Preparation of a novel bispecific antibody of tebufenpyrad and ochratoxin a recognition element and application in enzyme-linked immunosynchronous detection

Abstract

The application discloses a novel thiacloprid and ochratoxin A recognition element bispecific antibody preparation and application in enzyme-linked immunosorbent assay synchronous detection, and belongs to the technical field of molecular biology. The application provides a thiacloprid and ochratoxin A bispecific antibody, which is expressed in Expi293F cells and used for preparing an ELISA detection reagent, so that the bispecific detection of thiacloprid and ochratoxin A can be effectively realized. The linear ranges of the bispecific antibody provided by the application for detecting thiacloprid and ochratoxin A are 0.05-1000 ng / mL and 0.05-500 ng / mL respectively, and the IC50 is 17.10 ng / mL and 16.69 ng / mL respectively.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of thiacloprid and ochratoxin A novel recognition element bispecific antibody preparation and application in enzyme-linked immunosorbent simultaneous detection, belong to molecular biology technical field. BACKGROUND

[0002] Pesticide residues and mycotoxin pollution are important hidden troubles in the production, storage, processing and marketing of agricultural products. Thiacloprid (TCL) is widely used in agricultural production due to its broad-spectrum and high efficiency, and has high residues. Ochratoxin A (OTA) is a typical mycotoxin with strong harmfulness and wide pollution range, and has residues in cereal and fruit and vegetable agricultural products. However, the immune detection method for simultaneous detection of the two harmful substances usually uses multiple single-specific antibodies in combination, which has the defects of high detection cost. Therefore, it is very important to establish a simultaneous detection method for the two pollutants based on bispecific antibodies.

[0003] Due to the complex correlation between the environmental system and the food supply chain system, thiacloprid in the environment will inevitably enter the human food chain, so the detection of thiacloprid residues in food is of great significance. At present, the detection of TCL in food mainly includes large instrument methods such as chromatography and mass spectrometry, and immune analysis methods such as enzyme-linked immunosorbent assay and fluorescence immunoassay. Ochratoxin A has multiple transmission routes in the food chain, so there are many types of food contaminated by ochratoxin A. In order to reduce the risk of human exposure to OTA and reduce the transmission of OTA in the food chain, more and more researchers are committed to the development of OTA detection technology, including instrument analysis and immune analysis. However, the current common rapid detection products are mainly based on traditional immune antibodies for single component detection, which cannot realize the simultaneous detection of pesticides and toxins, and the antibody preparation is complex and time-consuming. The bispecific antibody recognition element prepared based on genetic engineering recombination technology can solve the above problems, and can be expressed rapidly in vitro, realizing the simultaneous detection of cross-class harmful substances. SUMMARY

[0004] The first object of the present application is to provide a thiacloprid and ochratoxin A detection antibody, whose amino acid sequence is shown as SEQ ID NO. 6.

[0005] In one embodiment, the thiacloprid detection antibody comprises a single-chain antibody and a nanobody variable region, the amino acid sequence of the single-chain antibody is shown as SEQ ID NO. 4; and the sequence of the nanobody variable region is shown as SEQ ID NO. 5.

[0006] The present application also provides a gene encoding the thiacloprid and ochratoxin A detection antibody.

[0007] In an embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO. 3.

[0008] The present application also provides a recombinant vector containing the above-mentioned gene.

[0009] In an embodiment, pcDNA3.1 is used as the expression vector.

[0010] The present application also provides a cell containing the above-mentioned gene or the above-mentioned recombinant vector.

[0011] In an embodiment, the cell is a mammalian cell line.

[0012] In an embodiment, the cell is Expi293F cell.

[0013] The present application also provides a method for preparing a thiacloprid and ochratoxin A combined antibody, which comprises expressing the nucleotide sequence shown as SEQ ID NO. 3 in Expi293F cell.

[0014] In an embodiment, pcDNA3.1 is used as the expression vector to express the coding gene of the thiacloprid and ochratoxin A combined antibody.

[0015] In an embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO. 3.

[0016] The present application also provides the application of the thiacloprid and ochratoxin A combined antibody in detecting thiacloprid and ochratoxin A.

[0017] The present application also provides a kit for combined detection of thiacloprid and ochratoxin A, which contains the thiacloprid and ochratoxin A combined antibody, or the recombinant vector, or the cell.

[0018] In an embodiment, the kit further contains any one of (a)-(c) as follows:

[0019] (a) a conjugate of the hapten of thiacloprid and ochratoxin A and carrier protein, and an enzyme-labeled thiacloprid and ochratoxin A detection antibody;

[0020] (b) a conjugate of the hapten of thiacloprid and ochratoxin A and carrier protein, and an enzyme-labeled antibody of the thiacloprid and ochratoxin A detection antibody;

[0021] (c) an enzyme-labeled thiacloprid and ochratoxin A detection antibody.

[0022] In an embodiment, the kit further contains a thiacloprid and ochratoxin A standard.

[0023] The application also provides the use of the gene, the recombinant vector, the cell or the kit in detecting thiacloprid and ochratoxin A, and the use is not for the purpose of diagnosis and / or treatment of diseases.

[0024] Beneficial effects:

[0025] The application provides a bispecific antibody of thiacloprid and ochratoxin A, which is composed of a single-chain antibody and a nanobody variable region, the amino acid sequence of the single-chain antibody is shown in SEQ ID NO. 4, and the nucleotide sequence of the nanobody variable region is shown in SEQ ID NO. 5. The linear range of the bispecific antibody provided by the application for detecting thiacloprid and ochratoxin A is 0.05-1000 ng / mL and 0.05-500 ng / mL respectively, the LOD is 0.337 ng / mL and 0.788 ng / mL respectively, and the IC50 is 17.10 ng / mL and 16.69 ng / mL respectively. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The application is designed for the purpose of thiacloprid-ochratoxin A gene;

[0027] Figure 2 The application is designed for the purpose of thiacloprid-ochratoxin A gene;

[0028] Figure 3 The application is designed for the purpose of thiacloprid-ochratoxin A gene; 450 Numerical values;

[0029] Figure 4 The application is designed for the purpose of thiacloprid-ochratoxin A gene; DETAILED DESCRIPTION

[0030] In the following examples, the experimental methods are all conventional methods unless otherwise specified.

[0031] Unless otherwise specified, the cells, culture media, transformation reagents and the like used in the application are all normal procurement channels.

[0032] Expi293 in the application TMExpression medium was purchased from Gibco, Opti-MEM I was purchased from thermofisher website, Transfection-reagent was purchased, Enhancer was purchased from thermofisher website, DON-BSA antigen was purchased from thermofisher website.

[0033] Example 1: Antibody expression plasmid construction

[0034] The heavy chain of nucleotide sequence as shown in SEQ ID NO. 3 was constructed into pcDNA3.1 vector between EcoRI and BamHI enzyme cutting sites, obtaining a plasmid for expressing thiacloprid and ochratoxin A bispecific antibody.

[0035] Example 2: Construction of eukaryotic expression system

[0036] The purchased Expi293F cells (1 x 10 7 ) were thawed at room temperature 37℃, then centrifuged quickly under the condition of 350g, the excess supernatant was removed, and transferred to fresh Expi293 TM (Thermo Fisher Scientific) expression medium, cultured at 37℃, 5-8% CO2. The time of each passage of the cells after resuscitation was 3-4 days, 1-2 generations after resuscitation, and the two generations of cells before thawing may observe slower growth rate and lower cell viability. After 3-4 passages, a certain amount of cells were harvested and transferred to fresh Expi293 TM Expression medium was prepared for transfection.

[0037] 24h before transfection, the cells were counted, and the cell density was adjusted to 2.5 x 10 TM cells / mL of medium using Expi293 6 Expression medium. This example carried out 30mL transfection, 75 x 10 6 cells were added to a new 125mL flask,

[0038] On the day of transfection, the cells were counted and the viability was determined. 75 x 10 6 cells were added to a new 125mL flask, and the volume was adjusted to 25.5mL using Expi293 TM Expression medium, according to the volume of transfection medium,

[0039] Example: According to the transfection volume of 30 mL, in order to make the light chain and heavy chain better assembly, the ratio between the two is set to 3:2, take 30 μg of thiamethoxam and ochratoxin A plasmid, dissolved in 1.5 mL of Opti-MEM I, add 81 μL of Transfection-reagent to 1.5 mL of Opti-MEM I, and stand for 10 min, then mix and stand for 10 min, then slowly add to the culture medium, cultivate at 125 rpm for 16-22 h, then add 80 μL of enhancer I and 1.5 mL of enhancer II for continuous cultivation.

[0040] Example 3: Protein purification

[0041] When designing the target gene, the C-terminal of the heavy chain gene is designed with a 6×His tag for easy purification. The designed gene is shown in Figure 1 , and the nucleotide sequence is shown in SEQ ID NO. 3.

[0042] The transfected Expi293F cells were cultured for 7 days, and the entire culture system was collected. The culture system was centrifuged at 3200 r / min for 20 min, and the supernatant was filtered with a 0.22 μm filter membrane. The filtered supernatant was passed through a nickel column 3-5 times to allow the 6×His-tagged bispecific antibody to bind to the nickel column. After washing with a 25 mM imidazole-containing washing solution to remove non-specifically bound impurities, the target protein bound to the nickel column was eluted with a 250 mM imidazole-containing elution solution, and the protein concentration was 50 μg / mL.

[0043] SDS-PAGE was performed on the eluted target protein, and a clear bispecific antibody protein band was observed on the protein gel. Figure 2 ).

[0044] Example 4: ELISA detection of antibody activity

[0045] 1. Determine the linear range of detection

[0046] (1) Coating: Take 100 μL / well of TCL-BSA and OTA-BSA antigen dissolved in phosphate buffer solution for coating, and adjust the concentration of TCL-BSA and OTA-BSA to 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL and 7.5 μg / mL respectively, and incubate at 37°C for 2 h.

[0047] (2) Washing: Pour out the solution in the plate, and wash with PBST 4-6 times, then pat dry.

[0048] (3) Blocking: After washing and drying the 96-well plate, 250 μL of 5% (V / V) skim milk powder blocking solution was added to each well, and the plate was incubated at 37°C for 2 h. After washing, the plate was dried and stored for later use.

[0049] (4) Antibody incubation: The purified antibody of Example 3 was added to the coated wells, and 100 μL of the antibody was added to each well, and the plate was incubated at 37°C for 1 h. Then the plate was washed 4-6 times and dried.

[0050] (5) Secondary antibody incubation: The enzyme-labeled secondary antibody (HRP Conjugated Anti His-Tag Mouse Monoclonal Antibody) was diluted to an appropriate concentration, and 100 μL of the enzyme-labeled secondary antibody was added to each well. The plate was incubated at 7°C for 30 min, and then washed with PBST for 4-6 times and dried.

[0051] (6) Color development: 50 μL of TMB color developing solution A and B was added to each well, and the plate was incubated at 37°C for 15 min in the dark.

[0052] (7) Termination and determination: 50 μL of termination solution was added to each well to terminate the reaction, and then the OD450nm value of each well was determined using an enzyme-labeled instrument. Figure 3 )。

[0053] The IC50 of the expressed antibody for thiamethoxam and ochratoxin A was determined by IC-ELISA, and the IC50 values for thiamethoxam and ochratoxin A were 17.10 ng / mL and 16.69 ng / mL, respectively, and the linear ranges were 0.05-1000 ng / mL and 0.05-500 ng / mL, respectively. This indicates that the antibody has a wide linear range for thiamethoxam and ochratoxin A and can be used for the immunoassay detection of thiamethoxam and ochratoxin A. Figure 4

[0054] 2. Spiked recovery experiment

[0055] In order to evaluate the accuracy of the developed simultaneous quantitative OTA and TCL ELISA kit, 0.1, 0.5, and 1 mg / kg of OTA and TCL standard solutions were added, respectively. The ELISA kit developed in this study was used for detection, and the instrument analysis method was used for verification, and the results are shown in Table 1.

[0056] Table 1 Detection results of the sample by the bi-specific antibody

[0057]

[0058] Comparative Example:

[0059] ​In view of the difference between the protein modification ability of E. coli and that of animal cells, the single-chain antibody and nanobody belong to the small molecule antibody which is easy to express, so the antibodies of thiacloprid and ochratoxin A are expressed in E. coli.

[0060] E. coli expression: the expression vector of PET-28a(+) is used, the expression strain is E. coli BL21, the antibody with the nucleotide sequence as shown in SEQ ID NO. 7 and SEQ ID NO. 8 is expressed, the constructed recombinant E. coli cells are cultured in LB medium until the OD is 0.6, the final concentration of 0.1 mM IPTG is used for induction at 16 DEG C for 18 h, the fermentation broth is collected, and the protein is purified by using a purification kit.

[0061] The verification step for the antibody recognition ability refers to embodiment 4.

[0062] The results show that the affinity constant of the antibody is 2.3 x 10 5 L / mol, which indicates that the antibody recognition ability is very weak, the binding ability is poor, and it cannot be applied to actual detection.

[0063] Although the present application has been disclosed with the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A bispecific antibody for joint detection of acetamiprid and ochratoxin A, characterized in that, The amino acid sequence of the antibody is shown as SEQ ID NO.

6.

2. A gene encoding the antibody of claim 1.

3. The gene of claim 2, wherein, The nucleotide sequence of the gene is shown as SEQ ID NO.

3.

4. A recombinant vector carrying the gene of claim 2 or 3.

5. A cell expressing the antibody of claim 1.

6. A method of preparing a bispecific antibody of tebufenpyrad and ochratoxin A, characterized by, The nucleotide sequence shown as SEQ ID NO. 3 is expressed in Expi293F cells using pcDNA3.1 as an expression vector.

7. The method of claim 6, wherein, The antibody produced by the cell is also purified.

8. Use of the antibody according to claim 1 for the detection of thiacloprid and ochratoxin A, characterized in that, The application is not for the purpose of diagnosis and / or treatment of a disease.

9. A kit for the combined detection of acrinathrin and ochratoxin A, characterized in that, Any one of the following (a)~(b) is contained: (a) a conjugate of a hapten of thiacloprid and ochratoxin A and a carrier protein, an enzyme label of the antibody of claim 1; (b) an enzyme label of the antibody of claim 1.

10. Use of the kit of claim 9 in detecting thiacloprid and ochratoxin A, which is not for the purpose of diagnosis and / or treatment of a disease.

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