阅读说明：本技术 一种通用型的***病毒结构蛋白抗体及其阻断elisa检测试剂盒 (General type foot-and-mouth disease virus structural protein antibody and blocking ELISA detection kit thereof ) 是由 付元芳 李坤 卢曾军 曹轶梅 刘在新 王省 包慧芳 李平花 白兴文 孙普 马雪青 于 2019-10-25 设计创作，主要内容包括：本发明提供了通用型的口蹄疫病毒结构蛋白抗体及其阻断ELISA检测试剂盒,属于病毒检测技术领域。通用型口蹄疫病毒结构蛋白阻断ELISA抗体检测试剂盒,包括以下组成：包被抗原的酶标板、浓缩生物素标记单抗；浓缩生物素标记单抗中的单抗为单克隆抗体E32；包被抗原的酶标板为通过单克隆抗体F104间接包被FMDV抗原。E32和F104抗体特异性结合在FMDV结构蛋白VP2蛋白中的型间保守抗原表位,灵敏度和特异性均高,适用于O、A与Asia1型FMDV感染或灭活疫苗免疫后结构蛋白抗体的检测,是一种FMD非免疫动物群体感染状况监测的新方法,为FMDV结构蛋白VP2表位缺失的标记疫苗提供配套鉴别诊断方法。(The invention provides a general foot-and-mouth disease virus structural protein antibody and a blocking ELISA detection kit thereof, belonging to the technical field of virus detection. The universal foot-and-mouth disease virus structural protein blocking ELISA antibody detection kit comprises the following components: an antigen-coated ELISA plate and a concentrated biotin-labeled monoclonal antibody; monoclonal antibody in the concentrated biotin labeled monoclonal antibody is monoclonal antibody E32; the ELISA plate coated with the antigen is used for indirectly coating FMDV antigen through a monoclonal antibody F104. The E32 and F104 antibodies are specifically combined with the intertype conserved epitope in FMDV structural protein VP2 protein, the sensitivity and the specificity are high, the method is suitable for detecting the structural protein antibodies after O, A and Asia1 FMDV infection or inactivated vaccine immunization, the method is a novel method for monitoring the infection condition of FMD non-immune animal groups, and a matched differential diagnosis method is provided for the FMDV structural protein VP2 epitope deleted marker vaccine.)

1. A monoclonal antibody E32 of a general foot-and-mouth disease virus structural protein comprises a heavy chain variable region HV1 and a light chain variable region LV1, and is characterized in that the amino acid sequence of the heavy chain variable region HV1 is shown as SEQ ID No. 3; the amino acid sequence of the light chain variable region LV1 is shown in SEQ ID No. 4.

2. A monoclonal antibody composition of a general-type foot-and-mouth disease virus structural protein, comprising the monoclonal antibody E32 of claim 1 and a monoclonal antibody F104 of a general-type foot-and-mouth disease virus structural protein;

the monoclonal antibody F104 comprises a heavy chain variable region HV2 and a light chain variable region LV 2; the amino acid sequence of the heavy chain variable region HV2 is shown as SEQ ID No. 1; the amino acid sequence of the light chain variable region LV2 is shown in SEQ ID No. 2.

3. A general type foot-and-mouth disease virus structural protein blocks ELISA antibody detection kit based on bovine single B cell antibody comprises the following components: an antigen-coated ELISA plate, 100 times concentrated biotin-labeled monoclonal antibody, 100 times concentrated ELISA-labeled avidin, 25 times concentrated washing solution, substrate solution, stop solution, positive control serum and negative control serum, wherein the monoclonal antibody in the 100 times concentrated biotin-labeled monoclonal antibody is the monoclonal antibody E32 of claim 1;

the preparation method of the antigen-coated ELISA plate comprises the steps of firstly coating the monoclonal antibody F104 in the monoclonal antibody composition of claim 2 on the ELISA plate, then capturing the foot-and-mouth disease virus antigen by using the monoclonal antibody F104, adding a solid-phase antigen stabilizing solution, incubating, removing the supernatant, drying the ELISA plate by blowing, and sealing; the foot and mouth disease virus antigen comprises O, A and/or Asia1 type foot and mouth disease virus antigen.

4. The kit of claim 3, wherein the working concentration of the 100 x concentrated biotin-labeled mab is 1: 8000 to 32000.

5. The kit according to claim 3, wherein the monoclonal antibody F104 is coated at a concentration of 1 μ g/mL to 10 μ g/mL.

6. The kit of claim 3, wherein said foot and mouth disease virus antigen is present at a working concentration of 1: 4 to 16.

7. The kit of claim 6, wherein said type O foot and mouth disease virus antigen is present at a working concentration of 1: 16;

the working concentration of the A-type foot-and-mouth disease virus antigen is 1: 8;

the working concentration of the Asia1 type foot-and-mouth disease virus antigen is 1: 4.

8. the kit according to claim 3 or 7, wherein the foot-and-mouth disease virus antigen is Asia1 type foot-and-mouth disease virus antigen.

9. Use of the monoclonal antibody E32 of claim 1 or the monoclonal antibody composition of claim 2 for preparing a reagent for detecting the infection status of O, A or Asia1 type foot-and-mouth disease virus in a non-immune background.

10. Use of the monoclonal antibody E32 of claim 1 or the monoclonal antibody composition of claim 2 for preparing a detection reagent for structural protein antibodies after O, A or Asia1 type foot-and-mouth disease virus infection or immunization of animals.

Technical Field

The invention belongs to the technical field of virus detection, and particularly relates to a general foot-and-mouth disease virus structural protein antibody and a blocking ELISA detection kit thereof.

Background

Foot-and-mouth disease (FMD) is a virulent infectious disease of artiodactyl animals caused by FMDV (foot-and-mouth disease virus) infection. The disease incidence is extremely high, and the feed additive has serious influence on the breeding industry and the international livestock product trade, so that the prevention and control of the disease are highly emphasized in various countries. The key to prevent and control foot-and-mouth disease is the application of high-efficiency vaccine and accurate diagnosis method. At present, the prevention and treatment of foot-and-mouth disease in China mainly depends on inactivated vaccine immunization, and under the background, the detection of FMDV non-structural protein (NSP) antibodies is an effective method for identifying infected animals and immunized animals at present, is also an FMDV infection condition monitoring and evaluating method without distinguishing serotypes, and a general FMDV antibody detection method based on structural proteins is not reported yet. The research of the general detection method based on the structural protein has important application value for the investigation of FMDV infection conditions in the non-immune background, and because the antibody response to the structural protein is usually faster and has higher response level than that of the non-structural protein, the detection of the structural protein antibody can discover the infection conditions earlier, and has important significance for the prevention and control of FMDV. Meanwhile, the research of the marker vaccine based on the epitope deletion of the structural protein is still blank, the research of the epitope deletion marker vaccine based on the structural protein can possibly promote the birth of cheap and efficient FMDV inactivated vaccine, and the research of the epitope deletion negative marker vaccine based on the structural protein and an ELISA method for detecting a deleted epitope antibody can provide a low-cost and efficient prevention and control technology and product for the prevention and control of foot-and-mouth disease.

FMDV has seven serotypes, O, A, C, SAT1, SAT2, SAT3 and AsiaI, with no cross-protection between the types, but serologically cross-reactive. At present, the type-O FMDV and the type-A FMDV are mainly popular in China. FMDV antigen structure is complex, different serotypes, genotypes and isolates have different antigenic sites and epitopes, wide antigenic variation exists, a conservative antigen structure determining virus species characteristics also exists, a broad-spectrum foot-and-mouth disease virus antibody detection kit is developed, broad-spectrum specific monoclonal antibodies for identifying conserved epitopes among types need to be screened, but the existing broad-spectrum specific monoclonal antibodies are rare, and certain obstacle is brought to establishment of a broad-spectrum FMDV antibody detection method.

Disclosure of Invention

In view of the above, the present invention aims to provide a general-purpose aftosa structural protein antibody, which can recognize a broad-spectrum specific monoclonal antibody of an intervarietal conserved epitope and has high specificity.

The invention also aims to provide a general foot-and-mouth disease virus structural protein blocking ELISA antibody detection kit prepared based on the general foot-and-mouth disease virus structural protein antibody, which is used for monitoring the FMDV infection condition of animals under the non-immune background and has the characteristics of good specificity and high sensitivity.

The invention provides a universal monoclonal antibody E32 of foot-and-mouth disease virus structural protein, which comprises a heavy chain variable region HV1 and a light chain variable region LV1, wherein the amino acid sequence of the heavy chain variable region HV1 is shown as SEQ ID No. 3; the amino acid sequence of the light chain variable region LV1 is shown in SEQ ID No. 4.

The invention provides a monoclonal antibody composition of a general type foot-and-mouth disease virus structural protein, which comprises a monoclonal antibody E32 and a monoclonal antibody F104 of the general type foot-and-mouth disease virus structural protein;

the monoclonal antibody F104 comprises a heavy chain variable region HV2 and a light chain variable region LV 2; the amino acid sequence of the heavy chain variable region HV2 is shown as SEQ ID No. 1; the amino acid sequence of the light chain variable region LV2 is shown in SEQ ID No. 2.

The invention provides a general type foot-and-mouth disease virus structural protein blocking ELISA antibody detection kit based on bovine single B cell antibody, which comprises the following components: antigen-coated ELISA plate, 100 times concentrated biotin-labeled monoclonal antibody, 100 times concentrated ELISA-labeled avidin, 25 times concentrated washing solution, substrate solution, stop solution, positive control serum and negative control serum;

the monoclonal antibody in the 100 multiplied concentrated biotin labeled monoclonal antibody is the monoclonal antibody E32;

the preparation method of the antigen-coated ELISA plate comprises the steps of firstly coating the monoclonal antibody F104 in the monoclonal antibody composition on the ELISA plate, then capturing the foot-and-mouth disease virus antigen by using the monoclonal antibody F104, adding a solid-phase antigen stabilizing solution, incubating, sucking and discarding the supernatant, drying the ELISA plate by blowing, and sealing; the foot and mouth disease virus antigen comprises O, A and/or Asia1 type foot and mouth disease virus antigen.

Preferably, the working concentration of the 100 × concentrated biotin-labeled mab is 1: 8000 to 32000.

Preferably, the coating concentration of the monoclonal antibody F104 is 1 to 10 mu g/mL.

Preferably, the working concentration of the foot-and-mouth disease virus antigen is 1: 4 to 16.

Preferably, the working concentration of the type-O foot-and-mouth disease virus antigen is 1: 16;

the working concentration of the A-type foot-and-mouth disease virus antigen is 1: 8;

the working concentration of the Asia1 type foot-and-mouth disease virus antigen is 1: 4.

preferably, the foot-and-mouth disease virus antigen is Asia1 type foot-and-mouth disease virus antigen.

The invention provides application of the monoclonal antibody E32 or the monoclonal antibody composition in preparing a detection reagent for detecting the infection condition of O, A or Asia1 type foot-and-mouth disease virus under a non-immune background.

The invention provides application of the monoclonal antibody E32 or the monoclonal antibody composition in preparing a detection reagent of a structural protein antibody after O, A or Asia1 type foot-and-mouth disease virus infection or animal immunization.

The invention provides a universal monoclonal antibody E32 of foot-and-mouth disease virus structural protein, which comprises a heavy chain variable region HV1 and a light chain variable region LV1, wherein the amino acid sequence of the heavy chain variable region HV1 is shown as SEQ ID No. 3; the amino acid sequence of the light chain variable region LV1 is shown in SEQ ID No. 4. The monoclonal antibody E32 is an FMDV universal monoclonal antibody prepared by utilizing a single B cell antibody technology, can recognize FMDVVP2 protein epitopes and also can recognize conserved antigen epitopes among serotypes, and therefore, the specific recognition and detection of universal foot and mouth disease viruses can be realized.

The invention provides a monoclonal antibody composition of a general type foot-and-mouth disease virus structural protein, which comprises the monoclonal antibody E32 and a monoclonal antibody F104 of the general type foot-and-mouth disease virus structural protein. The monoclonal antibody F104 is a O, A or Asia1 FMDV universal monoclonal antibody prepared by a bovine single B cell antibody technology, the recognized epitope is positioned in FMDVVP2 protein and is a serotype-conserved epitope, and therefore, the monoclonal antibody F104 and the monoclonal antibody E32 are the same and are FMDV structural protein universal monoclonal antibodies. The monoclonal antibody E32 in the monoclonal antibody composition is used as a detection antibody, and the monoclonal antibody F104 is used as a capture antibody and applied to the preparation of a kit for detecting the foot-and-mouth disease virus antibody.

The invention provides a general foot-and-mouth disease virus structural protein blocking ELISA antibody detection kit based on bovine single B cell antibody, wherein a monoclonal antibody F104 is coated on an ELISA plate to be used as a capture antibody for capturing FMDV inactivated antigen; the monoclonal antibody E32 marked by biotin is taken as a detection antibody, competes with an antibody which recognizes the same epitope in a sample to be detected to bind an antigen, and then the specific binding of biotin and avidin is taken as an indication system to display the abundance of the antibody in the sample, so that the kit has the characteristics of good specificity, high sensitivity and simple reagent composition, and can be used for detecting the serum of animals infected by different serotype viruses. The specificity and sensitivity experiment result shows that the detection sensitivity of the kit to 121 cattle infected by 3-230 days is 100 percent; the detection sensitivity of 79 parts of sheep serum 12 days to 1 year after infection reaches 91.1 percent; the detection sensitivity to 80 parts of pig serum 3 days to 6 months after infection reaches 81.3 percent; the specificity to serum from clinical healthy cattle, sheep and pig is 100%.

Drawings

FIG. 1 is a graph showing the results of comparative analyses of the positive standard serum blocking rates at various dilutions;

FIG. 2 is a graph showing the results of comparative analyses of the standard positive serum blocking rates under the respective reaction conditions;

FIG. 3 is a graph showing the results of the detection of negative and positive sera in cattle;

FIG. 4 is a graph showing the results of the detection of negative and positive sera in sheep;

FIG. 5 is a diagram showing the results of the detection of negative serum and positive serum in swine.

Detailed Description

The invention provides a universal monoclonal antibody E32 of foot-and-mouth disease virus structural protein, which comprises a heavy chain variable region HV1 and a light chain variable region LV1, wherein the amino acid sequence of the heavy chain variable region HV1 is shown in MNPLWTLLFVLSAPRGVLSQVQLRESGPSLVKPSQTLSLTCTVSGFSLSNYALNWVRQAPGKALECLGGIGPSGHTDYNPALKSRLIITKDNSKNQVSLSVSSVTPEDTATYICARDSGIYGTSGWGCIGGFDDNYIDAWGHGLLVTVSS (SEQ ID No. 3); the amino acid sequence of the light chain variable region LV1 is shown in MSTMAWSPLFLTLVAVYTGSWAQAVLTQPSSVSGSLGQRVSITCSGSSNNIGAHGVGWYQQIPGSGLRTIIYNNSKRPSGVPDRFSGSKSGNTATLTISSLQAGDEADYFCATSDYSTRSSAFGSGTTLTVLGQPKSAPSVTLFPPSKEELSANK (SEQ ID No. 4).

In the present invention, the preparation method of the monoclonal antibody E32 preferably comprises the following steps: the coding sequences of the heavy chain variable region and the light chain variable region of the antibody E32 are respectively connected with the constant region sequences of a bovine IgG2 heavy chain and a bovine IgG2 light chain and then are respectively inserted into a pcDNA3.4 eukaryotic expression vector, heavy chain and light chain expression plasmids are mixed according to a proportion and transfected into CHO suspension culture cells to obtain a complete IgG2 subtype antibody through expression, and the antibody is obtained through affinity chromatography purification. The preparation method of the antibody E32 is described in a patent with the patent reference number CN 201810929067.1.

The invention provides a monoclonal antibody composition of a general type foot-and-mouth disease virus structural protein, which comprises a monoclonal antibody E32 and a monoclonal antibody F104 of the general type foot-and-mouth disease virus structural protein; the monoclonal antibody F104 comprises a heavy chain variable region HV2 and a light chain variable region LV 2; the amino acid sequence of the heavy chain variable region HV2 is shown as MNPLWTLLFVLSAPRGVLSQVQLRESGPSLVKPSQTLSLTCTVSGFSLSSYDVGWVRQAPGKGLECLGGIGNRGNTKYNPALKSRLSITKDNSKSQVSLSLSSVTSEDTATYYCTKSYGGNDVYDCYDSEYWGQGLLVTVSS (SEQ ID No. 1); the amino acid sequence of the light chain variable region LV2 is shown in MAWSPLFLTLVAVYTGSWAQAVLTQPSSVSGSLGQRVSITCSGSSNNIGRYSVGWYQQVPGSGLRTIIYISSSRPSGVPDRFSGSKSGNTATLTISSLQAEDEADYFCATNDYSSDTTIFGSGTTLTVLGQPKS (SEQ ID No. 2).

In the present invention, the monoclonal antibody F104 was prepared in the same manner as the above-described monoclonal antibody E32. In the monoclonal antibody composition, the ratio of the monoclonal antibody F104 to the monoclonal antibody E32 is not particularly limited, and the concentration of the two antibodies used in combination is determined according to the actual concentration of the reagents used in the preparation.

The invention provides a general type foot-and-mouth disease virus structural protein blocking ELISA antibody detection kit based on bovine single B cell antibody, which comprises the following components: antigen-coated ELISA plate, 100 × concentrated biotin-labeled monoclonal antibody, 100 × concentrated ELISA-labeled avidin, 25-fold concentrated washing solution, substrate solution, stop solution, positive control serum and negative control serum.

In the present invention, the monoclonal antibody of the 100 × concentrated biotin-labeled monoclonal antibody is the monoclonal antibody E32 described above. The working concentration of the 100 × concentrated biotin-labeled monoclonal antibody is preferably 1: 8000 to 32000, more preferably 1: 16000. the preparation method of the biotin-labeled monoclonal antibody is not particularly limited, and a labeling method known in the art can be used.

The preparation method of the antigen-coated ELISA plate comprises the steps of firstly coating a monoclonal antibody F104 in the monoclonal antibody composition on the ELISA plate, then capturing foot-and-mouth disease virus antigen by using the monoclonal antibody F104, adding a solid phase antigen stabilizing solution (PBS (phosphate buffered saline) containing sucrose with the mass concentration of 5%), incubating, removing supernatant, drying the ELISA plate by blowing, and sealing; the foot and mouth disease virus antigen comprises O, A and/or Asia1 type foot and mouth disease virus antigen.

The coating concentration of the monoclonal antibody F104 is preferably 1 to 10 mu g/mL, more preferably 2 to 8 mu g/mL, and most preferably 5 mu g/mL. The working concentration of the foot-and-mouth disease virus antigen is preferably 1: 4 to 16. According to the different types of serotypes of the foot-and-mouth disease virus, the working concentration is different, and the working concentration is as follows: the working concentration of the O-type foot-and-mouth disease virus antigen is preferably 1: 16; the working concentration of the A-type foot-and-mouth disease virus antigen is 1: 8; the working concentration of the Asia1 type foot-and-mouth disease virus antigen is 1: 4. the virus antigens of different serotypes show different blocking rates, and the antigen with the highest positive detection rate is used as the optimal capture antigen, and the foot-and-mouth disease virus antigen is preferably Asia1 type foot-and-mouth disease virus antigen.

In the present invention, the kit comprises 100 × concentrated enzyme-labeled avidin. The 100 × concentrated enzyme-labeled avidin is preferably horseradish peroxidase (HRP) -labeled avidin. The source of HRP-labeled avidin is not particularly limited as long as HRP-labeled avidin known in the art is used.

In the present invention, the kit comprises 25-fold concentrated washing solution. The preparation method of the 25-fold concentrated washing solution is preferably as follows: adding NaCl 200g, KCl 5g and Na into 500mL of ultrapure water₂HPO₄·12H₂O 72.5g、KH₂PO ₄5g and 12.5mL of Tween 20, adding ultrapure water to a constant volume of 1000mL, adjusting the pH value to 7.4, autoclaving, and storing at room temperature for later use.

In the present invention, the kit comprises a substrate solution. The kind of the substrate solution is determined according to the kind of enzyme in the enzyme-labeled avidin. When HRP labels avidin, the substrate solution is 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) substrate.

In the present invention, the kit comprises a stop solution. The stop solution is preferably 0.3mol/L H₂SO₄And (3) solution.

In the present invention, the kit comprises a positive control serum and a negative control serum. The positive control serum is a cattle, sheep or pig which is collected 1-3 months after the foot-and-mouth disease virus infection, the non-structural protein antibody and the structural protein antibody are positive through detection, and the positive control serum is prepared by adding a preservative after being subjected to sterile treatment. The negative control serum is collected from non-immune healthy cattle, sheep or pigs, is negative by detecting foot-and-mouth disease virus structural protein and non-structural protein antibodies, and is preserved by adding a preservative after being subjected to sterile treatment.

In the present invention, the method of using the kit preferably comprises the steps of:

A1. diluting the serum to be detected by 1 xPBST, sucking the diluted serum to be detected, adding the diluted serum to an ELISA plate capturing antigens, adding 100 mu L of the diluted serum to each hole, and acting for 1h at room temperature; the dilution of the serum is preferably 1: 1;

A2. washing the plate with 1 × PBST washing solution, repeatedly washing for 5 times, and drying for the last time;

A3. 100 × concentrated biotin-labeled monoclonal antibody was added to the mixture according to the ratio of 1: diluting 100 to 1 xPBST, adding an enzyme label plate, adding 100 mu L of enzyme label plate into each hole, and acting for 30min at room temperature (18-25 ℃);

A4. the washing was performed as above, and the final patting was done with HRP-labeled avidin at 1: diluting 100 to 1 xPBST, adding an enzyme label plate, adding 100 mu L of enzyme label plate into each hole, and acting for 15min at room temperature (18-25 ℃);

A5. adding 100 mu L of substrate solution into each hole, and acting for 10-15 min at room temperature (18-25 ℃);

A6. adding stop solution 100 μ L per well, mixing under gentle vibration, and reading light absorption value (OD) of 450nm with microplate reader within 10min_450nm)；

A7. And (3) calculating the blocking rate: blocking Rate (PI) ═ 1-sample OD_450nmValue/negative control OD_450nmValue) × 100%;

A8. conditions for test establishment: negative control OD_450nmvalue-Positive control OD_450nmThe value is more than or equal to 1.0, and the positive control blocking rate is more than or equal to 50 percent;

A9. and (3) judging standard: and (3) judging whether the foot-and-mouth disease virus antibodies exist or not by calculating PI of each serum sample: the PI value is more than or equal to 50 percent, and the FMDV antibody is judged to be positive; the PI value is less than 50%, and the FMDV antibody is judged to be negative.

The invention provides application of the monoclonal antibody E32 or the monoclonal antibody composition in preparing a detection reagent for detecting the infection condition of O, A or Asia1 type foot-and-mouth disease virus under a non-immune background. The detection reagent comprises the universal foot-and-mouth disease virus structural protein blocking ELISA antibody detection kit.

The invention provides application of the monoclonal antibody E32 or the monoclonal antibody composition in preparing a detection reagent of a structural protein antibody after O, A or Asia1 type foot-and-mouth disease virus infection or animal immunization. The detection reagent comprises an in vitro diagnostic reagent prepared based on a competition method, and comprises a colloidal gold in vitro diagnostic reagent, a chemiluminescence in vitro diagnostic reagent or an ELISA detection reagent and the like.

The present invention provides a general-purpose aftosa structural protein antibody and its blocking ELISA detection kit, which will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.