CN117706085A - African swine fever virus antibody detection kit - Google Patents
African swine fever virus antibody detection kit Download PDFInfo
- Publication number
- CN117706085A CN117706085A CN202211119109.8A CN202211119109A CN117706085A CN 117706085 A CN117706085 A CN 117706085A CN 202211119109 A CN202211119109 A CN 202211119109A CN 117706085 A CN117706085 A CN 117706085A
- Authority
- CN
- China
- Prior art keywords
- swine fever
- african swine
- fever virus
- seq
- antibody detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 85
- 241000701386 African swine fever virus Species 0.000 title claims abstract description 51
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 19
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 17
- 229920001184 polypeptide Polymers 0.000 claims abstract description 15
- 239000012472 biological sample Substances 0.000 claims abstract description 12
- 239000007790 solid phase Substances 0.000 claims abstract description 12
- 101001105440 Chlamydomonas reinhardtii Photosystem I reaction center subunit IV, chloroplastic Proteins 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 241000282898 Sus scrofa Species 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 claims description 2
- 210000002381 plasma Anatomy 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 241000282887 Suidae Species 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000004140 cleaning Methods 0.000 description 7
- 208000007407 African swine fever Diseases 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical group 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 101900228950 African swine fever virus Phosphoprotein p30 Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102100027303 Interferon-induced protein with tetratricopeptide repeats 2 Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102100021409 Probable ATP-dependent RNA helicase DDX17 Human genes 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000005441 aurora Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application discloses an African swine fever virus antibody detection kit. The African swine fever virus antibody detection kit comprises an antibody detection chip; the antibody detection chip comprises a solid phase carrier and SEQ ID NO independently fixed on the solid phase carrier: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide shown in the formula 4. The African swine fever virus antibody detection kit can be used for detecting African swine fever virus antibodies in biological samples, and can realize high sensitivity and high specificity at the same time.
Description
Technical Field
The invention mainly relates to the technical field of biological detection. In particular, the invention relates to an African swine fever virus antibody detection kit.
Background
African swine fever is an acute, hemorrhagic and virulent infectious disease caused by infection of domestic pigs and various wild pigs (such as African wild pigs, european wild pigs and the like) by African swine fever virus (African Swine fever virus, ASFV), and the prevention and control of the African swine fever mainly depend on detection and screening, so that the detection kit becomes an indispensable tool for preventing and controlling the African swine fever epidemic situation.
The detection kits developed at present can be classified into two types according to detection targets, one type is nucleic acid for detecting viruses, and the other type is antibody for detecting viruses. However, these test kits have the following disadvantages, so that the requirements of epidemic prevention and clinical work cannot be fully satisfied: the specificity of the nucleic acid detection kit is close to 100%, but the sensitivity is lower due to the reasons of difficult sampling, intermittent detoxification of pigs with toxicity and the like, namely more missed detection exists; the sensitivity of the antibody detection kit using ASFV component proteins such as P54 protein and P72 protein can reach more than 95%, but the sensitivity must be sacrificed for realizing high sensitivity due to principle defects (particularly, some non-ASFV antibodies generated in pigs aiming at vaccines of other epidemic diseases can be recognized by the component proteins), namely, more false detection exists.
Therefore, it is necessary to develop an african swine fever virus antibody detection kit having high detection sensitivity and specificity.
Disclosure of Invention
An epitope is a region of a protein antigen to which an antibody binds, and multiple epitopes may be present in a single protein antigen. The epitope peptide is a polypeptide containing an epitope, which can be obtained by artificial synthesis, and can be used for detecting an antibody capable of binding to the epitope peptide.
Through intensive research, the inventor finds a group of antigen epitope peptides from African swine fever virus constituent proteins and the combination of African swine fever virus P30 proteins, and when the antigen epitope peptides are used for detecting African swine fever virus antibodies, the sensitivity and the specificity can be realized at the same time by more than 95%.
Namely, the present invention includes:
1. an african swine fever virus antibody detection kit comprising an antibody detection chip;
the antibody detection chip comprises a solid phase carrier and SEQ ID NO independently fixed on the solid phase carrier: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO: 4:
SEQ ID NO：1(PFTHLLIEKACKDHNYEVIK，MGF-17)，
SEQ ID NO：2(PVTGRPATNRPATNKPVTDN，p54-11)，
SEQ ID NO:3 (PVTDRLVMATGGPAAAPAAA, p 54-13), and
SEQ ID NO：4(SNIKNVNKSYGKPDPEPTLS，p72-4)。
2. the african swine fever virus antibody detection kit of item 1, which is used for detecting whether an african swine fever virus antibody is contained in a biological sample derived from a pig. Here, the biological sample may include african swine fever virus antibodies, and may be, for example, whole blood, plasma, or serum, but is not limited thereto.
3. The african swine fever virus antibody detection kit of item 1, wherein the solid phase carrier is further independently immobilized with the P30 protein of african swine fever virus.
4. The application of the antibody detection chip in preparing an African swine fever virus antibody detection kit,
the antibody detection chip comprises a solid phase carrier, and SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide shown in the formula 4.
5. The use according to item 4, wherein the african swine fever virus antibody detection kit is used for detecting whether an african swine fever virus antibody is contained in a biological sample of porcine origin.
6. The use according to item 4, wherein the solid support has independently immobilized thereon the P30 protein of African swine fever virus.
7. A detection reagent comprising SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide shown in the formula 4.
8. The test agent according to item 7, wherein the test agent further comprises P30 protein of African swine fever virus.
9. The use of the detection reagent according to item 7 or 8 for preparing an african swine fever virus antibody detection kit.
Detailed Description
The African swine fever virus antibody detection kit is used for detecting whether an African swine fever virus antibody is contained in a biological sample derived from pigs. Specifically, the number of the types of the polypeptides responsive to the biological sample among the polypeptides on the antibody detection chip is referred to as DMI (Pep), the number of the types of the proteins responsive to the biological sample among the proteins on the antibody detection chip is referred to as DMI (Pro), and DMI (Pep) +DMI (Pro) is referred to as DMI (total), then, for a certain biological sample,
when DMI (total) is less than or equal to 1, the detection result is judged to be negative, namely, the sample does not contain African swine fever virus antibody;
when DMI (total) > 2 and DMI (Pro) =0, the detection result is judged as negative, i.e., the sample does not contain african swine fever virus antibody; and is also provided with
When DMI (total) > 2 and DMI (Pro) =1, the detection result was judged to be positive, i.e., african swine fever virus antibody was contained in the sample.
In this specification, "response" means that the signal value of a positive spot read with a reading device is significantly different from the signal value of a negative spot. For example, the signal value of the positive spot 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 the negative spot 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 idetora biotechnology limited, su.
In the present specification, the solid support may be one or more, but is preferably one, that is, all polypeptides are independently linked to the same solid support. In the present invention, the solid carrier is not particularly limited as long as it is a carrier that is a solid or insoluble material. Attachment of the polypeptide to the solid support may be carried out using methods of attachment of polypeptides to solid supports known to those skilled in the art.
EXAMPLE 1 preparation and confirmation of Polypeptides
SEQ ID NO: 1-4 by gold srey biosynthesis and confirmed by mass spectrometry; the P30 protein of african swine fever virus recombinantly expressed by escherichia coli was purchased from aurora. Wherein,
SEQ ID NO：1(PFTHLLIEKACKDHNYEVIK，MGF-17)，
SEQ ID NO：2(PVTGRPATNRPATNKPVTDN，p54-11)，
SEQ ID NO：3(PVTDRLVMATGGPAAAPAAA，p54-13)，
SEQ ID NO：4(SNIKNVNKSYGKPDPEPTLS，p72-4)。
example 2 preparation of antibody detection chip (kit)
The above SEQ ID NOs were spotted on a conventional ELISA solid phase carrier by a conventional method: 1-4, and spotting a pig IgG as a positive quality control point and a PB buffer solution point as a negative quality control point, thereby preparing an antibody detection chip.
Correspondingly, the African swine fever virus antibody detection kit is prepared, and comprises reagents for antibody detection, such as concentrated cleaning liquid, sample diluent, enzyme-labeled antibody solution, TMB color development liquid and the like, besides the antibody detection chip.
EXAMPLE 3 detection of antibodies Using the kit
Detection step
(1) 20 Xconcentrated washing solution (TBST: 0.4M Tris-HCl,2.74M NaCl, 2%Tween20,pH7.2.+ -. 0.2) was washed with purified water at 1:20, diluting to obtain the cleaning solution. To completely infiltrate the surface of the test chip, about 100. Mu.L of the cleaning solution was applied to the surface of the test chip using a pipette, and the test chip was immersed for a certain period of time.
(2) Serum samples to be tested were assayed in accordance with 1 with sample dilutions (0.05 MPBS,1% BSA,0.2% PVP,0.5% Tween20, pH 7.2.+ -. 0.2): and 50 dilution.
(3) For the detection chip from which the cleaning liquid was discarded, 100. Mu.L of the diluted serum sample was aspirated and added to the detection chip in a state in which the surface was completely wetted.
(4) The detection chip was incubated in a constant temperature shaker at 500 rpm for 30 minutes at 37 degrees celsius.
(5) The serum sample is discarded, and the surface of the detection chip is cleaned by a cleaning liquid.
(6) After washing, 100. Mu.L of enzyme-labeled antibody solution (rabbit anti-pig IgG-HRP, sigma, A5670) diluted 1:7500 was added to the detection chip, and the detection chip was incubated in a thermostatted shaker at 500 rpm for 30 minutes at 37 ℃.
(7) Discarding the enzyme-labeled antibody solution, and cleaning the surface of the detection chip by using a cleaning solution.
(8) After the washing was completed, 90. Mu.L of TMB color development solution (Thermo, prod # 37574) was added to the detection chip, and the detection chip was allowed to stand still at 37℃for incubation for 30 minutes under dark conditions.
(9) The TMB color developing solution was discarded, rinsed 3 times with pure water, and dried/blown dry.
(10) And photographing the detection chip by using a microarray chip imager, and judging the result.
(11) And (3) result judgment:
for each serum, the signal value of an epitope polypeptide point or a protein point on the antibody detection chip is more than or equal to 10 (the signal value of a negative control point is less than 1), and the antibody detection chip is "response"; otherwise, it is non-response.
-comparing said SEQ ID NO: 1-4, wherein the number of the types of the polypeptides responsive to the serum is referred to as DMI (Pep), the number of the types of the proteins responsive to the biological sample among the proteins on the antibody detection chip is referred to as DMI (Pro) (since P30 is the only protein, the value is 0 or 1), and DMI (Pep) +DMI (Pro) is referred to as DMI (total),
when DMI (total) is less than or equal to 1, the detection result is judged to be negative, namely, the sample does not contain African swine fever virus antibody;
when DMI (total) > 2 and DMI (Pro) =0, the detection result is judged as negative, i.e., the sample does not contain african swine fever virus antibody; and is also provided with
When DMI (total) > 2 and DMI (Pro) =1, the detection result was judged to be positive, i.e., african swine fever virus antibody was contained in the sample.
Example 4 antibody detection results
For 89 positive samples (the detection results of the nucleic acid detection for determining that pigs are infected with African swine fever virus and other antibody detection methods are positive), the detection results of the African swine fever antibody detection kit are as follows: 88 parts of positive and 1 part of negative.
For the determined 150 negative samples (the detection results of the nucleic acid detection for determining that pigs are not infected with African swine fever virus and other antibody detection methods are negative), the detection results of the African swine fever antibody detection kit are as follows: positive 2 parts, negative 148 parts.
Therefore, the sensitivity of the African swine fever antibody detection kit is 88/89=98.9%, and the specificity is 148/150=98.7%, and high sensitivity and high specificity are realized at the same time.
Claims (10)
1. An african swine fever virus antibody detection kit comprising an antibody detection chip;
the method is characterized in that: the antibody detection chip comprises a solid phase carrier and SEQ ID NO independently fixed on the solid phase carrier: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide shown in the formula 4.
2. The african swine fever virus antibody detection kit of claim 1, wherein: the kit is used for detecting whether the biological sample of pig origin contains African swine fever virus antibodies.
3. The african swine fever virus antibody detection kit of claim 2, wherein: the biological sample comprises whole blood, plasma or serum.
4. The african swine fever virus antibody detection kit of claim 1, wherein: the solid phase carrier is also independently fixed with P30 protein of African swine fever virus.
5. The application of the antibody detection chip in preparing an African swine fever virus antibody detection kit,
the antibody detection chip comprises a solid phase carrier, and SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide shown in the formula 4.
6. Use according to claim 5, characterized in that: the African swine fever virus antibody detection kit is used for detecting whether an African swine fever virus antibody is contained in a biological sample of pig origin.
7. Use according to claim 5, characterized in that: the solid phase carrier is also independently fixed with P30 protein of African swine fever virus.
8. A detection reagent comprising SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide shown in the formula 4.
9. The reagent according to claim 8, further comprising P30 protein of African swine fever virus.
10. Use of the detection reagent according to any one of claims 8 to 9 for the preparation of an african swine fever virus antibody detection kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211119109.8A CN117706085A (en) | 2022-09-14 | 2022-09-14 | African swine fever virus antibody detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211119109.8A CN117706085A (en) | 2022-09-14 | 2022-09-14 | African swine fever virus antibody detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117706085A true CN117706085A (en) | 2024-03-15 |
Family
ID=90161215
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211119109.8A Pending CN117706085A (en) | 2022-09-14 | 2022-09-14 | African swine fever virus antibody detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117706085A (en) |
-
2022
- 2022-09-14 CN CN202211119109.8A patent/CN117706085A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112229994B (en) | Novel coronavirus antibody detection kit based on magnetic particle chemiluminescence | |
| US5595915A (en) | Method of determining antigenically active amino acid sequences | |
| US7785773B1 (en) | Method of diagnosis and kit therefor | |
| AU2018374469B2 (en) | Target interference suppressed anti-drug antibody assay | |
| JP3958796B2 (en) | Antigen-specific IgG detection | |
| CN111856027B (en) | New coronavirus antibody detection kit suitable for examination of patients without obvious symptoms | |
| Howell et al. | An evaluation of in vitro protein–protein interaction techniques: assessing contaminating background proteins | |
| US7399644B2 (en) | Immunoassay, reagent for immunoassay, and production method of the same | |
| JP5836569B2 (en) | Reagent for HCV antigen-antibody combination assay | |
| KR930004055B1 (en) | Peptide and its use | |
| CN111978377B (en) | COVID-19 antigen, preparation method and application | |
| JP6808178B2 (en) | Simultaneous detection method and kit for human parvovirus B19 antigen and antibody | |
| JP3374101B2 (en) | Diagnosing Feline Immunodeficiency Virus Infection Using ENV / GAG Polypeptide Markers | |
| CN117706085A (en) | African swine fever virus antibody detection kit | |
| CN110178031A (en) | The determination of serology of zika virus infection | |
| JP2003202345A (en) | Reagent for simultaneous detection of hcv core antigen and antibody | |
| CN104302660B (en) | A kind of method for detecting hepatitis C virus infection | |
| CN111781349B (en) | Diagnostic kit capable of predicting prognosis of COVID-19 patient | |
| US8257938B2 (en) | Hemolysin and its protein fragments in sero-detection of Anaplasma phagocytophilum | |
| CN109836478B (en) | Preparation method and application of African swine fever virus P11.5 protein specific polyclonal antibody | |
| CN112213493B (en) | Peste des petits ruminants detection kit capable of being used for distinguishing vaccine immunity from natural infection | |
| CN108948174B (en) | Citrulline modified peptide and application thereof | |
| CN110498844B (en) | Peste des petits ruminants diagnostic kit | |
| CN113512097A (en) | Detection reagent and detection kit for polypeptide, trimer and SARS-CoV-2 neutralizing antibody | |
| CN110498845B (en) | Peste des petits ruminants diagnostic kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |