A nano antibody, detection kit and detection method for microcystin LR

Abstract

The present invention provides a microcystin LR nanobody, a detection kit and an immunodetection method, wherein the amino acid sequence of the nanobody includes three variable regions CDR1, CDR2 and CDR3. The nanobody of the present invention can detect microcystin LR, especially microcystin LR in water, and can identify a variety of microcystins, including microcystin LR, microcystin YR and microcystin RR; based on the nanobody, the present invention establishes a direct competitive ELISA detection method, the measurement result is highly accurate, has good stability, and can be widely used in the detection of algae toxins in drinking water.

Description

Technical Field

[0001] The invention relates to a nano antibody, a detection kit and an immunodetection method of microcystin LR, and belongs to the field of biotechnology. Background Art

[0002] Cyanobacteria are the most toxic and widely distributed type of algae. Cyanobacteria are photoautotrophic prokaryotes. When water bodies become eutrophic due to the injection of industrial wastewater or domestic sewage, they will cause the massive reproduction of harmful algae such as cyanobacteria. These algae groups will form algae slurry visible to the naked eye, which is also known as water bloom. Although cyanobacteria will reproduce in eutrophic water bodies, because the nutrients in the water body are limited, these large amounts of cyanobacteria will also die in large areas under nutrient deficiency. During the death of cyanobacteria, the decomposition of the algae will consume a large amount of dissolved oxygen in the water body, leading to the death of other aquatic organisms and causing foul odor in the water body. The most serious situation is that the disintegration of a large number of cyanobacterial cells will cause the cyanobacterial toxins inside them to be released into the water body, affecting the safety of water quality. Common cyanobacterial toxins in water bodies are mainly microcystins and pseudocylindrosporin. These toxins mainly act on the liver, posing health risks to residents.

[0003] Microcystin (MC) is the most common of many cyanobacterial toxins. It is usually secreted by algae such as Microcystis, Anabaena, Aphanizomenon, Oscillatoria, Gelatocystis and Nodularia during outbreaks, among which Microcystis is the main secretion. Microcystin is also the most harmful type of hepatotoxin to humans. Currently, there are as many as 90 confirmed microcystin isomers. Among these discovered isomers, Microcystin-LR (MC-LR), Microcystin-RR (MC-RR) and Microcystin-YR (MC-YR) are the most toxic. MC-LR is a cyclic heptapeptide with a molecular weight of 995.2Da. It is easily soluble in water, non-volatile, and has extremely stable chemical properties. It cannot be destroyed by high temperature, light, protease, etc. It cannot be effectively removed by coagulation, sedimentation and filtration in water treatment processes. It can be degraded under the irradiation of ultraviolet rays and the action of certain bacteria in water. Microcystins are not only soluble in water, but also in a variety of organic reagents, and are resistant to acid and alkali. These chemical properties make them difficult to degrade in nature. In 2017, the World Health Organization (WHO) has classified MC-LR as a Class 2B carcinogen.

[0004] There are various methods for detecting microcystins, which mainly include gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay, high performance liquid chromatography, and high-performance liquid chromatography-tandem mass spectrometry. Although the instrumental method has high accuracy, it is expensive, has high detection cost, is inconvenient to move, and cannot be used for on-site surveys. The immunoassay method (ELISA) based on antibodies has the advantages of being rapid, sensitive, simple, and low-cost, but the monoclonal antibodies and polyclonal antibodies used in the market today have the disadvantages of poor stability and easy inactivation. At present, the detection method for microcystins is still imperfect, and the detection speed and accuracy need to be improved. Therefore, the development of a new microcystin detection method is a technical problem that needs to be solved in this field. Summary of the invention

[0005] The present invention provides a nano antibody that can specifically bind to microcystin LR, and can be used to detect microcystin LR in water. The nano antibody of the present invention has high affinity with the antigen and high detection sensitivity.

[0006] To achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0007] In the first aspect, the present invention provides a nanobody against microcystin LR, the amino acid sequence of the nanobody comprising three variable regions CDR1, CDR2, and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 3.

[0008] The nano-antibody of the microcystin LR of the present invention is folded into a specific region that binds to the antigen through the amino acid sequences of the specific variable regions CDR1, CDR2, and CDR3, can specifically recognize the antigen microcystin LR, and also has a certain affinity for other isomers MC-RR and MC-YR, so it can detect MC-LR, MC-RR, and MC-YR in water.

[0009] Preferably, the Nanobody further comprises four framework regions FR1, FR2, FR3 and FR4; the amino acid sequence of the FR1 is shown in SEQ ID NO: 4, the amino acid sequence of the FR2 is shown in SEQ ID NO: 5, the amino acid sequence of the FR3 is shown in SEQ ID NO: 6, and the amino acid sequence of the FR4 is shown in SEQ ID NO: 7.

[0010] The amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the nanoantibody of the microcystin LR of the present invention have more hydrophobic amino acids, which enhances the binding force between the antibody and the fixed carrier, improves the stability of the solid-phase antibody, and improves the sensitivity of the immunoassay method.

[0011] In a second aspect, the present invention also provides a nucleic acid sequence encoding the anti-microcystin LR nanobody, and the nucleic acid sequence is shown in SEQ ID NO:9.

[0012] In a third aspect, the present invention also provides a microcystin LR immunoassay kit, the kit comprising:

[0013] (1) Solid phase carrier: a solid phase carrier coated with microcystin LR nanobody (MC-LR-VHH-1) at a concentration of 0.1 to 10 μg / mL;

[0014] (2) Microcystin-LR standard solution: concentrations of 0 to 100 ng / mL;

[0015] (3) Microcystin-LR peroxidase marker (MC-LR-HRP) solution, concentration 0.01-10 μg / mL;

[0016] (4) enzyme-labeled reaction substrate colorimetric solution;

[0017] (5) reaction termination solution;

[0018] (6) washing liquid;

[0019] (7) Sample diluent.

[0020] Preferably, the preparation method of the solid phase carrier coated with MC-LR-VHH-1 is as follows:

[0021] A: Add MC-LR-VHH-1 solution to carbonate buffer to make the final concentration of the antibody 1-2 μg / mL, mix well, and prepare antibody coating solution;

[0022] B: Add the above antibody coating solution to the solid phase carrier, keep warm at 25-37°C for 1-3 hours, discard the excess coating solution, wash with phosphate buffered saline (PBS), block, discard the excess blocking solution, wash and dry, to obtain a solid phase carrier coated with MC-LR-VHH-1.

[0023] Preferably, the carbonate buffer has a concentration of 0.01 M and a pH value of 9.6.

[0024] Preferably, the phosphate buffer has a concentration of 0.05 M and a pH value of 7.2.

[0025] Preferably, the blocking solution is PBS containing 0.05%-0.5% (m / v) bovine serum albumin (BSA), with a concentration of 0.05M and a pH value of 7.4.

[0026] Preferably, the blocking step is to add the blocking solution to the solid phase carrier and place the solid phase carrier at 4°C overnight.

[0027] Preferably, the concentrations of the MC-LR standard solutions are 1.56 ng / mL, 3.13 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively.

[0028] Preferably, the preparation method of the microcystin LR peroxidase (MC-LR-HRP) is as follows:

[0029] a: Dissolve the MC-LR standard in dimethyl sulfoxide (DMSO), then add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to obtain a mixed solution A, in which the molar ratio of MC-LR to NHS and EDC is: MC-LR:NHS:EDC=1:1:2.

[0030] b: Add mixed solution A and glutaraldehyde solution to phosphate buffer, dialyze to remove glutaraldehyde, and obtain the MC-LR-HRP.

[0031] Preferably, the concentration of the glutaraldehyde solution is 1% (v / v);

[0032] Preferably, the volume ratio of the added mixed solution A to the glutaraldehyde solution is 1:1.

[0033] Preferably, the phosphate buffer has a concentration of 0.05 M and a pH value of 7.2.

[0034] Preferably, the dialysis temperature is 4° C. and the time is 10 to 12 hours.

[0035] Preferably, 100 mL of enzyme-labeled reaction substrate colorimetric solution in the kit contains components with the following concentrations: 0.5-0.7% (m / v) 3,3',5,5'-tetramethylbenzidine (TMB), 1-3% (m / v) citric acid, 1-4% (v / v) dimethyl sulfoxide (DMSO), and 100 mL of distilled water.

[0036] Preferably, the reaction termination solution in the kit is 2M sulfuric acid solution.

[0037] Preferably, the washing solution in the kit is a 0.25M phosphate buffer at pH 7.4, containing 0.1% (v / v) polysorbate 20.

[0038] Preferably, the sample diluent is a 0.25 M phosphate buffer at pH 7.2, containing 0.05% Tween-20 (v / v).

[0039] Preferably, the solid phase carrier is a porous plate made of polystyrene.

[0040] In a fourth aspect, the present invention also provides an immunoassay method for microcystin LR, comprising the following steps:

[0041] S1 Sample processing: centrifuge the water sample, take the supernatant, dilute it, and obtain the sample dilution solution;

[0042] S2: adding microcystin-LR-peroxidase (MC-LR-HRP) solution and microcystin-LR (MC-LR) series standard solution to the solid phase carrier standard well; adding MC-LR-HRP solution and the sample diluent of step S1 to the solid phase carrier sample well; placing the solid phase carrier at 25-37°C and incubating for 10-30 minutes;

[0043] S3 washing: discard the excess liquid of the solid phase carrier and wash with buffer;

[0044] S4 color development reaction: add color development substrate to the solid phase support in step S3, and incubate at 25-37°C for 10-30 minutes;

[0045] S5 reaction termination: adding a reaction termination solution to the solid phase support in step S4;

[0046] S6 detection: Place the solid phase carrier on a microplate reader to measure OD 450 The absorbance is calculated according to formula 1:

[0047]

[0048] In formula 1: B is the average absorbance value of the MC-LR standard solution or sample solution; B0 is the average absorbance value of the blank liquid without MC-LR; a standard curve is drawn with the logarithm of the mass concentration of the MC-LR standard solution as the abscissa (X) and the inhibition rate as the ordinate (Y), and the content of MC-LR in the sample is measured.

[0049] In a fifth aspect, the present invention also provides the use of the nanobody or the immunoassay kit in detecting algal toxins.

[0050] Specific embodiment

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0052] Source of reagents:

[0053] Phage vector pComb3XSS: purchased from Beijing Huayueyang Biological, product number VECT59073, product name: pcomb3xss;

[0054] Competent cells TG1: purchased from Shanghai Lianmai Biotechnology Co., Ltd. (LMAl Bio), product name: TG1 thermosensitive competent cells;

[0055] Microcystin LR antigen: purchased from Shanghai Yuduo Biotechnology Co., Ltd., catalog number: YDD300, specification: 1 mg / tube;

[0056] Microcystin LR monoclonal antibody: purchased from Wuhan Aimijie Technology Co., Ltd., product number MAB8473, specification 1 mg / tube, antibody subtype: mouse IIgG.

[0057] Preparation method of microcystin LR monoclonal antibody-horseradish peroxidase complex (MC-LR-IIgG-HRP): MC-LR-IIgG-HRP was prepared by glutaraldehyde one-step cross-linking method.

[0058] Glutaraldehyde is a commonly used homobifunctional crosslinking agent, which combines with the amino groups of HRP and antibody protein through its two aldehyde groups to form HRP-glutaraldehyde-antibody protein conjugate. It can be carried out in a buffer solution with a temperature range of 4-40°C and a pH of 6.0-8.0. The enzyme and the antibody to be labeled are mixed, and glutaraldehyde is added at the same time for crosslinking reaction.

[0059] Example 1 Preparation of Algal Toxin Nanobodies

[0060] 1. Preparation of immune antigen microcystin LR-BSA complex (MC-LR-BSA)

[0061] 1) Activation of MC-LR: 1 mg of MC-LR standard was dissolved in 0.1 mL of dimethyl sulfoxide (DMSO), and N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to obtain a mixed solution A, in which the molar ratio of MC-LR to NHS and EDC was: MC-LR to NHS and EDC = 1:1:2.

[0062] 2) Coupling: Add the mixed solution A dropwise into a 5% bovine serum albumin (BSA) solution for reaction. The reaction product is dialyzed in a 0.05 mol / L PBS buffer solution with a pH value of 7.0 at 4°C for 24 hours to obtain the antigen MC-LR-BSA.

[0063] 2. Immunity

[0064] Take 200 μg of antigen MC-LR-BSA and emulsify it with an equal volume of Freund's complete adjuvant. Each time, inject it subcutaneously on the left and right sides near the alpaca's cervical lymph nodes. Each side is injected at 2 points, and each point is injected with about 0.4 mL of emulsified antigen. Immunization is performed once every 2 weeks, and at least 4 immunizations are performed. Before each antigen immunization, blood is collected for immune evaluation, and 5 mL of blood is collected each time; the blood is centrifuged at 4000 rpm for 10 minutes in a pre-cooled 25°C centrifuge on the same day to separate and freeze the upper serum for subsequent antibody titer detection. 50 mL of blood is collected from the alpaca's neck vein at intervals of 5-7 days after the fourth immunization, and the total RNA of PBMC cells in the blood sample with the best serum inhibition is extracted using the trizol method.

[0065] 3. Establishment of phage display library

[0066] 3.1 Amplification of antibody VHH nucleic acid sequence fragments

[0067] 1) Reverse transcription of cDNA: Using RNA as a template, synthesize the first strand of cDNA according to the instructions of the first strand reverse transcription kit of TARAKA.

[0068] 2) PCR amplification of the VHH heavy chain variable region: using cDNA as a template for the first round of PCR amplification to obtain the first round of amplification products;

[0069] Table 1. First round amplification reaction system

[0070]

[0071] Table 2 First round amplification reaction program

[0072]

[0073]

[0074] (3) Perform a second round of PCR amplification using the first round amplification product as a template to obtain the second round amplification product:

[0075] Table 3 Second round amplification reaction system

[0076]

[0077] Table 4 Second round amplification reaction program

[0078] temperature time 98℃ 30s 98℃ 10s 55℃ 2min 72℃ 10min cycle 20

[0079] The second round of PCR products were purified by agarose gel purification. The product showed a target band at the 600 bp position. The target band at this position was cut and recovered as the VHH gene required for library construction.

[0080] 3.2 Construction of phage library

[0081] The second round amplification product obtained above was double digested with sfiI by the phage vector pComb3XSS, and then the linear second round amplification product was purified and recovered and connected to the vector pComb3XSS to obtain the expression vector pComb3XSS-VHH expressing the nanobody.

[0082] 10 μL (100 ng) of expression vector pComb3XSS-VHH was added to 100 μL competent cells TG1, and electroporated in a pre-cooled electroporation cup. After electroporation, 900 μL SOC medium was added, and then transferred to a 1 mL centrifuge tube. After shaking culture at 37°C and 200-220 rpm for 3 h, an appropriate amount of culture was spread on an LB plate containing ampicillin, and the plate was inverted and cultured at 37°C overnight. The number of single colonies was calculated, and the library capacity of the transformation library was greater than 10 9 The library was used for the construction of phage library.

[0083] 3.3 Phage library amplification and purification

[0084] Scrape off the bacteria on the plate in the previous step and mix them well to make the number of bacteria about 10 8 The bacteria were transferred into 100 mL of 2×YT culture medium pre-added with ampicillin antibiotics and cultured at 37°C and 220 rpm until OD 600 The value reached 0.5. After adding helper phage at a ratio of 10:1 between helper phage and bacterial cell number, continue to culture at 37℃ for 30min. Add kanamycin at a final concentration of 40μg / mL and culture at 30℃ overnight on a shaker. Centrifuge the overnight cultured bacteria at 4℃ and 12000rpm for 10min, transfer the supernatant to a new centrifuge tube, add pre-cooled 5×PEG8000 / NaCl, and incubate on ice for 20min. Centrifuge at 4℃ and 12000rpm for 15min, remove the supernatant, and add 1mL PBS buffer to dissolve the precipitate. Add 250μl 5×PEG8000 / NaCl again and incubate on ice for 20min. Centrifuge at 4℃ and 15000g for 20min, remove the supernatant, and dissolve the precipitate in 1mL PBS to obtain the phage library.

[0085] 3.4 Antibody screening and identification

[0086] 3.4.1 Antibody Screening:

[0087] Coating antigen: Add 100 μg antigen to 4 mL PBS and add to the immunotube, incubate overnight at 4°C with slow rotation;

[0088] Blocking: Add 2 mL of 3% BSA to the coated immunotubes, incubate with rotation at room temperature for 1 hour, and wash the blocked immunotubes three times with PBS containing 0.01% Tween, 5 minutes each time;

[0089] Antigen and phage incubation: Add appropriate amount of amplified and purified phage into the immunotube and incubate with rotation at room temperature for 2 hours;

[0090] Washing: Wash the incubated immunotube 20 times with PBS containing 0.1% Tween;

[0091] Elution: discard the excess liquid in the immunotube, add 1mL of 0.25mg / mL Trypsin solution, rotate and elute at room temperature for 30 minutes, add 10μL of 10% AEBSF to terminate the elution, and transfer the solution in the immunotube to a new 1.5mL centrifuge tube, which is the phage eluate for the first round of screening;

[0092] Phage eluate titer detection: Take 10 μL of the first round of phage eluate, dilute it 10 times in a centrifuge tube, dilute it 10 times in total, add 90 μL OD 600 The TG1 bacterial solution was 0.4-0.5, mixed and incubated at 37°C for 30 minutes, and each gradient of bacterial solution was spread on 2×YT solid medium (Amp), inverted and cultured at 37°C overnight, and the number of single colonies on the culture plate was counted on the next day and the titer of the phage eluate was calculated;

[0093] Amplification of the first round of phage eluate: Take 500μl of the phage eluate obtained after the first round of screening to 5mL of bacterial solution with OD600 of 0.5-0.55, continue to culture at 37°C, 250rpm for 30 minutes, and evenly spread all the bacterial solution on a solid culture plate containing 100μg / mL Amp and 2% glucose and 2% agarose, and culture overnight at 37°C. The next day, scrape the colonies on the culture plate and collect them in a centrifuge tube, which is the amplified bacterial sub-library. According to the phage library amplification and purification method, repeat the screening process twice, gradually halve the amount of antigen coated immune tubes, and obtain the eluted phage after 3 screenings. The eluted phage can be sequenced by NGS to obtain a candidate nanoantibody DNA sequence library that binds to the antigen.

[0094] 3.4.2 Antibody ELISA identification:

[0095] After gradient dilution of the phages obtained in the last round of screening, 10 μL of each was added to the TG1 bacterial solution with an OD600nm of 0.5, cultured at 37°C for 30 minutes, coated on a 2x YT culture plate containing ampicillin, and cultured overnight at 37°C to obtain monoclonal colonies on the second day; at least 180 single colonies were randomly selected for detection, and colonies with a large ratio of absorbance values ​​between the antigen-coated wells and the corresponding control wells were selected for sequencing (two independent phage-Elisa analyses) to obtain the gene sequence of the candidate nanoantibody.

[0096] Example 2 MC-LR-VHH antibody affinity test

[0097] Based on the positive monoclonal sequencing results and NGS sequencing results, 8 nanoantibodies were selected for yeast expression to obtain VHH antibody proteins, and the affinity of these 8 antibodies to the MC-LR antigen was tested by ELISA. The specific operation is: the MC-LR-VHH antibody protein of the present invention is expressed using a yeast expression system with a his tag, and the expressed antibody VHH is labeled with a his tag, so that the VHH is combined with the anti-his probe, and the labeled antibody is combined with different concentrations of antigen protein, and the antigen protein concentrations are: 400nM, 200nM, 100nM, 50nM, 25nM and 12.5nM. The binding force of different VHH antibodies is as follows:

[0098] Table 5 Antibody binding capacity of different MC-LR-VHH

[0099]

[0100]

[0101] Note: E stands for 10

[0102] The results of ELISA antibody affinity test showed that MC-LR-VHH-1, MC-LR-VHH-2, MC-LR-VHH-5, and MC-LR-VHH-8 had strong binding ability. MC-LR-VHH-1, MC-LR-VHH-2, MC-LR-VHH-5, and MC-LR-VHH-8 were selected for further testing.

[0103] Example 3 Detection of the binding stability of MC-LR-VHH to a solid phase carrier

[0104] In order to detect the stability of the binding of the four antibodies obtained in Example 2 to the solid phase carrier, the following method was used:

[0105] 1) Preparation of solid phase antibodies:

[0106] A: Add MC-LR-VHH solutions (prepared from MC-LR-VHH-1, MC-LR-VHH-2, MC-LR-VHH-5, and MC-LR-VHH-8, respectively) to 0.01 M carbonate buffer (pH 9.6) to make the final concentration of H antibody 2 μg / mL, mix well, and prepare antibody coating solution;

[0107] B: Add 200 μl of the above antibody coating solution to each well of the solid phase carrier (which can be an ELISA plate in this embodiment), incubate at room temperature overnight, discard the excess coating solution, wash with 0.01M pH 7.4 phosphate buffer (PBS), add 200 μl of 0.05M pH 7.4 PBS containing 20 g / L bovine serum albumin (BSA), incubate at 4°C overnight, wash three times, and dry to obtain a solid phase carrier coated with MC-LR-VHH, with an antibody concentration of 1 μg / mL, and obtain a solid phase antibody.

[0108] 2) The prepared solid phase antibodies were stored at 50° C., 37° C. and 4° C., respectively. During the storage process, the solid phase antibodies were placed on a shaker at 220 rpm for 20 min every 48 hours. After 1 week, 2 weeks, 4 weeks and 8 weeks of storage, their binding properties with the solid phase carrier were measured, and the measured results were compared with those at the beginning of storage, with the binding effect at the beginning of storage being set as 100%.

[0109] In this example, three identical ELISA plates were prepared, each of which contained four different nanoantibodies: MC-LR-VHH-1, MC-LR-VHH-2, MC-LR-VHH-5, and MC-LR-VHH-8. The three ELISA plates were then placed at 50°C, 37°C, and 4°C, respectively, to determine the binding performance of each nanoantibody to the solid phase carrier.

[0110] Determination method: ELISA method was used for detection. 100 μl of 50 nM microcystin-LR antigen was added to the solid phase antibody placed at different times and temperatures, incubated at 37°C for 20 min, the excess liquid in the well was discarded, and the well was washed three times with phosphoric acid solution; then 100 μl of 50 nM MC-LR-IIgG-HRP was added, incubated at 37°C for 20 min, the excess liquid in the well was discarded, and the well was washed three times with phosphoric acid solution; 100 μL of chromogenic substrate solution TMB was added, and the color was developed at 37°C for 15 min; 50 μL of 2M sulfuric acid solution was added to terminate the reaction, and the OD450 value was read with an enzyme reader. The results are as follows:

[0111] Table 6: Storage of different MC-LR-VHH at 50°C for different time periods and shaking results

[0112]

[0113] Table 7: Storage of different MC-LR-VHH at 37°C for different time periods and shaking results

[0114]

[0115] Table 8: Storage of different MC-LR-VHH at 4°C for different time periods and shaking results

[0116]

[0117] As can be seen from the above table, among the above four MC-LR-VHH antibodies, although MC-LR-VHH-2 and MC-LR-VHH8 have the best affinity with the MC-LR antigen, their binding force with the solid phase carrier is relatively poor. At 50°C and 37°C in the 8th week, the binding force of MC-LR-VHH-2 with the solid phase carrier is only 80%, and the binding force of MC-LR-VHH-8 with the solid phase carrier is only 76%. Although MC-LR-VHH-1 has a slightly lower binding force with the antigen than MC-LR-VHH-2 and MC-LR-VHH-8, it has the strongest binding force with the solid phase carrier, and the binding effect with the solid phase carrier is still more than 90% at 50°C and 37°C in the 8th week. Therefore, MC-LR-VHH-1 is selected as the nanobody for immunodetection of MC-LR in the present invention.

[0118] NGS sequencing of MC-LR-VHH-1 showed that the sequences of the three variable regions CDR1, CDR2, and CDR3 of its amino acid sequence were shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the sequences of the four framework regions FR1, FR2, FR3, and FR4 of MC-LR-VHH-1 were shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; the nucleic acid sequence encoding MC-LR-VHH-1 was shown in SEQ ID NO: 9.

[0119] CDR1 amino acid sequence: WWFSCYKR (SEQ ID NO: 1)

[0120] CDR2 amino acid sequence: NMACTTWKHP (SEQ ID NO: 2)

[0121] CDR3 amino acid sequence: AWSSHRSWCYSCLV (SEQ ID NO: 3)

[0122] FR1 amino acid sequence: GGQLVESGGGLVQVGGSLVLSCAIGL (SEQ ID NO: 4)

[0123] FR2 amino acid sequence: LVWMGRQPPGKQRELAEVFV (SEQ ID NO: 5)

[0124] FR3 amino acid sequence: FGVKGRFTISRDNAVNTIPLQMNSLKPEDTAVYYC (SEQ ID NO: 6)

[0125] FR4 amino acid sequence: WGQGTQVTVSS (SEQ ID NO: 7)

[0126] The full-length amino acid sequence of MC-LR-VHH-1:

[0127] GGQLVESGGGLVQVGGSLVLSCAIGLWWFSCYKRLGWMGRQPPGKQRELAEVFVNMACT

[0128] TWKHPFGVKGRFTISRDNAVNTIPLQMNSLKPEDTAVYYCAWSSHRSWCYSCLVWGQGTQ VTVSS(SEQ ID NO: 8)

[0129] Nucleic acid sequence encoding MC-LR-VHH-1:

[0130] GGTGGCCAGCTTGTTGAATCTGGCGGAGGTTTGGTGCAAGTGGGTGGCAGTCTCGTATCGTGCGCGGCCTGGTGGTTTAGTTGCTACAAAAGGACTGGGTGATGGGTAGGCAACCTGGAAAGCAACGGGAACTGCTGAGGTCTTTGTTAATGGCTGCTACTACTTGGAAAACATTCC TGGTGTTAAAGGGTTCACTATTAGTCGTGATAATGCTGTTAACTATTCCTGCTGCAAATGAATAGTACTAAAACCCGAATGATACTGCTTATTTATTGCAGCAAGCTAGCAGACATAGAAGCTGGTATTGCAGCTGCTGGTTTGGGGACAAGGAACTCAAGTAACTGTAAGTAGT(SEQ ID NO:9)

[0131] Example 4 An immunoassay kit for anti-algal toxins

[0132] This embodiment provides an immunoassay kit for anti-algal toxins, the kit comprising:

[0133] (1) ELISA plate: a solid phase carrier coated with MC-LR-VHH-1 (i.e., microcystin-LR-nanobody-1), with an antibody concentration of 0.1 to 10 μg / mL;

[0134] (2) MC-LR standard solution: concentrations of 1.56 ng / mL, 3.13 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL;

[0135] (3) MC-LR peroxidase marker solution, concentration 0.01-10 μg / mL;

[0136] (4) Color developing solution: prepared by mixing 70 mg of 3,3',5,5'-tetramethylbenzidine (TMB), 2.55 g of citric acid, 4 mL of dimethyl sulfoxide (DMSO) and 96 mL of distilled water;

[0137] (5) Reaction termination solution: 2M sulfuric acid solution;

[0138] (6) 50-fold concentrated washing solution: PBS solution containing 0.1% polysorbate 20, pH 7.4;

[0139] (7) Antigen diluent: PBS solution containing 0.8% BSA.

[0140] The preparation method of the solid phase carrier coated with MC-LR-VHH-1 in this example is as follows:

[0141] A: Add MC-LR-VHH-1 solution to 0.01 M carbonate buffer (pH 9.6) to make the final concentration of the antibody 2 μg / mL, mix well, and prepare the antibody coating solution;

[0142] B: Add 100 μl of the above-mentioned antibody coating solution to each well of the solid phase carrier (which can be an ELISA plate in this embodiment), incubate at room temperature overnight, discard the excess coating solution, wash with 0.01M pH 7.4 phosphate buffer (PBS), add 200 μl of 0.05M pH 7.4 PBS containing 20 g / L bovine serum albumin (BSA), incubate at 4°C overnight, wash three times, and dry to obtain a solid phase carrier coated with MC-LR-VHH-1.

[0143] The preparation method of microcystin-LR peroxidase (MC-LR-HRP) in this example is as follows:

[0144] a: Activation of MC-LR: Dissolve 1 mg of MC-LR standard in 0.1 mL of dimethyl sulfoxide, and add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to obtain a mixed solution A, in which the molar ratio of MC-LR to NHS, EDC is: MC-LR to NHS, EDC = 1:1:2.

[0145] b: Dissolve 10 mg of HRP in 2 mL of phosphate buffer (0.05 M pH 7.2) containing 5 mg of activated MC-LR, add 3 mL of 1% (v / v) glutaraldehyde solution after dissolution, let stand at room temperature for 3 to 4 hours, then dialyze at 4°C to remove glutaraldehyde to obtain MC-LR-HRP (i.e., microcystin-LR-peroxidase marker).

[0146] In this embodiment, the solid phase carrier coated with MC-LR-VHH-1 has a MC-LR-VHH-1 antibody concentration range of 0.1 to 10 μg / mL, which can be 0.1 μg / mL, 0.5 μg / mL, 0.8 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL and any concentration within these numerical values ​​and numerical ranges, and those skilled in the art can adjust according to actual needs.

[0147] In this embodiment, the concentration range of the MC-LR peroxidase marker is 0.01 to 10 μg / mL, and can be any concentration of 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 0.8 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL and any concentration of these numerical values ​​and numerical ranges, and those skilled in the art can adjust according to actual needs.

[0148] Example 4 A method for detecting MC-LR

[0149] This embodiment provides a MC-LR enzyme-linked immunosorbent assay method, which specifically includes the following steps:

[0150] S1. Sample treatment: centrifuge the water sample at 12000 rpm for 15 min, take the supernatant and dilute it 10 times;

[0151] S2. Use the kit of Example 3

[0152] S2.1 Sample addition: Add 100 μL MC-LR-HRP solution and 100 μL MC-LR series standard solution to the standard wells of the ELISA plate; add 100 μL MC-LR-HRP solution and 100 μL MC-LR sample solution to the sample wells of the ELISA plate; incubate the ELISA plate at 37°C for 30 min;

[0153] S2.2 Washing: Discard excess liquid in the wells and wash three times with phosphoric acid buffer;

[0154] S2.3 Color development and termination: Add 100 μL of color development substrate to each reaction well, incubate at 37°C for 20 min, and then add 100 μL of 2 M sulfuric acid stop solution;

[0155] S2.4 Detection: Place the ELISA plate in an ELISA reader to measure the absorbance at OD value 450. The result is calculated as follows:

[0156]

[0157] In the formula: B is the average absorbance value of the standard solution or sample solution; B0 is the average absorbance value of the 0 concentration blank.

[0158] The corresponding concentration of 10% inhibition rate was used as the minimum detection limit, and the inhibition rate of 20% to 80% was used as the quantitative linear detection range. The logarithm of the mass concentration of the MC-LR standard solution was used as the abscissa (X), and the inhibition rate was used as the ordinate (Y) to draw a standard curve, and the content of MC-LR in the sample was measured.

[0159] Example 5 Method Sensitivity and Precision Determination

[0160] Sensitivity determination: The logarithmic value of the mass concentration of the MC-LR standard solution (0 ng / mL, 1.56 ng / mL, 3.13 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL) was used as the abscissa (X), and the inhibition rate was used as the ordinate (Y) to draw a standard curve. The sensitivity (IC 50 ) was 2.87 ng / mL, R 2 =0.998, with good linear relationship, the minimum detection limit is 0.422 ng / mL, and the linear range is 0.9~12.89 ng / mL.

[0161] Precision determination: The intra-plate variation coefficient of 10 ELISA plates did not exceed 6%, and the intra-plate and inter-plate variation coefficients did not exceed 10%, indicating that the method of the present invention has good precision and high stability.

[0162] Example 6 Determination of cross-reaction rate

[0163] The structural analogs of MC-LR were prepared into solutions with the same mass concentration as the test objects, and their IC50 was determined by the method of Example 4. The structural analogs of MC-LR include microcystin-RR (MC-RR), microcystin-YR (MC-YR), ochratoxin (OTA), and zearalenone (ZEN). The results are shown in the following table:

[0164] Table 9: Cross-reactivity of MC-LR-VHH-1 with structural analogs of MC-LR

[0165] Microcystins IC50(ng / mL) Cross-reaction rate MC-LR 8.28 100％ MC-RR 9.68 85.0％ MC-YR 25.22 32.8％ OTA —— 0％ ZEN —— 0％

[0166] It can be seen from the above table that the cross-reaction rates of MC-LR-VHH-1 with MC-RR and MC-YR are 85% and 32.8% respectively, and there is basically no cross-reaction with OTA and ZEN, indicating that the antibody has good specificity and can identify MC-LR, MC-YR, and MC-RR. Therefore, it can detect microcystins in water.

[0167] Comparative Example

[0168] The commercially available MC-LR antibody and the antibody of the present invention MC-LR-VHH-1 were selected to compare the binding performance with the solid phase carrier, and the test process was as follows:

[0169] The commercially available MC-LR antibody (the commercially available antibody is synthesized by Nanjing Zhongding Biotechnology Co., Ltd. using recombinant antibody protein expression technology) and the MC-LR-VHH-1 antibody prepared by the present invention are respectively dissolved in 0.01M carbonate buffer (pH 9.6) and mixed evenly to make the final concentration of the antibody 2μg / mL to prepare an antibody coating solution; 100μl of the antibody coating solution is added to each well, incubated at room temperature overnight, and the excess coating solution is discarded. The wells are washed with 0.01M pH 7.4 phosphate buffer (PBS), and 200μl of 0.05M pH 7.4 PBS containing 20g / L bovine serum albumin (BSA) is added, incubated at 4°C overnight, washed, and dried to prepare solid phase carriers 1 to 4 coated with different MC-LR antibodies, wherein solid phase carrier-1 is coated with MC-LR-VHH-1 antibody, solid phase carrier-2 is coated with commercially available antibody 1, solid phase carrier-3 is coated with commercially available antibody 2, and solid phase carrier-4 is coated with commercially available antibody 3. The binding stability of the antibody to the solid phase carrier was tested by referring to the method of Example 3. The results are shown in the following table:

[0170] Table 10: Binding stability of different antibodies to solid phase carriers at 37°C

[0171]

[0172] Table 11: Binding stability of different antibodies and solid phase carriers at 4°C

[0173]

[0174] As can be seen from the results in the above table, the stability of commercial antibody 1 after being placed at 4°C and 37°C for 8 weeks is 70%, the stability of commercial antibodies 2 and 3 after being placed at 4°C and 37°C for 8 weeks is less than 80%, and the stability of MC-LR-VHH-1 of the present invention after being placed at 4°C and 37°C for 8 weeks is still maintained at 90%. This shows that MC-LR-VHH-1 can stably and firmly bind to the solid phase carrier. By comparing the amino acid sequences of the nano antibody of the present invention with those of the commercially available antibodies, it is found that the amino acid sequence of the nano antibody of the present invention contains more hydrophobic amino acids, such as glycine, alanine, valine, leucine and proline, etc., which further increases the number of disulfide bonds with the solid surface, thereby improving the binding ability with the solid phase carrier.

[0175] Amino acid sequence of commercially available antibody 1:

[0176] EVQLLESGGSVQAGGSLRSQTVSGSISRNHDSRWCGYWFRQVPGKEREGVA

[0177] GAFHMGVREYYYADSVKDRHDNAMNTMYLQMNFLNPEDTAMYYCAAHRTERWWQCGANGSEYKYWGQGTLVTVA (SEQ ID NO: 10);

[0178] Amino acid sequence of commercially available antibody 2:

[0179] QVQLVESGGGEVQPGGSLRLSCAAAGFTFSTEPMDWVRQAPGKGLEWVSSIS

[0180] SDGGRTLYRDSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYFCARSDGEARGQGTQVSVSSAHHSEDPHGQAGQ (SEQ ID NO: 11);

[0181] Amino acid sequence of commercially available antibody 3:

[0182] EVQVVESMGGLVQTMNSLRLSCGISGPTNTIRRIGWARQAPDAQRTWVASVR

[0183] TNGTATYTESVTGRFTISRDNAGKTMVLQMNNLEPADTAIYRCYVRRLMYH ARIPQITWGEGTQVTVLA (SEQ ID NO: 12).

[0184] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A microcystin LR nanobody, characterized in that: The amino acid sequence of the nanobody includes three variable regions CDR1, CDR2, and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:

3.

2. The Nanobody according to claim 1, characterized in that The nanobody also includes four framework regions FR1, FR2, FR3 and FR4; the amino acid sequence of the FR1 is shown in SEQ ID NO: 4, the amino acid sequence of the FR2 is shown in SEQ ID NO: 5, the amino acid sequence of the FR3 is shown in SEQ ID NO: 6, and the amino acid sequence of the FR4 is shown in SEQ ID NO:

7.

3. A nucleic acid sequence encoding a Nanobody according to claim 1 or 2, characterized in that: The nucleic acid sequence is shown in SEQ ID NO:

9.

4. A microcystin LR immunoassay kit, characterized in that: The kit comprises: (1) Solid phase carrier: coated with the Nanobody according to claim 1 or 2, wherein the antibody concentration is 0.1-10 μg / mL; (2) Microcystin LR standard solution: concentration 0.1~100 ng / mL; (3) Microcystin LR peroxidase marker solution, concentration 0.01~10 μg / mL; (4) Enzyme-labeled reaction substrate colorimetric solution; (5) Reaction termination solution; (6) Detergent; (7) Sample diluent.

5. The detection kit according to claim 4, characterized in that The preparation method of the solid phase carrier coated with the microcystin LR nanobody is as follows: A: Add the microcystin LR nanoantibody solution according to claim 1 or 2 to a carbonate buffer solution to make the final antibody concentration 1-2 μg / mL, mix well, and prepare an antibody coating solution; B: Add the antibody coating solution to the solid phase carrier, keep warm at 25-37°C for 1-3 hours, discard the excess coating solution, wash with phosphate buffer, block, discard the excess blocking solution, wash and dry, and obtain a solid phase carrier coated with microcystin LR nanoantibody.

6. The detection kit according to claim 5, characterized in that The carbonate buffer has a concentration of 0.01 M and a pH value of 9.6; the phosphate buffer has a concentration of 0.05 M and a pH value of 7.2; and the blocking solution is a phosphate buffer containing 0.05%-0.5% (m / v) bovine serum albumin, a concentration of 0.05 M and a pH value of 7.

4.

7. The detection kit according to claim 4, characterized in that The preparation method of the microcystin LR peroxidase marker solution is as follows: a: Dissolve the microcystin LR (MC-LR) standard in dimethyl sulfoxide, and then add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to obtain a mixed solution A, wherein the molar ratio of MC-LR to NHS and EDC in the mixed solution A is: MC-LR:NHS:EDC=1:1:2; b: Add mixed solution A and glutaraldehyde solution into phosphate buffer, dialyze to remove glutaraldehyde, and obtain the microcystin LR peroxidase marker.

8. The detection kit according to claim 4, characterized in that 100 mL of enzyme-labeled reaction substrate colorimetric solution in the kit contains the following components: 0.5-0.7% (m / v) 3,3',5,5'-tetramethylbenzidine (TMB), 1-3% (m / v) citric acid, 1-4% (v / v) dimethyl sulfoxide, and 100 mL of distilled water; The reaction termination solution is 2M sulfuric acid solution; The washing solution is a phosphate buffer with a concentration of 0.25M and a pH of 7.4, which contains 0.1% (v / v) polysorbate-20; The sample diluent is a phosphate buffer solution with a concentration of 0.25 M and a pH of 7.2, which contains 0.05% Tween-20 (v / v).

9. Use of the nanobody according to any one of claims 1 to 2 or the detection kit according to any one of claims 5 to 8 in detecting microcystin LR.

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