Bimodal immunoassay method for mc-lr toxin

Abstract

The present application relates to the technical field of microcystin detection, and particularly relates to a double-mode immunodetection method of MC-LR toxin. The method comprises the following steps: synthesizing MS@Au-Pt NZs, connecting A23-SBP recombinant antibody tag to obtain NZs@Ab; mixing NZs@Ab and a solution containing MC-LR to be detected, and then incubating and magnetically separating; removing supernatant, and then adding carbonate coating buffer solution for mixing; fixing MC-LR-BSA in a microplate, and then adding the mixed solution; after incubation, the microplate is a compound of MC-LR and NZs@Ab, and the microplate is washed with washing buffer solution; adding 3,3',5,5'-tetramethylbenzidine and H2O2 solution for color development for 20-30 min, and then adding a stop solution to detect absorbance; subsequently, adding AuNPs solution, and then detecting SERS signal after incubation for 10-35 min. The method has the advantages of high detection sensitivity and good accuracy.

Description

Technical Field

[0001] This invention relates to the field of microcystin detection technology, and more particularly to a dual-modal immunoassay method for MC-LR toxin. Background Technology

[0002] Eutrophication in freshwater bodies leads to the production of large amounts of microcystins (MCs) from toxic cyanobacterial blooms. MC-LR toxin is the most common MC toxin. Excessive ingestion of MC-LR toxin can cause diarrhea, vomiting, and even death in humans. Consuming seafood contaminated with MC-LR toxin is a significant route for its entry into the human body. Furthermore, MC-LR toxin is often present in the biological tissues and cells of seafood and is not digested or dissolved in the seafood itself. Therefore, to ensure food safety, researching a rapid detection method for MC-LR toxin in edible seafood is particularly important.

[0003] Currently, analytical methods for detecting MC-LR toxins include bioanalysis, fluorescence detection, cytotoxicity assays, enzyme-linked immunosorbent assays (ELISA), high-performance liquid chromatography (HPLC), and electrochemical analysis. However, these methods have limitations, such as poor specificity and the arduousness and low sensitivity of bioanalysis methods. Furthermore, previous studies have largely focused on the detection of MC-LR toxins in the environment or drinking water, with only a few addressing the detection of MC-LR toxins in aquatic products, primarily due to severe sample matrix interference. Therefore, there is a need to develop more robust and sensitive methods for analyzing and detecting trace amounts of MC-LR toxins in complex sample matrices.

[0004] Surface-enhanced Raman spectroscopy (SERS) is a powerful spectroscopic analytical technique capable of analyzing both the structure of substances and detecting substances at the single-molecule level. However, the molecular structures of MC-LR toxins differ only slightly from those of other macromolecules (MCs), resulting in highly complex Raman bands. Furthermore, interference from complex matrices is severe when directly detecting MC-LR toxins in aquatic products. Therefore, directly detecting trace amounts of MC-LR toxins in different fish species using label-free Raman methods is extremely challenging.

[0005] To improve the sensitivity of MC-LR toxin detection and meet the need for rapid analysis and detection of trace amounts of MC-LR toxin in aquatic products, this study introduces antigen-antibody assays for specific recognition of MC-LR toxin, utilizes nanozymes for signal amplification, and combines ultraviolet spectroscopy and surface-enhanced Raman spectroscopy data for complementary advantages, thereby improving the accuracy and quality of detection results. Although sensitive determination of MC-LR toxin has been achieved, the poor accuracy of these rapid analytical methods in single-mode response readings has hindered their development. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a dual-modal immunoassay method for MC-LR toxin.

[0007] The primary objective of this invention is to provide a dual-modal immunoassay method for MC-LR toxin, specifically comprising the following steps:

[0008] S1. Synthesis of magnetic silicon-based functional nanoenzyme material MS@Au-Pt NZs:

[0009] S2. Magnetic silicon-based functional nanozyme material MS@Au-Pt NZs is linked to the A23-SBP recombinant antibody tag to obtain NZs@Ab;

[0010] S3. Mix NZs@Ab and the test solution, incubate at 35-38℃ and 80-150rpm for 20-40min, then magnetically separate, remove the supernatant, add carbonate coating buffer, and mix.

[0011] S4. Immobilize the microcystin-bovine serum albumin conjugate MC-LR-BSA in a microplate and seal the unimmobilized sites in the microplate with BSA.

[0012] S5. After fixation, add the mixture coated in step S3 and incubate at 35-38℃ for 1.5-2.5h. After incubation, the MC-LR and NZs@Ab complex coated on the microplate is obtained; wash the microplate with washing buffer.

[0013] S6. Add 3,3',5,5'-tetramethylbenzidine and H2O2 solution for color development for 20-30 min, then add stop solution to terminate the reaction and measure absorbance.

[0014] S7. After detection, add AuNPs solution with a concentration of 1-4 mg / mL, and detect the SERS signal after 10-35 min; analyze the measured absorbance data and SERS data.

[0015] Preferably, step S1 specifically includes the following sub-steps:

[0016] S101. Tetraethyl orthosilicate reagent is mixed with Fe3O4 MNP magnetic nanoparticles and reacted to grow a layer of SiO2 on the surface of Fe3O4 MNP to obtain Fe3O4@SiO2.

[0017] S102, adding 3-aminopropyltriethoxysilane reagent to aminate the Fe3O4@SiO2 surface to obtain MS-NH2;

[0018] S103, chloroauric acid, chloroplatinic acid and sodium borohydride are added to reduce MS-NH2 to form magnetic silicon-based functional nanoenzyme material MS@Au-Pt NZs.

[0019] Preferably, the incubation temperature in step S4 is 37°C and the incubation time is 30 min; the carbonate coating buffer has a pH of 9.6 and a carbonate concentration of 0.05 mol / L.

[0020] Preferably, the co-incubation time in step S5 is 2 hours and the temperature is 37°C; the washing buffer is a PBS buffer solution containing 0.05% Tween-20 with pH = 7.4.

[0021] Preferably, the stop solution in step S6 is a 2 mol / L H2SO4 solution; the colorimetric reaction time is 30 min. The absorbance is measured at 452 nm; the SERS data in step S7 is 1603 cm⁻¹. -1 Intensity value at the characteristic peak.

[0022] Preferably, in step S7, the concentration of the AuNPs solution is 2 mg / mL, and the particle size of the AuNPs particles is 30–32 nm. The conditions for detecting the SERS signal are: laser power: 35 mW; range: 200–3000 cm⁻¹. -1 Integration time: 2s; Cumulative count: 1; Detection time is 15min after adding AuNPs solution.

[0023] The second objective of this invention is to provide a dual-modal immunoassay kit for MC-LR toxin, comprising: magnetic silicon-based functional nanozyme material MS@Au-Pt NZs, A23-SBP recombinant antibody tag, and microcystin-bovine serum albumin conjugate (MC-LR-BSA); and to detect MC-LR toxin in the test sample using a dual-modal immunoassay method for MC-LR toxin.

[0024] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0025] 1) Magnetic nanozymes with recognition, separation and catalysis functions can be used to amplify the MC-LR toxin detection signal and improve the sensitivity of MC-LR toxin detection;

[0026] 2) By utilizing the catalytic TMB color development phenomenon of magnetic nanozymes, the simultaneous output of ultraviolet spectral and surface-enhanced Raman spectral signals can be achieved. The two signals can be mutually verified, improving the accuracy of MC-LR detection.

[0027] 3) Compared with single-modal biodetection methods, the dual-modal biodetection method of the present invention can significantly improve the reliability and analytical performance of MC-LR toxin detection due to its resistance to interference from both external and internal environments. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the principle of the dual-modal immunoassay method for MC-LR toxin provided in an embodiment of the present invention.

[0029] Figure 2 These are transmission electron microscope (TEM) images provided according to embodiments of the present invention; A is a TEM image of Fe3O4@SiO2; B is a TEM image of NZs@Ab+MC-LR; C is a TEM image of AuNPs; and D is a high-magnification TEM image of AuNPs.

[0030] Figure 3 These are ultraviolet and Raman spectra provided according to embodiments of the present invention; A is the ultraviolet spectrum of microplates with unfixed MC-LR-BSA and microplates with fixed MC-LR-BSA after catalytic colorimetric reaction and termination; in B, a is TMB, b is TMB+MS@Au-Pt NZs, and c is the Raman spectrum of TMB+MS@Au-Pt NZs+Au NPs.

[0031] Figure 4 The graphs provided in this embodiment of the invention show the absorbance curves of different concentrations of MC-LR at (A) 452 nm, (B) a linear fit plot of the absorbance at 452 nm, and (C) at 1603 cm⁻¹. -1 Raman intensity curve at (D) 1603cm -1 Linear fitting plot of Raman intensity at the location.

[0032] Figure 5 The results show the effects of different conditions on SERS signals according to embodiments of the present invention: (A) SERS signals at different detection times; (B) SERS signals measured by gold nanoparticles of different sizes; (C) SERS signals of Au NPs solutions of different concentrations. Detailed Implementation

[0033] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0035] Example 1

[0036] A dual-modal immunoassay for MC-LR toxin Figure 1 The principle is illustrated, and the specific steps include the following:

[0037] S1. Synthesis of magnetic silicon-based functional nanoenzyme material MS@Au-Pt NZs:

[0038] S101. Tetraethyl orthosilicate (TEOS) reagent is mixed and reacted with magnetite nanoparticles (Fe3O4 MNP) to grow a layer of SiO2 on the surface of Fe3O4 MNP, thus obtaining Fe3O4@SiO2. Figure 2 A);

[0039] S102, adding 3-aminopropyltriethoxysilane (APTES) reagent to aminate the Fe3O4@SiO2 surface to obtain MS-NH2;

[0040] S103, with the addition of chloroauric acid, chloroplatinic acid, and sodium borohydride, reduces MS-NH2 to form the magnetic silicon-based functional nanoenzyme material MS@Au-Pt NZs. Figure 2 A);

[0041] S2. Magnetic silicon-based functional nanozyme material MS@Au-Pt NZs is linked to the A23-SBP recombinant antibody tag (purchased from Jiangxi Zhongde Biotechnology Co., Ltd.) to obtain NZs@Ab( Figure 2 B);

[0042] S3. Mix NZs@Ab and the test solution, place them in a constant temperature shaker, incubate at 37℃ and 100rpm for 30min, then magnetically separate, remove the supernatant, and add 50μL of coating buffer (0.05mol / L carbonate buffer, pH=9.6).

[0043] S4. Immobilize the microcystin-bovine serum albumin conjugate (MC-LR-BSA) in 96-well microplates and seal the unimmobilized sites in the 96-well microplates with BSA to avoid non-specific adsorption.

[0044] S5. After fixation, add the mixture coated in step S3 and incubate in a 37°C oven for 2 hours. After incubation, the MC-LR and NZs@Ab complex coated on the plate is obtained. Wash the 96 microwells with washing buffer (PBS containing 0.05% Tween-20, pH=7.4).

[0045] S6. Add 50 μL each of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 solution for color development for 30 min. Add 20 μL of stop solution (2 mol / L H2SO4) to terminate the reaction. Detect the absorbance using an ELISA reader (instrument parameters: select [GRE96ft]-Greiner 96-well planar transparent plate; measurement wavelength: 400nm-800nm; step size: 2nm; readout-flash count: 5 times; settling time: 0ms).

[0046] S7. After detection, add a 2 mg / mL AuNPs solution to each of the 96 microwells. Figure 2 100 μL of CD was added, and the SERS signal was detected after 15 min (laser power: 35 mW; range: 200–3000 cm⁻¹). -1 Integration time: 2s; Cumulative count: 1); Analyze the measured absorbance and SERS data.

[0047] Figure 2 In the transmission electron microscope image, A represents Fe3O4@SiO2, which exhibits a distinct core-shell structure and good dispersion among the particles. This indicates that SiO2 was successfully coated on the surface of Fe3O4 MNPs. Figure 2 B is NZs@Ab+MC-LR. Morphologically, a transparent thin film is observed on the NZs surface, possibly due to the addition of the MC-LR toxin, causing the NZs@Ab particles to adhere together. Additionally, numerous fine particles are present on the NZs surface, which may be gold-platinum nanoparticles. The transmission electron microscopy (TEM) images confirm the successful preparation of the NZs@Ab material. Figure 2 C and Figure 2 D represents Au NPs. It can be seen that the synthesized Au NPs are basically spherical and have good dispersion, which can be used for subsequent Raman signal enhancement.

[0048] In the TMB colorimetric experiment, micropores without fixed MC-LR-BSA were used as a control, and the UV absorbance was measured. In Raman signal detection, TMB was used as a blank control, and a solution without Au NPs particles was used as a negative control; the Raman spectra were measured. The results are as follows: Figure 3 As shown. By Figure 3 As shown in A, the MC-LR-BSA immobilized on the microplate can increase the absorbance of the solution at 452 nm. This indicates that MC-LR-BSA can effectively bind to the antibody on NZs@Ab, allowing NZs@Ab to be immobilized in the 96 microwells and catalyze more TMB, resulting in a higher absorbance at 452 nm. Figure 3In Figure B, it can be seen that the Raman characteristic peak intensity of TMB is very weak. When MS@Au-Pt NZs is added, the Raman characteristic peak of TMB is enhanced, but the intensity of the characteristic peak is not high. After adding MS@Au-Pt NZs and Au NPs particles, the Raman characteristic peak of TMB is significantly enhanced. This indicates that the simultaneous addition of MS@Au-Pt NZs and Au NPs particles has the best Raman enhancement effect on TMB.

[0049] Example 2

[0050] Plot the ultraviolet absorbance curve and the Raman spectrum curve. The specific measurement method is as follows:

[0051] S1. Synthesis of magnetic silicon-based functional nanoenzyme material MS@Au-Pt NZs;

[0052] S2. The magnetic silicon-based functional nanozyme material MS@Au-Pt NZs is linked to the A23-SBP recombinant antibody tag to obtain NZs@Ab (see Example 1 for the above steps);

[0053] S3. Take 50 μL of MC-LR at different concentrations (0 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL) and mix them with 50 μL of NZs@Ab solution respectively. After mixing, place the mixture in a constant temperature shaker and incubate at 37℃ and 100 rpm for 30 min. Then, perform magnetic separation, remove the supernatant, and add 50 μL of coating buffer (0.05 mol / L carbonate buffer, pH=9.6) to each mixture.

[0054] S4. Immobilize the microcystin-bovine serum albumin conjugate (MC-LR-BSA) in 96-well microplates and seal the unimmobilized sites in the 96-well microplates with BSA to avoid non-specific adsorption.

[0055] S5. After fixation, add the mixture coated in step S3 and incubate in a 37°C oven for 2 hours. After incubation, the MC-LR and NZs@Ab complex coated on the plate is obtained. Wash the 96 microwells with washing buffer (PBS containing 0.05% Tween-20, pH=7.4).

[0056] S6. Add 50 μL each of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 solution for color development for 30 min. Add 20 μL of stop solution (2 mol / L H2SO4) to terminate the reaction. Detect the absorbance using an ELISA reader. Analyze the measured absorbance data and plot standard curves for different MC-LR concentrations.

[0057] S7. After detection, add 100 μL of AuNPs solution with a concentration of 2 mg / mL to each 96-well microtube. Detect the SERS Raman signal after 15 min (laser power: 35 mW; range: 200–3000 cm⁻¹). -1 Integration time: 2s; Cumulative count: 1); Analyze the measured absorbance and SERS data and plot standard curves for different MC-LR concentrations.

[0058] The UV and Raman spectra of the catalytic TMB solution were measured, and the linear relationship between the signal intensity and the concentration of MC-LR toxin was established. The results are as follows: Figure 4 As shown. The linear range for detecting MC-LR toxin is 10–500 ng / mL, and the linear fitting equations are Y = -0.12lgX + 0.47R. 2 =0.9999 and Y = -533.32lgX + 2800.47R 2 =0.9527, with detection limits of 5.2 ng / mL (S / N = 3) and 6.7 ng / mL (S / N = 3), respectively. Comparison of this bimodal immunoassay method with other immunoassay methods showed that this method has high sensitivity and good stability.

[0059] Example 3

[0060] The optimization experiments for the dual-modal immunoassay method for MC-LR toxin specifically include:

[0061] I. Optimization of TMB color development time

[0062] Three parallel experiments were conducted. First, 50 μL of 50 ng / mL MC-LR was mixed with 50 μL of MS@Au-Pt NZs-Ab solution and incubated on a shaker for 30 min. After magnetic separation, the supernatant was discarded, and 50 μL of coating solution was added. Then, the above solution was added to 50 μL of pre-coated and dried 10 μg / mL MC-LR-BSA solution in each well of a 96-well plate and incubated for 2 h. Finally, 50 μL of TMB and 50 μL of hydrogen peroxide solution were added for color development, and absorbance was measured every 5 min.

[0063] Experiments have shown that the visible light spectrum is most effective when measured with an ELISA reader 30 minutes after color development.

[0064] II. Optimization of measurement time after adding Au NPs

[0065] Mix 50 μL of 0.1 ng / mL MC-LR with 50 μL of MS@Au-Pt NZs solution and incubate on a shaker for 30 min. Perform magnetic separation, discard the supernatant, and add 50 μL of coating buffer. Then, add the solution to each well of a 96-well plate coated with and dried 50 μL of 10 μg / mL MC-LR-BSA solution and incubate for 2 h. Add 50 μL of solutions A and B to each well for color development. After 30 min of color development, add 20 μL of stop solution to each well. Finally, detect the SERS signal at different time points (10, 15, 20, 25, 30, 35 min) after adding 100 μL of Au NPs solution to the well. Figure 5 A). Experiments showed that the SERS intensity was highest 15 minutes after the addition of Au NPs.

[0066] III. Optimization of Au NPs Particle Size

[0067] Five experimental groups were set up, with three replicates per group. 100 μL of MS@Au-Pt NZs, 50 μL of TMB, and 50 μL of AuNPs with different particle sizes (18.89 nm, 31.46 nm, 39.14 nm, and 44.24 nm) were added to the well plates. The absorbance and SERS signal of each group were measured (see...). Figure 5 B). Experiments showed that the highest SERS intensity was obtained using gold nanoparticles at a density of 31.46 nm.

[0068] IV. Optimization of Au NPs Concentration

[0069] Mix 10 μL of MS@Au-Pt NZs diluted 10-fold with 20 μL of TMB. After 30 min, add 20 μL of stop solution and 100 μL of concentrated Au NPs solutions of different concentrations (0.25, 0.5, 1, 2, 4 mg / mL). Detect the SERS signal after 15 min (see [link to sample]). Figure 5 C). Experiments have shown that the SERS signal is superior at 2 mg / mL Au NPs.

[0070] The signal detected under optimal conditions has the best effect.

[0071] In summary, this invention addresses the challenge of sensitive and accurate Raman detection of MC-LR toxin by designing a dual-modal immunoassay method based on magnetic functional nanozymes. Its key innovative technology lies in employing an antigen-antibody competitive binding recognition mechanism for MC-LR toxin. The magnetic functional nanozyme catalyzes TMB color development to generate both UV and Raman signals, achieving the same recognition mode while outputting different detection signals. Compared to single-mode detection methods, this method offers significantly higher accuracy.

[0072] This method achieves rapid and sensitive detection of the target MC-LR by using a dual-modal sensor combining colorimetry and Raman spectroscopy to achieve the same recognition mode while outputting different detection signals, compared to single detection methods. It employs a competitive binding mechanism between antibodies and the coated antigen; part of the antibody specifically binds to the antigen, while the remaining antibody binds to the antigen coated on the well plate. Magnetic functional nanozymes exhibit the catalytic effect of natural enzymes; when the antibody binds to the antigen coated on the well plate, it catalyzes the development of TMB colorimetry, generating a UV signal, which is then detected colorimetrically. After terminating the reaction with a stop solution (2 mol / L H₂SO₄), the absorbance is measured using a microplate reader. Au NPs are then added for incubation, and the Raman signal is measured using a Raman spectrometer. The content is analyzed based on a standard curve.

[0073] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0074] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A dual-modal immunoassay method for MC-LR toxin, characterized in that, Specifically, the steps include the following: S1. Synthesis of magnetic silicon-based functional nanoenzyme material MS@Au-Pt NZs: S101. Tetraethyl orthosilicate reagent is mixed with Fe3O4MNP magnetic nanoparticles to grow a layer of SiO2 on the surface of Fe3O4MNP, thus obtaining Fe3O4@SiO2. S102, adding 3-aminopropyltriethoxysilane reagent to aminate the Fe3O4@SiO2 surface to obtain MS-NH2; S103, chloroauric acid, chloroplatinic acid and sodium borohydride are added to reduce MS-NH2 to form magnetic silicon-based functional nanoenzyme material MS@Au-Pt NZs; S2. Magnetic silicon-based functional nanozyme material MS@Au-Pt NZs is linked to the A23-SBP recombinant antibody tag to obtain NZs@Ab; S3. Mix NZs@Ab and the test solution, incubate at 35~38℃ and 80~150rpm for 20~40min, then perform magnetic separation, remove the supernatant, add carbonate coating buffer with pH=9.6 and carbonate concentration of 0.05mol / L, and mix. S4. Immobilize the microcystin-bovine serum albumin conjugate (MC-LR-BSA) in a microplate and seal the unimmobilized sites in the microplate with BSA. S5. After fixation, add the mixture coated in step S3 and incubate at 35~38℃ for 1.5~2.5h. After incubation, the MC-LR and NZs@Ab complex coated on the microplate is obtained; wash the microplate with washing buffer. S6. Add 3,3',5,5'-tetramethylbenzidine and H2O2 solution for color development reaction for 20-30 min, add stop solution to terminate reaction, and detect absorbance at 452 nm. S7. After detection, add AuNPs solution with a concentration of 1~4 mg / mL, and detect the SERS signal after 10~35 min; analyze the measured absorbance data and SERS data.

2. The dual-modal immunoassay method for MC-LR toxin according to claim 1, characterized in that: In step S7, the concentration of the AuNPs solution is 2 mg / mL, and the particle size of the AuNPs particles is 30~32 nm.

3. The dual-modal immunoassay method for MC-LR toxin according to claim 2, characterized in that: The conditions for detecting the SERS Raman signal in step S7 are: laser power: 35mW; Range: 200~3000cm -1 Integration time: 2s; Cumulative count: 1; Detection time is 15min after adding the AuNPs solution.

4. The dual-modal immunoassay method for MC-LR toxin according to any one of claims 1-3, characterized in that: The termination solution in step S6 is a 2 mol / L H2SO4 solution; the color development takes 30 minutes.

5. The dual-modal immunoassay method for MC-LR toxin according to claim 4, characterized in that: The co-incubation time in step S5 is 2 hours and the temperature is 37°C; the washing buffer is a PBS buffer solution containing 0.05% Tween-20 with pH=7.

4.

6. The dual-modal immunoassay method for MC-LR toxin according to claim 5, characterized in that: In step S4, the incubation temperature is 37°C and the incubation time is 30 minutes.

7. The dual-modal immunoassay method for MC-LR toxin according to claim 6, characterized in that: In step S7, the detected Raman signal is 1603 cm⁻¹. -1 .

8. A dual-modal immunoassay kit for MC-LR toxin, comprising: The magnetic silicon-based functional nanoenzyme material MS@Au-PtNZs, the A23-SBP recombinant antibody tag, and the microcystin-bovine serum albumin conjugate MC-LR-BSA are characterized by the use of the dual-modal immunoassay method for MC-LR toxin as described in claim 1 to detect MC-LR toxin in the test sample.

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