A kit for detecting bovine leukemia virus IgG antibodies, and its detection method and application

Abstract

The present invention belongs to the technical field of chemiluminescence detection, and specifically relates to a kit for detecting bovine leukemia virus IgG antibodies, its detection method and application. The sample processing liquid in the kit includes: ProteinA, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, casein, mercaptoethanol, Tween-20, Proclin; the labeling of magnetic beads adopts PEG4000 and ethanolamine to jointly block. The blocking method provided by the present invention is more than the traditional single protein blocking, removes the interference of exogenous proteins, reduces nonspecific binding in the sample, reduces the background, and improves the stability, sensitivity and specificity of the detection; compared with the traditional large-scale diluted sample, the sample processing liquid removes excess IgG in the sample, does not require the equipment dilution module dilution process during the detection process, and does not require manual pre-dilution process, thereby improving the detection efficiency.

Description

Technical Field

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

[0002] Bovine leukemia virus (BLV) is a virus that infects only cattle. It is closely related to human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2), belonging to the same family as HTLV-1 and the genus deltaretrovirus in the family Retroviridae. It is an RNA virus containing single-stranded RNA. BLV replicates by budding, whereby buds form on the cell surface and are released to form new individuals. BLV is vulnerable to environmental factors and can be inactivated by ultraviolet light and repeated freeze-thaw cycles. It is heat-sensitive and can be inactivated by 0.5% carbolic acid and formaldehyde solutions. BLV is the pathogen that causes bovine leukemia, a disease characterized by persistent B lymphocytosis and systemic lymphadenopathy. Clinical symptoms are subtle during the incubation period (generally 4-5 years), but the mortality rate is high.

[0003] Enzootic bovine leukemia (BLV) is classified as a Category B animal disease. Infected cattle and latently infected cattle are the most important sources of infection and carry the virus throughout their lives. Natural reservoirs include zebu cattle, buffalo, sheep, and capybaras, but under experimental conditions, BLV can also infect goats, alpacas, rabbits, rats, and chickens. Researchers have even found BLV nucleic acid in human mammary tissue. Horizontal transmission occurs primarily through contact with blood, body fluids, and secretions, and through mechanical bites by blood-sucking insects. Currently, there are no commercially available BLV vaccines or effective treatments. Most cattle farms control BLV through testing and culling. Diagnosis is fundamental to BLV prevention and control. Since most BLV-infected cattle do not display any clinical symptoms, this poses a significant obstacle to clinical diagnosis. Regular monitoring of antigen or antibody levels in cattle is crucial.

[0004] Serological testing for BLV antibodies is a common and widely used method. Enzyme-linked immunosorbent assay (ELISA) involves adsorbing antigens or antibodies onto a solid-phase carrier and utilizing the specific reaction between them to achieve qualitative or quantitative analysis of the target. However, ELISA experiments are time-consuming and complex, requiring manual labor throughout the entire process, which can easily lead to contamination and false negatives or positives. Furthermore, it is particularly inconvenient for large-scale screening.

[0005] In view of the shortcomings of existing technologies and market demand, there is an urgent need to develop a simple and effective method for detecting bovine leukemia virus. Summary of the Invention

[0006] To address these shortcomings, the present invention provides a chemiluminescent detection kit and sample processing fluid for bovine leukemia virus IgG antibodies, as well as a detection method and application. This method overcomes practical challenges encountered by other methodologies, such as the cumbersome operational procedures, the need for machine-based dilution modules or manual dilution, the time-consuming and high-cost nature of the experiments, and the inconvenience of widespread screening. It has significant application value in regularly monitoring antibody levels in cattle herds to prevent BLV infection.

[0007] The technical solution of the present invention is:

[0008] In one aspect, the present invention provides a kit for detecting bovine leukemia virus IgG antibodies, the kit comprising a sample processing solution and a blocking agent.

[0009] Specifically, the sample processing solution consists of, by weight percentage, 0.00001%-0.001% of Protein A, 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, 0.1%-0.5% of casein, 0.01%-0.1% of mercaptoethanol, 0.01%-0.1% of Tween-20, 0.01%-0.1% of Proclin 300, and the remainder of solvent.

[0010] Preferably, in some embodiments, the sample processing solution consists of, by weight percentage, 0.0001% Protein A, 0.686% disodium hydrogen phosphate dodecahydrate, 0.048% potassium dihydrogen phosphate, 0.04% potassium chloride, 1.6% sodium chloride, 0.1% casein, 0.1% mercaptoethanol, 0.05% Tween-20, 0.05% Proclin 300, and water.

[0011] In some embodiments, the sample processing solution consists of, by weight percentage, 0.0001% Protein A, 1.5% disodium hydrogen phosphate dodecahydrate, 0.01% potassium dihydrogen phosphate, 0.1% potassium chloride, 1% sodium chloride, 0.5% casein, 0.01% mercaptoethanol, 0.1% Tween-20, 0.01% Proclin 300, and water.

[0012] In some embodiments, the sample processing solution consists of, by weight percentage, 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.

[0013] Specifically, the blocking agent includes PEG4000 and ethanolamine.

[0014] Preferably, the concentration of the PEG4000 may be 0.1 wt%-1 wt%; the concentration of the ethanolamine may be 10 mM-100 mM.

[0015] Preferably, the concentration of the PEG4000 may be 0.1 wt%-0.5 wt%; the concentration of the ethanolamine may be 10 mM-50 mM.

[0016] Preferably, the concentration of PEG4000 may be 0.1 wt%-0.2 wt%; the concentration of ethanolamine may be 10 mM-20 mM.

[0017] Preferably, the concentration of PEG4000 may be 0.1 wt %; the concentration of ethanolamine may be 10 mM.

[0018] Specifically, the kit further comprises an M diluent for diluting the magnetic bead-bovine leukemia virus antigen complex and / or an R diluent for diluting the IgG-chemiluminescent marker complex.

[0019] More specifically, the M diluent is composed, by weight percentage, 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 remainder solvent.

[0020] Preferably, in some embodiments, the M diluent is composed, by weight percentage, 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.

[0021] Preferably, in some embodiments, the M diluent is composed, by weight percentage, of 1.5% disodium hydrogen phosphate dodecahydrate, 0.01% potassium dihydrogen phosphate, 0.1% potassium chloride, 1% sodium chloride, 2% fish skin gelatin, 0.1% bovine serum albumin, 0.1% Tween-20, 0.01% Proclin 300, and water.

[0022] Preferably, in some embodiments, the M diluent is composed, by weight percentage, of 0.5% disodium hydrogen phosphate dodecahydrate, 0.1% potassium dihydrogen phosphate, 0.01% potassium chloride, 5% sodium chloride, 0.5% fish skin gelatin, 1% bovine serum albumin, 0.01% Tween-20, 0.1% Proclin 300 and water.

[0023] To be more specific, the R diluent is composed, by weight percentage, 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 water.

[0024] Preferably, in some embodiments, the R diluent consists, by weight percentage, of 0.686% disodium hydrogen phosphate dodecahydrate, 0.048% potassium dihydrogen phosphate, 0.04% potassium chloride, 1.6% sodium chloride, 1% sucrose, 3% trehalose, 0.2% EDTA-2Na, 0.05% Tween-20, 0.05% Proclin 300, and water.

[0025] Preferably, in some embodiments, the R diluent consists, by weight percentage, of 1.5% disodium hydrogen phosphate dodecahydrate, 0.01% potassium dihydrogen phosphate, 0.1% potassium chloride, 1% sodium chloride, 5% sucrose, 1% trehalose, 0.5% EDTA-2Na, 0.01% Tween-20, 0.1% Proclin 300, and water.

[0026] Preferably, in some embodiments, the R diluent consists, by weight percentage, of 0.5% disodium hydrogen phosphate dodecahydrate, 0.1% potassium dihydrogen phosphate, 0.01% potassium chloride, 5% sodium chloride, 1% sucrose, 5% trehalose, 0.1% EDTA-2Na, 0.1% Tween-20, 0.01% Proclin 300, and water.

[0027] 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.

[0028] Preferably, the magnetic beads in the magnetic bead-bovine leukemia virus antigen complex may be carboxyl magnetic beads.

[0029] Specifically, the viral antigens in the magnetic bead-bovine leukemia virus antigen complex include but are not limited to: natural bovine leukemia virus, bovine leukemia virus protein gp51, and bovine leukemia virus protein p24.

[0030] Preferably, the viral antigen in the magnetic bead-bovine leukemia virus antigen complex may be bovine leukemia virus protein gp51.

[0031] Further preferably, the gene sequence of the viral antigen in the magnetic bead-bovine leukemia virus antigen complex is referenced from UniProt (Universal Protein Resource), and its amino acid sequence is as follows:

[0032] SEQ ID NO.1:

[0033] MHHHHHHWRCSSLSLGNQQWMTAYNQEAKFSISINQILEAHNQSPFCAKSPRYTLDSVNGYPKIYWPPPQGRRRFGARAMVTYDCEPRCPYVGADRFDCPHWDNASQADQGSFYVNHQILFLHLKQCHGIFTLTWEIWG YDPLITFSLHKIPDPPQPDFPQLNSDWVPSVRSWALLLNQTARAFPDCAICWEPSPPWAPEILVYNKTISSSGPGLALPDAQIFWVNTSSFNTTQGWHHPSQRLLFNVSQGNALLLPPISLVNLSTASSAPPTRVRRS*

[0034] Specifically, the chemiluminescent marker in the IgG-chemiluminescent marker complex includes, but is not limited to, acridinium ester, luminol, horseradish peroxidase or alkaline phosphatase.

[0035] Preferably, the chemiluminescent marker in the IgG-chemiluminescent marker complex may be an acridinium ester.

[0036] 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.

[0037] Preferably, the IgG in the IgG-chemiluminescent marker complex can be goat anti-bovine IgG, rabbit anti-bovine IgG or mouse anti-bovine IgG.

[0038] Specifically, the kit further includes calibrators and / or quality control products.

[0039] In another aspect, the present invention provides a method for detecting bovine leukemia virus IgG antibodies, wherein the sample is detected using the aforementioned kit, and the specific steps are as follows:

[0040] S1. The sample and the sample treatment solution are mixed to obtain A;

[0041] S2, incubating A with the magnetic beads-bovine leukemia virus antigen complex to obtain B;

[0042] S3, adding IgG-chemiluminescent marker complex to B to obtain C;

[0043] S4, add substrate to C and measure the luminescence value;

[0044] Specifically, the sample in step S1 is whole blood, serum or plasma.

[0045] Specifically, the magnetic beads in the magnetic bead-bovine leukemia virus antigen complex in step S2 can be carboxyl magnetic beads.

[0046] Specifically, the chemiluminescent marker in the IgG-chemiluminescent marker complex in step S3 can be an acridinium ester.

[0047] Specifically, the IgG in the IgG-chemiluminescent marker complex in step S3 can be goat anti-bovine IgG, rabbit anti-bovine IgG or mouse anti-bovine IgG.

[0048] In another aspect, the present invention provides the use of the aforementioned kit method or detection method in the preparation of a product for detecting bovine leukemia virus IgG antibodies.

[0049] The beneficial effects of the present invention are:

[0050] (1) The detection method provided by the present invention can effectively reduce nonspecific binding in the sample and reduce the occurrence of false positives.

[0051] (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 detection sample.

[0052] (3) The sample processing solution provided by the present invention does not require pre-dilution during the detection process, which reduces the detection steps and improves the detection efficiency. DETAILED DESCRIPTION

[0053] The present invention will be further clarified and fully described below by way of examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely for illustration. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0054] Example 1

[0055] Preparation of sample treatment solution: Protein A 0.001g, disodium hydrogen phosphate dodecahydrate 6.86g, potassium dihydrogen phosphate 0.48g, potassium chloride 0.4g, sodium chloride 16g, casein 1g, mercaptoethanol 1g, Tween-20 0.5g, Proclin 3000 0.5g, dilute to 1L with water.

[0056] Preparation of M diluent: 6.86 g disodium hydrogen phosphate dodecahydrate, 0.48 g potassium dihydrogen phosphate, 0.4 g potassium chloride, 16 g sodium chloride, 10 g fish skin gelatin, 5 g bovine serum albumin, 0.5 g Tween-20, 0.5 g Proclin 300, and dilute to 1 L with water.

[0057] Preparation of R diluent: 6.86 g disodium hydrogen phosphate dodecahydrate, 0.48 g potassium dihydrogen phosphate, 0.4 g potassium chloride, 16 g sodium chloride, 10 g sucrose, 30 g trehalose, 2 g EDTA-2Na, 0.5 g Tween-20, 0.5 g Proclin 300, and make up to 1 L with water.

[0058] Preparation method of complex of carboxyl magnetic beads and bovine leukemia virus antigen:

[0059] (1) Wash 1 mL of 10 mg / mL carboxyl magnetic beads with 25 mM MES (pH 6.4) for three times, remove the supernatant, and re-dissolve.

[0060] (2) Activate 1 mL of 10 mg / mL carboxyl magnetic beads with 20 mM EDC and 2 mM NHS, oscillate and mix for 30 minutes, and then wash with 25 mM MES (pH 6.4) after activation. Wash three times, remove the supernatant, and redissolve.

[0061] (3) Add 0.1 mg of bovine leukemia antigen for reaction and shake for 3 hours to mix; use 0.1 M Tris-HCl for easy washing, wash three times, remove the supernatant and re-dissolve.

[0062] (4) Block the mixture with 0.1% PEG4000 and 10 mM ethanolamine by shaking for 2 h.

[0063] (5) Wash with 0.1 M Tris-HCl solution for three times, remove the supernatant, and then re-dissolve with M diluent.

[0064] Preparation of M reagent: Use the prepared M diluent to dilute the carboxyl magnetic beads-bovine leukemia virus antigen complex to 0.5 mg / mL.

[0065] Preparation of R reagent: Use the prepared R diluent to dilute the goat anti-bovine IgG-acridinium ester complex to 1 μg / mL.

[0066] The test principle of this kit is as follows: 10 μL serum or plasma sample and 90 μL sample treatment solution are added to the reaction cup, and the sample treatment solution with the sample is incubated and combined with 30 μL of reagent (carboxyl magnetic beads-bovine leukemia virus antigen complex) to form a complex of bovine leukemia antibody-bovine leukemia antigen-carboxyl magnetic beads, followed by the first step of washing (① the magnetic field adsorbs the complex of leukemia antibody-bovine leukemia antigen-carboxyl magnetic beads, and the supernatant is removed; ② 300 μL 0.01M PBST is added to the reaction cup and shaken to mix; ③ the magnetic field adsorbs the complex of leukemia antibody-bovine leukemia antigen-carboxyl magnetic beads, and the supernatant is removed, and steps ①②③ are repeated three times). After washing, the complex remains in the reaction cup due to the force of the magnetic field, and then 100 μL is added. R reagent (sheep anti-bovine IgG-acridinium ester complex) forms a complex of goat anti-bovine IgG aridinium ester, bovine leukemia antibody, bovine leukemia antigen, and carboxyl magnetic beads. A second wash step is then performed (① The complex of goat anti-bovine IgG aridinium ester, bovine leukemia antibody, bovine leukemia antigen, and carboxyl magnetic beads is adsorbed by the magnetic field, and the supernatant is removed; ② 300 μl of 0.01 M PBST is added to the reaction cup and shaken to mix; ③ The complex of goat anti-bovine IgG aridinium ester, bovine leukemia antibody, bovine leukemia antigen, and carboxyl magnetic beads is adsorbed by the magnetic field, and the supernatant is removed. Steps ①, ②, and ③ are repeated three times). After washing, the complex remains in the reaction cup due to the force of the magnetic field. After adding 100 μl of pre-excitation solution and 100 μl of excitation solution, the luminescence value is obtained by photometry, and the test result is obtained.

[0067] Example 2

[0068] The difference from Example 1 is that the configuration of the R reagent uses an acridinium ester complex labeled with rabbit anti-bovine IgG. Other steps are the same as in Example 1.

[0069] Example 3

[0070] The difference from Example 1 is that the R reagent is configured with an acridinium ester complex labeled with mouse anti-bovine IgG. Other steps are the same as in Example 1.

[0071] Example 4

[0072] The differences from Example 1 are as follows, and the rest are the same as Example 1.

[0073] Preparation of sample treatment solution: Protein A 0.0001g, disodium hydrogen phosphate dodecahydrate 15g, potassium dihydrogen phosphate 0.1, potassium chloride 1g, sodium chloride 10g, casein 5g, mercaptoethanol 0.1g, Tween-20 1g, Proclin 300 0.1g, dilute to 1L with water.

[0074] Preparation of M diluent: 15 g disodium hydrogen phosphate dodecahydrate, 0.1 g potassium dihydrogen phosphate, 1 g potassium chloride, 10 g sodium chloride, 20 g fish skin gelatin, 1 g bovine serum albumin, 1 g Tween-20, 0.1 g Proclin 300, and dilute to 1 L with water.

[0075] Preparation of R diluent: 15 g disodium hydrogen phosphate dodecahydrate, 0.1 g potassium dihydrogen phosphate, 1 g potassium chloride, 10 g sodium chloride, 50 g sucrose, 10 g trehalose, 5 g EDTA-2Na, 0.1 g Tween-20, 1 g Proclin 300, and dilute to 1 L with water.

[0076] Example 5

[0077] The differences from Example 1 are as follows, and the rest are the same as Example 1.

[0078] Preparation of sample treatment solution: Protein A 0.01g, disodium hydrogen phosphate dodecahydrate 5g, potassium dihydrogen phosphate 1g, potassium chloride 0.1g, sodium chloride 50g, casein 1g, mercaptoethanol 1g, Tween-20 0.1g, Proclin 300 1g, dilute to 1L with water.

[0079] Preparation of M diluent: 5g disodium hydrogen phosphate dodecahydrate, 1g potassium dihydrogen phosphate, 0.1g potassium chloride, 50g sodium chloride, 5g fish skin gelatin, 10g bovine serum albumin, 0.1g Tween-20, 1g Proclin 300, and dilute to 1L with water.

[0080] 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, and 0.1 g of Proclin 300. Add water to make up to 1 L.

[0081] Comparative Example 1

[0082] The difference from Example 1 is that no sample processing liquid is used. Other aspects are the same as Example 1.

[0083] Comparative Example 2

[0084] The difference from Example 1 is that the blocking agents PEG4000 and ethanolamine are not used in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen.

[0085] Comparative Example 3

[0086] The difference from Example 1 is that only PEG4000 is used as a blocking agent in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen.

[0087] Comparative Example 4

[0088] The difference from Example 1 is that only ethanolamine is used as a blocking agent in the preparation of the complex of carboxyl magnetic beads and bovine leukemia virus antigen.

[0089] Comparative Example 5

[0090] The difference from Example 1 is that only the commonly used blocking agent BSA is used to prepare the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other steps are the same as in Example 1.

[0091] Comparative Example 6

[0092] The difference from Example 1 is that only skimmed milk powder, a common blocking agent, is used to prepare the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other aspects are the same as in Example 1.

[0093] Comparative Example 7

[0094] The difference from Example 1 is that the common blocking agents BSA and PEG4000 are used as blocking agents to prepare the complex of carboxyl magnetic beads and bovine leukemia virus antigen. Other procedures are the same as in Example 1.

[0095] Comparative Example 8

[0096] The difference from Example 1 is that the complex of carboxyl magnetic beads and bovine leukemia virus antigen is prepared using common blocking agents BSA and ethanolamine as blocking agents. Other procedures are the same as in Example 1.

[0097] Comparative Example 9

[0098] The difference from Example 1 is that the complex of carboxyl magnetic beads and bovine leukemia virus antigen is prepared using commonly used blocking agents, skim milk powder and PEG4000. Other procedures are the same as in Example 1.

[0099] Comparative Example 10

[0100] The difference from Example 1 is that the complex of carboxyl magnetic beads and bovine leukemia virus antigen is prepared using commonly used blocking agents, skim milk powder and ethanolamine. Other aspects are the same as in Example 1.

[0101] Comparative Example 11

[0102] The difference from Example 1 is that the PEG4000 used in preparing the complex of carboxyl magnetic beads and bovine leukemia virus antigen is replaced by polyacrylamide. Other steps are the same as in Example 1.

[0103] Comparative Example 12

[0104] The difference from Example 1 is that the ethanolamine used in preparing the complex of carboxyl magnetic beads and bovine leukemia virus antigen is replaced by tris(hydroxymethyl)aminomethane. Other details are the same as in Example 1.

[0105] Comparative Example 13

[0106] The difference from Example 1 is that the sample is diluted in advance using a sample processing solution before being loaded onto the machine. Other steps are the same as in Example 1.

[0107] Comparative Example 14

[0108] The difference from Example 1 is that the sample is diluted in advance with physiological saline before being loaded onto the machine. Other details are the same as in Example 1.

[0109] Comparative Example 15

[0110] The difference from Example 1 is that the sample is diluted in advance with PBS before being loaded onto the machine. Other details are the same as in Example 1.

[0111] Effect Example 1

[0112] 80 serum samples that had been tested for nucleic acid were selected, including 50 negative serum samples and 30 positive serum samples. The 80 clinical serum samples were tested using Examples 1 to 5 and Comparative Examples 1 to 15, respectively. The results of Examples 1 to 5 were compared with those of Comparative Examples 1 to 15. The results are shown in Table 1:

[0113] Table 1

[0114]

[0115] Example 1 tested 30 positive sera determined by nucleic acid, 2 were negative sera, 28 were positive sera, and the positive compliance rate was 93.3%; tested 50 negative sera determined by nucleic acid, 2 were positive sera, 48 were negative sera, the negative compliance rate was 96.0%, and the total compliance rate was 95.0%.

[0116] Example 2 tested 30 positive sera determined by nucleic acid, 2 were negative sera, 28 were positive sera, and the positive compliance rate was 93.3%; tested 50 negative sera determined by nucleic acid, 2 were positive sera, 48 were negative sera, the negative compliance rate was 96.0%, and the total compliance rate was 95.0%.

[0117] Example 3 tested 30 positive sera determined by nucleic acid, 2 were negative sera, 28 were positive sera, and the positive compliance rate was 93.3%; tested 50 negative sera determined by nucleic acid, 2 were positive sera, 48 were negative sera, the negative compliance rate was 96.0%, and the total compliance rate was 95.0%.

[0118] Example 4 tested 30 positive sera determined by nucleic acid, 2 were negative sera, 28 were positive sera, and the positive compliance rate was 93.3%; tested 50 negative sera determined by nucleic acid, 2 were positive sera, 48 were negative sera, the negative compliance rate was 96.0%, and the total compliance rate was 95.0%.

[0119] Example 5 tested 30 positive sera determined by nucleic acid, 2 were negative sera, 28 were positive sera, and the positive compliance rate was 93.3%; tested 50 negative sera determined by nucleic acid, 2 were positive sera, 48 were negative sera, the negative compliance rate was 96.0%, and the total compliance rate was 95.0%.

[0120] In comparative example 1, 30 cases of positive sera determined by nucleic acid were tested, 1 case was negative serum, 29 cases were positive sera, and the positive compliance rate was 96.7%; 50 cases of negative sera determined by nucleic acid were tested, 35 cases were positive sera, 15 cases were negative sera, the negative compliance rate was 30.0%, and the total compliance rate was 55.0%.

[0121] In comparative example 2, 30 cases of positive sera determined by nucleic acid were tested, 0 cases were negative sera, 30 cases were positive sera, and the positive compliance rate was 100.0%; 50 cases of negative sera determined by nucleic acid were tested, 50 cases were positive sera, 0 cases were negative sera, the negative compliance rate was 0.0%, and the total compliance rate was 37.5%.

[0122] In comparative example 3, 30 cases of positive sera determined by nucleic acid were tested, 12 cases were negative sera, 18 cases were positive sera, and the positive compliance rate was 60.0%; 50 cases of negative sera determined by nucleic acid were tested, 7 cases were positive sera, 43 cases were negative sera, the negative compliance rate was 86.0%, and the total compliance rate was 76.3%.

[0123] In comparative example 4, 30 cases of positive sera determined by nucleic acid were tested, 11 cases were negative sera, 19 cases were positive sera, and the positive compliance rate was 63.3%; 50 cases of negative sera determined by nucleic acid were tested, 5 cases were positive sera, 45 cases were negative sera, the negative compliance rate was 90.0%, and the total compliance rate was 80.0%.

[0124] In comparative example 5, 30 cases of positive sera determined by nucleic acid were tested, 18 cases were negative sera, 12 cases were positive sera, and the positive compliance rate was 40.0%; 50 cases of negative sera determined by nucleic acid were tested, 0 cases were positive sera, 50 cases were negative sera, the negative compliance rate was 100.0%, and the total compliance rate was 77.5%.

[0125] Comparative Example 6 tested 30 cases of positive sera determined by nucleic acid, 10 cases were negative sera, 20 cases were positive sera, and the positive compliance rate was 66.7%; tested 50 cases of negative sera determined by nucleic acid, 1 case was positive serum, 49 cases were negative sera, the negative compliance rate was 98.0%, and the total compliance rate was 86.3%.

[0126] Comparative Example 7 tested 30 cases of positive sera determined by nucleic acid, 6 cases were negative sera, 24 cases were positive sera, and the positive compliance rate was 80.0%; tested 50 cases of negative sera determined by nucleic acid, 5 cases were positive sera, 45 cases were negative sera, the negative compliance rate was 90.0%, and the total compliance rate was 86.3%.

[0127] In comparison example 8, 30 cases of positive sera determined by nucleic acid were tested, 8 cases were negative sera, 22 cases were positive sera, and the positive compliance rate was 73.3%; 50 cases of negative sera determined by nucleic acid were tested, 4 cases were positive sera, 46 cases were negative sera, the negative compliance rate was 92.0%, and the total compliance rate was 85.0%.

[0128] In comparative example 9, 30 cases of positive sera determined by nucleic acid were tested, 7 cases were negative sera, 23 cases were positive sera, and the positive compliance rate was 76.7%; 50 cases of negative sera determined by nucleic acid were tested, 4 cases were positive sera, 46 cases were negative sera, the negative compliance rate was 92.0%, and the total compliance rate was 86.3%.

[0129] In comparison example 10, 30 cases of positive sera determined by nucleic acid were tested, 7 cases were negative sera, 23 cases were positive sera, and the positive compliance rate was 76.7%; 50 cases of negative sera determined by nucleic acid were tested, 6 cases were positive sera, 44 cases were negative sera, the negative compliance rate was 88.0%, and the total compliance rate was 83.8%.

[0130] Comparative Example 11 tested 30 cases of positive sera determined by nucleic acid, 11 of which were negative sera and 19 were positive sera, with a positive compliance rate of 63.3%; tested 50 cases of negative sera determined by nucleic acid, 5 of which were positive sera and 45 were negative sera, with a negative compliance rate of 90.0% and an overall compliance rate of 80.0%.

[0131] In comparison example 12, 30 cases of positive sera determined by nucleic acid were tested, 18 cases were negative sera, 12 cases were positive sera, and the positive compliance rate was 40.0%; 50 cases of negative sera determined by nucleic acid were tested, 5 cases were positive sera, 45 cases were negative sera, the negative compliance rate was 90.0%, and the total compliance rate was 71.3%.

[0132] Comparative Example 13 tested 30 cases of positive sera determined by nucleic acid, 2 cases were negative sera, 28 cases were positive sera, and the positive compliance rate was 93.3%; tested 50 cases of negative sera determined by nucleic acid, 2 cases were positive sera, 48 cases were negative sera, the negative compliance rate was 96.0%, and the total compliance rate was 95.0%.

[0133] Comparative Example 14 tested 30 cases of positive sera determined by nucleic acid, 0 cases were negative sera, 30 cases were positive sera, and the positive compliance rate was 100.0%; tested 50 cases of negative sera determined by nucleic acid, 9 cases were positive sera, 41 cases were negative sera, the negative compliance rate was 82.0%, and the total compliance rate was 88.8%.

[0134] In comparison example 15, 30 cases of positive sera determined by nucleic acid were tested, 0 cases were negative sera, 30 cases were positive sera, and the positive compliance rate was 100.0%; 50 cases of negative sera determined by nucleic acid were tested, 9 cases were positive sera, 41 cases were negative sera, the negative compliance rate was 82.0%, and the total compliance rate was 88.8%.

[0135] As shown in the table above, the positive / negative coincidence rates and overall coincidence rates for Examples 1-5 and Comparative Example 13 were all above 90%, meeting the test conditions. The remaining comparative examples had lower positive / negative coincidence rates and did not meet the test conditions. Comparative Example 13 used sample processing fluid to pre-dilute the sample before loading it onto the instrument. While this achieved the same test results, it added an additional reaction step. Therefore, Examples 1-5 are more preferred.

[0136] Effect Example 2

[0137] Deionized water was used to test the background luminescence values ​​of Examples 1 to 5 and Comparative Examples 1 to 15, and the results were repeated three times. See Table 2:

[0138] Table 2

[0139]

[0140] As can be seen from the table above, the background luminescence values ​​of deionized water tested in Examples 1-5 and Comparative Example 13 were all below 5000, meeting the detection requirements. All of the above Examples and Comparative Examples employed non-protein blocking methods, including a combination of PEG4000 and ethanolamine. The background values ​​of the remaining Comparative Examples were high and did not meet the detection requirements. Comparative Example 13 used sample treatment solution to pre-dilute the sample before loading. While achieving the same detection effect, it added a reaction step. Therefore, Examples 1-5 are more preferred.

[0141] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A kit for detecting bovine leukemia virus IgG antibodies, characterized in that: The kit includes a sample processing solution, a magnetic bead-bovine leukemia virus antigen complex, an IgG-chemiluminescent marker complex, and a blocking agent; The sample treatment solution consists of, by weight percentage, 0.00001%-0.001% of Protein A, 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, 0.1%-0.5% of casein, 0.01%-0.1% of mercaptoethanol, 0.01%-0.1% of Tween-20, 0.01%-0.1% of Proclin 300, and the balance of solvent; The blocking agents include 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, wherein Also included is an M diluent for diluting the magnetic bead-bovine leukemia virus antigen complex and / or an R diluent for diluting the IgG-chemiluminescent marker complex; The M diluent is composed 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, 0.5%-2% of fish skin gelatin, 0.1%-1% of bovine serum albumin, 0.01%-0.1% of Tween-20, 0.01%-0.1% of Proclin 300, and the balance of a solvent by weight percentage; The R diluent is composed, by weight percentage, 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 solvent.

4. The kit according to claim 3, wherein 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 marker in the IgG-chemiluminescent marker complex is acridinium ester, luminol, horseradish peroxidase or alkaline phosphatase.

6. The kit according to claim 5, characterized in that The IgG in the IgG-chemiluminescent marker 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 to 6, characterized in that The kit also includes calibrators and / or quality control products.

8. A method for detecting bovine leukemia virus IgG antibodies for non-disease diagnosis purposes, characterized in that: The sample is tested using the kit according to any one of claims 1 to 7, and the specific steps are as follows: S1. The sample and the sample treatment solution are mixed to obtain A; S2, incubating A with the magnetic beads-bovine leukemia virus antigen complex to obtain B; S3, adding IgG-chemiluminescent marker complex to B to obtain C; S4. Add 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 marker in the IgG-chemiluminescent marker 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 to 7 in the preparation of a product for detecting bovine leukemia virus IgG antibodies.

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