A method for simultaneous determination of microcystins and nodularins

Abstract

The application discloses a method for simultaneously determining microcystin and nodularin, and comprises the following steps: (1) preparing an artificial antigen; (2) preparing Eu 3+ labeling antigen and Sm 3+ labeling antigen; (3) coating goat anti-mouse IgG antibody on a solid carrier, and then preparing a series of mixed standard solutions; (4) adding reaction solutions in the solid carrier coated with the goat anti-mouse IgG antibody to perform direct competitive immune reaction; (5) respectively determining fluorescence intensities by using a time-resolved fluorescence detector; and (6) calculating concentrations of the microcystin and the nodularin in samples according to a standard curve. The determination method can realize single-time testing of simultaneously quantifying the two toxin concentrations in a short time, and significantly improves detection efficiency. The method does not need complex sample pretreatment and expensive instruments, is simple in operation, low in cost, high in detection sensitivity and strong in specificity, and is suitable for routine monitoring of environmental water samples and emergency detection of blue-green algae blooms.

Description

Technical Field

[0001] This invention relates to the field of environmental monitoring and immunoassay technology, and in particular to a method for the simultaneous determination of microcystin and columnar cystin. Background Technology

[0002] Cyanobacterial toxins are a large class of toxic chemicals produced by cyanobacteria and widely found in freshwater bodies. They possess hepatotoxicity, neurotoxicity, and cytotoxicity, posing a serious threat to human health. Developing efficient and reliable detection methods is one of the effective means of preventing and warning of cyanobacterial toxin risks. Microcystins (MCs) are the most widely distributed and extremely harmful cyanobacterial toxins globally. In recent years, with the rapid invasion of Cyclostome from tropical to temperate regions, Cyclostome toxins (CYNs) are gradually becoming another type of cyanobacterial toxin threatening the water supply security of many countries.

[0003] In natural water bodies, the dominant species in cyanobacterial blooms are often not a single species. The coexistence of multiple cyanobacteria leads to the widespread coexistence of multiple cyanobacterial toxins in the water. In recent years, some large lakes and reservoirs in my country have seen cases where microcystin and cylindricalotoxin can be detected simultaneously. Therefore, the compound pollution of cyanobacterial toxins is a new challenge facing freshwater bodies across the country.

[0004] Monitoring cyanobacterial toxins has always been a research hotspot internationally. Utilizing their physiological and chemical properties, such as molecular weight, chromophores, and reactivity, a series of methods have been developed for determining the concentration of cyanobacterial toxins in water bodies. Most of these methods are for single toxin types.

[0005] Studies have employed liquid chromatography-tandem mass spectrometry (LC-MS / MS) to analyze multiple cyanobacterial toxins, including microcystins and columnar phycotoxins, in a single test. However, this method requires very expensive equipment, complex sample preparation, and must be operated by professionals. Immunoassays based on polyclonal or monoclonal antibodies are also widely used for the detection of cyanobacterial toxins. Among them, enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical mode in practice. Compared with chemical analysis methods, ELISA requires less sample volume, does not require sample pretreatment, and the test results can be obtained within 2 hours. Therefore, it can easily determine the concentration of cyanobacterial toxins in water samples and plays an important role in the emergency monitoring of toxic cyanobacterial blooms. However, ELISA is also only for single toxin types.

[0006] Time-resolved fluoroimmunoassay (TRFIA) uses rare earth lanthanide ions as markers and is an ultra-micro quantitative immunoassay technique superior to ELISA. Because different rare earth lanthanides have significant differences in wavelength and decay time, it can realize multi-channel detection of different substances in the same sample. Currently, dual-labeled TRFIA is mainly used in the field of clinical diagnosis, and there are no reports of its application in the field of environmental monitoring.

[0007] Therefore, there is currently a lack of a method that requires low operator skill and can simultaneously and rapidly determine two types of cyanobacterial toxins. There is an urgent need to develop new and efficient detection methods to obtain the concentrations of two toxins in a single measurement, thereby providing basic data for water supply safety more quickly and effectively, and meeting the needs of environmental practitioners and scientific researchers in conducting health management and risk assessment. Therefore, this invention proposes a method for the simultaneous determination of microcystin and columnar cyanobacterial toxins to solve the problems existing in the prior art. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to propose a method for the simultaneous determination of microcystin and Cyclophyta toxin. This method, for the first time, applies dual-label time-resolved fluorescence immunoassay to the joint detection of two cyanobacterial toxins in environmental water bodies. It enables the simultaneous quantification of the concentrations of both toxins in a single test within a short time, significantly improving detection efficiency. It eliminates the need for complex sample pretreatment and expensive instruments, is simple to operate, has low cost, and exhibits high detection sensitivity and specificity. It is suitable for routine monitoring of environmental water samples and emergency detection of cyanobacterial blooms.

[0009] To achieve the objectives of this invention, the invention is implemented through the following technical solution: a method for simultaneous determination of microcystin and pterocytoxin, comprising the following steps: Step 1: Prepare microcystin-bovine serum albumin conjugate and cytosporin-bovine serum albumin conjugate separately; Step 2: Using lanthanide elements Eu 3+ Labeling microcystin-bovine serum albumin conjugates to obtain Eu 3+ Labeling antigens with lanthanide elements Sm 3+ Labeling of *Cyclophorus toxin*-bovine serum albumin conjugate yields Sm 3+ Labeled antigens; Step 3: Coat the goat anti-mouse IgG antibody onto a solid-phase carrier, block it, and set it aside for later use. Then prepare a series of mixed standard solutions containing microcystin and columnar cytotoxin. Step 4: Add the reaction solution sequentially to the solid-phase carrier coated with goat anti-mouse IgG antibody, and carry out a direct competitive immune reaction at room temperature for 30-60 minutes; Step 5: After the reaction is complete, wash the sample, add the enhancement solution, and use a time-resolved fluorescence detector to detect fluorescence in Eu. 3+ and Sm 3 + The fluorescence intensity was measured at the characteristic emission wavelength, and the concentrations of microcystin and columnar cystin in the sample were calculated based on the standard curve.

[0010] A further improvement is that the preparation of the microcystin-bovine serum albumin conjugate in step one involves first modifying bovine serum albumin with ethylenediamine to obtain EDA-BSA, then reacting the microcystin with EDA-BSA in the presence of carbodiimide, followed by dialysis and lyophilization to obtain the final product.

[0011] A further improvement is that the preparation of the *Strombyx moritoxin*-bovine serum albumin conjugate in step one involves dissolving bovine serum albumin in phosphate buffer, adding *Strombyx moritoxin* and formaldehyde, stirring the mixture in the dark, and then dialysis to obtain the conjugate.

[0012] A further improvement is made in the following: the preparation method of the two labeled antigens in step two involves mixing Eu at a mass ratio of 2-3:1. 3+ The labeling reagent was mixed with the microcystin-bovine serum albumin conjugate at a mass ratio of 1:0.8-1.2, and Sm was added. 3+ The labeling reagent was mixed with the *Strombyx mori* toxin-bovine serum albumin conjugate and reacted overnight at room temperature. The high-fluorescence signal fraction was collected after gel chromatography purification, and a stabilizer was added for storage to obtain Eu. 3+ Labeled antigens and Sm 3+ Labeling antigens.

[0013] A further improvement is that the coating concentration of goat anti-mouse IgG antibody in step three is 5 μg / mL, and the coating conditions are overnight at 37°C or overnight at 4°C.

[0014] A further improvement is that the concentrations of microcystin and pterocytoxin in the series of mixed standard solutions in step three are 0.1 ng / mL, 0.3 ng / mL, 1 ng / mL, 2 ng / mL and 4 ng / mL, respectively.

[0015] A further improvement is that the reaction solution in step four is the sample to be tested or a standard solution, Eu. 3+ Labeled antigen, Sm 3+ Labeled antigens and a mixture containing monoclonal antibodies against microcystin and monoclonal antibodies against cytotoxin; The working dilution of the microcystin monoclonal antibody is 1:10000-20000, and the working dilution of the columnar cystin monoclonal antibody is 1:5000-15000.

[0016] The further improvement lies in: in step five, Eu 3+ The characteristic emission wavelength is 610-620nm, Sm 3+ The characteristic emission wavelength is 640-650 nm; the detection parameters of the time-resolved fluorescence detector are set to Eu. 3+ The channel delay is 0.30-0.50ms, and the window time is 0.30-0.50ms; Sm 3+ The channel delay is 0.04-0.06ms, and the window time is 0.08-0.12ms.

[0017] A further improvement is that the standard curve fitting in step five uses a semi-logarithmic method, with the ratio of fluorescence values ​​of each standard to the negative control as the ordinate and the logarithm of the standard concentration as the abscissa for linear fitting.

[0018] The beneficial effects of this invention are as follows: This invention is the first to apply dual-label time-resolved fluorescence immunoassay technology to the joint detection of two cyanobacterial toxins in environmental water bodies. It can achieve simultaneous quantification of the concentration of two toxins in a single test within a short time, which significantly improves the detection efficiency. It does not require complex sample pretreatment and expensive instruments, is simple to operate, has low cost, high detection sensitivity and strong specificity, and is suitable for routine monitoring of environmental water samples and emergency detection of cyanobacterial blooms. It provides an efficient and reliable technical means for risk assessment of cyanobacterial toxin compound pollution and water supply safety assurance. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the TRFIA method for simultaneously detecting CYN and MC according to the present invention.

[0020] Figure 2 This is a graph showing the fluorescence value and binding rate (B / B0) of the MCLR monoclonal antibody at different dilutions according to the present invention.

[0021] Figure 3 This is a graph showing the fluorescence value and binding rate (B / B0) at different N8 dilutions of the present invention.

[0022] Figure 4 This is a standard curve of MCLR and CYN in the dual-label time-resolved fluorescence immunoassay of the present invention. Detailed Implementation

[0023] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention. Example

[0024] according to Figure 1 As shown, this embodiment provides a method for the simultaneous determination of microcystin and pterocytoxin, including the following steps: Step 1: Prepare microcystin-bovine serum albumin conjugate and cytosporin-bovine serum albumin conjugate separately; The specific preparation of the microcystin-bovine serum albumin conjugate involves first modifying bovine serum albumin with ethylenediamine to obtain EDA-BSA, then reacting the microcystin with EDA-BSA in the presence of carbodiimide, followed by dialysis and lyophilization. The preparation of the *Cyclophorus toxin*-bovine serum albumin conjugate involves dissolving bovine serum albumin in phosphate buffer, adding *Cyclophorus toxin* and formaldehyde, stirring the mixture in the dark, and then dialysis to obtain the conjugate.

[0025] Step 2: Using lanthanide elements Eu 3+ Labeling microcystin-bovine serum albumin conjugates to obtain Eu 3+ Labeling antigens with lanthanide elements Sm 3+ Labeling of *Cyclophorus toxin*-bovine serum albumin conjugate yields Sm 3+ Labeled antigens; Eu 3+ The method for preparing the labeled antigen is to mix Eu at a mass ratio of 2:1. 3+ The labeling reagent was mixed with the microcystin-bovine serum albumin conjugate and reacted overnight at room temperature. The high fluorescence signal component was collected after purification by gel chromatography and stored with a stabilizer. Sm 3+ The method for preparing the labeled antigen is to mix Sm at a mass ratio of 1:0.8. 3+ The labeling reagent was mixed with the *Strombyx moritoxin*-bovine serum albumin conjugate and reacted overnight at room temperature. The high-fluorescence signal fraction was collected after purification by gel chromatography and stored with a stabilizer.

[0026] Step 3: Coat the goat anti-mouse IgG antibody onto a solid-phase carrier, block it, and set it aside for later use. Then prepare a series of mixed standard solutions containing microcystin and columnar cytotoxin. The coating concentration of goat anti-mouse IgG antibody was 5 μg / mL, and the coating conditions were overnight at 37°C or overnight at 4°C. The concentrations of microcystin and pterostilbene in the series of mixed standard solutions were 0.1 ng / mL, 0.3 ng / mL, 1 ng / mL, 2 ng / mL and 4 ng / mL.

[0027] Step 4: Add the reaction solution sequentially to the solid-phase carrier coated with goat anti-mouse IgG antibody, and carry out a direct competitive immune reaction at room temperature for 50 minutes. The reaction solution is the sample to be tested or a standard solution, Eu. 3+ Labeled antigen, Sm 3+ Labeled antigens and a mixture containing monoclonal antibodies against microcystin and monoclonal antibodies against cytotoxin; The working dilution of the microcystin monoclonal antibody is 1:15000, and the working dilution of the columniformis monoclonal antibody is 1:10000.

[0028] Step 5: After the reaction is complete, wash the sample, add the enhancement solution, and use a time-resolved fluorescence detector to detect fluorescence in Eu. 3+ and Sm 3 + The fluorescence intensity was measured at the characteristic emission wavelength, and the concentrations of microcystin and columnar cystin in the sample were calculated based on the standard curve. Eu 3+ The characteristic emission wavelength is 615 nm, and the detection parameters of the time-resolved fluorescence detector are set to Eu. 3+ The channel delay is 0.40ms, and the window time is 0.40ms. Sm 3+ The characteristic emission wavelength is 643 nm, and the detection parameters of the time-resolved fluorescence detector are set to Sm 3+ The channel delay is 0.05ms and the window time is 0.1ms.

[0029] The standard curve was fitted using a semi-logarithmic method, with the ratio of fluorescence values ​​of each standard to the negative control as the ordinate and the logarithm of the standard concentration as the abscissa for linear fitting.

[0030] Application examples according to Figures 1-4 As shown in the figure, this embodiment provides a method for the simultaneous determination of microcystin and pterocytoxin, as detailed below: I. Selection of Materials and Instruments MCLR monoclonal antibody was purchased from Beijing DianShi KeChuang Co., Ltd.; CYN monoclonal antibody N8 was obtained from the CYN-M010 hybridoma cell line (China Center for Type Culture Collection, Wuhan University, Wuhan, China, submitted for deposit on November 14, 2019, accession number CCTCC NO: C2019253); BSA (bovine serum albumin) was a product of Sigma-Aldrich; CYN standards and MCLR standards were purchased from Alexis Laboratories; goat anti-mouse secondary antibody was purchased from Rockland, USA; DTTA-Eu... 3+ and DTTA-Sm 3+Purchased from Platinum Elmo, 96-well microplates from Thermo Scientific, enhancement solution from Darui Biotechnology Co., Ltd., and other related reagents were domestic or imported analytical grade. High-speed refrigerated centrifuge, semi-automatic TRFIA detector, and plate washer were also used.

[0031] II. Reaction Principle Rare earth elements Sm and Eu, which have large Stokes shifts and stable properties, were selected as labeling materials and conjugated with BSA-CYN and BSA-MCLR artificial antigens. (See attached instructions.) Figure 1 As shown, dual-label time-resolved immunofluorescence analysis is based on the direct competitive immune response between antigen and antibody.

[0032] Labeled antigens, samples or standards, and CYN and MCLR monoclonal antibodies were sequentially added to a 96-well plate for the reaction. The labeled antigens and samples competitively bind to their corresponding monoclonal antibodies. The monoclonal antibodies bind to the solid-coated goat anti-rabbit secondary antibody to form immune complexes. The free antigens and antibodies were removed by washing the plate, and enhancement solution was added. The analytes were quantitatively detected by a time-resolved detector. The fluorescence values ​​of MCLR and CYN in the sample were inversely proportional.

[0033] III. Measurement Method 1. Preparation of BSA-CYN The preparation of BSA-CYN involves first dissolving bovine serum albumin (BSA) in phosphate-buffered saline (PBS), then adding CYN and formaldehyde, stirring the mixture at room temperature in the dark, and finally dialyzing the conjugate through 0.01M phosphate buffer and storing it frozen at -20°C for later use.

[0034] 2. Preparation of BSA-MCLR The connection between MCLR and the vector is as follows: (1) Preparation of DEA-BSA Dissolve 300 mg BSA in 15 ml double-distilled water, add 400 mg EDPC while stirring, and after dissolving, add 1 ml 10% EDA (ethylenediamine). Adjust the pH to 5.5 with 1 M HCl. Stir the mixture at room temperature for 2 h, then add 200 mg EDPC and adjust the pH to 5.5 with 1 M HCl. Incubate the mixture at 4 °C overnight, dialyze against PBS (7.2) for 48 h, freeze dry, and store at -20 °C.

[0035] (2) MCLR-EDA-BSA 0.5 mg MCLR was dissolved in 8 μl of anhydrous ethanol and then diluted with 32 μl of double-distilled water. This solution was mixed with EDA-BSA (2 mg EDA-BSA dissolved in 8 μl of double-distilled water), and the pH was adjusted to 5.0 with a few drops of 0.2 M NaOH. Then, this solution was added dropwise to EDPC solution (0.132 g EDPC dissolved in 150 μl of double-distilled water). The mixture was stirred continuously throughout the process, and the pH was maintained at 5.0. The reaction was carried out at room temperature for 5 h. The reaction mixture was dialyzed against 0.1 M NaCl, lyophilized, and stored at -20 °C.

[0036] 3. Analysis and identification of coupling results The indirect competitive ELISA method was used for identification and analysis. BSA-MCLR (or BSA-CYN) was coated with MCLR and anti-MCLR monoclonal antibody (or CYN and CYN monoclonal antibody N8), a blank control was set up, and finally the enzyme-labeled secondary antibody HRP-IgG was added. The detection was performed using an ELISA reader. Based on the principle of antigen-antibody specific binding, the OD value was observed to determine whether the detection antigens BSA-MCLR and BSA-CYN were successfully linked.

[0037] 4. Sm 3+ and Eu 3+ The mark (1) Eu 3+ Preparation of labeled antigens After washing the BSA-MCLR antigen in ultrafiltration centrifuge tubes, mix thoroughly with europium-labeled reagent at a mass ratio of 2.5:1 and react overnight at room temperature (25°C). The next day, purify the solution using a Sephadex™ G-50 gel chromatography column, collecting the liquid before and after peak elution (1 mL / tube). Pipette 1 μL from each tube into a 96-well plate, add 200 μL of enhancement buffer, shake at room temperature for 5 min, and perform detection. Combine the tubes with the highest fluorescence signal values, add 1% BSA stabilizer, and store at -20°C.

[0038] (2) Sm 3+ Preparation of labeled antigens After washing the BSA-CYN antigen in ultrafiltration centrifuge tubes, mix it thoroughly with samarium-labeled reagent at a mass ratio of 1:1 and react overnight at room temperature (25°C). The next day, purify the solution using a Sephadex™ G-50 gel chromatography column, collecting the liquid before and after the peak (1 mL / tube). Pipette 1 μl from each tube into a 96-well plate, add 200 μL of enhancement solution, shake at room temperature for 5 min, and perform detection. Combine the liquids from the tubes with the highest fluorescence signal values, add 1% BSA stabilizer, and store at -20°C.

[0039] 5. Preparation of solid-phase coated secondary antibodies Goat anti-mouse IgG at a concentration of 5 μg / mL was aliquoted into 96-well microplates and incubated overnight at 37°C. The coating solution was then poured off and the plates were washed. Blocking solution was added and the plates were incubated overnight at 4°C. The blocking solution was then poured off and the plates were washed. The plates were then vacuum-dried and stored at -20°C for later use.

[0040] 6. Preparation of Standards Prepare a series of MCLR solutions with concentrations of 0, 0.2, 0.6, 2, 4, and 8 ng / mL using a 1.5% standard diluent; prepare a series of CYN solutions with concentrations of 0, 0.2, 0.6, 2, 4, and 8 ng / mL using a 1.5% standard diluent. Then, mix the two stock solutions separately at a 1:1 ratio from low to high concentrations to obtain a final series of standard concentrations of 0, 0.1, 0.3, 1, 2, and 4 ng / mL. Store at 4°C for later use.

[0041] 7. Establishment of the dual-label TRFIA detection method and fitting of the standard curve As per the instruction manual Figure 1 As shown, the dual-label time-resolved fluorescence immunoassay system uses a one-step method. 50 μL of standard or 50 μL of blank sample is added to each well coated with mouse secondary antibody, followed by 50 μL of Eu-containing sample. 3+ -MCLR、Sm 3+ Add 50 μL of diluent containing MCLR monoclonal antibody and CYN monoclonal antibody to the CYN analysis buffer, incubate at room temperature for 45 min, wash the plate 6 times, add 150 μL of enhancement buffer to each well, shake at room temperature for 5 min, and then measure the signal using the Eu / Sm dual labeling program in the DR6606 time-resolved instrument.

[0042] Eu 3+ The measurement conditions were as follows: excitation wavelength 34nm; emission wavelength 615nm; delay time: 0.40ms; window time: 0.40ms; cycle time: 1.0ms; while Sm 3+ The excitation wavelength and cycle time remained unchanged, while the delay time, emission wavelength, and window time were fixed at 0.05 ms, 643 nm, and 0.1 ms, respectively.

[0043] The standard curve was fitted using a semi-logarithmic method. First, the average fluorescence values ​​B and B0 of each standard and negative control were calculated. Then, the binding rate, i.e., the ratio of B / B0, was calculated. The standard curve was then plotted based on the relationship between the B / B0 value of the standard sample (Y-axis) and the logarithmic value of the corresponding standard concentration (X-axis).

[0044] IV. Results Analysis 1. Determination of the optimal dilution factor for the conjugate and monoclonal antibody In Eu 3+-BSA-MCLR at a dilution of 1:10, as per the instructions attached. Figure 2 It can be seen that the B / B0 ratio decreases with decreasing MCLR monoclonal antibody concentration, with the highest fluorescence value at a monoclonal antibody dilution of 1:10000 and the lowest B / B0 value at a monoclonal antibody dilution of 1:15000; in Sm 3+ -BSA-CYN at a dilution of 1:40, as per the instructions attached. Figure 3 It can be seen that both the fluorescence value and B / B0 decrease with the decrease of N8 concentration. The highest value is at an N8 dilution of 1:5000, and the lowest value is at an N8 dilution ratio of 1:20000. The overall fluorescence value of samarium-labeled antigens is lower than that of europium-labeled antigens.

[0045] Since low fluorescence values ​​are not conducive to the experiment, the optimal dilution of monoclonal antibodies in the dual-label time-resolved immunofluorescence assay is 1:15000 for MCLR monoclonal antibody and 1:10000 for N8 monoclonal antibody.

[0046] 2. Establishment of the double-marked TRFIA standard curve The optimized direct competitive dual-label time-resolved immunofluorescence assay was performed. Linear fitting using the semi-logarithmic method revealed good linearity for both microcystin and *Cyclopodiazetoxin* within the range of 0.1–4 ng / mL, as per the instructions. Figure 4 As shown, Figure 4 In Figure a, the MCLR standard curve (Eu-labeled) and in Figure b, the CYN standard curve (Sm-labeled) show that it is feasible to obtain the concentrations of two cyanobacterial toxins in a single test.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for the simultaneous determination of microcystin and pterostilbenetoxin, characterized in that, Includes the following steps: Step 1: Prepare microcystin-bovine serum albumin conjugate and cytosporin-bovine serum albumin conjugate separately; Step 2: Using lanthanide elements Eu 3+ Labeling microcystin-bovine serum albumin conjugates to obtain Eu 3+ Labeling antigens with lanthanide elements Sm 3+ Labeling of *Cyclophorus toxin*-bovine serum albumin conjugate yields Sm 3+ Labeled antigens; Step 3: Coat the goat anti-mouse IgG antibody onto a solid-phase carrier, block it, and set it aside for later use. Then prepare a series of mixed standard solutions containing microcystin and columnar cytotoxin. Step 4: Add the reaction solution sequentially to the solid-phase carrier coated with goat anti-mouse IgG antibody, and carry out a direct competitive immune reaction at room temperature for 30-60 minutes; Step 5: After the reaction is complete, wash the sample, add the enhancement solution, and use a time-resolved fluorescence detector to detect fluorescence in Eu. 3+ and Sm 3+ The fluorescence intensity was measured at the characteristic emission wavelength, and the concentrations of microcystin and columnar cystin in the sample were calculated based on the standard curve.

2. The method for simultaneous determination of microcystin and pterocytoxin according to claim 1, characterized in that: The preparation of the microcystin-bovine serum albumin conjugate in step one involves first modifying bovine serum albumin with ethylenediamine to obtain EDA-BSA, then reacting the microcystin with EDA-BSA in the presence of carbodiimide, followed by dialysis and lyophilization.

3. The method for simultaneous determination of microcystin and pterocytoxin according to claim 1, characterized in that: The preparation of the *Strombyx moritoxin*-bovine serum albumin conjugate in step one involves dissolving bovine serum albumin in phosphate buffer, adding *Strombyx moritoxin* and formaldehyde, stirring the mixture in the dark, and then dialysis to obtain the conjugate.

4. The method for simultaneous determination of microcystin and pterocytoxin according to claim 1, characterized in that: The preparation method of the two labeled antigens in step two is to mix Eu at a mass ratio of 2-3:

1. 3+ The labeling reagent was mixed with the microcystin-bovine serum albumin conjugate at a mass ratio of 1:0.8-1.2, and Sm was added. 3+ The labeling reagent was mixed with the *Strombyx mori* toxin-bovine serum albumin conjugate and reacted overnight at room temperature. The high-fluorescence signal fraction was collected after gel chromatography purification, and a stabilizer was added for storage to obtain Eu. 3+ Labeled antigens and Sm 3+ Labeling antigens.

5. The method for simultaneous determination of microcystin and columnar polycystin according to claim 1, characterized in that: In step three, the coating concentration of goat anti-mouse IgG antibody is 5 μg / mL, and the coating conditions are overnight at 37°C or overnight at 4°C.

6. The method for simultaneous determination of microcystin and pterocytoxin according to claim 1, characterized in that: In step three, the concentrations of microcystin and columnar polycystin in the series of mixed standard solutions are 0.1 ng / mL, 0.3 ng / mL, 1 ng / mL, 2 ng / mL, and 4 ng / mL, respectively.

7. The method for simultaneous determination of microcystin and pterocytoxin according to claim 1, characterized in that: In step four, the reaction solution is the sample to be tested or a standard solution, Eu. 3+ Labeled antigen, Sm 3+ Labeled antigens and a mixture containing monoclonal antibodies against microcystin and monoclonal antibodies against cytotoxin; The working dilution of the microcystin monoclonal antibody is 1:10000-20000, and the working dilution of the columnar cystin monoclonal antibody is 1:5000-15000.

8. The method for simultaneous determination of microcystin and pterocytoxin according to claim 1, characterized in that: In step five, Eu 3+ The characteristic emission wavelength is 610-620nm, Sm 3+ The characteristic emission wavelength is 640-650 nm; the detection parameters of the time-resolved fluorescence detector are set to Eu. 3+ The channel delay is 0.30-0.50ms, and the window time is 0.30-0.50ms; Sm 3+ The channel delay is 0.04-0.06ms, and the window time is 0.08-0.12ms.

9. The method for simultaneous determination of microcystin and pterocytoxin according to claim 1, characterized in that: In step five, the standard curve is fitted using a semi-logarithmic method, with the ratio of fluorescence values ​​of each standard to the negative control as the ordinate and the logarithm of the standard concentration as the abscissa for linear fitting.

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