A single-chain antibody against dextran and use thereof

Abstract

The application discloses an anti-dextran single-chain antibody and application. The amino acid sequence of the anti-dextran single-chain antibody is shown as SEQ ID NO. 6, and further, a COMP sequence is added at the C terminal of the single-chain antibody to form a derivative variant A48-COMP, and the amino acid sequence of the derivative variant of the single-chain antibody is shown as SEQ ID NO. 8. The derivative variant A48-COMP can produce turbidity with dextran, and can be directly applied to an immune turbidimetry method for measuring dextran. Compared with a general immune turbidimetry method, the application uses a genetic engineering antibody, is easy to prepare, has low detection cost, and has the advantages of simplicity and rapidness.

Description

Technical Field

[0001] This invention relates to the field of sugar production, and particularly to the variable region sequence of anti-dextran antibody and the preparation and application of its recombinant single-chain antibody. Background Technology

[0002] Dextran is a poly-D-glucose compound whose glucose residues are linked by α-1,6-glycosidic bonds to form the main chain, while side chains are formed by glucose residues or glucose chains linked to the main chain by α-1,2-, α-1,3-, or α-1,4-glycosidic bonds. Dextran is a common impurity in the sugar production process, causing various adverse effects. Detection of dextran is crucial for assessing sugar quality and applications, the severity of dextran problems in sugar production, and the effectiveness of prevention and removal measures.

[0003] Previously, methods for detecting dextran included the alcohol Haze method, GPC method, Robert-copper method, and immunoturbidimetric method. The alcohol Haze method and GPC method are nonspecific and easily affected by substances commonly found in the sugar production process, such as starch and pectin; the Robert-copper method is complicated to operate, time-consuming, and subject to interference from other colors during colorimetric analysis.

[0004] Immunoturbidimetry, which detects antigen-antibody complexes via light scattering, offers advantages such as high specificity, ease of operation, and rapid detection. However, the high cost of preparing monoclonal antibodies based on hybridoma technology limits its widespread use in the sugar industry. Preparing recombinant antibodies using exogenous protein expression systems can reduce production costs. However, recombinant single-chain antibodies often suffer from insufficient affinity and small molecular size; although they can bind to antigens, they fail to produce sufficient turbidity, thus precluding their application in immunoturbidimetry. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an anti-dextran single-chain antibody and its derivative variants. This invention is the first to utilize an immunoturbidimetric assay based on this anti-dextran single-chain antibody derivative to determine dextran. Compared to conventional immunoturbidimetric methods, it uses genetically engineered antibodies, requires less antibody, and has lower detection costs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention first obtains a monoclonal antibody against dextran using hybridoma technology. The antibody gene was cloned, and its sequence was obtained. Through genetic engineering, the corresponding single-chain antibody was obtained through exogenous expression. Although this single-chain antibody has a relatively high titer and can specifically bind to dextran, it cannot react with dextran to produce turbidity, and therefore cannot be directly used for the immunoturbidimetric determination of dextran.

[0008] A derivative variant, A48-COMP, is created by adding a COMP sequence to the C-terminus of a single-chain antibody. The COMP sequence acts as a polymer, thus aggregating the previously isolated single-chain antibodies. This derivative variant, A48-COMP, can produce turbidity with dextran and can be directly applied to the immunoturbidimetric determination of dextran.

[0009] On one hand, the present invention provides a single-chain antibody against dextran, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2.

[0010] Accordingly, the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.3; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.1.

[0011] The light chain variable region and heavy chain variable region of the single-chain antibody described in this invention are linked by a linker polypeptide, and the amino acid sequence of the single-chain antibody is shown in SEQ ID NO.6.

[0012] Accordingly, the nucleotide sequence of the single-chain antibody is shown in SEQ ID NO.5.

[0013] On the other hand, the present invention also provides a derivative variant of a single-chain antibody against dextran, comprising the above-mentioned single-chain antibody against dextran and an amino acid sequence COMP, the amino acid sequence of which is shown in SEQ ID NO.8.

[0014] The present invention also provides a gene encoding a derivative variant of the above-described anti-dextran single-chain antibody, the nucleotide sequence of which is shown in SEQ ID NO.7.

[0015] The present invention also provides a vector containing the coding gene of the above-described derivative variant.

[0016] The present invention also provides a recombinant cell containing the above-described carrier.

[0017] The present invention also provides the use of the above-described dextran single-chain antibody derivatives in the preparation of dextran detection kits.

[0018] Preferably, the kit is an immunoturbidimetric assay kit.

[0019] The present invention also provides a kit comprising a derivative variant of the above-described anti-dextran single-chain antibody.

[0020] This invention offers the following advantages: It discloses for the first time a derivative variant of an anti-dextran single-chain antibody, applicable to the immunoturbidimetric detection of dextran. Compared to conventional immunoturbidimetry, it utilizes genetically engineered antibodies, is easy to prepare, has low detection costs, and offers the advantages of simplicity and speed. Detailed Implementation

[0021] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0022] Example 1: Generation of anti-dextran monoclonal antibody

[0023] 1. Immunizing mice

[0024] Dextran T-40 and Dextran T-40-BSA cross-links were mixed with an equal volume of complete Freund's adjuvant and thoroughly emulsified. The mixture was then injected intradermally into mice via the back of the mouse at a dose of 50 μg per mouse. Two weeks later, a second immunization was administered with an equal volume of incomplete Freund's adjuvant. Booster immunizations were then given every two weeks thereafter. After three immunizations, serum antibody titers were measured.

[0025] 2. Serum antibody detection

[0026] Indirect ELISA was used. Dextran T-40-OVA cross-linked material was diluted with carbonate buffer and coated onto 96-well plates at 100 μL / well, incubated overnight at 4°C. The plates were blocked with 1% gelatin for 2 h, washed three times with PBST, and 100 μL of serially diluted serum was added to each well. Positive and negative controls were also included. The plates were incubated at 37°C for 1 h, washed three times with PBST, and then incubated with goat anti-mouse enzyme-labeled secondary antibody at 37°C for 1 h, washed three more times with PBST, and then OPD substrate solution was added. The plates were incubated at 37°C in the dark for 15 min, and the reaction was terminated with stop solution. Results were read at 490 nm using a microplate reader.

[0027] 3. Cell fusion, screening, and cloning

[0028] Three days after the initial immunization, mouse spleen cell suspensions were prepared and logarithmically growing SP2 / 0 cells were collected. Spleen cells and SP2 / 0 cells were mixed at a 5:1 ratio and fused using 50% PEG-mediated fusion. The cells were then screened and cultured in HAT medium. Hybridoma clones formed after one week. The hybridoma supernatant was screened using indirect ELISA, and positive cells were subjected to three consecutive subcloning experiments using limiting dilution. After indirect ELISA screening and cloning, stable antibody-secreting hybridoma cell lines were obtained.

[0029] 4. Antibody preparation

[0030] Liquid paraffin was injected intraperitoneally into each mouse (0.5 mL). Seven days later, diluted hybridoma cells were injected intraperitoneally. The ascites fluid was observed daily after a five-day interval. If the abdomen became significantly distended, ascites fluid was collected using a needle. The ascites fluid was centrifuged at 3000 rpm for 5 minutes, the supernatant was collected, its titer was determined, and the cells were stored at -20°C.

[0031] 5. Antibody purification

[0032] Affinity chromatography:

[0033] (1) Equilibrate the column with Buffer A (0.05 mol / L boric acid, 4.0 mol / L NaCl, pH 9.0, solvent is water).

[0034] (2) Add a small amount of ascites fluid and rinse the column with Buffer A.

[0035] (3) Then elute with Buffer B (0.05mol / L sodium phosphate, 0.05mol / L sodium citrate, 0.3mol / L NaCl, pH 3.0, solvent is water).

[0036] (4) Collect the protein and add 5% (v / v) of 1.0 mol / L Tris-HCl, adjusting the pH to 8.0. The purified antibody was analyzed by SDS-PAGE, and its heavy chain molecular weight was approximately 50 kDa, and its light chain molecular weight was approximately 23 kDa.

[0037] 6. Antibody titer determination

[0038] Indirect ELISA method for determining antibody titer

[0039] Coating: The Dextran T-40-OVA crosslinker was dissolved in buffer (0.05M carbonate buffer, pH 9.6) to a final concentration of 5 μg / mL and incubated overnight at 4°C in a 96-well plate.

[0040] Blocking: After washing three times with 0.01M PBST (containing 0.05% Tween-20, pH 7.4), block with 2% BSA (200 μL per well) at 37°C for 2 hours, and then wash again with PBST.

[0041] Primary antibody incubation: Add monoclonal antibody (MAb) diluted in PBS to each well (100 μL per well), incubate at 37°C for 1 hour, and then wash.

[0042] Secondary antibody incubation: Add goat anti-mouse IgG-HRP secondary antibody diluted to 1:10,000, incubate at 37°C for 1 hour, and then wash.

[0043] Color development: Add OPD substrate solution (50 μL per well) and incubate at 37°C for 10 minutes. Terminate the reaction by adding 2M sulfuric acid solution (50 μL per well), and then measure the OD value at 492 nm using a microplate reader (Model 680, Bio-Rad).

[0044] Antibody titers were determined by indirect ELISA, and the antibody A48 with the highest titer was found to be 5.12 × 10⁻⁶. 6 .

[0045] Example 2: Cloning and determination of the anti-dextran monoclonal antibody gene

[0046] 1. Total RNA was extracted and purified from hybridoma cells using an RNA extraction kit (RNAiso Plus, Takara). Following the instructions of the kit (PrimeScript 1st Strand cDNA Synthesis Kit, Takara), the RNA was reverse transcribed into first-strand cDNA using Oligo dT Primer.

[0047] The cDNA synthesis reaction solution was used directly as a template for the PCR reaction, and PCR amplification was performed using the high-fidelity polymerase TransGen HiFi (Beijing TransGen Biotech Co., Ltd.).

[0048] in,

[0049] Antibody heavy chain PCR primers:

[0050] Upstream primer: 5'-GCCGCCACAGTTCCTGAAGACACTG-3';

[0051] Downstream primer: 5'-CATGGTCCCAGGCATTGCTGGGTGCT-3';

[0052] The PCR conditions were: denaturation at 95℃ for 5 min; denaturation at 94℃ for 1 min; annealing at 50℃ for 1 min; extension at 72℃ for 2 min, for 31 cycles; and a total extension at 72℃ for 10 min.

[0053] Antibody light chain PCR primers:

[0054] Upstream primer: 5'-GGCACCATGGAGWCACAKWCTCAGGTCTTTRTA-3';

[0055] Downstream primer: 5'-TCAAGTGCAAAGACTCACTTTATTGAA-3';

[0056] The PCR conditions were: denaturation at 95℃ for 5 min; denaturation at 94℃ for 1 min; annealing at 50℃ for 1 min; extension at 72℃ for 1 min, for 31 cycles; and a total extension at 72℃ for 10 min.

[0057] After PCR, Taq polymerase was added and incubated at 72°C for 10 minutes. The reaction products were subjected to agarose gel electrophoresis, and the target fragments were excised and recovered from the gel to purify the PCR products. The PCR products were ligated into T vectors (TA cloning kit, TransGold), and positive clones were selected for sequencing and analysis.

[0058] 2. Analyze the antibody nucleic acid sequences obtained from sequencing using the IMGT website (https: / / www.imgt.org / ). Obtain sequence information for the variable regions of the antibody heavy and light chains:

[0059] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO.1:

[0060] CAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGG

[0061] TGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTACTTGATAGAGTGGGTAAAGCAGA

[0062] GGCCTGGACAGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTGAC

[0063] TACAATGAGAGGTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTG

[0064] CCTACATGCACCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGAG

[0065] GGGTAGCTTACTATGGTACACCTCTCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0066] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2:

[0067] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTDY

[0068] NERFKGKATLTADKSSSTAYMHLSSLTSDDSAVYFCARGVAYYGTPLWYFDVWGAGTTVT VSS.

[0069] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO.3:

[0070] AACATTCTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACC

[0071] TTGAGCTGCAAGGCCAGTGAGAATGTGGGTACTTATGTATCCTGGTTTCAACAGAAACC

[0072] AGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCG

[0073] ATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAG

[0074] GCTGAAGACCTTGCAGATTATCACTGTGGACAGAGTTACAGCTATCCCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA.

[0075] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.4:

[0076] NILMTQSPKMSMSVGERVTLSCKASENVGTYVSWFQQKPEQSPKLLIYGASNRYTGVPDR FTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPTFGGSGTKLEIK.

[0077] Example 3 Expression and purification of single-chain antibodies

[0078] 1. Construction of single-chain antibody A48HL

[0079] Based on the previously obtained sequence information of the antibody heavy and light chains, a single-chain antibody was designed with the overall structure as follows: heavy chain variable region - linker - light chain variable region.

[0080] The nucleic acid sequence of the synthetic single-chain antibody A48HL is shown in SEQ ID NO.5: CAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTACTTGATAGAGTGGGTAAAGCAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTGACTACAATGAGAGGTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCACCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGAGGGGTAGCTTACTATGGTACACCTCTCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGTGGTTCTGGAGGTGGTGGATCTGGTGGAGGTGGATCTAACATTCTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGAGCTGCAAGGCCAGTGAGAATGTGGGTACTTATGTATCCTGGTTTCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGAGTTACAGCTATCCCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACATCATCATCATCATCATTGA。

[0081] The amino acid sequence of the single-chain antibody A48HL is shown in SEQ ID NO.6:

[0082] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTDY

[0083] NERFKGKATLTADKSSSTAYMHLSSLTSDDSAVYFCARGVAYYGTPLWYFDVWGAGTTVT

[0084] VSSGGGGSGGGGSGGGGSNILMTQSPKMSMSVGERVTLSCKASENVGTYVSWFQQKPEQ

[0085] SPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPTFGGSGTKLEIKHHHHHH.

[0086] The synthesized sequence was cloned into the vector pUC57.

[0087] Using the synthesized sequence as a template, PCR amplification was performed.

[0088] Upstream primer: 5'-TCTCTCGAGAAAAGACAGGTCCAGCTGCAGCAGTCT-3';

[0089] Downstream primer: 5'-TGCTCTAGATCAATGATGATGATGATGATGTTTTAT-3';

[0090] The conditions were: denaturation at 95℃ for 5 min; denaturation at 94℃ for 1 min; annealing at 53℃ for 1 min; extension at 72℃ for 2 min, for 30 cycles; and a total extension at 72℃ for 10 min. The PCR product was recovered by gel electrophoresis to obtain the gene fragment.

[0091] The obtained gene fragment and plasmid pGAPZαA (purchased from Invitrogen) were double-digested with XhoI and XbaI, respectively. The digestion products were ligated with T4 DNA ligase and transformed into competent *E. coli* JM109 (Beijing TransGen Biotech Co., Ltd.). Positive clones were screened on LB agar plates (containing 25 μg / mL bleomycin). Several clones were selected for colony PCR identification and sequencing to obtain the recombinant expression vector.

[0092] The recombinant expression vector was digested with AvrII restriction enzyme overnight at 37°C. The digestion product was purified using a DNA fragment purification kit (purchased from Shanghai Sangon Biotech Co., Ltd.) to obtain a linearized vector. Competent Pichia pastoris KM71H cells were prepared according to the Invitrogen Pichia pastoris expression manual. The obtained linearized vector was mixed with 80 μL of competent Pichia pastoris KM71H cells and placed in a 1 mm electroporation cuvette. The mixture was incubated on ice for 5 min before electroporation. The electroporation parameters were: 1600 V, 25 μF, 200 Ω. After transformation, the yeast cells were plated on YPD-resistant plates containing 100 μg / mL bleomycin (containing 100 μg / mL bleomycin, 1% yeast extract, 2% peptone, 2% agar, and water as solvent) and incubated at 30°C for 3-4 days. Positive clones were selected, and the engineered strains were obtained by colony PCR and sequencing verification.

[0093] Shake flask culture of engineered bacteria: The activated strains from step (1) were transferred into liquid culture medium (culture medium components: yeast powder 10g / L, peptone 20g / L, 5% (w / v) glycerol, solvent is water) and cultured under the following conditions: 40mL liquid in a 250mL Erlenmeyer flask, temperature 30℃, rotation speed 250r / min, culture for 120 hours.

[0094] The fermentation product was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and purified using the HisTrap FFCrude (GE Healthcare) protein purification kit to obtain the single-chain antibody A48HL.

[0095] Example 4: Expression and purification of single-chain antibody variants

[0096] Construction of the single-chain antibody A48HL-COMP:

[0097] Based on the aforementioned single-chain antibody A48HL, a helical domain (COMP48, Asp29-Gln76) of human chondrocyte oligomeric matrix protein is linked to the C-terminus of the single-chain antibody. Its amino acid sequence is (DLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACGMQQ). The overall structure of A48HL-COMP is: heavy chain variable region - linker - light chain variable region - linker - COMP.

[0098] Artificially synthesized sequence (SEQ ID NO.7):

[0099] TCTCTCGAGAAAAGACAAGTCCAGCTTCAGCAATCCGGTGCCGAATTGGTTAGACCTGG

[0100] TACATCAGTCAAAGTTTCCTGCAAAGCAAGTGGTTACGCATTTACCAACTACTTGATTGA

[0101] ATGGGTTAAGCAAAGACCTGGTCAGGGACTTGAGTGGATTGGTGTCATCAACCCTGGAT

[0102] CTGGTGGAACAGATTACAACGAAAGATTCAAGGGTAAAGCTACCTTGACTGCCGACAAA

[0103] TCTTCCTCAACCGCTTACATGCATTTGAGTTCTCTTACTTCTGATGACTCCGCCGTTTATT

[0104] TTTGTGCAAGAGGTGTCGCTTACTATGGAACTCCATTGTGGTACTTCGATGTTTGGGGTG

[0105] CCGGAACTACAGTTACAGTCTCCTCAGGTGGAGGTGGATCTGGTGGAGGTGGTTCCGGT

[0106] GGAGGTGGATCAAATATCTTGATGACTCAATCTCCTAAGTCCATGTCAATGAGTGTTGGT

[0107] GAAAGAGTCACACTTTCATGCAAAGCTAGTGAGAACGTTGGTACCTATGTCAGTTGGTT

[0108] CCAACAGAAGCCAGAACAGTCTCCTAAATTGCTTATCTACGGTGCATCCAATAGATATAC

[0109] TGGAGTTCCAGATAGATTCACTGGTTCTGGTTCCGCTACAGACTTCACATTGACCATCAG

[0110] TTCTGTTCAAGCCGAGGATCTTGCAGACTACCACTGTGGACAGTCATATAGTTACCCTAC

[0111] CTTCGGATCAGGAACAAAGTTGGAAATCAAGGGTGGTGGTGGTTCAGGTGGTGGTGGT

[0112] TCAGGTGGTGGTGGTTCCGATTTGGGTCCTCAGATGCTTAGAGAGCTTCAAGAAACAAA

[0113] CGCTGCCTTGCAAGATGTTAGAGAATTGCTTAGACAACAGGTCAGAGAGATTACTTTTCT

[0114] TAAGAACACAGTTATGGAATGCGACGCCTGCGGAATGCAACAAGGTAGTCATCATCATCACCACCACTAATCTAGAGCA.

[0115] The synthesized sequence was cloned into the vector pUC57.

[0116] The amino acid sequence of the single-chain antibody A48HL-COMP is shown in SEQ ID NO.8:

[0117] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTDY

[0118] NERFKGKATLTADKSSSTAYMHLSSLTSDDSAVYFCARGVAYYGTPLWYFDVWGAGTTVT

[0119] VSSGGGGSGGGGSGGGGSNILMTQSPKMSMSVGERVTLSCKASENVGTYVSWFQQKPEQ

[0120] SPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPTFGGSGTKLEI

[0121] KGGGGSGGGGSGGGGSDLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACGMQQGSHHHHHH.

[0122] The synthesized gene fragment and plasmid pGAPZαA (Invitrogen) were double-digested with XhoI and XbaI, respectively. The digestion products were ligated with T4 DNA ligase and transformed into competent *E. coli* JM109 (purchased from TransGen Biotech, Beijing). Positive clones were screened on LB agar plates (containing 25 μg / mL bleomycin). Several clones were selected for colony PCR identification and sequencing to obtain the recombinant expression vector.

[0123] The recombinant expression vector was digested with AvrII restriction enzyme overnight at 37°C. The digestion product was purified using a DNA fragment purification kit (purchased from Shanghai Sangon Biotech Co., Ltd.) to obtain a linearized vector. Competent Pichia pastoris KM71H cells were prepared according to the Invitrogen Pichia pastoris expression manual. The obtained linearized vector was mixed with 80 μL of competent Pichia pastoris KM71H cells and placed in a 1 mm electroporation cuvette. The mixture was incubated on ice for 5 min before electroporation. The electroporation parameters were: 1600 V, 25 μF, 200 Ω. After transformation, the yeast cells were plated on YPD-resistant plates containing 100 μg / mL bleomycin (containing 100 μg / mL bleomycin, 1% yeast extract, 2% peptone, 2% agar, and water as solvent) and incubated at 30°C for 3-4 days. Positive clones were selected, and the engineered strains were obtained by colony PCR and sequencing verification.

[0124] Shake flask culture of engineered bacteria: The activated strains from step (1) were transferred into liquid culture medium (culture medium components: yeast powder 10g / L, peptone 20g / L, 5% (w / v) glycerol, solvent is water) and cultured under the following conditions: 40mL liquid in a 250mL Erlenmeyer flask, temperature 30℃, rotation speed 250r / min, culture for 120 hours.

[0125] The fermentation product was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and purified using the HisTrap FFCrude (GE Healthcare) protein purification kit to obtain the single-chain antibody A48HL-COMP.

[0126] Example 5: Validation of the titer of single-chain antibodies

[0127] Dextran T-40-OVA crosslinking was diluted with carbonate buffer and coated onto 96-well plates at 100 μL / well, incubated overnight at 4°C. The plates were blocked with 1% gelatin for 2 h, washed three times with PBST, and 100 μL of serially diluted test antibody was added to each well. Positive and negative controls were also included. The plates were incubated at 37°C for 1 h, washed five times with PBST, and then mouse anti-6×His antibody was added. The plates were incubated at 37°C for 1 h, washed three times with PBST, and then goat anti-mouse enzyme-labeled secondary antibody was added. The plates were incubated at 37°C for 45 min, washed five times with PBST, and then OPD substrate solution was added. The plates were incubated at 37°C in the dark for 15 min. The reaction was terminated with stop solution, and the results were read at 490 nm using a microplate reader.

[0128] The titers of single-chain antibody A48HL and single-chain antibody A48HL-COMP are shown in Table 1:

[0129] Table 1. Validation of the titer of single-chain antibodies

[0130] Antibody concentration (μg / mL) A48HL(OD490) A48HL-COMP(OD490) 100 2.275 2.718 50 2.024 2.200 25 1.755 2.026 12.5 1.242 1.780 6.25 0.805 1.353 3.125 0.677 1.027 1.56 0.434 0.716 0.781 0.209 0.404 0.390 0.122 0.231 0 0.098 0.110

[0131] Example 6: Turbidimetric reaction of single-chain antibodies

[0132] Dextran standard stock solution (1 mg / mL): Weigh out dextran standard ([C6H) 10 [O5]n, CAS No.: 9004-54-0, purity >99%, molecular weight 1,500,000~2,800,000) approximately 2g (weighed to an accuracy of 0.1mg) was placed in a pre-weighed weighing bottle and dried in a drying oven at 105℃ for 3 hours. After drying, it was removed, placed in a desiccator, cooled to room temperature, and weighed (accurate to 0.1mg). The moisture content of the dextran standard was calculated. Based on the moisture content of the dextran standard, approximately 0.1000g of undried dextran was quickly weighed to obtain approximately 0.1000g of dextran containing no moisture. Approximately 2mL of water was added to dissolve it into a paste. The mixture was left to stand for approximately 10 minutes, stirring occasionally to ensure uniform hydration of the particles. When gelation occurred, small amounts of water were added repeatedly until approximately 25mL was reached to dissolve the gel. The solution was then transferred to a 100mL volumetric flask, and water was added to the mark. The mixture was stirred thoroughly. This solution should be prepared immediately before use.

[0133] Prepare dextran solutions with concentrations of 100, 200, 400, 600, 800, and 1000 ppm by taking the standard stock solution of dextran.

[0134] Pipette 1.5 mL of single-chain antibody solution (0.2 mg / mL) into a test dish, place it in a turbidimeter, cap it, and accurately read the turbidity value (N) after 1 minute. 样0Remove the test dish and use a pipette to add 15 μL of sample solution to the antibody solution. Start the stopwatch. Cover the test dish with the lid and invert it 6 times to mix thoroughly, avoiding the formation of air bubbles during mixing. Place the test dish in the turbidimeter, close the lid, and accurately read the turbidity value (N) after 1 minute. 样 ), calculate the turbidity increment (ΔNTU) of the reaction system. 样 =N 样 -N 样0 ).

[0135] Table 2. Turbidimetric reaction of single-chain antibodies

[0136] Dextran concentration (ppm) A48HL(△NTU) A48HL-COMP(△NTU) 0 0.2 0.3 100 0.2 1.5 200 0.3 5.4 400 0.4 12.4 600 0.3 39.7 800 0.5 46.3 1000 0.9 50.2

[0137] Example 7: Determination of dextran content in samples

[0138] Dextran standard series solutions: Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of dextran standard stock solution (1 mg / mL) into 10 mL volumetric flasks and dilute to volume. The mass concentrations of this dextran standard series solutions are 0 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L, respectively. Dextran standard series solutions of appropriate mass concentrations can be prepared according to the actual dextran content in the sample, with a mass concentration not exceeding 600 mg / L.

[0139] Pipette 1.5 mL of single-chain antibody (A48HL-COMP) solution (0.2 mg / mL) into a test dish, place it in a turbidimeter, cap it, and accurately read the turbidity value (N) after 1 minute. 标0 Remove the test dish and use a pipette to add 15 μL of dextran standard series solution to the antibody solution. Start the stopwatch. Cover the test dish with the lid and mix well, avoiding the formation of air bubbles during mixing. Place the test dish in the turbidimeter, close the lid, and accurately read the turbidity value (N) after 1 minute. 标 ), calculate the turbidity increase (ΔNTU) of the reaction solution system. 标 A standard curve was obtained by plotting the dextran concentration on the x-axis and the turbidity increment on the y-axis.

[0140] Pipette 1.5 mL of single-chain antibody solution (0.2 mg / mL) into a test dish, place it in a turbidimeter, cap it, and accurately read the turbidity value (N) after 1 minute. 样0 Remove the test dish and use a pipette to add 15 μL of sample solution to the antibody solution. Start the stopwatch. Cover the test dish with the lid and invert it 6 times to mix thoroughly, avoiding the formation of air bubbles during mixing. Place the test dish in the turbidimeter, close the lid, and accurately read the turbidity value (N) after 1 minute. 样 ), calculate the turbidity increment (ΔNTU) of the reaction system.样 =N 样 -N 样0 ).

[0141] Table 3. Determination of dextran content in samples

[0142] Test sample Dextran content (ppm) maltose Not detected Isomaltose Not detected fructose Not detected glucose Not detected BSA Not detected OVA Not detected white sugar 182 brown sugar 335 First Pressed Sugar Juice 90

[0143] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A single-chain antibody against dextran, characterized in that, Include: The light chain variable region, whose amino acid sequence is shown in SEQ ID NO.4; The heavy chain variable region has the amino acid sequence shown in SEQ ID NO.

2.

2. The single-chain antibody against dextran according to claim 1, characterized in that, The amino acid sequence of the single-chain antibody is shown in SEQ ID NO.

6.

3. A derivative variant of a single-chain antibody against dextran, characterized in that, The single-chain antibody against dextran as described in claim 1 or 2 and an amino acid sequence COMP, the amino acid sequence of which is shown in SEQ ID NO.

8.

4. A gene encoding a derivative variant of the single-chain antibody against dextran as described in claim 3.

5. The gene according to claim 4, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

7.

6. A carrier, characterized in that, It contains the gene described in claim 4.

7. A recombinant cell, characterized in that, It contains the carrier as described in claim 6.

8. The use of a derivative variant of the anti-dextran single-chain antibody according to claim 3 in the preparation of a dextran detection kit.

9. The application according to claim 8, characterized in that, The kit is an immunoturbidimetric assay kit.

10. A reagent kit, characterized in that, Derivative variants comprising the single-chain antibody against dextran as described in claim 3.