Bispecific anti-idiotypic nanobodies and uses thereof

Abstract

The application provides a kind of bispecific anti-idiotype nanobody and its application, bispecific anti-idiotype nanobody is selected from any one of the following: (I) by the amino acid sequence of the vomit toxin anti-idiotype nanobody of SEQ ID NO:2 is connected with aflatoxin anti-idiotype nanobody or corn smut enone anti-idiotype nanobody by connecting peptide connection obtains;(II) at least one of tag sequence, enzyme cutting site sequence or signal sequence is obtained by adding at one end or both ends of (I) amino acid sequence.This bispecific anti-idiotype nanobody can simultaneously recognize vomit toxin and aflatoxin / corn smut enone monoclonal antibody, can be used as the green alternative antigen of the above two groups of fungal toxin combination;It can be used for the detection of combined pollution of fungal toxin, improves the efficiency of fungal toxin detection.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a bispecific anti-idiotype nanobody and its applications. Background Technology

[0002] In nature, filamentous fungi can produce secondary metabolites such as mycotoxins under suitable conditions. Aflatoxin B1 (AFB1) is the most potent carcinogen among mycotoxins, while vomitoxin (DON) contamination is the most prevalent and has the highest detection rate. An 8-year global feed product analysis showed that the positive rate for mycotoxins was 72%, with detection rates of 27%, 36%, and 55% for aflatoxins (AFTs), zearalenone (ZEN), and vomitoxin (DON) in the samples, respectively. Mycotoxin contamination is quite common in grains such as corn, rice, and wheat, and these toxins first cause harm to livestock in the food chain. Studies have shown that the combined toxicity of mycotoxins on rat livers is enhanced when they are present simultaneously, with a significant synergistic effect. Therefore, it is necessary to establish a technology for the simultaneous detection of mycotoxins. According to China's national food safety standard GB2761-2017, the limit for aflatoxin B1 in different grains is 5~20 μg / kg; the limit for vomitoxin in poultry compound feed is ≤5000 μg / kg; and the limit for vomitoxin in food is ≤1000 μg / kg for corn, cornmeal (residue, flakes), and ≤1000 μg / kg for barley, wheat, oatmeal, and wheat flour. GB2761-2011, "Limits of Mycotoxins in Food," requires that the content of zearalenone in grains and their products should be less than 60 μg / kg.

[0003] For the detection of mycotoxins, various detection methods have been established, including physicochemical analysis methods and immunoassay methods. Physicochemical analysis methods mainly include thin-layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and near-infrared spectroscopy (NIR). These methods have high sensitivity and good repeatability; however, they require expensive instruments, skilled operators, and cumbersome sample pretreatment, which limits their widespread application. With the development of monoclonal antibodies, immunoassay based on the specific binding of antigens and antibodies has rapidly developed. Commonly used immunoassay methods include enzyme-linked immunosorbent assay (ELISA) and immunochromatography, which have advantages such as sensitivity, speed, specificity, and simplicity. In recent years, immunochromatographic methods for multiplex and combined detection of biotoxins have emerged. Pure toxins, as competitive antigens, compete with antigens in the sample for monoclonal antibodies. This requires large quantities of pure toxins, which can be harmful to operators and the environment. Furthermore, the monoclonal antibodies used need to be labeled individually, increasing the workload.

[0004] High-quality, non-toxic alternatives to biotoxins can avoid harm to operators and the environment. Anti-idiotypic nanobodies are specific antibodies cloned from camelids and cartilaginous fish that target the antigenic determinants (idiotypes) of the variable regions of antibodies. They are small, highly stable, easily genetically engineered, and possess antigenicity, having the molecular basis to mimic the binding of natural antigens to their corresponding antibodies. They can block the binding of natural antigens to antibodies and are used as substitutes for natural antigens in immunoassays. While using anti-idiotypic nanobodies as non-toxic alternatives to biotoxins avoids harm to operators and the environment, the simultaneous detection of multiple biotoxins requires separate labeling of monoclonal antibodies, which is labor-intensive and complex. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention provides a bispecific anti-idiotypic nanobody and its application, which is specific to both vomitoxin and aflatoxin / zearalenone and can be used for the combined immunoassay analysis of two fungal toxins, vomitoxin and aflatoxin / zearalenone.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] In a first aspect, the present invention provides a bispecific anti-idiotype nanobody, selected from any of the following:

[0008] (I): The anti-idiotypic nanobody of vomitoxin is obtained by linking anti-idiotypic nanobody of aflatoxin or zearalenone through a linker peptide. The amino acid sequence of the anti-idiotypic nanobody of vomitoxin is shown in SEQ ID NO:2.

[0009] (II): Obtained by adding at least one of a tag sequence, restriction enzyme site sequence or signal sequence to one or both ends of the amino acid sequence of (I).

[0010] According to the above scheme, the linker peptide is (G4S).

[0011] According to the above scheme, the amino acid sequences of the aflatoxin anti-idiotype nanobody and the zearalenone anti-idiotype nanobody are shown in SEQ ID NO:1 and SEQ ID NO:3, respectively.

[0012] Secondly, the present invention provides a nucleic acid molecule encoding the above-mentioned bispecific anti-idiotype nanobody.

[0013] Thirdly, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecules.

[0014] Fourthly, the present invention provides a host bacterium for transforming or transfecting the above-mentioned expression vector.

[0015] Fifthly, the present invention provides a method for preparing the above-mentioned bispecific anti-idiotype nanobody, using the above-mentioned host bacteria to produce the above-mentioned bispecific anti-idiotype nanobody.

[0016] Sixthly, the present invention provides the application of the above-mentioned bispecific anti-idiotype nanobody in the combined immunoassay detection of two fungal toxins, vomitoxin and aflatoxin / zearalenone.

[0017] According to the above scheme, the immunoassay detection includes enzyme-linked immunosorbent assay (ELISA), fluorescence polar immunoassay, immunochromatography, electrochemical immunosensing, bioimmunosensing, nanozyme immunoassay, nanoimmunosensing, immunodot hybridization, and latex immunoturbidimetry.

[0018] According to the above scheme, the bispecific anti-unique nanobody, as an alternative to fungal toxins, is used in immunoassay detection in the form of competitive antigens, coating antigens, calibrators, or standards.

[0019] In a seventh aspect, the present invention provides a kit for the joint detection of fungal toxins, comprising the above-mentioned bispecific anti-idiotype nanobody, the kit being used for the joint detection of vomitoxin and aflatoxin / zearalenone.

[0020] According to the above scheme, the kit is a dual-detection immunochromatographic kit or a dual-detection enzyme-linked immunosorbent assay kit;

[0021] The dual-detection immunochromatographic kit includes a test strip and a probe. The test strip includes a base plate, a sample pad, an NC membrane, and an absorbent pad arranged sequentially on the base plate. The NC membrane has two detection lines, T1 and T2, which are respectively coated with monoclonal antibodies against two fungal toxins, namely vomitoxin and aflatoxin / zearalenone. The two detection lines are mutual control lines. The probe is obtained by coupling europium oxide latex microspheres with the above-mentioned bispecific anti-idiotype nanoantibody.

[0022] The dual-detection enzyme-linked immunosorbent assay kit includes the bispecific anti-idiotype nanobody and monoclonal antibodies against two fungal toxins, vomitoxin and aflatoxin / zearalenone. The two monoclonal antibodies are labeled with different markers, which are distinguishable from each other and do not affect each other.

[0023] The beneficial effects of this invention are as follows: This invention provides a highly sensitive and specific anti-idiotypic nanobody for vomitoxin, the amino acid sequence of which is shown in SEQ ID NO:2. Based on this, this invention further connects the anti-idiotypic nanobody for vomitoxin with either an aflatoxin anti-idiotypic nanobody or a zearalenone anti-idiotypic nanobody via a linker peptide to construct a bispecific anti-idiotypic nanobody. This nanobody can simultaneously recognize both vomitoxin and aflatoxin / zearalenone as monoclonal antibodies, serving as a green alternative antigen for both vomitoxin and aflatoxin / zearalenone. It can also be used for the detection of co-contamination by mycotoxins, improving the efficiency of mycotoxin detection. Attached Figure Description

[0024] Figure 1 This is an optimized diagram for AD bispecific nanobody ELISA detection, in which... Figure 1 A is the optimized VELISA diagram when the AD bispecific nanobody is used as a substitute antigen for aflatoxin B1. Figure 1 B is the optimized VELISA diagram when the AD bispecific nanobody is used as a substitute antigen for vomiting toxin.

[0025] Figure 2 The results show the specificity detection of AD bispecific nanobodies against fungal toxins. Figure 2 A represents the specificity detection result of the AD bispecific nanobody against aflatoxin B1 after the addition of aflatoxin B1 monoclonal antibody 1C11. Figure 2 B represents the specificity detection result of the AD bispecific nanobody against vomitoxin after the addition of vomitoxin monoclonal antibody;

[0026] Figure 3 This is a standard curve for the AD bispecific nanobody ELISA method, where... Figure 3 A represents the standard curve of the AD bispecific nanobody as a competitive antigen for AFB1. Figure 3 B represents the standard curve of AD bispecific nanobody as a competitive antigen for vomitoxin;

[0027] Figure 4 This is a schematic diagram of the principle of the dual-detection immunochromatographic test strip;

[0028] Figure 5 Graph showing the parameter optimization of dual-detection immunochromatographic test strips;

[0029] Figure 6 This is a graph showing the specificity and standard curve of the dual-detection immunochromatographic assay kit. Figure 6 A represents the specific detection result of the dual-detection immunochromatographic kit for aflatoxin B1. Figure 6B represents the specific detection result of the dual-detection immunochromatographic kit for vomitoxin. Figure 6 C represents the standard curve for the detection of aflatoxin B1 using this method. Figure 6 D represents the standard curve for detecting vomitoxin using this detection method. Detailed Implementation

[0030] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] Anti-idiotype antibodies are specific antibodies that target the antigenic determinants (idiotypes) of the variable region of an antibody. Since they are all antigenic, anti-idiotype antibodies have the molecular basis to mimic the binding of natural antigens to their corresponding antibodies. They can block the binding of natural antigens to antibodies and can be used as substitutes for natural antigens in immunoassays.

[0036] Heavy chain antibodies (HCAbs) are antibodies found in the bodies of camels (camels, dromedary camels, and llamas) that consist only of heavy chain dimers. Their antigen-binding site consists of a single domain called the VHH region, hence these antibodies are also called single-domain antibodies (sdAbs). Because these antibodies are variable regions after removing the constant region, their molecular weight is only 12-15 kDa, and their diameter is approximately 10 nanometers, hence they are also called nanobodies (Nbs). Compared to conventional tetrachain antibodies (scFvs), nanobodies have advantages in solubility, stability, resistance to aggregation, foldability, expression yield, and ease of DNA manipulation, library construction, and 3D structure determination.

[0037] In order to obtain alternatives to mycotoxins for use in immunoassay detection, the inventors constructed an alpaca-derived phage-display nanobody library using phage display technology. Through optimized affinity screening conditions, they obtained anti-unique nanobodies that can specifically bind to monoclonal antibodies against mycotoxins, including but not limited to vomitoxin, aflatoxin, and zearalenone.

[0038] In some specific embodiments of the present invention, anti-idiotypic nanobodies against vomitoxin, aflatoxin, and zearalenone are provided respectively.

[0039] The amino acid sequence of the anti-idiotypic nanobody DO-7, which specifically binds to the monoclonal antibody against vomitoxin (DON), is shown in SEQ ID NO:2. DO-7 exhibits high sensitivity, with an IC50 of [missing information - likely related to DON]. 50 The value was 49.2645 ng / mL, and there was no cross-reactivity with ochratoxin A (OTA), T-2 toxin (T-2), serpentine toxin (DAS), fumonisin B1 (FB1), and aflatoxin (AFB1), indicating good specificity.

[0040] MKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAASGGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNANNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAHHSEDPHGQAGQ (SEQ ID NO: 2)

[0041] In some specific embodiments, the amino acid sequence of the aflatoxin anti-idiotype antibody VH2-5 is shown in SEQ ID NO:1, and its sensitivity IC50 against aflatoxin B1 (AFB1) is [not specified]. 50 The concentration was 0.16 ng / mL.

[0042] QLQLVESGGGLVQPGGSLRLSCAASGSAFDYYHIGWFRQAPGKEREGVSCISSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADNLPPIQQMCVMTPYYSRDYWGKGTLVTVSSEPKTPKPQD (SEQ IDNO:1)

[0043] In some specific embodiments, the amino acid sequence of the zearalenone anti-idiotype nanobody 8# is shown in SEQ ID NO:3, and its sensitivity to zearalenone (ZEN) is IC50. 50 It was 0.118 ng / mL.

[0044] AQAAQLQLVESGGGLVQPGESLRLSCAASGFTLDDYTIGWWRRAPGKELEGISCISRDGSTTYADSVKGRFTASRDNAKNTVYLQMNSLKPEDTAVYYCATSLSCTVVAGPYDYWGQGTQVTVSSEPKTPKPQDGQAGQ (SEQ ID NO: 3)

[0045] This invention further constructs a bispecific anti-idiotypic nanobody by linking a vomitoxin anti-idiotypic nanobody with an aflatoxin anti-idiotypic nanobody or a zearalenone anti-idiotypic nanobody via a linker peptide. This bispecific anti-idiotypic nanobody exhibits specificity against both vomitoxin and monoclonal antibodies against aflatoxin / zearalenone. It can specifically bind to monoclonal antibodies against both vomitoxin and aflatoxin, or monoclonal antibodies against both vomitoxin and zearalenone, and shows no cross-reactivity with monoclonal antibodies against other fungal toxins. 。 In subsequent embodiments, the present invention is illustrated by linking a vomiting toxin anti-idiotypic nanobody and an aflatoxin anti-idiotypic nanobody via a linker peptide.

[0046] In this invention, a "linking peptide" refers to a peptide that links two polypeptides. The length of a linking peptide can be 1-30 amino acids or 1-40 amino acids. In some embodiments, the linking peptide can be 1-25, 1-20, or 1-18 amino acids in length.

[0047] In a specific embodiment of this application, the linker peptide is (G4S), that is, its amino acid sequence is: GGGGS.

[0048] In this invention, the anti-idiotypic nanobody of mycotoxins should also include: deletion, insertion or substitution of several amino acids, and addition or deletion of one or more amino acids at the C-terminus and / or N-terminus, but maintaining the activity and function of the anti-idiotypic nanobody of mycotoxins of this invention. For example, in the art, substitution with amino acids with similar or comparable properties usually does not change the function of the protein. Adding one or more amino acids at the C-terminus and / or N-terminus, such as adding tag sequences, restriction enzyme site sequences, signal sequences or secretion signal sequences, usually does not change the function of the resulting protein.

[0049] Aflatoxin / zearalenone anti-idiotypic nanobodies and vomitoxin anti-idiotypic nanobodies are linked by linker peptides. The N-terminus of the aflatoxin / zearalenone anti-idiotypic nanobodies can be linked to the C-terminus of the vomitoxin anti-idiotypic nanobodies via a linker peptide, or the N-terminus of the vomitoxin anti-idiotypic nanobodies can be linked to the C-terminus of the aflatoxin / zearalenone anti-idiotypic nanobodies via a linker peptide.

[0050] In some specific embodiments of the present invention, taking the N-terminus of aflatoxin anti-idiotype nanobody VH2-5 linked to the C-terminus of vomitoxin anti-idiotype nanobody DO-7 via a linker peptide (G4S) as an example, the amino acid sequence of the constructed bispecific anti-idiotype nanobody AD (A represents aflatoxin anti-idiotype nanobody, D represents vomitoxin anti-idiotype nanobody) is shown in SEQ ID NO:4.

[0051] QLQLVESGGGLVQPGGSLRLSCAASGSAFDYYHIGWFRQAPGKEREGVSCISSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADNLPPIQQMCVMTPYYSRDYWGKGTLVTVSSEPKTPKPQDGGGGSMKKTAIAIA VALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAASGGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNANNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAHHSEDPHGQAGQ (SEQ ID NO:4)

[0052] In one or more embodiments of the present invention, in order to facilitate protein purification, affinity tags, such as histidine tags, can be added to one or both ends of the bispecific anti-idiotype nanobody.

[0053] The amino acid sequence of the bispecific anti-idiotype nanobody DZ obtained by linking the N-terminus of the vomitoxin anti-idiotype nanobody to the C-terminus of the zearalenone anti-idiotype nanobody via a linker peptide is shown in SEQ ID NO:5.

[0054] MKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAASGGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNANNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAHHSEDPHG QAGQGGGGSAQAAQLQLVESGGGLVQPGESLRLSCAASGFTLDDYTIGWWRRAPGKELEGISCISSRDGSTTYADSVKGRFTASRDNAKNTVYLQMNSLKPEDTAVYYCATSLSCTVVAGPYDYWGQGTQVTVSSEPKTPKPQDGQAGQ (SEQ ID NO:5)

[0055] Those skilled in the art can obtain the nucleic acid molecule encoding the bispecific anti-idiotype nanobody based on its amino acid sequence. As is well known, the expression results of the same nucleotide sequence in different microbial hosts often vary greatly. In order to optimally express the bispecific anti-idiotype nanobody in different genetically engineered bacteria, the expression genes of these anti-idiotype nanobody can be codon optimized.

[0056] Nucleic acid molecules encoding the aforementioned bispecific anti-idiotype nanoantibodies are also within the scope of protection of this invention. The nucleic acid molecules of this invention can be in DNA or RNA form; the DNA form includes cDNA or artificially synthesized DNA, and the DNA can be a coding strand or a non-coding strand.

[0057] Expression vectors containing the aforementioned nucleic acid molecules, and host bacteria produced using the expression vectors through genetic engineering means (transfection or transformation), are all within the scope of protection of this invention.

[0058] The host microorganism of the present invention can be any microorganism suitable for expressing the above expression vector, such as bacteria or fungi.

[0059] The bispecific anti-idiotype nanobody of the present invention can be synthesized artificially or produced using the aforementioned host bacteria through genetic engineering. For example, a recombinant expression vector containing the aforementioned nucleic acid molecules can be transformed or transferred to a suitable host bacteria, the host bacteria can be cultured in a suitable culture medium and induced to express the nanobody. The soluble protein form of the nanobody can then be isolated and purified from the culture medium or cells. When preparing the antibody using genetic engineering, affinity tags, such as histidine tags, can be added to one or both ends of the bispecific anti-idiotype nanobody to facilitate protein purification.

[0060] The amino acid sequence of the aflatoxin B1 and vomitoxin bispecific anti-idiotype nanobody AD after adding the tag sequence and enzyme cleavage site sequence is shown in SEQ ID NO:6.

[0061] MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSQLQLVESGGGLVQPGGSLRLSCAASGSAFDYYHIGWFRQAPGKEREGVSCISSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADNLPPIQQMCVMTPYYSRDYWGKGTLVTVSSEP KTPKPQDGGGGSMKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAASGGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNANNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAHHSEDPHGQAGQLEHHHHHH (SEQ ID NO:6)

[0062] This invention provides the application of the above-mentioned bispecific anti-idiotype nanobody in the combined immunoassay detection of fungal toxins.

[0063] Furthermore, the immunoassays include enzyme-linked immunosorbent assay (ELISA), fluorescence polar immunoassay, immunochromatography (such as colloidal gold immunochromatography, fluorescence immunochromatography, and time-resolved fluorescence immunochromatography), electrochemical immunosensing, bioimmunosensing, nanozyme immunoassay, nanoimmunosensing, immunodot hybridization, and latex immunoturbidimetry.

[0064] Specifically, the bispecific anti-unique nanobody is used as an alternative to mycotoxins in immunoassay detection in the form of a competitive antigen, coating antigen, calibrator, or standard.

[0065] The amino acid sequence of the bispecific anti-idiotype nanobody of the present invention can be used as a precursor and modified by random mutation or site-directed mutagenesis to obtain mutants with better properties (affinity, stability, etc.).

[0066] The bispecific anti-idiotype nanobody of the present invention can be linked to various signal materials (such as fluorescent proteins, fluoresceins, enzymes, etc.) by chemical or biological conjugation, and used as a detection antigen in immunoassay.

[0067] The present invention also provides a kit for the combined detection of fungal toxins, comprising the above-mentioned bispecific anti-idiotype nanobody, the kit being used for the combined detection of vomitoxin and aflatoxin, or the combined detection of vomitoxin and zearalenone.

[0068] According to the above scheme, the kit is a dual-detection immunochromatographic kit or a dual-detection enzyme-linked immunosorbent assay kit.

[0069] According to the above scheme, the dual-detection enzyme-linked immunosorbent assay kit includes the above-mentioned bispecific anti-idiotype nanobody and corresponding monoclonal antibodies against two fungal toxins. The two monoclonal antibodies are labeled with different markers, and the two markers are distinguishable from each other and do not affect each other.

[0070] According to the above scheme, the dual-detection immunochromatographic kit includes a test strip and a probe. The test strip includes a base plate, a sample pad, an NC membrane, and an absorbent pad arranged sequentially on the base plate. The NC membrane has two detection lines, T1 and T2, which are respectively coated with monoclonal antibodies against two fungal toxins. The two detection lines serve as control lines for each other. The probe is obtained by coupling europium oxide latex microspheres with the above-mentioned bispecific anti-idiotype nanoantibody.

[0071] The molecular biology experiments in the following examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation. These were primarily conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook et al., published by Science Press. Specific experimental conditions could be determined through simple experiments if necessary. PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments could be made through simple experiments if necessary.

[0072] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0073] Monoclonal antibodies against aflatoxin B1 and vomitoxin can be either homemade or commercially available.

[0074] Example 1: Establishment of an A-D Bispecific Nanobody ELISA Detection Method

[0075] Bispecific anti-idiotype nanobody AD can be used as a substitute antigen for aflatoxin B1 or vomitoxin, or as a combined substitute antigen for aflatoxin B1 and vomitoxin for VELISA detection.

[0076] The following describes the VELISA procedure using AD as a substitute antigen for aflatoxin B1 in a VEISA (VELISA) as an example. The procedure is as follows:

[0077] (1) The AD bispecific nanobody of Example 1 was diluted with PBS and used as an antigen for coating on an enzyme-labeled plate. The plate was coated overnight at 4°C and blocked the next day with 0.8% skim milk powder. (2) Aflatoxin B1 monoclonal antibody (using monoclonal antibody 1C11, secreted by hybridoma cell line 1C11 with accession number CCTCC NO.C201013 in patent document CN 101993855 B) dilution solution or monoclonal antibody dilution solution and standard dilution solution were added to the enzyme-labeled wells. (3) Horseradish peroxidase-labeled goat anti-mouse secondary antibody was diluted with PBST1:5000. (4) TMB single-component chromogenic solution was added, and the chromogenic reaction was stopped immediately after 15 min at 37°C. Finally, the OD was measured by an enzyme-labeled plate reader. 450 .

[0078] The AD bispecific nanobody was used as a substitute antigen for aflatoxin B1 or vomitoxin to optimize the conditions of VELISA, and the specificity of the AD bispecific nanobody was detected to establish its standard curve.

[0079] 1. VELISA Condition Optimization

[0080] Four different concentrations were diluted with the antibody before VELISA detection. Figure 1 A is the optimized VELISA diagram when the AD bispecific nanobody is used as a substitute antigen for aflatoxin B1. Figure 1 B shows the optimized VELISA diagram when the AD bispecific nanobody is used as a substitute antigen for vomitoxin. The coating concentration and working concentration with an OD value of 1 after binding with the anti-aflatoxin monoclonal antibody 1C11 and the vomitoxin monoclonal antibody were taken. The optimal coating concentrations of the AD bispecific nanobody were 0.2 µg / mL and 1 µg / mL, respectively, and the optimal working concentrations of the two monoclonal antibodies were 1:8000 and 1:2000, respectively.

[0081] 2. Specificity detection and standard curve establishment of AD bispecific nanobodies

[0082] The specificity of the AD bispecific nanobody for the detection of aflatoxin B1 and vomitoxin was determined separately. The optimal coating concentration was selected, and the specificity of the nanobody was verified in step (2) of VELISA using toxins such as ochratoxin (OTA), zearalenone (ZEN), aflatoxin B1 (AFB1), vomitoxin (DON), and fumonisin B1 (FB1). Figure 2 The results of the specific detection of the nanobody were shown. When 1C11 and toxins OTA, ZEN, AFB1, DON or FB1 were added in step (2) of VELISA, the AD bispecific nanobody only competed for AFB1. When the vomiting toxin monoclonal antibody and toxins OTA, ZEN, AFB1, DON or FB1 were added in step (2) of VELISA, the AD bispecific nanobody only competed for DON. This indicates that in addition to having a good competitive effect on aflatoxin B1 (AFB1) and DON, the nanobody has no obvious cross-reactivity with other toxins and has good specificity. It can be used to detect AFB1 and DON toxins at the same time. Figure 3 The curves show the competition between nanobodies and AFB1 and DON, IC50. 50 The concentrations were 0.47 μg / L and 149 μg / L, respectively.

[0083] Example 2

[0084] This embodiment utilizes the aforementioned bispecific anti-idiotype nanobody for enzyme-linked immunosorbent assay (ELISA) to simultaneously detect two fungal toxins: vomitoxin and aflatoxin B1 / zearalenone. The specific method is as follows:

[0085] 1) After coating an ELISA plate with a bispecific anti-idiotype nanobody as an antigen, the plate was blocked.

[0086] 2) The monoclonal antibodies of the two corresponding mycotoxins are labeled with different markers and added to the enzyme label wells along with the sample to be tested. The two markers used are distinguishable from each other and do not affect each other.

[0087] 3) Detect the signals of the two markers to determine whether the above two fungal toxins are present in the sample and quantify them.

[0088] The two markers mentioned above can be two fluorescent markers with different emission wavelengths, such as fluorescein isothiocyanate (FITC) and Cy3, or one can be a fluorescent marker (such as fluorescent quantum dot microspheres) and the other can be a colorimetric marker (such as manganese dioxide nanozyme). The above are just examples. Any existing markers that can be distinguished and do not interfere with each other can be used as markers for the monoclonal antibodies of this invention to achieve simultaneous detection of two fungal toxins.

[0089] Example 3

[0090] Establishment of a dual-detection immunochromatographic reagent kit

[0091] This embodiment utilizes the aforementioned bispecific anti-idiotype nanobody to prepare a fluorescent microsphere dual-detection immunochromatographic kit for the simultaneous detection of two fungal toxins: vomitoxin and aflatoxin B1 / zearalenone.

[0092] The dual-detection immunochromatographic kit includes a test strip and a probe. The test strip includes a base plate, a sample pad, an NC membrane, and an absorbent pad arranged sequentially on the base plate. The NC membrane has two detection lines, T1 and T2, which are coated with monoclonal antibodies against two different fungal toxins, respectively. The two detection lines serve as control lines for each other. The probe is obtained by coupling europium oxide latex microspheres with the bispecific anti-idiotype nanobody prepared above.

[0093] The following example uses the prepared bispecific anti-idiotype nanobody AD to illustrate the preparation, application, and detection method of the fluorescent microsphere dual-detection immunochromatographic kit.

[0094] 1. Assembly of double-check test strips

[0095] After cutting the sample pads, immerse them in blocking solution for 15 minutes and then dry them in a 37°C oven for later use. The test strips consist of a base plate, an NC membrane, a sample pad, and an absorbent pad. First, using an XYZ3050 spot membrane instrument, aflatoxin monoclonal antibody 1C11 and vomitoxin monoclonal antibody are uniformly coated at a concentration of 0.5 mg / mL onto the NC membrane already assembled to the base plate, serving as the T1 and T2 lines, respectively, with a distance of 5 mm between the two lines. The membrane is then dried at 37°C for 30 minutes. Next, the sealed sample pad and absorbent pad are assembled onto the base plate, overlapping by 1 mm. Finally, using a CM4000 strip cutter, the assembled base plate is cut into uniform 4 mm wide test strips, collected, dried, and protected from light for later use. Figure 4 This is a schematic diagram illustrating the detection principle of the dual-detection immunochromatographic test strip.

[0096] 2. Probe preparation

[0097] Add 500 μL of BB buffer solution (pH 8.18) to a 2 mL centrifuge tube. Take 50 μL of europium oxide latex microspheres and add 15 μL (15 mg / mL) of EDC. Vortex to mix and centrifuge at 13000 rpm (4 °C) for 10 min. Discard the supernatant and reconstitute with 500 μL of 0.5% BSA-BB buffer. Then add 200 μL (0.15 mg / mL) of AD nanobody, vortex to mix and sonicate for 5 min. Wrap the centrifuge tube with aluminum foil to protect it from light and vortex for 12 h. Centrifuge and discard the supernatant. Reconstitute with 500 μL of 5% BSA-BB, mix well by pipetting, vortex for 3 h, and store at 4 °C for later use. Transmission electron microscopy (TEM) revealed that the average particle size of the europium oxide latex microspheres was 122.48 ± 0.089 nm. These microspheres exhibited two UV absorption peaks at 295 nm and 345 nm, with stronger fluorescence intensity at 295 nm; therefore, subsequent imaging and analysis were performed at 295 nm. The microspheres showed the strongest excitation brightness at 614 nm.

[0098] 3. Condition Optimization

[0099] To improve the sensitivity and repeatability of the test strip, the following parameters were optimized for the europium oxide latex microsphere immunochromatographic test strip: sample pad, NC membrane, probe sustained-release solution formulation, T-line antigen spraying concentration, probe working concentration, and detection line blocking reagent and concentration.

[0100] First, chromatographic experiments were conducted using three existing sample pad sizes (Fusion3, Fusion5) and glass fiber membranes (glass fiber). Glass fiber was selected as the optimal sample pad. Further optimization was performed using two different NC membrane sizes, NC95 and NCFF120HP, with the best chromatographic result (NC95) selected as the optimal NC membrane. Next, the antibody coating reagent for the detection line was optimized. Three formulations were selected for coating the T-line: PBS, PBS+BSA, and PBS+OVA. The concentrations of the three coating reagents (0.5%, 1%, and 2%) were then optimized, with the coating reagent producing the brightest T-line and minimal background interference after chromatography being selected as the optimal coating reagent formulation. Finally, seven different ratios of sustained-release solutions were optimized, with the optimal sustained-release solution selected based on moderate chromatography speed, no significant sample pad clogging, low NC membrane background value, and clear T-line fluorescence bands. Then, the spraying volume of the test strip was optimized for 16 concentrations (1:2000, 0.125, 0.25, 0.5), and the antigen concentration with the lowest concentration that did not affect the reading of the T-line fluorescence was determined to be the optimal antigen spraying concentration. Finally, 16 concentration gradients from 0.2 μL to 2.4 μL were set to optimize the optimal working volume of the test strip probe. The group with clear test line chromatography and the lowest background influence was selected for establishing the test strip standard curve and specificity detection.

[0101] Figure 5 To optimize the detection method, glass fiber was ultimately chosen as the blocking solution for the sample pad. 1:2000 DON and 1C11 monoclonal antibodies were sprayed onto the NC95. The bispecific nanobody was diluted with 0.5% BSA+PBS, and 1.4 μL of probe was added for chromatography.

[0102] 4. Establishment of standard curve and specificity evaluation of dual-detection immunochromatographic reagent kit

[0103] To further verify the specificity of the dual-detection immunochromatographic assay kit, AFB1, DON, ZEN, OTA, and T-2 toxins were diluted to 100 ng / mL. 50 μL of each of the dual-detection probes and the aforementioned toxins were added to the reaction wells. An equal volume of the test solution was used as a negative control. After chromatography, fluorescence signals were collected by taking photos at 295 nm using a mobile phone, and the T-line signal was observed visually. ImageJ grayscale processing and data analysis were used to verify whether there was cross-reactivity with other fungal toxins.

[0104] Figure 6 In the figure, A and B represent the specificity evaluation of this detection method. The results show that, in addition to significant inhibitory effects on AFB1 and DON, there is no significant cross-reactivity with several other toxins.

[0105] The AFB1 standard solution was serially diluted with chromatography solvent to 0.01, 0.05, 0.1, 0.25, 1, 2, 5, 10, 20, 50, 80, and 100 ng / mL (final concentrations). Under optimal conditions, a standard curve was fitted with the logarithm of the AFB1 concentration as the x-axis and the fluorescence intensity of the corresponding concentration test strips as the y-axis. Figure 6 C and D in the diagram represent the standard curves established for this detection method. The fitting equations for AFB1 and DON are respectively: Y = -1967.54336 + 6728.21476X (R²) 2 =0.99046) and Y=-1605.8123+3951.93955X(R 2 =0.99478), detection limits were 0.025 μg / L and 0.021 mg / L, respectively.

[0106] When used for sample testing, the specific detection method is as follows: After diluting the sample with diluent, add the probe, insert the test strip into the sample cup and incubate at 37°C for 5~10 min. Then, take a picture with a mobile phone under 295 nm ultraviolet light to collect the fluorescence signal. Use ImageJ to analyze the fluorescence intensity and finally calculate the content of AFB1 and DON in the sample to be tested.

[0107] Traditional immunochromatographic test strips for multiple or dual detection of fungal toxins involve conjugating monoclonal antibodies against multiple fungal toxins with tracers, and then spraying multiple antigens onto the NC membrane of the test strip. This method involves one-step conjugation of bispecific nanobodies with fluorescent microspheres, and then spraying anti-AFB1 and anti-DON monoclonal antibodies onto the NC membrane, respectively, as T1 and T2 detection lines. These two detection lines can serve as mutual control lines.

[0108] This bispecific nanobody can also be applied to other immunochromatographic methods, such as colloidal gold immunochromatography and multimodal immunochromatographic strip methods. Simply replace the fluorescent microspheres in this embodiment with colloidal gold or a multimodal tracer conjugated to the bispecific nanobody.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bispecific anti-idiotype nanobody, characterized in that, Choose from any of the following: (I): The anti-idiotype nanobody of vomitoxin and anti-idiotype nanobody of aflatoxin are linked by a linker peptide. The amino acid sequence of the anti-idiotype nanobody of vomitoxin is shown in SEQ ID NO:2, and the amino acid sequence of the anti-idiotype nanobody of aflatoxin is shown in SEQ ID NO:

1. (II): Obtained by adding at least one of a tag sequence, an enzyme restriction site sequence, and a signal sequence to one or both ends of the amino acid sequence in (I); The linker peptide has a length of 1-40 amino acids.

2. The bispecific anti-idiotype nanobody according to claim 1, characterized in that, The linker peptide is (G4S).

3. A nucleic acid molecule encoding the bispecific anti-idiotype nanobody as described in claim 1 or 2.

4. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 3.

5. Transformation or transfection of the host bacteria of the expression vector as described in claim 4.

6. The method for preparing bispecific anti-idiotype nanobodies as described in claim 1 or 2, characterized in that, The bispecific anti-idiotype nanobody is produced using the host bacteria described in claim 5.

7. The application of the bispecific anti-idiotype nanobody as described in claim 1 or 2 in the combined immunoassay detection of two fungal toxins, vomitoxin and aflatoxin, for non-diagnostic purposes.

8. The application according to claim 7, characterized in that, The immunoassays include enzyme-linked immunosorbent assay (ELISA), fluorescence polar immunoassay, immunochromatography, bioimmunosensing, nanozyme immunoassay, immunodot hybridization, and latex immunoturbidimetry.

9. The application according to claim 8, characterized in that, The bioimmunosensing method includes electrochemical immunosensing and nanoimmunosensing.

10. The application according to any one of claims 7-9, characterized in that, The bispecific anti-idiotype nanobody, as an alternative to mycotoxins, is used in immunoassay detection in the form of a competitive antigen, coating antigen, calibrator, or standard.

11. A kit for the combined detection of mycotoxins, characterized in that, The kit includes the bispecific anti-idiotype nanobody as described in claim 1 or 2, and is used for the combined detection of vomitoxin and aflatoxin.

12. The kit for combined detection of mycotoxins according to claim 11, characterized in that, The kit is a dual-detection immunochromatographic kit or a dual-detection enzyme-linked immunosorbent assay kit. The dual-detection immunochromatographic kit includes a test strip and a probe. The test strip includes a base plate, a sample pad, an NC membrane, and an absorbent pad arranged sequentially on the base plate. The NC membrane has two detection lines, T1 and T2, which are respectively coated with monoclonal antibodies against two fungal toxins, vomitoxin and aflatoxin. The two detection lines serve as control lines for each other. The probe is obtained by coupling europium oxide latex microspheres with the bispecific anti-idiotype nanobody described in claim 1 or 2. The dual-detection enzyme-linked immunosorbent assay kit includes the bispecific anti-idiotype nanobody and monoclonal antibodies against two fungal toxins, vomitoxin and aflatoxin. The two monoclonal antibodies are labeled with different markers, which are distinguishable from each other and do not affect each other.

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