CN102653558B - Single-chain antibody and application thereof in detecting aflatoxin - Google Patents

Abstract

The invention discloses a single-chain antibody and its application in detecting aflatoxins. The single-chain antibody provided by the present invention is a polypeptide consisting of a light chain variable region, a connecting peptide and a heavy chain variable region; the connecting peptide is located between the light chain variable region and the heavy chain variable region ; The light chain variable region is shown in sequence 1 from N-terminal 1 to 110 amino acid residues in the sequence listing; the heavy chain variable region is shown in sequence 1 from N-terminal 131 to 248 amino acids Residues are shown. The single-chain antibody provided by the invention has a good cross-reactivity rate, high affinity and high sensitivity to a variety of aflatoxins, is suitable for rapid immunological detection of a variety of multi-residual aflatoxins, and will play an important role in the detection of aflatoxins .

Description

A kind of single-chain antibody and its application in detecting aflatoxin

technical field

The invention relates to a single-chain antibody and its application in detecting aflatoxins.

Background technique

Aflatoxin (Aflatoxin, AFT) is a mycotoxin that poses a huge threat to human health and agricultural production. Its chemical structure is similar, and it is a derivative of dihydrofuranocoumarin. The molecular weight is about 312-346. Secondary metabolites produced by Aspergillus flavus (A. parasiticus). As many as 250,000 people die of mycotoxin-induced cancers around the world every year. Mycotoxin contamination also leads to the destruction of a large number of agricultural products, causing economic losses of hundreds of millions of dollars every year. The occurrence of aflatoxins in food and feed is highest in hot and humid regions.

Aflatoxin is a highly toxic and highly toxic substance that can damage the liver tissue of humans and animals, and can lead to liver cancer or even death in severe cases. In 1993, aflatoxin was classified as a Class 1 carcinogen by the Cancer Research Institute of the World Health Organization (WHO). At present, more than 20 kinds of aflatoxins have been isolated and identified, mainly including B ₁ , B ₂ , G ₁ , G ₂ , M ₁ , M ₂ , P ₁ , Q, H ₁ , GM, B _2a and toxic alcohol, etc. . Aflatoxin B ₁ is the most common in naturally contaminated food, and its toxicity and carcinogenicity are also the strongest. Due to the great toxicity of aflatoxin, more than 70 countries and regions have imposed limits on the content of AFT in food. In 2006, the WHO Codex Alimentarius Commission recommended that the maximum allowable amount of aflatoxin in food and feed be the total amount (the sum of AFB ₁ , AFB ₂ , AFG ₁ , and AFG ₂ ) to be less than 15 μg/kg; the maximum allowable amount of AFM ₁ in milk is 0.5 μg/kg. The European Union re-evaluated the level of mycotoxins in grains and grain products in 2008, and established stricter upper limit standards: AFB ₁ limit 2μg/kg, AFT total limit 4μg/kg. In November 1990, the Ministry of Health of China issued the "Administrative Measures for the Prevention of Food Contamination by Aflatoxin", which stipulated that the limit of AFB ₁ in different types of samples was 5-204 μg/kg.

Methods for the quantitative determination of AFT fall into two categories. One is a physical and chemical analysis method based on chromatography, which includes thin-layer chromatography (TLC), liquid chromatography (LC), high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry with fluorescence detection. Continuous use detection (LC-MASS). The other is immunochemical methods, such as radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA). Among the above detection technologies, immunological methods, especially ELISA technology, are more and more widely used in the field of aflatoxin analysis and detection due to its characteristics of rapidity, low cost, and high-throughput detection suitable for a large number of samples. At present, the antibodies used in domestic ELISA kits are mainly polyclonal antibodies and monoclonal antibodies. The high cost and limited output of these two antibody preparation techniques greatly limit the further development of immunological methods in detection.

Contents of the invention

The purpose of the present invention is to provide a single-chain antibody and its application in detecting aflatoxin.

The single-chain antibody provided by the present invention is a polypeptide consisting of a light chain variable region, a connecting peptide and a heavy chain variable region; the connecting peptide is located between the light chain variable region and the heavy chain variable region ; The light chain variable region is shown in sequence 1 from N-terminal 1 to 110 amino acid residues in the sequence listing; the heavy chain variable region is shown in sequence 1 from N-terminal 131 to 248 amino acids Residues are shown.

The connecting peptide can be as shown in the 111th to 130th amino acid residues from the N-terminal of Sequence 1 in the sequence listing.

The single-chain antibody can specifically be the polypeptide shown in Sequence 1 of the Sequence Listing or the polypeptide shown in Sequence 4 of the Sequence Listing.

The present invention also protects the gene encoding the single-chain antibody, which consists of the coding sequence of the light chain variable region, the coding sequence of the connecting peptide and the coding sequence of the heavy chain variable region.

The coding sequence of the light chain variable region can be as shown in the 9th to 338th nucleotides from the 5' end of Sequence 2 in the sequence listing. The coding sequence of the heavy chain variable region can be as shown in the 399th to 752nd nucleotides from the 5' end of Sequence 2 in the sequence listing.

The coding sequence of the connecting peptide can be as shown in the 339th to 398th nucleotides from the 5' end of Sequence 2 in the sequence listing.

The gene can specifically be the following (a) or (b): (a) the DNA molecule shown in the 9th to 752nd nucleotides from the 5' end of sequence 2 in the sequence listing; (b) the sequence 2 in the sequence listing from The DNA molecule indicated by nucleotides 9 to 791 of the 5' end.

Expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the genes all belong to the protection scope of the present invention.

The recombinant vector can specifically be the recombinant plasmid obtained by inserting the gene into the pET-22b (+) plasmid, more specifically, the gene can be inserted between the NcoI and XhoI restriction sites of the pET-22b (+) plasmid The obtained recombinant plasmid.

Specifically, the recombinant bacteria can be recombinant bacteria obtained by introducing the recombinant plasmid into Escherichia coli, and more specifically can be recombinant plasmids obtained by introducing the recombinant plasmid into Escherichia coli BL21 (DE3) strain.

The invention also protects a method for preparing the single-chain antibody, which is to ferment and cultivate the recombinant bacteria to obtain the single-chain antibody.

The method specifically includes the following steps: (1) When the recombinant bacteria are cultivated to OD600=1.0, IPTG is added to make the concentration 0.5mM, and then vibrated at 25°C and 250rpm for 8 hours, and the bacterial precipitate is collected by centrifugation; (2 ) Disrupting the cells obtained in step (1), and centrifuging to collect the supernatant; (3) Dialyzing and concentrating the supernatant obtained in step (2) in sequence to obtain a single-chain antibody solution.

The present invention also protects the application of any one of the above single-chain antibodies in the detection of aflatoxins.

The aflatoxin can specifically be AFB ₁ (aflatoxin B ₁ ), AFB ₂ (aflatoxin B ₂ ), AFM ₁ (aflatoxin M ₁ ), AFM ₂ (aflatoxin M ₂ ), AFG ₁ (aflatoxin G ₁ ) or AFG ₂ (aflatoxin G ₂ ).

Specifically, the single-chain antibody may be a single-chain antibody prepared by the method described above.

Recombinant antibody is a newly developed third-generation antibody preparation technology. Through efficient screening of antibody libraries, antibody fragments with excellent characteristics can be obtained. By expressing the antibody fragment through an efficient expression system, the antibody fragment can be prepared in large quantities at low cost. Aiming at the shortcomings of current monoclonal antibodies and polyclonal antibodies, the present invention uses AFM _1- specific monoclonal antibody hybridoma as a source to prepare single-chain antibody (scFv), and uses protein in vitro evolution technology and phage display antibody library technology to perform its performance Optimization to improve its affinity and cross-reactivity to different AFTs.

The single-chain antibody provided by the invention has a good cross-reactivity rate, high affinity and high sensitivity to a variety of aflatoxins, is suitable for rapid immunological detection of a variety of multi-residual aflatoxins, and will play an important role in the detection of aflatoxins .

Description of drawings

Figure 1 is the spatial structure model of aflatoxin scFv; A: schematic diagram of scFv main chain structure; B: solution-accessible surface of scFv; red is HCDRs, and blue is LCDRs.

Figure 2 is the computer simulation of the interaction between scFv and AFM ₁ ; A: AFM ₁ binding site (yellow is HCDR3, orange is HCDR2, red is HCDR3, blue is LCDR3); B: 2D schematic diagram of antigen-antibody interaction (purple is Amino acid residues that make polar contacts, green ones that make nonpolar contacts, and blue dotted lines that show hydrogen bonding).

Figure 3 is a graph made using aflatoxin _B1 .

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. AFB ₁ (aflatoxin B ₁ ), AFM ₁ (aflatoxin M ₁ ), AFM ₂ (aflatoxin M ₂ ), AFB ₂ (aflatoxin B ₂ ), AFG ₁ (aflatoxin G ₁ ) and AFG ₂ (aflatoxin G ₂ ) was purchased from Sigma, USA.

The PBS buffers used in the examples are all pH 7.4, 0.01M PBS buffers unless otherwise specified.

The carbonate buffer used in the embodiment is the sodium carbonate buffer of pH9.6, 0.05mol/L.

Bovine serum albumin is referred to as BSA.

N,N-dimethylformamide (DMF) is represented by formula (I).

Formula (I)

N-hydroxysuccinimide (NHS) is represented by formula (II).

Formula (Ⅱ)

1-Ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is represented by formula (III).

Formula (Ⅲ)

Carboxymethyl hydroxylamine hemihydrochloride is shown in formula (IV).

Formula (Ⅳ)

Embodiment 1, discovery of single-chain antibody

1. Extraction of hybridoma mRNA and construction of full-length scFv gene

Take 1×10 ⁶ hybridoma cells with good homogeneity and purity (secreting AFM _1- specific monoclonal antibody), extract total RNA and purify to obtain mRNA, and reverse transcribe to obtain cDNA. Using a full set of primers, cDNA was used as a template to amplify the heavy chain variable region (VH) and light chain variable region (VL) genes, respectively, cloned into the pCR 2.1 vector and sequenced, after computer analysis and database BLAST comparison , VH and VL genes with potential immunocompetence and correct expression cassettes were identified. Overlap extension PCR (SOE-PCR) primers were designed according to the above-identified genes, and the full-length scFv gene was constructed by splicing. The scFv gene was sequenced and analyzed, and cloned into the pCR 2.1 vector for preservation and amplification.

2. scFv expression and identification

The above-mentioned scFv was cloned from the pCR 2.1 vector into the pET series vector for scFv expression, and the expression conditions were adjusted so that the target scFv protein was located in the peripheral cavity. Gentle osmotic shock method was used to extract the periluminal protein of the expressed host bacteria, and it was subjected to SDS denatured polyacrylamide gel electrophoresis (SDS page) and immunoblotting analysis (immunoblotting) to evaluate the expression product and its expression efficiency. Using AFM1-BSA as the coating antigen, the periluminal scFv was analyzed by indirect competition ELISA to evaluate the immune activity of the scFv.

3. Homology modeling and molecular docking of aflatoxin scFv

(1) Homologous modeling

Submit the VL and VH sequences in the scFv sequence to the protein database (Protein Data Bank, Brookhaven National Laboratory), and obtain the protein crystal structure with the highest homology through BLAST alignment. Use the Modeler algorithm in Discovery Studio 2.5.5 (DS 2.5.5) to align the above VL and VH sequences with their homologous crystal structures (Alignment), and use the antibody variable region framework region in the crystal structure template as the basis Build homology models. Due to the high variability of antibody CDR regions, on the basis of the above homology model, the Looper algorithm in DS 2.5.5 was used to define the CDR regions in the VL and VH models obtained, and the PDB database search was performed for the CDR regions to obtain the highest homology CDR crystal structure template and reconstruct the CDR region. For the H CDR3 region with the highest variability, since the homology of its optimal template is less than 50%, the de novo algorithm was used to calculate its reasonable conformation from scratch. After constructing and obtaining the homology models of VH and VL respectively, the protein main chain structure was overlapped by the least square method to finally construct the Fv structure, and the 200-step steepest descent energy minimization (steepestdescent energy minimization) and 200-step total Cojugate Gradient energy minimization. The resulting scFv spatial structure model is shown in Figure 1.

(2) Molecular docking

The chemical structures of aflatoxins were retrieved from the NCBI PubChem database. The flexible docking of the aflatoxin-specific scFv to AFM ₁ described above was performed using DS 2.5.5 and CCDCGold suite 4.2. 10 docking results were obtained, and Gold score was used to evaluate the conformation of small molecules in all the docking results, and the results were further analyzed using the ligand analysis module (Analyze bidding site tool) in DS 2.5.5, the results are shown in Figure 2.

4. Site-directed mutation and screening of scFv hypervariable regions (CDRs)

In the combination of antibody and hapten, the CDRs region in the antibody structure, especially the heavy chain CDR3 (H CDR3) and light chain CDR3 (LCDR3) play the most important role. At the same time, due to the extremely small size of the hapten molecule, it can only be in contact with a few of the six CDR regions. Therefore, it is necessary to use molecular simulation to guide subsequent optimization and select relatively effective mutation regions and evolution strategies.

The above computer molecular simulation results show that in the binding of scFv to AFM ₁ , H59Tyr in the CDR2 region of the heavy chain plays the most important role, forming hydrogen bonds and strong π-π interactions. In addition, the light chain CDR3 makes a relatively major contribution to the hydrophobic interaction cavity. Guided by this analysis, degenerate primers were designed according to the known scFv gene sequence, and mutations were introduced in H CDR3 and L CDR3 by PCR technology. In this way, the hydrogen bond in the original antigen-antibody binding can be kept unaffected to the greatest extent. At the same time, in order to avoid too many clones that are inactive or unable to express normally, the adjusted "NNS" degenerate codon is used to introduce mutations, and a 50% random mutation probability is generated at each amino acid mutation point. Due to the limitation of the library capacity of the phage display library (about 10 ⁹ ), random mutations of 6-7 amino acids were performed each time, and about 6.4×10 ⁷ mutants were generated. Therefore, for the 13 amino acids that are in contact with the hapten in the above binding analysis, two phage mutation display libraries need to be constructed respectively (the library capacity is 1.0×10 ^{7 ~ 8} ), and two mutation screenings are performed consecutively.

5. Active clone expression and identification

After constructing the mutant phage display library, the active clones in the library were screened by pure liquid-phase immunoaffinity enrichment using biotin-labeled hapten and streptavidin-coated nano-magnetic beads. A total of 4-5 rounds were carried out, and in each round of enrichment, the concentration of antigen used for screening was gradually reduced, and different evolutionary targets were replaced to enrich and screen the mutant library (see Table 1) to improve the cross-reactivity rate and affinity of antibodies to drugs of the same family .

Table 1 Enrichment and screening of phage mutation display library

Number of enrichments Antigen Concentration phage input Phage output 1 AFM ₁ 1μg/mL 1.0×10 ¹² 2.3×10 ⁵ 2 AFM ₁ 0.1μg/mL 1.0×10 ¹² 3.8×10 ⁶ 3 AFB ₁ 10μg/mL 1.0×10 ¹² 1.9×10 ⁴ 4 AFB ₁ 1μg/mL 1.0×10 ¹² 3.4×10 ⁵ 5 AFB ₁ 0.1μg/mL 1.0×10 ¹² 6.1×10 ⁶

After the last round of enrichment, individual colony clones were randomly selected for secretory expression of scFv, and peritoneal cavity extracts were prepared for SDS-Page and ELISA identification.

The obtained scFv sequence is shown as sequence 1 in the sequence listing. In Sequence 1, amino acid residues 1 to 110 from the N-terminal are light chain variable regions, amino acid residues 111 to 130 are connecting peptides, and amino acid residues 131 to 248 are heavy chain variable regions.

Embodiment 2, the preparation of single-chain antibody

1. Construction of recombinant plasmids

1. Synthesize the double-stranded DNA molecule shown in Sequence 2 of the sequence listing (the 1st to 6th nucleotides from the 5' end are the NcoI restriction recognition sequence, and the 9th to 338th nucleotides are the light chain variable region Coding sequence, the 339th to 398th nucleotides are the coding sequence of the connecting peptide, the 399th to 752nd nucleotides are the coding sequence of the heavy chain variable region, and the 762nd to 791st nucleotides are the C-myc tag tag The coding sequence, the 792nd to 797th nucleotides are the XhoI restriction recognition sequence).

2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

F1: 5'- CCATGG CACAGACGGTTG-3';

R1: 5'- CTCGAG CAGGTCTTCTTC-3'.

3. Digest the PCR amplification product of step 2 with restriction endonucleases NcoI and XhoI, and recover the digested product.

4. Digest the pET-22b(+) plasmid (Novagen, Beijing) with restriction endonucleases NcoI and XhoI, and recover the vector backbone (about 5424bp).

5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a recombinant plasmid. According to the sequencing results, the structure of the recombinant plasmid is described as follows: a double-stranded DNA molecule shown in sequence 2 of the sequence table is inserted between the NcoI and XhoI restriction sites of the pET-22b(+) plasmid. In the recombinant plasmid, the 5' end of the double-stranded DNA molecule shown in Sequence 2 of the sequence table is fused with the coding sequence of the guide peptide pelB on the vector backbone, and the 3' end is fused with the coding sequence of the His-tag tag on the vector backbone, Express the fusion protein.

2. Construction of recombinant bacteria

The recombinant plasmid constructed in step 1 was introduced into Escherichia coli BL21(DE3) strain (Novagen, Beijing) to obtain recombinant bacteria.

3. Expression and purification of single chain antibody

1. Inoculate the recombinant bacteria constructed in step 2 into the Super Broth medium, so that the initial concentration of the recombinant bacteria is OD600=0.1, shake culture at 37°C and 250rpm until OD600=1.0; add IPTG to make the concentration 0.5mM, 25°C , 250rpm shaking culture for 8 hours; then centrifuge the bacterial solution at 6000g for 15min, collect the bacterial sediment, and carry out cell disruption (parameters for ultrasonic disruption: ultrasonic probe is 6Φ, power 400W, and the whole working time is 30 minutes, wherein, each cycle ultrasonic 3 Second interval 5 seconds), centrifuge (12000g, 30min) to collect the supernatant, then dialyze the supernatant in PBS buffer, filter with 0.22μm filter membrane after dialysis, collect the filtrate and store it at 4°C for later use.

2. Dissolve 1g of powdered Ni-IDA resin (MACHEREY-NAGEL, Germany) in 5mL of ultrapure water, and then add it to the chromatography column to make it settle under the action of gravity to form a stable column bed. When the water level in the chromatography column drops to the column bed level, slowly add 5 times the column volume of pH 8.0 PBS buffer to balance the purification column. Then add the filtrate collected in step 1, make it pass through the purification column under gravity, when its liquid level drops to the column bed surface, add the pH 8.0 PBS damping fluid containing 20mM imidazole of 10 times column volume to wash, remove foreign protein . Finally, 3 times the column volume of PBS buffer containing 100 mM imidazole at pH 8.0 was added to elute the target protein, and the post-column eluate was collected. Finally, the 40g/100mL PEG8000 aqueous solution was used to concentrate the eluate after passing through the column (4°C) to obtain a single-chain antibody solution.

Fourth, the preparation of the control solution

1. Synthesize the double-stranded DNA molecule shown in sequence 3 of the sequence list (the 1st to 6th nucleotides from the 5' end are the NcoI restriction recognition sequence, and the 18th to 47th nucleotides are the C-myc tag tag Coding sequence, the 48th to 53rd nucleotides are the XhoI restriction recognition sequence).

Steps 2 to 4 are the same as Step 1 to 2 to 4.

5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a control plasmid. According to the sequencing results, the structure of the control plasmid is described as follows: a double-stranded DNA molecule shown in sequence 3 of the sequence table is inserted between the NcoI and XhoI restriction sites of the pET-22b(+) plasmid.

6. Introduce the control plasmid obtained in step 5 into Escherichia coli BL21(DE3) strain (Novagen, Beijing) to obtain control bacteria.

7. Substitute the control bacteria for the recombinant bacteria to perform step 3 to obtain a control solution.

Embodiment 3, the application of single-chain antibody

1. Preparation of AFB ₁ -BSA

1. Synthesis of AFB ₁ -BSA

(1) Dissolve 5mg of aflatoxin B ₁ in 5ml of pyridine, add 25mg of carboxymethylhydroxylamine hemihydrochloride, stir overnight at room temperature in the dark, spin dry at 40°C; dissolve with a small amount of methanol, separate with a TLC scraper, and develop The reagent is methanol: chloroform = 1:9, take the fluorescent band of the product with RF = 0.2, extract it with 2ml ethyl acetate, add 500μl methanol and 200μl DMF to assist in the extraction, filter, and rotate the filtrate at 40°C to obtain 300μl of the product solution.

(2) Take 300 μl of the above product solution, add 300 μl DMF and 300 μl water, add 8 mg EDC and 8 mg NHS, activate for 1 hour, react in the dark, and obtain solution Ⅰ;

(3) Dissolve 10mg BSA in 1ml carbonate buffer (0.1M, PH=9.51), fully dissolve, and stir at room temperature, which is solution II.

(4) Add solution I to solution II, stir slowly for 24 hours, put it into a dialysis bag, dialyze in normal saline at 4°C for 72 hours (change the water 6 times in the middle), then centrifuge at 8000rmp for 30 minutes at 4°C, take the upper Clear, namely AFB ₁ -BSA solution, divided into ampoule bottles and stored at -20°C.

(5) After diluting the AFB ₁ -BSA solution with PBS buffer solution, measure the spectrophotometric values at 280nm and 260nm, calculate the protein concentration in the diluent according to the formula, and multiply the measured protein concentration value by its dilution factor. AFB ₁ -BSA concentration in the original AFB ₁ -BSA solution. Protein concentration (mg/ml) = 1.45 x OD ₂₈₀ -0.74 x OD ₂₆₀ . The concentration of AFB ₁ -BSA in the AFB ₁ -BSA solution was 6.1 mg/ml.

2. Characterization of AFB ₁ -BSA

Dilute the AFB ₁ -BSA solution with PBS buffer (to make the concentration of AFB ₁ -BSA 5mg/mL) as solution A; use the PBS buffer containing 5mg/mL AFB ₁ as solution B; make the solution containing 5mg/mL BSA PBS buffer as solution C. The solution A, solution B and solution C were subjected to ultraviolet (200-380nm) spectral scanning. Compared with solution C, the UV spectrum of solution A changed significantly, indicating that the compound was successfully coupled with BSA.

2. Application of single chain antibody

1. The AFB ₁ -BSA prepared in Example 2 (concentration adjusted by carbonate buffer) was used for coating, 100 μL/well, and the coating concentration of AFB ₁ -BSA was 0.5 μg/mL.

2. Incubate at 4°C for 16 hours.

3. Seal and wash the plate.

4. Add 50 μL of aflatoxin B ₁ to each well (composed of aflatoxin B ₁ and PBS buffer; the concentrations of aflatoxin B ₁ are 0.0001ng/ml, 0.001ng/ml, 0.01ng/ml, 0.05ng respectively /ml, 0.1ng/ml, 0.5ng/ml, 0.8ng/ml, 1ng/ml, 5ng/ml, 10ng/ml; the wells only added with PBS buffer were used as control wells); each concentration set 3 replicates hole.

5. Add 50 μL of the single-chain antibody solution prepared in Example 2 to each well.

6. Incubate at room temperature for 2 hours and wash the plate.

7. Add 100 μL horseradish peroxidase-labeled anti-C-myc antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., TA-1) to each well and incubate at room temperature for 2 hours.

8. Wash the board.

9. Add TMB color developing solution, and develop color in the dark for 15 minutes.

10. Add 100 μL 2mol/L sulfuric acid to each well to stop the reaction; read the OD ₄₅₀ value.

Divide the absorbance value obtained by using various concentrations of aflatoxin B ₁ (the average value of three replicate wells) by the absorbance value of the control well as the vertical axis, and draw the aflatoxin B ₁ concentration (ng/ml) as the horizontal axis Graph. The results are shown in Figure 3. According to the graph, the corresponding aflatoxin B ₁ concentration (μg/L) when the ordinate value is equal to 50% is obtained, that is, the IC ₅₀ value.

The IC ₅₀ value of scFv for detection of aflatoxin B ₁ was 0.125ng/ml, and the detection limit was 0.01ng/ml.

3. Controlled test

The single-chain antibody solution was replaced by the control solution, and the others were the same as step 2. The absorbance values of each aflatoxin B ₁ concentration were equal, and no ELISA occurred.

4. Cross-reactivity rate of single chain antibody

1 to 3 are the same as 1 to 3 in step 2.

4. Add 50 μL of aflatoxin to each well (composed of aflatoxin and PBS buffer; the concentration of aflatoxin is 0.0001ng/ml, 0.001ng/ml, 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, respectively. ml, 0.5ng/ml, 0.8ng/ml, 1ng/ml, 5ng/ml, 10ng/ml; the wells only added with PBS buffer were used as control wells); each concentration was set up with 3 replicate wells.

The following aflatoxins were used: AFB ₁ , AFM ₁ , AFM ₂ , AFB ₂ , AFG ₁ , AFG ₂ .

5 to 10 are the same as 5 to 10 in step 2.

Divide the absorbance value (average value of three duplicate wells) obtained by using each concentration of specific aflatoxin by the absorbance value of the control well as the vertical axis, and draw the curve with the specific aflatoxin concentration (ng/ml) as the horizontal axis . According to the graph, the specific aflatoxin concentration (μg/L) corresponding to the ordinate value equal to 50% is obtained, that is, the IC ₅₀ value.

Use the following formula to calculate the cross-reactivity rate of the single-chain antibody to aflatoxin.

The cross-reactivity rate of scFv to detect aflatoxin M ₁ was 91.3%. The cross-reactivity rate of scFv to detect aflatoxin _M2 was 45%. The cross-reactivity rate of scFv to detect aflatoxin _B2 was 95%. The cross-reactivity rate of single chain antibody to detect aflatoxin G ₁ was 136%. The cross-reactivity rate of scFv to detect aflatoxin _G2 was 50%. It meets the requirements for the detection of the total amount of Aspergillus flavus.

Claims (10)

1. single-chain antibody, the polypeptide of being formed by variable region of light chain, connection peptides and variable region of heavy chain; Described connection peptides is between described variable region of light chain and described variable region of heavy chain; Described variable region of light chain as the sequence 1 of sequence table from shown in N-terminal the 1st to the 110 amino acids residue; Described variable region of heavy chain as the sequence 1 of sequence table from shown in N-terminal the 131st to the 248 amino acids residue.

2. single-chain antibody as claimed in claim 1 is characterized in that: described connection peptides as the sequence 1 of sequence table from shown in N-terminal the 111st to the 130 amino acids residue.

3. single-chain antibody as claimed in claim 2 is characterized in that: described single-chain antibody is the polypeptide shown in the sequence 4 of the polypeptide shown in the sequence 1 of sequence table or sequence table.

4. the gene of the described single-chain antibody of coding claim 1 is made up of the encoding sequence of the encoding sequence of described variable region of light chain, described connection peptides and the encoding sequence of described variable region of heavy chain.

5. gene as claimed in claim 4 is characterized in that: the encoding sequence of described variable region of light chain as the sequence 2 of sequence table from shown in 5 ' terminal the 9th to 338 Nucleotide; The encoding sequence of described variable region of heavy chain as the sequence 2 of sequence table from shown in 5 ' terminal the 399th to 752 Nucleotide.

6. gene as claimed in claim 5 is characterized in that: the encoding sequence of described connection peptides as the sequence 2 of sequence table from shown in 5 ' terminal the 339th to 398 Nucleotide.

7. gene as claimed in claim 6 is characterized in that: described gene is following (a) or (b):

(a) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 9th to 752 Nucleotide;

(b) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 9th to 791 Nucleotide.

8. contain arbitrary described expression of gene box, recombinant vectors, reorganization bacterium or transgenic cell line in the claim 4 to 7.

9. the method for preparing arbitrary described single-chain antibody in the claim 1 to 3 is the described reorganization of fermentation culture claim 8 bacterium, obtains described single-chain antibody.

10. the application of arbitrary described single-chain antibody in detecting aflatoxin in the claim 1 to 3.

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