Bispecific anti-idiotype nano antibody and application thereof

Abstract

The invention provides a bispecific anti-idiotypic nano antibody and an application of the bispecific anti-idiotypic nano antibody. The bispecific anti-idiotypic nano antibody is selected from any one of the following items: (I) the bispecific anti-idiotypic nano antibody is obtained by connecting a vomitoxin anti-idiotypic nano antibody with an amino acid sequence of SEQ ID NO: 2 with an aflatoxin anti-idiotypic nano antibody or a zearalenone anti-idiotypic nano antibody through a connecting peptide; and (II) adding at least one of a tag sequence, a restriction enzyme cutting site sequence or a signal sequence to one end or two ends of the amino acid sequence of (I). The bispecific anti-idiotypic nano antibody disclosed by the invention can be used for simultaneously recognizing vomitoxin and aflatoxin / zearalenone monoclonal antibodies, and can be used as a green alternative antigen of the combination of the two groups of fungaltoxins; the method can be used for detecting fungaltoxin combined pollution, and the fungaltoxin detection efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a bispecific anti-idiotypic nano antibody and application thereof. Background Art

[0002] In nature, filamentous fungi can produce secondary metabolites such as mycotoxins under suitable environments. Among them, aflatoxin B1 (AFB1) is the most toxic carcinogen among mycotoxins, while vomitoxin (DON) contamination is the most common and has the highest detection rate. An 8-year global feed product analysis showed that the positive rate of mycotoxins was 72%, and the detection rates of aflatoxins (AFTs), zearalenone (ZEN) and vomitoxin (DON) in samples were 27%, 36% and 55%, respectively. Corn, rice, wheat and other grains are quite commonly contaminated by mycotoxins, which first toxicate livestock when entering the food chain. Studies have shown that when mycotoxins are present at the same time, they show enhanced comprehensive toxicity to rat livers, and the combined effect is obvious. In view of this phenomenon, it is necessary to establish a synchronous detection technology for mycotoxins. According to China's national food safety standard GB2761-2017, the limit standard for aflatoxin B1 in different grains is 5-20μg / kg; the limit standard for vomitoxin in poultry compound feed is ≤5000μg / kg, and the limit standard for vomitoxin in food is ≤1000μg / kg for corn, cornmeal (dregs, flakes), and ≤1000μg / kg for barley, wheat, oatmeal, and wheat flour; "GB 2761-2011 Limits of Mycotoxins in Food" requires that the content of zearalenone in grains and their products should be less than 60μg / kg.

[0003] For the detection of mycotoxins, a variety of detection methods have been established, including physical and chemical analysis methods and immunoassay analysis methods. The physical and chemical 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 are highly sensitive and reproducible. However, they require expensive instruments, skilled operators and cumbersome sample pretreatment, which limits the wide application of these methods. With the emergence of monoclonal antibodies, immunoassay analysis based on the specific binding of antigens and antibodies has developed rapidly. Commonly used immunoassay analysis methods include enzyme-linked immunosorbent assay (ELISA), immunochromatography, etc., which have the advantages of sensitivity, rapidity, specificity and simplicity. In recent years, immunochromatographic methods for multiple and joint detection of biological toxins have continued to emerge. Pure toxins are used as competitive antigens to compete with antigens in samples for monoclonal antibodies. This requires a large amount of pure toxins, which will harm operators and the environment. At the same time, the monoclonal antibodies used need to be labeled separately, which increases the workload.

[0004] High-quality non-toxic substitutes for biological toxins can avoid harm to operators and the environment. Anti-idiotypic nanoantibodies are specific antibodies against antigenic determinants (idiotypic) in the variable region of antibodies cloned from camelids and cartilaginous fish. They are small in size, highly stable, and easy to be modified by genetic engineering. They are antigenic and have a molecular basis for simulating the binding of natural antigens to corresponding antibodies. They can block the binding of natural antigens to antibodies and are used as substitutes for natural antigens in immunoassays. Using anti-idiotypic nanoantibodies as non-toxic substitutes for biological toxins can avoid harm to operators and the environment. However, for the joint detection of multiple biological toxins, monoclonal antibodies need to be labeled separately, which has the problems of large workload and complex operation. Summary of the invention

[0005] In order to solve the problems existing in the background technology, the present invention provides a bispecific anti-idiotypic nanoantibody and its application, which is specific to both vomitoxin and aflatoxin / zearalenone and can be used for the combined immunodetection analysis of two fungal toxins, vomitoxin and aflatoxin / zearalenone.

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

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

[0008] (I): obtained by connecting the anti-idiotypic nanobody of vomitoxin with the anti-idiotypic nanobody of aflatoxin or the anti-idiotypic nanobody of zearalenone through a connecting peptide, wherein 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, a restriction site sequence or a signal sequence to one or both ends of the amino acid sequence of (I).

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

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

[0012] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned bispecific anti-idiotypic Nanobody.

[0013] In a third aspect, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0014] In a fourth aspect, the present invention provides a host bacteria for transformation or transfection of the above expression vector.

[0015] In a fifth aspect, the present invention provides a method for preparing the above-mentioned bispecific anti-idiotypic nanoantibodies, using the above-mentioned host bacteria to produce the above-mentioned bispecific anti-idiotypic nanoantibodies.

[0016] In a sixth aspect, the present invention provides the use of the above-mentioned bispecific anti-idiotypic nanoantibody 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, fluorescent polarity immunoassay, immunochromatography, electrochemical immunosensor, biological immunosensor, nanoenzyme immunoassay, nanoimmunosensor, immunospot hybridization and latex immunoturbidimetry.

[0018] According to the above scheme, the bispecific anti-idiosyncratic nanobody is used as a substitute for fungal toxins in the form of competing antigens, coated antigens, calibrators or standards in immunoassay detection.

[0019] In a seventh aspect, the present invention provides a kit for the combined detection of mycotoxins, comprising the above-mentioned bispecific anti-idiotypic nanoantibody, and the kit is used for the combined 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 comprises a test strip and a probe, wherein the test strip comprises a bottom plate, a sample pad, an NC membrane and a water-absorbing pad sequentially arranged on the bottom plate, and the NC membrane is provided with two detection lines T1 and T2, which are respectively coated with monoclonal antibodies of two fungal toxins, vomitoxin and aflatoxin / zearalenone, and the two detection lines serve as quality control lines for each other, and the probe is obtained by coupling europium oxide latex microspheres with the above-mentioned bispecific anti-idiotypic nanoantibody;

[0022] The dual-detection enzyme-linked immunosorbent assay kit includes the bispecific anti-idiotypic nanoantibody and monoclonal antibodies for two fungal toxins, namely, vomitoxin and aflatoxin / zearalenone. The two monoclonal antibodies are labeled with different markers, and the two markers can be distinguished from each other without affecting each other.

[0023] The beneficial effects of the present invention are as follows: the present invention provides a vomitoxin anti-idiotypic nanoantibody with high sensitivity and good specificity, and its amino acid sequence is shown in SEQ ID NO: 2. On this basis, the present invention further connects the vomitoxin anti-idiotypic nanoantibody with the aflatoxin anti-idiotypic nanoantibody or the zearalenone anti-idiotypic nanoantibody through a connecting peptide to construct a bispecific anti-idiotypic nanoantibody, which can simultaneously recognize monoclonal antibodies of two fungal toxins, vomitoxin and aflatoxin / zearalenone, and can be used as a green alternative antigen for two fungal toxins, vomitoxin and aflatoxin / zearalenone; it can be used for the detection of combined mycotoxin contamination to improve the efficiency of mycotoxin detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the optimization diagram for AD bispecific nanoantibody ELISA detection, where Figure 1 A is the VELISA optimization diagram when AD bispecific nanoantibody is used as a substitute antigen for aflatoxin B1. Figure 1 B is the VELISA optimization diagram when AD bispecific nanobody is used as a surrogate antigen for vomitoxin;

[0025] Figure 2 is the specific detection result of AD bispecific nanoantibody to fungal toxins, among which Figure 2 A is the specific detection result of AD bispecific nanoantibody against aflatoxin B1 after adding aflatoxin B1 monoclonal antibody 1C11. Figure 2 B is the specific detection result of AD bispecific nanoantibody to vomitoxin after adding vomitoxin monoclonal antibody;

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

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

[0028] Figure 5 This is a parameter optimization diagram for dual-detection immunochromatographic test strips;

[0029] Figure 6 is the specificity and standard curve of the dual detection immunochromatographic kit, where Figure 6 A is the specific detection result of aflatoxin B1 by the dual detection immunochromatographic kit. Figure 6B is the specific detection result of DON by the dual detection immunochromatographic kit. Figure 6 C is the standard curve for aflatoxin B1 detected by this detection method, Figure 6 D is the standard curve of the detection method for vomitoxin. DETAILED DESCRIPTION

[0030] The principles and features of the present invention are described below in conjunction with the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0035] Anti-idiotype antibodies are specific antibodies produced against the antigenic determinants (idiotypes) in the variable region of antibodies. They are all antigenic, so anti-idiotype antibodies have the molecular basis to simulate the binding of natural antigens to corresponding antibodies, can block the binding of natural antigens to antibodies, and can be used as a substitute for natural antigens in immunoassays.

[0036] Heavy chain antibodies (HCAbs) are antibodies with only heavy chain dimers that exist in the camelid family (camels, dromedaries and llamas). Their antigen binding site consists of a domain, called the VHH region, so this type of antibody is also called a single domain antibody (sdAb). Since this type of antibody is the variable region sequence after the constant region is removed, the molecular weight is only 12 to 15 kD and the diameter is about 10 nanometers, it is also called a nanobody (Nbs). Compared with the scFv of conventional four-chain antibodies, nanobodies have advantages in solubility, stability, resistance to aggregation, foldability, expression yield, and ease of DNA manipulation, library construction and 3-D structure determination.

[0037] In order to obtain substitutes for fungal toxins used in immunoassay detection, the inventors constructed an alpaca-derived phage-displayed nanoantibody library through phage display technology, and screened out anti-unique nanoantibodies of monoclonal antibodies that can specifically bind to fungal toxins through optimized affinity selection conditions. The fungal toxins include but are 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 screened anti-idiotypic nanoantibody DO-7 that can specifically bind to the DON monoclonal antibody is shown in SEQ ID NO: 2. DO-7 has high sensitivity, and its IC 50 The value was 49.2645 ng / mL, and there was no cross reaction with ochratoxin A (OTA), T-2 toxin (T-2), diatoxin (DAS), fumonisin B1 toxin (FB1) and aflatoxin (AFB1), with good specificity.

[0040] MKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAASGGNLDIDD MGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISSRDNANNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAHHSEDPHGQAGQ(SEQ ID NO:2)

[0041] In some specific embodiments, the amino acid sequence of the aflatoxin anti-idiotypic antibody VH2-5 is as shown in SEQ ID NO: 1, and its sensitivity IC to aflatoxin B1 (AFB1) is 50 It is 0.16ng / mL.

[0042] QLQLVESGGGLVQPGGSLRLSCAASGSAFDYYHIGWFRQAPGKEREGVSCISSSGS TNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADNLPPIQQMCVMTPYY SRDYWGKGTLVTVSSEPKTPKPQD(SEQID NO:1)

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

[0044] AQAAQLQLVESGGGLVQPGESLRLSCAASGFTLDDYTIGWWRRAPGKELEGISCI SSRDGSTTYADSVKGRFTASRDNAKNTVYLQMNSLKPEDTAVYYCATSLSCTVVAGPY DYWGQGTQVTVSSEPKTPKPQDGQAGQ(SEQ ID NO:3)

[0045] The present invention further connects the anti-idiotypic nanoantibody of vomitoxin with the anti-idiotypic nanoantibody of aflatoxin or the anti-idiotypic nanoantibody of zearalenone through a connecting peptide, and constructs a bispecific anti-idiotypic nanoantibody that is specific for both the monoclonal antibody of vomitoxin and aflatoxin / zearalenone, which can specifically bind to the monoclonal antibody of vomitoxin and aflatoxin, or the monoclonal antibody of vomitoxin and zearalenone, and has no cross-reaction with the monoclonal antibody of other fungal toxins. In the subsequent examples, the present invention is exemplified by connecting the anti-idiotypic nanoantibody of vomitoxin with the anti-idiotypic nanoantibody of aflatoxin through a connecting peptide.

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

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

[0048] In the present invention, the anti-idiotypic nanoantibodies to fungal toxins should also include: deletion, insertion or substitution of several amino acids, and addition or deletion of one or several amino acids at the C-terminus and / or N-terminus, but the derivatives formed by maintaining the activity and function of the anti-idiotypic nanoantibodies to fungal toxins of the present invention, for example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and adding one or several amino acids at the C-terminus and / or N-terminus, such as adding a tag sequence, a restriction site sequence, a signal sequence or a secretion signal sequence, usually does not change the function of the resulting protein.

[0049] The aflatoxin / zearalenone anti-idiotypic nanoantibody and the vomitoxin anti-idiotypic nanoantibody are connected by a connecting peptide. The N-terminus of the aflatoxin / zearalenone anti-idiotypic nanoantibody is connected to the C-terminus of the vomitoxin anti-idiotypic nanoantibody through a connecting peptide, or the N-terminus of the vomitoxin anti-idiotypic nanoantibody is connected to the C-terminus of the aflatoxin / zearalenone anti-idiotypic nanoantibody through a connecting peptide.

[0050] In some specific embodiments of the present invention, taking the N-terminus of aflatoxin anti-idiotypic nanoantibody VH2-5 connected to the C-terminus of vomitoxin anti-idiotypic nanoantibody DO-7 through a connecting peptide (G4S) as an example, the amino acid sequence of the constructed bispecific anti-idiotypic nanoantibody AD (A represents aflatoxin anti-idiotypic nanoantibody, and D represents vomitoxin anti-idiotypic nanoantibody) 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, an affinity tag, such as a histidine tag, may be added to one or both ends of the bispecific anti-idiotypic nanobody.

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

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

[0055] A person skilled in the art can obtain a nucleic acid molecule encoding the above-mentioned bispecific anti-idiotypic Nanobody based on its amino acid sequence. It is well known that the expression results of the same nucleotide sequence in different microbial hosts are often very different. In order to optimally express the above-mentioned bispecific anti-idiotypic Nanobody in different genetically engineered bacteria, the expression genes of these anti-idiotypic Nanobodies can be codon optimized.

[0056] The nucleic acid molecules encoding the above-mentioned bispecific anti-idiotypic nanobodies are also within the scope of protection of the present invention. The nucleic acid molecules of the present invention can be in the form of DNA or RNA, the DNA form includes cDNA or artificially synthesized DNA, and the DNA can be a coding strand or a non-coding strand.

[0057] The expression vector containing the above-mentioned nucleic acid molecule, and the host bacteria produced by genetic engineering means (transfection or transformation) using the expression vector are all within the protection scope of the present invention.

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

[0059] The bispecific anti-idiotypic nanobodies of the present invention can be produced by artificial synthesis or by genetic engineering using the above-mentioned host bacteria, such as transforming or transferring the recombinant expression vector containing the above-mentioned nucleic acid molecule to a suitable host bacteria, culturing the host bacteria in a suitable culture medium and inducing expression, and separating and purifying the nanobodies in the form of soluble proteins from the culture medium or cells. When preparing antibodies by genetic engineering, affinity tags, such as histidine tags, can be added to one or both ends of the bispecific anti-idiotypic nanobodies to facilitate protein purification.

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

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

[0062] The present invention provides the use of the above-mentioned bispecific anti-idiotypic nanobody in the combined immunoassay detection of fungal toxins.

[0063] Furthermore, the immunoassay detection includes enzyme-linked immunosorbent assay, fluorescent polar immunoassay, immunochromatography (such as colloidal gold immunochromatography, fluorescent immunochromatography, time-resolved fluorescent immunochromatography), electrochemical immunosensor, biological immunosensor, nanoenzyme immunoassay, nanoimmunosensor, immunospot hybridization and latex immunoturbidimetry.

[0064] Specifically, the bispecific anti-idiosyncratic nanobody is used as a substitute for fungal toxins in the form of competing antigens, coated antigens, calibrators or standards in immunoassay detection.

[0065] The amino acid sequence of the bispecific anti-idiotypic 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-idiotypic nanobody of the present invention can be connected to various signal materials (such as fluorescent protein, fluorescein, enzyme, etc.) by chemical coupling or biological coupling, and used as a detection antigen in immunoassay.

[0067] The present invention also provides a kit for the combined detection of mycotoxins, comprising the above-mentioned bispecific anti-idiotypic nanoantibody, wherein the kit is 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 immunochromatography kit or a dual-detection enzyme-linked immunosorbent assay kit.

[0069] According to the above scheme, the dual-detection ELISA kit includes the above-mentioned bispecific anti-idiotypic nanoantibody and the corresponding monoclonal antibodies of two fungal toxins. The two monoclonal antibodies are labeled with different markers, and the two markers can be distinguished from each other without affecting each other.

[0070] According to the above scheme, the dual-detection immunochromatography kit includes a test strip and a probe. The test strip includes a bottom plate, a sample pad, an NC membrane and a water-absorbing pad arranged in sequence on the bottom plate. Two detection lines T1 and T2 are provided on the NC membrane, which are respectively coated with monoclonal antibodies of two fungal toxins. The two detection lines serve as quality control lines for each other. The probe is obtained by coupling europium oxide latex microspheres with the above-mentioned bispecific anti-idiotypic nanoantibody.

[0071] The molecular biology experiments in the following examples include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., mainly with reference to "Molecular Cloning Laboratory Guide" (3rd Edition), edited by J. Sambrook et al., Science Press, and the specific experimental conditions can be determined by simple experiments when necessary. PCR amplification experiments are carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier, and can be adjusted by simple experiments when necessary.

[0072] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

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

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

[0075] The bispecific anti-idiotypic nanoantibody AD can be used as a surrogate antigen for aflatoxin B1 or vomitoxin, respectively, or as a combined surrogate antigen for aflatoxin B1 and vomitoxin for VELISA detection.

[0076] The following is an example of ELISA (VELISA) using AD as a surrogate antigen for aflatoxin B1 to explain the VELISA operation process. The operation process is:

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

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

[0079] 1. VELISA Condition Optimization

[0080] When coating the antibody, dilute it to 4 different concentrations for VELISA testing. Figure 1 A is the VELISA optimization diagram when AD bispecific nanoantibody is used as a substitute antigen for aflatoxin B1. Figure 1 B is the VELISA optimization diagram when the AD bispecific nanoantibody is used as a surrogate antigen for vomitoxin. The coating concentration and working concentration at which the OD value is 1 after binding with the anti-aflatoxin monoclonal antibody 1C11 and the vomitoxin monoclonal antibody are taken. The optimal coating concentrations of the AD bispecific nanoantibody are 0.2 μg / mL and 1 μg / mL, respectively, and the optimal working concentrations of the two monoclonal antibodies are 1:8000 and 1:2000, respectively.

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

[0082] The specificity of the AD bispecific nanobody for aflatoxin B1 and vomitoxin was determined, and the optimal coating concentration was selected. Ochratoxin (OTA), zearalenone (ZEN), aflatoxin B1 (AFB1), vomitoxin (DON), fumonisin B1 (FB1) and other toxins were used to verify the specificity of the nanobody in step (2) of VELISA. Figure 2 The specific detection results of the nanoantibody are shown. When 1C11 and toxins OTA, ZEN, AFB1, DON or FB1 are added in step (2) of VELISA, the AD bispecific nanoantibody only competes with AFB1. When vomitoxin monoclonal antibody and toxins OTA, ZEN, AFB1, DON or FB1 are added in step (2) of VELISA, the AD bispecific nanoantibody only competes with DON. This shows that in addition to having a good competitive effect on aflatoxin B1 (AFB1) and DON, the nanoantibody has no obvious cross-reaction with other toxins, has good specificity, and can be used to simultaneously detect AFB1 and DON. Figure 3 is the competition curve of nanoantibodies against AFB1 and DON, IC 50 They were 0.47 μg / L and 149 μg / L respectively.

[0083] Example 2

[0084] This example uses the above-mentioned bispecific anti-idiotypic nanoantibodies for ELISA to simultaneously detect two mycotoxins, DON and aflatoxin B1 / zearalenone. The specific method is as follows:

[0085] 1) The bispecific anti-idiotypic nanobody is used as an antigen to coat the ELISA plate and then blocked;

[0086] 2) The monoclonal antibodies of the corresponding two fungal toxins are labeled with different markers, and added to the enzyme-labeled wells together with the sample to be tested, and the two markers used can be distinguished from each other and do not affect each other;

[0087] 3) Detect the two marker signals to determine whether the two mycotoxins mentioned above are present in the sample and quantify them.

[0088] The above two markers 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 marker quantum dot microspheres) and the other can be a colorimetric marker (such as nanozyme manganese dioxide). The above are just examples. As long as the existing markers can be distinguished and do not affect each other, they can be used as markers of the present invention to mark the monoclonal antibody to achieve simultaneous detection of two fungal toxins.

[0089] Example 3

[0090] Establishment of dual detection immunochromatographic kit

[0091] In this example, the above-mentioned bispecific anti-idiotypic nanoantibodies are used 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 immunochromatography kit includes a test strip and a probe. The test strip includes a bottom plate, a sample pad, an NC membrane and a water absorbent pad arranged in sequence on the bottom plate. Two detection lines T1 and T2 are arranged on the NC membrane, which are respectively coated with monoclonal antibodies of two fungal toxins. The two detection lines serve as quality control lines for each other. The probe is obtained by coupling europium oxide latex microspheres with the bispecific anti-idiotypic nanoantibody prepared above.

[0093] The following specifically takes the prepared bispecific anti-idiotypic nanobody AD as an example to illustrate the preparation, application and detection method of the fluorescent microsphere dual-detection immunochromatography kit.

[0094] 1. Dual test strip assembly

[0095] The cut sample pad was soaked in the blocking solution for 15 minutes and then dried in a 37°C oven. It was taken out for use. The test strip consists of a base plate, an NC membrane, a sample pad, and an absorbent pad. First, the aflatoxin monoclonal antibody 1C11 and the vomitoxin monoclonal antibody were uniformly coated on the NC membrane assembled on the base plate at a concentration of 0.5 mg / mL using the XYZ 3050 dot film instrument as the T1 line and the T2 line. The distance between the two lines was 5 mm and dried at 37°C for 30 minutes. Then, the closed sample pad and the absorbent pad were assembled on the base plate, overlapping each other by 1 mm. Finally, the assembled base plate was cut into uniform 4 mm wide test strips using a CM 4000 strip cutter, which were collected and dried in a dark place for use. Figure 4 This is a schematic diagram of the detection principle of the dual-detection immunochromatographic test strip.

[0096] 2. Probe Preparation

[0097] Add 500 μL BB buffer solution (PH 8.18) to a 2 ml centrifuge tube, take 50 μL europium oxide latex microspheres, add 15 μL (15 mg / mL) of EDC, shake and mix and use an oscillator for 15 min, centrifuge at 13000 r / min (4°C) for 10 min, discard the supernatant, add 500 μL 0.5% BSA-BB buffer for reconstitution, then add 200 μL (0.15 mg / mL) of AD nanoantibody, shake and mix for 5 min, wrap the centrifuge tube with tin foil to avoid light and shake for 12 h, centrifuge and discard the supernatant, reconstitute with 500 μL 5% BSA-BB, blow evenly, vortex for 3 h, and store at 4°C for use. The average particle size of the europium oxide latex microspheres observed under a transmission electron microscope is 122.48±0.089nm. The europium oxide latex microspheres have two ultraviolet absorption peaks, 295nm and 345nm, respectively. The fluorescence intensity is stronger at 295nm, so the subsequent photo detection is carried out at 295nm. The microspheres have the strongest excitation brightness at 614nm.

[0098] 3. Condition Optimization

[0099] In order to make the test strips have high sensitivity and good repeatability, the following parameters of europium oxide latex microsphere immunochromatographic test strips were optimized; sample pad, NC membrane, probe sustained-release solution formula, T-line antigen spraying concentration, probe working concentration, detection line blocking reagent and its concentration.

[0100] First, three existing sample pads Fusion 3, Fusion 5 and glass fiber membrane (glass fiber) in the laboratory were selected for chromatography experiments, and the glass fiber was selected as the best sample pad. Two different specifications of NC membranes, NC 95 and NC FF 120HP, were further optimized as NC membranes, and the best chromatography result (NC95) was selected as the best NC membrane. Then, the detection line antibody coating reagent was optimized, and three formulas of PBS, PBS+BSA, and PBS+OVA were selected to spray T lines, and then three coating reagents (0.5%, 1%, and 2%) were selected for optimization. The coating reagent with the brightest T line brightness and the smallest background effect after chromatography was the best coating reagent formula. Then, 7 different ratios of sustained-release solutions were optimized, and the sustained-release solution with moderate chromatography speed, no obvious blockage of the sample pad, low NC membrane background value, and obvious T-line fluorescence bands was selected as the best sustained-release solution. Then, the spraying amount of the test line of 16 concentrations was optimized (1:2000, 0.125, 0.25, 0.5), and the antigen concentration with the lowest test line concentration and the T line fluorescence intensity not affecting the reading was the optimal antigen spraying concentration. Finally, 16 concentration gradients were set from 0.2μL to 2.4μL to optimize the optimal working volume of the test strip probe, and the group with clear test line chromatography and the lowest background impact was selected for the establishment of the test strip standard curve and specificity detection.

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

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

[0103] In order to further verify the specificity of the dual detection immunochromatographic kit, AFB1, DON, ZEN, OTA and T-2 toxins were diluted to 100 ng / mL. 50 μL of the dual detection probe and the above toxins were added to the reaction wells, and an equal volume of the test liquid sample slow-release solution was taken as the negative control solution. After the chromatography, the fluorescence signal was collected by taking a mobile phone photo at 295 nm, and the T line signal was observed by naked eyes. The imageJ grayscale processing data analysis results were used to verify whether there was a cross reaction with other fungal toxins.

[0104] Figure 6 (A) and (B) are the specificity evaluations of the detection method. The results showed that, except for the obvious inhibitory effects on AFB1 and DON, there was no obvious cross-reaction with other toxins.

[0105] The AFB1 standard solution was diluted with the chromatography solution to 0.01, 0.05, 0.1, 0.25, 1, 2, 5, 10, 20, 50, 80 and 100 ng / mL (final concentration). Under the optimal conditions, the standard curve was fitted with the logarithmic value of the AFB1 concentration as the abscissa and the fluorescence intensity of the corresponding concentration test strip as the ordinate. Figure 6 (C) and (D) are the standard curves of the detection method. The fitting equations for AFB1 and DON are: Y = -1967.54336 + 6728.21476X (R 2 =0.99046) and Y = -1605.8123 + 3951.93955X (R 2 =0.99478), and the detection limits were 0.025μg / L and 0.021mg / L respectively.

[0106] When used for sample detection, 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 to 10 minutes, then take a photo with a mobile phone under 295nm ultraviolet light to collect the fluorescence signal, use ImageJ analysis to obtain the fluorescence intensity, and finally convert it to obtain the content of AFB1 and DON in the sample to be tested.

[0107] Traditional mycotoxin multi-test or dual-test immunochromatographic test strips couple multiple mycotoxin monoclonal antibodies with tracers, and spray multiple antigens on the NC membrane of the test strip. This method couples bispecific nanoantibodies with fluorescent microspheres in one step, and sprays anti-AFB1 and DON monoclonal antibodies on the NC membrane respectively as T1 and T2 test lines, and the two test lines can serve as quality control lines for each other.

[0108] The bispecific nanobody can also be applied to other immunochromatography methods, such as colloidal gold immunochromatography, multimodal immunochromatography test strip method, etc. That is, the fluorescent microspheres in this embodiment are replaced with colloidal gold or multimodal tracers coupled with 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A bispecific anti-idiotypic nanobody, characterized in that: Select from any of the following: (I): obtained by connecting the anti-idiotypic nanobody of vomitoxin with the anti-idiotypic nanobody of aflatoxin or the anti-idiotypic nanobody of zearalenone through a connecting peptide, wherein the amino acid sequence of the anti-idiotypic nanobody of vomitoxin is shown in SEQ ID NO: 2; (II): obtained by adding at least one of a tag sequence, a restriction site sequence and a signal sequence to one or both ends of the amino acid sequence of (I).

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

3. The bispecific anti-idiotypic nanobody according to claim 1, characterized in that The amino acid sequences of the aflatoxin anti-idiotypic nanobody and the zearalenone anti-idiotypic nanobody are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively.

4. A nucleic acid molecule encoding the bispecific anti-idiotypic Nanobody according to any one of claims 1 to 3.

5. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 4.

6. Transform or transfect the host bacteria with the expression vector as claimed in claim 5.

7. The method for preparing a bispecific anti-idiotypic nanobody according to any one of claims 1 to 3, characterized in that: The bispecific anti-idiotypic nanobody is produced using the host bacteria described in claim 6.

8. Use of the bispecific anti-idiotypic nanobody according to any one of claims 1 to 3 in the combined immunoassay detection of two mycotoxins, vomitoxin and aflatoxin / zearalenone.

9. The use according to claim 8, characterized in that: The immunoassay detection includes enzyme-linked immunosorbent assay, fluorescent polar immunoassay, immunochromatography, electrochemical immunosensor, biological immunosensor, nanoenzyme immunoassay, nanoimmunosensor, immunospot hybridization and latex immunoturbidimetry.

10. The use according to claim 8 or 9, characterized in that: The bispecific anti-idiotypic nanobody is used as a substitute for fungal toxins in the form of competing antigens, coated antigens, calibrators or standards in immunoassay detection.

11. A kit for combined detection of mycotoxins, characterized in that: The kit comprises the bispecific anti-idiotypic nanobody according to any one of claims 1 to 3, and is used for the combined detection of vomitoxin and aflatoxin / zearalenone.

12. The kit for combined detection of mycotoxins according to claim 11, characterized in that: The kit is a dual-detection immunochromatography kit or a dual-detection enzyme-linked immunosorbent assay kit; The dual-detection immunochromatographic kit comprises a test strip and a probe, wherein the test strip comprises a bottom plate, a sample pad, an NC membrane and a water-absorbing pad sequentially arranged on the bottom plate, and two detection lines T1 and T2 are arranged on the NC membrane, which are respectively coated with monoclonal antibodies of two fungal toxins, namely, vomitoxin and aflatoxin / zearalenone, and the two detection lines serve as quality control lines for each other, and the probe is obtained by coupling europium oxide latex microspheres with the bispecific anti-idiotypic nanoantibody according to any one of claims 1 to 3; The dual-detection enzyme-linked immunosorbent assay kit includes the bispecific anti-idiotypic nanoantibody and monoclonal antibodies for two fungal toxins, namely, vomitoxin and aflatoxin / zearalenone. The two monoclonal antibodies are labeled with different markers, and the two markers can be distinguished from each other without affecting each other.

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