Nanometer antibody for detecting beta-conglycinin, ELISA (enzyme-linked immuno sorbent assay) kit and application of nanometer antibody

Abstract

The invention discloses a nano antibody for detecting beta-conglycinin, an ELISA kit and application of the nano antibody and the ELISA kit, and belongs to the technical field of beta-conglycinin detection. Wherein the nano antibody comprises a nano antibody 1 and a nano antibody 2 which can be respectively used as a coating antibody and a detection antibody (biotin label) in the ELISA kit; the ELISA kit can also comprise reagents such as enzyme-labeled streptavidin, a color developing solution and the like, and the quantitative analysis of the beta-conglycinin in a to-be-detected sample is realized by specifically utilizing a sandwich ELISA method and a color developing reaction. According to the scheme, the specificity is high, the detection limit is low, beta-conglycinin residues in products such as fermented soybeans, enzymolysis soybeans, soybean meal and feed can be accurately and sensitively detected, meanwhile, the defects that in traditional instrument detection, the sample pretreatment process is tedious, operation is complex and time-consuming are overcome, and the detection cost is greatly saved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of β-conglycinin detection, and particularly relates to a nanobody for detecting β-conglycinin, an enzyme-linked immunoassay (ELISA) kit and application thereof. BACKGROUND

[0002] β-conglycinin is a major storage protein in soybeans, belonging to the 7S globulin family. Although this protein has some nutritional value for humans and other mammals, it can also cause some negative physiological reactions, especially in young animals. In young animals such as weaned piglets, calves, and rats, β-conglycinin can cause allergic reactions, manifesting as diarrhea, decreased growth performance, and even death; this may be because the digestive system of young animals has not fully developed and cannot effectively process this protein, triggering an immune response. In addition, β-conglycinin has been shown to cause damage to pig small intestinal epithelial cells by activating the p38 / JNK MAPK signaling pathway, which can affect intestinal barrier function and nutrient absorption, thereby affecting the overall health and growth of animals. Therefore, it is of practical significance to establish a method for detecting β-conglycinin.

[0003] The conventional detection method for β-conglycinin is mainly instrumental analysis, including gas chromatography (GC), high-performance liquid chromatography (HPLC), gas / liquid chromatography-mass spectrometry (GC / LC-MS), etc. Although these analysis methods have high accuracy, they require expensive instruments, and the sample pre-treatment process is complex, including separation, extraction, purification, derivatization, etc. The analysis speed is slow, the detection sensitivity is low, and it is difficult to achieve on-site rapid detection. With the rapid increase in the amount of samples to be detected, especially the requirement for on-site rapid detection, traditional instrumental analysis methods are difficult to meet the requirements. Therefore, it is urgent to develop a high-efficiency and convenient β-conglycinin detection method.

[0004] Immunoassay is a method for detecting microorganisms, proteins, and other substances based on the specific reaction of antigens and antibodies. Compared with traditional instrumental detection, immunoassay has the advantages of simple operation, high sensitivity, strong specificity, and the ability to achieve on-site rapid detection, which can provide a high-efficiency and convenient approach for β-conglycinin sample detection. SUMMARY

[0005] Therefore, the present application aims to develop a β-conglycinin detection scheme based on immunoassay, providing a more efficient and convenient approach for detecting β-conglycinin samples.

[0006] To achieve the above-mentioned purpose, the present application specifically adopts the following technical solutions: The first aspect of the present application provides nanobodies for detecting β-conglycinin, specifically including the following two: a nanobody 1, having an amino acid sequence of: a). as shown in SEQ ID NO. 1; or, b). an amino acid sequence obtained after connecting a tag to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO. 1; or, c). an amino acid sequence having the same function obtained after substituting, deleting and / or adding one or more amino acid residues in the amino acid sequence of a) or b); a nanobody 2, having an amino acid sequence of: d). as shown in SEQ ID NO. 2; or, e). an amino acid sequence obtained after connecting a tag to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO. 2; or, f). an amino acid sequence having the same function obtained after substituting, deleting and / or adding one or more amino acid residues in the amino acid sequence of d) or e).

[0007] It can be understood that, in the above-mentioned nanobodies, the tag connected to the N-terminus and / or C-terminus does not change the function of the nanobody, and is only used to assist in the expression and purification, detection and tracking of the polypeptide, etc.

[0008] The second aspect of the present application provides biological materials related to the above-mentioned nanobodies, at least including the following: i). nucleic acid molecules encoding the nanobody 1 and the nanobody 2; in some embodiments of the present application, the nucleotide sequence of the nucleic acid molecule encoding the nanobody 1 is shown in SEQ ID NO. 3, and the nucleotide sequence of the nucleic acid molecule encoding the nanobody 2 is shown in SEQ ID NO. 4; ii). recombinant DNA, expression cassette, transposon, plasmid vector, bacteriophage vector, viral vector, engineered bacteria or transgenic cell line, etc. containing the nucleic acid molecule in i).

[0009] Based on the above-mentioned nanobodies, the third aspect of the present application provides an ELISA reagent or kit for detecting β-conglycinin.

[0010] In the ELISA kit of the present application, either of the nanobody 1 and the nanobody 2 serves as a coating antibody, and the other nanobody is labeled with biotin to serve as a detection antibody. It can be understood that, specifically including the following two cases: the nanobody 1 serves as a coating antibody coated on an enzyme-labeled plate, and the nanobody 2 is labeled with biotin to serve as a detection antibody; the nanobody 2 serves as a coating antibody coated on an enzyme-labeled plate, and the nanobody 1 is labeled with biotin to serve as a detection antibody.

[0011] Preferably, in the above-mentioned ELISA kit, at least the following are included: a box body and an enzyme-labeled plate arranged in the box body in a detachable manner, wherein each well of the enzyme-labeled plate is coated with a nanobody, and the coating concentration is 60-120 ng / mL; a detection antibody, i.e., another nanobody labeled with biotin; an enzyme-labeled streptavidin, wherein the enzyme is specifically a peroxidase, such as horseradish peroxidase; a color developing solution containing tetramethylbenzidine.

[0012] More preferably, in the above-mentioned ELISA kit, the coating concentration of the nanobody is 80-100 ng / mL.

[0013] More preferably, in the above-mentioned ELISA kit, the following reagents can also be included: buffer PBS, washing liquid PBST, β-conglycinin standard or standard solution, reaction termination liquid, etc.

[0014] More preferably, in the above-mentioned ELISA kit, the color developing solution includes A liquid and B liquid, wherein the A liquid includes urea peroxide, citric acid, Na2HPO4, Tween-20 and distilled water, and the B liquid includes tetramethylbenzidine, DMSO, citric acid and distilled water. In some embodiments of the present application, the A liquid is prepared from urea peroxide 1 g, citric acid 10.3 g, Na2HPO4·12H2O 35.8 g, Tween-20 100 μL and distilled water 1000 mL, and the pH value thereof is 5; the B liquid is prepared from tetramethylbenzidine 700 mg, DMSO 40 mL, citric acid 10.3 g and distilled water 1000 mL, and the pH value thereof is 7.4.

[0015] The fourth aspect of the present application provides an application of the above-mentioned nanobody, ELISA reagent or kit in detecting β-conglycinin residues, which can specifically include the following steps: S1. Adding a sample to be tested to each well of the enzyme-labeled plate coated with a nanobody, incubating and then washing; S2. Adding a detection antibody for incubation and then washing; S3. Adding an enzyme-labeled streptavidin for incubation and then washing; S4. Adding a color developing solution for color development in the dark for 10-15 min, then terminating the reaction, and determining the OD value of each well at 450 nm.

[0016] In the above detection process, after adding the sample to be detected into each well of the enzyme label plate coated with nanobodies, the solid-phase coated nanobodies and the β-conglycinin in the sample to be detected are combined, and then the detection antibody (i.e. biotinylated nanobody) added is also combined with the β-conglycinin. Since the content of the coated antibody in each well is consistent, when the concentration of the β-conglycinin in the sample to be detected is high, the combined detection antibody is more, and then the enzyme-labeled streptavidin combined with the fixed antibody is more, and the enzyme is combined with the color developing agent in the color developing liquid added last to perform color developing reaction. Specifically, the OD value detected by the enzyme label instrument is high, indicating that the content of the β-conglycinin in the sample is high; on the contrary, when the concentration of the β-conglycinin in the sample to be detected is low, the measured OD value is low, indicating that the content of the β-conglycinin in the sample is low. According to the standard curve drawn by detecting the standard solution of the β-conglycinin with a known concentration, the concentration of the β-conglycinin in the sample to be detected can be calculated.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application develops two kinds of nanobodies suitable for ELISA detection of β-conglycinin. Compared with conventional antibodies, the nanobodies have the characteristics of small molecular weight, easy expression and low production cost, and the two kinds of nanobodies have low cross reactivity, which is more conducive to precise detection of β-conglycinin based on immune analysis.

[0018] Based on the two kinds of nanobodies and the sandwich ELISA method, the present application further provides an ELISA kit suitable for detecting β-conglycinin, which can accurately and sensitively detect the residual β-conglycinin in fermented soybeans, enzymatically hydrolyzed soybeans, soybean meal and feed, etc. The kit has the advantages of simple sample pretreatment process, less time consumption, good specificity, high sensitivity (the minimum detection limit EC 20 is 5.23 ng / mL), can simultaneously detect a large number of samples, and the sample detection cost is much lower than that of the traditional instrument detection method, which has important significance for solving the on-site detection technology of a large number of samples. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The standard curve drawn for Example 4 of the present application. DETAILED DESCRIPTION

[0020] 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 application belongs. The terminology used in the description of the application and the claims herein is intended to be interpreted in accordance with the principles of etymology and the dictionary definitions available. The term "comprising" and any variation thereof used in the specification and claims herein is intended to cover the inclusion of a feature, item, component or step, but not the exclusion of any other feature, item, component or step.

[0021] In view of the shortcomings of the current β-conglycinin residue analysis method, such as high cost, complex pretreatment, poor specificity, low sensitivity and difficulty in realizing on-site detection, and since antibody selection is the core problem of immunoassay method, the application develops a pair of anti-β-conglycinin nanobodies capable of detecting β-conglycinin, and then provides an ELISA detection kit and a detection method suitable for β-conglycinin analysis based on the pair of nanobodies, so that the final detection scheme has the characteristics of high specificity, high sensitivity, high accuracy, high precision, simple operation method, and can be used for rapid detection of a large number of samples.

[0022] The application specifically adopts the following strategy to obtain two anti-β-conglycinin nanobodies: β-conglycinin is used as an immunogen to immunize a llama, total RNA of peripheral blood lymphocytes is extracted, a variable region gene fragment of heavy chain antibody is cloned through reverse transcription and overlap PCR, the gene fragment is cloned into a phagemid vector through enzyme digestion and ligation, high-efficiency electroporation is performed on Escherichia coli, and a phage nanobody library is constructed through helper phage rescue to screen specific β-conglycinin nanobodies.

[0023] Further, the two nanobodies are used as coating antibodies and detection antibodies respectively, and an ELISA detection kit is constructed by using the color development reaction of peroxidase and tetramethylbenzidine, which at least includes an enzyme-labeled plate on which the nanobodies are fixed, a detection antibody (biotinylated nanobody), peroxidase-labeled streptavidin and a color developing solution containing tetramethylbenzidine. When the kit is used for detection, the nanobodies coated on the wall of the enzyme-labeled plate capture β-conglycinin, and then the biotinylated nanobodies targeting β-conglycinin and peroxidase-labeled streptavidin are added to form a sandwich ELISA method, and the result is obtained through the color development reaction of peroxidase and tetramethylbenzidine.

[0024] The technical solutions of the application will be described clearly and completely in combination with specific examples. It should be understood that the examples described herein are only used to illustrate and explain the application, and are not used to limit the application. If not specifically indicated, the examples are in accordance with the conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or the conditions suggested by the manufacturer's instructions.

[0025] Example 1 This example provides nanobodies for detecting β-conglycinin, specifically: a nanobody 1 with an amino acid sequence as shown in SEQ ID NO. 1, and a nanobody 2 with an amino acid sequence as shown in SEQ ID NO. 2.

[0026] The present application constructs a phage nanobody library and obtains the above-mentioned nanobody through screening, and the specific operation is as follows: (1) Construction of a β-conglycinin phage display nanobody library.

[0027] β-conglycinin (1 mg) was dissolved in sterile normal saline, mixed with an equal volume of Freund's complete adjuvant (priming) or Freund's incomplete adjuvant (secondary and subsequent), and after being fully emulsified, multiple point injections were performed at the muscles of the alpaca. Then, the alpaca was immunized once every 14 days, and a total of 6 times. Seven days after the last immunization, the peripheral blood lymphocytes of the alpaca were extracted, total RNA was extracted, and VHH gene fragments were cloned through reverse transcription PCR and overlap PCR. The VHH gene fragments were connected to the phagemid pComb3x through restriction enzyme I, and then high-efficiency electroporation was performed into Escherichia coli ER2738 to construct a β-conglycinin phage nanobody library. It was determined that the capacity of the primary library reached 10 Sfi I The pComb3X plasmid vector and the scFv gene fragment were subjected to enzyme digestion, the VHH gene fragment was connected to the phagemid pComb3x through T4 ligase, and then high-efficiency electroporation was performed into Escherichia coli ER2738 to construct a β-conglycinin phage nanobody library. It was determined that the capacity of the primary library reached 10 8 cfu, and helper phage (multiplicity of infection 20:1) M13KO7 was added for rescue to obtain a phage nanobody library (phage display VHH library) with a library capacity of 10 13 pfu / mL, and the library had good diversity.

[0028] The reverse transcription kit used in the above-mentioned reverse transcription PCR is an M-MLV first-strand cDNA synthesis kit (purchased from OMEGA Company), the reverse transcription reaction system is shown in Table 1, and the reverse transcription is performed at 42°C for 30 min.

[0029] Table 1 Reverse transcription reaction system

[0030] The above-mentioned overlap PCR includes the following two steps: The first round of PCR has a reaction system as shown in Table 2, and the reaction program is as follows: 95°C pre-denaturation for 3 min; 94°C for 30 s, 57°C for 30 s, 72°C for 45 s, 25 cycles; 72°C for 10 min; and 4°C storage.

[0031] Table 2 First round of PCR reaction system

[0032] The second round of PCR has a reaction system as shown in Table 3, and the reaction program is as follows: 95°C pre-denaturation for 3 min; 94°C for 30 s, 57°C for 30 s, 72°C for 45 s, 30 cycles; 72°C extension for 10 min, and 4°C storage.

[0033] Table 3 Second round of reaction system

[0034] Specifically, the sequences of the PCR primers used in Table 2 and Table 3 are as follows (R represents base A / G, W represents base A / T, and K represents base G / T): GSP-RT: 5'-CGCCATCAATRTACCAGTTGA-3' (SEQ ID NO. 5); LP-leader: 5'-GTGGTCCTGGCTGCTCTW-3' (SEQ ID NO. 6); Primer F: 5'-CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC-3' (SEQ ID NO. 7); Primer R: 5'-CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO. 8).

[0035] (2) Screening of β-conglycinin-specific nanobodies.

[0036] Coat β-conglycinin in the first well of a 96-well enzyme-labeled plate at a concentration of 100 ng / mL overnight at 4°C; the next day, pour out the coating solution, wash three times with PBST, and add 150 μL of 1% gelatin to the first two wells and 150 μL of 1% BSA solution to the last four wells, and incubate at room temperature for 1 h.

[0037] Take 110 μL of the phage display VHH library of step (1), add 110 μL of 3% BSA solution, and shake (25°C, 220 rpm) for 1 h to allow the phages to bind to the BSA sufficiently, thereby removing non-specific phage antibodies; discard the liquid, and wash three times with PBST for 1 min each time.

[0038] In the first two wells of a 96-well enzyme-coated plate, 100 μL of the premixed library was added, and the reaction was shaken at room temperature for 2 h to allow the phage to bind to the coating agent; the liquid was discarded, and the wells were washed with PBST three times, each for 1 min; 100 μL of Gly-HCl (0.2 M, pH 2.2) was added, and the wells were shaken for 10 min; then, 20 μL of Tris-HCl (2 M, pH 8.0) was added to the wells, and the wells were shaken for 10 min to elute the phage antibodies with strong binding ability to the β-conglycinin standard; the phage eluate in the wells was collected, 10 μL of which was diluted and used to determine the titer, and the rest was used for amplification.

[0039] The phage eluate was added to fresh E. coli ER2738 bacterial solution, which was allowed to stand at 37°C for 15 min; carbenicillin and SB medium were added, and the solution was cultured at 37°C and 220 rpm for 2 h; helper phage M13KO7 (multiplicity of infection MOI = 20:1) and kanamycin were added, and the solution was cultured overnight; the next day, the supernatant was obtained by centrifugation, and PEG-NaCl solution was added to precipitate and purify the phage.

[0040] The amplification product was subjected to the next round of screening, the amount of coating agent was reduced by 2 times, the titer of each round was calculated, and a single clone was selected for amplification and ELISA identification. After 4 rounds of screening, a positive single clone was obtained.

[0041] Example 2 This example provides a preparation method of a nanobody 1 and a biotinylated nanobody 2, which comprises the following steps: (1) Preparation of nanobody 1.

[0042] A recombinant plasmid containing the sequence shown in SEQ ID NO. 3 was constructed, which was transformed into E. coli TOP10F' competent cells, and after recovery, the cells were plated on solid medium and cultured overnight. The next day, a single colony was selected and cultured in LB-carbenicillin medium to the logarithmic phase, IPTG (final concentration 1 mM) was added, and induction was performed overnight (37°C, 220 r / min). The next day, the bacterial pellet was collected by centrifugation at 8000 r / min for 5 min, the pellet was washed twice with PBS phosphate buffer, and then 3-5 mL of PBS was used to resuspend the pellet, the cells were lysed with an ultrasonic disrupter in an ice bath environment, and the lysis was stopped when the bacterial solution was clear and transparent. The pellet and supernatant were separated by centrifugation at 8000 r / min for 5 min. The supernatant was placed in a dialysis bag and dialyzed with PBS, and the liquid was changed every 6 h for a total of 5-6 times to obtain high-purity anti-β-conglycinin nanobody. Amino acid sequencing analysis showed that the amino acid sequence of the obtained β-conglycinin nanobody was as shown in SEQ ID NO. 1.

[0043] (2) Preparation of biotinylated nanobody 2.

[0044] Sac I and Hind III restriction sites were introduced into SEQ ID NO. 4 by PCR amplification, and a pAC6-VHH recombinant plasmid was constructed, which was transformed into E. coli CVB101 competent cells and plated on 2YT-Amp plates for overnight culture. The next day, a single colony was picked and transferred to 1 L of 2YT (Amp, 100 μg / ml) liquid medium, and the bacteria were cultured at 220 r / min and 37°C until the logarithmic phase. Then, D-biotin at a final concentration of about 50 μmol and inducer IPTG at a final concentration of about 1 mmol / L were added for overnight induction of expression. The next day, the bacterial pellet was collected, sonicated, and the VHH-biotin recombinant protein was purified using a nickel column. Amino acid sequencing analysis showed that the amino acid sequence of the obtained β-conglycinin nanobody was as shown in SEQ ID NO. 2.

[0045] Example 3 Based on the nanobody 1 and biotinylated nanobody 2 prepared in Example 2, this example provides an ELISA kit, which comprises: ① a box body; ② a 96-well enzyme-labeled plate arranged in the box body and detachable, wherein the enzyme-labeled plate is coated with nanobody 1; ③ a detection antibody, i.e., biotinylated nanobody 2; ④ a β-conglycinin standard; ⑤ horseradish peroxidase-labeled streptavidin at a concentration of 0.1 μg / mL; ⑥ a color developing solution composed of A liquid and B liquid, wherein the A liquid (pH 5) is prepared from urea peroxide 1 g, citric acid 10.3 g, Na2HPO4·12H2O 35.8 g, Tween-20 100 μL, and distilled water 1000 mL, and the B liquid (pH 7.4) is prepared from tetramethyl benzidine 700 mg, DMSO 40 mL, citric acid 10.3 g, and distilled water 1000 mL; ⑦ a reaction termination solution, i.e., 2 M sulfuric acid solution; ⑧ buffer PBS; ⑨ washing liquid PBST.

[0046] Example 4 Based on the ELISA kit in Example 3, this example provides a method for detecting β-conglycinin, which comprises the following steps: Coat the nanobody 1 on a 96-well enzyme-labeled plate, with a concentration of 100 ng / mL per well, and react overnight at 4°C; the next day, shake off the liquid in the wells, wash 3 times with PBST containing 0.05% Tween, and pat dry the enzyme-labeled plate by inverting it on absorbent paper; add blocking solution, incubate at 37°C for 30 minutes, shake off the liquid in the wells, wash 3 times with 0.05% PBST, and pat dry the enzyme-labeled plate by inverting it on absorbent paper.

[0047] Add 100 μL of β-conglycinin standard solution (solutions with concentrations of 0 ng / mL, 1 ng / mL, 4 ng / mL, 12 ng / mL, 37 ng / mL, 111 ng / mL, 333 ng / mL, and 1000 ng / mL, respectively, prepared from a β-conglycinin standard) or a sample to be tested to each well (2-4 replicates), and incubate at 37°C for 30 minutes; shake off the liquid in the wells, wash 3 times with PBST, and pat dry the enzyme-labeled plate by inverting it on absorbent paper.

[0048] Add 100 μL of biotin-labeled nanobody 2 (1:2000, v / v), and incubate at 37°C for 30 minutes; shake off the liquid in the wells, wash 3 times with PBST, and pat dry the enzyme-labeled plate by inverting it on absorbent paper.

[0049] Add horseradish peroxidase-labeled streptavidin, and incubate at 37°C for 30 minutes; shake off the liquid in the wells, wash the plate 3 times with PBST, and pat dry; mix equal volumes of A and B liquids, add 100 μL per well, develop color in the dark for 15 minutes, add a stop solution to terminate the reaction, and measure the OD value of each well at a wavelength of 450 nm on an enzyme-labeled instrument.

[0050] Subtract the value of the blank well from the OD value of each standard well, and take the concentration of the corresponding standard as the abscissa to draw a β-conglycinin sample standard inhibition curve. Figure 1 According to the regression equation of the curve, the concentration of the sample to be tested can be calculated, and the concentration EC 50 of the β-conglycinin sample can also be calculated. According to the curve equation (y=y0+a / [1+(x / x0) b ]), the EC 50 is calculated to be 38.26 ng / mL, and the linear range (EC 20 -EC 80 ) is 5.23-417.15 ng / mL. In the curve equation, y0=0.038, a=2.3418, b=-0.8327, and x0=35.2588.

[0051] Example 5 The cross-reactivity is selected to evaluate the specificity of the detection scheme of the present application for β-conglycinin samples, and the specific operation is as follows: Coat the nanobody 1 on the 96-well enzyme-labeled plate, each well is coated at a concentration of 100 ng / mL, and the reaction is carried out overnight at 4°C; the next day, the liquid in the well is shaken off, and the plate is washed with PBST containing 0.05% Tween for 3 times, and then the plate is inverted on the absorbent paper and dried; the blocking solution is added, and the plate is incubated at 37°C for 30 minutes, the liquid in the well is shaken off, and the plate is washed with 0.05% PBST for 3 times, and then the plate is inverted on the absorbent paper and dried.

[0052] Prepare 0 ng / mL, 1 ng / mL, 4 ng / mL, 12 ng / mL, 37 ng / mL, 111 ng / mL, 333 ng / mL and 1000 ng / mL of the β-conglycinin sample, glycinin, Gly m Bd 28K and trypsin inhibitor standard solution respectively. Add 100 μL of the standard sample to each well (2-4 repeats are carried out); incubate at 37°C for 30 minutes; shake off the liquid in the well, wash with PBST for 3 times, and then invert the plate on the absorbent paper and dry.

[0053] Add 100 μL of biotin-labeled nanobody 2 (1:2000, v / v), and incubate at 37°C for 30 minutes; shake off the liquid in the well, wash with PBST for 3 times, and then invert the plate on the absorbent paper and dry; add the enzyme-labeled streptavidin, and incubate at 37°C for 30 minutes; shake off the liquid in the well, wash the plate with PBST for 3 times, and then dry.

[0054] Mix equal volumes of A and B liquids, add 100 μL to each well, and develop color in the dark for 15 minutes; add the termination solution to terminate the reaction, and then measure the OD value of each well at a wavelength of 450 nm on the enzyme-labeled instrument.

[0055] Calculate the EC value of each kind of analog to be tested, and calculate the cross-reactivity by using the formula (cross-reactivity = [EC (β-conglycinin sample) / EC (analog)] × 100%). 50 50 50

[0056] The experimental results are shown in Table 4, and the cross-reactivity of the β-conglycinin sample analog analyzed by the detection scheme of the present application is less than 0.5%, which indicates that the ELISA method has good specificity for the glycinin sample.

[0057] Table 4: Specificity detection results

[0058] ​​​In conclusion, the two kinds of nanobodies provided by the application can be used for detection of β-conglycinin, and the ELISA kit and method developed based on the nanobodies have the advantages of good specificity, high sensitivity, less time consumption, etc., and are suitable for on-site detection of soybean protein residue in large batches of samples.

[0059] It should be noted that the above examples are only part of the embodiments of the present application, not all the embodiments, and are only used to illustrate the technical solutions of the present application, not to limit; based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor belong to the scope of protection of the present application.

Claims

1. Nanobodies for the detection of β-conglycinin, characterized in that, The following two are included: Nanobody 1, the amino acid sequence of which is any of the following: a). as shown in SEQ ID NO. 1; b). an amino acid sequence obtained after connecting a tag to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO. 1; c). an amino acid sequence with the same function obtained after substitution, deletion and / or addition of one or more amino acid residues in the amino acid sequence in a) or b); Nanobody 2, the amino acid sequence of which is any of the following: d). as shown in SEQ ID NO. 2; e). an amino acid sequence obtained after connecting a tag to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO. 2; f). an amino acid sequence with the same function obtained after substitution, deletion and / or addition of one or more amino acid residues in the amino acid sequence in d) or e).

2. A nucleic acid molecule encoding the nanobody of claim 1.

3. The nucleic acid molecule of claim 2, wherein The nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO. 3 and the nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO.

4.

4. An expression cassette, transposon or recombinant vector comprising the nucleic acid molecule of claim 2 or 3.

5. A recombinant cell comprising the recombinant vector of claim 4.

6. Use of the nanobody of claim 1 in the preparation of a β-conglycinin detection reagent or kit.

7. An ELISA kit for detecting β-conglycinin, characterized by, Either of the nanobody 1 and nanobody 2 of claim 1 is used as a coating antibody, and the other nanobody is used as a detection antibody.

8. The ELISA kit according to claim 7, characterized in that, The detection antibody is labeled with biotin, and the ELISA kit further comprises peroxidase-labeled streptavidin and a color developing solution, wherein tetramethyl benzidine is used as a color developing substance in the color developing solution.

9. The ELISA kit according to claim 8, characterized in that, The color developing solution comprises A solution and B solution, wherein the A solution comprises urea peroxide, citric acid, Na2HPO4, Tween-20 and distilled water, and the B solution comprises tetramethyl benzidine, DMSO, citric acid and distilled water.

10. Use of the nanobody of claim 1 or the ELISA kit of any one of claims 7-9 in the detection of β-conglycinin.

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