Fluorescent immunolayer kit for detecting canine parvovirus, canine coronavirus and giardia virus and its preparation method and application

Abstract

This application belongs to the field of in vitro detection technology, specifically relating to a fluorescent immunochromatographic kit for detecting canine parvovirus, canine coronavirus, and Giardia virus, as well as its preparation method and application. This application provides a fluorescent immunochromatographic kit for detecting canine parvovirus, canine coronavirus, and Giardia virus. Through a triple detection system design, it can simultaneously detect three important pathogens: canine parvovirus, canine coronavirus, and Giardia virus, improving detection efficiency and providing more comprehensive information for clinical diagnosis. Furthermore, by adding metal chelating agents and lysis agents to the sample diluent, metal ion impurities in the sample that cause non-specific binding can be specifically chelated, effectively eliminating interference and significantly improving the detection rate of the target pathogens while enhancing detection sensitivity, thus meeting the needs of rapid on-site screening in canine clinics.

Description

Technical Field

[0001] This application belongs to the field of in vitro detection technology, specifically relating to a fluorescent immunoassay kit for detecting canine parvovirus, canine coronavirus, and Giardia virus, as well as its preparation method and application. Background Technology

[0002] Canine parvovirus (CPV) belongs to the family Parvoviridae and genus Parvovirus in etiological classification. This pathogen is highly contagious via the fecal-oral route, causing acute systemic infection in canines. Clinical statistics show that during outbreaks, the infection rate approaches 90%, and the mortality rate in infected puppies can exceed 70%. It is listed as one of the major infectious diseases in dogs by the World Organisation for Animal Health (OIE). Canine coronavirus (CCV) is classified into the order Nidovirales, family Coronaviridae, and subfamily Coronavirinae. This pathogen has a high prevalence in canine populations and can infect dogs of any age, but puppies under six months old are generally more susceptible. Giardia lamblia is a widely distributed zoonotic parasitic protozoan disease characterized by diarrhea and weight loss, and is prevalent worldwide.

[0003] Current diagnostic methods for canine viral infections include virus isolation, culture and identification, serological detection, and molecular biological detection. Virus isolation and culture can take 3-7 days, are complex, and require highly specialized laboratory conditions. PCR technology requires specialized equipment and technicians, with a detection cycle of 1.5-4 hours; RNA samples are easily degraded and pose a risk of cross-contamination. While colloidal gold immunochromatography is a rapid screening method, it only provides qualitative detection, has low sensitivity, and is prone to false negatives in the early stages of the disease. Clinically, during the incubation period of CPV infection and the early stages of the disease (1-2 days), the viral antigen load in canine feces is low. The detection limits of current mainstream CPV fluorescent detection reagents are generally higher than those of nucleic acid detection technologies, making it difficult to capture samples with low viral loads in the early stages of infection, leading to diagnostic delays.

[0004] Therefore, developing rapid, sensitive detection technologies applicable to the early stages of infection is of great significance for the early prevention and control of canine diseases. Summary of the Invention

[0005] Based on this, one embodiment of this application provides a fluorescent immunoassay kit for detecting canine parvovirus, canine coronavirus, and Giardia virus, as well as its preparation method and application.

[0006] This application provides a fluorescent immunochromatographic detection kit for canine parvovirus, canine coronavirus, and Giardia virus, comprising: immunochromatographic test strips and sample diluent;

[0007] The immunochromatographic test strip includes a base plate and a sample pad, a conjugate pad, a coating membrane, and an absorbent pad disposed on the base plate;

[0008] The sample pad, conjugate pad, coating membrane, and absorbent pad are sequentially connected to each other in contact.

[0009] The conjugate pad contains fluorescent microspheres labeled with canine parvovirus antibody, fluorescent microspheres labeled with canine coronavirus antibody, and fluorescent microspheres labeled with Giardia lamblia antibody.

[0010] The immunochromatographic test strips detect canine parvovirus antigen, canine coronavirus antigen, and Giardia lamblia antigen using a double antibody sandwich method.

[0011] The sample diluent comprises the following components: 0.01 M-0.05 M sodium dihydrogen phosphate, 0.01 M-0.05 M disodium hydrogen phosphate, 0.2 M-0.4 M sodium chloride, 0.001 M-0.005 M EDTA-Na2, 0.5 w / v%-1 w / v% Triton-100, 0.1 w / v%-1 w / v% polyvinylpyrrolidone, 0.01%-0.1% SDS, 0.001 mM-0.005 mM tetrasodium iminodisuccinate, and 0.01 w / v%-0.02 w / v% preservative, with a pH of 7.5-8.5.

[0012] In some embodiments, the sample pad contains: 0.5 mg / mL to 1 mg / mL of mouse IgG, 0.5 w / v% to 1 w / v% of Evans blue, 0.5 mg / mL to 2 mg / mL of the blocker HIRE-R-R001, 0.5 mg / mL to 2 mg / mL of erythrocytes, 5 w / v% to 10 w / v% of trehalose, 0.5 w / v% to 3 w / v% of Tween-20, and 2 w / v% to 5 w / v% of BSA.

[0013] In some embodiments, the conjugate pad contains: 0.5 w / v%-3 w / v% of Tween-20, 2 w / v%-5 w / v% of BSA, 5 w / v%-10 w / v% of trehalose, 8 w / v%-10 w / v% of canine parvovirus fluorescent microsphere markers, 8 w / v%-10 w / v% of canine coronavirus fluorescent microsphere markers, 8 w / v%-10 w / v% of Giardia lamblia fluorescent microsphere markers, and 0.5 w / v%-1 w / v% of chicken IgY fluorescent microsphere markers.

[0014] In some embodiments, the coating membrane is provided with detection lines and control lines;

[0015] The detection lines include a first detection line, a second detection line, and a third detection line;

[0016] In some embodiments, the first detection line is coated with canine coronavirus monoclonal antibody.

[0017] In some embodiments, the second detection line is coated with canine parvovirus monoclonal antibody.

[0018] In some embodiments, the third detection line is coated with Giardia lamblia monoclonal antibody.

[0019] The quality control line is coated with sheep anti-chicken antibody.

[0020] In some embodiments, the coating concentration of the canine coronavirus monoclonal antibody is 0.8 mg / mL to 1.2 mg / mL.

[0021] In some embodiments, the coating concentration of the canine parvovirus monoclonal antibody is 0.8 mg / mL to 1.2 mg / mL.

[0022] The coating concentration of the Giardia lamblia monoclonal antibody is 0.4 mg / mL to 0.6 mg / mL.

[0023] In some embodiments, the coating concentration of the goat anti-chicken antibody is 0.4 mg / mL to 0.6 mg / mL.

[0024] Another aspect of this application provides a method for preparing the aforementioned fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus, comprising:

[0025] Prepare the sample dilution by mixing 0.01 M-0.05 M sodium dihydrogen phosphate, 0.01 M-0.05 M disodium hydrogen phosphate, 0.2 M-0.4 M sodium chloride, 0.001 M-0.005 M EDTA-Na2, 0.5 w / v%-1 w / v% Triton-100, 0.1 w / v%-1 w / v% polyvinylpyrrolidone, 0.01%-0.1% SDS, 0.001 mM-0.005 mM tetrasodium iminodisuccinate, and 0.01 w / v%-0.02 w / v% preservative, with a pH of 7.5-8.5.

[0026] The canine parvovirus antibody fluorescent microsphere marker, the canine coronavirus antibody fluorescent microsphere marker, and the Giardia lamblia antibody fluorescent microsphere marker were prepared using a conjugation activation solution; and the above antibody fluorescent microsphere markers were sprayed onto the conjugate pad.

[0027] The first detection antibody, the second detection antibody, and the third detection antibody are sprayed onto the coating membrane to form the detection line;

[0028] The goat anti-chicken antibody was sprayed onto the coating membrane to form the control line;

[0029] Assemble the sample pad, the conjugation pad, the coating membrane, and the absorbent pad onto the base plate.

[0030] In some embodiments, the coupling activation solution comprises:

[0031] 0.05 w / v%–0.2 w / v% of N-lauroyl glutamate sodium, 0.01 w / v%–0.1 w / v% of sulfobetaine-12 and 0.05 M–0.1 M of 2-(N-morpholino)ethanesulfonic acid, pH 5.5–6.5.

[0032] In some embodiments, the method for preparing the antibody fluorescent microsphere label includes:

[0033] Fluorescent microspheres were dispersed in the coupling activation solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added for activation to prepare activated microspheres;

[0034] The activated microspheres were mixed with antibodies and then coupled to prepare antibody-fluorescent microsphere labels; and

[0035] The antibody fluorescent microsphere label is mixed with a blocking solution. The inert blocking agent in the blocking solution covers the unbound sites on the surface of the microspheres, quenches residual activating groups, eliminates non-specific adsorption, reduces background fluorescence, protects antibody activity, and prevents microsphere aggregation.

[0036] Another aspect of this application provides a method for detecting canine parvovirus, canine coronavirus, and Giardia virus, comprising: using the aforementioned fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus to detect the sample to be tested.

[0037] This application provides a fluorescent immunoassay kit for detecting canine parvovirus, canine coronavirus, and Giardia virus. Through a triple detection system design, it can simultaneously detect three important pathogens—canine parvovirus, canine coronavirus, and Giardia—improving detection efficiency and providing more comprehensive information for clinical diagnosis. Furthermore, by adding metal chelating agents and lysis agents to the sample diluent, metal ion impurities in the sample that cause non-specific binding can be specifically chelated, effectively eliminating interference and significantly improving the detection rate of the target pathogens while enhancing detection sensitivity, thus meeting the needs of rapid on-site screening in canine clinics. Other components in the sample diluent, such as sodium dihydrogen phosphate and disodium hydrogen phosphate, form a buffer system to maintain pH stability in the detection environment; sodium chloride provides suitable ionic strength; tetrasodium iminodisuccinate (IDS) and EDTA-Na2 further chelate metal ions; Triton-100 and polyvinylpyrrolidone act as surfactants to reduce non-specific binding, improve the signal-to-noise ratio, and further enhance detection sensitivity. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 The CPV Ag reference concentration-T / C detection results for Examples 1-6 provided in this application;

[0040] Figure 2 The CCV Ag reference concentration-T / C detection results for Examples 1-6 provided in this application;

[0041] Figure 3 The T / C test results of GIA Ag reference concentration for Examples 1-6 provided in this application. Detailed Implementation

[0042] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0043] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0044] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0045] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0046] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0047] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0048] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0049] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0050] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0051] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0052] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0053] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0054] In this application, % (w / w) and w / v% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0055] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0056] The basic principle of the fluorescence immunochromatographic kit in this application is a double-antibody sandwich method. It uses a dry fluorescence immunoassay analyzer to quantitatively detect fluorescence signals, providing a highly sensitive, highly specific, and simple-to-operate (15 min) kit for accurate quantitative detection of canine parvovirus, canine coronavirus, and Giardia lamblia viruses in feces. Specifically, the analyte in the sample binds to monoclonal antibodies labeled with canine parvovirus, canine coronavirus, and Giardia lamblia viruses dispersed on the conjugate pad, forming a complex. Under chromatography, the complex diffuses forward along the reaction membrane and is captured by the corresponding monoclonal capture antibodies for canine parvovirus, canine coronavirus, and Giardia lamblia viruses fixed on the detection line of the reaction membrane. The more analyte in the sample, the more complex is formed, the more complex accumulates on the detection line, and the stronger the fluorescence signal displayed by the fluorescence instrument. The intensity of the fluorescence signal reflects the amount of analyte captured.

[0057] This application provides a fluorescent immunochromatographic detection kit for canine parvovirus, canine coronavirus, and Giardia virus, comprising: immunochromatographic test strips and sample diluent;

[0058] The immunochromatographic test strip includes a base plate and a sample pad, a conjugate pad, a coating membrane, and an absorbent pad disposed on the base plate;

[0059] The sample pad, conjugate pad, coating membrane, and absorbent pad are sequentially connected to each other in contact.

[0060] The conjugate pad contains fluorescent microspheres labeled with canine parvovirus antibody, fluorescent microspheres labeled with canine coronavirus antibody, and fluorescent microspheres labeled with Giardia lamblia antibody.

[0061] The immunochromatographic test strips detect canine parvovirus antigen, canine coronavirus antigen, and Giardia lamblia antigen using a double antibody sandwich method.

[0062] The sample diluent comprises the following components: 0.01 M-0.05 M sodium dihydrogen phosphate, 0.01 M-0.05 M disodium hydrogen phosphate, 0.2 M-0.4 M sodium chloride, 0.001 M-0.005 M EDTA-Na2, 0.5 w / v%-1 w / v% Triton-100, 0.1 w / v%-1 w / v% polyvinylpyrrolidone, 0.001 mM-0.005 mM tetrasodium iminodisuccinate (IDS), and 0.01 w / v%-0.02 w / v% preservative, with a pH of 7.5-8.5.

[0063] For example, sodium dihydrogen phosphate concentrations are 0.01 M, 0.02 M, 0.03 M, 0.04 M, and 0.05 M, or any intermediate value; disodium hydrogen phosphate concentrations are 0.01 M, 0.02 M, 0.03 M, 0.04 M, and 0.05 M, or any intermediate value; sodium chloride concentrations are 0.2 M, 0.25 M, 0.3 M, 0.35 M, and 0.4 M, or any intermediate value; EDTA-Na2 concentrations are 0.001 M, 0.002 M, 0.003 M, 0.004 M, and 0.005 M, or any intermediate value; and Triton-100 concentrations are 0.5 w / v%, 0.6 w / v%, 0.7 w / v%, 0.8 w / v%, 0.9 w / v%, and 1 w / v%, or any intermediate value.

[0064] The concentrations of polyvinylpyrrolidone are 0.1 w / v%, 0.2 w / v%, 0.3 w / v%, 0.4 w / v%, 0.5 w / v%, 0.6 w / v%, 0.7 w / v%, 0.8 w / v%, 0.9 w / v%, and 1 w / v%, or any value in between; the concentrations of tetrasodium iminodisuccinate (IDS) are 0.001 mM, 0.002 mM, 0.003 mM, 0.004 mM, and 0.005 mM, or any value in between; the concentrations of preservatives are 0.01 w / v%, 0.012 w / v%, 0.014 w / v%, 0.016 w / v%, 0.018 w / v%, and 0.02 w / v%, or any value in between.

[0065] This application significantly improves the detection sensitivity of samples with low viral load by optimizing the sample diluent formulation, particularly by introducing tetrasodium iminodisuccinate (IDS) as a key component. IDS possesses excellent metal ion chelating ability, effectively eliminating interference from metal ions in the sample on the immune response. Simultaneously, it stabilizes the viral antigen structure, preventing degradation, thereby significantly improving detection sensitivity and enabling accurate detection of samples with low viral load in the early stages of infection. Sodium dihydrogen phosphate and disodium hydrogen phosphate form a buffer system to maintain pH stability in the detection environment; sodium chloride provides suitable ionic strength; EDTA-Na2 further chelates metal ions; Triton-100 and polyvinylpyrrolidone act as surfactants, reducing non-specific binding, improving the signal-to-noise ratio, and further enhancing detection sensitivity.

[0066] In some embodiments, the sample pad contains: 0.5 mg / mL-1 mg / mL mouse IgG, 0.5 w / v%-1 w / v% Evans blue, 0.5 mg / mL-2 mg / mL HIRE-R-R001 (an inhibitor), 0.5 mg / mL-2 mg / mL erythrocytes, 5 w / v%-10 w / v% trehalose, 0.5 w / v%-3 w / v% Tween-20, and 2 w / v%-5 w / v% BSA. These components on the sample pad effectively reduce interference from nonspecific substances in the sample, improving the specificity and sensitivity of the detection. Mouse IgG and the inhibitor HIRE-R-R001 block nonspecific binding sites; Evans blue serves as a tracer dye, facilitating observation of sample flow; RBCs remove interference from erythrocytes in the sample; trehalose acts as a stabilizer, protecting antibody activity; and Tween-20 and BSA further reduce nonspecific adsorption, improving the signal-to-noise ratio.

[0067] For example, the concentrations of mouse IgG were 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, and 1 mg / mL; the concentrations of Evans blue were 0.5 w / v%, 0.6 w / v%, 0.7 w / v%, 0.8 w / v%, 0.9 w / v%, and 1 w / v%; the concentrations of the inhibitor R-001 were 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL; the concentrations of erythrocytes were 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL; the concentrations of trehalose were 5 w / v%, 6 w / v%, 7 w / v%, 8 w / v%, 9 w / v%, and 10 w / v%; the concentrations of Tween-20 were 0.5 w / v%, 1 w / v%, 1.5 w / v%, 2 w / v%, 2.5 w / v%, and 3 w / v%; and the concentration of BSA was 2... w / v%, 2.5 w / v%, 3 w / v%, 3.5 w / v%, 4 w / v%, 4.5 w / v%, 5 w / v%.

[0068] In some embodiments, the conjugate pad contains: 0.5 w / v%-3 w / v% of Tween-20, 2 w / v%-5 w / v% of BSA, 5 w / v%-10 w / v% of trehalose, 8 w / v%-10 w / v% of canine parvovirus fluorescent microsphere markers, 8 w / v%-10 w / v% of canine coronavirus fluorescent microsphere markers, 8 w / v%-10 w / v% of Giardia lamblia fluorescent microsphere markers, and 0.5 w / v%-1 w / v% of chicken IgY fluorescent microsphere markers.

[0069] To address the shortcomings of insufficient sensitivity of canine parvovirus reagents and the tendency to miss low-load samples, this study focuses on improving labeling titer and signal recognition. It optimizes the formulation of the buffer solution on the binding pad and innovatively adds signal amplifiers and surfactants to enhance the target binding signal and reduce non-specific matrix interference, thereby comprehensively improving the reagent's detection sensitivity.

[0070] For example, the concentrations of Tween-20 are 0.5 w / v%, 1 w / v%, 1.5 w / v%, 2 w / v%, 2.5 w / v%, and 3 w / v%; BSA: 2 w / v%, 2.5 w / v%, 3 w / v%, 3.5 w / v%, 4 w / v%, 4.5 w / v%, 5 w / v%, and any values ​​in between; the concentrations of trehalose are: 5 w / v%, 6 w / v%, 7 w / v%, 8 w / v%, 9 w / v%, 10 w / v%, and any values ​​in between; the concentrations of canine parvovirus fluorescent microsphere markers are: 8 w / v%, 8.5 w / v%, 9 w / v%, 9.5 w / v%, 10 w / v%, and any values ​​in between; the concentrations of canine coronavirus fluorescent microsphere markers are: 8 w / v%, 8.5 w / v%, 9 w / v%, 9.5 w / v%, 10 w / v%. w / v% and any value in between; the concentration of Giardia lamblia fluorescent microsphere labeling is: 8 w / v%, 8.5 w / v%, 9 w / v%, 9.5 w / v%, 10 w / v% and any value in between; the concentration of chicken IgY fluorescent microsphere labeling is: 0.5 w / v%, 0.6 w / v%, 0.7 w / v%, 0.8 w / v%, 0.9 w / v%, 1 w / v% and any value in between.

[0071] In some embodiments, the coating membrane is provided with detection lines and control lines.

[0072] The detection lines include a first detection line, a second detection line, and a third detection line.

[0073] In some embodiments, the first detection line is coated with canine coronavirus monoclonal antibody.

[0074] In some embodiments, the second detection line is coated with canine parvovirus monoclonal antibody.

[0075] In some embodiments, the third detection line is coated with Giardia lamblia monoclonal antibody.

[0076] In some embodiments, the control line is coated with goat anti-chicken antibody.

[0077] In some embodiments, the coating concentration of the canine coronavirus monoclonal antibody is between 0.8 mg / mL and 1.2 mg / mL. For example, the coating concentration of the canine coronavirus monoclonal antibody is 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, or 1.2 mg / mL, or any value in between.

[0078] In some embodiments, the coating concentration of the canine parvovirus monoclonal antibody is between 0.8 mg / mL and 1.2 mg / mL. For example, the coating concentration of the canine parvovirus monoclonal antibody is 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, or 1.2 mg / mL, or any value in between.

[0079] In some embodiments, the coating concentration of the Giardia lamblia monoclonal antibody is 0.4 mg / mL to 0.6 mg / mL. For example, the coating concentration of the Giardia lamblia monoclonal antibody is 0.4 mg / mL, 0.5 mg / mL, or 0.6 mg / mL, or any value in between.

[0080] In some embodiments, the coating concentration of the goat anti-chicken antibody is 0.4 mg / mL to 0.6 mg / mL. For example, the coating concentration of the goat anti-chicken antibody is 0.4 mg / mL, 0.5 mg / mL, or 0.6 mg / mL, or any value in between.

[0081] Another aspect of this application provides a method for preparing the aforementioned fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus, comprising:

[0082] Provide 0.01 M-0.05 M sodium dihydrogen phosphate, 0.01 M-0.05 M disodium hydrogen phosphate, 0.2 M-0.4 M sodium chloride, 0.001 M-0.005 M EDTA-Na2, 0.5 w / v%-1 w / v% Triton-100, 0.1 w / v%-1 w / v% polyvinylpyrrolidone, 0.01%-0.1% SDS, 0.001 mM-0.005 mM tetrasodium iminodisuccinate (IDS), and 0.01 w / v%-0.02 w / v% preservative, and mix them to a pH of 7.5-8.5 to prepare the sample dilution.

[0083] The canine parvovirus antibody fluorescent microsphere marker, the canine coronavirus antibody fluorescent microsphere marker, and the Giardia lamblia antibody fluorescent microsphere marker were prepared using a conjugation activation solution; and the above antibody fluorescent microsphere markers were sprayed onto the conjugate pad.

[0084] The first detection antibody, the second detection antibody, and the third detection antibody are sprayed onto the coating membrane to form the detection line;

[0085] The goat anti-chicken antibody was sprayed onto the coating membrane to form the control line;

[0086] Assemble the sample pad, the conjugation pad, the coating membrane, and the absorbent pad onto the base plate.

[0087] In some embodiments, the coupling activation solution comprises: 0.05 w / v%-0.2 w / v% of N-lauroyl glutamate sodium salt, 0.01 w / v%-0.1 w / v% of sulfobetaine-12, and 0.05 M-0.1 M of 2-(N-morpholino)ethanesulfonic acid, with a pH of 5.5-6.5. The optimized coupling activation solution can significantly improve the coupling efficiency between the antibody and the fluorescent microspheres, enhance the fluorescence signal intensity, and thus improve detection sensitivity. N-lauroyl glutamate sodium salt, as an amino acid-type anionic surfactant, possesses both mildness, hydrophilicity, and hydrophobicity, playing a role in dispersing, stabilizing, and resisting non-specific adsorption throughout the microsphere labeling process; 2-methylimidazole can stabilize the activation intermediate, reduce hydrolysis losses, and significantly improve coupling efficiency.

[0088] For example, the concentration of tetrasodium iminodisuccinate (IDS) in the sample diluent can be selected from any value among 0.001 mM, 0.002 mM, 0.003 mM, 0.004 mM, and 0.005 mM.

[0089] For example, the concentration of sodium N-lauroyl glutamate in the coupling activation solution can be selected from any value among 0.05 w / v%, 0.1 w / v%, 0.15 w / v%, and 0.2 w / v%; the concentration of sulfobetaine-12 can be selected from any value among 0.01 w / v%, 0.02 w / v%, 0.04 w / v%, 0.08 w / v%, and 0.1 w / v%.

[0090] In some embodiments, the method for preparing the antibody fluorescent microsphere label includes:

[0091] Fluorescent microspheres were dispersed in the coupling activation solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added for activation to prepare activated microspheres;

[0092] The activated microspheres were mixed with antibodies and then coupled to prepare antibody-fluorescent microsphere labels; and

[0093] The antibody fluorescent microsphere label was mixed with the blocking solution and then blocked.

[0094] Another aspect of this application provides a method for detecting canine parvovirus, canine coronavirus, and Giardia virus, comprising: using the aforementioned fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus.

[0095] The testing methods can be used for disease diagnosis and treatment purposes or for non-disease diagnosis and treatment purposes, such as studies on virus distribution or diversity, laboratory studies, etc.

[0096] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0097] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0098] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0099] Example 1

[0100] This embodiment provides a method for preparing a fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus, comprising the following steps:

[0101] I. Sample diluent: Sodium dihydrogen phosphate 0.01M, disodium hydrogen phosphate 0.01M, sodium chloride 0.3M, EDTA-Na2 0.005M, Triton-100 0.5 w / v %, polyvinylpyrrolidone 1 w / v %, SDS 0.1 w / v %, tetrasodium iminodisuccinate (IDS) 0.005mM, preservative 0.02 w / v %, pH = 7.5-8.5.

[0102] II. Preparation method of immunochromatographic test strips:

[0103] 1. Preparation of sample pad

[0104] The sample pad contained 1 mg / mL mouse IgG, 1 wt Evans blue, 2 mg / mL HIRE-R001 (blocker), 1 mg / mL erythrocytes, 5 w / v trehalose, 0.5 wt% Tween 20, and 2 wt% BSA. It was sprayed onto a glass fiber pad at a rate of 6 μL / cm and dried in a drying oven at 50°C for 24 hours.

[0105] 2. Preparation of fluorescent microsphere-labeled antibodies

[0106] Take 1 mL of 1% (10 mg / mL) fluorescent microspheres, shake well, then pick up the desired labeled fluorescent microspheres, centrifuge, and discard the supernatant. Add 1 mL of coupling activation solution, sonicate to disperse, centrifuge, and discard the supernatant.

[0107] (1) Preparation of mouse anti-CPV monoclonal antibody 3H6 fluorescent microsphere conjugate

[0108] Equilibrate EDC and NHS activators to room temperature. Weigh 10 mg of each activator and dissolve them in the conjugation activating solution to a concentration of 50 mg / mL. Prepare the activators fresh each time. Add 0.8 mL of the conjugation activating solution and sonicate. Then add 0.1 mL of 50 mg / mL EDC solution and 0.1 mL of 50 mg / mL NHS solution, and vortex for 20 minutes. Centrifuge to remove the supernatant. Add 1 mL of the conjugation activating solution and sonicate. Repeat centrifugation twice. Add the conjugation activating solution (original microsphere volume minus labeled antibody volume) and sonicate. Add mouse anti-CPV monoclonal antibody 3H6 to a concentration of 0.3 g / mL. Mix in the dark for 2 hours, then centrifuge to remove the supernatant. Add 1 mL of blocking solution (tris(hydroxymethyl)aminomethane, sodium casein, pH=8.0), sonicate, and mix in the dark at room temperature for 1 hour (or overnight at 4°C). Centrifuge to remove the supernatant. Add 1 mL of blocking solution (tris(hydroxymethyl)aminomethane, sodium casein, pH=8.0) and sonicate. Store at 4℃ for later use.

[0109] The coupling activation solution is: 0.2% N-lauroyl glutamate sodium, 0.02 w / v % sulfobetaine-12, and 0.1M 2-(N-morpholine) ethanesulfonic acid (pH = 5.5-6.5).

[0110] (2) Preparation of mouse anti-CCV monoclonal antibody 14A5-fluorescent microsphere conjugate

[0111] Equilibrate EDC and NHS activators to room temperature. Weigh 10 mg of each activator and dissolve them in the conjugation activating solution to a concentration of 50 mg / mL. Prepare the activators fresh each time. Add 0.8 mL of the conjugation activating solution and sonicate. Then add 0.1 mL of 50 mg / mL EDC solution and 0.1 mL of 50 mg / mL NHS solution, and vortex for 20 minutes. Centrifuge to remove the supernatant. Add 1 mL of the conjugation activating solution and sonicate. Repeat centrifugation twice. Add the conjugation activating solution (original microsphere volume minus labeled antibody volume) and sonicate. Add mouse anti-CCV monoclonal antibody 14A5 to a concentration of 0.3 g / mL, mix in the dark for 2 hours, and centrifuge to remove the supernatant. Add 1 mL of blocking solution (tris(hydroxymethyl)aminomethane, sodium casein, pH=8.0), sonicate, mix in the dark at room temperature for 1 hour (or overnight at 4°C), and centrifuge to remove the supernatant. Add 1 mL of blocking solution (tris(hydroxymethyl)aminomethane, sodium casein, pH=8.0) and sonicate. Store at 4℃ for later use.

[0112] (3) Preparation of mouse anti-GIA monoclonal antibody 14H7 fluorescent microsphere conjugate

[0113] Equilibrate EDC and NHS activators to room temperature. Weigh approximately 10 mg of each activator and dissolve them in the conjugation activating solution to a concentration of 50 mg / mL. Prepare the activators fresh each time. Add 0.8 mL of the conjugation activating solution and sonicate. Then add 0.1 mL of 50 mg / mL EDC solution and 0.1 mL of 50 mg / mL NHS solution, and vortex for 20 minutes. Centrifuge to remove the supernatant. Add 1 mL of the conjugation activating solution and sonicate. Repeat centrifugation twice. Add the conjugation activating solution (original microsphere volume minus labeled antibody volume) and sonicate. Add mouse anti-GIA monoclonal antibody 14H7 to a concentration of 0.3 mg / mL, mix in the dark for 2 hours, and centrifuge to remove the supernatant. Add 1 mL of blocking solution (tris(hydroxymethyl)aminomethane, sodium casein, pH=8.0), sonicate, mix in the dark at room temperature for 1 hour (or overnight at 4°C), and centrifuge to remove the supernatant. Add 1 mL of blocking solution (tris(hydroxymethyl)aminomethane, sodium casein, pH=8.0) and sonicate. Store at 4℃ for later use.

[0114] (4) Preparation of quality control line chicken IgY fluorescent microsphere conjugate

[0115] Equilibrate EDC and NHS activators to room temperature. Weigh 10 mg of each activator and dissolve them in the conjugation activating solution to a concentration of 50 mg / mL. Prepare the activators fresh each time. Add 0.8 mL of the conjugation activating solution and sonicate. Then add 0.1 mL of 50 mg / mL EDC solution and 0.1 mL of 50 mg / mL NHS solution, and vortex for 20 minutes. Centrifuge to remove the supernatant. Add 1 mL of the conjugation activating solution and sonicate. Repeat centrifugation twice. Add the conjugation activating solution (original microsphere volume minus labeled antibody volume) and sonicate. Add the control line chicken IgY antibody to a concentration of 0.2 mg / mL, mix in the dark for 2 hours, and centrifuge to remove the supernatant. Add 1 mL of blocking solution (tris(hydroxymethyl)aminomethane, sodium casein, pH=8.0), sonicate, mix in the dark at room temperature for 1 hour (or overnight at 4°C), and centrifuge to remove the supernatant. Add 1 mL of blocking solution (tris(hydroxymethyl)aminomethane, sodium casein, pH=8.0) and sonicate. Store at 4°C for later use.

[0116] 3. Preparation of binding pad

[0117] The conjugate pad contains 3 wt% Tween 20, 5 wt% BSA, 5% trehalose, 10% CPV fluorescent microsphere labeling, 8% CCV fluorescent microsphere labeling, 8% GIA fluorescent microsphere labeling, and 0.5% chicken IgY fluorescent microsphere labeling.

[0118] 4. Preparation of coating membrane

[0119] (1) Preparation of control lines: The sheep anti-chicken was diluted to 0.5 mg / mL with Tris buffer (trehalose 0.5%, NaCl 1%) at pH=8.0 and streaked on the nitrocellulose membrane at 1.0 μL / cm to obtain the control lines. The nitrocellulose membrane was then placed in a drying oven and dried at 50℃ for 72h.

[0120] (2) Preparation of the first detection line: The canine coronavirus monoclonal antibody was diluted to 1.0 mg / mL with Tris buffer (1% trehalose, 1% NaCl) at pH=8.0, and the line was streaked on the nitrocellulose membrane at 1.0 μL / cm to obtain the detection line 1. The nitrocellulose membrane was then placed in a drying oven and dried at 50°C for 72 h.

[0121] (3) Preparation of the second detection line: The canine parvovirus monoclonal antibody was diluted to 1.0 mg / mL with Tris buffer (1% trehalose, 1% NaCl) at pH 8.0, and the line was streaked on the nitrocellulose membrane at 1.0 μL / cm to obtain the detection line 2. The nitrocellulose membrane was then placed in a drying oven and dried at 50°C for 72 h.

[0122] (4) Preparation of the third detection line: The Giardia lamblia monoclonal antibody was diluted to 0.5 mg / mL with Tris buffer (1% trehalose, 0.5% NaCl) at pH=8.0, and the line was streaked on the nitrocellulose membrane at 1.0 μL / cm to obtain the detection line 3. The nitrocellulose membrane was then placed in a drying oven and dried at 50°C for 72 h.

[0123] 5. Preparation of absorbent pads

[0124] Cut the absorbent paper into strips measuring 30cm x 2.5cm each.

[0125] 6. Assembly

[0126] The coating film, absorbent pad, and sample pad are attached to a PVC base plate, and the attached intermediate material is cut into test strips of a certain width using a cutting machine.

[0127] Example 2

[0128] The difference between Example 2 and Example 1 is as follows:

[0129] In step 2, the coupling activation solution is 0.2% N-lauroyl glutamate sodium and 0.1M 2-(N-morpholine) ethanesulfonic acid (pH=5.5-6.5).

[0130] Example 3

[0131] The difference between Example 3 and Example 1 is as follows:

[0132] In step 2, the coupling activation solution is 0.5% lecithin, 0.02 w / v % sulfobetaine-12, and 0.1M 2-(N-morpholino)ethanesulfonic acid (pH = 5.5-6.5).

[0133] Example 4

[0134] The difference between Example 4 and Example 1 is as follows:

[0135] In step 2, the coupling activation solution is 0.1% alkyl glycoside (APG), 0.02 w / v % sulfobetaine-12, and 0.1M 2-(N-morpholino) ethanesulfonic acid (pH = 5.5-6.5).

[0136] Example 5

[0137] The difference between Example 5 and Example 1 is that in step 2, the coupling activation solution is 0.2 w / v % N-lauroyl glutamate sodium, 0.1 w / v % sulfobetaine-12, and 0.1 M 2-(N-morpholine) ethanesulfonic acid (pH = 5.5-6.5).

[0138] Example 6

[0139] The difference between Example 6 and Example 1 is as follows:

[0140] In step 2, the coupling activation solution is 0.2 w / v % N-lauroyl glutamate sodium, 0.01 w / v % sulfobetaine-12, and 0.5 M 2-(N-morpholine) ethanesulfonic acid (pH = 5.5-6.5).

[0141] Comparative Example 1

[0142] This comparative example is basically the same as Example 1, except that the sample diluent is:

[0143] Sodium dihydrogen phosphate 0.01M, disodium hydrogen phosphate 0.01M, sodium chloride 0.3M, EDTA-Na2 0.005M, Triton-100 1 w / v%, polyvinylpyrrolidone 1 w / v %, SDS 0.1 w / v %, 5mM hydroxyethyl bisphosphonic acid (HEPD), preservative 0.02 w / v %, pH=7.5-8.5.

[0144] Comparative Example 2

[0145] This comparative example is basically the same as Example 1, except that the sample diluent is:

[0146] Sodium dihydrogen phosphate 0.01M, disodium hydrogen phosphate 0.01M, sodium chloride 0.3M, EDTA-Na2 0.005M, Triton-100 1 w / v %, polyvinylpyrrolidone 1 w / v %, 0.005mM tetrasodium iminodisuccinate (IDS), preservative 0.02 w / v %, pH = 7.5-8.5.

[0147] Comparative Example 3

[0148] This comparative example is basically the same as Example 1, except that the sample diluent is:

[0149] Sodium dihydrogen phosphate 0.01M, disodium hydrogen phosphate 0.01M, sodium chloride 0.3M, EDTA-Na2 0.005M, Triton-100 1w / v%, polyvinylpyrrolidone 1w / v%, 1w / v% Tween 20, 0.005mM tetrasodium iminodisuccinate (IDS), preservative 0.02w / v%, pH=7.5-8.5.

[0150] Comparative Example 4

[0151] This comparative example is basically the same as Example 1, except that the sample diluent is:

[0152] Sodium dihydrogen phosphate 0.01M, disodium hydrogen phosphate 0.01M, sodium chloride 0.3M, EDTA-Na2 0.005M, S9 1w / v%, polyvinylpyrrolidone 1w / v%, 1w / v% Tween-20, 0.005mM tetrasodium iminodisuccinate (IDS), preservative 0.02w / v, pH=7.5-8.5.

[0153] Result verification:

[0154] 1. Reagent strips were prepared using six different coupling activation solution formulations (Examples 1, 2, 3, 4, 5, and 6) to detect CPV Ag (canine parvovirus) reference standard and blank dilution at concentrations of 8000 ng / mL, 4000 ng / mL, 1500 ng / mL, 500 ng / mL, 100 ng / mL, 40 ng / mL, and 20 ng / mL. A scatter plot was constructed with T / C as the ordinate and the reference standard concentration as the abscissa. The results are shown in Table 1 and... Figure 1 As shown:

[0155] Table 1: CPV Ag Reference Test - T / C Value

[0156]

[0157] 2. Reagent strips were prepared using six different coupling activation solution formulations (Examples 1, 2, 3, 4, 5, and 6) to detect CCV Ag (canine coronavirus) reference standard and blank dilution at concentrations of 35 µg / mL, 20 µg / mL, 15 µg / mL, 10 µg / mL, 6 µg / mL, and 2 µg / mL. The values ​​were plotted as follows: T / C ratio on the ordinate and reference standard concentration on the abscissa. The results are shown in Table 2. Figure 2 As shown:

[0158] Table 2: CCV Ag Reference Test - T / C Value

[0159]

[0160] 3. Six coupling activation solution formulations (Examples 1, 2, 3, 4, 5, and 6) were used to label reagent strips. The values ​​of GIA Ag (Giardia lamblia) reference standard and blank dilution with concentrations of 35 µg / mL, 20 µg / mL, 15 µg / mL, 10 µg / mL, 6 µg / mL, and 2 µg / mL were measured. A scatter plot was drawn with T / C as the ordinate and the reference standard concentration as the abscissa. The results are shown in Table 3. Figure 3 As shown.

[0161] Table 3: GIA Ag Reference Test - T / C Value

[0162]

[0163] Experimental conclusion:

[0164] As can be seen from the experimental data, in the triple detection (CPV / CCV / GIA Ag), the linearity R between the concentration of the reference sample in Example 1 and T / C is [not specified]. 2 >0.99 indicates the highest upper limit of T / C and the best gradient. Examples 2, 3, 4, 5, and 6 demonstrate linear R... 2<0.99. 2-(N-morpholino)ethanesulfonic acid is a commonly used and suitable buffer component for protein microsphere labeling, specifically designed to stabilize the fluorescent reaction environment. Sodium N-lauroyl glutamate is an amino acid-type anionic surfactant, possessing both mildness, hydrophilicity, and hydrophobicity. It plays a crucial role in the entire process of microsphere (fluorescent microspheres, latex microspheres, etc.) labeling (antigen / antibody conjugation), acting as a dispersant, stabilizer, and anti-nonspecific adsorption agent. It is well-suited to the practical needs of veterinary fluorescent immunochromatographic microsphere labeling, being non-irritating and not degrading biological activity, making it a preferred adjuvant in labeling systems. Sulfobetaine-12 is an amphoteric surfactant with good biocompatibility and low toxicity, used to stabilize proteins, reduce nonspecific adsorption, and lower background signals in bioanalysis. The combined use of 2-(N-morpholino)ethanesulfonic acid, N-lauroyl glutamate sodium salt, and sulfobetaine-12 resulted in the optimal upper limit and gradient of T / C in Example 1. This also meets the practical requirements for labeling veterinary fluorescent immunochromatographic microspheres, solving the technical pain points of existing coupling systems such as poor stability, numerous non-specific interferences, and insufficient detection accuracy. Compared with single components or combinations of any two components, it has more significant technical advantages and application value.

[0165] 4. Five CPV-positive samples and five CPV-negative samples were tested using five optimized sample dilutions (Comparative Examples 1, 2, 3, and 4, and Example 1), with PCR results serving as controls. Following the manufacturer's instructions, the CPV Ag detection range was 16-8000 ng / mL; the reference range was >60 ng / mL for positive. The results are shown in Table 4.

[0166] Table 4: CPV Ag test values ​​in clinical samples (unit: ng / mL)

[0167]

[0168] "+" indicates a positive test result, and "-" indicates a negative test result.

[0169] 5. Five CCV-positive samples and five CCV-negative samples were tested using five optimized sample dilutions (Comparative Examples 1, 2, 3, and 4, and Example 1), with PCR results serving as controls. Following the manufacturer's instructions, the CCV Ag detection range was 0-35 µg / mL; the reference range was >4 µg / mL for positive. The results are shown in Table 5.

[0170] Table 5: CCV Ag test values ​​in clinical samples (unit: µg / mL)

[0171]

[0172] "+" indicates a positive test result, and "-" indicates a negative test result.

[0173] 6. Five GIA-positive samples and five GIA-negative samples were tested using five optimized sample dilutions (Comparative Examples 1, 2, 3, and 4, and Example 1), with PCR results serving as controls. Following the manufacturer's instructions, the GIA Ag detection range was 0-35 µg / mL, and the reference range was >4 µg / mL for a positive result. The results are shown in Table 6.

[0174] Table 6: GIA Ag Clinical Sample Tests (Unit: µg / mL)

[0175]

[0176] "+" indicates a positive test result, and "-" indicates a negative test result.

[0177] A comprehensive analysis of the experimental data from Comparative Examples 1, 2, 3, and 4 and Example 1 revealed that the sample dilution formulation of Example 1 exhibited significant advantages. Comparative Examples 1 and 2 showed varying degrees of false negatives; specifically, sample 6 from Comparative Examples 1 and 2 showed a false negative in the CPV Ag clinical test; sample 12 from Comparative Examples 1 and 2 showed a false negative in the CCV Ag clinical test; and sample 21 from Comparative Example 1 showed a false negative in the GIA Ag clinical test. The sensitivity of Comparative Examples 3, 4, and 5 was not as good as that of Example 1. Example 1, consistent with the PCR control results, showed significantly higher specificity and sensitivity than Comparative Examples 1, 2, 3, and 4. The combination of components in this formulation effectively enhanced the specific binding signal and suppressed non-specific reactions, thus achieving a better signal-to-noise ratio and a wider detection dynamic range. Tetrasodium iminodisuccinate (IDS), as a green, mild, and highly efficient metal ion chelating agent, effectively chelates Ca in the sample. 2+ Mg 2+ Fe 2+ / 3+ Cu 2+ Mn 2+ They form stable water-soluble complexes. Combined use with EDTA is particularly effective for Fe... 3+ Cu 2+ Enhanced chelating power makes it more suitable for detecting canine infectious diseases in bloody fecal samples. It improves the false negative phenomenon in canine parvovirus and canine coronavirus cases caused by blood cells on the mucus membrane in the sample due to bloody stool symptoms, significantly increasing the detection rate of canine infectious diseases. SDS is a strong anionic surfactant with strong cleavage, solubilization, and interference removal effects. It can cleave and release viral antigens within proteins, making it easier for antibodies to bind to targets. It can also dissolve lipids, remove mucus interference, reduce sample viscosity, reduce non-specific binding of proteins and impurities, resulting in lower chromatographic background and fewer false negatives and false positives.

[0178] Compared with Comparative Examples 1, 2, 3, and 4, Example 1 demonstrates higher detection sensitivity and specificity. The gradient between positive and negative thresholds is more pronounced, the critical region is more clearly defined, and the gray area is reduced. The upper limit for high values ​​is higher, the line range is wider, high-concentration samples are not missed, and the applicability is broader.

[0179] 7. Using four optimized sample dilutions (Comparative Examples 1, 2, 3, and 4), one CPV-positive sample and one CPV-negative sample were tested respectively, compared with Example 1. PCR results were used as controls. The tests were repeated five times. The CPV Ag detection range was 16-8000 ng / mL, and the reference range was >60 ng / mL for positive. The coefficient of variation (CV) should be ≤15%. The results are shown in Table 7.

[0180] Table 7: CPV Ag clinical sample repeatability (unit: ng / mL)

[0181]

[0182] 8. Using four optimized sample dilutions (Comparative Examples 1, 2, 3, and 4), one CCV-positive sample and one CCV-negative sample were tested respectively, compared with Example 1. PCR results were used as controls. The tests were repeated five times. The CCV Ag detection range was 0-35 µg / mL, and the reference range was >4 µg / mL for positive. The coefficient of variation (CV) should be ≤15%. The results are shown in Table 8.

[0183] Table 8: Repeatability of CCV Ag in Clinical Samples (Unit: µg / mL)

[0184]

[0185] 9. Using four optimized sample dilutions (Comparative Examples 1, 2, 3, and 4), one GIA-positive sample and one GIA-negative sample were tested respectively, compared with Example 1. PCR results were used as controls. The tests were repeated five times. The detection range for GIA Ag was 0-35 µg / mL, and the reference range was >4 µg / mL for positive. The coefficient of variation (CV) should be ≤15%. The results are shown in Table 9.

[0186] Table 9: Repeatability of GIA Ag in Clinical Samples (Unit: µg / mL)

[0187]

[0188] Experimental results showed that, compared with Example 1, Comparative Examples 1, 2, 3, and 4 all had coefficients of variation ≥15%, indicating poor repeatability. Example 1 showed variation ≤15% in both positive and negative samples, indicating that EDTA in Example 1 broadly chelates Ca. 2+ Mg 2+Fe 3+ Cu 2+ For heavy metals, IDS complements EDTA by strongly complexing heavy metals and transition metals, synergistically resisting metal ion interference. The compound surfactant: Triton X-100 0.5% + SDS 0.1% (nonionic + anionic surfactant blend) desorbs, reduces matrix interference, and prevents aggregation. This effectively reduces CV (volume-dependent characterization) and comprehensively improves the repeatability of sample testing in fluorescence immunoassay.

[0189] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A fluorescent immunoassay kit for detecting canine parvovirus, canine coronavirus, and Giardia virus, characterized in that, include: Immunochromatographic test strips and sample diluent; The immunochromatographic test strip includes a base plate and a sample pad, a conjugate pad, a coating membrane, and an absorbent pad disposed on the base plate; The sample pad, conjugate pad, coating membrane, and absorbent pad are sequentially connected to each other in contact. The conjugate pad contains fluorescent microspheres labeled with canine parvovirus antibody, fluorescent microspheres labeled with canine coronavirus antibody, and fluorescent microspheres labeled with Giardia lamblia antibody. The immunochromatographic test strips detect canine parvovirus antigen, canine coronavirus antigen, and Giardia lamblia antigen using a double antibody sandwich method. The sample diluent comprises the following components: 0.01 M-0.05 M sodium dihydrogen phosphate, 0.01 M-0.05 M disodium hydrogen phosphate, 0.2 M-0.4 M sodium chloride, 0.001 M-0.005 M EDTA-Na2, 0.5 w / v%-1 w / v% Triton-100, 0.1 w / v%-1 w / v% polyvinylpyrrolidone, 0.01%-0.1% SDS, 0.001 mM-0.005 mM tetrasodium iminodisuccinate, and 0.01 w / v%-0.02 w / v% preservative, with a pH of 7.5-8.

5.

2. The fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus according to claim 1, characterized in that, The sample pad contains: 0.5 mg / mL-1 mg / mL mouse IgG, 0.5 w / v%-1 w / v% Evans blue, 0.5 mg / mL-2 mg / mL HIRE-R-R001 blocker, 0.5 mg / mL-2 mg / mL erythrocytes, 5 w / v%-10 w / v% trehalose, 0.5 w / v%-3 w / v% Tween-20, and 2 w / v%-5 w / v% BSA.

3. The fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus according to claim 1, characterized in that, The conjugate pad contains: 0.5 w / v%-3 w / v% Tween-20, 2 w / v%-5 w / v% BSA, 5 w / v%-10 w / v% trehalose, 8 w / v%-10 w / v% canine parvovirus fluorescent microsphere marker, 8 w / v%-10 w / v% canine coronavirus fluorescent microsphere marker, 8 w / v%-10 w / v% Giardia lamblia fluorescent microsphere marker, and 0.5 w / v%-1 w / v% chicken IgY fluorescent microsphere marker.

4. The fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus according to claim 1, characterized in that, The coating membrane is provided with detection lines and quality control lines; The detection lines include a first detection line, a second detection line, and a third detection line; The first detection line is coated with canine coronavirus monoclonal antibody; and / or The second detection line is coated with canine parvovirus monoclonal antibody; and / or The third detection line is coated with Giardia lamblia monoclonal antibody; and / or The quality control line is coated with sheep anti-chicken antibody.

5. The fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus according to claim 4, characterized in that, The coating concentration of the canine coronavirus monoclonal antibody is 0.8 mg / mL-1.2 mg / mL; and / or The coating concentration of the canine parvovirus monoclonal antibody is 0.8 mg / mL-1.2 mg / mL; and / or The coating concentration of the Giardia lamblia monoclonal antibody is 0.4 mg / mL to 0.6 mg / mL.

6. The fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus according to claim 4, characterized in that, The coating concentration of the goat anti-chicken antibody is 0.4 mg / mL-0.6 mg / mL.

7. The method for preparing the fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus according to any one of claims 1 to 6, characterized in that, include: Prepare the sample diluent by mixing 0.01 M-0.05 M sodium dihydrogen phosphate, 0.01 M-0.05 M disodium hydrogen phosphate, 0.2 M-0.4 M sodium chloride, 0.001 M-0.005 M EDTA-Na2, 0.5 w / v%-1 w / v% Triton-100, 0.1 w / v%-1 w / v% polyvinylpyrrolidone, 0.01%-0.1% SDS, 0.001 mM-0.005 mM tetrasodium iminodisuccinate, and 0.01 w / v%-0.02 w / v% preservative, with a pH of 7.5-8.

5. The canine parvovirus antibody fluorescent microsphere label, the canine coronavirus antibody fluorescent microsphere label, and the Giardia lamblia antibody fluorescent microsphere label were prepared using a coupling activation solution. The aforementioned antibody fluorescent microsphere labeling was then sprayed onto the conjugation pad; The first detection antibody, the second detection antibody, and the third detection antibody are sprayed onto the coating membrane to form the detection line; The goat anti-chicken antibody was sprayed onto the coating membrane to form the control line; Assemble the sample pad, the conjugation pad, the coating membrane, and the absorbent pad onto the base plate.

8. The method for preparing the fluorescent immunoassay kit for canine parvovirus, canine coronavirus, and Giardia virus according to claim 7, characterized in that, The coupling activation solution comprises: 0.05 w / v%-0.2 w / v% of N-lauroyl glutamate sodium, 0.01 w / v%-0.1 w / v% of sulfobetaine-12 and 0.05 M-0.1 M of 2-(N-morpholino)ethanesulfonic acid, with a pH of 5.5-6.

5.

9. The method for preparing the fluorescent immunochromatographic reagent kit for detecting canine parvovirus, canine coronavirus, and Giardia virus according to claim 8, characterized in that, The method for preparing the antibody fluorescent microsphere label includes: Fluorescent microspheres were dispersed in the coupling activation solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added for activation to prepare activated microspheres; The activated microspheres are mixed with antibodies and then coupled to prepare antibody fluorescent microsphere labels; the antibody fluorescent microsphere labels are mixed with blocking solutions, and the inert blocking agents in the blocking solutions are used to cover the unbound sites on the surface of the microspheres, quench residual activated groups, eliminate non-specific adsorption, reduce background fluorescence, protect antibody activity, and prevent microsphere aggregation.

10. A method for detecting canine parvovirus, canine coronavirus, and Giardia lamblia virus, characterized in that, include: The fluorescent immunoassay kits for canine parvovirus, canine coronavirus, and Giardia virus as described in any one of claims 1 to 6 were used to detect the samples to be tested.

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