A canine adenovirus, canine distemper virus and canine parvovirus immunoprotection evaluation chip and method

Abstract

The present application relates to the field of biological products for veterinary immunization effect detection and protein chip technology, and particularly relates to a canine adenovirus, canine distemper virus and canine parvovirus immune protection evaluation chip and method. The chip fixes the function-keeping antigen and function-loss mutant antigen of each virus, wherein the canine distemper virus sets two detection channels of receptor binding region and non-receptor type neutralizing epitope. After the canine serum to be tested reacts with the chip, the paired binding signal is obtained by using fluorescently labeled anti-canine IgG, the difference response of the function-keeping antigen and the function-loss mutant antigen is calculated after background subtraction and antigen fixation amount correction, and compared with the preset threshold. The scheme can reduce the interference of non-functional region binding antibodies and antigen fixation differences, and realize the synchronous evaluation of the antibody levels related to the immune protection of the three viruses.

Description

Technical Field

[0001] This invention relates to the field of immunization efficacy testing of veterinary biological products and protein chip technology, specifically to a chip and method for assessing immune protection against canine adenovirus, canine distemper virus, and canine parvovirus. Background Technology

[0002] Canine adenovirus, canine distemper virus, and canine parvovirus are important pathogens endangering canine health. Canine adenovirus infection can cause infectious hepatitis or respiratory diseases in dogs; canine distemper virus infection can affect the respiratory, digestive, and nervous systems; and canine parvovirus infection can cause severe hemorrhagic enteritis or myocardial damage. Vaccination is the primary measure to prevent these viral infections, but there are differences among dogs in age, maternal antibody levels, immune status, vaccine type, and immunization schedule. The antibody levels produced after vaccination and the actual protective immune capacity also vary among individuals. Therefore, it is necessary to test the antibody response after vaccination to assess the immunization effect and provide a basis for subsequent immunization programs.

[0003] Current methods for evaluating vaccine immunization efficacy mainly include enzyme-linked immunosorbent assay (ELISA), immunochromatographic assay, hemagglutination inhibition assay, and virus neutralization assay. Virus neutralization assays can directly reflect the ability of antibodies to inhibit viral infection, but they usually require cell culture and virus manipulation, have a long testing cycle, and demand high standards for experimental conditions, biosafety facilities, and operators, making them unsuitable for rapid testing of large numbers of samples. ELISA and immunochromatographic assays are relatively simple to perform, but their detection signals typically reflect the overall binding level of the sample's antibodies to viral antigens, making it difficult to directly distinguish between antibodies targeting viral invasion functional regions and antibodies binding to other regions.

[0004] Patent document CN102175874A discloses the preparation and use of a protein chip for evaluating vaccine immunization efficacy. This protein chip immobilizes protective antigens of multiple pathogens or antigenic substances obtained from the corresponding vaccines onto a substrate. By detecting the binding signal between antibodies and corresponding antigens in the sample, it achieves simultaneous detection of antibodies from multiple vaccines. This technology improves antibody detection throughput, but it uses a detection mode of direct antigen-antibody binding, and the detection results mainly reflect the overall binding level of the sample antibody to the immobilized antigen. Since the same viral protein usually contains multiple antigenic epitopes, this method has difficulty distinguishing between antibodies targeting viral receptor-binding regions or other invasion-related neutralizing epitopes and antibodies binding to non-functional regions. Furthermore, it does not establish a joint evaluation system for canine adenovirus, canine distemper virus, and canine parvovirus.

[0005] Patent document CN118795137A discloses a canine triple antibody chromatographic test strip and its preparation method. It uses quantum dots with different emission wavelengths to label canine distemper virus H protein, canine parvovirus VP2 protein, and canine infectious hepatitis virus Hexon protein, respectively. The presence of corresponding viral antibodies in the sample is determined based on the fluorescence values ​​at the corresponding wavelengths. This test strip can simultaneously detect three viral antibodies; however, it still relies on the overall binding signal between the antibody and the corresponding viral protein as the basis for judgment. It is mainly used for detecting antibody positivity or positivity or overall levels, and it is difficult to further reflect whether the detected antibody can block the binding of the virus to host cell receptors, interfere with viral membrane fusion, or inhibit viral entry into host cells.

[0006] Not all antibodies produced after vaccination have neutralizing or protective effects. While some antibodies can bind to viral proteins, the antigenic epitopes they recognize do not participate in viral receptor binding, membrane fusion, or cell entry. Therefore, evaluating immunization efficacy solely based on the overall antibody binding signal to viral antigens may result in a high overall antibody signal but insufficient actual virus neutralization capacity. Furthermore, canine distemper virus neutralizing antibodies may exert their effects by blocking the binding of the H protein to the host receptor or by interfering with the synergistic effect of the H and F proteins to inhibit viral membrane fusion; detection of a single functional pathway may still underestimate the protective effects of some antibodies.

[0007] Therefore, the existing technology lacks a high-throughput immunoprotection assessment technology that can simultaneously detect canine adenovirus, canine distemper virus and canine parvovirus in the same detection system, reduce interference from antibodies binding to non-functional regions, and more accurately reflect the level of specific antibodies against viral invasion of functional regions. Summary of the Invention

[0008] This invention aims to provide a chip and method for assessing the immune protection of canine adenovirus, canine distemper virus, and canine parvovirus. By comparing the antibody binding signals of functionally intact antigens and functionally lost mutant antigens, and reducing the interference of antibodies binding to non-functional regions, it enables the simultaneous detection of antibodies specific to the functional regions of the three viruses and the evaluation of the level of immune protection.

[0009] Firstly, in order to achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0010] A chip for assessing immune protection against canine adenovirus, canine distemper virus, and canine parvovirus includes a chip substrate and at least one reaction region disposed on the chip substrate, each reaction region comprising:

[0011] The canine adenovirus detection unit includes a canine adenovirus functional retention antigen point and a canine adenovirus functional loss mutation antigen point. The canine adenovirus functional retention antigen contains the amino acid sequence shown in SEQ ID NO: 1. The canine adenovirus functional loss mutation antigen is formed by mutating arginine at position 24 of SEQ ID NO: 1 to glutamic acid or alanine.

[0012] A canine distemper virus detection unit includes receptor-binding paired antigen sites and non-receptor-binding neutralizing epitope paired antigen sites; the functionally intact antigen in the receptor-binding paired antigen sites comprises the amino acid sequence shown in SEQ ID NO: 2, and the functionally lost mutant antigen is formed by mutating at least two of the following in SEQ ID NO: 2: tyrosine at position 467, aspartic acid at position 468, and arginine at position 471 to alanine; the functionally intact antigen in the non-receptor-binding neutralizing epitope paired antigen sites comprises the DIEREFD sequence shown in positions 180-186 of SEQ ID NO: 2, and the functionally lost mutant antigen is formed by replacing aspartic acid and / or arginine in the DIEREFD sequence.

[0013] The canine parvovirus detection unit includes a functional virus-like particle antigen spot formed by the VP2 protein as shown in SEQ ID NO: 3, and a functional loss-of-function mutant virus-like particle antigen spot formed by the VP2 protein of SEQ ID NO: 3 with asparagine mutated to lysine at position 93 and asparagine mutated to aspartic acid at position 323.

[0014] In each paired antigen, the function-preserving antigen and the function-loss mutant antigen are fixed in the same way and with the same molar amount. The chip is configured to determine the specific antibody level of the corresponding viral invasion functional region based on the difference in the correction antibody binding signals of the two antigens.

[0015] As a further improvement, the canine adenovirus loss-of-function mutant antigen is formed by mutating arginine at position 24 of SEQ ID NO: 1 to glutamic acid.

[0016] The canine distemper virus receptor-binding loss-of-function mutant antigen is formed by mutating tyrosine at position 467, aspartic acid at position 468, and arginine at position 471 of SEQ ID NO: 2 to alanine.

[0017] The canine distemper virus non-receptor-binding neutralizing epitope loss-of-function mutant antigen is formed by the mutation of aspartic acid at position 180 to tyrosine and arginine at position 183 to glycine in SEQ ID NO: 2, and contains the YIEGEFD sequence.

[0018] As a further improvement, for canine adenovirus paired antigen, canine distemper virus receptor-binding paired antigen, and canine parvovirus paired antigen, the binding signal of the loss-of-function mutant antigen to the corresponding host receptor is no higher than 40% of the binding signal of the function-maintaining antigen to the host receptor, and the binding signal of the loss-of-function mutant antigen to the conformational antibody that recognizes the non-target region is no lower than 80% of the binding signal of the function-maintaining antigen to the conformational antibody.

[0019] As a further improvement, the host receptor corresponding to the canine adenovirus pairing antigen is the Coxsackievirus and adenovirus receptor CAR; the host receptor corresponding to the canine distemper virus receptor-binding pairing antigen is the canine SLAM receptor; and the host receptor corresponding to the canine parvovirus pairing antigen is the canine transferrin receptor.

[0020] As a further improvement, the canine distemper virus non-receptor-binding neutralizing epitope function-maintaining antigen and function-loss mutant antigen respectively include spacer sequences disposed at both ends of the epitope and a directional immobilization group disposed at one end away from the epitope; the binding signal of the neutralizing antibody recognizing the DIEREFD sequence to the function-loss mutant antigen is not higher than 20% of its binding signal to the function-maintaining antigen.

[0021] As a further improvement, the particle size of the canine parvovirus functional retention virus-like particles and the loss-of-function mutant virus-like particles is 20-30 nm, and the difference in their main particle size does not exceed 15%. The binding signal of the loss-of-function mutant virus-like particles to the canine transferrin receptor is 10%-40% of the corresponding binding signal of the functional retention virus-like particles.

[0022] As a further improvement, each antigen has the same biotinylated tag at the end away from the target functional region, and the chip substrate is a streptavidin-modified substrate, with each antigen being oriented and fixed to the chip substrate by the biotinylated tag.

[0023] As a further improvement, each antigen is provided with at least 3 replicates; each reaction zone also includes a canine IgG positive control point, a bovine serum albumin negative control point, a blank control point, and an antigen fixed amount control point, wherein the signal variation coefficient of the replicates does not exceed 15%.

[0024] As a further improvement, the ratio of the fixed amount control signal of the functional retention antigen and the functional loss mutation antigen of the same paired antigen is 0.85 to 1.15; the chip includes 8, 16, 24 or 48 reaction regions that are isolated from each other.

[0025] Secondly, the present invention also provides a method for detecting functional region-specific antibodies against canine adenovirus, canine distemper virus, and canine parvovirus in canine in vitro samples, employing the immune protection assessment chip described herein, comprising:

[0026] S1, after diluting the canine in vitro sample, add it to the reaction area of ​​the immune protection assessment chip, so that the antibodies in the sample bind to each functional maintenance antigen and functional loss mutation antigen respectively;

[0027] S2, clean the immune protection assessment chip, add labeled anti-canine immunoglobulin detection antibody, incubate, and then clean again;

[0028] S3, collect the detection signals of each antigen site, subtract the local background signal, and use the antigen fixation control signal or conformation control signal to correct the detection signals of functionally maintained antigens and functionally lost mutant antigens, respectively.

[0029] S4. Calculate the difference between the correction signal of the functional preservation antigen and the correction signal of the functional loss mutation antigen for each paired antigen to obtain the antibody score of the canine adenovirus functional region, the antibody score of the canine distemper virus receptor-binding region, the antibody score of the canine distemper virus non-receptor-binding neutralizing epitope, and the antibody score of the canine parvovirus functional region.

[0030] As a further improvement, the canine in vitro sample is canine serum or canine plasma, and the canine in vitro sample is diluted 50 to 200 times for single-point detection; the sample and the chip are incubated at 37°C for 20 to 40 minutes, and the labeled anti-canine immunoglobulin detection antibody is a fluorescently labeled anti-canine IgG antibody.

[0031] As a further improvement, the functional region antibody score of the i-th paired antigen... Determined according to the following formula:

[0032] ;

[0033] In the formula, The functional region antibody score corresponding to the i-th paired antigen; The detection signal is for the antigen that preserves the function of the sample to be tested; This is the detection signal for the sample to be tested and the loss-of-function mutant antigen; A fixed amount or conformational control signal for maintaining the function of the antigen; It is a fixed amount or conformational control signal for loss-of-function mutant antigens.

[0034] As a further improvement, the canine in vitro samples are serially diluted, and the highest sample dilution factor in which the difference between the correction signal of the functional retention antigen and the correction signal of the functional loss mutation antigen is still higher than the corresponding detection threshold is determined as the functional region-specific antibody titer of the corresponding virus; when the detection signal of the functional retention antigen is lower than the detection limit, the corresponding functional region-specific antibody is determined to be undetectable.

[0035] As a further improvement, evaluation thresholds for antibody scores in each functional region are established using canine serum with reference test results; the reference test for canine adenovirus and canine distemper virus is the virus neutralization test, and the reference test for canine parvovirus is the virus neutralization test or the hemagglutination inhibition test, and the evaluation thresholds are determined based on the receiver operating characteristic curves of the functional region antibody scores and the reference test results.

[0036] As a further improvement, when the canine adenovirus functional region antibody score reaches the canine adenovirus evaluation threshold, the canine distemper virus receptor-binding region antibody score and the non-receptor-binding neutralizing epitope antibody score reach the corresponding evaluation threshold, and the canine parvovirus functional region antibody score reaches the canine parvovirus evaluation threshold, the evaluation result is output that the functional antibodies of the three viruses have reached the set level; when the functional region antibody score of any virus does not reach the corresponding evaluation threshold, the evaluation result is output that the functional antibody of that virus has not reached the set level.

[0037] This invention, based on viral antigens shown in SEQ ID NO: 1-3, sets specific amino acid mutations at receptor binding sites or neutralizing epitopes to form functionally maintained antigens and loss-of-function mutant antigens corresponding to the sequence range, fixation method, and molar fixation amount. The difference in the corrected binding signals between these two antigens is used to extract specific antibody responses to the receptor binding region or neutralizing epitope, thereby reducing the influence of binding antibodies against non-functional regions, differences in antigen fixation amount, and non-specific background on the detection results. This makes the evaluation results more reflective of the antibody's ability to block viral receptor binding or interfere with the viral invasion process. Specifically, the canine distemper virus detection unit simultaneously sets receptor-binding paired antigens and non-receptor-binding neutralizing epitope paired antigens, which can take into account both receptor blocking and membrane fusion interference neutralization mechanisms, reducing the risk of missed detection caused by a single detection pathway. The three viral detection units are integrated into a single chip, enabling the analysis of multiple functional antibodies in a single test. This offers advantages such as low sample volume, high throughput, good repeatability, and short detection cycle, and reduces reliance on live virus culture and high-level biosafety conditions. It is suitable for evaluating vaccine immunization efficacy, dynamic monitoring of functional antibodies, and optimization of immunization programs. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the structure and dot array arrangement of the immune protection assessment chip for canine adenovirus, canine distemper virus and canine parvovirus of the present invention.

[0039] Figure 2 This is a schematic diagram illustrating the principle of detecting functional region-specific antibodies using the pairing signal of functionally intact antigens and functionally lost mutant antigens.

[0040] Figure 3 This is a flowchart of the immune protection assessment method of the present invention.

[0041] Figure 4Figure showing the quality control results for functional preservation antigens and loss-of-function mutant antigens of CAV, CDV, and CPV.

[0042] Figure 5 This is a graph showing the performance deviation rate results obtained by converting the repeatability and storage stability of the immune protection evaluation chip of this invention.

[0043] Figure 6 This is a comparison graph showing the sensitivity, specificity, and area under the curve when the present invention detects CAV, CDV, and CPV.

[0044] Figure 7 This graph shows the comparison results between the method of the present invention and detection methods that use only functional preservation antigens, do not perform antigen fixation amount correction, use whole denatured antigens, and use different fixation methods for paired antigens. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the methods for molecular cloning, protein expression and purification, antigen fixation, immunological detection, cell culture and statistical analysis are all performed using conventional methods in the art; the reagents and instruments used can be commercially available products with the same functions, unless otherwise specified by the manufacturer.

[0046] The canine adenovirus referred to in this invention is abbreviated as CAV, preferably canine adenovirus type 2; canine distemper virus is abbreviated as CDV; and canine parvovirus is abbreviated as CPV.

[0047] The functional preservation antigen referred to in this invention refers to an antigen that maintains the corresponding viral receptor binding function, key neutralizing epitope antibody binding function, or virus-like particle conformation; the loss-of-function mutant antigen refers to an antigen that has the same or substantially the same viral source sequence range and overall conformation as the corresponding functional preservation antigen, but whose target receptor binding function or target neutralizing antibody binding function is significantly reduced through one or more amino acid mutations.

[0048] In the following embodiments, the relative binding activity of the loss-of-function mutant antigen to the target receptor or target neutralizing antibody is no higher than 40% of that of the corresponding functionally intact antigen, and the relative binding activity to non-target conformational antibodies is no less than 80% of that of the corresponding functionally intact antigen. Through the above quality control, the signal difference between paired antigens mainly reflects the antibodies in the test sample targeting the viral invasion functional region or key neutralizing epitopes, rather than the signal difference caused by overall antigen denaturation, differences in antigen immobilization, or non-specific adsorption.

[0049] Example 1: Preparation of CAV function-preserving antigen and loss-of-function mutant antigen

[0050] The nucleic acid sequence encoding the head domain of the CAV-2 spike protein was designed based on SEQ ID NO:1, and codons were optimized according to the codon preferences of the E. coli expression system. Recombinant expression vectors encoding SEQ ID NO:1, the R24E mutant, and the R24A mutant were constructed, respectively.

[0051] The 24th amino acid of SEQ ID NO:1 corresponds to the 384th amino acid of the full-length CAV-2 spike protein sequence. A flexible linker peptide, AviTag, and a hexahistidine purification tag are sequentially linked to the carboxyl terminus of the antigen coding sequence. The flexible linker peptide, AviTag, and hexahistidine purification tag are not part of the viral antigen sequence itself.

[0052] The correctly sequenced expression vectors were transformed into E. coli BL21(DE3) competent cells, and single colonies were picked and inoculated into liquid culture medium containing the corresponding antibiotics and cultured at 37°C and 200 rpm until OD500. 600 The concentration was 0.6–0.8. Isopropyl-β-D-thiogalactoside was added to a final concentration of 0.2 mmol / L, and expression was induced at 18°C ​​for 16 hours.

[0053] The bacterial cells were collected by centrifugation and resuspended in lysis buffer containing 20 mmol / L Tris-HCl, 300 mmol / L NaCl, and 20 mmol / L imidazole at pH 8.0. After sonication, the cells were centrifuged at 12000×g for 30 minutes at 4°C, and the supernatant was collected.

[0054] The supernatant was purified by nickel affinity chromatography, and the target protein was eluted with a buffer containing 250 mmol / L imidazole. The eluted fraction was further purified by molecular sieve chromatography with a molecular sieve equilibration buffer of 20 mmol / L HEPES, 150 mmol / L NaCl, pH 7.4.

[0055] SDS-PAGE grayscale analysis showed that the purities of the SEQ ID NO:1 functional retention antigen, R24E mutant antigen, and R24A mutant antigen were 94.8%, 93.7%, and 94.1%, respectively. Molecular sieve chromatography results showed that the proportions of the trimer main peaks of the three antigens were 92.6%, 90.8%, and 91.3%, respectively, with no obvious macromolecular aggregation peaks observed.

[0056] The purified antigens were treated with biotin ligase to induce site-specific biotinylation of AviTag. Antibiotin binding assays showed that the biotinylation rates of all three antigens were not less than 85%.

[0057] Receptor binding activity was detected using a canine CAR-Fc fusion protein composed of the extracellular regions of canine Coxsackievirus and adenovirus receptors and a canine IgG Fc fragments. Canine CAR-Fc was used at concentration gradients of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, and 8 μg / mL, and the binding response of each antigen was detected using a biomembrane interferometry method.

[0058] The maximum binding response value of the antigen maintained by SEQ ID NO:1 was 100%. The relative canine CAR binding activity of the R24E mutant antigen was 2.8%, and the relative canine CAR binding activity of the R24A mutant antigen was 24.6%.

[0059] Further detection was performed using a conformational monoclonal antibody that recognizes the non-receptor-binding region of the CAV-2 spike protein. The relative binding activities of the R24E and R24A mutant antigens to the conformational monoclonal antibody were 91.6% and 94.2%, respectively.

[0060] The quality control results of CAV paired antigens are shown in Table 1.

[0061] Table 1. Quality control results of CAV paired antigens

[0062]

[0063] As shown in Table 1, the target receptor binding activity of both R24E and R24A mutant antigens is less than 40%, while the non-target conformation retention rate is greater than 80%, making them suitable as loss-of-function mutant antigens. Among them, the receptor binding function of the R24E mutant antigen is reduced more fully, therefore, the R24E mutant antigen is the preferred CAV loss-of-function mutant antigen for subsequent chip fabrication.

[0064] like Figure 4 As shown, the functional antigen of SEQ ID NO:1 exhibits a clear canine CAR concentration-dependent binding; the canine CAR binding response of the R24E mutant antigen is close to the background; the R24A mutant antigen still retains weak binding, but the relative binding activity is within the preset functional loss range.

[0065] Example 2: Preparation of CDV receptor binding function-maintaining antigens and loss-of-function mutant antigens

[0066] The extracellular coding sequence of the CDVH protein was designed based on SEQ ID NO:2. A mammalian cell secretion signal peptide was placed at the N-terminus of the coding sequence, and a trimerization domain, a flexible linker peptide, an AviTag, and a hexahistidine purification tag were sequentially placed at the C-terminus.

[0067] Site-directed mutagenesis was used to construct Y467A / D468A / R471A triple mutant, Y467A / D468A double mutant, Y467A / R471A double mutant, and D468A / R471A double mutant, respectively. Positions 467, 468, and 471 of SEQ ID NO:2 correspond to positions 525, 526, and 529 of the full-length CDVH protein sequence, respectively.

[0068] The correctly sequenced vector was transfected into HEK293F suspension cells. The cell density at transfection was 1.5 × 10⁶ cells / year. 6 Cells per mL, with a viability of not less than 95%. After transfection, cells were cultured at 37°C, 5% CO2, and 120 rpm for 6 days.

[0069] The culture supernatant was collected, centrifuged at 5000×g for 20 minutes at 4°C to remove cells and cell debris, and then filtered through a 0.22μm filter membrane. The filtered culture supernatant was then purified sequentially by nickel affinity chromatography and molecular sieve chromatography.

[0070] The purities of the functional preservation antigen, triple mutation antigen, Y467A / D468A double mutation antigen, Y467A / R471A double mutation antigen, and D468A / R471A double mutation antigen were 92.9%, 91.8%, 92.2%, 91.5%, and 93.0%, respectively; the proportions of the soluble oligomer main peak were 89.7%, 87.9%, 88.4%, 86.8%, and 89.1%, respectively.

[0071] Receptor binding activity was detected using the canine SLAM extracellular domain-Fc fusion protein. The maximum binding response value of the antigen maintained by SEQ ID NO:2 was set at 100%. The detection results for each mutant antigen are shown in Table 2.

[0072] Table 2. Quality control results of CDV receptor binding pairing antigens

[0073]

[0074] As shown in Table 2, the relative canine SLAM binding activity of the four mutant antigens is no higher than 40%, and the relative binding activity of the non-receptor binding region conformational monoclonal antibody is no less than 80%. Therefore, they all meet the quality control requirements for loss-of-function mutant antigens.

[0075] Subsequent embodiments preferably use the Y467A / D468A / R471A triple mutant antigen. The target receptor binding activity of the triple mutant antigen is only 3.1% of that of the functionally intact antigen, while the non-target conformation retention rate is 88.7%, which can significantly reduce canine SLAM binding while maintaining the non-target conformation of the CDVH protein.

[0076] like Figure 4 As shown, the functional antigen of SEQ ID NO:2 produces a clear canine SLAM binding curve, the binding response of the triple mutant antigen and the two double mutant antigens containing Y467A is close to the background, and the D468A / R471A double mutant antigen retains a small amount of canine SLAM binding activity.

[0077] Example 3: Preparation of CDV vaccine lineage-associated non-receptor type neutralizing epitope pairing antigen

[0078] This embodiment is used to detect lineage-associated non-receptor neutralizing epitope antibodies formed after immunization with the America-1 lineage vaccine.

[0079] The following two epitope peptides were synthesized respectively:

[0080] Functional epitope peptide: Biotin-GGG-DIEREFD-GGG;

[0081] Loss-of-function mutant epitope peptide: Biotin-GGG-YIEGEFD-GGG.

[0082] DIEREFD corresponds to amino acids 180 to 186 of SEQ ID NO:2; YIEGEFD is formed by mutations of D180Y and R183G. The amino and carboxyl terms of both epitope peptides are provided with identical flexible spacer sequences to reduce the impact of the chip substrate on the accessibility of short peptide epitopes.

[0083] Both epitope peptides were prepared using the same solid-phase synthesis, purification, and quantification methods. High-performance liquid chromatography (HPLC) analysis showed that the purities of the functionally intact epitope peptide and the loss-of-function mutant epitope peptide were 97.6% and 97.2%, respectively; mass spectrometry results were consistent with the theoretical molecular weights.

[0084] The binding activity of two epitope peptides was detected using a 2D12 neutralizing monoclonal antibody that recognizes the DIEGEFD epitope. The binding signal of the functionally intact epitope peptide was set at 100%, and the relative binding activity of the YIEGEFD loss-of-function mutant epitope peptide was 3.9%.

[0085] The immobilization amounts of two epitope peptides were determined by fluorescently labeled avidin detection molecules. The signal ratio of the immobilized amount of the mutant epitope peptide to the immobilized amount of the functionally intact epitope peptide was 1.04, which was in the range of 0.85 to 1.15.

[0086] like Figure 4 As shown, the DIEGEFD functional epitope peptide showed significant binding to the 2D12 neutralizing monoclonal antibody, while the binding signal of the YIEGEFD mutant epitope peptide was close to that of the negative control.

[0087] It should be noted that the DIEREFD / YIEGEFD paired detection pathway is mainly used to evaluate lineage-related neutralizing epitope antibodies formed after immunization with America-1 lineage vaccines such as Onderstepoort. For samples that may be infected with other CDV lineage viruses, this epitope pathway is not used as a general protective indicator on its own, but is evaluated in conjunction with the CDV receptor binding pathway in Example 2.

[0088] Example 4: Preparation of CPV function-preserving virus-like particles and loss-of-function mutant virus-like particles

[0089] The mature CPVVP2 protein coding sequence was designed based on SEQ ID NO:3 and optimized according to the codon preferences of the baculovirus expression system. Site-directed mutagenesis was used to mutate asparagine at position 93 of SEQ ID NO:3 to lysine and asparagine at position 323 to aspartic acid, yielding the N93K / N323D double-mutant VP2 coding sequence.

[0090] Recombinant baculoviruses expressing SEQ ID NO:3 and the N93K / N323D double mutant VP2 were prepared. The Sf9 cell density was adjusted to 2.0 × 10⁶ cells / year. 6 Cells were inoculated at a density of 1 / mL with a cell viability of not less than 95%, and inoculated with recombinant baculovirus at a multiplicity of infection of 3, and cultured at 27°C for 72 hours.

[0091] Cells were collected, resuspended and lysed in a buffer solution of 20 mmol / L HEPES, 150 mmol / L NaCl, pH 7.4. After removing cell debris by low-speed centrifugation, virus-like particles were purified sequentially by sucrose density gradient centrifugation, ultrafiltration, and molecular sieve chromatography.

[0092] SDS-PAGE analysis showed that the VP2 purity of the functionally preserved virus-like particle (SEQ ID NO:3) and the N93K / N323D mutant virus-like particle was 93.5% and 92.8%, respectively.

[0093] The morphology of virus-like particles was observed using transmission electron microscopy, and the particle size was determined using dynamic light scattering. The results are shown in Table 3.

[0094] Table 3. Quality control results of CPV-paired virus-like particles

[0095]

[0096] As shown in Table 3, the average particle size difference between the two types of virus-like particles was 3.2%, and both formed nearly spherical particles with a particle size of 20–30 nm. The canine transferrin receptor binding activity of the N93K / N323D mutant virus-like particles decreased to 17.8%, while the binding activity against conformational monoclonal antibodies that recognize the conserved non-receptor binding region of VP2 remained at 88.9%.

[0097] like Figure 4 As shown, the particle size distributions of the two types of virus-like particles are basically the same, while the canine transferrin receptor binding curve of the N93K / N323D mutant virus-like particles is significantly lower than that of the functionally preserved virus-like particles.

[0098] Example 5: Fabrication of an immune protection assessment chip

[0099] like Figure 1 As shown, a low autofluorescence glass substrate coated with streptavidin was used to divide the chip substrate into 16 isolated reaction zones. Each reaction zone was used to detect one canine serum or plasma sample.

[0100] Each reaction zone contains CAV functional preservation antigen points, CAVR24E functional loss mutation antigen points, CDV receptor binding functional preservation antigen points, CDVY467A / D468A / R471A functional loss mutation antigen points, CDVDIEREFD functional preservation epitope sites, CDVYIEGEFD functional loss mutation epitope sites, CPV functional preservation virus-like particle points, CPVN93K / N323D functional loss mutation virus-like particle points, as well as canine IgG positive control points, negative control points, fixed-amount correction points, and blank points.

[0101] Three parallel replicates were set up for each detection site. Functional preservation antigens and loss-of-function mutant antigens within the same pair were prepared at equimolar concentrations. CPV virus-like particles were prepared at molar equivalents of the VP2 subunit, and the spot concentration was further adjusted using a fixed-volume correction signal.

[0102] After spotting, the chip was placed in an environment with a relative humidity of 60% for 2 hours to allow the biotinylated antigen to fully bind with streptavidin. Subsequently, it was blocked with phosphate buffer containing 1% bovine serum albumin and 0.05% Tween-20 for 1 hour, washed, dried under low humidity and light-protected conditions, and then sealed for storage.

[0103] Three batches of chips, C260601, C260608, and C260615, were prepared respectively. The immobilization amount of each paired antigen was determined using fluorescently labeled avidin-detecting molecules, and the results are shown in Table 4.

[0104] Table 4. Paired antigen fixation ratio of the three batches of chips

[0105]

[0106] The fixed signal ratios of each paired antigen in the three batches of chips were all in the range of 0.85 to 1.15, and the coefficients of variation at parallel points were all no greater than 15%.

[0107] like Figure 1As shown, functional preserving antigen sites and functional loss mutation antigen sites within the same pair are arranged adjacently, thereby reducing differences in local chip temperature, washing intensity, and scanning position.

[0108] Example 6: Chip Detection Method

[0109] Collect canine serum samples and dilute the serum 1:100 with phosphate buffer containing 1% bovine serum albumin and 0.05% Tween-20.

[0110] Add 80 μL of diluted sample to each reaction zone and incubate at 37°C for 45 minutes. After incubation, wash four times with phosphate buffer containing 0.05% Tween-20.

[0111] Add fluorescently labeled anti-canine IgG secondary antibody to each reaction zone and incubate at 37°C for 30 minutes. Wash four times again, rinse rapidly with purified water, dry, and scan using a fluorescence chip scanner. Scanning parameters were set according to the principle that the positive control point should be within the linear range of the detector.

[0112] like Figure 2 As shown, antibodies binding to non-functional regions can bind to both functionally intact antigens and loss-of-function mutant antigens simultaneously, and their corresponding signals are canceled out during the differential calculation process; antibodies targeting receptor-binding regions or key neutralizing epitopes mainly bind to functionally intact antigens, thereby forming differential signals between functionally intact antigen sites and loss-of-function mutant antigen sites.

[0113] like Figure 3 As shown, the scan data were sequentially processed through background subtraction, fixed amount correction, paired difference calculation, and immune protection-related antibody level assessment.

[0114] Let S be the sample signal after background subtraction of the functionally preserving antigen in the i-th detection. i,w The sample signal after background subtraction of the loss-of-function mutant antigen is S. i,m The fixed amount correction signal for maintaining the function of the antigen is C. i,w The fixed-amount correction signal for the loss-of-function mutant antigen is C. i,m The functional region-specific antibody response value Fᵢ is calculated according to the following formula:

[0115] F i =[(S i,w / C i,w )-(S i,m / C i,m )] / (S i,w / C i,w ).

[0116] When the background subtraction signal of the functional preserving antigen is below the predetermined limit of detection, the result is reported as below the limit of detection; when the calculated result is less than zero, it is reported as no functional region-specific difference signal is detected.

[0117] Taking CAV detection of sample S10 as an example, the original fluorescence value of the functionally preserved antigen point is 18420, the original fluorescence value of the functionally lost-function mutant antigen point is 6830, and the local background value is 310; the fixed-amount correction signal of the functionally preserved antigen is 12600, and the fixed-amount correction signal of the functionally lost-function mutant antigen is 12420. Calculations show that...

[0118] The functional preservation antigen correction signal is:

[0119] N w = (18420-310) / 12600 = 1.437;

[0120] The correction signal for loss-of-function mutant antigens is:

[0121] N_m=(6830-310) / 12420=0.525;

[0122] CAV functional region-specific antibody response value:

[0123] F_CAV=(1.437-0.525) / 1.437=0.635.

[0124] When calculating using the unrounded values ​​exported from the instrument, the final recorded value is 0.634.

[0125] Example 7: Optimization of Detection Conditions

[0126] The same group of high, medium, and low antibody level samples and negative samples were tested using serum dilutions of 1:50, 1:100, and 1:200, respectively. The results are shown in Table 5.

[0127] Table 5 Results of serum dilution optimization

[0128]

[0129] At a 1:50 dilution, the positive signal was strong, but the background of negative samples was significantly increased; at a 1:200 dilution, some low-level positive samples were close to the limit of detection; at a 1:100 dilution, positive samples maintained a high signal-to-noise ratio, while the negative background and the coefficient of variation for repeat points were low. Therefore, 1:100 was determined to be the preferred serum dilution factor.

[0130] Further comparisons were made of sample incubation times of 30 minutes, 45 minutes, and 60 minutes. With a positive sample signal at 45 minutes as 100%, the signal retention rates at 30 minutes, 45 minutes, and 60 minutes were 82.3%, 100.0%, and 104.6%, respectively, with parallel point variation coefficients of 8.7%, 6.1%, and 6.4%. Considering both detection time and signal stability, incubation at 37°C for 45 minutes was determined to be the optimal condition.

[0131] Example 8: Precision and Stability Test

[0132] Repeatability tests were conducted using canine serum samples with high, medium, and low levels of CAV, CDV, and CPV functional region-specific antibodies, as well as a reference sample with negative results for all three viruses.

[0133] In intra-chip repeatability testing, multiple repetitive reaction zones are set up within the same chip for the same sample; in inter-chip repeatability testing, different chips from the same batch are used to test the same sample; in inter-batch repeatability testing, chips from three independent batches, C260601, C260608, and C260615, are used to test the same sample.

[0134] The test results are shown in Table 6.

[0135] Table 6 Chip Precision Results

[0136]

[0137] As shown in Table 6, the coefficients of variation within and between chips are all no greater than 15%, and the coefficients of variation between batches are all no greater than 20%, indicating that the chips of the present invention have good repeatability and batch-to-batch consistency.

[0138] In the stability test, the chip was sealed and stored at 2-8℃, and tested at 0 months, 1 month, 3 months, 6 months, 9 months and 12 months respectively. The results are shown in Table 7.

[0139] Table 7. Chip Storage Stability Results

[0140]

[0141] After 12 months of storage, the average signal retention rate of the positive reference serum was 86.7%, which was higher than the initial value of 80%, and no false positive results exceeding the judgment threshold were found in the negative samples.

[0142] like Figure 5 As shown, the detection values ​​are concentrated within the chip, between chips, and between different batches; the signal slowly decreases as the storage time increases, but still meets the preset quality control requirements after 12 months.

[0143] Example 9: Methodological evaluation of canine serum samples

[0144] Ninety-six canine serum samples with vaccine immunization records were selected. Each sample was subjected to the chip detection method of this invention and a reference neutralization test.

[0145] In this study, the reference positive thresholds were defined as CAV neutralizing antibody titers of at least 1:32, CDV neutralizing antibody titers of at least 1:32, and CPV neutralizing antibody titers of at least 1:128. There were 62 CAV reference positive samples, 56 CDV reference positive samples, and 46 CPV reference positive samples.

[0146] The test results of some representative samples are shown in Table 8.

[0147] Table 8. Detection results of representative canine serum samples

[0148]

[0149] The combined CDV response value is the larger of the CDV receptor binding pathway response value and the CDV vaccine lineage-related epitope pathway response value.

[0150] Based on the results of the reference neutralization test, the subject operating characteristic curve was plotted, and the judgment threshold was determined according to the Youden index. The results are shown in Table 9.

[0151] Table 9. Results of the analysis of the receiver operating characteristic curves.

[0152]

[0153] As shown in Table 9, the areas under the curves for the CAV / CDV dual-pathway combination and CPV detection in this invention are 0.986, 0.987 and 0.974, respectively, indicating that the obtained functional region-specific antibody response values ​​can effectively distinguish between reference positive and reference negative samples.

[0154] When CDV uses only the receptor binding pathway, the area under the curve is 0.921; when it uses only the vaccine lineage-related epitope pathway, the area under the curve is 0.933; when the two pathways are combined, the area under the curve increases to 0.987, and the sensitivity and specificity reach 92.9% and 97.5%, respectively.

[0155] like Figure 6 As shown, the functional region-specific antibody response values ​​generally increase with the increase of the reference neutralizing antibody titer; the receiver operating characteristic curve is close to the upper left corner of the graph, indicating that the chip of the present invention has a good ability to distinguish the level of immune protection-related antibodies.

[0156] It should be noted that this embodiment evaluates the immune protection status related to the level of virus neutralizing antibodies. If the test results are to be used to directly predict clinical disease or animal survival after viral challenge, animal challenge experiments or prospective trials with clear clinical outcomes should be used for verification.

[0157] Example 10: Feasibility of different combinations of mutant antigens

[0158] To verify that different combinations of mutant antigens within the scope of the claims can be implemented, the following three types of chips were prepared respectively:

[0159] The first type of chip uses CAVR24E mutant antigen, CDVY467A / D468A / R471A triple mutant antigen, CDVYIEGEFD mutant epitope peptide, and CPVN93K / N323D mutant virus-like particles.

[0160] The second type of chip uses CAVR24A mutant antigen, CDVY467A / D468A double mutant antigen, CDVYIEGEFD mutant epitope peptide, and CPVN93K / N323D mutant virus-like particles.

[0161] The third type of chip uses CAVR24E mutant antigen, CDVD468A / R471A double mutant antigen, CDVYIEGEFD mutant epitope peptide, and CPVN93K / N323D mutant virus-like particles.

[0162] The same 48 representative serum samples were used to test the three types of chips. The overall concordance rates between the first, second, and third chips and the reference method were 93.8%, 91.7%, and 89.6%, respectively.

[0163] All three types of chips were able to distinguish between antibody levels in high, medium, and low functional regions, and the overall consistency rate difference did not exceed the preset range. This result indicates that as long as the mutant antigen meets the requirements of target binding activity not exceeding 40% of the functionally maintaining antigen and non-target conformation retention rate not less than 80% of the functionally maintaining antigen, different combinations of mutant antigens within the scope of the claims can be implemented and achieve essentially the same immunoprotective evaluation effect.

[0164] Comparative Example 1: Using only functional preservation antigens

[0165] A control chip was prepared in which only the functionally intact antigens of CAV, CDV, and CPV were immobilized, while the loss-of-function mutant antigens were not immobilized. Other preparation, incubation, washing, secondary antibody detection, and scanning conditions were the same as in Examples 5 and 6.

[0166] The total binding signal of the functionally intact antigen was used to directly evaluate the level of the corresponding antibody. The detection results showed that the areas under the curve for CAV, CDV, and CPV were 0.843, 0.823, and 0.846, respectively, which were significantly lower than the 0.986, 0.987, and 0.974 of the method of this invention.

[0167] Some samples with low neutralizing antibody titers but high total binding antibody levels were incorrectly identified as positive, indicating that using only functional preservation antigens cannot effectively eliminate interference caused by antibodies binding to non-functional regions.

[0168] Comparative Example 2: No antigen fixation correction performed

[0169] The same paired antigen chip as in Example 5 was used, but a fixed amount of correction signal was not used in data processing. Instead, the difference between the function-preserving antigen signal and the function-loss mutant antigen signal was used directly as the detection result.

[0170] Without fixed-quantity correction, the areas under the curves for CAV, CDV, and CPV detection were 0.902, 0.887, and 0.895, respectively, with inter-batch coefficients of variation ranging from 18.4% to 25.7%. After using the fixed-quantity correction of this invention, the inter-batch coefficients of variation were reduced to 7.2% to 11.8%.

[0171] This result indicates that when there is a small deviation between the actual fixation amount of functionally intact antigens and functionally lost mutant antigens, failure to perform fixation amount correction will incorrectly include the difference in antigen fixation amount in the functional region-specific antibody response value.

[0172] Comparative Example 3: Using whole denatured antigen as a loss-of-function control

[0173] The CAV, CDV, and CPV function-preserving antigens were treated at 95°C for 10 minutes, respectively, with the denatured antigens serving as a function-loss control. Other chip preparation and detection conditions were the same as in Examples 5 and 6.

[0174] Using non-target conformation monoclonal antibodies for detection, the non-target conformation retention rates of denatured CAV, CDV, and CPV antigens were 31.6%, 24.8%, and 28.5%, respectively, all significantly lower than 80%.

[0175] When denatured antigens were used as controls, the areas under the curves for CAV, CDV, and CPV were 0.806, 0.793, and 0.821, respectively. This indicates that overall denaturation not only destroys the target receptor binding region or the target neutralizing epitope, but also destroys a large number of non-target conformational epitopes, and therefore cannot be used to specifically eliminate antibody signals from non-functional regions.

[0176] Comparative Example 4: CDV uses only the receptor binding pathway

[0177] A chip containing only CDV receptor binding function retention antigen and Y467A / D468A / R471A loss-of-function mutant antigen was prepared, without setting DIEREFD and YIEGEFD epitope pairings.

[0178] When using only the receptor binding pathway, the sensitivity was 78.6%, the specificity was 92.5%, and the area under the curve was 0.921. When both the receptor binding pathway and the vaccine lineage-related non-receptor neutralizing epitope pathway were included, the sensitivity increased to 92.9%, the specificity increased to 97.5%, and the area under the curve increased to 0.987.

[0179] The results indicate that the composition of CDV functional antibodies in the serum of different dogs varies, and setting up two antibody detection pathways with different mechanisms of action can improve the coverage of different vaccine immune response types.

[0180] Comparative Example 5: Paired antigens were immobilized using different methods

[0181] Functional retention antigens were directionally immobilized using a biotin-streptavidin method, while loss-of-function mutant antigens were immobilized using a random covalent coupling method. Other conditions were the same as in Example 5.

[0182] After adopting different fixation methods, the negative reference sample showed a more obvious paired difference signal, with an inter-batch coefficient of variation of 17.5% to 24.4%, and the area under the curve for CAV, CDV and CPV detection decreased to 0.859, 0.835 and 0.847, respectively.

[0183] The results show that even if the total amount of fixation of functional retention antigens and loss-of-function mutant antigens is similar, different fixation directions and epitope accessibility will still produce systematic errors. Therefore, using the same fixation method within the same pair is beneficial to improve the accuracy and repeatability of differential signals.

[0184] The combined results of the method of the present invention and various comparative examples are shown in Table 10. Figure 7 As shown.

[0185] Table 10 Performance comparison of the method of the present invention and various comparative examples

[0186]

[0187] Depend on Figure 7 As shown in Table 10, using only functionally preserved antigens is susceptible to interference from antibodies binding to non-functional regions; without fixation amount correction, it is susceptible to the influence of differences in the actual fixation amount of paired antigens; using whole denatured antigens cannot maintain the consistency of non-target conformations; and using different fixation methods for paired antigens will introduce differences in antigen orientation and epitope accessibility.

[0188] In summary, this invention maintains the antigen and its corresponding site-specific loss-of-function mutant antigen by setting CAV, CDV, and CPV functions, so that antigens within the same pair use the same fixation method and essentially the same molar fixation amount, and evaluates functional region-specific antibodies based on the pairing difference signal after fixation amount correction. This can reduce interference caused by non-functional region binding antibodies, differences in antigen fixation amount, and differences in fixation direction.

Claims

1. A chip for assessing immune protection against canine adenovirus, canine distemper virus, and canine parvovirus, comprising a chip substrate and at least one reaction region disposed on the chip substrate, characterized in that, Each of the aforementioned reaction zones includes: The canine adenovirus detection unit includes a canine adenovirus function retention antigen point and a canine adenovirus function loss mutation antigen point. The amino acid sequence of the canine adenovirus function retention antigen is shown in SEQ ID NO:

1. The canine adenovirus function loss mutation antigen is formed by mutating arginine at position 24 of SEQ ID NO: 1 to glutamic acid or alanine. The canine distemper virus detection unit includes a receptor-binding paired antigen site and a non-receptor-binding neutralizing epitope paired antigen site. The amino acid sequence of the functionally intact antigen in the receptor-binding paired antigen site is shown in SEQ ID NO:

2. The loss-of-function mutant antigen is formed by mutating at least two of the following in SEQ ID NO: 2: tyrosine at position 467, aspartic acid at position 468, and arginine at position 471 to alanine. The amino acid sequence of the functionally intact antigen in the non-receptor-binding neutralizing epitope paired antigen site is the DIEGEFD sequence shown in positions 180-186 of SEQ ID NO:

2. The loss-of-function mutant antigen of the canine distemper virus non-receptor-binding neutralizing epitope is formed by mutating aspartic acid at position 180 to tyrosine and arginine at position 183 to glycine, and is the YIEGEFD sequence. The canine parvovirus detection unit comprises a functional virus-like particle antigen spot formed by the VP2 protein as shown in SEQ ID NO: 3, and a functional loss-of-function mutant virus-like particle antigen spot formed by the VP2 protein of SEQ ID NO: 3 with asparagine mutated to lysine at position 93 and asparagine mutated to aspartic acid at position 323. In each paired antigen, the function-preserving antigen and the function-loss mutant antigen are fixed in the same way and with the same molar amount. The chip is configured to determine the specific antibody level of the corresponding viral invasion functional region based on the difference in the correction antibody binding signals of the two antigens.

2. The immune protection assessment chip according to claim 1, characterized in that: The canine adenovirus loss-of-function mutant antigen is formed by mutating arginine at position 24 of SEQ ID NO: 1 to glutamic acid. The canine distemper virus receptor-binding loss-of-function mutant antigen is formed by mutating tyrosine at position 467, aspartic acid at position 468, and arginine at position 471 of SEQ ID NO: 2 to alanine.

3. The immune protection assessment chip according to claim 1, characterized in that, For canine adenovirus paired antigen, canine distemper virus receptor-binding paired antigen, and canine parvovirus paired antigen, the binding signal of the loss-of-function mutant antigen to the corresponding host receptor is not higher than 40% of the binding signal of the function-maintaining antigen to the host receptor, and the binding signal of the loss-of-function mutant antigen to the conformational antibody that recognizes non-target regions is not lower than 80% of the binding signal of the function-maintaining antigen to the conformational antibody.

4. The immune protection assessment chip according to claim 3, characterized in that, The host receptors corresponding to the canine adenovirus pairing antigens are Coxsackievirus and adenovirus receptors (CAR); the host receptors corresponding to the canine distemper virus receptor-binding pairing antigens are canine SLAM receptors; and the host receptors corresponding to the canine parvovirus pairing antigens are canine transferrin receptors.

5. The immune protection assessment chip according to claim 1, characterized in that, The canine distemper virus non-receptor-binding neutralizing epitope function-maintaining antigen and function-loss mutant antigen each include spacer sequences disposed at both ends of the epitope and a directional immobilization group disposed at one end away from the epitope; the binding signal of the neutralizing antibody recognizing the DIEREFD sequence to the function-loss mutant antigen is no higher than 20% of its binding signal to the function-maintaining antigen; And / or, the particle size of the canine parvovirus functional retention virus-like particles and the loss-of-function mutant virus-like particles is 20-30 nm, and the difference in their main particle size does not exceed 15%. The binding signal of the loss-of-function mutant virus-like particles to the canine transferrin receptor is 10%-40% of the corresponding binding signal of the functional retention virus-like particles.

6. The immune protection assessment chip according to claim 1, characterized in that, Each antigen has the same biotinylated tag at the end away from the target functional region. The chip substrate is a streptavidin-modified substrate. Each antigen is oriented and fixed to the chip substrate by the biotinylated tag. And / or, each antigen has at least three replicate sites; each reaction zone also includes a canine IgG positive control site, a bovine serum albumin negative control site, a blank control site, and an antigen fixed amount control site, wherein the signal variation coefficient of each replicate site does not exceed 15%; And / or, the ratio of the fixed amount control signal of the functional retention antigen to the functional loss mutation antigen of the same paired antigen is 0.85 to 1.15; the chip includes 8, 16, 24 or 48 reaction regions isolated from each other.

7. A method for detecting functional region-specific antibodies against canine adenovirus, canine distemper virus, and canine parvovirus in canine in vitro samples, wherein the method is not a disease diagnosis or treatment method, characterized in that, The immune protection assessment chip according to any one of claims 1 to 5 includes: S1, after diluting the canine in vitro sample, add it to the reaction area of ​​the immune protection assessment chip, so that the antibodies in the sample bind to each functional maintenance antigen and functional loss mutation antigen respectively; S2, clean the immune protection assessment chip, add labeled anti-canine immunoglobulin detection antibody, incubate, and then clean again; S3, collect the detection signals of each antigen site, subtract the local background signal, and use the antigen fixation control signal or conformation control signal to correct the detection signals of functionally maintained antigens and functionally lost mutant antigens, respectively. S4, calculate the difference between the correction signal of the functional preservation antigen and the correction signal of the functional loss mutation antigen for each paired antigen to obtain the antibody score of the functional region of canine adenovirus, the antibody score of the receptor binding region of canine distemper virus, the antibody score of the non-receptor binding neutralizing epitope of canine distemper virus, and the antibody score of the functional region of canine parvovirus. Functional region antibody score of the i-th paired antigen Determined according to the following formula: ； In the formula, The functional region antibody score corresponding to the i-th paired antigen; The detection signal is for the antigen that preserves the function of the sample to be tested; This is the detection signal for the sample to be tested and the loss-of-function mutant antigen; A fixed amount or conformational control signal for maintaining the function of the antigen; It is a fixed amount or conformational control signal for loss-of-function mutant antigens.

8. The detection method according to claim 7, characterized in that, The canine in vitro sample is canine serum or canine plasma. For single-point detection, the canine in vitro sample is diluted 50 to 200 times. The sample and the chip are incubated at 37°C for 20 to 40 minutes. The labeled anti-canine immunoglobulin detection antibody is a fluorescently labeled anti-canine IgG antibody.

9. The detection method according to claim 7, characterized in that, The canine in vitro samples were serially diluted, and the highest sample dilution factor in which the difference between the correction signal of the functional retention antigen and the correction signal of the functional loss mutation antigen was still higher than the corresponding detection threshold was determined as the functional region-specific antibody titer of the corresponding virus; when the detection signal of the functional retention antigen was lower than the detection limit, the corresponding functional region-specific antibody was determined to be undetectable. And / or, establish evaluation thresholds for antibody scores in each functional region using canine serum with reference test results; the reference test for canine adenovirus and canine distemper virus is a virus neutralization test, and the reference test for canine parvovirus is a virus neutralization test or a hemagglutination inhibition test, and determine the evaluation thresholds based on the receiver operating characteristic curves of functional region antibody scores and reference test results.

10. The detection method according to claim 9, characterized in that, When the canine adenovirus functional region antibody score reaches the canine adenovirus evaluation threshold, the canine distemper virus receptor-binding region antibody score and the non-receptor-binding neutralizing epitope antibody score reach the corresponding evaluation threshold, and the canine parvovirus functional region antibody score reaches the canine parvovirus evaluation threshold, the evaluation result is output that the functional antibodies of the three viruses have reached the set level; if the functional region antibody score of any virus does not reach the corresponding evaluation threshold, the evaluation result is output that the functional antibody of that virus has not reached the set level.

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