Anti-sulfadiazine nano antibody and application thereof

Abstract

The invention discloses an anti-sulfadiazine nano antibody and application thereof, and belongs to the technical field of nano antibodies. The invention aims to provide a nano antibody for detecting sulfadiazine, which has the advantages of small molecular weight, strong penetrating power, good stability and strong antigen binding capacity, and can recognize antigen epitopes which are difficult to recognize by conventional antibodies. The invention provides a nano antibody for detecting sulfadiazine. An amino acid sequence of a CDR1 of a variable region of the nano antibody is shown as SEQ ID NO.6, an amino acid sequence of a CDR2 of the variable region of the nano antibody is shown as SEQ ID NO.7, and an amino acid sequence of a CDR3 of the variable region of the nano antibody is shown as SEQ ID NO.8. The indirect competitive ELISA method established by the invention is a simple, rapid, sensitive and low-cost immunodetection method, and has a great application prospect in detection of sulfadiazine residues in animal-derived food.

Description

Technical Field

[0001] The present invention belongs to the field of nano antibodies, and in particular relates to an anti-sulfadiazine nano antibody and an application thereof. Background Art

[0002] Sulfadiazine (SD) is a common anti-systemic infection drug with a molecular formula of C 10 H 10 N4O2S, molecular weight 250.28Da, is white or off-white crystal or powder, gradually darkens in light, slightly soluble in ethanol or acetone, and almost insoluble in water. Its basic structure is similar to para-aminobenzoic acid (PABA), and it can compete with PABA for the dihydrofolate synthase of bacteria, affecting the folic acid metabolism and nucleotide synthesis of bacteria, thereby inhibiting the growth and reproduction of bacteria. SD has the characteristics of low toxicity, high potency, broad antibacterial spectrum, and easy absorption. It is widely used in the treatment of bacterial infectious diseases in aquaculture, medical treatment and animal husbandry. SD has a half-life of more than 10h and belongs to the medium-acting sulfadiazine class of drugs. Due to the irregular use and even abuse of SD, its residues in various animal-derived foods occur from time to time, which affects food safety and seriously threatens human health. Therefore, it is very important to strengthen the detection of SD residues.

[0003] At present, the analytical methods commonly used to detect sulfonamide drug residues mainly include high performance liquid chromatography, liquid chromatography-mass spectrometry and immunoassay. Among them, chromatography has high sensitivity and good accuracy, but this method has strict requirements on sample pretreatment, and it needs to go through standardized pretreatment steps before testing. Moreover, the professional instruments used are mostly large-scale equipment, which are expensive and require professional personnel to be trained and operated. The detection operation requirements are strict, and the number of equipment injections is limited, which cannot meet the actual needs of large-scale animal-derived food drug residue monitoring. Among immunoassays, enzyme-linked immunosorbent assay (ELISA) is the most common, which is simple and low-cost, and is more suitable for large-scale grassroots drug residue investigations. Therefore, it is more practical to establish an economical and efficient method for detecting sulfonamide drug residues. In terms of immunoassay analysis research, indirect competitive ELISA has been widely used in veterinary drug residue detection due to its high efficiency and convenience, but most of them use traditional polyclonal antibodies or monoclonal antibodies as detection antibodies, which have poor stability, weak antigen binding ability, and difficulty in identifying antigen epitopes. Summary of the invention

[0004] The purpose of the present invention is to provide a nano antibody for detecting sulfadiazine which has small molecular weight, strong penetration, good stability, strong antigen binding ability and can recognize antigen epitopes that are difficult to be recognized by conventional antibodies.

[0005] The present invention provides a nanobody for detecting sulfadiazine, wherein the amino acid sequence of CDR1 in the variable region of the nanobody is shown as SEQ ID NO.6, the amino acid sequence of CDR2 in the variable region of the nanobody is shown as SEQ ID NO.7, and the amino acid sequence of CDR3 in the variable region of the nanobody is shown as SEQ ID NO.8.

[0006] It is further defined that the Nanobody also includes four framework regions, the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.5.

[0007] The present invention provides a nucleotide encoding the above-mentioned nanobody.

[0008] The present invention provides a nano antibody for detecting sulfadiazine, and the amino acid sequence of the nano antibody is shown as SEQ ID NO.1.

[0009] The present invention provides a recombinant vector, which contains the above-mentioned nucleotides.

[0010] The present invention provides a recombinant microbial cell, which contains the above-mentioned nucleotide.

[0011] The present invention provides a kit for detecting sulfadiazine, wherein the kit contains the nano antibody.

[0012] The present invention provides a use of the nano antibody, the nucleotide sequence, the recombinant vector or the recombinant microbial cell in preparing a kit for detecting sulfadiazine.

[0013] The present invention is further defined as detecting sulfadiazine residues in the food field, the environmental field or the medical field.

[0014] The present invention provides a method for detecting sulfadiazine residues, which utilizes the nano antibody to perform indirect competitive ELISA.

[0015] Beneficial effects: The present invention uses the synthetic complete antigen SD-BSA to screen out the amino acid sequence of the anti-SD specific nanoantibody Nb1-B3 from the alpaca natural nanolibrary, recombines with the pET28a prokaryotic expression vector, and obtains the nanoantibody Nb1-B3 with higher purity after induction purification.

[0016] The IC50 value of the indirect competitive ELISA method established based on Nb1-B3 was 26.08 ng / mL, the linear detection range was between 3.57 and 169.88 ng / mL, the detection limit was 2.85 ng / mL, and the quantification limit was 3.46 ng / mL. This method has high specificity, accuracy and precision.

[0017] The indirect competitive ELISA method established by the present invention is a simple, rapid, sensitive and low-cost immunoassay method, and has great application prospects in the detection of sulfadiazine residues in animal-derived foods.

[0018] The present invention conducts a thermal stability analysis on the prepared nano antibody Nb1-B3, and it still has 90% activity after being heated at 95°C for 5 minutes, and still has 80% binding activity after being heated at 80°C for 1 hour. This shows that the nano antibody has high thermal stability.

[0019] The present invention conducts an organic solvent tolerance analysis on the prepared nano antibody Nb1-B3, and Nb1-B3 still has about 40% activity under the action of 40% methanol and 60% acetonitrile, and retains about 30% activity in 40% acetone and 60% DMSO. It can be seen that the nano antibody has good organic solvent tolerance.

[0020] Compared with monoclonal antibodies (150-200kDa), nanobodies (about 15kDa) have a smaller molecular weight, thus showing higher penetration. Due to their small size, nanobodies can bind to antigenic epitopes that are difficult for traditional antibodies to recognize, showing higher affinity activity. At the same time, nanobodies can still maintain binding ability with target antigens under high temperatures, organic solvents and different pH environments. The industrial large-scale production process of monoclonal antibodies is complex and costly. In contrast, nanobodies can be efficiently expressed in prokaryotic expression systems, with higher yields and lower production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the molecular structural formula of sulfadiazine;

[0022] Figure 2 Provide a synthetic route for SD-BSA and SD-OVA;

[0023] Figure 3 UV scanning images of SD-BSA and SD-OVA; A is SD-BSA protein, B is SD-OVA protein;

[0024] Figure 4This is the gel electrophoresis identification diagram of SD-BSA and SD-OVA; M is protein marker, 1 is BSA, 2 is SD-BSA, 3 is OVA, and 4 is SD-OVA;

[0025] Figure 5 It is the result of Phage ELISA identification;

[0026] Figure 6 is the result of PCR identification of positive clones; M is DNA molecular marker DL2000, 1 is ER2738 negative control, and 2 is 1-B3;

[0027] Figure 7 It is the double enzyme digestion verification of Nb-pET28a; M is the DNA molecular marker DL5000, 1 is pET28a, 2 is Nb1-B3-pET28a, and 3 is Nb1-B3-pET28a after enzyme digestion;

[0028] Figure 8 Western-blotting and SDS-PAGE analysis of nanoantibody Nb1-B3; A is Western-blotting analysis, B is SDS-PAGE analysis;

[0029] Fig. 9 For the analysis of the antigen binding activity of the nanobody Nb1-B3;

[0030] Fig.10 This is the thermal stability analysis of the nanobody Nb1-B3; A is Nb1-B3 incubated at 20-95°C for 5 min, and B is Nb1-B3 incubated at 80°C for 10-60 min;

[0031] Fig.11 This is the organic reagent tolerance analysis of nanobody Nb1-B3; A is methanol, B is acetonitrile, C is acetone, and D is dimethyl sulfoxide;

[0032] Fig.12 This is the standard curve for detecting sulfadiazine by indirect competitive ELISA based on Nb1-B3. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0034] Example 1. Synthesis and identification of sulfadiazine complete antigen

[0035] The chemical formula of sulfadiazine described in the following examples of the present invention is as follows Figure 1 shown.

[0036] 1. Synthesis of SD-BSA and SD-OVA artificial antigens

[0037] The present invention studies the synthesis of complete antigens SD-BSA and SD-OVA by diazotization. The synthesis route is shown in Figure 2 The specific operation method is as follows:

[0038] Weigh 10 mg SD and dissolve it in 5 mL 0.2 mol / L HCl. After cooling in an ice bath, add 1 mol / L NaNO2 dropwise until the starch potassium iodide test paper turns blue-black. After stirring at 4 ° C for 6 hours, slowly add 2 mL of 10 mg / mL bovine serum albumin (BSA) solution dropwise, adjust the pH to 8.5, and continue to react at 4 ° C overnight. The reaction product was dialyzed against PBS at 4 ° C for 3 days, and the solution was changed 3 times a day to remove small molecules that did not participate in the reaction. Finally, the complete antigen SD-BSA was obtained. The coating antigen SD-OVA was prepared in the same way and stored in small aliquots at -20 ° C.

[0039] 2. UV scanning identification

[0040] In order to identify whether the small molecule drug is successfully coupled to the protein, the SD, BSA, OVA, SD-BSA, and SD-OVA protein solutions were scanned at the full UV wavelength using a UV spectrophotometer to measure their UV absorption spectra and analyze the changes in characteristic absorption peaks. Figure 3 As shown, the UV scanning identification spectrum shows that SD has an absorption peak at 256nm, BSA and OVA have protein-specific absorption peaks at 278nm and 280nm, respectively, while the absorption peaks of SD-BSA and SD-OVA are shifted, which indicates that the UV characteristic absorption peaks of BSA and OVA change after binding with the drug. It can be inferred that after the carrier protein binds to the drug, the optical properties of the produced substance change, indicating that the coupling effect is good.

[0041] 3. SDS-PAGE identification

[0042] SDS-PAGE was used to analyze and identify BSA, OVA, SD-BSA and SD-OVA before and after coupling. Figure 4 It can be seen that the band sizes of BSA and OVA are around 66kDa and 45kDa, which are consistent with the theoretical values. In comparison, the electrophoresis bands of artificial synthetic antigens SD-BSA and SD-OVA are slightly higher and the molecular size is slightly increased, indicating that SD is successfully coupled with BSA and OVA.

[0043] Example 2. Screening and identification of sulfadiazine-specific nanobodies

[0044] 1. Screening of sulfadiazine nanoantibodies

[0045] Take 300 μL of natural nanoantibody library and add it to 100 mL of 2×YT (ATG) liquid culture medium, and culture at 37°C until OD 600 The ratio was 0.6. Helper phage M13KO7 was added at an MOI of 20:1, and the cells were gently shaken in a water bath at 37°C for 30 minutes, followed by shaking culture for 30 minutes. The bacterial solution was centrifuged at 3300×g at 4°C for 10 minutes, the bacterial pellet was resuspended in 100mL2×YT (ATK), and cultured at 30°C overnight. Two immunotubes were coated with 100μg / mL SD-BSA protein and BSA protein at 4°C overnight, respectively, with a coating volume of 2mL. The next day, the bacterial solution was centrifuged at 10000×g at 4°C for 10 minutes, the supernatant was aspirated and added with 1 / 4 volume of PEG / NaCl in an ice bath, ice bathed for 50 minutes, and centrifuged again at 10000×g at 4°C for 20 minutes, the supernatant was removed, 2.4mL PBS was added for resuspending, and 12000×g was centrifuged at 4°C for 10 minutes. The supernatant was the phage recombinant antibody library. Take 100 μL of phage supernatant and dilute it in gradient ratio, add it to the ER2738 host bacteria, shake it gently in a water bath at 37℃ for 30min, coat it on SOB (GTK) plates respectively, and calculate the input volume of the recombinant antibody library after overnight culture at 30℃. Pipette the recombinant antibody library and add it to the immunotube coated with BSA overnight in advance, and eliminate the reaction at room temperature for 30min. Then take 1.5mL of the liquid in the immunotube to the 5mL EP tube, add 1mL 3% MPBS to interfere for 20min. Pipette 2mL of the treated recombinant antibody library and add it to the SD-BSA coated immunotube, place it at 37℃ and shake it gently for 30min, and then let it stand for 90min. Discard the supernatant, wash it 3 times with PBST, and then wash it 3 times with PBS. After patting dry, add 2mL of 0.1mol / L triethylamine solution, shake it gently at 37℃ for 8min to elute, and immediately add an equal amount of Tris-HCl to mix and neutralize, which is the eluted phage. The eluted phage antibody library was added to the ER2738 host bacteria in the logarithmic growth phase, and gently shaken in a 37°C water bath for 30 minutes. 100 μL of gradient dilution was applied to SOB (ATG) plates to calculate the output of one round. The remaining bacterial solution was centrifuged at 3300×g for 10 minutes to resuspend the bacterial pellet, and the gradient dilution was applied to SOB (ATG) square culture plates. The colonies were scraped and collected the next day to obtain the primary recombinant antibody library.

[0046] Four rounds of panning were performed using the same method, with SD-BSA protein coating concentrations of 100 μg / mL, 75 μg / mL, 50 μg / mL, and 10 μg / mL, respectively. The number of PBST and PBS washes was increased by 3 times in each round, and a four-level recombinant antibody library was obtained.

[0047] Table 1 Four rounds of panning enrichment results

[0048] First round Round 2 Round 3 Round 4 enter <![CDATA[1.2×10 11 ]]> <![CDATA[1.3×10 11 ]]> <![CDATA[9.8×10 9 ]]> <![CDATA[1.2×10 10 ]]> Output <![CDATA[8.0×10 3 ]]> <![CDATA[1.4×10 4 ]]> <![CDATA[5.6×10 5 ]]> <![CDATA[3.8×10 5 ]]> Enrichment rate <![CDATA[6.7×10 -8 ]]> <![CDATA[1.1×10 -7 ]]> <![CDATA[5.7×10 -5 ]]> <![CDATA[3.1×10 -5 ]]>

[0049] Note: Enrichment rate = phage output (pfu) / phage input (pfu)

[0050] 2. Phage ELISA identification

[0051] 48 monoclonal clones obtained from the fourth round of enrichment were used to prepare phage supernatants, and the binding activity of recombinant phages to SD antigen was detected by ELISA. The clones with a ratio of OD values ​​of the experimental group coated with SD-OVA to the negative control group coated with OVA greater than 2.0 were considered positive clones. Figure 5 shown.

[0052] 3. PCR identification and sequencing analysis

[0053] The positive clone strain 1-B3 was selected to amplify the VHH gene, and the results of colony PCR identification were as follows: Figure 6 As shown, compared with the negative control, a band of about 450bp was amplified, indicating that the VHH gene of the selected clone strain was successfully inserted. The positive clone strain 1-B3 was sequenced and analyzed, and the amino acid sequence was shown in SEQ ID No.1, which had complete 4 FR framework regions and 3 CDR variable regions. The amino acid sequence of FR1 is shown in SEQ ID No.2, the amino acid sequence of FR2 is shown in SEQ ID No.3, the amino acid sequence of FR3 is shown in SEQ ID No.4, and the amino acid sequence of FR4 is shown in SEQ ID No.5; the amino acid sequence of CDR1 is shown in SEQ ID No.6, the amino acid sequence of CDR2 is shown in SEQ ID No.7, and the amino acid sequence of CDR3 is shown in SEQ ID No.8.

[0054] Example 3. Preparation of anti-sulfadiazine nanobody

[0055] 1. Construction of prokaryotic expression recombinant plasmid

[0056] The phagemid was extracted from the positive single clone for sequencing. After the sequencing was correct, it was used to construct a prokaryotic expression recombinant plasmid according to the sequence of the expression strain. BamH I and Xho I restriction sites were added to the 5' and 3' ends respectively, and the recombinant expression plasmid was synthesized by Heilongjiang Jiansu Gene Technology Co., Ltd. pET28a. The recombinant plasmid (in DH5α) was coated on an LB solid plate and inverted and cultured at 37°C overnight. The next day, a single clone was picked, and the extracted plasmid was identified by double enzyme digestion and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. Correct sequencing indicated that the prokaryotic expression vector was successfully constructed. The extracted plasmid was transformed into BL21 (DE3) competent medium for prokaryotic expression. The results after double enzyme digestion with BamH I and Xho I are shown in Figure 7Compared with the empty plasmid and the constructed prokaryotic expression vector, an inserted target fragment of about 450 bp appeared, proving that the prokaryotic expression recombinant plasmid was successfully constructed.

[0057] 2. Soluble expression and purification of nanobodies

[0058] After the glycerol strains were revived at 37°C, they were transferred to a large bottle of 500 mL LB-K liquid medium at a ratio of 1:100 and continued to be shaken and cultured until OD 600 When the bacterial solution reaches the logarithmic phase value, the bacterial solution is cooled in an ice bath and allowed to stand, and then IPTG inducer with a final concentration of 0.8mM is added, and low-temperature induction is carried out at 18℃ for 16h. The bacterial precipitate is collected the next day, weighed, and 30mL of bacterial active protein extraction reagent is added per gram of wet bacteria. At the same time, 2mg / mL of lysozyme and 100mM PMSF protease inhibitor are added to ensure the protein extraction efficiency. The protein is purified by nickel column affinity chromatography, and then the eluate is taken for Western blotting and SDS-PAGE verification. The results are as follows Figure 8 As shown, a single protein band appeared at about 15 kDa, indicating that the purified protein had a high purity.

[0059] The amino acid sequence of the obtained nanobody with specific anti-SD is shown in SEQ ID NO.1.

[0060] The antibody sequence includes four framework regions (FR1, FR2, FR3, FR4) and three complementary determining regions (CDR1, CDR2, CDR3). The arrangement order of the four framework regions and the three complementary determining regions is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0061] The amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.5; the amino acid sequence of CDR1 is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.8.

[0062] SEQ ID NO.1:

[0063] QVQLQESGGGLVQPGGSLTLSCVVSGSIFSSDAVVWYRQAPGSERELVAAMRSGGHT YYPDSVKDRFTISRDYDKNTIYLQMTSLKPDDTAMYYCAASRPGLKYYSDGYYSQSDFNL WGQGTQVTVSS;

[0064] SD nanobody gene sequence, SEQ ID NO.9:

[0065] caggtgcagctgcaggagtctgggggaggcttggtgcaacctggggggtctctgacactctcctgtgtagtctctggaagcatcttcagtagcgatgccgtggtctggtatcgtcaggctccagggagcgagcgcgagttggtcgcggctatgcgaagcggaggccacacatactaccctgattctgtgaaggaccgattcaccatctccagagactatgacaagaatacaatttatttgcaaatgaccagcctgaaacctgacgacacggccatgtattactgtgcggcatcccggcccggactgaaatactatagtgatggttactactcccaatctgactttaatttatggggccaggggacccaggtcaccgtctcctcac;

[0066] SEQ ID NO.2: QVQLQESGGGLVQPGGSLTLSCVVS;

[0067] FR1 gene sequence, SEQ ID NO.10:

[0068] caggtgcagctgcaggagtctgggggaggcttggtgcaacctggggggtctctgacactctcctgtgtagtctct;

[0069] SEQ ID NO.3: VVWYRQAPGSERELVAA;

[0070] FR2 gene sequence, SEQ ID NO.11:

[0071] gtggtctggtatcgtcaggctccagggagcgagcgcgagttggtcgcggct;

[0072] SEQ ID NO.4: YYPDSVKDRFTISRDYDKNTIYLQMTSLKPDDTAMYYC;

[0073] FR3 gene sequence, SEQ ID NO.12:

[0074] tactaccctgattctgtgaaggaccgattcaccatctccagagactatgacaagaatacaatttatttgcaaatgaccagcctgaaacctg acgacacggccatgtattactgt;

[0075] SEQ ID NO.5:WGQGTQVTVSS;

[0076] FR4 gene sequence, SEQ ID NO.13:

[0077] tggggccaggggacccaggtcaccgtctcctcac;

[0078] SEQ ID NO.6: GSIFSSDA;

[0079] CDR1 gene sequence, SEQ ID NO.14:

[0080] ggaagcatcttcagtagcgatgcc;

[0081] SEQ ID NO.7: MRSGGHT;

[0082] CDR2 gene sequence, SEQ ID NO.15:

[0083] atgcgaagcggaggccacaca;

[0084] SEQ ID NO.8:AASRPGLKYYSDGYYSQSDFNL.

[0085] CDR3 gene sequence, SEQ ID NO.16:

[0086] gcggcatcccggcccggactgaaatactatagtgatggttatactcccaatctgactttaattta.

[0087] Example 4. Biological properties of anti-sulfadiazine nanobody Nb1-B3

[0088] 1. Antigen binding ability analysis

[0089] Nb1-B3 was diluted to different gradients and added to the ELISA plate containing 5 μg / mL of SD-OVA. The OD values ​​of different concentrations of nanoantibodies binding to the coating were evaluated by ELISA. 450 The binding activity with target antigen is shown in Fig. 9 , indicating that it has good ability to bind to antigen.

[0090] 2. Thermal stability analysis

[0091] The nanobody was diluted to a working concentration (5 μg / mL) and heated in metal baths at 20°C, 30°C, 45°C, 60°C, 80°C and 95°C for 5 minutes. Another portion was placed at 80°C for 10 to 60 minutes, and samples were taken every 10 minutes. After the antibody returned to room temperature, the ELISA method was used to evaluate the binding efficiency of the antibody and the antigen. The binding efficiency of the untreated nanobody and the antigen was 100%, and the temperature tolerance of the nanobody was evaluated. The results are shown in Fig.10 In A, the activity of the nanoantibody remains stable as the heating temperature increases. After heating at 95°C for 5 minutes, it still has 90% activity. The binding activity of Nb1-B3 after incubation at 80°C for different time periods is shown in Figure 1. Fig.10 As shown in B, the binding activity is still 80% after heating at 80°C for 1 hour. It can be seen that the nanobody has a high thermal stability.

[0092] 3. Analysis of organic solvent tolerance

[0093] The nanobody was diluted with organic solvents (methanol, acetonitrile, acetone and dimethyl sulfoxide) of different concentrations (10%, 20%, 40% and 60%) to adjust the nanobody to an appropriate working concentration. The binding efficiency of the nanobody diluted with PBS to the antigen was 100%. The tolerance of the nanobody in different organic solvents and different concentrations of the same organic solvent was evaluated. The results are shown in Fig.11 Nb1-B3 still has about 40% activity in 40% methanol and 60% acetonitrile, and about 30% activity is retained in 40% acetone and 60% DMSO. This shows that nanobodies have good tolerance to organic solvents.

[0094] Example 5. Establishment of indirect competitive ELISA method for anti-sulfadiazine nanobody

[0095] Under the optimal working conditions, the logarithm of the SD standard with a series of concentration gradients is used as the horizontal axis, and the corresponding OD 450 The ratio of the reading value to the control well is the vertical axis, and the Logistic function is used for fitting to construct the standard curve of the anti-SD nanobody. Fig.12 Indirect competitive ELISA method based on Nb1-B3 IC 50 The value was 26.08 ng / mL, the linear detection range was between 3.57 and 169.88 ng / mL, the detection limit was 2.85 ng / mL, the quantification limit was 3.46 ng / mL, and the correlation coefficient was 0.993.

[0096] Example 6. Evaluation of the indirect competitive ELISA method with anti-sulfadiazine nanobodies

[0097] 1. Specificity analysis

[0098] Six common antibiotics were selected, including three commonly used sulfonamides: sulfadiazine (SD), sulfadimethoxine (SM2), sulfamethoxazole (SMZ) and three other common antibiotics: ampicillin (Amp), kanamycin (Kan), and enrofloxacin (ENR). An indirect competitive ELISA was used to draw a standard curve with the logarithm of drug concentration as the horizontal axis and the ratio of inhibition wells to control wells as the vertical axis, and the IC′ of each well was calculated. 50 The calculation formula is: Cross-reactivity (CR) = IC 50 (SD) / IC′ 50 (other drugs) × 100%. The results are shown in Table 2. The cross-reaction rates of the three non-sulfonamide drugs are all less than 0.1%, and there is basically no cross-reaction; the cross-reaction rates of the three sulfonamide drugs are 14.7%, 1.2% and 7.1%, respectively. This shows that the detection method has a low cross-reactivity only for sulfonamide drugs with a parent nucleus of sulfonamide, and at the same time proves that the nanoantibodies screened have good specificity.

[0099] Table 2 Cross-reaction rate determination

[0100]

[0101]

[0102] 2. Recovery rate determination

[0103] SPF pig urine and SPF eggs were used as matrices. Pig urine was directly diluted 20 times with PBS and then spiked. 5g of egg was homogenized and added to 20mL PBS, vortexed for 1min, shaken for 10min, 500μL of supernatant sample was taken, and then an equal amount of PBS buffer was added to mix thoroughly, centrifuged at 5000×g for 15min, and SD was added to the supernatant. The drug concentrations in the samples after addition were 25ng / mL, 50ng / mL and 100ng / mL, respectively. Three replicates were used in each group to calculate the recovery rate of the established indirect competitive ELISA method. Recovery rate = sample measured value / sample true value × 100%. The results are shown in Table 3. The recovery rate of drugs in pig urine as the matrix was 90.29% to 96.38%, and the recovery rate of drugs in eggs as the matrix was 84.08% to 93.60%. The content of the egg matrix was more complex than that of pig urine, so the recovery rate was lower than that of pig urine.

[0104] 3. Precision determination

[0105] The precision measurement standard is usually expressed by standard deviation or relative standard deviation, the latter is also called coefficient of variation (CV). The calculation formula is: CV (%) = standard deviation / measurement average × 100%. According to the general rules of the national enzyme-linked immunosorbent assay kit, the precision is evaluated by the coefficient of variation of the measurement results, and the coefficient of variation is not greater than 20%. The results are shown in Table 3. The coefficient of variation with pig urine as the matrix is ​​8.64% to 10.35%, and the coefficient of variation with eggs as the matrix is ​​10.37% to 11.41%. The coefficients of variation of the two matrices are both less than 12%, which shows that the established detection method has good precision.

[0106] Table 3 Determination of spike recovery

[0107]

Claims

1. A nanobody for detecting sulfadiazine, characterized in that: The amino acid sequence of CDR1 of the variable region of the Nanobody is shown in SEQ ID NO.6, the amino acid sequence of CDR2 of the variable region of the Nanobody is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 of the variable region of the Nanobody is shown in SEQ ID NO.

8.

2. The Nanobody according to claim 1, characterized in that The nanobody also includes four framework regions, the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.

5.

3. A nucleotide sequence encoding the Nanobody according to claim 1.

4. A nanobody for detecting sulfadiazine, characterized in that: The amino acid sequence of the nanobody is shown in SEQ ID NO.

1.

5. A recombinant vector, characterized in that: The recombinant vector contains the nucleotide sequence according to claim 3.

6. A recombinant microbial cell, characterized in that The recombinant microbial cell contains the nucleotide sequence of claim 3.

7. A kit for detecting sulfadiazine, characterized in that: The kit contains the Nanobody according to claim 1, 2 or 4.

8. Use of the nanobody according to claim 1, 2 or 4, the nucleotide sequence according to claim 3, the recombinant vector according to claim 5 or the recombinant microbial cell according to claim 6 in the preparation of a kit for detecting sulfadiazine.

9. The use according to claim 8, characterized in that: Detection of sulfadiazine residues in food, environmental or medical fields.

10. A method for detecting sulfadiazine residues, characterized in that: A method for indirect competitive ELISA using the Nanobody described in claim 1, 2 or 4.

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