A microcystin immunoassay based on α-type anti-idiotypic antibodies

Abstract

The present invention provides a novel immunoassay method for microcystin using α-type anti-idiotypic antibodies. This novel immunoassay method, established using an indirect competitive ELISA method and utilizing the anti-idiotypic antibodies represented by amino acid sequences 1 to 3, has the potential to be used for rapid screening and detection of microcystin in drinking water.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunological detection, and in particular relates to a microcystin immunoassay method based on α-type anti-idiotypic antibodies. Background Art

[0002] Hughes et al. first isolated a hepatotoxic "rapid death factor" from an extract of Microcystis aeruginosa NRC-1. This hepatotoxic factor, later renamed microcystins, has protein phosphatase inhibitory and potent hepatocarcinogenic properties. Microcystins (MCs) produce secondary metabolites that accumulate in lakes, oceans, and other water bodies, leading to a decline in water quality and a significant impact on the habitats of various aquatic organisms (fish and shrimp). This also threatens drinking water safety and the health of animals and humans. Numerous human and animal poisoning incidents caused by microcystins have been reported. Numerous methods for detecting microcystins have been developed, primarily instrumental analysis, bioassays, protein phosphatase inhibition, and immunoassays. Immunoassays, with their high sensitivity and high throughput, have become the fastest and most effective method for detecting microcystins.

[0003] Based on their functions and the serological reactivity between idiotypes and anti-idiotypes, Bona classifies anti-idiotypic antibodies into four subtypes: α, β, γ, and δ. Antibodies such as Ab2α recognize a unique site (i.e., the framework of the variable region) that is separate from the antigenic determinant, allowing it to bind to the antibody without interfering with hapten binding, making them hapten-inhibiting AIDs. Antibodies such as Ab2β bind to a unique site on the paratope and compete with the hapten for binding, making them hapten-inhibiting AIDs, known in immunology as antigen "internal images." Research has shown that certain Ab2β antibodies can be used as alternatives to highly toxic small molecule haptens to establish nontoxic immunoassays. Antibodies such as Ab2γ recognize idiotypic determinants associated with the antigen-binding site on the antibody molecule and compete or partially compete with the hapten for binding, but lack the "internal images" of Ab2β, making them hapten-inhibiting AIDs. Antibodies such as Ab2δ recognize the Fc fragment of an antibody and simultaneously bind to the antigen-binding site. Currently, the commonly used methods for preparing anti-idiotypic antibodies include polyclonal antibody technology, monoclonal antibody technology, and genetically engineered antibody technology.

[0004] The present invention is inspired by the report of Mu et al. (Mu X, Tong Z, Huang Q, et al. Nano-magneticimmunosensor based on staphylococcus protein a and the amplification effectof HRP-conjugated phage antibody[J]. Sensors, 2015, 15(2):3896-910.). Since the major phage capsid protein exists in multiple copies (such as the major capsid protein PVIII contains more than 2700 copies), it can bind to multiple anti-phage secondary antibodies, which may amplify the detection signal and thus improve the detection sensitivity. Inspired by this, a new MC immunoassay method based on α-type anti-idiotypic phage nanoantibodies was designed.

[0005] The inventors used the anti-MC-LR monoclonal antibody prepared by early laboratory screening as the target molecule, successfully screened out α-type anti-idiotypic antibodies in the camel-derived nanoantibody library, and established an indirect competitive ELISA detection method based on α-type anti-idiotypic antibodies. Summary of the Invention

[0006] The purpose of the present invention is to provide a novel MC immunoassay method based on α-type anti-idiotypic phage nanoantibodies to achieve more effective microcystin detection.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A microcystin immunoassay method based on α-type anti-idiotypic antibodies comprises the following steps:

[0009] 1) Coating: Coat the ELISA plate with diluted MC-LR-BSA;

[0010] 2) Blocking: Block the coated ELISA plate wells;

[0011] 3) Sample addition: Add a mixture of MC-LR and anti-MC-LR monoclonal antibody to the blocked wells of the ELISA plate and allow to react for a period of time;

[0012] 4) Add anti-idiotypic antibody: Add anti-idiotypic antibody to the wells of the ELISA plate and react for a period of time;

[0013] 5) Add secondary antibody: Add HRP-labeled secondary antibody to the wells of the ELISA plate after the reaction is completed;

[0014] 6) Color development: Add TMB substrate solution for color development, and read the OD value using a microplate reader after termination. 450 value,

[0015] Wherein, the anti-idiotypic antibody is selected from any one of amino acid sequences 1 to 3.

[0016] Specifically, in step 1) of the method of the present invention, the coating process includes: coating the wells of the ELISA plate with 2 μg / mL MC-LR-BSA diluted in CBS, with a coating volume of 100 μL / well, and coating at 4°C overnight.

[0017] Specifically, in step 2) of the method of the present invention, the blocking process includes: washing the coated ELISA plate wells with 0.05% PBST washing solution three times, blocking with 4% MPBS, 300 μL / well, and incubating at 37° C. for 1 hour.

[0018] Specifically, in step 3) of the method of the present invention, preferably, the mixture of MC-LR and anti-MC-LR monoclonal antibody is incubated for a period of 0.5-6 hours before being added to the wells of the ELISA plate. More preferably, the incubation time is 3-6 hours.

[0019] Specifically, in step 3) of the method of the present invention, the sample addition process includes: washing the blocked ELISA plate wells three times with 0.05% PBST washing solution, adding 50 μL of purified diluted monoclonal antibody solution and 50 μL of MC-LR standard solution diluted to different multiples, and incubating at 37°C for 1 hour, wherein the concentration of the diluted monoclonal antibody solution is 2.857 ng / mL, and the concentration of the MC-LR standard solution is 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, and 0 ng / mL.

[0020] Specifically, in step 4) of the method of the present invention, the process of adding the anti-idiotypic antibody includes: washing the wells of the ELISA plate with 0.05% PBST washing solution three times, adding 50 μL of 4% MPBS and 50 μL of anti-idiotypic phage nanoantibody rescue supernatant diluted to a concentration of 0.05~0.1 μg / mL, and incubating at 37°C for 1 hour.

[0021] Specifically, in step 5) of the method of the present invention, the process of adding the secondary antibody includes: washing the wells of the ELISA plate with 0.05% PBST washing solution three times, then diluting the HRP-labeled anti-M13 secondary antibody with 4% MPBS to a concentration of 498 ng / mL, adding it to the wells of the ELISA plate, 100 μL / well, and incubating at 37°C for 1 hour.

[0022] Specifically, in step 6) of the method of the present invention, the color development process includes: washing the wells of the ELISA plate with 0.05% PBST washing solution three times, adding the freshly prepared TMB substrate solution, 100 μL / well, developing at 37°C for 15 minutes, and then adding 2M H2SO4 to terminate the reaction, 50 μL / well, and reading the OD450 value with a microplate reader, wherein the TMB substrate solution is 10 mL CPBS + 100 μL TMB + 25 μL 0.65% hydrogen peroxide.

[0023] Compared with existing technologies, the present invention offers the following advantages: First, it provides a novel immunoassay for microcystin LR based on α-type anti-idiotypic antibodies. The established method was validated using spike-recovery experiments, and the results demonstrated that the novel immunoassay for microcystin LR is accurate and effective. The sensitivity of this novel immunoassay is 5.6 times higher than that of traditional indirect competitive ELISA, and this assay has potential commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of polyclonal ELISA (left) and monoclonal ELISA (right) for MC-LR anti-idiotypic antibodies are shown.

[0025] Figure 2 The following are the results of bacterial liquid PCR identification and amino acid sequence sequencing of 4 positive clones;

[0026] Figure 3 is the dose-response curve for different incubation times;

[0027] Figure 4 is the standard inhibition curve of the competition ELISA of phage clone E8;

[0028] Figure 5 It is a table showing the results of the cross-reactivity rate determination of MC-LR anti-idiotypic antibodies;

[0029] Figure 6 This is a table showing the results of the spike recovery experiment using ELISA and HPLC to detect MC-LR in tap water. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the specific embodiments and drawings of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] The present application adopts an indirect competitive ELISA method and uses the α-type anti-idiotypic antibody E8 to detect microcystin LR.

[0032] The present invention first provides a novel immunoassay for microcystin LR based on α-type anti-idiotypic antibodies. The coupled complex MC-LR-BSA is coated onto microtiter wells. A mixture of MC-LR and anti-MC-LR monoclonal antibody at varying gradient dilutions is added. Free MC-LR and immobilized MC-LR compete for the anti-MC-LR monoclonal antibody simultaneously. Phage nanobody supernatant is then added, followed by HRP-labeled anti-M13 secondary antibody. The reaction is developed using TMB substrate and terminated with 2 M H2SO4. The absorbance is read at 450 nm using a microplate reader. The color reaction confirms that phage antibody E8 specifically binds to the anti-MC-LR monoclonal antibody. Subsequently, different coating antigen and antibody concentrations and incubation times are optimized to establish an indirect competitive ELISA immunoassay for MCs based on α-type anti-idiotypic antibodies.

[0033] The established method was verified using a spike-recovery test, and the results showed that the established new microcystin immunoassay method was accurate and effective, and that this detection model had potential commercial value.

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0035] Sources of reagents and instruments involved in the examples:

[0036] Anti-MC-LR hybridoma cells (CCTCC No: C2019285) were prepared in our laboratory. Camel-derived phage nanobody library was purchased from Chengdu Apak Biotechnology Co., Ltd. The library capacity is 2×10 10 The phage display plasmid is pComb3XSS, the resistance is ampicillin (Amp), and the host bacteria is E. coli TG1.

[0037] MC-LR and MC-YR standards (purity 95%) were purchased from Abraxis, USA, and MC-RR and MC-LA standards (purity 95%) were purchased from the Environmental Protection Research and Testing Institute of the Ministry of Agriculture.

[0038] The 96-well ELISA plate was purchased from Corning, USA.

[0039] Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG was purchased from KPL, USA.

[0040] 12% precast gel, Tris-MOPS protein electrophoresis buffer, and Western blot TMB staining solution (ChromoSensor™ One-Solution TMB Substrate) were purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0041] Skim milk powder was purchased from Solaibao Biotechnology Co., Ltd.

[0042] All other chemical reagents and organic solvents used in the following examples were of domestic analytical grade.

[0043] Tabletop refrigerated centrifuge (Eppendoff), electrophoresis tank (Beijing Junyi JY-SCZ2+), electrophoresis apparatus (Beijing Liuyi Instrument Factory JY600E), small tabletop centrifuge (Eppendorff Centrifuge 5424R), microplate reader (Thermo Multiskan GO), plate washer (Thermo Wellwash), ultra-low temperature refrigerator (Haier), water purifier (Millipore Direct-Q 3UV), balance (Liangping Instrument FA2004), pH meter (Sartorius PB-10), clean bench (Sudong Antai Co., Ltd. SW-CJ-1F), constant temperature incubator (Jinghong Instrument Co., Ltd. DNP-9082).

[0044] Example 1 Screening of anti-idiotypic nanobodies against microcystin LR

[0045] Refer to the instructions of the commercial natural nanoantibody library for screening: the screening used a sterilized 96-well ELISA plate, 100 μL per well, at least 4 wells were prepared, and the number of phages put in was 10 9 about.

[0046] 1) Prepare a solution of purified monoclonal antibody diluted to a final concentration of 10 µg / mL in sterile PBS, coat at a volume of 100 µL / well, swirl carefully until the bottom of the microwells are completely wetted, and coat overnight at 4°C. 2) The next day, discard the coating solution, wash the plate three times with sterile PBS, fill each well with blocking solution (BSA-PBS), and incubate at 37°C for 1 hour. 3) Remove the blocking solution as described above, quickly wash the plate three times with PBS buffer, take 10 µL of phage library, dilute it with 390 µL of sterile PBS buffer, add it to the four coated enzyme-labeled wells, and incubate at 37°C for 2 hours. 4) Discard phage that have not bound to the target molecule and wash five times with sterile 0.05% PBST buffer as described in 3). 5) Add 100 µL of nonspecific buffer (0.1 M Glycine-HCl (pH 2.2-2.5) to each well and incubate at 37°C for 10 minutes. After eluting the bound phage for 10 min, the eluate was transferred to a sterile 1.5 mL centrifuge tube and neutralized with 30 μL of 1 M Tris-HCl (pH 9.1) buffer. 6) A small amount (10 μL) of the eluate was serially diluted for titer determination. A portion of the eluate was stored at 4°C for later use. The remaining eluate was pooled and amplified for the next round of affinity panning, resulting in three rounds of screening. 7) The library amplification results were subjected to the second and third rounds of panning, varying the panning conditions. Steps 1) to 5) were repeated, reducing the antigen coating concentration to 5 μg / mL and 2 μg / mL, respectively. BSA-PBS and OVA-PBS blocking solutions were used alternately, and the binding time was reduced to 1 hour and 0.5 hours, respectively. The number of PBST washes was increased to 10 and 15, respectively. The remaining steps were the same as above.

[0047] Example 2 Identification of Anti-Idiotypic Antibodies to Microcystin LR

[0048] Anti-idiotypic antibodies were identified using polyclonal ELISA and monoclonal ELISA. The specific steps for the polyclonal ELISA were as follows: 1) Coat a 96-well microtiter plate with purified monoclonal antibody at a concentration of 2 μg / mL, 100 μL / well, and incubate at 4°C overnight. 2) The next day, wash the plate three times with PBST and block with 4% MPBS at 300 μL / well for 1 hour at 37°C. 3) Wash the plate as above and add the same titer (2 × 10 6) The phage supernatant obtained by PEG / NaCl precipitation in each round of screening was added at 100 μL / well in triplicate and incubated at 37°C for 1 h; 4) Wash the plate as above, dilute the horseradish peroxidase (HRP)-labeled anti-M13 secondary antibody 5000-fold with 4% MPBS, add it to a 96-well plate at 100 μL / well, and incubate at 37°C for 1 h; 5) Wash the plate as above, add the freshly prepared substrate solution TMB (10 mL CPBS + 100 μL TMB + 25 μL 0.65% hydrogen peroxide), 100 μL / well, and develop at 37°C for 15 min; 6) Add 2 M H2SO4 to terminate the reaction at 50 μL / well, and read the OD450 value using a microplate reader.

[0049] The specific steps of monoclonal ELISA are as follows: 1) Coat a 96-well microtiter plate with purified monoclonal antibody at a coating concentration of 2 μg / mL and a coating volume of 100 μL / well, and incubate at 4°C overnight; 2) Wash the plate three times with PBST the next day, block with 4% MPBS, 300 μL / well, and incubate at 37°C for 1 h; 3) Wash the plate as above, add 50 μL of phage supernatant and 50 μL of PBS, mix well, and incubate at 37°C for 1 h; 4) Wash the plate as above, dilute horseradish peroxidase (HRP)-labeled anti-M13 secondary antibody 5000-fold with 4% MPBS, add to a 96-well plate, 100 μL / well, and incubate at 37°C for 1 h; 5) Wash the plate as above, add freshly prepared substrate solution TMB (10 mL CPBS + 100 μL TMB + 25 μL 0.65% hydrogen peroxide), 100 μL / well, and develop color at 37°C for 15 min. 6) Add 2M H2SO4 (50 μL / well) to terminate the reaction and read the OD450 value using a microplate reader.

[0050] Anti-idiotypic antibodies were further identified using culture PCR and DNA and amino acid sequencing. The specific steps were as follows: 10 μL of each culture of a single colony identified as positive by monoclonal ELISA was transferred to 2×TY-AG liquid medium. After overnight incubation, the cells were used for culture PCR and DNA and amino acid sequencing. The reaction system for a 20 μL culture PCR reaction was as follows:

[0051] Bacterial solution (template) 1 μL 2×Tap PCR Master Mix 10 μL Specific upstream primer 1 μL Specific downstream primers 1 μL ddH2O 7μL Total volume 20 μL

[0052] The PCR amplification reaction conditions were set as follows:

[0053]

[0054] The PCR products were identified using 1% agarose gel to verify whether the target fragment size of the PCR amplified product was consistent with the expectation.

[0055] DNA and amino acid sequence sequencing: positive clones were cultured overnight, and 500 μL of culture medium was aspirated the next day for labeling and sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0056] Figure 1 The results of polyclonal ELISA (left) and monoclonal ELISA (right) for MC-LR anti-idiotypic antibodies are shown. Figure 1 (Left) Results show that after three rounds of screening and enrichment, the ability of the phage nanobody to bind to the MC-LR monoclonal antibody gradually increased. After the third round of screening, the polyclonal ELISA results showed that it had the strongest binding ability. After the three rounds of screening, 300 single colonies were selected for monoclonal ELISA identification, and 4 positive clones were identified ( Figure 1 Right). Among them, F7 and E8 monoclonal antibodies had the highest positive reaction values. The positive clones screened in this experiment showed binding to the antibody without affecting the binding of MC-LR to the antibody, indicating no inhibitory effect. These four anti-idiotypic antibodies can be identified as Ab2α.

[0057] Figure 2 The following table shows the results of bacterial culture PCR identification and amino acid sequencing of four positive clones. After agarose gel electrophoresis, the PCR products of the four positive clones were placed on a gel imaging analyzer and photographed. Each clone showed a single band, consistent with the expected target gene (358 bp). Amino acid sequencing results showed that the amino acid sequences of F7 and E8 were identical. Ultimately, three positive clones were obtained, each with a different amino acid sequence.

[0058] Example 3 Optimization of incubation time

[0059] The indirect competitive ELISA method was used to detect microcystins. The specific steps are as follows:

[0060] 1) Coating: Coat a 96-well microtiter plate with 2 μg / mL MC-LR-BSA diluted in CBS (100 μL / well) at 4°C overnight.

[0061] 2) Blocking: The next day, wash the plate three times with 0.05% PBST, then block with 4% MPBS (300 μL / well) and incubate at 37°C for 1 hour.

[0062] 3) Sample addition: Wash the plate as above, then add 50 μL of purified diluted mAb pre-incubated for 0.5 h, 1 h, 3 h, and 6 h, and 50 μL of a mixture of MC-LR standards diluted to different multiples. Set up three replicates for each concentration, 100 μL / well, and set up a blank control (100 μL PBS). Incubate at 37°C for 1 h.

[0063] 4) Add secondary antibody: Wash the plate as above, dilute the horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody 5000-fold with 4% MPBS, add 100 μL / well to a 96-well plate, and incubate at 37°C for 1 h.

[0064] 5) Color development: Wash the plate as above, add freshly prepared TMB substrate solution (10 mL CPBS + 100 μL TMB + 25 μL 0.65% hydrogen peroxide) at 100 μL / well, and develop color at 37°C for 15 min.

[0065] 6) Termination: Add 2M H2SO4 to terminate the reaction, 50 μL / well, and read the OD450 value using a microplate reader.

[0066] The logarithm of MC-LR concentration was used as the horizontal axis and B / B0 as the vertical axis to generate the MC-LR standard inhibition curve. The inhibitory concentration IC50 was calculated using the four-parameter model. The changes in IC50 of several experimental groups were observed to determine the optimal incubation time.

[0067] Figure 3 Dose-response curves for different incubation times are shown. Assay sensitivity gradually increased with increasing incubation time. IC50 values ​​of approximately 4.61 ng / mL and 4.665 ng / mL were observed at 3 and 6 hours, respectively, representing 2.97-fold and 3.08-fold decreases compared to the IC50 values ​​obtained without incubation. However, the maximum response signal decreased by 50% when the incubation time reached 6 hours. No dose-dependent curve was obtained after overnight incubation, indicating a near-background signal. Therefore, a 3-hour incubation time is the optimal time for achieving optimal sensitivity.

[0068] Example 4 Establishment of a competitive ELISA method for detecting microcystins based on α-type anti-idiotypic antibodies

[0069] The indirect competitive ELISA method was used to detect microcystins. The specific steps are as follows:

[0070] 1) Coating: Coat a 96-well microtiter plate with 2 μg / mL MC-LR-BSA diluted in CBS (100 μL / well) at 4°C overnight.

[0071] 2) Blocking: The next day, wash the plate three times with 0.05% PBST, then block with 4% MPBS (300 μL / well) and incubate at 37°C for 1 hour.

[0072] 3) Sample addition: Wash the plate as above, then add 50 μL of purified diluted mAb pre-incubated for 3 h and 50 μL of a mixture of MC-LR standards diluted to different multiples. Set up three replicates for each concentration, 100 μL / well, and set up a blank control (100 μL PBS). Incubate at 37°C for 1 h.

[0073] 4) Add phage E8: Wash the plate as above, add 50 μL of 4% MPBS and 50 μL of anti-idiotypic phage nanobody E8 rescue supernatant, which is diluted approximately 10,000-fold to a concentration of 0.05-0.1 μg / mL, and incubate at 37°C for 1 hour.

[0074] 5) Add secondary antibody: Wash the plate as above, dilute horseradish peroxidase (HRP)-labeled anti-M13 secondary antibody 5000-fold with 4% MPBS, add 100 μL / well to a 96-well plate, and incubate at 37°C for 1 h.

[0075] 6) Color development: Wash the plate as above, add freshly prepared TMB substrate solution (10 mL CPBS + 100 μL TMB + 25 μL 0.65% hydrogen peroxide) at 100 μL / well, and develop color at 37°C for 15 min.

[0076] 7) Termination: Add 2M H2SO4 to terminate the reaction, 50 μL / well, and read the OD450 value using a microplate reader.

[0077] Figure 4 The inhibition curve for the competition ELISA of phage clone E8 is shown. The median inhibitory concentration (IC50) determined by the phage ELISA is 2.8 ng / mL, with a linear detection range of 1.2-6.9 ng / mL and an IC10 of 0.8 ng / mL. The IC10 of the phage ELISA is 0.8 ng / mL, which meets the WHO limit of 1 ng / mL for MC in drinking water.

[0078] Example 5 Determination of cross-reaction rate

[0079] Prepare serial dilution standard solutions of MC-LR and its structurally similar compounds. Use the E8-based indirect competitive ELISA assay protocol to establish a standard curve for MC-LR and its structurally similar compounds to calculate the IC50 of the inhibitory concentration and cross-reactivity ratio. The lower the cross-reactivity ratio, the greater the specificity of E8 for MC-LR.

[0080] In this study, the cross-reactivity rates of MC-RR, MC-YR, and MC-LA were determined. Figure 5 The IC50 values ​​of the three homologous compounds were 6.895 ng / mL, 6.33 ng / mL, and 14.3 ng / mL, respectively, with corresponding cross-reactivity rates of 99.19%, 107%, and 48%, respectively. These results indicate that E8 has similar recognition abilities for MC-RR and MC-YR, but weaker recognition for MC-LA.

[0081] Example 6 Addition and Recovery Test of MC-LR Toxin

[0082] (1) HPLC method for testing tap water samples

[0083] The determination of microcystins in source water was performed according to the Shanghai Municipal Standard DB31 / T 1178-2019. The following conditions were used: a C18 reversed-phase column (250 × 4.6 mm); a column temperature of 30°C; a mobile phase consisting of formic acid in acetonitrile (0.1% formic acid) and formic acid in water (0.1% formic acid); a flow rate of 0.4 mL / min; UV detection at 238 nm; and an injection volume of 20 μL. A microcystin LR standard was diluted with 20% methanol to concentrations of 10, 5, 2, 1, 0.5, 0.2, 0.1, and 0.01 μg / mL. Samples were injected sequentially according to the test conditions to obtain a microcystin LR calibration curve.

[0084] Laboratory tap water samples were processed through a membrane, and 10 mL of the water sample was spiked with microcystin LR standards at final concentrations of 4, 14, 30, and 60 ng / mL. The samples were freeze-dried to a powder, concentrated 10-fold in 1 mL of 20% methanol, and resuspended for instrumental determination of recovered MC-LR concentrations.

[0085] (2) ELISA method for testing tap water samples

[0086] To 10 mL of blank tap water sample after membrane treatment, add microcystin LR standard with final concentrations of 4, 14, 30, and 60 ng / mL, and mix on a rotary mixer for 3 hours; the mixed sample is directly detected by ELISA.

[0087] Figure 6 The results showed that the average recovery rates of ELISA and HPLC were between 89.28-99.29% and 97.1-114.34%, respectively. The coefficients of variation (CV) of ELISA and HPLC were between 2.85-11.97 and 1.62-4.12, respectively. The correlation coefficient was R 2 =0.9535. This result indicates a good correlation between the two. This new ELISA method has the potential to be used for the rapid screening of MC in drinking water.

[0088] Sequence Listing

[0089] Amino acid sequence 1 (E8 / F7):

[0090] QVQLVESGGGEVQPGGSLRLSCAAAGFTFSTEPMDWVRQAPGKGLEWVSSISSDGGRTLYRDSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYFCARSDGEARGQGTQVSVSSAHHSEDPHGQAGQ

[0091] Amino acid sequence 2 (B5):

[0092] EVQLVESGGGSVQTGGSLRLSCVTSGISTSRECLGWFRQSPGKGREGVAILDSSNEITDHADSVKGRFTISRDTAKNTMYLQMDSLNTEDTGVYTCARSSQGQGTQVTVSSAHHSEDPGQAGQ

[0093] Amino acid sequence 3 (D11):

[0094] EVQLVESGGGSVQAGGSLTLSCALSQYTSHCLAWFREAPGKEREGVAAMDPAGNTYYLDAIKGRFTISKDSANKRLDLQMNSLKPEDTATYKCAGSGGGQGTRVSVSSAHHSEDPGQAGQ

[0095] Anything not described in detail in the present invention is well known to those skilled in the art.

[0096] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified and replaced with equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention. SEQUENCE LISTING <110> Jiangsu University Jiangsu Academy of Agricultural Sciences <120> A microcystin immunoassay based on α-type anti-idiotypic antibodies <130> 123200004660115453 <160> 3 <170> PatentIn version 3.3 <210> 1 <211> 127 <212> PRT <213> Lama glama <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ala Gly Phe Thr Phe Ser Thr Glu 20 25 30 Pro Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Asp Gly Gly Arg Thr Leu Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Asp Gly Glu Ala Arg Gly Gln Gly Thr Gln Val Ser Val 100 105 110 Ser Ser Ala His His Ser Glu Asp Pro His Gly Gln Ala Gly Gln 115 120 125 <210> 2 <211> 123 <212> PRT <213> Lama glama <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Thr Ser Gly Ile Ser Thr Ser Arg Glu 20 25 30 Cys Leu Gly Trp Phe Arg Gln Ser Pro Gly Lys Gly Arg Glu Gly Val 35 40 45 Ala Ile Leu Asp Ser Ser Asn Glu Ile Thr Asp His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Asn Thr Glu Asp Thr Gly Val Tyr Thr Cys 85 90 95 Ala Arg Ser Ser Gln Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala 100 105 110 His His Ser Glu Asp Pro Gly Gln Ala Gly Gln 115 120 <210> 3 <211> 120 <212> PRT <213> Lama glama <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Leu Ser Gln Tyr Thr Ser His Cys Leu 20 25 30 Ala Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala 35 40 45 Met Asp Pro Ala Gly Asn Thr Tyr Tyr Leu Asp Ala Ile Lys Gly Arg 50 55 60 Phe Thr Ile Ser Lys Asp Ser Ala Asn Lys Arg Leu Asp Leu Gln Met 65 70 75 80 Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Lys Cys Ala Gly Ser 85 90 95 Gly Gly Gly Gln Gly Thr Arg Val Ser Val Ser Ser Ala His His Ser 100 105 110 Glu Asp Pro Gly Gln Ala Gly Gln 115 120

Claims

1. A microcystin immunoassay method based on α-type anti-idiotypic antibodies, characterized in that: The method comprises the following steps: 1) Coating: Coat the ELISA plate with diluted MC-LR-BSA; 2) Blocking: Block the coated ELISA plate wells; 3) Sample addition: Add a mixture of MC-LR and anti-MC-LR monoclonal antibody to the blocked wells of the ELISA plate and allow to react for a period of time; 4) Add anti-idiotypic antibody: Add anti-idiotypic antibody to the wells of the ELISA plate and react for a period of time; 5) Add secondary antibody: Add HRP-labeled secondary antibody to the wells of the ELISA plate after the reaction is completed; 6) Color development: Add TMB substrate solution for color development, and read the OD450 value with a microplate reader after termination. Wherein, the anti-idiotypic antibody is selected from any one of amino acid sequences 1 to 3.

2. The microcystin immunoassay method based on α-type anti-idiotypic antibodies according to claim 1, wherein: The coating process includes: coating the ELISA plate wells with 2 μg / mL MC-LR-BSA diluted in CBS, with a coating volume of 100 μL / well, and coating at 4° C. overnight.

3. The microcystin immunoassay method based on α-type anti-idiotypic antibodies according to claim 1, wherein: The blocking process includes: washing the coated ELISA plate wells with 0.05% PBST washing solution for 3 times, blocking with 4% MPBS, 300 μL / well, and incubating at 37° C. for 1 hour.

4. The microcystin immunoassay method based on α-type anti-idiotypic antibodies according to claim 1, wherein: The mixture of MC-LR and anti-MC-LR monoclonal antibody is incubated for a period of 0.5-6 hours before being added to the wells of the ELISA plate.

5. The microcystin immunoassay method based on α-type anti-idiotypic antibodies according to claim 4, characterized in that: The incubation time is 3-6h.

6. The microcystin immunoassay method based on α-type anti-idiotypic antibodies according to claim 1, wherein: The sample loading process includes: washing the blocked ELISA plate wells three times with 0.05% PBST solution, adding 50 μL of purified diluted monoclonal antibody solution and 50 μL of MC-LR standard solution diluted to different multiples, and incubating at 37°C for 1 hour, wherein the concentration of the diluted monoclonal antibody solution is 2.857 ng / mL, and the concentration of the MC-LR standard solution is 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, and 0 ng / mL.

7. The microcystin immunoassay method based on α-type anti-idiotypic antibodies according to claim 1, wherein: The process of adding anti-idiotypic antibodies includes: washing the wells of the ELISA plate with 0.05% PBST three times, adding 50 μL of 4% MPBS and 50 μL of anti-idiotypic phage nanoantibody rescue supernatant diluted to a concentration of 0.05~0.1 μg / mL, and incubating at 37°C for 1 hour.

8. The microcystin immunoassay method based on α-type anti-idiotypic antibodies according to claim 1, wherein: The process of adding secondary antibody includes: washing the wells of the ELISA plate with good reaction for 3 times with 0.05% PBST washing solution, diluting HRP-labeled anti-M13 secondary antibody to a concentration of 498 ng / mL with 4% MPBS, adding it to the wells of the ELISA plate, 100 μL / well, and incubating at 37°C for 1 hour.

9. The microcystin immunoassay method based on α-type anti-idiotypic antibodies according to claim 1, wherein: The color development process includes: washing the wells of the ELISA plate with 0.05% PBST three times, adding freshly prepared TMB substrate solution, 100 μL / well, developing at 37°C for 15 minutes, and then adding 2M H2SO4, 50 μL / well, to terminate the reaction, and reading the OD450 value using a microplate reader. The TMB substrate solution is 10 mL CPBS + 100 μL TMB + 25 μL 0.65% hydrogen peroxide.

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