, E. coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, hydatid, Lawsonia intracellularis and rabies virus. The detection results show that the brucellosis positive serum is positive, and the rest are negative. Therefore, the natural specific antigen carrying cell solid phase test paper card provided by the application has high specificity. The solid phase agglutination test reagent card for direct labeling of antigen is used to detect schistosome egg infection. The solid phase test paper card for soluble antigen of schistosome egg is prepared, and the rabbit serum positive for Japanese schistosome egg, brucellosis, E. coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, hydatid and Lawsonia intracellularis is detected. The detection results show that except for the Japanese schistosome egg positive serum which is positive, the rest of the serum is negative. Therefore, the solid phase agglutination test reagent card for direct labeling of antigen provided by the application has high specificity. The solid phase agglutination test reagent card for expressing heterologous antigen by gene recombination is used to detect rabies virus infection. The solid phase test paper card for rabies virus is prepared, and the rabbit serum positive for rabies virus, brucellosis, Japanese schistosome egg, E. coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, hydatid and Lawsonia intracellularis is detected. The detection results show that except for the rabies virus positive serum which is positive, the rest of the serum is negative. Therefore, the solid phase agglutination test reagent card for expressing heterologous antigen by gene recombination provided by the application has high specificity.

[0061] In some modes, the solid-phase detection test paper card carrying the specific antigen of the cell is used to detect brucellosis, the solid-phase agglutination test reagent card directly labeled with the antigen is used to detect schistosome egg infection, and the solid-phase agglutination test reagent card using the heterologous antigen expressed by gene recombination is used to detect rabies virus infection. The positive serum of the cow, sheep, rabbit and mouse of the brucellosis, schistosome egg and rabies virus is detected at three different times (one month interval), and the detection results at the three different times are consistent. Therefore, the solid-phase detection test paper card has good repeatability.

[0062] In some modes, the solid-phase detection test paper card carrying the specific antigen of the cell is used to detect brucellosis, and the detection results of the ten positive clinical serum samples of the cow by the prepared brucellosis rose solid-phase detection test paper card, the rose plate agglutination test, the iELISA commercial detection kit (IDVET) and the cELISA detection kit (Keqian) are compared, and it is shown that the detection accuracy of the prepared brucellosis rose solid-phase detection test paper card is significantly higher than that of the rose plate agglutination test.

[0063] In some modes, the solid-phase agglutination test reagent card using the heterologous antigen expressed by gene recombination is used to detect rabies virus infection, the solid-phase detection test paper card of the rabies virus is prepared, and the clinical negative / positive serum samples of the mouse and rabbit are detected, and the result shows that the detection accuracy can reach 100%, which indicates that the solid-phase agglutination test reagent card using the heterologous antigen expressed by gene recombination has high detection accuracy.

[0064] Further, the sample dilution multiple, sample addition amount, reaction time and observation time of the solid-phase detection test paper card are optimized.

[0065] In some modes, the solid-phase detection test paper card carrying the specific antigen of the cell is used to detect brucellosis, the brucellosis rose solid-phase detection test paper card is prepared, the blood of the rabbit, sheep, mouse and cow infected with the brucellosis is collected and the serum thereof is separated, the serum is diluted according to the multiple of 1: (10-320), and then the detection is performed by the test paper card. The result shows that when the serum is diluted according to the multiple of 1:10, the detection results of the four animals are all positive. Therefore, when the sample to be detected is serum, the dilution multiple of the serum is preferably 10 times.

[0066] In some modes, the cell solid-phase detection test paper card naturally carrying specific antigens is taken as an example for detecting brucellosis, a brucellosis rose solid-phase detection test paper card is prepared, blood of rabbits infected with brucellosis is collected and serum thereof is separated, after dilution, 5 μL, 10 μL and 20 μL of sample adding amount are used for detection respectively, the sample adding amount of the detection reagent is the same as that of the serum, and the results show that the sample adding amount of the three systems can effectively determine the results, and therefore, from the perspective of saving samples and detection reagents, the sample adding amount of the serum and the detection reagent is preferably 5 μL.

[0067] In some modes, the cell solid-phase detection test paper card naturally carrying specific antigens is taken as an example for detecting brucellosis, a brucellosis rose solid-phase detection test paper card is prepared, blood of rabbits, sheep, mice and cattle infected with brucellosis is collected and serum thereof is separated, after dilution, the serum is added to two reaction wells respectively, the serum in one reaction well is reacted with the detection reagent for 10 s, and then the washing liquid is added, and the serum in the other reaction well is reacted with the detection reagent for 3 min, and then the washing liquid is added. The results show that the detection results of the two reaction times are consistent, and therefore, from the perspective of saving time, the reaction time is preferably 10-20 s, and the observation time is set to be within 3 min.

[0068] In some modes, the cell solid-phase detection test paper card naturally carrying specific antigens is taken as an example for detecting brucellosis, a brucellosis rose solid-phase detection test paper card is prepared, blood of rabbits, sheep, mice and cattle infected with brucellosis is collected and serum thereof is separated, after dilution, the serum is added to two reaction wells respectively, the serum in one reaction well is reacted with the detection reagent for 10 s, and then the washing liquid is added, and the serum in the other reaction well is reacted with the detection reagent for 3 min, and then the washing liquid is added. The results show that the detection results of the two reaction times are consistent, and therefore, from the perspective of saving time, the reaction time is preferably 10-20 s, and the observation time is set to be within 3 min.

[0069] Further, the material and the number of layers of the reaction film and the dialysis film are screened.

[0070] Further, the formula of the washing liquid is also screened.

[0071] Further, the capture protein of the present application is streptococcal protein G, and it is optimized, and the optimization method is to construct a recombinant streptococcal protein G so that its ability to bind IgG is stronger.

[0072] Further, when preparing the solid-phase agglutination detection test reagent card using gene recombinant expression of heterologous antigens, the vector, cell and sequence for expressing the heterologous antigens are optimized.

[0073] In some embodiments, the solid-phase agglutination test reagent card for detecting rabies virus by using gene recombination to express heterologous antigens is optimized, the optimal expression system and the carrier used for rabies virus-G protein are screened, the expression system is E. coli expression system, the carrier is pET-28a(+), and the nucleotide sequence of rabies virus-G protein is optimized to improve the expression amount of rabies virus-G protein and the detection sensitivity and specificity.

[0074] Further, in the preparation of the solid-phase agglutination test reagent card directly labeled by antigens, the inert carrier coupled with the antigens is screened.

[0075] In another aspect, the application provides a solid-phase agglutination test paper card for detecting rabies virus by using gene recombination to express heterologous antigens, which comprises a capture protein and a test reagent, the test reagent comprises particulate antigens, the particulate antigens are cells carrying specific antigens, the specific antigens specifically bind to the antibody to be detected, and the cells carrying specific antigens are cells expressing and displaying specific antigens by gene recombination.

[0076] Further, the cells carrying specific antigens are prokaryotic cells or eukaryotic cells.

[0077] Further, the cells carrying specific antigens include any one or more of bacteria, yeasts, and animal cells.

[0078] Further, the capture protein is a ligand of the antibody to be detected or a monoclonal antibody or a polyclonal antibody targeting the antibody to be detected.

[0079] Further, the cells carrying specific antigens have color or do not have color, when they do not have color, they need to be dyed by dyes, or a color-developing substance is expressed in the cells by transgenic means, or a color-developing substrate is added to the culture medium of the cells to release the color-developing substance after the enzyme decomposes the substrate to produce color.

[0080] Further, the solid-phase agglutination test paper card for detecting rabies virus by using gene recombination to express heterologous antigens further comprises a reaction membrane, a filtration membrane, a water-absorbing pad, and a shell, the reaction membrane is embedded with the capture protein, the shell is provided with a sample addition hole, and the pore size of the reaction membrane and the filtration membrane is the same, which is 3-8 μm.

[0081] Further, the solid-phase agglutination test paper card for detecting rabies virus by using gene recombination to express heterologous antigens further comprises a washing liquid.

[0082] In another aspect, the application provides a use method of the solid-phase agglutination test paper card for detecting rabies virus by using gene recombination to express heterologous antigens, which comprises the following steps:

[0083] (1) embedding the capture protein into the reaction membrane;

[0084] (2) assembling the reaction membrane, the filtration membrane, the filtration membrane, the water absorption pad and the shell into a solid phase agglutination test paper card;

[0085] (3) diluting or not diluting the sample to be tested;

[0086] (4) adding the sample to be tested and the detection reagent into the sample addition hole of the solid phase agglutination test paper card in sequence for reaction, adding the washing liquid after reaction; observing whether the sample addition hole is colored or not.

[0087] Further, the sample to be tested in step (3) is any one or more of whole blood, serum and plasma, and the dilution multiple is 0-64.

[0088] Further, the sample to be tested and the detection reagent in step (4) are added in the same amount of 5-20 μL; and the reaction time is 10-180 seconds.

[0089] The present application has the following beneficial effects:

[0090] 1. The present application provides a solid phase agglutination test paper card directly labeled with antigens, wherein the reaction area of the solid phase agglutination test paper card comprises a reaction membrane, the reaction membrane is coated with a capture protein, the capture protein can capture the antibody to be tested in the sample to be tested, the capture protein is combined with the antibody to be tested and then combined with the particulate antigen in the detection reagent to form an agglutination complex, and the agglutination complex is intercepted in the reaction membrane because it exceeds the pore size of the reaction membrane and cannot infiltrate;

[0091] 2. The particulate antigen is a conjugate of a specific antigen and an inert carrier, and the specific antigen is a non-protein antigen, so that the solid phase agglutination test paper card directly labeled with antigens is used for detecting the antibody of the non-protein antigen;

[0092] 3. The inert carrier itself has color or has luminescence performance, or has color through dyeing;

[0093] 4. The solid phase agglutination test paper card directly labeled with antigens provided by the present application has the advantages of simple detection steps, short detection time, high sensitivity, accurate detection results and strong specificity, and can be used not only for the detection of various diseases and the monitoring of antibody levels, but also has important significance in the fields of infectious disease prevention and control and medical diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0094] Figure 1 It is the physical map and detection principle schematic diagram of the solid phase test paper card of Example 1.

[0095] Figure 2The positive and negative judgment criteria for the test paper card of the test for brucellosis of Example 1 were detected for samples diluted 10 times, 100 times and 1000 times;

[0096] Figure 3 The schematic diagram of the insertion of the nucleotide sequence of the recombinant rabies virus-G protein of Example 1 into the pET-28a(+) vector;

[0097] Figure 4 The expression of the recombinant rabies virus-G protein of Example 1 by E. coli at different induction times;

[0098] Figure 5 The solubility of the recombinant rabies virus-G protein of Example 1;

[0099] Figure 6 The purification of the recombinant rabies virus-G protein of Example 1;

[0100] Figure 7 The results of the Western Blot experiment of the recombinant rabies virus-G protein of Example 1;

[0101] Figure 8 The positive and negative judgment criteria for the test paper card of the test for rabies virus of Example 1 were detected for samples diluted 10 times, 100 times and 1000 times;

[0102] Figure 9 The positive and negative judgment criteria for the test paper card of the test for the soluble antigen of the eggs of schistosoma of Example 1 were detected for samples diluted 10 times, 100 times and 1000 times;

[0103] Figure 10 The sensitivity detection results of the test paper card of the test for brucella of Example 2 and the test for brucella on the plate;

[0104] Figure 11 The detection results of the titer of the rabbit polyclonal serum of brucella by the test for brucella on the plate, the test paper card of the test for brucella and iELISA of Example 2;

[0105] Figure 12 The sensitivity detection results of the test paper card of the test for the soluble antigen of the eggs of schistosoma of Example 2;

[0106] Figure 13 The sensitivity detection results of the test paper card of the test for rabies virus of Example 2;

[0107] Figure 14 The specificity detection results of the test paper card of the test for brucellosis of Example 2;

[0108] Figure 15Results of the repeated detection of the test paper card for the solid phase agglutination test of Brucella rubrum for Example 2;

[0109] Figure 16 Results of the detection of the test paper card for the solid phase agglutination test of Brucella rubrum for Example 2 for whole blood samples with negative / positive results;

[0110] Figure 17 Results of the detection of the test paper card for the solid phase agglutination test of Brucella rubrum and the plate agglutination test of Brucella rubrum for Example 2 for clinical samples with negative / positive results of bovine Brucella;

[0111] Figure 18 Influence of different sample amounts of the samples to be detected on the detection results of the test paper card for the solid phase agglutination test for Example 3;

[0112] Figure 19 Influence of different reaction times on the detection results of the test paper card for the solid phase agglutination test for Example 3;

[0113] Figure 20 Structural diagram of the streptococcal G protein for Example 6. DETAILED DESCRIPTION

[0114] The application will be further described in detail below in combination with the drawings and examples, and it should be noted that the following examples are intended to facilitate the understanding of the application and do not limit the application in any way.

[0115] Example 1, preparation of the test reagent card for the solid phase detection agglutination test

[0116] The 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 the proteins on the surfaces of microorganisms (including bacteria, viruses, etc.), the proteins on the surfaces of animal cells (including red blood cells, tumor cells, etc.) and the proteins on the surfaces of plant cells (including pollens, etc.), and 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 test reagent card for the solid phase detection agglutination test provided by the application can detect whether the body has produced antibodies of the above biological antigens and non-biological antigens.

[0117] Among the above antigens, some protein antigens are specific antigens naturally carried by certain cells (such as proteins on the surface of bacteria, red blood cells, etc.), so these cells can be directly used as particulate antigens in the detection reagent. The size of the cells is smaller than the pore size of the reaction membrane and the filtration membrane, and when the specific antigens carried on the surface of the cells agglutinate with the antibodies to be detected, the size of the complex formed is larger than the pore size of the reaction membrane and the filtration membrane, and is trapped in the reaction membrane. Some antigens are proteins, but no cells naturally carrying the corresponding specific antigens can be found (such as viruses), so it is necessary to express and display the specific antigens on the surface of prokaryotic or eukaryotic cells by genetic recombination, and then use the cells carrying the specific antigens as particulate antigens in the detection reagent to agglutinate with the antibodies to be detected. In addition, for non-protein antigens (such as soluble antigens of schistosome eggs), it is not possible to express and display the specific antigens on the surface of prokaryotic or eukaryotic cells by genetic recombination, so it is necessary to obtain the antigens by additional physical or chemical methods, and then couple the obtained antigens with inert carriers to form conjugates as particulate antigens of the detection reagent to agglutinate with the antibodies to be detected.

[0118] The protein antigens are taken as examples of proteins carried on the surface of bacteria and viruses, and the non-protein antigens are taken as examples of parasite eggs. The bacteria are specifically selected as Brucella, the virus is selected as rabies virus, and the parasite egg is selected as Japanese schistosome egg, and solid-phase detection test paper cards for detecting the above three kinds of organisms are prepared. The solid-phase detection test paper card is shown in the physical map and detection principle diagram as shown in Figure 1 The agglutination principle of the solid-phase detection test paper card is small-pore solid-phase agglutination, which can increase the color development intensity of the reaction. The solid-phase detection test paper card comprises three layers of glass fiber membranes, the uppermost layer of glass fiber membrane is used as a reaction membrane embedding streptococcal G protein, and the lower two layers of glass fiber membranes are used as filtration membranes. The sample is added from the sample well, the streptococcal G protein on the reaction membrane captures and enriches the target protein, improves the detection sensitivity, and then the detection reagent (containing particulate antigens) and the washing liquid are added in turn. If the target protein exists, the target protein and the particulate antigens form an agglutination complex and develop color in the sample well, and the washing liquid can reduce the interference of other components and non-specific agglutination on the detection, and improve the determination accuracy.

[0119] 1. Preparation of a tiger red solid-phase detection test paper card for brucellosis (a cell solid-phase detection test paper card naturally carrying specific antigens)

[0120] The Brucella is a particulate antigen, the size of a single Brucella is smaller than the pore size of the reaction membrane and the filtration membrane, and the size of the agglutination complex formed after the specific antigens carried on the surface of the Brucella combine with the antibodies to be detected is larger than the pore size of the reaction membrane and the filtration membrane, so it can be trapped in the reaction membrane to develop color, and therefore the Brucella can be directly used as a detection reagent.

[0121] The specific steps are as follows:

[0122] (1) Embed streptococcus G protein on the reaction membrane: use colloidal gold spraying system to spray streptococcus G protein on the reaction membrane (Shanghai Jieyi Biotechnology Co., Ltd., GF2-S), the spraying concentration is 0.1 mg / mL, and the spraying amount is 10 μL / cm 2 After spraying, the reaction membrane is placed at 37℃ for drying;

[0123] (2) Assemble the reaction membrane 1 layer, the filtration membrane 2 layer (Shanghai Jieyi Biotechnology Co., Ltd., GL-B02), the water absorption pad (Shanghai Jieyi Biotechnology Co., Ltd., H5072), and the card shell to prepare a solid-phase detection test paper card, wherein the pore sizes of the reaction membrane and the filtration membrane are the same, being 3 μm-8 μm;

[0124] (3) Prepare a detection reagent, which is Brucella dyed by Rose Bengal reagent, and the preparation method is as follows: 1) preparation of bacterial solution: make Brucella culture into bacterial suspension, heat to kill at 70-80℃, and collect bacterial bodies by centrifugation at 2000 rpm for 5 min; 2) preparation of buffer solution: take 120 g of sodium hydroxide and add to 2000 mL of 0.5% picric acid physiological saline, after dissolution, add 540 mL of concentrated lactic acid, and then add 0.5% picric acid physiological saline to a total amount of 6000 mL, sterilize at 121℃ for 30 min; 3) preparation of Rose Bengal dye solution: take 4 g of Rose Bengal dye (Rose Bengal-tetrachlorotetraiodofluorescein sodium salt), add 396 mL of sterilized distilled water, and fully oscillate to dissolve, and store at 4℃ for standby; 4) preparation of granular antigen solution: weigh the precipitated bacterial bodies, and prepare bacterial suspension by adding 0.5% picric acid physiological saline 22.5 mL per gram of bacterial bodies, stir for 30 min by a magnetic stirrer, then add 1 mL of Rose Bengal dye solution per 35 mL of bacterial suspension, stir for 30 min by a magnetic stirrer, filter through gauze, discard the supernatant by centrifugal precipitation, weigh the wet weight of the dyed bacterial bodies, and add buffer solution according to the proportion of 4-6 mL per gram of dyed bacterial bodies, and stir for 30 min by a magnetic stirrer; 5) preparation of detection reagent (standardization of granular antigen): standardize the antigen with 5 portions of IgG antibody serum, and prepare 5 portions of IgG serum into 25 international agglutinin units / mL, 50 international agglutinin units / mL, 100 international agglutinin units / mL, and 200 international agglutinin units / mL with negative serum. The antigen dilution liquid with 25 units / mL serum showing “-” reaction, 50 units / mL serum showing “+” reaction, 100 units / mL serum showing “++” reaction, and 200 units / mL serum showing “+++” reaction is the optimal concentration, and then dilute the granular antigen solution according to the proportion;

[0125] (4) Prepare a washing solution: 0.01 mol / L phosphate buffer solution + 1% Tween 20 + 1% triton 100.

[0126] The positive and negative judgment criteria of the test paper card for detecting brucellosis are shown in the following table. Figure 2

[0127] 2. Preparation of rabies virus solid phase test paper card (using solid phase agglutination test reagent card of heterologous antigen expressed by gene recombination)

[0128] Rabies virus belongs to particulate antigen, but the complex formed after its binding with the antibody to be detected is still smaller than the pore size of the reaction membrane and the filtration membrane, so it cannot be used as a test reagent. However, the surface antigen of rabies virus stimulates the body to produce corresponding antibodies, so the rabies virus surface antigen is expressed and displayed on the cell surface by gene recombination, and the cells carrying the rabies virus surface antigen are used as particulate antigens in the test reagent.

[0129] The specific steps are as follows:

[0130] (1) Embedding streptococcal G protein on the reaction membrane: using a colloidal gold spraying system to spray streptococcal G protein on the reaction membrane, the spraying concentration is 0.1 mg / mL, the spraying amount is 10 μL / cm 2 , and the reaction membrane is dried at 37°C after spraying;

[0131] (2) Assembling the reaction membrane 1 layer, the filtration membrane 2 layers, the water absorption pad, and the card shell to prepare a solid phase test paper card, the pore size of the reaction membrane and the filtration membrane is the same, which is 3 μm-8 μm;

[0132] (3) Prepare the test reagent, express and display the rabies virus-G protein on the surface of bacteria, yeast or animal cells by gene recombination, for example, using Escherichia coli, the specific method is as follows: 1) Preparation of Escherichia coli capable of expressing and displaying rabies virus-G protein: insert the nucleotide sequence of rabies virus-G protein (the nucleotide sequence is shown in SEQ ID NO. 2, and the corresponding protein sequence is shown in SEQ ID NO. 1) into pET-28a(+) vector (as shown in Figure 3 ), introduce the recombinant plasmid into Escherichia coli and induce expression (as shown in Figures 4-7 ​E. coli successfully expressed and displayed rabies virus surface antigens); 2) Preparation of antigen diluent: prepare the A solution (add NaCl to the mixture of 0.05M Na2CO3 and 0.5% phenol to a final concentration of 2%) and the B solution (add NaCl to the mixture of 0.1M NaHCO3 and 0.5% phenol to a final concentration of 2%), take 1.5 parts of the A solution and 8.5 parts of the B solution, mix, and the pH of the solution is 5.9; 3) Preparation of rose Bengal (RB) staining solution: rose Bengal 1g, add sterilized physiological saline to 100mL, dissolve thoroughly, filter, seal, and store at 4°C; 4) Preparation of detection reagent (particulate antigen solution): inoculate the E. coli prepared above which can express and display rabies virus surface antigens, cultivate at 37°C for 2-24h, add IPTG to induce the expression of recombinant antigens, collect the cells after induction for 2-8h, inactivate in a water bath at 70-80°C for 30min, centrifuge at 5000r / min for 5-30min, discard the supernatant, wash the sedimented cells with sterilized physiological saline and centrifuge 3 times, and finally discard the supernatant; add 10mL of sterilized physiological saline per gram of wet cells, shake thoroughly, and then stir at a constant temperature of 4°C for 30min on a magnetic stirrer, centrifuge, discard the supernatant, add an equal amount of sterilized physiological saline to the sedimented cells, mix the cell suspension with 1% rose Bengal (RB) staining solution at a volume ratio of 30:1, shake thoroughly, and place at 4°C for 24h, shake multiple times during the period, centrifuge at 5000r / min for 30min, and discard the supernatant; add 10mL of antigen diluent per gram of wet cells, shake thoroughly, and the solution is the detection reagent;

[0133] (4) Prepare the washing solution: 0.01mol / L phosphate buffer + 1% Tween 20 + 1% triton 100.

[0134] It should be noted that the above is an example of bacteria, and the bacteria that can express and display rabies virus-G protein (the above is E. coli), in addition to bacteria, cells can also be yeast (such as Pichia pastoris), animal cells (such as insect cells Sf9, mammalian cells HEK239, CHO, etc.). Different cells express and display antigens require different expression vectors.

[0135] The positive and negative judgment criteria for detecting samples diluted by 10 times, 100 times and 1000 times using the solid-phase detection test paper card of the rabies virus are shown in Figure 8 .

[0136] 3. Preparation of a solid-phase detection test paper card of schistosome egg soluble antigen (antigen directly labeled solid-phase agglutination detection reagent card)

[0137] Although the schistosome egg belongs to granular antigen, the size of single schistosome egg has already exceeded the pore size of reaction membrane and filtration membrane, so the schistosome egg cannot be used as the detection reagent. In addition, the soluble antigens carried by the schistosome egg stimulate the body to produce corresponding antibodies, and these soluble antigens are not all proteins, so the soluble antigens carried by the schistosome egg are coupled with inert carriers as the granular antigens in the detection reagent to occur agglutination reaction with the antibody to be detected.

[0138] The specific steps are as follows:

[0139] (1) Embedding streptococcus G protein on the reaction membrane: using the colloidal gold spraying system to spray the streptococcus G protein on the reaction membrane, the spraying concentration is 0.1 mg / mL, and the spraying amount is 10 μL / cm 2 After spraying, the reaction membrane is placed at 37℃ for drying;

[0140] (2) Assembling the solid-phase detection test paper card by using the reaction membrane 1 layer, the filtration membrane 2 layers, the water absorption pad and the card shell, wherein the pore sizes of the reaction membrane and the filtration membrane are the same, which is 3-8 μm;

[0141] (3) Obtaining the soluble antigens of the schistosome egg: 1) Infection of animals: the mice are infected with the cercaria of the schistosome japonicum by the tail vein injection method; 2) Obtaining the liver eggs: the mice are sacrificed after being anesthetized for 6-8 weeks after infection, the liver is taken out, the liver is cut into pieces, and the liver is grinded into homogenate in a tissue grinder by adding appropriate amount of physiological saline. Then the homogenate is filtered through multiple layers of gauze to remove tissue fragments to obtain a suspension containing eggs; 3) Egg purification: the egg suspension is carefully laid on a sucrose solution (30%-50% sucrose solution), after centrifugation, the eggs will concentrate in a specific sucrose density layer, the liquid in the layer is collected, and the eggs are washed several times with physiological saline to obtain relatively pure schistosome japonicum eggs; 4) Egg breaking: the collected pure eggs are suspended with appropriate amount of PBS, and then broken by using an ultrasonic breaking instrument; 5) Centrifugal separation: the broken egg suspension is centrifuged at 12000 r / min for 15-20 min, and the supernatant is collected, which is the crude schistosome japonicum egg antigen; 6) Antigen purification: in order to obtain higher purity antigen, a Sephadex G-200 gel filtration chromatography column is used, the crude antigen is loaded, and then eluted with PBS, different antigen components are separated according to the molecular weight, the elution peak containing the target antigen is collected, and the purified schistosome japonicum egg antigen is obtained.

[0142] (4) Preparing the detection reagent:

[0143] 1) The soluble antigen of Schistosoma japonicum eggs is coupled with colloidal gold / laticolour microspheres to prepare the granular antigen stock solution; 2) Preparation of detection reagent (standardization of granular antigen): 5 portions of serum containing specific IgG antibody against soluble antigen of Schistosoma japonicum eggs are used to standardize the antigen, and 5 portions of IgG serum are diluted by 2, 4, 8, 16, 32, 64 times respectively with negative serum. The antigen dilution with 64 times dilution showing “++” reaction is the optimal concentration, and then the granular antigen stock solution is diluted in the same proportion.

[0144] (5) Preparation of washing solution: 0.01 mol / L phosphate buffer + 1% Tween 20 + 1% triton 100.

[0145] The positive and negative judgment criteria of the samples diluted by 10 times, 100 times and 1000 times are detected by the solid-phase detection test paper card using the soluble antigen of Schistosoma eggs as shown in Figure 9 .

[0146] The above-mentioned streptococcal G protein can be replaced by protein A of Staphylococcus aureus, IgE-enriching binding protein galectin-3, Sm22.6 and SmTAL1 of Schistosoma mansoni, ABA-1 produced by Ascaris suum, Anis1 and Anis2 produced by Heterakis sp., human FcεRI, FcεRII, etc., or can be replaced by monoclonal or polyclonal antibodies produced against the constant region of the antibody to be detected.

[0147] When the granular antigen in the detection reagent is a cell carrying specific antigen, it has agglutination reaction with the antibody to be detected to develop color. In addition to the above-mentioned staining of Brucella using tiger red reagent and Escherichia coli expressing and displaying rabies virus-G protein, the cell carrying specific antigen itself can also have color, such as red blood cells, Staphylococcus aureus, etc. In addition, color developing substrates can be added to the culture medium of the cell to allow the enzyme produced by the metabolism of the cell carrying specific antigen to decompose the substrate and release color developing substances to achieve the purpose of staining, for example, X-gal in Escherichia coli shows blue color, CHROMagar in Candida shows color, and Staphylococcus aureus shows pink or green color through color developing medium. In addition, the color developing substances can be expressed inside the cell carrying specific antigen by transgenic method to form specific color, for example, the green fluorescent protein gene is transferred into Escherichia coli to express EGFP, RFP, etc., so that the cell carrying specific antigen can form green fluorescence, red fluorescence, etc. under excitation light irradiation after binding with the antibody to be detected.

[0148] When the particulate antigen in the detection reagent is a specific antigen conjugated with an inert carrier, the inert carrier includes, but is not limited to, magnetic microspheres, fluorescent microspheres, carbon powder, and the like, and the inert carrier itself has no color and can be colored by a dye to have a color.

[0149] The embodiment only gives specific preparation of the corresponding solid-phase agglutination detection reagent card and detection method for three pathogenic organisms. In addition to the solid-phase agglutination detection reagent card for detecting the three pathogenic organisms, the present application can also prepare solid-phase agglutination detection reagent cards for detecting diseases including, but not limited to, anthrax, toxoplasmosis, and the like, diseases of the second category, diseases of the third category such as E. coli disease, fowl cholera, coccidiosis, rabbit coccidiosis, chicken white dysentery, bovine tuberculosis Mycobacterium tuberculosis, and the like, and diseases such as plague, cholera, anthrax (pulmonary anthrax requires Class A management), tuberculosis, typhoid / paratyphoid, pertussis, diphtheria, neonatal tetanus, scarlet fever, brucellosis, gonorrhea, syphilis, malaria, leprosy, typhus, kala-azar, hydatid disease, and filariasis. In addition, the solid-phase agglutination detection reagent card provided by the present application can be applied to detection of diseases including, but not limited to, autoimmune diseases, pregnancy detection, drug transfusion and transplantation, drug monitoring, and forensic medicine.

[0150] The method for using the solid-phase detection test paper card is as follows:

[0151] 1. Dilute the sample to be tested by 0-64 times (preferably 10 times), and the sample to be tested is one of whole blood, serum, and plasma.

[0152] 2. Add 5-20 μL (preferably 5-10 μL) of the sample to be tested to the sample well of the solid-phase detection test paper card, immediately add the same volume of the detection reagent, and wait for 10-180 s for reaction.

[0153] 3. Add 3 drops of washing solution, and after waiting for 1 min, observe whether the sample well develops color; positive development of color is positive, and no development of color is negative.

[0154] It should be noted that when whole blood is used for detection, fresh blood can be quickly decolorized, and positive and negative samples can be judged by the naked eye, but the negative sample has a slightly red background, and the positive sample has a dark red patch.

[0155] Example 2, Performance detection of the solid-phase detection agglutination test reagent card

[0156] 1. Sensitivity detection

[0157] 1) Sensitivity of the solid-phase detection test paper card of cells naturally carrying specific antigens

[0158] For example, the blood of a rabbit infected with Brucella was collected, and positive polyclonal antibody serum was separated. The titer was detected by iELISA, and negative serum was used as a control.

[0159] The iELISA detection method is as follows: the Brucella granular antigen prepared in Example 1 is diluted to an appropriate concentration with coating buffer, and 100 μL is added to each well of the ELISA plate; the ELISA plate is placed in a 4°C refrigerator overnight or in a 37°C incubator for 2 hours to allow the antigen to adsorb to the surface of the plate wells; the liquid in the wells is discarded, and the ELISA plate is washed with washing solution 3 times, each time for 1 minute, and then dried on absorbent paper to remove unbound antigens and impurities; 5% BSA is added for blocking, and incubated in a 37°C incubator for 1 hour. The serum to be tested is appropriately diluted with diluent, generally starting with a 1:100 dilution, and then added to the ELISA plate wells coated with the antigen, 100 μL per well, with negative control wells; the ELISA plate is placed in a 37°C incubator for 1 hour to allow the specific antibodies in the serum to fully bind to the coated antigen; the liquid in the wells is discarded, and the ELISA plate is washed with washing solution 3 times, each time for 1 minute, and then dried on absorbent paper to remove unbound serum proteins and other impurities; enzyme-labeled secondary antibody diluted with diluent is added, 100 μL per well, and the dilution of the enzyme-labeled secondary antibody is adjusted according to its instructions; the ELISA plate is placed in a 37°C incubator for 1 hour to allow the enzyme-labeled secondary antibody to bind to the specific antibodies bound to the antigen; the ELISA plate is washed with washing solution 4 times, each time for 1 minute, and then dried on absorbent paper to remove unbound enzyme-labeled secondary antibody; substrate solution is added, 100 μL per well, and after gentle mixing, the ELISA plate is placed in a 37°C incubator for 30 minutes to allow the enzyme on the enzyme-labeled secondary antibody to catalyze the substrate to develop color; when the color development reaches an appropriate level, stop solution is added, 100 μL per well, to stop the enzyme-catalyzed reaction; the absorbance value (OD value) of each well is measured at 450 nm using an enzyme-labeled instrument.

[0160] The iELISA detection results are shown in Table 1 below.

[0161] Table 1: iELISA titer detection results of Brucella rabbit positive polyclonal antibody serum

[0162]

[0163] According to the data in Table 1, the iELISA titer of the rabbit Brucella positive polyclonal antibody serum can reach 1:200,000.

[0164] Further, a Brucella disease rose solid-phase detection test paper card was prepared according to the method in Example 1, and the detection sensitivity of the test paper card and the rose plate agglutination test was compared.

[0165] The detection method of the Rose Bengal plate agglutination test is operated according to the Rose Bengal plate agglutination kit (Qingdao Lijian) instruction, and specifically as follows: before the test, the serum and antigen are placed at room temperature for 30-60 min; the glass plate is marked with the number of the detected serum, and negative serum and positive serum controls are set; then 0.03 mL of the corresponding detected serum is added; 0.03 mL of the Rose Bengal plate agglutination antigen is added beside the detected serum; the serum and antigen are stirred with a sterilized toothpick to fully mix, and the result is observed within 5 min.

[0166] The detection results are shown in Table 1 and Figure 10 and Figure 11 As shown, the rabbit serum detected by the brucellosis Rose Bengal solid-phase detection test paper card prepared in the application can still be effectively determined as positive at a dilution of 2048 times, while the Rose Bengal plate agglutination test can still observe agglutination at a dilution of 64 times, but cannot effectively observe agglutination at a dilution of 128 times. Therefore, the natural antigen-carrying cell solid-phase detection test paper card provided by the application can effectively improve the detection sensitivity, which is more than 20 times higher than that of the plate agglutination test.

[0167] 2) Sensitivity of the antigen directly labeled solid-phase agglutination detection test reagent card

[0168] Taking the detection of schistosome egg infection as an example, the schistosome egg soluble antigen solid-phase detection test paper card, including the colloidal gold solid-phase detection test paper card and the latex solid-phase detection test paper card, is prepared according to the method in Example 1. The positive serum of a cow is diluted and diluted at different ratios, and the detection is performed according to the method in Example 1, with negative serum as a control. The detection results are shown in Table 2 and Figure 12 .

[0169] Table 2, detection results of different proportions of negative / positive serum of the schistosome egg soluble antigen of a cow

[0170]

[0171] According to the results in Table 2 and Figure 12 , the positive serum can still be distinguished by the naked eye at a dilution of 512 times.

[0172] Further, the positive sample is continuously diluted, and it can still be distinguished by the naked eye at a dilution of 640 times. Therefore, the antigen directly labeled solid-phase agglutination detection test reagent card provided by the application has high sensitivity.

[0173] 3) Sensitivity of the solid-phase agglutination detection test reagent card using gene recombinant expression of heterologous antigen

[0174] For example, for detecting rabies virus infection, the solid-phase detection test paper card of rabies virus was prepared according to the method in Example 1. The positive serum of the rabbit immunized with rabies was collected, diluted and diluted by different multiples, detected according to the method in Example 1 and compared with the detection by the iELISA method, and the negative serum was used as a control. The detection results are shown in Table 3, and the positive serum results detected by the solid-phase detection test paper card are shown in Figure 13 .

[0175] Table 3, detection results of different dilution ratios of positive and negative serum of rabbits with rabies virus

[0176]

[0177] According to the results in Table 3 and Figure 13 , the sensitivity of the positive serum detected by the iELISA method is very high, and the positive sample diluted to 204800 times is recognized as positive. Although the sensitivity of the positive serum detected by the solid-phase detection test paper card is not as high as that of the iELISA method, the positive and negative can still be distinguished by the naked eye when diluted by 512 times, which shows that the solid-phase agglutination detection reagent card provided by the application using gene recombinant expression of heterologous antigen still has high sensitivity. In addition, compared with the iELISA method, the solid-phase agglutination detection reagent card provided by the application using gene recombinant expression of heterologous antigen does not need to be operated by professional technicians through professional equipment, and can realize primary detection and large-scale screening out of the laboratory environment, and the detection time is fast, and the detection time of a single sample is not more than ten minutes. In addition, compared with the iELISA method, the solid-phase agglutination detection reagent card provided by the application using gene recombinant expression of heterologous antigen does not need to be operated by professional technicians through professional equipment, and can realize primary detection and large-scale screening out of the laboratory environment, and the detection time is fast, and the detection time of a single sample is not more than ten minutes.

[0178] 2, specific detection

[0179] 1) Specificity of the solid-phase detection test paper card of cells naturally carrying specific antigens: For example, for detecting Brucella infection, the solid-phase detection test paper card of Brucella was prepared according to the method in Example 1. The test paper card was used to detect Brucella, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, hydatid, Lawsonia intracellularis and positive rabbit serum of rabies virus according to the detection method in Example 1. The detection results are shown in Figure 14 , which shows that except for the positive serum of Brucella which is positive, the rest of the serum is negative, so the solid-phase detection test paper card provided by the application which naturally carries specific antigens has high specificity.

[0180] 2) The specificity of the antigen directly labeled solid phase agglutination test reagent card: taking the detection of schistosome egg infection as an example, the solid phase test paper card of schistosome egg soluble antigen was prepared according to the method in Example 1, including colloidal gold solid phase test paper card and latex solid phase test paper card. The positive rabbit serum of Japanese blood fluke egg, Brucella, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, hydatid, Lawsonia intracellularis was detected by using the test paper card according to the detection method in Example 1. The detection results showed that except the positive serum of Japanese blood fluke egg was positive, the other sera were negative, so the antigen directly labeled solid phase agglutination test reagent card provided by the application has high specificity.

[0181] 3) The specificity of the solid phase agglutination test reagent card using heterologous antigen expressed by gene recombination: taking the detection of rabies virus infection as an example, the solid phase test paper card of rabies virus was prepared according to the method in Example 1. The positive rabbit serum of rabies virus, Brucella, Japanese blood fluke egg, Escherichia coli, Clostridium perfringens, Haemophilus paragallinarum, Cryptosporidium, hydatid, Lawsonia intracellularis was detected by using the test paper card according to the detection method in Example 1. The detection results showed that except the positive serum of rabies virus was positive, the other sera were negative, so the solid phase agglutination test reagent card using heterologous antigen expressed by gene recombination provided by the application has high specificity.

[0182] 3、Repeatability test

[0183] 1) The repeatability of the solid phase test paper card of cells naturally carrying specific antigens: taking the detection of Brucella infection as an example, the solid phase test paper card of Brucella disease was prepared according to the method in Example 1. The positive serum of Brucella of cattle, sheep, rabbits and mice was collected and detected three times at the same interval according to the method in Example 1, and the time interval was 1 month. The detection results are shown in Table 1. Figure 15 As shown in Table 1, the positive serum of cattle and rabbits was positive in three detections at different times, which proved that the solid phase test paper card of cells naturally carrying specific antigens provided by the application had good repeatability.

[0184] 2) The repeatability of the antigen directly labeled solid phase agglutination test reagent card: taking the detection of schistosome egg infection as an example, the solid phase test paper card of schistosome egg soluble antigen was prepared according to the method in Example 1. The positive serum of schistosome egg of cattle, sheep, rabbits and mice was collected and detected by using the same method as in Example 1). The detection results showed that the positive serum of different animals was positive in three detections at different times, which proved that the antigen directly labeled solid phase agglutination test reagent card provided by the application had good repeatability.

[0185] 3) The specificity of the solid phase agglutination test reagent card for expressing heterologous antigens by genetic recombination: taking the detection of rabies virus infection as an example, the solid phase test paper card for rabies virus was prepared according to the method in Example 1. The positive serum of the rabies virus of the rabbit and the mouse was collected and detected by the same method as in Example 1). The detection results showed that the positive serum of different animals was positive in three detections at different times, which proved that the solid phase agglutination test reagent card for expressing heterologous antigens by genetic recombination provided by the application had good repeatability.

[0186] 4) Detection of whole blood samples

[0187] Taking the detection of brucellosis as an example. The positive and negative whole blood and serum of the mouse and the rabbit were detected by comparison. After blood collection, the whole blood (not diluted) was added with 10 μL for detection, and the separated serum (not diluted) was also added with 10 μL for detection. The other operation steps were consistent with Example 1, and the detection results were shown in Table 2. Figure 16 (B: whole blood; S: serum; N: negative; P: positive) as shown, the positive and negative of the whole blood and serum of the mouse and the rabbit could be clearly distinguished, indicating that the brucellosis rose solid phase test paper card provided by the application could be used for direct detection of whole blood. The whole blood detection is an important way to improve the convenience of detection, and the brucellosis rose solid phase test paper card can successfully detect the blood of the brucellosis positive animal and distinguish the infection. This result not only provides the possibility for whole blood detection, but also provides a new idea for future screening of brucellosis in wild animals and free-range animals.

[0188] 5) Comparison of the accuracy of clinical sample detection

[0189] 1) Detection of clinical samples by solid phase test paper card for cells naturally carrying specific antigens

[0190] Taking detection of brucellosis as an example, the brucellosis rose solid-phase detection test paper card is prepared according to the method in Example 1. The detection results of 10 positive clinical serum samples and 10 negative serum samples of cattle by the brucellosis rose solid-phase detection test paper card prepared in the application and the rose plate agglutination kit (Qingdao Lijian), iELISA preliminary detection (detection of clinical sample OD value), iELISA commercial detection kit (purchased from IDVET) and cELISA detection kit (purchased from Wuhan Keqian Biological Co., Ltd.) are compared. The detection method of the brucellosis rose solid-phase detection test paper card refers to Example 1, the rose plate agglutination test and iELISA preliminary detection method refer to Example 2, the detection method of the iELISA commercial detection kit and the cELISA detection kit refers to the respective instructions. The negative / positive judgment criteria of iELISA preliminary detection are as follows: the OD450 average value (X) and the standard deviation (SD) of a plurality of negative control samples are calculated, and X+2SD is taken as the critical value, and the critical value calculation result is 0.17; OD450 nm>0.17, positive; OD450 nm≤0.17, negative. The negative / positive judgment criteria of IDVET-iELISA are as follows: S / P%≤110%, negative; 110%<S / P%<120%, suspicious; S / P%≥120%, positive. The negative / positive judgment criteria of Keqian 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.

[0191] The detection results of 10 positive clinical serum samples and 10 negative serum samples of cattle are shown in Table 4, wherein the solid-phase detection test kit and the rose plate detection test results of the 10 positive clinical serum samples of cattle are shown in Table 4. Figure 17

[0192] Table 4, detection results of different methods for positive clinical serum samples of cattle

[0193]

[0194]

[0195] According to Figure 17 ​The 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.

[0196] 2) Antigen-labeled solid-phase agglutination assay kit for detecting clinical samples.

[0197] 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.

[0198] Table 5. Negative / positive serum detection results of sheep schistosomiasis eggs

[0199]

[0200] Table 6. Negative / positive serum detection results of bovine schistosomiasis eggs

[0201]

[0202] 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%.

[0203] 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.

[0204] 3) Detection of clinical samples using a solid-phase agglutination assay kit for recombinant expression of heterologous antigens.

[0205] For example, the rabies virus antibody is detected, and the solid-phase detection test paper card of the rabies virus is prepared according to the method in Example 1. The negative / positive serum of the rabies virus inactivated vaccine immunization and non-immunization is collected, wherein the positive serum is 40 cases in total, 15 cases of mice and 25 cases of rabbits; the negative serum is 30 cases in total, 15 cases of mice and 15 cases of rabbits. The serum is detected according to the use method in Example 1, the serum is diluted by 1:10, and the sample amount is 10 μL. The detection results are shown in Tables 7-8.

[0206] Table 7, detection results of negative / positive serum of mouse rabies virus

[0207]

[0208] Table 8, detection results of negative / positive serum of rabbit rabies virus

[0209]

[0210] According to the results in Tables 7-8, the sensitivity and specificity of the detection of the negative / positive serum of the rabies virus in mice and rabbits are both 100%, and the coincidence rate is 100%. Therefore, the solid-phase agglutination detection reagent card provided by the application has high detection accuracy.

[0211] Example 3, optimization of detection conditions of the solid-phase detection reagent card

[0212] 1. Optimization of dilution ratio of the sample to be detected

[0213] For example, the detection of Brucella infection is detected, and the solid-phase detection test paper card of Brucella disease is prepared according to the method in Example 1. The blood of rabbits, sheep, mice and cattle infected with Brucella is collected, and the positive serum is separated, which is diluted by 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320, and then detected, and the detection method is referred to Example 1, and the detection results are shown in Table 9.

[0214] Table 9, detection results of different dilution ratios of positive serum of Brucella in different animals

[0215]

[0216] According to the data in Table 9, the results show that when the serum is diluted by 1:10, the detection results of the four kinds of animals are all positive, and therefore when the sample to be detected is serum, the dilution ratio of the serum is preferably 10 times.

[0217] Further, the whole blood and plasma of the four kinds of animals are diluted according to the dilution ratio, and the detection results show that when the whole blood and plasma are diluted by 1:10, the detection results of the four kinds of animals are all positive.

[0218] 2. Optimization of sample volume of sample to be tested and detection reagent

[0219] For example, for detection of Brucella infection, the Brucella test paper card was prepared according to the method of Example 1. The blood of rabbits infected with Brucella was collected and the positive serum was separated. After being diluted by 16 times and 64 times, 5 μL, 10 μL and 20 μL of sample volume were used for detection, respectively. The sample volume of the detection reagent was the same as that of the serum. The detection method was according to Example 1, and the negative serum was used as a control. The detection results are shown in Table 1. Figure 18 As shown in Table 1, the detection results of the three systems with different sample volumes were all effective, and therefore, from the perspective of saving samples and detection reagents, the sample volume of the serum and the detection reagent was preferably 5 μL.

[0220] 3. Optimization of reaction time and observation time

[0221] 1) Optimization of reaction time and observation time of Brucella test paper card

[0222] For example, for detection of Brucella infection, the Brucella test paper card was prepared according to the method of Example 1. The blood of rabbits infected with Brucella was collected and the positive serum was separated. After being diluted by 16 times and 64 times, 5 μL, 10 μL and 20 μL of sample volume were used for detection, respectively. The sample volume of the detection reagent was the same as that of the serum. The detection method was according to Example 1, and the negative serum was used as a control. The detection results are shown in Table 1. Figure 19 As shown in Table 1, the detection results of the three systems with different sample volumes were all effective, and therefore, from the perspective of saving samples and detection reagents, the sample volume of the serum and the detection reagent was preferably 5 μL.

[0223] 2) Soluble antigen of schistosome egg solid-phase test paper card

[0224] For example, for detection of schistosome egg infection, the colloidal gold solid-phase test paper card and the latex solid-phase test paper card of the soluble antigen of schistosome egg were prepared according to the method of Example 1. The positive serum of rabbits and mice was collected, and after being diluted by 10 times, 10 μL of sample volume was added to 6 reaction wells, respectively. The samples in the 6 reaction wells were reacted with the detection reagent for 10 s, 20 s, 30 s, 60 s, 2 min and 3 min, respectively, and then the washing liquid was added. The rest of the detection conditions were the same as those of Example 1. The results showed that the colloidal gold solid-phase test paper card and the latex solid-phase test paper card could distinguish the positive and negative samples under the above 6 reaction times. From the perspective of saving time, the sample was reacted with the detection reagent for 10-60 s, and then the washing liquid was added. From the perspective of preventing sample from being dried and standardizing the detection, the reaction time of the sample with the detection reagent was preferably 10-20 s, and the observation time was within 1-10 min.

[0225] Example 4, screening of reaction membranes and diafiltration membranes

[0226] 1. Screening of reaction membranes and diafiltration membrane materials

[0227] The types of reaction membranes and diafiltration membranes are shown in Table 10 below, both the reaction membranes and the diafiltration membranes are purchased from Shanghai Jieyi Biotechnology Co., Ltd. Different types of reaction membranes and diafiltration membrane materials and textures are different, which will affect the non-specific binding and diafiltration speed in the detection process, and thus affect the detection accuracy, so it is necessary to screen the optimal combination of reaction membranes and diafiltration membranes.

[0228] Table 10, types of reaction membranes and diafiltration membranes

[0229]

[0230] Taking the detection of Brucella infection as an example, the different reaction membranes and diafiltration membranes in Table 10 above are paired according to the method in Example 1 to prepare Brucellosis Rose Red Solid-phase Detection Test Paper Card, and then the diafiltration of different reaction membranes and diafiltration membrane combinations for detecting positive and negative serum is detected. 20 cases of rabbit positive serum and 20 cases of rabbit negative serum were detected respectively, and the positive accuracy and negative accuracy were calculated, and the detection results are shown in Table 11 below.

[0231] Table 11, positive accuracy and negative accuracy of different reaction membranes and diafiltration membrane combinations for detection

[0232]

[0233] According to the data in Table 11, only the reaction membrane 1 and the diafiltration membrane 1 of combination 1 can reduce the false positive rate and the false negative rate to the lowest, while the rest of the reaction membrane and the diafiltration membrane combination will result in a higher false positive rate and a false negative rate. The false negative phenomenon and the false positive phenomenon in the detection of negative samples and positive samples are the result of the joint action of different reaction membranes and diafiltration membranes. If the reaction membrane material has stronger non-specific adsorption capacity, it will result in an increase in false positive rate; If the pore size of the diafiltration membrane is too small, the sample diafiltration speed will be too slow, the time of the sample staying in the reaction membrane will increase, and thus the risk of non-specific adsorption will increase, resulting in an increase in false positive rate; If the pore size of the reaction membrane and the diafiltration membrane is too large or the pore size of the diafiltration membrane is too large, the sample diafiltration speed will be too fast, and thus the sample to be tested will not react with the reaction membrane, resulting in a decrease in positive signal. Therefore, when the reaction membrane is reaction membrane 1 and the diafiltration membrane is diafiltration membrane 1, the effect of detecting the sample is the best, the non-specific adsorption of the reaction membrane is reduced to the lowest, and the diafiltration speed is the most appropriate.

[0234] 2. Screening of reaction membrane and diafiltration membrane layers

[0235] Take the detection of Brucella infection as an example, change the number of layers of the reaction membrane and the percolation membrane in Example 1, the number of layers of the reaction membrane is 1-2 layers, and the number of layers of the percolation membrane is 1-3 layers respectively, combine the reaction membrane and the percolation membrane with different layers according to the method in Example 1 to prepare the Brucella test paper card, and detect 20 positive rabbit sera and 20 negative rabbit sera respectively, calculate the positive correct rate and the negative correct rate, and the detection results are shown in the following Table 12.

[0236] Table 12, positive correct rate and negative correct rate of different reaction membrane and percolation membrane layer combinations for detection

[0237]

[0238]

[0239] According to the data in Table 12, comparing the results of combinations 1-3, when the reaction membrane is 1 layer, too few layers of the percolation membrane will increase the false negative rate, because the percolation speed is too fast, the antibodies in the sample do not have sufficient contact with the reaction membrane, and the positive signal is weakened; too many layers of the percolation membrane will increase the false positive rate, because the percolation speed is too slow, sample residues can cause an increase in non-specific reactions, resulting in false positive results; therefore, when the reaction membrane is 1 layer and the percolation membrane is 2 layers, the false positive rate and the false negative rate are the lowest. Comparing the results of combinations 4-6, when the reaction membrane is 2 layers, whether the number of layers of the percolation membrane is too many or too few, serious false negatives and false positives will occur.

[0240] Based on the above analysis, the reaction membrane is preferably 1 layer, and the percolation membrane is preferably 2 layers.

[0241] Example 5, washing liquid formula screening

[0242] Different washing liquid formula combinations are shown in Table 13.

[0243] Table 13, different washing liquid formula combinations

[0244] Formulation Component Formulation 1 0.01 mol / L Phosphate buffer Formulation 2 0.01 mol / L Phosphate buffer + 1% Tween 20 + 1% Triton 100 Formulation 3 0.02 mol / L Tris-base Formulation 4 0.02 mol / L Tris-base + 0.5% PEG 20000

[0245] Take the detection of Brucella infection as an example, change the number of layers of the reaction membrane and the percolation membrane in Example 1, the number of layers of the reaction membrane is 1-2 layers, and the number of layers of the percolation membrane is 1-3 layers respectively, combine the reaction membrane and the percolation membrane with different layers according to the method in Example 1 to prepare the Brucella test paper card, and detect 20 positive rabbit sera and 20 negative rabbit sera respectively, calculate the positive correct rate and the negative correct rate, and the detection results are shown in the following Table 12.

[0246] Table 14, positive correct rate and negative correct rate of different formula washing liquids for detection

[0247]

[0248] According to the results in Table 14, the accuracy of washing liquid formula 2 for detecting positive serum and negative serum is the highest among all the washing liquid formulas, which indicates that the use of washing liquid formula 2 for detection can minimize the probability of false negative and false positive. If the elution capacity of the washing liquid is too weak, the non-specific substances cannot be sufficiently eluted and remain in the reaction membrane to interfere with the detection results, resulting in false positives. If the elution capacity of the washing liquid is too strong, on the one hand, it can change the structure of non-specific proteins or other biological molecules in the sample to be tested, exposing more antigenic determinants or binding sites to combine with the capture protein on the reaction membrane and the particulate antigen in the detection reagent, thereby producing false positives. On the other hand, the elution capacity of the washing liquid that is too strong can elute the antibodies to be tested that have combined with the capture protein and the particulate antigen on the reaction membrane, thereby producing false negatives. Therefore, the elution capacity of the washing liquid cannot be too weak or too strong, and the elution capacity of washing liquid formula 2 is just right, which is the preferred one.

[0249] Example 6, Streptococcus Protein G Optimization

[0250] Streptococcus protein G (SPG) can bind to the Fc end of human and various animal antibodies IgG, which is similar to staphylococcus protein A (SPA), but compared with SPA, SPG has stronger binding force and wider binding spectrum with IgG. In this embodiment, a recombinant streptococcus protein G is constructed to have stronger binding capacity with IgG.

[0251] The structure of SPG is as follows: Figure 20As shown, from the N-terminal, there are three homologous structure regions A1, A2 and A3, each of which consists of 24 amino acids, and the three homologous structures are separated by homologous regions B1, B2 consisting of 51 amino acids, followed by a spacer region S, followed by homologous structure regions C1, C2 and C3 consisting of 55 amino acids, which are separated by D1 and D2 regions, and the C3 region is followed by a hydrophilic region W, and finally by the M region. Studies have shown that the three homologous amino acid sequences C1, C2 and C3 regions of SPG are related to the binding of the Fc end of antibody IgG, and the C1 and C2 regions only differ by 2 amino acid sequences, the C1 and C3 regions differ by 6 amino acid sequences, and the binding capacity of the C3 region to the antibody IgG is equivalent to 7 times that of the C1 region. In view of this, the embodiment reconstructs the IgG binding fragment of the SPG gene, only retains the C region which can specifically bind to the Fc end of the antibody IgG, and replaces the gene fragments of the C1 and C2 regions with the gene fragments of the C3 region, to obtain a C3-D1-C3-D2-C3 (rSPG) gene sequence. At the same time, it is detected whether the rSPG sequence contains rare codons of Escherichia coli (frequency of use <10%). If it contains rare codons, replace them with Escherichia coli preferred codons that encode the same amino acid, and finally add a TAA termination sequence at the 3' end of the sequence to construct a pET-28a(+)-rSGP recombinant plasmid. After induction and expression, the purified rSPG solution is dialyzed in PBS overnight and used.

[0252] Taking the detection of Brucella infection as an example, the purified rSPG above is prepared into a Brucella rose solid-phase detection test paper card according to the method in Example 1, different dilutions of rabbit positive serum are detected, and a Brucella rose solid-phase detection test paper card prepared from the non-optimized SPG sequence is compared, and the detection results are shown in Table 15.

[0253] Table 15, results of streptococcal G protein before and after optimization for detecting rabbit positive serum

[0254]

[0255] According to the data in Table 15, the Brucella rose solid-phase detection test paper card prepared from the rSPG after sequence optimization can still detect positive at a dilution of 2048 times of positive serum, while the Brucella rose solid-phase detection test paper card prepared from the non-optimized SPG sequence cannot detect positive at a dilution of 512 times of positive serum. This shows that through the optimization of the C region sequence, the enrichment capacity of SPG for IgG in the detection sample can be significantly improved, and since the antibody to be detected is also IgG, the enrichment capacity for the antibody to be detected is further improved, thereby improving the detection sensitivity.

[0256] Further, the purified rSPG was diluted with PBS to different spraying concentrations, 0, 0.05, 0.1, 0.2, 0.5 mg / mL, respectively, and the brucellosis test paper card was prepared according to the method in Example 1, and the rabbit positive serum was detected, and the detection results are shown in Table 16.

[0257] Table 16, results of different concentrations of rSPG for detecting rabbit positive serum

[0258]

[0259] According to the results in Table 16, the brucellosis test paper card prepared by spraying rSPG at a concentration of 0.05-0.5 can effectively determine the positive serum diluted 2048 times as positive, but considering the detection effect and cost saving, the most suitable spraying concentration is 0.1 mg / mL.

[0260] Example 7, optimization of solid phase agglutination test reagent card for heterologous antigen expressed by gene recombination

[0261] Taking the detection of rabies virus as an example, the solid phase test paper card for rabies virus prepared in Example 1 was optimized.

[0262] 1, optimization of cells carrying specific antigens

[0263] In Example 1, the rabies virus-G protein was expressed and displayed on the surface of E. coli by using E. coli prokaryotic expression system (vector pET-28a(+)). In this experiment, the rabies virus-G protein was expressed on the surface of other cells by 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.

[0264] The rabies virus-G protein sequence in Example 1 was connected to the above different vectors and introduced into the corresponding expression system and expressed and displayed. The rabies virus-G protein prepared by the four different expression systems was detected by Western blot method. The results showed that the content of the rabies virus-G protein expressed and displayed by the E. coli prokaryotic expression system was the most.

[0265] Further, the four kinds of cells prepared above were prepared into particulate antigen solution according to the method in Example 1, and then diluted by the same multiple for detecting different dilution multiples of rabbit positive serum, and the detection results are shown in Table 17.

[0266] Table 17, results of different expression systems for detecting different dilution multiples of rabbit positive serum

[0267]

[0268]

[0269] According to the data in Table 17, it is shown that the cells carrying the rabies virus-G protein prepared by four different expression systems all have good sensitivity and specificity, but compared with each other, the cells prepared by the E. coli expression system have higher sensitivity and specificity. Considering the production cost and culture conditions, the production cost of E. coli is low and the culture conditions are simple, which is preferred.

[0270] Therefore, the expression system is preferably the E. coli expression system.

[0271] 2. Screening of vectors

[0272] In this experiment, the vectors used in Example 1 are pET-28a(+), and this experiment will compare the effects of different vectors on the expression and display of rabies virus-G protein by E. coli. Different vectors include pET-29a(+), pET-30a(+), pGEX-4T-1 and pAIDA-I. After connecting the rabies virus-G protein sequence to the above four different vectors, expression and display are performed, and then the expression content of the four cells is detected by Western blot method. The results show that the content of rabies virus-G protein expressed and displayed using the pET-28a(+) vector is the most.

[0273] Further, the four E. coli cells expressed using different vectors are prepared into particulate antigen solution according to the method in Example 1, and then diluted by the same fold to detect rabbit positive serum of different dilution folds, and the detection results are shown in Table 18.

[0274] Table 18, detection results of different dilution folds of rabbit positive serum by using different vectors expressed E. coli to prepare detection reagents

[0275] 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 + + + + + + + + - -

[0276] According to the data analysis in Table 18, it is shown that the detection of different dilution folds of rabbit positive serum by using different vectors expressed E. coli to prepare detection reagents all have good sensitivity and specificity, but compared with each other, the rabies virus-G protein expressed by the pET-28a(+) vector has higher sensitivity and specificity. Therefore, the vector of the E. coli expression system is preferably pET-28a(+).

[0277] 3. Optimization of protein sequence

[0278] 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 preferred codons of E. coli. In this experiment, the protein expression content of E. coli before and after optimization of the sequence will be compared. The nucleotide sequence before optimization is shown in SEQ ID NO. 3. The two different sequences are linked to the pET-28a(+) vector and then introduced into E. coli for induction expression. The expression content of the four cells is detected by Western blot method. The results show that the pET-28a(+) vector linked to SEQ ID NO. 1 sequence is used for expression and display of the rabies virus-G protein with the highest content.

[0279] Further, the E. coli cells expressing the rabies virus-G protein using the above two different nucleotide sequences are prepared into a particulate antigen solution, which is then diluted by the same factor to detect rabbit positive serum of different dilution factors. The detection results are shown in Table 19.

[0280] Table 19. Results of detecting rabbit positive serum of different dilution factors using E. coli prepared by different nucleotide sequences as detection reagents

[0281] 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 + + + + + + + + - -

[0282] According to the data in Table 19, it is shown that the optimized nucleotide sequence can improve the sensitivity and specificity of E. coli expressing the rabies virus-G protein for detection.

[0283] Therefore, by optimizing the codons, the expression amount of heterologous antigens and the detection sensitivity and specificity can be further improved.

[0284] Example 8. Optimization of solid-phase agglutination test reagent card using directly labeled antigens

[0285] 1. Screening of specific non-protein antigens and inert carriers

[0286] Taking the detection of Schistosoma japonicum egg infection as an example, the solid-phase test paper card of Schistosoma egg soluble antigen prepared in Example 1 has a inert carrier of colloidal gold or latex color microspheres. The inert carrier is replaced with red blood cells, magnetic microspheres, and carbon powder. The rabbit positive serum is detected, the serum is diluted by 10 times, the sample amount is 10 μL, and the rest of the detection conditions are referred to Example 1.

[0287] The specific preparation method of Schistosoma egg soluble antigen labeled with red blood cells is as follows:

[0288] (1) Collection and treatment of red blood cells: ① Collect blood from the jugular vein of healthy adult sheep into a triangular flask containing glass beads; ② Shake for 15-20 minutes; ③ Add an equal volume of Al's solution; ④ Place in a 4℃ refrigerator for 3 days;

[0289] (2) Red blood cell washing: ① The stable defibrillation blood was filtered in a triangular funnel with a small amount of defatted cotton between double-layer gauze to remove blood clots; ② An appropriate amount of red blood cell filtrate was taken in a centrifuge tube, and 10 times the volume of 0.01M pH 7.2 phosphate buffer was added; ③ Centrifugation was performed at 2000 rpm for 10 minutes; ④ The supernatant was aspirated with a pipette with a rubber tube; ⑤ The precipitate was resuspended with an appropriate amount of PBS solution, and centrifugation was performed again; ⑥ Steps ②-⑤ were repeated 5 times, and a 5% red blood cell suspension was obtained, which was stored in a refrigerator at 4°C;

[0290] (3) Red blood cell sensitization: 1% red blood cells were added to 200 mL of PBS solution (pH 6.4); 0.6 mL of SEA antigen (1 mg / mL) was added; 37°C water bath oscillation for 45 min; centrifugation at 3500 rpm for 5 min, discard the supernatant; resuspend with 50 mL PBS (pH 7.2), add 0.5 mL of rabbit negative serum blocking (serum final concentration 1%); centrifugation at 3500 rpm for 1-5 min, discard the supernatant; add 50 mL PBS (pH 7.2) and 0.5 mL of rabbit negative serum blocking (serum final concentration 1%); test the effect of the developed reagent (red blood cell concentration is 4%); store at 4°C.

[0291] The detection results show that the schistosome egg soluble antigen labeled with colloidal gold, latex color microspheres, magnetic microspheres and carbon powder can be used for detection and can distinguish between positive and negative; the sensitized red blood cell labeled solid phase test paper card cannot distinguish between positive and negative, because the freeze-dried sensitized red blood cell labeled antigen cannot be washed by the washing solution, and the negative serum develops color as the positive serum. The possible reason for the analysis is that the freeze-dried red blood cell labeled antigen has serious non-specific adsorption with the reaction membrane.

[0292] Further, the schistosome egg soluble antigen labeled with colloidal gold, latex color microspheres, magnetic microspheres and carbon powder was used to detect rabbit negative serum, 20 negative samples were detected for each, and the false positive rate was calculated, and the detection results are shown in Table 20.

[0293] Table 20, detection results of different inert carriers labeled schistosome egg soluble antigen on rabbit negative serum

[0294]

[0295] According to the data in Table 20, it is shown that the false positive rate of the schistosome egg soluble antigen labeled with colloidal gold and latex color microspheres for detecting rabbit negative serum is 0%, while magnetic microspheres and carbon powder appear false positive, and the false positive rate of carbon powder is more than 50%, and the non-specific adsorption is serious. Therefore, the preferred inert carrier is colloidal gold and latex color microspheres.

[0296] 2. Screening of other non-protein antigens and inert carriers

[0297] The non-protein antigen material in the above-mentioned Experiment 1 is soluble antigen of Schistosoma japonicum egg, which includes protein, polysaccharide, glycoprotein and other substances. In this experiment, other kinds of solid phase agglutination test reagent cards directly labeled with antigens are prepared for detecting antibodies produced by the body in response to non-protein antigens such as saccharides, toxins and drugs. The saccharides are capsular polysaccharide of Streptococcus pneumoniae and capsular polysaccharide of Haemophilus influenzae, the toxins are toxin A and toxin B of Clostridium difficile, and the drug is amoxicillin. The preparation method is referred to the preparation of solid phase agglutination test reagent cards directly labeled with antigens in Example 1. The inert antibody can be selected from colloidal gold, latex color microspheres, magnetic microspheres and carbon powder.

[0298] The positive sera of Streptococcus pneumoniae, Haemophilus influenzae, Clostridium difficile and amoxicillin are detected by the corresponding solid phase test reagent cards according to the method in Example 1, and all of them are detected positive.

[0299] Further, the solid phase agglutination test reagent cards for detecting different antigens labeled with different inert antibodies are used to detect the corresponding negative sera, each of which is used to detect 20 negative samples to calculate the false positive rate. The detection results are shown in Table 21.

[0300] Table 21. False positive rate of solid phase agglutination test reagent cards for detecting negative samples

[0301]

[0302] According to the data in Table 21, the use of colloidal gold and latex color microspheres for labeling polysaccharides, toxins and small molecule drugs for detection will not cause false positive, so the preferred inert carrier is colloidal gold and latex color microspheres.

[0303] Although the present application is disclosed as above, the present application is not limited thereto. Any person skilled in the art, without departing from the spirit and scope of the present application, can make various changes and modifications, therefore the protection scope of the present application should be subject to the scope defined by the claims.

[0304] SEQ ID NO. 1

[0305] Rabies virus amino acid sequence LGPWSPIDIHHLSCPNNLVVEDEGCTNLSEFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVTDLDPYDKSLHSRVFPGGNCSGITVSSTYCSTNHDYTIWMPENLRLGTSCDIFTNSRGKRASKGGKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSDETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGSDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEVEDFVEVHLPDVHEQVSGVE SEQ ID NO. 2

[0306]

[0307]

Claims

1. An antigen directly labeled solid phase agglutination test strip card, characterized by, The test kit comprises a capture protein and a detection reagent, the detection reagent comprises a particulate antigen, the particulate antigen is a conjugate of a specific antigen and an inert carrier, the specific antigen specifically binds to the antibody to be detected.

2. The antigen directly labeled solid phase agglutination test strip card according to claim 1, wherein, The specific antigen is a non-protein antigen.

3. The antigen directly labeled solid phase agglutination test strip card according to claim 2, wherein, The specific antigen is obtained by physical or chemical methods.

4. The antigen directly labeled solid phase agglutination test strip card according to claim 3, wherein, The capture protein is a ligand of the antibody to be detected or a monoclonal antibody targeting the antibody to be detected.

5. The antigen directly labeled solid phase agglutination test strip card according to claim 4, wherein, The inert carrier itself has color or has luminescent properties or itself has no color, when it itself has no color, it needs to be dyed by a dye.

6. The direct antigen-labeled solid phase agglutination test strip card according to claim 1, wherein, It also comprises a reaction membrane, a percolation membrane, a water absorption pad and a shell; the reaction membrane embeds the capture protein, the shell is provided with a sample addition hole; the pore size of the reaction membrane and the percolation membrane is the same, being 3-8 μm.

7. The antigen directly labeled solid phase agglutination test strip card according to claim 1, wherein, It also comprises a washing solution.

8. The method of using an antigen directly labeled solid phase agglutination test strip card according to any one of claims 1 to 7, wherein, The test kit comprises the following steps: (1) embedding the capture protein into the reaction membrane; (2) assembling the reaction membrane, the percolation membrane, the percolation membrane, the water absorption pad and the shell into a solid-phase agglutination test paper card; (3) diluting the sample to be detected; (4) sequentially adding the sample to be detected and the detection reagent into the sample addition hole of the solid-phase agglutination test paper card for reaction, then adding the washing solution after the reaction; observing whether the sample addition hole shows color.

9. The method of use of claim 8, wherein, The sample to be detected in step (3) is any one or more of whole blood, serum and plasma, and the dilution multiple is 10-64.

10. The method of use of claim 9, wherein, The sample addition amount of the sample to be detected and the detection reagent in step (4) is the same, being 5-20 μL; the reaction time is 10-180 seconds.

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