African swine fever virus antibody test kit

Abstract

This application discloses an African swine fever virus antibody detection kit. The African swine fever virus antibody detection kit of the present invention comprises an antibody detection chip; the antibody detection chip comprises a solid-phase support and polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 independently immobilized on the solid-phase support. The African swine fever virus antibody detection kit of the present invention can be used for the detection of African swine fever virus antibodies in biological samples, achieving both high sensitivity and high specificity.

Description

Technical Field

[0001] This invention primarily relates to the field of biological detection technology. Specifically, this invention relates to an African swine fever virus antibody detection kit. Background Technology

[0002] African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and various wild boars (such as African wild boar and European wild boar). Its prevention and control mainly rely on detection and screening, so test kits have become an indispensable tool for the prevention and control of ASF.

[0003] Currently developed diagnostic kits can be divided into two categories based on their detection targets: those that detect viral nucleic acids and those that detect viral antibodies. However, these kits still have the following shortcomings, which prevent them from fully meeting the needs of disease prevention and clinical work: Nucleic acid detection kits have a specificity close to 100%, but their sensitivity is low due to difficulties in sampling and the intermittent viral shedding of infected pigs, resulting in a relatively high number of false negatives; while antibody detection kits using ASFV component proteins such as P54 and P72 proteins can achieve a sensitivity of over 95%, but due to inherent flaws in their design, achieving high sensitivity inevitably sacrifices specificity (especially since some non-ASFV antibodies produced in pigs by vaccines against other diseases can also be recognized by these component proteins), resulting in a relatively high number of false positives.

[0004] Therefore, it is necessary to develop an antibody detection kit for African swine fever virus with high sensitivity and specificity. Summary of the Invention

[0005] An antigenic epitope is a region of a protein antigen that binds to an antibody; a single protein antigen may contain multiple antigenic epitopes. An antigenic epitope peptide is a polypeptide containing an antigenic epitope, which can be obtained artificially. Antigenic epitope peptides can also be used to detect antibodies that can bind to them.

[0006] Through in-depth research, the inventors found a combination of antigenic epitope peptides from the constituent proteins of African swine fever virus and the African swine fever virus P30 protein. When used for the detection of African swine fever virus antibodies, it can achieve a sensitivity and specificity of more than 95% at the same time.

[0007] That is, the present invention includes:

[0008] 1. An African swine fever virus antibody detection kit, comprising an antibody detection chip;

[0009] The antibody detection chip comprises a solid-phase carrier and polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, which are independently immobilized on the solid-phase carrier.

[0010] SEQ ID NO: 1 (PFTHLLIEKACKDHNYEVIK, MGF-17),

[0011] SEQ ID NO: 2(PVTGRPATNRPATNKPVTDN, p54-11),

[0012] SEQ ID NO: 3 (PVTDRLVMATGGPAAAPAAA, p54-13), and

[0013] SEQ ID NO: 4 (SNIKNVNKSYGKPDPEPTLS, p72-4).

[0014] 2. The African swine fever virus antibody detection kit according to item 1, which is used to detect whether a biological sample of pig origin contains African swine fever virus antibodies. Here, the biological sample only needs to be capable of containing African swine fever virus antibodies, and can be, for example, whole blood, plasma, serum, but is not limited thereto.

[0015] 3. The African swine fever virus antibody detection kit according to item 1, wherein the P30 protein of African swine fever virus is independently immobilized on the solid phase support.

[0016] 4. Application of antibody detection chips in the preparation of African swine fever virus antibody detection kits.

[0017] The antibody detection chip includes a solid-phase carrier and polypeptides represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 respectively immobilized on the solid-phase carrier.

[0018] 5. The use according to item 4, wherein the African swine fever virus antibody detection kit is used to detect whether a biological sample of pig origin contains African swine fever virus antibodies.

[0019] 6. The use according to item 4, wherein the P30 protein of African swine fever virus is also independently immobilized on the solid support.

[0020] 7. A detection reagent comprising the polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0021] 8. The detection reagent according to item 7, wherein the detection reagent further comprises the P30 protein of African swine fever virus.

[0022] 9. Use of the detection reagent according to item 7 or 8 in the preparation of an African swine fever virus antibody detection kit. Detailed Implementation

[0023] The African swine fever virus antibody detection kit of the present invention is used to detect whether African swine fever virus antibodies are present in biological samples of pig origin. Specifically, the number of polypeptides on the antibody detection chip that respond to the biological sample is denoted as DMI(Pep), the number of proteins on the antibody detection chip that respond to the biological sample is denoted as DMI(Pro), and DMI(Pep) + DMI(Pro) is denoted as DMI(Total). Therefore, for a given biological sample,

[0024] When DMI (total) ≤ 1, the test result is considered negative, meaning that the sample does not contain African swine fever virus antibodies;

[0025] When DMI(total) ≥ 2 and DMI(Pro) = 0, the test result is considered negative, meaning that the sample does not contain African swine fever virus antibodies; and

[0026] When DMI(total)≥2 and DMI(Pro)=1, the test result is considered positive, meaning that the sample contains African swine fever virus antibodies.

[0027] In this specification, "response" refers to a signal value read by the reading device that is significantly different from the signal value of a negative point. For example, the signal value of a positive point read by the reading device is greater than or equal to 10, preferably greater than or equal to 20, more preferably greater than or equal to 30; while the signal value of a negative point read by the reading device is less than 10, preferably less than 5, more preferably less than 1. The reading device may be, for example, a microarray chip imager manufactured by Suzhou Abtop Biotechnology Co., Ltd.

[0028] In this specification, there may be one or more solid supports, but preferably one, meaning that all peptides are independently linked to the same solid support. There are no special limitations on the solid support in this invention, as long as it is a solid or insoluble material. The linking of peptides to the solid support can be performed using methods known to those skilled in the art.

[0029] Example 1. Preparation and confirmation of polypeptides

[0030] The polypeptides SEQ ID NO: 1–4 were synthesized by Genscript Biotech and confirmed by mass spectrometry; the P30 protein of African swine fever virus recombinantly expressed in *E. coli* was purchased from Genscript Biotech.

[0031] SEQ ID NO: 1 (PFTHLLIEKACKDHNYEVIK, MGF-17),

[0032] SEQ ID NO: 2(PVTGRPATNRPATNKPVTDN, p54-11),

[0033] SEQ ID NO: 3(PVTDRLVMATGGPAAAPAAA, p54-13),

[0034] SEQ ID NO: 4 (SNIKNVNKSYGKPDPEPTLS, p72-4).

[0035] Example 2. Preparation of antibody detection chip (reagent kit)

[0036] An antibody detection chip was prepared by spotting solutions of the polypeptides and P30 protein shown in SEQ ID NO: 1-4 onto a conventional ELISA solid-phase carrier using conventional methods. In addition, a porcine IgG was spotted as a positive control point and a PB buffer was spotted as a negative control point.

[0037] Accordingly, an African swine fever virus antibody detection kit was prepared, which, in addition to the antibody detection chip, also includes concentrated cleaning solution, sample diluent, enzyme-labeled antibody solution, TMB chromogenic solution, and other reagents for antibody detection.

[0038] Example 3. Antibody detection using a kit

[0039] Detection steps

[0040] (1) Dilute the 20× concentrated cleaning solution (TBST: 0.4M Tris-HCl, 2.74M NaCl, 2% Tween 20, pH 7.2±0.2) with purified water at a ratio of 1:20 to obtain the cleaning solution. To ensure complete wetting of the detection chip surface, add approximately 100 μL of the cleaning solution to the detection chip surface using a pipette and soak the detection chip for a certain period of time.

[0041] (2) Dilute the serum sample to be tested at a ratio of 1:50 with sample dilution buffer (0.05MPBS, 1% BSA, 0.2% PVP, 0.5% Tween 20, pH 7.2±0.2).

[0042] (3) For the detection chip that has been cleaned, add 100 μL of diluted serum sample to the detection chip while the surface is completely wet.

[0043] (4) Incubate the detection chip in a constant temperature shaker at 500 rpm for 30 minutes at a temperature of 37 degrees Celsius.

[0044] (5) Discard the serum sample and clean the surface of the detection chip with cleaning solution.

[0045] (6) After cleaning, add 100 μL of enzyme-labeled antibody solution (rabbit anti-pig IgG-HRP, Sigma, A5670) diluted at 1:7500 to the detection chip, and incubate the detection chip in a constant temperature shaker at 500 rpm for 30 minutes at a temperature of 37 degrees Celsius.

[0046] (7) Discard the enzyme-labeled antibody solution and clean the surface of the detection chip with cleaning solution.

[0047] (8) After cleaning, add 90 μL of TMB colorimetric solution (Thermo, Prod#37574) to the detection chip, and incubate the detection chip in the dark for 30 minutes at a temperature of 37 degrees Celsius.

[0048] (9) Discard the TMB colorimetric solution, rinse 3 times with pure water, and air dry / blow dry.

[0049] (10) Take a picture of the detection chip with a microarray chip imager and determine the result.

[0050] (11) Result determination:

[0051] For each serum sample, a "response" is indicated when the signal value of a specific antigen epitope polypeptide or protein spot on the antibody detection chip is greater than or equal to 10 (the signal value of the negative control spot is less than 1); otherwise, there is no response.

[0052] The number of polypeptides in the polypeptides of SEQ ID NO: 1-4 that respond to the serum is denoted as DMI(Pep), and the number of proteins on the antibody detection chip that respond to the biological sample is denoted as DMI(Pro) (since there is only one protein, P30, this value is 0 or 1). DMI(Pep) + DMI(Pro) is denoted as DMI(Total).

[0053] When DMI (total) ≤ 1, the test result is considered negative, meaning that the sample does not contain African swine fever virus antibodies;

[0054] When DMI(total) ≥ 2 and DMI(Pro) = 0, the test result is considered negative, meaning that the sample does not contain African swine fever virus antibodies; and

[0055] When DMI(total)≥2 and DMI(Pro)=1, the test result is considered positive, meaning that the sample contains African swine fever virus antibodies.

[0056] Example 4. Antibody detection results

[0057] For the 89 confirmed positive samples (the pigs were confirmed to be infected with African swine fever virus by nucleic acid testing and the results of other antibody testing methods were positive), the results of testing with the above-mentioned African swine fever antibody test kit were: 88 positive and 1 negative.

[0058] For the 150 identified negative samples (where nucleic acid testing confirmed that the pigs were not infected with African swine fever virus and other antibody testing methods were negative), the results of testing with the above-mentioned African swine fever antibody test kit were: 2 positive and 148 negative.

[0059] Therefore, the above-mentioned African swine fever antibody detection kit has a sensitivity of 88 / 89 = 98.9% and a specificity of 148 / 150 = 98.7%, achieving both high sensitivity and high specificity.

Claims

1. An African swine fever virus antibody detection kit, comprising an antibody detection chip; Its features are, The antibody detection chip includes: solid support The polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the P30 protein of African swine fever virus, were independently immobilized on the solid support.

2. The African swine fever virus antibody detection kit according to claim 1, characterized in that: The kit is used to detect whether pig-derived biological samples contain antibodies against African swine fever virus.

3. The African swine fever virus antibody detection kit according to claim 2, characterized in that: The biological samples include whole blood, plasma, or serum.

4. The use of an antibody detection chip in the preparation of an African swine fever virus antibody detection kit, wherein the antibody detection chip comprises: solid support The polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the P30 protein of African swine fever virus were respectively immobilized on the solid support.

5. The use according to claim 4, characterized in that: The African swine fever virus antibody detection kit is used to detect whether pig-derived biological samples contain African swine fever virus antibodies.

6. A detection reagent, characterized in that, Includes the polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, as well as the P30 protein of African swine fever virus.

7. Use of the detection reagent according to claim 6 in the preparation of an African swine fever virus antibody detection kit.