Kit for detecting bovine leukemia virus IgG antibody as well as detection method and application thereof

Abstract

The invention belongs to the technical field of chemiluminescence detection, and particularly relates to a kit for detecting a bovine leukemia virus IgG antibody as well as a detection method and application of the kit. A sample treatment solution in the kit is prepared from Protein A, disodium hydrogen phosphate dodecahydrate, monopotassium phosphate, potassium chloride, sodium chloride, casein, mercaptoethanol, Tween-20 and Proclin; and the mark of the magnetic bead is sealed by combining PEG4000 and ethanolamine. Compared with traditional single protein sealing, the sealing mode provided by the invention has the advantages that interference of foreign protein is removed, non-specific binding in a sample is reduced, the background is reduced, and the stability, sensitivity and specificity of detection are improved; compared with a traditional large-proportion diluted sample, the sample treatment liquid has the advantages that excessive IgG in the sample is removed, dilution treatment of an equipment dilution module is not needed in the detection process, manual pre-dilution treatment is also not needed, and the detection efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemiluminescence detection, and particularly relates to a kit for detecting bovine leukemia virus IgG antibody, a detection method thereof and an application thereof. Background Art

[0002] Bovine leukemia virus (BLV) is a virus that only infects cattle. It is similar to human T-cell lymphotropic virus type 1 and type 2 (HTLV-1 and HTLV-2), belongs to the same family as HTLV-1, and belongs to the genus deltaretrovirus of the family retroviridae. It is an RNA virus containing single-stranded RNA. BLV replicates by budding proliferation, that is, budding and proliferating on the cell surface and releasing to form another new individual. BLV is not resistant to the outside world and can be inactivated by ultraviolet rays and repeated freezing and thawing. It is not heat-resistant, and 0.5% phenol and formaldehyde solution can also inactivate it. Bovine leukemia virus is the pathogen that causes bovine leukemia. The characteristics of this disease mainly include persistent B lymphocyte proliferation and generalized lymphadenopathy. The clinical symptoms are not obvious during the incubation period (generally 4-5 years), and the mortality rate is relatively high after the onset.

[0003] Enzootic bovine leukemia is classified as a Class B animal epidemic disease. Infected cattle and latently infected cattle are the most important sources of infection and carry the virus for life. Its natural infection hosts include zebu, buffalo, sheep, capybara, etc., but it can also infect goats, alpacas, rabbits, rats and chickens under artificial experimental conditions. Some scholars even found BLV nucleic acid in human breast tissue. Horizontal transmission is mainly carried out through contact with blood, body fluids and secretions and mechanical bites of blood-sucking insects. At present, there is still no commercial vaccine and specific therapeutic drug for bovine leukemia virus. Most cattle farms mainly control bovine leukemia virus through detection, culling and other methods. In the prevention and control work of bovine leukemia virus, the diagnosis of bovine leukemia virus is the basis of prevention and control work. Since most cattle infected with bovine leukemia virus do not show any clinical symptoms, this poses a serious obstacle to clinical diagnosis. It is particularly important to regularly monitor the antigen or antibody level of the cattle herd.

[0004] Serological testing of BLV antibody is a relatively conventional and widely used method. Among them, enzyme-linked immunosorbent assay technology adsorbs antigens or antibodies on the surface of solid-phase carriers and uses the specific reaction of antigen and antibody to achieve qualitative or quantitative analysis of the target analyte. However, the enzyme-linked immunosorbent assay technology has a long experimental time and a cumbersome procedure. All processes in the experiment are manually operated, which is extremely easy to cause contamination, resulting in false negatives or false positives, and it is particularly inconvenient for a large number of screening operations.

[0005] In view of the deficiencies of the prior art and market demands, there is an urgent need to develop a simple and effective method for detecting bovine leukemia virus. Summary of the Invention

[0006] In view of the above deficiencies, the present invention provides a chemiluminescence detection kit for bovine leukemia virus IgG antibody, a sample treatment solution, a detection method and an application thereof. This method solves the problems in practical applications such as the cumbersome operation process of other methodologies that require a machine dilution module or manual dilution, long experimental time, high cost, and inconvenience for extensive screening, and has important application value for regularly monitoring the antibody level of cattle herds to prevent BLV infection.

[0007] The technical solution of the present invention is as follows: On the one hand, the present invention provides a kit for detecting bovine leukemia virus IgG antibody, and the kit includes a sample treatment solution and a blocking agent.

[0008] Specifically, the sample treatment solution is composed of Protein A 0.00001%-0.001%, disodium hydrogen phosphate dodecahydrate 0.5%-1.5%, potassium dihydrogen phosphate 0.01%-0.1%, potassium chloride 0.01%-0.1%, sodium chloride 1%-5%, casein 0.1%-0.5%, mercaptoethanol 0.01%-0.1%, Tween-20 0.01%-0.1%, Proclin 300 0.01%-0.1% and the balance of solvent by weight percentage.

[0009] Preferably, in some embodiments, the sample treatment solution is composed of Protein A 0.0001%, disodium hydrogen phosphate dodecahydrate 0.686%, potassium dihydrogen phosphate 0.048%, potassium chloride 0.04%, sodium chloride 1.6%, casein 0.1%, mercaptoethanol 0.1%, Tween-20 0.05%, Proclin 300 0.05% and water by weight percentage.

[0010] In some embodiments, the sample treatment solution is composed of Protein A 0.0001%, disodium hydrogen phosphate dodecahydrate 1.5%, potassium dihydrogen phosphate 0.01%, potassium chloride 0.1%, sodium chloride 1%, casein 0.5%, mercaptoethanol 0.01%, Tween-20 0.1%, Proclin 300 0.01% and water by weight percentage.

[0011] In some embodiments, the sample treatment solution, by weight percentage, consists of 0.001% Protein A, 0.5% disodium hydrogen phosphate dodecahydrate, 0.1% potassium dihydrogen phosphate, 0.1% potassium chloride, 5% sodium chloride, 0.1% casein, 0.1% mercaptoethanol, 0.01% Tween-20, 0.1% Proclin 300 and water.

[0012] Specifically, the blocking agent includes PEG4000 and ethanolamine.

[0013] Preferably, the concentration of PEG4000 can be 0.1 wt% - 1 wt%; the concentration of ethanolamine can be 10 mM - 100 mM.

[0014] Preferably, the concentration of PEG4000 can be 0.1 wt% - 0.5 wt%; the concentration of ethanolamine can be 10 mM - 50 mM.

[0015] Preferably, the concentration of PEG4000 can be 0.1 wt% - 0.2 wt%; the concentration of ethanolamine can be 10 mM - 20 mM.

[0016] Preferably, the concentration of PEG4000 can be 0.1 wt%; the concentration of ethanolamine can be 10 mM.

[0017] Specifically, the kit further includes an M diluent for diluting the magnetic bead-bovine leukemia virus antigen complex and / or an R diluent for diluting the IgG-chemiluminescent label complex.

[0018] More specifically, the M diluent, by weight percentage, consists of 0.5% - 1.5% disodium hydrogen phosphate dodecahydrate, 0.01% - 0.1% potassium dihydrogen phosphate, 0.01% - 0.1% potassium chloride, 1% - 5% sodium chloride, 0.5% - 2% fish skin gelatin, 0.1% - 1% bovine serum albumin, 0.01% - 0.1% Tween-20, 0.01% - 0.1% Proclin 300 and the balance of the solvent.

[0019] Preferably, in some embodiments, the M diluent, by weight percentage, consists of 0.686% disodium hydrogen phosphate dodecahydrate, 0.048% potassium dihydrogen phosphate, 0.04% potassium chloride, 1.6% sodium chloride, 1% fish skin gelatin, 0.5% bovine serum albumin, 0.05% Tween-20, 0.05% Proclin 300 and water.

[0020] Preferably, in some embodiments, the M diluent, by weight percentage, consists of 1.5% of disodium hydrogen phosphate dodecahydrate, 0.01% of potassium dihydrogen phosphate, 0.1% of potassium chloride, 1% of sodium chloride, 2% of fish skin gelatin, 0.1% of bovine serum albumin, 0.1% of Tween-20, 0.01% of Proclin 300 and water.

[0021] Preferably, in some embodiments, the M diluent, by weight percentage, consists of 0.5% of disodium hydrogen phosphate dodecahydrate, 0.1% of potassium dihydrogen phosphate, 0.01% of potassium chloride, 5% of sodium chloride, 0.5% of fish skin gelatin, 1% of bovine serum albumin, 0.01% of Tween-20, 0.1% of Proclin 300 and water.

[0022] More specifically, the R diluent, by weight percentage, consists of 0.5%-1.5% of disodium hydrogen phosphate dodecahydrate, 0.01%-0.1% of potassium dihydrogen phosphate, 0.01%-0.1% of potassium chloride, 1%-5% of sodium chloride, 1%-5% of sucrose, 1%-5% of trehalose, 0.1%-0.5% of EDTA-2Na, 0.01%-0.1% of Tween-20, 0.01%-0.1% of Proclin 300 and water.

[0023] Preferably, in some embodiments, the R diluent, by weight percentage, consists of 0.686% of disodium hydrogen phosphate dodecahydrate, 0.048% of potassium dihydrogen phosphate, 0.04% of potassium chloride, 1.6% of sodium chloride, 1% of sucrose, 3% of trehalose, 0.2% of EDTA-2Na, 0.05% of Tween-20, 0.05% of Proclin 300 and water.

[0024] Preferably, in some embodiments, the R diluent, by weight percentage, consists of 1.5% of disodium hydrogen phosphate dodecahydrate, 0.01% of potassium dihydrogen phosphate, 0.1% of potassium chloride, 1% of sodium chloride, 5% of sucrose, 1% of trehalose, 0.5% of EDTA-2Na, 0.01% of Tween-20, 0.1% of Proclin 300 and water.

[0025] Preferably, in some embodiments, the R diluent, by weight percentage, consists of 0.5% of disodium hydrogen phosphate dodecahydrate, 0.1% of potassium dihydrogen phosphate, 0.01% of potassium chloride, 5% of sodium chloride, 1% of sucrose, 5% of trehalose, 0.1% of EDTA-2Na, 0.1% of Tween-20, 0.01% of Proclin 300 and water.

[0026] Specifically, the magnetic beads in the magnetic bead-bovine leukemia virus antigen complex include, but are not limited to: carboxyl magnetic beads, amino magnetic beads, streptavidin magnetic beads, epoxy magnetic beads or silane magnetic beads.

[0027] Preferably, the magnetic beads in the magnetic bead-bovine leukemia virus antigen complex can be carboxyl magnetic beads.

[0028] Specifically, the viral antigens in the magnetic bead-bovine leukemia virus antigen complex include, but are not limited to: bovine leukemia natural virus, bovine leukemia virus protein gp51, and bovine leukemia virus protein p24.

[0029] Preferably, the viral antigen in the magnetic bead-bovine leukemia virus antigen complex can be bovine leukemia virus protein gp51.

[0030] More preferably, the gene sequence of the viral antigen in the magnetic bead-bovine leukemia virus antigen complex is cited from UniProt (Universal Protein Resource), and its amino acid sequence is as follows: SEQ ID NO.1: MHHHHHHWRCSLSLGNQQWMTAYNQEAKFSISINQILEAHNQSPFCAKSPRYTLDSVNGYPKIYWPPPQGRRRFGARAMVTYDCEPRCPYVGADRFDCPHWDNASQADQGSFYVNHQILFLHLKQCHGIFTLTWEIWGYDPLITFSLHKIPDPPQPDFPQLNSDWVPSVRSWALLLNQTARAFPDCAICWEPSPPWAPEILVYNKTISSSGPGLALPDAQIFWVNTSSFNTTQGWHHPSQRLLFNVSQGNALLLPPISLVNLSTASSAPPTRVRRS* Specifically, the chemiluminescent markers in the IgG-chemiluminescent marker complex include, but are not limited to: acridinium ester, luminol, horseradish peroxidase, or alkaline phosphatase.

[0031] Preferably, the chemiluminescent marker in the IgG-chemiluminescent marker complex can be acridinium ester.

[0032] Specifically, the IgG in the IgG-chemiluminescent marker complex includes, but is not limited to: goat anti-bovine IgG, rabbit anti-bovine IgG, mouse anti-bovine IgG, horse anti-bovine IgG, pig anti-bovine IgG, or chicken anti-bovine IgG.

[0033] Preferably, the IgG in the IgG-chemiluminescent marker complex can be goat anti-bovine IgG, rabbit anti-bovine IgG, or mouse anti-bovine IgG.

[0034] Specifically, the kit further includes a calibrator and / or a quality control product.

[0035] In another aspect, the present invention provides a method for detecting bovine leukemia virus IgG antibody, which detects a sample through the aforementioned kit. Specifically, the steps are as follows: S1. Mix the sample with the sample treatment solution to obtain A; S2. Incubate A with the magnetic bead-bovine leukemia virus antigen complex to obtain B; S3. Add the IgG-chemiluminescent label complex to B to obtain C; S4. Add a substrate to C and measure the luminescence value; Specifically, the sample in step S1 is whole blood, serum or plasma.

[0036] Specifically, the magnetic bead in the magnetic bead-bovine leukemia virus antigen complex in step S2 can be a carboxyl magnetic bead.

[0037] Specifically, the chemiluminescent label in the IgG-chemiluminescent label complex in step S3 can be acridinium ester.

[0038] Specifically, the IgG in the IgG-chemiluminescent label complex in step S3 can be goat anti-bovine IgG, rabbit anti-bovine IgG or mouse anti-bovine IgG.

[0039] In another aspect, the present invention provides the application of the aforementioned kit method or detection method in the preparation of a product for detecting bovine leukemia virus IgG antibody.

[0040] The beneficial effects of the present invention are as follows: (1) The detection method provided by the present invention can effectively reduce non-specific binding in the sample and reduce the occurrence of false positives.

[0041] (2) The blocking method provided by the present invention reduces the background, improves the stability and sensitivity of the reagent, and effectively improves the specificity of the detected sample.

[0042] (3) The sample treatment solution provided by the present invention does not require pre-dilution treatment during the detection process, reduces the detection steps, and improves the detection efficiency. Specific Embodiments

[0043] The present invention will be further clearly and completely described below through examples. The following examples are only a part of the examples of the present invention, and are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following examples are all conventional experiments unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0044] Example 1 Preparation of sample processing solution: 0.001 g of Protein A, 6.86 g of disodium hydrogen phosphate dodecahydrate, 0.48 g of potassium dihydrogen phosphate, 0.4 g of potassium chloride, 16 g of sodium chloride, 1 g of casein, 1 g of mercaptoethanol, 0.5 g of Tween-20, 0.5 g of Proclin 300, and make up to 1 L with water.

[0045] Preparation of M diluent: 6.86 g of disodium hydrogen phosphate dodecahydrate, 0.48 g of potassium dihydrogen phosphate, 0.4 g of potassium chloride, 16 g of sodium chloride, 10 g of fish skin gelatin, 5 g of bovine serum albumin, 0.5 g of Tween-20, 0.5 g of Proclin 300, and make up to 1 L with water.

[0046] Preparation of R diluent: 6.86 g of disodium hydrogen phosphate dodecahydrate, 0.48 g of potassium dihydrogen phosphate, 0.4 g of potassium chloride, 16 g of sodium chloride, 10 g of sucrose, 30 g of trehalose, 2 g of EDTA-2Na, 0.5 g of Tween-20, 0.5 g of Proclin 300, and make up to 1 L with water.

[0047] Preparation method of the complex of carboxyl magnetic beads and bovine leukemia virus antigen: (1) Wash 1 mL of 10 mg / mL carboxyl magnetic beads with 25 mM MES at pH 6.4, wash three times, discard the supernatant, and then redissolve.

[0048] (2) Activate 1 mL of 10 mg / mL carboxyl magnetic beads with 20 mM EDC and 2 mM NHS, mix well by shaking for 30 min. After activation, wash with 25 mM MES at pH 6.4, wash three times, discard the supernatant, and then redissolve.

[0049] (3) Add 0.1 mg of bovine leukemia antigen for reaction, mix well by shaking for 3 h; wash with 0.1 M Tris-HCl solution, wash three times, discard the supernatant, and then redissolve.

[0050] (4) Block with 0.1% (mass percentage) PEG4000 and 10 mM ethanolamine for 2 h, mix well by shaking.

[0051] (5) Wash with 0.1 M Tris-HCl solution, wash three times, discard the supernatant, and then redissolve with M diluent.

[0052] Preparation of M reagent: Use the prepared M diluent to dilute the carboxyl magnetic bead-bovine leukemia virus antigen complex to 0.5 mg / mL.

[0053] Preparation of R reagent: Use the prepared R diluent to dilute the goat anti-bovine IgG-acridinium ester complex to 1 μg / mL.

[0054] Testing principle of the kit: Add 10 μL of serum or plasma sample and 90 μL of sample treatment solution into the reaction cup. The sample treatment solution with the sample is incubated and combined with 30 μL of M reagent (carboxyl magnetic beads - bovine leukemia virus antigen complex) to form a complex of bovine leukemia antibody - bovine leukemia antigen - carboxyl magnetic beads. Then, the first washing is carried out (① Magnetically adsorb the complex of bovine leukemia antibody - bovine leukemia antigen - carboxyl magnetic beads and aspirate the supernatant; ② Add 300 μl of 0.01 M PBST into the reaction cup and mix well by shaking; ③ Magnetically adsorb the complex of bovine leukemia antibody - bovine leukemia antigen - carboxyl magnetic beads and aspirate the supernatant. Repeat ①②③ three times). After washing, the complex remains in the reaction cup due to the action of the magnetic field. Then, add 100 μL of R reagent (goat anti - bovine IgG - acridinium ester complex) to form a complex of goat anti - bovine IgG acridinium ester - bovine leukemia antibody - bovine leukemia antigen - carboxyl magnetic beads. Then, the second washing is carried out (① Magnetically adsorb the complex of goat anti - bovine IgG acridinium ester - bovine leukemia antibody - bovine leukemia antigen - carboxyl magnetic beads and aspirate the supernatant; ② Add 300 μl of 0.01 M PBST into the reaction cup and mix well by shaking; ③ Magnetically adsorb the complex of goat anti - bovine IgG acridinium ester - bovine leukemia antibody - bovine leukemia antigen - carboxyl magnetic beads and aspirate the supernatant. Repeat ①②③ three times). After washing, the complex remains in the reaction cup due to the action of the magnetic field. After adding 100 μL of pre - excitation solution and 100 μL of excitation solution, the luminescence value is obtained by measuring the light, and then the test result is obtained.

[0055] Example 2 The difference from Example 1 is that: The R reagent is prepared using acridinium ester complex labeled with rabbit anti - bovine IgG. Others are the same as in Example 1.

[0056] Example 3 The difference from Example 1 is that: The R reagent is prepared using acridinium ester complex labeled with mouse anti - bovine IgG. Others are the same as in Example 1.

[0057] Example 4 The differences from Example 1 are as follows, and others are the same as in Example 1.

[0058] Preparation of the sample treatment solution: 0.0001 g of Protein A, 15 g of disodium hydrogen phosphate dodecahydrate, 0.1 g of potassium dihydrogen phosphate, 1 g of potassium chloride, 10 g of sodium chloride, 5 g of casein, 0.1 g of mercaptoethanol, 1 g of Tween - 20, 0.1 g of Proclin 300, and make up the volume to 1 L with water.

[0059] Preparation of M diluent: 15 g of disodium hydrogen phosphate dodecahydrate, 0.1 g of potassium dihydrogen phosphate, 1 g of potassium chloride, 10 g of sodium chloride, 20 g of fish skin gelatin, 1 g of bovine serum albumin, 1 g of Tween-20, 0.1 g of Proclin 300, and make up to 1 L with water.

[0060] Preparation of R diluent: 15 g of disodium hydrogen phosphate dodecahydrate, 0.1 g of potassium dihydrogen phosphate, 1 g of potassium chloride, 10 g of sodium chloride, 50 g of sucrose, 10 g of trehalose, 5 g of EDTA-2Na, 0.1 g of Tween-20, 1 g of Proclin 300, and make up to 1 L with water.

[0061] Example 5 The differences from Example 1 are as follows, and the others are the same as Example 1.

[0062] Preparation of sample treatment solution: 0.01 g of Protein A, 5 g of disodium hydrogen phosphate dodecahydrate, 1 g of potassium dihydrogen phosphate, 0.1 g of potassium chloride, 50 g of sodium chloride, 1 g of casein, 1 g of mercaptoethanol, 0.1 g of Tween-20, 1 g of Proclin 300, and make up to 1 L with water.

[0063] Preparation of M diluent: 5 g of disodium hydrogen phosphate dodecahydrate, 1 g of potassium dihydrogen phosphate, 0.1 g of potassium chloride, 50 g of sodium chloride, 5 g of fish skin gelatin, 10 g of bovine serum albumin, 0.1 g of Tween-20, 1 g of Proclin 300, and make up to 1 L with water.

[0064] Preparation of R diluent: 5 g of disodium hydrogen phosphate dodecahydrate, 1 g of potassium dihydrogen phosphate, 0.1 g of potassium chloride, 50 g of sodium chloride, 10 g of sucrose, 50 g of trehalose, 1 g of EDTA-2Na, 1 g of Tween-20, 0.1 g of Proclin 300, and make up to 1 L with water.

[0065] Comparative Example 1 The difference from Example 1 is that the sample treatment solution is not used. The others are the same as Example 1.

[0066] Comparative Example 2 The difference from Example 1 is that the complex of carboxyl magnetic beads and bovine leukemia virus antigen is not prepared using the blocking agents PEG4000 and ethanolamine. The others are the same as Example 1.

[0067] Comparative Example 3 The difference from Example 1 is that only PEG4000 is used as the blocking agent in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. The others are the same as Example 1.

[0068] Comparative Example 4 The difference from Example 1 is that only ethanolamine is used as the blocking agent in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other conditions are the same as those in Example 1.

[0069] Comparative Example 5 The difference from Example 1 is that only the common blocking agent BSA is used in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other conditions are the same as those in Example 1.

[0070] Comparative Example 6 The difference from Example 1 is that only the common blocking agent skim milk powder is used in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other conditions are the same as those in Example 1.

[0071] Comparative Example 7 The difference from Example 1 is that the common blocking agent BSA and PEG4000 are used as the blocking agent in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other conditions are the same as those in Example 1.

[0072] Comparative Example 8 The difference from Example 1 is that the common blocking agent BSA and ethanolamine are used as the blocking agent in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other conditions are the same as those in Example 1.

[0073] Comparative Example 9 The difference from Example 1 is that the common blocking agent skim milk powder and PEG4000 are used as the blocking agent in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other conditions are the same as those in Example 1.

[0074] Comparative Example 10 The difference from Example 1 is that the common blocking agent skim milk powder and ethanolamine are used as the blocking agent in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other conditions are the same as those in Example 1.

[0075] Comparative Example 11 The difference from Example 1 is that polyacrylamide is used to replace PEG4000 in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other conditions are the same as those in Example 1.

[0076] Comparative Example 12 The difference from Example 1 is that ethanolamine is replaced by tris(hydroxymethyl)aminomethane in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other conditions are the same as those in Example 1.

[0077] Comparative Example 13 The difference from Example 1 is that the sample is pre-diluted with the sample treatment solution before the sample is loaded onto the machine. Other conditions are the same as those in Example 1.

[0078] Comparative Example 14 The difference from Example 1 is that the sample is pre-diluted with normal saline before being loaded onto the instrument, and the rest is the same as in Example 1.

[0079] Comparative Example 15 The difference from Example 1 is that the sample is pre-diluted with PBS before being loaded onto the instrument, and the rest is the same as in Example 1.

[0080] Effect Example 1 Eighty serum samples that had been tested by nucleic acid were selected, including 50 negative serum samples and 30 positive serum samples. Examples 1 - 5 and Comparative Examples 1 - 15 were respectively used to detect the 80 clinical serum samples, and the compliance of the results of Examples 1 - 5 and Comparative Examples 1 - 15 was compared. The results are shown in Table 1: Table 1

[0081] In Example 1, 30 serum samples determined to be positive by nucleic acid were detected. Among them, 2 were negative serum samples and 28 were positive serum samples, and the positive compliance rate was 93.3%. 50 serum samples determined to be negative by nucleic acid were detected. Among them, 2 were positive serum samples and 48 were negative serum samples, and the negative compliance rate was 96.0%. The total compliance rate was 95.0%.

[0082] In Example 2, 30 serum samples determined to be positive by nucleic acid were detected. Among them, 2 were negative serum samples and 28 were positive serum samples, and the positive compliance rate was 93.3%. 50 serum samples determined to be negative by nucleic acid were detected. Among them, 2 were positive serum samples and 48 were negative serum samples, and the negative compliance rate was 96.0%. The total compliance rate was 95.0%.

[0083] In Example 3, 30 serum samples determined to be positive by nucleic acid were detected. Among them, 2 were negative serum samples and 28 were positive serum samples, and the positive compliance rate was 93.3%. 50 serum samples determined to be negative by nucleic acid were detected. Among them, 2 were positive serum samples and 48 were negative serum samples, and the negative compliance rate was 96.0%. The total compliance rate was 95.0%.

[0084] In Example 4, 30 serum samples determined to be positive by nucleic acid were detected. Among them, 2 were negative serum samples and 28 were positive serum samples, and the positive compliance rate was 93.3%. 50 serum samples determined to be negative by nucleic acid were detected. Among them, 2 were positive serum samples and 48 were negative serum samples, and the negative compliance rate was 96.0%. The total compliance rate was 95.0%.

[0085] In Example 5, 30 serum samples determined to be positive by nucleic acid were detected. Among them, 2 were negative serum samples and 28 were positive serum samples, and the positive compliance rate was 93.3%. 50 serum samples determined to be negative by nucleic acid were detected. Among them, 2 were positive serum samples and 48 were negative serum samples, and the negative compliance rate was 96.0%. The total compliance rate was 95.0%.

[0086] For Comparative Example 1, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 1 was negative and 29 were positive, with a positive coincidence rate of 96.7%. Also, 50 serum samples determined to be negative by nucleic acid testing were examined. Among them, 35 were positive and 15 were negative, with a negative coincidence rate of 30.0% and an overall coincidence rate of 55.0%.

[0087] For Comparative Example 2, 30 serum samples determined to be positive by nucleic acid testing were examined. None was negative and 30 were positive, with a positive coincidence rate of 100.0%. Also, 50 serum samples determined to be negative by nucleic acid testing were examined. All 50 were positive and none was negative, with a negative coincidence rate of 0.0% and an overall coincidence rate of 37.5%.

[0088] For Comparative Example 3, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 12 were negative and 18 were positive, with a positive coincidence rate of 60.0%. Also, 50 serum samples determined to be negative by nucleic acid testing were examined. Among them, 7 were positive and 43 were negative, with a negative coincidence rate of 86.0% and an overall coincidence rate of 76.3%.

[0089] For Comparative Example 4, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 11 were negative and 19 were positive, with a positive coincidence rate of 63.3%. Also, 50 serum samples determined to be negative by nucleic acid testing were examined. Among them, 5 were positive and 45 were negative, with a negative coincidence rate of 90.0% and an overall coincidence rate of 80.0%.

[0090] For Comparative Example 5, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 18 were negative and 12 were positive, with a positive coincidence rate of 40.0%. Also, 50 serum samples determined to be negative by nucleic acid testing were examined. None was positive and 50 were negative, with a negative coincidence rate of 100.0% and an overall coincidence rate of 77.5%.

[0091] For Comparative Example 6, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 10 were negative and 20 were positive, with a positive coincidence rate of 66.7%. Also, 50 serum samples determined to be negative by nucleic acid testing were examined. Among them, 1 was positive and 49 were negative, with a negative coincidence rate of 98.0% and an overall coincidence rate of 86.3%.

[0092] For Comparative Example 7, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 6 were negative and 24 were positive, with a positive coincidence rate of 80.0%. Also, 50 serum samples determined to be negative by nucleic acid testing were examined. Among them, 5 were positive and 45 were negative, with a negative coincidence rate of 90.0% and an overall coincidence rate of 86.3%.

[0093] In Comparative Example 8, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 8 were negative and 22 were positive. The positive coincidence rate was 73.3%. For 50 serum samples determined to be negative by nucleic acid testing, 4 were positive and 46 were negative. The negative coincidence rate was 92.0%, and the overall coincidence rate was 85.0%.

[0094] In Comparative Example 9, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 7 were negative and 23 were positive. The positive coincidence rate was 76.7%. For 50 serum samples determined to be negative by nucleic acid testing, 4 were positive and 46 were negative. The negative coincidence rate was 92.0%, and the overall coincidence rate was 86.3%.

[0095] In Comparative Example 10, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 7 were negative and 23 were positive. The positive coincidence rate was 76.7%. For 50 serum samples determined to be negative by nucleic acid testing, 6 were positive and 44 were negative. The negative coincidence rate was 88.0%, and the overall coincidence rate was 83.8%.

[0096] In Comparative Example 11, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 11 were negative and 19 were positive. The positive coincidence rate was 63.3%. For 50 serum samples determined to be negative by nucleic acid testing, 5 were positive and 45 were negative. The negative coincidence rate was 90.0%, and the overall coincidence rate was 80.0%.

[0097] In Comparative Example 12, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 18 were negative and 12 were positive. The positive coincidence rate was 40.0%. For 50 serum samples determined to be negative by nucleic acid testing, 5 were positive and 45 were negative. The negative coincidence rate was 90.0%, and the overall coincidence rate was 71.3%.

[0098] In Comparative Example 13, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 2 were negative and 28 were positive. The positive coincidence rate was 93.3%. For 50 serum samples determined to be negative by nucleic acid testing, 2 were positive and 48 were negative. The negative coincidence rate was 96.0%, and the overall coincidence rate was 95.0%.

[0099] In Comparative Example 14, 30 serum samples determined to be positive by nucleic acid testing were examined. Among them, 0 were negative and 30 were positive. The positive coincidence rate was 100.0%. For 50 serum samples determined to be negative by nucleic acid testing, 9 were positive and 41 were negative. The negative coincidence rate was 82.0%, and the overall coincidence rate was 88.8%.

[0100] For Comparative Example 15, 30 positive sera determined by nucleic acid detection were tested, 0 were negative sera, and 30 were positive sera, with a positive coincidence rate of 100.0%; 50 negative sera determined by nucleic acid detection were tested, 9 were positive sera, and 41 were negative sera, with a negative coincidence rate of 82.0% and a total coincidence rate of 88.8%.

[0101] As can be seen from the above table, the positive and negative coincidence rates and the total coincidence rate of Examples 1 - 5 and Comparative Example 13 are all above 90%, meeting the detection conditions. The positive and negative coincidence rates of the remaining comparative examples are relatively low and do not meet the detection conditions. For Comparative Example 13, the sample was pre-diluted with a sample treatment solution before sample loading. Although the same detection effect was achieved, the reaction steps were increased. Therefore, Examples 1 - 5 are more preferred.

[0102] Effect Example 2 The background luminescence values of Examples 1 - 5 and Comparative Examples 1 - 15 were tested using deionized water and repeated three times. The results are shown in Table 2: Table 2

[0103] As can be seen from the above table, the results of the background luminescence values of deionized water tested in Examples 1 - 5 and Comparative Example 13 are all lower than 5000, meeting the detection conditions. The above examples and comparative examples all adopt a non-protein blocking method including combined blocking with PEG4000 and ethanolamine. The background values of the remaining comparative examples are all too high and do not meet the detection conditions. For Comparative Example 13, the sample was pre-diluted with a sample treatment solution before sample loading. Although the same detection effect was achieved, the reaction steps were increased. Therefore, Examples 1 - 5 are more preferred.

[0104] The above detailed description is a specific description of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A kit for detecting bovine leukemia virus IgG antibody, characterized in that, The kit includes a sample treatment solution, a magnetic bead-bovine leukemia virus antigen complex, an IgG-chemiluminescent label complex, and a blocking agent; The sample treatment solution, by weight percentage, consists of 0.00001%-0.001% Protein A, 0.5%-1.5% disodium hydrogen phosphate dodecahydrate, 0.01%-0.1% potassium dihydrogen phosphate, 0.01%-0.1% potassium chloride, 1%-5% sodium chloride, 0.1%-0.5% casein, 0.01%-0.1% mercaptoethanol, 0.01%-0.1% Tween-20, 0.01%-0.1% Proclin 300, and the balance of a solvent; The blocking agent includes PEG4000 and ethanolamine.

2. The kit according to claim 1, wherein The concentration of the PEG4000 is 0.1wt%-1wt%; the concentration of the ethanolamine is 10mM-100mM.

3. The kit according to claim 1, characterized in that, It also includes an M diluent for diluting the magnetic bead-bovine leukemia virus antigen complex and / or an R diluent for diluting the IgG-chemiluminescent label complex; The M diluent, by weight percentage, consists of 0.5%-1.5% disodium hydrogen phosphate dodecahydrate, 0.01%-0.1% potassium dihydrogen phosphate, 0.01%-0.1% potassium chloride, 1%-5% sodium chloride, 0.5%-2% fish skin gelatin, 0.1%-1% bovine serum albumin, 0.01%-0.1% Tween-20, 0.01%-0.1% Proclin 300, and the balance of a solvent; The R diluent, by weight percentage, consists of 0.5%-1.5% disodium hydrogen phosphate dodecahydrate, 0.01%-0.1% potassium dihydrogen phosphate, 0.01%-0.1% potassium chloride, 1%-5% sodium chloride, 1%-5% sucrose, 1%-5% trehalose, 0.1%-0.5% EDTA-2Na, 0.01%-0.1% Tween-20, 0.01%-0.1% Proclin 300, and the balance of a solvent.

4. The kit according to claim 3, characterized in that The magnetic beads in the magnetic bead-bovine leukemia virus antigen complex are carboxyl magnetic beads, amino magnetic beads, streptavidin magnetic beads, epoxy magnetic beads, or silane magnetic beads.

5. The kit according to claim 4, characterized in that, The chemiluminescent label in the IgG-chemiluminescent label complex is acridinium ester, luminol, horseradish peroxidase, or alkaline phosphatase.

6. The kit according to claim 5, wherein The IgG in the IgG-chemiluminescent label complex is goat anti-bovine IgG, rabbit anti-bovine IgG, mouse anti-bovine IgG, horse anti-bovine IgG, pig anti-bovine IgG, or chicken anti-bovine IgG.

7. The kit according to any one of claims 1-6, characterized in that, The kit also includes a calibrator and / or a quality control product.

8. A method for detecting bovine leukemia virus IgG antibody for non-disease diagnosis purposes, characterized in that, Using the kit according to any one of claims 1-7 to detect a sample, the specific steps are as follows: S1. Mix the sample with the sample treatment solution to obtain A; S2. Incubate A with the magnetic bead-bovine leukemia virus antigen complex to obtain B; S3. Add the IgG-chemiluminescent label complex to B to obtain C; S4. Add a substrate to C and measure the luminescence value.

9. The detection method according to claim 8, characterized in that, The sample in step S1 is whole blood, serum or plasma; the magnetic beads in the magnetic bead-bovine leukemia virus antigen complex in step S2 are carboxyl magnetic beads; the chemiluminescent label in the IgG-chemiluminescent label complex in step S3 is acridinium ester, and the IgG is goat anti-bovine IgG, rabbit anti-bovine IgG or mouse anti-bovine IgG.

10. Use of the kit according to any one of claims 1-7 in the preparation of a product for detecting bovine leukemia virus IgG antibody.

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