Preparation method and application of a rapid detection test strip for paralytic shellfish poison

Abstract

The application relates to a preparation method and application of a paralytic shellfish poison rapid detection test strip, belonging to the field of red tide algae toxin immunochromatographic detection. A first paralytic shellfish poison monoclonal antibody coated with colloidal gold markers is combined on a pad, the first paralytic shellfish poison monoclonal antibody is obtained by immunization, cell fusion and screening with chain naked dinoflagellate whole cells as immunogens; a detection line and a quality control line are arranged on a nitrocellulose membrane in a chromatographic direction in sequence, the detection line is close to one side of the pad and coated with a second paralytic shellfish poison monoclonal antibody, the second paralytic shellfish poison monoclonal antibody and the first paralytic shellfish poison monoclonal antibody recognize different epitopes of paralytic shellfish poison; and the quality control line is close to one side of a water absorption pad and coated with a sheep anti-mouse IgG antibody. The application has the advantages of intuitive results and simple operation, and is suitable for on-site rapid screening of paralytic shellfish poison in shellfish tissues, water bodies and algae liquid.

Description

Technical Field

[0001] This invention relates to the immunochromatographic detection of red tide algal toxins, and particularly to the immunochromatographic detection and analysis test strips for paralytic shellfish poisoning, their preparation and detection methods, and the field of shellfish poisoning concentration monitoring. Background Technology

[0002] Paralytic shellfish toxins (PSTs) are among the most dangerous red tide algal toxins, accumulating in shellfish and posing a significant threat to human health and aquaculture. Currently common detection methods, such as the mouse bioassay, high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA), are complex, time-consuming, and require specialized personnel and equipment, making them unsuitable for rapid on-site screening during peak red tide seasons.

[0003] Colloidal gold immunochromatographic test strips have shown great potential for rapid on-site testing due to their speed, simplicity, and lack of the need for complex instruments. Existing technologies include test strips targeting single components of PSTs (such as scimitar toxin STX), but their antibodies only target specific toxin analogs and may not be able to comprehensively detect the diverse structural PST complexes present in nature, posing a risk of false negatives. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned problems in the prior art and to provide a rapid detection colloidal gold test strip capable of detecting multiple paralytic shellfish poison components, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A rapid detection colloidal gold immunochromatographic test strip for paralytic shellfish poisoning includes a PVC backing and a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and an absorbent pad sequentially overlapped and adhered to the PVC backing.

[0007] The conjugate pad is coated with a first anti-paralytic shellfish poison monoclonal antibody labeled with colloidal gold. The first anti-paralytic shellfish poison monoclonal antibody is obtained by immunization, cell fusion and screening using whole cells of Gymnodinium catenatum as an immunogen.

[0008] The nitrocellulose membrane has a detection line and a control line arranged sequentially along the chromatography direction. The detection line is near the conjugate pad and is coated with a second anti-paralytic shellfish monoclonal antibody. The second anti-paralytic shellfish monoclonal antibody and the first anti-paralytic shellfish monoclonal antibody recognize different epitopes of paralytic shellfish. The control line is near the absorbent pad and is coated with goat anti-mouse IgG antibody.

[0009] The whole cell of *Gymnodinium chaineri* includes algal cells in the logarithmic growth phase and the stationary phase.

[0010] The preparation method of the colloidal gold immunochromatographic test strip for rapid detection of paralytic shellfish poisoning includes the following steps:

[0011] 1) Antigen preparation and animal immunization: Algal cells of Gynostemma pentaphyllum were collected, washed, resuspended, and mixed with adjuvant to emulsify and obtain immunogens, which were then used to immunize Balb / c mice.

[0012] 2) Monoclonal antibody preparation: Spleen cells from immunized mice were fused with myeloma cells, and hybridoma cell lines secreting anti-paralytic shellfish poisoning monoclonal antibodies were obtained through screening. Two anti-paralytic shellfish poisoning monoclonal antibodies recognizing different epitopes were prepared and purified, and were designated as the first anti-paralytic shellfish poisoning monoclonal antibody and the second anti-paralytic shellfish poisoning monoclonal antibody.

[0013] 3) Colloidal gold labeling: Colloidal gold solution was prepared by the trisodium citrate reduction method. After adjusting the pH, the first anti-paralytic shellfish monoclonal antibody obtained in step 2) was added for labeling to prepare a colloidal gold-labeled antibody complex. The complex was then coated on a glass fiber membrane or polyester fiber membrane and dried to obtain a conjugate pad.

[0014] 4) Nitrocellulose membrane coating: The second anti-paralytic shellfish poison monoclonal antibody and the goat anti-mouse IgG antibody obtained in step 2) are coated onto a nitrocellulose membrane to form a detection line and a control line, and then dried for later use;

[0015] 5) Test strip assembly: The sample pad, conjugate pad, coated nitrocellulose membrane, and absorbent pad are sequentially overlapped and adhered to the PVC backing, and then cut to obtain the test strip.

[0016] The algal cells of *Gymnodinium chaineri* mentioned in step 1) include algal cells in the logarithmic growth phase and the stationary phase. These algal cells are washed with PBS and resuspended to a concentration of 1 × 10⁻⁶. 5 ~1×10 6 The concentration was 100 cells / mL, and then fully emulsified with an equal volume of Freund's adjuvant.

[0017] The colloidal gold solution described in step 3) has a particle size of 20~50nm. After adjusting the pH to 8.0~9.0, the first anti-paralytic shellfish poisoning monoclonal antibody is added. After stirring and reacting, BSA is added for blocking. After centrifugation, the solution is resuspended in a reconstitution solution and then sprayed onto a glass fiber membrane.

[0018] The application of the aforementioned colloidal gold immunochromatographic test strip for rapid detection of paralytic shellfish poisoning in the detection of paralytic shellfish poisoning.

[0019] Specifically, the sample solution to be tested is dropped onto the sample pad of the test strip. After capillary chromatography, the color development of the detection line and the control line is observed. If both the detection line and the control line develop color, the result is positive. If only the control line develops color, the result is negative. If the control line does not develop color, the result is invalid.

[0020] In this invention, the amount of sample solution added is 80~100μL, and the colorimetric results are observed after 3~15 minutes.

[0021] The sample to be tested is the supernatant of shellfish tissue homogenate, water sample, or algal liquid sample.

[0022] The application also includes comparing the colorimetric results with a standard colorimetric card to make a semi-quantitative estimate of the paralytic shellfish poisoning content.

[0023] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0024] 1. This invention uses whole cells of Gynostemma pentaphyllum as an immunogen, and the resulting antibodies can recognize multiple PSTs components, avoiding the false negatives that may be caused by single toxin antibodies, making it highly practical.

[0025] 2. The detection process of this invention only takes 3 to 15 minutes, which is fast and convenient. It does not require complicated instruments or professional operators and is suitable for rapid on-site screening.

[0026] 3. The test strip of this invention has good specificity for PSTs and can effectively distinguish between positive and negative samples.

[0027] 4. The test strip preparation process of this invention is mature, the cost of a single test is low, and it is conducive to large-scale promotion and application. Attached Figure Description

[0028] Figure 1 This invention provides the results of using test strips to detect the visceral tissue fluid of *Gymnodinium gracilis* infected with *Gymnodinium gracilis* in the laboratory; wherein, Figure 1 (a) shows the laboratory infection of jade mussels with *Gymnodinium cladomorphum* for 3 hours; Figure 1 (b) shows the laboratory infection of jade mussels with *Gymnodinium cladomorphum* for 6 hours; Figure 1 (c) shows the laboratory infection of jade mussels with *Gymnodinium fasciatus* for 12 hours.

[0029] Figure 2 This invention provides the results of using test strips to detect the visceral tissue fluid of triangular shellfish at a red tide site; wherein, Figure 2 (a) is a sample of aquacultured triangular clam in the mudflats of Fuzhou City, with paralytic shellfish toxicity (191.1 mouse units by mouse biological method) diluted 10 times; Figure 2 (b) is a sample of triangular clam cultured in the mudflats of Fuzhou City, diluted 10 times with paralytic shellfish toxicity (210.0 mouse units by mouse biological method).

[0030] Figure 3 This is the specificity test result of the test strip of the present invention. Detailed Implementation

[0031] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0032] Example 1: Preparation of Monoclonal Antibodies

[0033] 1. Antigen preparation: Culture *Gymnodinium chaineri*, collect algal cells from the logarithmic and stationary phases respectively, wash with PBS and resuspend, adjusting the concentration to 1×10⁻⁶. 6 cells / mL, fully emulsified with an equal volume of complete Freund's adjuvant.

[0034] 2. Animal immunization: Emulsified algal cell suspension was used to immunize 6-8 week old female Balb / c mice. The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant.

[0035] 3. Cell fusion and screening: Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells and selectively cultured in HAT medium. Positive clones were screened by indirect ELISA to obtain hybridoma cell lines that stably secrete anti-PSTs monoclonal antibodies.

[0036] 4. Antibody production and purification: Hybridoma cells were injected into the peritoneal cavity of mice pretreated with liquid paraffin, and ascites fluid was collected. Monoclonal antibodies in the ascites fluid were purified using the caprylic acid-ammonium sulfate method.

[0037] Example 2: Preparation of colloidal gold test strips

[0038] 1. Preparation of colloidal gold: A colloidal gold solution with a particle size of about 40 nm was prepared by reducing chloroauric acid with trisodium citrate.

[0039] 2. Preparation of gold-labeled antibody: Adjust the pH of colloidal gold to 8.5 with 0.1M K2CO3, add purified anti-PSTs monoclonal antibody while stirring, continue stirring, add BSA for stabilization, centrifuge and resuspend to obtain gold-labeled antibody complex. Spray it evenly onto a glass fiber membrane, dry it to form a binding pad.

[0040] 3. Coating the NC membrane: Spray another anti-PSTs monoclonal antibody (recognizing different epitopes) and goat anti-mouse IgG antibody at a concentration of 1 mg / mL onto the NC membrane using a membrane coating instrument to form a detection line (T line) and a control line (C line), and then dry them for later use.

[0041] 4. Assembly: On the PVC backing, sequentially attach the sample pad, bonding pad, coated NC membrane, and absorbent pad. Cut to an appropriate width, insert into a plastic cartridge with a sample application port and observation window, vacuum seal, and store in a dry, cool place.

[0042] Example 3: Test strip performance testing and application

[0043] 1. Actual sample testing:

[0044] a. Laboratory simulated samples: After infecting jade mussels with *Gymnodinium fasciatus* for 3h, 6h, and 12h, the visceral tissue of the mussels was homogenized and centrifuged. 80-100μL of the supernatant was collected for analysis. Results were displayed after 3 minutes and stabilized after 15 minutes. The results showed that the longer the infection time, the darker the T-line (corresponding to...). Figure 1 ).

[0045] b. Field Samples: Triangular shellfish from areas in the Fuzhou tidal flats where *Gymnodinium chainense* red tides had previously occurred were collected. 80-100 μL of the supernatant from the homogenate of their visceral tissue was used for testing. Results were displayed in 3 minutes and stabilized after 15 minutes. The test strip showed a positive result, consistent with the results obtained using the mouse biological method (corresponding to...). Figure 2 ).

[0046] c. Specificity Detection: This test strip was used to detect the algal toxins of *Karenella mikimotoi* and *Alexandrium tamarense*. The results showed that the test strip only showed a T-line for the paralytic shellfish toxicity of *Gymnodinium chaineri*, indicating a positive result; while no color was observed for *Karenella mikimotoi* and *Alexandrium tamarense*, indicating a negative result. This demonstrates that the test strip has excellent specificity (corresponding to...). Figure 3 ); Figure 3 (a) shows the test results for *Gymnodinium chaineri*; (b) shows the test results for *Karenella mikimotoi*; and (c) shows the test results for *Alexandrium tamarense*.

Claims

1. A colloidal gold immunochromatographic test strip for rapid detection of paralytic shellfish poisoning, characterized in that: Includes a PVC backing and a sample pad, a bonding pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped and adhered to the PVC backing; The conjugate pad is coated with a first anti-paralytic shellfish poison monoclonal antibody labeled with colloidal gold. The first anti-paralytic shellfish poison monoclonal antibody is obtained by immunization, cell fusion and screening using whole cells of Gymnodinium catenatum as an immunogen. The nitrocellulose membrane has a detection line and a control line arranged sequentially along the chromatography direction. The detection line is near the conjugate pad and is coated with a second anti-paralytic shellfish monoclonal antibody. The second anti-paralytic shellfish monoclonal antibody and the first anti-paralytic shellfish monoclonal antibody recognize different epitopes of paralytic shellfish. The control line is near the absorbent pad and is coated with goat anti-mouse IgG antibody.

2. The rapid detection colloidal gold immunochromatographic test strip for paralytic shellfish poisoning as described in claim 1, characterized in that: The whole cell of *Gymnodinium chaineri* includes algal cells in the logarithmic growth phase and the stationary phase.

3. A method for preparing a colloidal gold immunochromatographic test strip for rapid detection of paralytic shellfish poisoning as described in any one of claims 1 to 2, characterized in that, Includes the following steps: 1) Antigen preparation and animal immunization: Algal cells of Gynostemma pentaphyllum were collected, washed, resuspended, and emulsified with adjuvant to obtain immunogens, which were then used to immunize Balb / c mice. 2) Monoclonal antibody preparation: Spleen cells from immunized mice were fused with myeloma cells, and hybridoma cell lines secreting anti-paralytic shellfish poisoning monoclonal antibodies were obtained through screening. Two anti-paralytic shellfish poisoning monoclonal antibodies recognizing different epitopes were prepared and purified, and were designated as the first anti-paralytic shellfish poisoning monoclonal antibody and the second anti-paralytic shellfish poisoning monoclonal antibody. 3) Colloidal gold labeling: Colloidal gold solution was prepared by the trisodium citrate reduction method. After adjusting the pH, the first anti-paralytic shellfish monoclonal antibody obtained in step 2) was added for labeling to prepare a colloidal gold-labeled antibody complex. The complex was then coated on a glass fiber membrane or polyester fiber membrane and dried to obtain a conjugate pad. 4) Nitrocellulose membrane coating: The second anti-paralytic shellfish poison monoclonal antibody and the goat anti-mouse IgG antibody obtained in step 2) are coated onto a nitrocellulose membrane to form a detection line and a control line, and then dried for later use; 5) Test strip assembly: The sample pad, conjugate pad, coated nitrocellulose membrane, and absorbent pad are sequentially overlapped and adhered to the PVC backing, and then cut to obtain the test strip.

4. The preparation method according to claim 3, characterized in that: The algal cells of *Gymnodinium chaineri* mentioned in step 1) include algal cells in the logarithmic growth phase and the stationary phase. These algal cells are washed with PBS and resuspended to a concentration of 1 × 10⁻⁶. 5 ~1×10 6 The concentration was 100 cells / mL, and then fully emulsified with an equal volume of Freund's adjuvant.

5. The preparation method according to claim 3, characterized in that: The colloidal gold solution described in step 3) has a particle size of 20~50nm. After adjusting the pH to 8.0~9.0, the first anti-paralytic shellfish poisoning monoclonal antibody is added. After stirring and reacting, BSA is added for blocking. After centrifugation, the solution is resuspended in a reconstitution solution and then sprayed onto a glass fiber membrane.

6. The application of a colloidal gold immunochromatographic test strip for rapid detection of paralytic shellfish poisoning as described in any one of claims 1 to 2 in the detection of paralytic shellfish poisoning.

7. The application as described in claim 6, characterized in that: Add the sample solution to be tested to the sample pad of the test strip. After capillary chromatography, observe the color development of the test line and the control line. If both the test line and the control line show color, the result is positive. If only the control line shows color, the result is negative.

8. The application as described in claim 7, characterized in that: Add 80-100 μL of the sample solution to be tested, and observe the color development results after 3-15 minutes.

9. The application as described in claim 7, characterized in that: The sample to be tested is the supernatant of shellfish tissue homogenate, water sample, or algal liquid sample.

Images