CN116338168A - Magnetic separation detection kit and method for detecting aflatoxin B1 - Google Patents
Magnetic separation detection kit and method for detecting aflatoxin B1 Download PDFInfo
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- CN116338168A CN116338168A CN202210930823.9A CN202210930823A CN116338168A CN 116338168 A CN116338168 A CN 116338168A CN 202210930823 A CN202210930823 A CN 202210930823A CN 116338168 A CN116338168 A CN 116338168A
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- alkaline phosphatase
- antigen
- aflatoxin
- magnetic beads
- fusion protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses a magnetic separation detection kit and a magnetic separation detection method for detecting aflatoxin B1, and belongs to the technical field of biology. The magnetic separation detection kit comprises alkaline phosphatase nano antibody fusion protein and antigen-modified magnetic beads, wherein the amino acid sequence of the alkaline phosphatase nano antibody fusion protein is shown in SEQ ID NO:1, wherein the antigen-modified magnetic beads are modified on the surfaces of epoxy resin magnetic beads by adopting complete antigen ABF 1. The invention develops a magnetic separation competitive chemiluminescence immunoassay method for detecting aflatoxin B1, which is simpler and faster to operate, based on alkaline phosphatase nanometer antibody fusion protein and antigen-modified epoxy resin magnetic beads. The method can realize high-specificity and high-sensitivity detection of aflatoxin B1, and simultaneously provides a rapid, simple and reliable strategy for high-sensitivity analysis of food pollutants, and has important significance.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a magnetic separation detection kit and a magnetic separation detection method for detecting aflatoxin B1.
Background
Aflatoxin B1 (AFB 1) is one of the most predominant and toxic of the 20 aflatoxins found at present, and its pollution occurs mainly in tropical and subtropical areas. However, the distribution of AFB1 contaminated areas may increase in the next few years due to changes in the climate environment, which also means that the contamination may occur in many of the hitherto safe environments. Studies have shown that prolonged exposure to AFB1 can lead to cancer, chronic poisoning, birth defects and even genetic alterations in humans, with a consequent great economic loss. Accumulation of food chains is the main route to the above problems due to the lack of efficient and stable detoxification means. This enhances the need for rapid detection capability, which is of great importance in providing timely monitoring or early warning.
By detecting AFB1 using high performance liquid chromatography, chromatography and mass spectrometry, a great deal of work has been done with some success. However, the time-consuming pretreatment steps and the need for specialized operators make the detection of AFB1 challenging. Immunoassays that utilize antibodies and antigen specific binding are widely accepted and used because of their general applicability, high sensitivity, simplicity and low cost advantages. However, a major drawback of current immunoassays is that separate reagents are required for recognition and signal generation. This makes the labeling, immobilization or washing procedure unavoidable, which further increases the analysis time of the detection. In addition, labeling and complex analytical steps also add to batch variability and analytical errors. Antibodies are the core of molecular recognition and detection, and among the most widely used polyclonal antibodies (pAb) and monoclonal antibodies (mAb), there is a need to overcome several key disadvantages including large molecular weight, expensive preparation process, poor reproducibility and easy inactivation. Many one-step immunoassays, such as fumonisin B1, porcine circovirus type 2, and ochratoxin a, are currently constructed using fusion of alkaline phosphatase (ALP) with nanobodies. Although one-step immunoassays simplify the detection process, immobilization of the target or recognition antibody is still time consuming, and thus the target requirements for establishing a "plug-and-play" immunoassay remain unmet. Based on the above considerations, there is a need to provide a "plug and play" and rapid readout immunoassay method for the detection of AFB1.
Disclosure of Invention
The invention aims to provide a magnetic separation detection kit and a magnetic separation detection method for detecting aflatoxin B1, which are used for solving the problems of the prior art, and the magnetic separation competitive chemiluminescence immunoassay method based on the nano antibody with high tolerance and stability is used for sensitively and rapidly detecting the aflatoxin B1, thereby providing a rapid, simple, convenient and reliable strategy for high-sensitivity analysis of food pollutants and having important significance.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a magnetic separation detection kit for detecting aflatoxin B1, which comprises the following components:
(1) Alkaline phosphatase nanometer antibody fusion protein, the amino acid sequence of the alkaline phosphatase nanometer antibody fusion protein is shown in SEQ ID NO:1 is shown as follows:
*MTPEMPVLENRAAQGDITAPGGARRLTGDQTAALRDSLSDKPAKNIILLIGDGMGDSEITAARNYAEGAGGFFKGIDALPLTGQYTHYALNKKTGKPDYVTDSAASATAWSTGVKTYNGALGVDIHEKDHPTILEMAKAAGLATGNVSTAELQDATPAALVAHVTSRKCYGPSATSEKCPGNALEKGGKGSITEQLLNARADVTLGGGAKTFAETATAGEWQGKTLREQAQARGYQLVSDTASLNSVTEANQQKPLLGLFADGNMPVRWQGPKATYHGNIDKPAVTCTPNPQRNDSVPTLAQMTDKAIELLSKNEKGFFLQVEGASIDKQDHAANPCGQIGETVDLDEAVQRALEFAKKDGNTLVIVTADHAHASQIVAPDTKAPGLTQALNTKDGAVMVMSYGNSEEDSQEHTGSQLRIAAYGPHAANVVGLTDQTDLFYTMKAALGLKGGGGSGGGGSGGGGSVLAALLQGVQAQVQLVDSGGGSVQAGGSLRLSCVASGYTLSNYCMGWFRQVSGKEREGVAGIWTGGGSIWYADSVKGRFTISQDKDKKTLYLQMNSLKPEDTAVYYCAARWSGSWSGCSGRDYNYWGQGTQVTVSSLEHHHHHH*。
(2) The magnetic bead modified by the antigen is formed by modifying the surface of an epoxy resin magnetic bead by adopting a complete antigen ABF 1-BSA.
Preferably, the method for synthesizing the alkaline phosphatase nano antibody fusion protein comprises the following steps: nanobody Nb gene and alkaline phosphatase gene were passed through a linker (G) 4 S) 3 And (3) connecting to form a recombinant gene, cloning the recombinant gene to an expression vector, and transferring the recombinant gene into escherichia coli for expression to obtain the alkaline phosphatase nano-antibody fusion protein.
Preferably, the nucleotide sequence of the recombinant gene is shown as SEQ ID NO:2 is shown as follows:
TAAATGACACCAGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTACTGCACCCGGCGGTGCTCGCCGCTTAACGGGTGATCAGACCGCCGCTCTGCGTGATTCTCTTAGCGATAAACCTGCAAAAAATATTATTTTGCTGATTGGCGATGGGATGGGGGACTCGGAAATTACTGCCGCACGCAATTATGCCGAAGGTGCGGGCGGCTTTTTTAAAGGTATCGATGCCTTACCGCTTACCGGGCAATACACTCACTATGCGCTGAATAAAAAAACCGGCAAACCGGACTACGTCACCGACTCGGCTGCATCAGCAACCGCCTGGTCAACTGGTGTCAAAACCTATAACGGCGCGCTGGGCGTCGATATTCACGAAAAAGATCACCCAACGATTCTGGAAATGGCAAAAGCCGCAGGTCTGGCGACCGGTAACGTTTCTACCGCAGAGTTGCAGGATGCCACGCCCGCTGCGCTGGTGGCGCATGTGACCTCGCGCAAATGCTACGGTCCGAGTGCGACCAGTGAAAAATGTCCGGGTAACGCTCTGGAAAAAGGCGGAAAAGGATCGATTACCGAACAGCTGCTTAACGCCCGTGCCGATGTTACGCTTGGCGGCGGTGCAAAAACCTTTGCTGAAACGGCAACCGCCGGTGAATGGCAGGGAAAAACGCTGCGTGAACAGGCACAGGCGCGTGGTTATCAGTTGGTGAGCGATACTGCCTCACTGAATTCGGTGACGGAAGCGAATCAGCAAAAACCCCTATTAGGACTGTTTGCTGACGGCAATATGCCAGTGCGCTGGCAAGGACCGAAAGCAACGTACCACGGTAATATAGATAAGCCCGCAGTCACCTGTACGCCTAATCCGCAACGTAATGACAGTGTACCGACCCTGGCGCAGATGACCGACAAAGCCATTGAATTGTTGAGTAAAAATGAGAAAGGCTTTTTCCTGCAAGTTGAAGGTGCGTCAATCGATAAACAGGATCACGCTGCGAATCCTTGTGGGCAAATTGGCGAGACGGTCGATCTCGATGAAGCCGTACAACGGGCGCTGGAATTCGCTAAAAAGGATGGTAACACGTTGGTCATAGTCACCGCTGATCACGCCCATGCCAGCCAGATAGTTGCGCCAGATACCAAAGCTCCGGGCCTCACCCAGGCGCTAAATACCAAAGATGGCGCAGTGATGGTGATGAGTTACGGGAACTCCGAAGAGGATTCACAAGAACATACCGGCAGTCAGTTGCGTATTGCGGCGTATGGCCCGCATGCCGCCAATGTTGTTGGACTGACCGACCAGACCGATCTCTTCTACACCATGAAAGCCGCCCTGGGGCTGAAAGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGTCCTGGCTGCTCTTCTACAAGGTGTCCAGGCTCAGGTGCAACTGGTGGACTCTGGGGGGGGCTCAGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGATACACCTTGAGTAACTACTGCATGGGCTGGTTCCGCCAGGTTTCAGGGAAGGAGCGCGAGGGGGTCGCAGGTATTTGGACTGGCGGTGGTAGCATATGGTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCTCAAGACAAGGACAAGAAGACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCGTGTATTACTGTGCGGCAAGGTGGTCAGGCTCTTGGTCTGGGTGTTCAGGAAGGGACTATAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCACTCGAGCACCACCACCACCACCACTGA。
preferably, the preparation method of the antigen-modified magnetic beads comprises the following steps: and (3) reacting the epoxy resin magnetic beads with the aflatoxin B1-bovine serum albumin connector, vertically suspending for 10-15h at room temperature, blocking the residual active sites by using a magnetic bead blocking buffer solution, and obtaining the antigen modified magnetic beads.
Preferably, the diameter of the epoxy resin magnetic beads is 0.3-2.6 μm;
the mass ratio of the epoxy resin magnetic beads to the aflatoxin B1-bovine serum albumin connector is (10-20): 1.
the invention also provides a method for detecting aflatoxin B1, which comprises the following steps:
(1) And (3) establishing a detection system: mixing the alkaline phosphatase nanobody fusion protein, the antigen-modified magnetic beads and an aflatoxin B1 standard substance in PBS, incubating at 37 ℃, collecting an alkaline phosphatase-nanobody-aflatoxin B1 complete antigen-magnetic bead complex through magnetic adsorption, adding a luminescent substrate, and detecting the chemiluminescent intensity of the alkaline phosphatase nanobody fusion protein combined with the antigen-modified magnetic beads to obtain a standard curve;
(2) And (3) combining the standard curve obtained by adopting the same method in the step (1) to obtain the content of aflatoxin B1 in the sample to be detected.
Preferably, in the step (1), the volume ratio of the alkaline phosphatase nanobody fusion protein, the antigen-modified magnetic beads and the aflatoxin B1 standard is 3:4:3; the dilution ratio of the alkaline phosphatase nanometer antibody fusion protein is (1:250) - (1:2000), the concentration of the antigen modified magnetic beads is 0.0625-0.5mg/mL, and the concentration of the aflatoxin B1 is 1 multiplied by 10 0 -1×10 5 pg/mL。
Preferably, the particle size of the antigen-modified magnetic beads is 0.3 μm, and the dilution factor of the alkaline phosphatase nanobody is 1:250, wherein the concentration of the antigen modified magnetic beads is 0.25mg/mL.
Preferably, in step (1), the incubation time is 20min;
the method further comprises the step of washing the alkaline phosphatase-nanobody-aflatoxin B1 complete antigen-magnetic bead complex with a PBST buffer solution for 1 time before adding the luminescent substrate; the luminescent substrate is APS-5.
The invention also provides application of the magnetic separation detection kit in detecting aflatoxin B1 in food. More preferably, the food product comprises corn, oat, milk and oil.
The invention discloses the following technical effects:
the invention discloses a magnetic separation competitive chemiluminescent immunoassay (MS-CLIA) for detecting aflatoxin B1 (AFB 1) with simpler and faster operation based on alkaline phosphatase nano antibody fusion protein (ALP-Nb) and antigen modified epoxy resin magnetic beads (AFB 1-MBs) (the detection principle and the detection flow chart are shown in figure 9). Specifically, the Anti-AFB1 nanobody which can be specifically identified with the AFB1 is screened from the immune camel, and the fusion protein of the AFB1-MBs and the ALP-Nb is beneficial to realizing one-step detection of 'plug and play', so that the total detection time of MS-CLIA is shortened to 30min from 2h of the traditional competitive ELISA. Meanwhile, in order to improve the performance of MS-CLIA, the detection method is optimized, and tolerance experiments are carried out on complex conditions. Finally, the MS-CLIA realizes the sensitive detection of the AFB1, and the detection limit is at least0.743pg/mL，IC 50 =0.33 ng/mL, the linear range is 7.23pg/mL to 12.38ng/mL, and shows strong tolerance and practicality to complex matrix environments in sample detection. Therefore, the MS-CLIA can realize the AFB1 detection with high specificity and high sensitivity, and simultaneously provides a rapid, simple and reliable strategy for the high-sensitivity analysis of food pollutants, thereby having important significance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a SEM image and TEM image (A) of an epoxy magnetic bead, a SEM image and TEM image (B) of AFB1-MBs, and a SEM image and TEM image (C) of ALP-Nb-AFB 1-MBs;
FIG. 2 is a graph showing conditions for optimizing detection of AFB1 (1 ng/mL) at different particle sizes of AFB1-MBs and at different dilutions of ALP-Nb; a: the concentration of AFB1-MBs with the particle size of 0.3 μm and ALP-Nb with different dilutions are optimized; b: the concentration of AFB1-MBs with the particle size of 1.0 μm and ALP-Nb with different dilutions are optimized; c: the concentration of AFB1-MBs with the particle size of 2.6 μm and ALP-Nb with different dilutions are optimized;
FIG. 3 is a standard curve; a: establishing a standard curve by using AFB1-MBs with the particle size of 0.3 mu m; b: a standard curve established by using the mAFB1-MBs with the particle size of 1.0 mu; c: standard curves established with AFB1-MBs with particle size of 2.6 μm;
FIG. 4 is a diagram of detection condition optimization in a detection system; a: the detection reactions were incubated at 37℃for a period of 5, 10, 20, 40 and 80 min; b: washing times (1, 2, 3);
FIG. 5 is a diagram of detection condition optimization in a detection system; a: incubation at 75 ℃ for 0, 10, 20, 30, 40 and 50min; b: PBS buffer solutions with different pH values are used as diluting reagents; c: PBS buffer solutions with different concentrations of NaCl are used as diluting reagents;
FIG. 6 is a standard curve for different methanol concentrations as diluents; a: a standard curve was established with PBS buffer (10%) containing 10% methanol as the dilution reagent; b: a standard curve was established with PBS buffer containing 30% methanol as the dilution reagent; c: a standard curve was established with a PBS buffer containing 60% methanol as the dilution reagent;
FIG. 7 is a standard curve and specificity evaluation; a: standard curve of AFB1 detection, concentration ranging from 0 to 10 6 pg/mL; b: specific evaluation of magnetic separation immunoassays, including AFB2, AFG1, AFG2, AFM1, FB1, DON, ZEN, and T2 (all 50ng mL-1);
FIG. 8 shows the matrix effect of different foods; a: the matrix effect of corn; b: the matric effect of oat; c: the matrix effect of the oil; d: a matrix effect of milk;
FIG. 9 is a schematic diagram of the detection principle and detection flow of aflatoxin B1 by magnetic separation competitive chemiluminescent immunoassay;
FIG. 10 shows the amino acid sequence of the AFB1 nanobody Nb 10E;
FIG. 11 is a pET-22b-ALP-Nb construction map.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1. Experimental method
1.1 Preparation of ALP-Nb fusion proteins
To obtain a pool of specific nanobodies against AFB1, healthy camels were subjected to three subcutaneous immunizations (three immunizations spaced for one week, 1mg each) with complete antigen (AFB 1-BSA) (purchased from shandong green all biotechnology limited). Peripheral blood mononuclear cells were collected from 20mL blood samples 4-5 days after the last immunization. cDNA prepared from total RNA (reverse transcription of RNA into cDNA by reverse transcription kit PrimeScript) TM IV 1st strand cDNA Synthesis Mix, purchased from TaKaRa), heavy chain antibody genes were obtained using cDNA as template, primers: call-1:GTCCTGGGCTGCTCTTCTACAAGG, call-2:GGTACGTGCTGTTGAACTGTTCC; the heavy chain antibody gene is taken as a template to obtain a nano antibody gene primer: nb-F cggggtaccccGTCCTGGCTGCTCTTCTACA, nb-R, tccccccggggTGAGGAGAGAYGGGACCWGGGT, degenerate primer: y=c/T; w=a/T, a recombinant vector (designated Nb-pcatab) was constructed for constructing nanobody phage display libraries. By four rounds of phage display biopanning, a nanobody library carrying a recombinant vector Nb-pCantab is infected with M13 phage, and the nanobody is expressed on phage by culturingThe surface, fix AFB1 complete antigen, express phage of nanometer antibody and AFB1 complete antigen, use PBST to elute and remove unbound phage, first, two rounds of elutriation uses glycine (0.2M) and Tris-HCl to elute and express phage of nanometer antibody bound with complete antigen, phage that each elution obtains is used for next round of elutriation, third, four rounds use AFB1 standard to compete and elute phage of expressing nanometer antibody bound with complete antigen. VHH clones that have good competitive binding to AFB1 were selected from a library of phage VHH (also called Nanobody). The VHH gene was PCR amplified using pET-22b-Nb-F and pET-22b-Nb-R primers. The recombinant plasmid, designated pET-22b-VHH vector, the PCR product was digested at the same restriction site and ligated into pET-22b vector. The VHH protein was expressed by E.coli (E.coli) strain Trans B (DE 3) and induced overnight at 16℃with isopropyl-B-D-thiogalactoside (IPTG, 0.1 mM). The VHH protein was purified directly by Ni-NTA column. To assess the binding capacity of the expressed and purified products, an indirect competitive enzyme-linked immunosorbent assay of anti-His-tagged mAb-HRP was used (iceelisa results show that Nb10E exhibits better binding activity and specificity to AFB1.
ALP-Nb fusion proteins were constructed by SOE-PCR fusion of Nb, (G4S) 3 linker and ALP (derived from E.coli). Then, as described above, the recombinant gene of the alkaline phosphatase nanobody fusion protein was cloned into pET-22 b. The pET-22B-ALP-Nb plasmid was transferred into E.coli Trans B (DE 3). As described above, the expression and purity of ALP-Nb were determined. Finally, ALP-Nb was identified by SDS-PAGE. Good binding and competitive activity of ALP-Nb was demonstrated by a one-step indirect competitive chemiluminescent immunoassay (icCLIA) using the intensity of the chemiluminescent reaction of ALP and APS-5 as signal outputs.
Preparation of 1.2AFB1-BSA immunomagnetic beads
0.2mL of epoxy Magnetic Beads (MBs) (20 mg.mL -1 ) Injected into a 1.5mL tube. The tube was subjected to a magnetic field to separate it. MBs were washed three times with 0.5mL of PBS buffer (0.1mM,1mM EDTA,pH 8.5). MBs were then suspended in a suspension containing 0.15mg AFB1-BSA and 185mg Na 2 SO 4 In 1mL PBS buffer, while establishingA control group without AFB1-BSA was designated as negative magnetic beads (N-MBs). The two mixtures were gently shaken at room temperature for 12 hours and washed three times with 0.5mL of PBS buffer. Thereafter, blocking was performed with a magnetic bead blocking buffer for 6 hours, and washing was performed 6 times with 1mL of PBST (0.1% Tween-20)/PBS buffer to obtain AFB1-BSA-MBs (AFB 1-MBs) and N-MBs.
1.3 establishment of detection System
AFB1-MBs were titrated with a range of concentrations of ALP-Nb. Mixing target (AFB1 standard), ALP-Nb and AFB1-MBs in PBS, adding 30uL AFB1 standard +30uLALP-Nb+40uLAFB1-MBs into a white ELISA plate with low specific adsorption, incubating at 37 ℃, collecting the ALP-Nb-AFB1-MBs by magnetic adsorption, and washing free AFB1 and ALP-Nb-AFB1 by PBST. After that, 100. Mu.L of APS-5 was added to the reaction cell, and the chemiluminescent signal (CL) intensity of ALP-Nb bound to AFB1-MBs was monitored by SpectraMax-5M (Molecular devices. USA).
1.4 optimization of detection System
To achieve optimal performance, experimental conditions were studied using a one-factor test. Experimental conditions were optimized according to literature. Specifically, the diameter of the epoxy beads (0.3,1.0,2.6 μm), the concentration of AFB1-MBs (0.0625, 0.125,0.25,0.5 mg. Multidot.mL) -1 ) Different antibody dilutions (VHH 1:250,1:500,1:1000 and 1:2000), reaction times (5, 10, 20, 40 and 80 min), wash times (1, 2, 3), thermostability of ALP-Nb (0, 10, 20, 30, 40, 50min at 75 ℃), methanol content of the target dilutions (10%, 30%, 60%), reaction buffer pH (PBS with ph=1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0), concentration of NaCl (PBS with 0, 25, 50, 100, 200 and 400 mM), by comparison of 0.5ng·ml -1 CL intensity and inhibition rate of AFB1 were optimized and screened. Nonspecific adsorption of MBs to ALP-Nb was analyzed using blocked N-MBs as a negative control.
1.5 specificity
Specifically, expressed in terms of Inhibition Rate (IR), structural analogs such as aflatoxin B2 (AFB 2), aflatoxin G1 (AFG 1), aflatoxin G2 (AFG 2), aflatoxin M1 (AFM 1), fumonisin B1 (FB 1), deoxynivale (DON), zearalenone (ZEN) and trichothecene-2 (T2) were identified by evaluation. IR (%) is expressed as the inhibition rate of the target analyte and the like at the same concentration.
1.6 magnetic separation competitive immunoassay method in samples
The accuracy of the detection system is determined by the recovery rate and the coefficient of variation of the standard additive. First, the effect of the matrix on the detection system is evaluated. To 5 g oat/corn was added 20ml of a PBS solution containing 60% methanol, to 1 g milk/oil was added 5ml of a PBS solution containing 60% methanol, and the mixture was vigorously shaken at room temperature for 30min, centrifuged at 6000 g for 10min, and the supernatant was collected. The supernatant was then diluted 1-fold with PBS as AFB1 diluent and the supernatant from the different samples was compared with a PBS solution containing 30% methanol to 1ng mL in the detection system -1 Influence of CL intensity and inhibition of AFB1. AFB1 standards (0.08, 0.4, 0.8, 8 and 80. Mu.g.kg) -1 ) Added to oat/milk/oil/corn samples (IC according to the test system standard curve 50 、IC 20 And IC 80 ) And respectively adding PBS solution containing 60% methanol for extraction. The addition recovery rate (R,%) and the coefficient of variation (CV,%) were calculated according to the following formulas, respectively. Recovery (%) = (measured value/added value) ×100%, coefficient of variation (%) = (standard deviation/average value) ×100%.
2. Results and analysis
2.1 obtaining anti-AFB1 nanobodies
Nanobodies have been widely reported in immunoassays and are readily available through several rounds of biopanning sieves. After three immunizations with AFB1-BSA, the anti-AFB1 antibody titres reached 1:5000. total cellular RNA was isolated from about 20mL of peripheral blood, reverse transcribed into cDNA, and amplified to a fragment of about 750bp in the first round of PCR. Then, a target band of about 450bp was amplified in the second round of PCR, and finally, a phage display library against AFB1 was constructed, the capacity of which reached about 6X 10 7 CFU·mL -1 . Confirm the insertion of VHH gene and diversity of library, 10 individual clones were randomly selected for detection using colony PCR, indicating that the insertion rate was exceeded90% is passed. Sequencing results showed tremendous diversity. These results indicate that a reliable phage display library was successfully developed for screening nanobodies against AFB1.
During the four rounds of biopanning, the encapsulation concentration of AFB1-BSA antigen was reduced round by round, and specific phage binding to AFB1 antigen was eluted with Tris-HCl in the first and second rounds of biopanning, followed by competitive elution with AFB1 standard in the third and fourth rounds of biopanning. Positive clones were sequenced using the M13 primer and subsequently classified according to the amino acid sequence of the Complementarity Determining Regions (CDRs). Finally, a specific anti-AFB1 nanobody was obtained by screening and named Nb10E (fig. 10). Furthermore, nb10E was found to exhibit high binding activity to AFB1 by iceelisa.
2.2 preparation of ALP-Nb
The construction of pET-22b-ALP-Nb is shown in FIG. 11. Positive recombinant plasmids were confirmed by sequencing and the correct plasmid was transformed into E.coli Trans B (DE 3). A colony was selected from the 2YT agar plate, and cultured in a shaking incubator at 37℃and 220 rpm. When the OD of the culture 600 When the value reached between 0.4 and 0.6, IPTG was added to induce expression of ALP-Nb fusion proteins. After purification by Ni-NTA column, the ALP-Nb fusion protein was analyzed by SDS-PAGE. SDS-PAGE analysis showed that the fusion protein was soluble and had a band of approximately 70 kDa. ALP-Nb was successfully prepared.
2.3AFB1-BSA modified epoxy resin magnetic beads
The epoxy groups [ -CH (O) CH- ] have the ability to react with polyfunctional compounds to form cured products having a crosslinked structure, they are capable of undergoing a ring-opening reaction with mercapto groups, binding thioethers under mild conditions, due to the high tension present in the three-membered ring. Bovine Serum Albumin (BSA), a free thiol group is located at position 34 of the BSA peptide chain. Thus, AFB1-BSA can be modified on the surface of epoxy-based magnetic beads. As shown in FIGS. 1A and B, field emission Scanning Electron Microscope (SEM) images show that the epoxy-based magnetic beads are in the form of clusters with a rough surface having an average diameter of about 0.3 μm, and that the rough surface of the magnetic beads is advantageous for labeling biological macromolecules such as proteins, however, AFB1-MBs are monodisperse and uniform and have a smooth surface. The detailed structure in FIGS. 1A, B shows that by field emission Transmission Electron Microscopy (TEM), the edges of MBs are sharp, the surface is uneven, and the edges of AFB1-MBs are blurred. TEM images showed similar dimensions and surface morphology, consistent with SEM results. ALP of E.coli is a dimer composed of the same monomers. Thus, FIG. 1C shows that a gap of a fixed size is formed at the junction of AFB1-MBs and ALP-Nb, which is more evident in TEM images.
2.4 optimization of detection systems
2.4.1AFB1-MBs concentration and varying dilutions of ALP-Nb.
The dilution of ALP-Nb represents the concentration series of ALP-Nb in the detection system. A low ALP-Nb content results in insufficient CL intensity, whereas a low detection sensitivity may be due to a high ALP-Nb content, as well as the effect of the concentration of AFB1-MBs on the sensitivity, so that appropriate AFB1-MBs and ALP-Nb contents are critical to the reaction system (FIGS. 2A, B, C). Our results indicate that the concentration of AFB1-MBs is 0.25 mg.multidot.mL -1 The sensitivity of the detection system was optimal at an ALP-Nb dilution of 1:250, and the inhibition rate of AFB1 (1.0 ng mL-1) was all the best for the three particle sizes of magnetic beads at the above proportional concentrations.
2.4.2 epoxy magnetic bead size
The size of the epoxy-based magnetic beads affects CL strength and stability of antibody/antigen binding. Thus, MBs of optimal diameter and surface area bind the substance, and a sufficient CL signal is more readily detected. After determining the ratio of MBs to ALP-Nb in the detection system, three standard curves were established for different MBs particle sizes (FIGS. 3A, B, C). Our results show that MBs of 0.3 μm achieve the best sensitivity, (IC) 50 Is 0.422 ng.mL -1 The linear range is 0.0372-4.314 ng.mL -1 ) (FIG. 1A).
2.4.3 reaction time and washing times
In order to improve the sensitivity of the detection and reduce the detection time, the reaction time and the washing times are optimized. The reaction system was incubated at 37℃for 5, 10, 20, 40 and 80min. The results show that the reaction reached maximum CL strength at 20min and exhibited excellent sensitivity, still stable at 40 and 80min (fig. 4A). Thus, the incubation time of the reaction was set to 20min, saving a lot of time compared to conventional ELISA and other methods. Then, after 20min incubation, the washing times after the reaction were optimized, free samples were eluted, ALP-Nb-AFB1-MBs were adsorbed to the bottom of the reaction cell by magnetic enrichment, and PBST was used for eluting 1,2, and 3 times, respectively. The results showed that CL intensity and detection sensitivity were better when the number of washes was 1 (fig. 4B). In conclusion, the method ensures better sensitivity, and simultaneously greatly shortens the detection time, and only takes 30 minutes from sample addition to result acquisition. In addition, the operation process is simple and convenient.
2.4.4 stability and sensitivity
Nanobodies have unique physical and chemical advantages over traditional antibodies, such as high tolerance to temperature, methanol, salt concentration and pH. In order to make the detection system capable of coping with complex uncontrollable detection environments after fusion expression of Nb with ALP, it was verified whether the advantage of Nb itself was affected or not, and the tolerance of ALP to the above conditions was examined. We performed a series of reaction conditions and tolerability experiments. We found in the results of the thermal stability experiments that ALP-Nb still has high activity, and that the binding capacity of Nb to the target and ALP catalytic chemiluminescent activity of APS-5 are relatively stable when incubated at 75℃for 20min (FIG. 5A). Methanol is a common target extraction solvent in crop sample detection. To make our method more practical and better for sample detection, we established standard curves in three different concentrations of methanol solution, as shown in fig. 6A-C. The results show that ALP-Nb has good tolerance, high activity and detection sensitivity in PBS buffer containing 60% methanol. IC in PBS buffer containing 10% and 30% methanol 50 Closer (IC in PBS buffer with 10% methanol) 50 ＝0.39ng·mL -1 IC in PBS buffer containing 30% methanol 50 ＝0.46ng·mL -1 ) But the linear range of detection was greater in PBS buffer containing 30% methanol. Finally, the pH value and the salt concentration are optimized, HCl and NaOH are added into PBS buffer solution to adjust the pH value, and ALP-Nb is added into the PBS buffer solution in different modesThe pH reaction conditions show a certain tolerance to strong acids and strong bases. Whereas CL intensity is maximum at pH8.0 in PBS buffer (fig. 5B), which may be determined by the nature of ALP itself. Due to the nature of Nb, alkaline conditions do not affect the activity of the target for Nb binding. As above, naCl was added to the PBS buffer to prepare salt PBS buffers of different concentrations. Our method has excellent sensitivity in 200mM NaCl-PBS buffer (FIG. 5C). The salt tolerance of this assay is very valuable because of the high concentration of NaCl in many samples. In summary, the strong tolerance to temperature, acid-base and salt concentration enables our method to be better applied to sample detection, and also shows a strong application prospect.
2.5 AFB1 magnetic separation immunoassay based on ALP-Nb10E
A standard curve for the detection of AFB1 was established under optimized experimental conditions (30% methanol, pH8.0, 200mM NaCl-PBS buffer) (FIG. 7A). Optimized immunoassay with LOD of 0.743 pg.mL -1 (IC 90 )，IC 50 Is 0.33 ng.mL -1 The linear range is 7.23pg mL-1 to 12.38 ng.mL -1 (IC 20 -IC 80 ). Compared with the traditional ELISA, the MS-icCLIA greatly improves the detection sensitivity of the AFB1. ALP-Nb10E, IC based on icCLIA under the same reaction conditions 50 1.05 ng/ml -1 The linear range was 35.13 pg.mL -1 ～28.205ng·mL -1 The method comprises the steps of carrying out a first treatment on the surface of the Nb10E, IC based on ICELISA 50 1.36ng mL -1 The linear range was 179.64pg mL -1 ～13.33ng·mL -1 。
2.6 Cross-reactivity
To evaluate the selectivity of our immunoassays we used AFB2, AFG1, AFG2, AFM1, FB1, DON, ZEN and T2 (50 ng. ML -1 ) They are similar to each other to determine selectivity. Cross-reactivity was determined by the analog (50 ng mL -1 ) Is calculated by the inhibition ratio of (c). The above experiments show that the cross-reactivity with structural analogues of AFB1 is low (fig. 7B).
2.7 matrix Effect
Most samples contain complex matrix compounds, such as proteinsFat, sugar and pigment. These substances may lead to unavoidable and unexpected matrix effects of direct detection. Considering that the sample matrix may affect the immunoassay, we first examined the effect of the sample matrix on our method. Adding a PBS solution containing 60% methanol into the sample, shaking vigorously, and centrifuging to collect supernatant, namely a blank extracting solution. The blank extract was diluted 1, 4 and 10 times, respectively, and contained 0.5ng mL -1 AFB1. The CL strength and sensitivity of the different matrix solutions were compared with those of PBS solution containing 30% methanol. As a result, it was found that the CL intensities and detection sensitivities remained relatively stable when the sample matrix was diluted 1, 4, 10-fold (FIG. 8A, B, C, D). In order to simplify the detection procedure and shorten the detection time, the dilution factor is finally determined to be 1 time.
2.8 sample testing
Recovery test was performed by analysis of the addition of six levels of AFB1 (0, 0.08, 0.4, 0.8, 8, 80. Mu.g kg) -1 ) Is performed on corn, oat, milk and oil samples. Five parallel experiments were performed for each sample. As shown in Table 1, different amounts of AFB1 were added to the samples, the average recovery of corn was between 81.75% and 120.30%, and CV was between 2.99 and 7.26%. Different amounts of AFB1 were added to the samples, the average recovery of oat varied from 88.95% -103.96% and CV was between 2.24-11.91%. Different amounts of AFB1 were added to the samples, the average recovery of oil was 98.56% -122.25% and CV values were between 2.24-11.91%. For different numbers of AFB1 samples, the average recovery rate of milk is 96.24% -123.37%, and the CV value is between 1.48-9.66%. These results indicate that the established immunoassay has good accuracy and specificity and can detect AFB1 in different substrates (corn, oat, milk and oil samples).
Table 1 recovery analysis of AFB1 added to samples (corn, oat, edible oil, milk) using magnetic separation competitive immunoassay (n=5)
In summary, we have developed an ultrasensitive, specific and stable magnetic separation immunoassay method for AFB1 based on ALP-Nb10E, which achieves the one-step target, wherein the detection procedure is significantly simplified. The detection sample and the detection reagent are added into the reaction tank at the same time, the reaction time is 20min, and the detection can be completed within 30min after cleaning once. The chemiluminescent intensity is used as an output and thus the detection signal is amplified, showing better sensitivity than conventional ELISA. In addition, since Nb is easy to prepare, express on a large scale, and modify gene, our detection method has the advantage of low cost, easy mass production. The developed nano antibody shows high binding affinity to AFB1, and the sensitivity can meet the detection requirement of AFB1. Recombinant proteins used in immunoassays can be mass-produced by prokaryotic expression, and one can store them for a long period of time only by preserving their DNA sequence information. In particular, our method shows very high sensitivity to AFB1 with LOD of 0.734 pg.mL -1 ，IC 50 ＝0.33ng·mL -1 . More valuable, our detection method shows a strong ability to cope with complex test conditions. This also means that the magnetic separation immunoassay based on ALP-Nb10E shows good application prospect in the field of rapid detection of AFB1.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. A magnetic separation assay kit for detecting aflatoxin B1 comprising:
(1) Alkaline phosphatase nanometer antibody fusion protein, the amino acid sequence of the alkaline phosphatase nanometer antibody fusion protein is shown in SEQ ID NO:1 is shown in the specification;
(2) The magnetic bead modified by the antigen is formed by modifying the surface of an epoxy resin magnetic bead by adopting a complete antigen ABF 1-BSA.
2. The magnetic separation assay kit of claim 1, wherein the method of synthesizing the alkaline phosphatase nanobody fusion protein comprises the steps of: nanobody Nb gene and alkaline phosphatase gene were passed through a linker (G) 4 S) 3 And (3) connecting to form a recombinant gene, cloning the recombinant gene to an expression vector, and transferring the recombinant gene into escherichia coli for expression to obtain the alkaline phosphatase nano-antibody fusion protein.
3. The magnetic separation assay kit of claim 2, wherein the nucleotide sequence of the recombinant gene is as set forth in SEQ ID NO: 2.
4. The magnetic separation assay kit of claim 1, wherein the method of preparing antigen-modified magnetic beads comprises the steps of: and (3) reacting the epoxy resin magnetic beads with the aflatoxin B1-bovine serum albumin connector, vertically suspending for 10-15h at room temperature, and sealing active sites of the magnetic beads by using a magnetic bead sealing buffer solution to obtain antigen-modified magnetic beads.
5. The magnetic separation assay kit of claim 4 wherein the epoxy magnetic beads have a diameter of 0.3 μm to 2.6 μm;
the mass ratio of the epoxy resin magnetic beads to the aflatoxin B1-bovine serum albumin connector is (10-20): 1.
6. a method for detecting aflatoxin B1, comprising the steps of:
(1) And (3) establishing a detection system: mixing the alkaline phosphatase nanobody fusion protein, the antigen-modified magnetic beads and the aflatoxin B1 standard substance in PBS, incubating at 37 ℃, collecting alkaline phosphatase-nanobody-aflatoxin B1 complete antigen-magnetic bead complex through magnetic adsorption, adding alkaline phosphatase chemiluminescent substrate, and detecting the chemiluminescent intensity of the alkaline phosphatase nanobody fusion protein combined with the antigen-modified magnetic beads to obtain a standard curve;
(2) And (3) combining the standard curve obtained by adopting the same method in the step (1) to obtain the content of aflatoxin B1 in the sample to be detected.
7. The method of claim 6, wherein in step (1), the alkaline phosphatase nanobody fusion protein, the antigen-modified magnetic beads, and aflatoxin B1 standard are in a 3:4:3 ratio by volume; the dilution ratio of the alkaline phosphatase nanometer antibody fusion protein is (1:250) - (1:2000), the concentration of the antigen modified magnetic beads is 0.0625-0.5mg/mL, and the concentration of the aflatoxin B1 is 1 multiplied by 10 0 -1×10 5 pg/mL。
8. The method of claim 7, wherein in step (1), the antigen-modified magnetic beads have a particle size of 0.3 μm and the alkaline phosphatase nanobody dilution factor is 1:250, wherein the concentration of the complete antigen modified magnetic beads is 0.25mg/mL.
9. The method of claim 6, wherein in step (1), the incubation time is 20 minutes;
the method further comprises the step of washing the alkaline phosphatase-nanobody-aflatoxin B1 complete antigen-magnetic bead complex with a PBST buffer solution for 1 time before adding the luminescent substrate; the luminescent substrate is APS-5.
10. Use of the magnetic separation detection kit according to any one of claims 1-5 for detecting aflatoxin B1 in food.
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| CN117347621B (en) * | 2023-08-25 | 2024-03-12 | 广东省农业科学院农业生物基因研究中心 | Method for detecting aflatoxin B1 by using protein mimic antigen-nano antibody |
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