Anti-aflatoxin b1 monoclonal antibody and application

Abstract

The application provides an anti-aflatoxin B1 monoclonal antibody and application, and particularly relates to an anti-aflatoxin B1 monoclonal antibody containing only a heavy chain variable region, a nucleotide coding the antibody, an expression vector carrying the monoclonal antibody or a nucleotide sequence coding the monoclonal antibody. The application screens and prepares an antibody for aflatoxin B1 from a phage nanobody synthesis library and applies the antibody to detection or identification of aflatoxin B1. The affinity constant of the antibody is 1.96E-07, the antibody can effectively detect aflatoxin B1, and can be identified from other common fungal toxins.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an aflatoxin B1 antibody, and more specifically to an aflatoxin B1-specific monoclonal antibody and its applications. Background Technology

[0002] Biotoxins are metabolites produced in nature by various organisms, including animals, plants, and microorganisms. They are diverse, primarily consisting of proteins, polypeptides, or small molecule compounds. The most common biotoxins are marine paralytic shellfish toxins and various fungal toxins, all of which are small molecule compounds (molecular weight less than 1 kDa). Their toxicity is severe and their sources are widespread, causing significant economic losses to industries such as agriculture, animal husbandry, and aquaculture, and also posing a threat to human health and safety. Therefore, research on the detection and treatment of biotoxins is of great importance.

[0003] Aflatoxin B1 (AFB1) is a secondary metabolite produced by toxin-producing strains of Aspergillus flavus and Aspergillus parasiticus, and is widely found in moldy grains and their products. AFB1 is chemically stable but possesses strong toxicity, carcinogenicity, teratogenicity, and mutagenicity, making it one of the most toxic mycotoxins and posing a significant threat to human health. AFB1 exhibits hepatotropism in the body and, along with hepatitis B virus infection, is considered one of the two most important risk factors for liver cancer, classified as a Group 1 carcinogen by the World Health Organization. AFB1 contamination is widespread, and despite the abundance of chemical antifungal agents, their effectiveness is not ideal, and they often pose potential toxicity to humans and animals. Currently, there is no specific treatment for AFB1 poisoning; treatment focuses on liver protection, detoxification, and symptomatic relief. Therefore, detecting AFB1 to prevent AFB1 poisoning is of great significance.

[0004] In recent years, phage antibody display technology has developed rapidly, greatly improving the yield of antibodies against biotoxins. Currently established antibody libraries include three categories: natural antibody libraries, immune antibody libraries, and synthetic or semi-synthetic antibody libraries. Among them, immune antibody libraries have high specificity but still require animal immunization; both they and the antibodies in natural antibody libraries need further humanization before they can be used for the treatment of biotoxin poisoning. Synthetic antibody libraries, on the other hand, are artificially synthesized based on antibody sequences, avoiding both the humanization process and the risk of failure due to biotoxin immunization in animals. They also offer high antibody diversity and will become an important tool for the research and development of biotoxin antibodies.

[0005] Aflatoxin B1 specific recognition antibodies have an extremely low yield rate and strong cross-reactivity. Specific recognition antibodies are difficult to obtain from natural antibody libraries and immune antibody libraries, and there is also a lack of relevant methods for artificially synthesizing specific antibodies. Summary of the Invention

[0006] The purpose of this invention is to develop monoclonal antibodies that specifically recognize aflatoxin B1. Addressing the extremely low yield of aflatoxin B1-specific antibodies and the strong cross-reactivity of the obtained antibodies, this invention utilizes a phage nanobody total synthesis library to screen for aflatoxin B1 and designs experiments to obtain specific antibodies. First, a phage nanobody artificial synthesis library is designed; second, positive monoclonal antibodies specifically recognizing aflatoxin B1 are obtained through screening; and finally, the affinity and cross-reactivity of the monoclonal antibodies are verified.

[0007] In a first aspect, the present invention provides an anti-aflatoxin B1 monoclonal antibody containing only a heavy chain variable region, the amino acid sequence of which is shown below:

[0008] EVQLVESGGGLVQPGGSLRLSCAASGSTFSYYTMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAARTFRYRWYPYDYWGQGTQVTVSS (SEQ ID NO. 1).

[0009] In a second aspect, the present invention provides a nucleotide encoding the aforementioned anti-aflatoxin B1 monoclonal antibody, for encoding the heavy chain variable region, the nucleotide sequence of which is shown below:

[0010] GAGGTGCAGCTGGTGGAGAGCGGTGGTGGTCTCGTGCAGCCCGGCGGTAGTCTGCGCCTCAGCTGTGCCGCCAGCGGTAGCACCTTTAGCTATTATACCATGGGCTGGTTTCGCCAAGCCCCCGGCAAAGGTCGCGAACTGGTGGCCGCCATTTCCCGCACGGGCGGTAGCACCTACTACCCA GATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACGCCAAGCGCATGGTGTATCTGCAGATGAACAGTCTGCGCGCCGAGGACACCGCCGTGTATTATTGTGCCGCCCGCACCTTTCGCTATCGCTGGTATCCATACGATTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGC (SEQ IDNO.2).

[0011] In a third aspect, the present invention provides an expression vector carrying the above-mentioned anti-aflatoxin B1 monoclonal antibody or a nucleotide sequence encoding the same.

[0012] A fourth aspect of the present invention provides the use of an anti-aflatoxin B1 monoclonal antibody, the first use being in the preparation of a medicine for the prevention or treatment of aflatoxin B1 infection; and the second use being in the preparation of aflatoxin B1 detection or identification reagents or detection kits.

[0013] For use in the preparation of remedies for the prevention or treatment of aflatoxin B1 infection, a fifth aspect of the present invention provides a pharmaceutical composition for treating aflatoxin B1 infection, comprising an active ingredient and a pharmaceutically acceptable pharmaceutical carrier, said active ingredient comprising an anti-aflatoxin B1 monoclonal antibody, a nucleotide encoding the antibody, or a carrier carrying an anti-aflatoxin B1 monoclonal antibody or encoding the antibody nucleotide.

[0014] For the purpose of use in aflatoxin B1 detection or identification reagents or kits, the present invention provides an aflatoxin B1 infection detection or identification kit, the kit comprising a blood sample processing reagent and a labeled complex obtained by bio-labeling or chemical labeling of an anti-aflatoxin B1 monoclonal antibody.

[0015] SDS-PAGE analysis revealed a distinct protein band between 15 and 20 kDa. Figure 2 After removing the solvent imidazole, the purified antibody protein was obtained. Using the ELISA method, a standard curve was plotted with the antibody concentration as the x-axis and the OD450 value of the reaction between aflatoxin B1 and the antibody as the y-axis. The affinity constant of the antibody was calculated based on the curve and the formula (n[Ab1]-[Ab]) / (n-1), and its affinity value was 1.96E-07. Attached Figure Description

[0016] Figure 1 Aflatoxin B1, aflatoxin B1-BSA conjugates, and BSA curves under different UV wavelengths.

[0017] Figure 2 The image shows SDS-PAGE analysis of antibody soluble expression and purification. Lane 1 is the cell lysis buffer before IPTG induction; lane 2 is the supernatant after IPTG induction; lane 3 is the cell lysis buffer after IPTG induction; lanes 5-8 are the 40 nM imidazole washing elution buffer; lanes 9-13 are the 500 mM imidazole elution buffer.

[0018] Figure 3 This is a graph showing the affinity analysis between positive antibodies and aflatoxin B1. Detailed Implementation

[0019] The following embodiments and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. In addition, the embodiments do not include a detailed description of conventional methods.

[0020] I. Preparation and Identification of Aflatoxin B1 Complete Antigen

[0021] Aflatoxin B1 was conjugated to bovine serum albumin (BSA) using the glutaraldehyde method. Prepared AFB1 and BSA were dissolved separately in PBS (10 mg / mL), thoroughly mixed, and then glutaraldehyde was added. The mixture was incubated on ice for 15 min, then transferred to room temperature and reacted for another 1.5 h. One-tenth of a volume of 1 M Tris-HCl (pH 8.0) was added to block unreacted glutaraldehyde. After mixing at room temperature for 15 min, the mixture was dialyzed against PBS and concentrated. The conjugation results were confirmed by full-wavelength scanning using a UV-Vis spectrophotometer. (See [link to relevant documentation]). Figure 1 .

[0022] II. Enrichment and Screening of Phage Antibody Libraries

[0023] Antibody library screening employed a solid-phase antigen immunosorbent assay (SIA). Aflatoxin B1 complete antigen was diluted to 10 μg / ml with phosphate buffer. Following the method described by Raffi et al. in *Nature Protocols*, 2007, 1368-1386, the antibody library underwent a four-round "adsorption-elution-amplification" screening process. The input / output ratio (enrichment rate) of phages in each round was calculated as an indicator for enrichment screening. Finally, 96 clones were randomly selected from the fourth round of colonies, inoculated into culture medium, and induced overnight with helper phage M13K07. The supernatant was collected for further analysis of phage antibody specificity.

[0024] III. Specificity Detection of Aflatoxin B1 Complete Antigen by Phage Antibodies

[0025] The aflatoxin B1 complete antigen was coated with 2 μg / ml and 0.5% bovine serum albumin was used as a control. The supernatant of 96 phages was incubated at room temperature for 1 h, and then incubated with HRP-anti M13K07 secondary antibody for 1 h. After color development, the OD450 value was detected by microplate reader. The monoclonal antibody with better binding activity to aflatoxin B1 complete antigen than the control group was selected.

[0026] IV. Specificity detection of aflatoxin B1 by phage antibodies

[0027] Considering the impact of the coupling process on the structure of aflatoxin B1, and the competitive ELISA detection of the competitive effect of aflatoxin B1 on positive clones, aflatoxin B1 was added during phage supernatant incubation to competitively inhibit the binding of antibodies to the complete antigen, thereby selecting monoclonal clones with higher competitiveness.

[0028] V. Soluble Expression and Identification of Anti-Aflatoxin B1 Nanobodies

[0029] The antibody sequence was loaded into the pET-25b(+) plasmid and transformed into BL21 competent cells. The antibody was expressed under IPTG induction and then purified by affinity. The purified protein was eluted with 500 mM imidazole elution buffer and analyzed by SDS-PAGE. A clear protein band appeared in the 15-20 kDa range. Figure 2 After removing imidazole, the purified antibody protein is obtained.

[0030] VI. Positive Antibody Affinity Analysis

[0031] Using the ELISA method, a standard curve was plotted with antibody concentration on the x-axis and the OD450 value of the reaction between aflatoxin B1 and antibody on the y-axis. Figure 3 The affinity constant of the antibody was calculated based on the curve and the formula (n[Ab1]-[Ab]) / (n-1), and its affinity value was 1.96E-07.

[0032] In summary, this antibody exhibits high in vitro binding affinity, solubility, and specificity, and has the potential to be developed as an anti-aflatoxin B1 infection preparation or as an in vitro detection or identification kit.

[0033] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

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Claims

1. A monoclonal antibody against aflatoxin B1, characterized in that, This monoclonal antibody contains only the heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. A polynucleotide encoding the anti-aflatoxin B1 monoclonal antibody of claim 1, characterized in that, The polynucleotide sequence is shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, The expression vector carries the polynucleotide encoding the anti-aflatoxin B1 monoclonal antibody as described in claim 2.

4. The use of the anti-aflatoxin B1 monoclonal antibody according to claim 1 in the preparation of aflatoxin B1 detection or identification reagents or kits.

5. The use according to claim 4, characterized in that: in, The detection or identification reagent includes a biolabeled or chemically labeled anti-aflatoxin B1 monoclonal antibody; The kit includes a labeled complex obtained by biolabeling or chemical labeling of an anti-aflatoxin B1 monoclonal antibody.

6. A kit for detecting or identifying aflatoxin B1, characterized in that, The kit includes blood sample processing reagents and a labeled complex obtained by biolabeling or chemical labeling of the anti-aflatoxin B1 monoclonal antibody as described in claim 1.

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