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en:aree:in_collaborazione:tossine_algali:metodi [2023/02/15 19:42] Gianluca Frustagli created |
en:aree:in_collaborazione:tossine_algali:metodi [2023/03/07 14:38] (current) Gianluca Frustagli |
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| - | <WRAP todo> | ||
| - | ==== Under construction ==== | ||
| - | </WRAP> | ||
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| **Services and tools in collaboration** | **Services and tools in collaboration** | ||
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| Microcystin contamination in fish tissue is a recent detection in Italian lakes and its monitoring requires fast and precise analysis techniques, easy to apply and capable of providing results in real time. Instrumental chemical analysis is mostly used to confirm and identify the different variants of microcystins. | Microcystin contamination in fish tissue is a recent detection in Italian lakes and its monitoring requires fast and precise analysis techniques, easy to apply and capable of providing results in real time. Instrumental chemical analysis is mostly used to confirm and identify the different variants of microcystins. | ||
| - | Given the majority percentage of toxins directly combined with the cells in the tissues (largely invisible to instrumental methods) compared to the free ones, and the need to use different standards for the detection of the single compounds, the simultaneous use of the ELISA analysis guarantees a rapid quantitative measurement of the total amount of MCs present, useful from the point of view of risk assessment. Studies performed on Italian lakes have shown that ELISA test results showed MC concentrations 3 to 8 times higher than the ones calculated from LC-MS/MS analyses ((Bruno et al., 2009)). | + | Given the majority percentage of toxins directly combined with the cells in the tissues (largely invisible to instrumental methods) compared to the free ones, and the need to use different standards for the detection of the single compounds, the simultaneous use of the ELISA analysis guarantees a rapid quantitative measurement of the total amount of MCs present, useful from the point of view of risk assessment. Studies performed on Italian lakes have shown that ELISA test results showed MC concentrations 3 to 8 times higher than the ones calculated from LC-MS/MS analyses((Bruno et al., 2009)). |
| ===== 1. Chlorophyll a extraction from water ===== | ===== 1. Chlorophyll a extraction from water ===== | ||
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| The currently available test (ABRAXIS, USA) is a direct competitive ELISA based on the recognition of BMAA by specific antibodies. BMAA, if present in a sample, and a BMAA-HRP analogue compete for the binding sites of rabbit anti-BMAA antibodies in solution. The BMAA antibodies are then bound by a second immobilized antibody on the wells of the microtitre plate. After a wash step and addition of substrate solution, a color signal is generated. The intensity of the blue color is inversely proportional to the concentration of BMAA present in the sample. The color reaction is stopped after a specified time and the color is evaluated using an ELISA microplate reader. Sample concentrations are determined by interpolation using the standard curve constructed with each test. The LOD for this test, based on MC-LR, is 4 μg/L. The coefficients of variation (CV%) for the standards are less than 10%; for samples below 15%. | The currently available test (ABRAXIS, USA) is a direct competitive ELISA based on the recognition of BMAA by specific antibodies. BMAA, if present in a sample, and a BMAA-HRP analogue compete for the binding sites of rabbit anti-BMAA antibodies in solution. The BMAA antibodies are then bound by a second immobilized antibody on the wells of the microtitre plate. After a wash step and addition of substrate solution, a color signal is generated. The intensity of the blue color is inversely proportional to the concentration of BMAA present in the sample. The color reaction is stopped after a specified time and the color is evaluated using an ELISA microplate reader. Sample concentrations are determined by interpolation using the standard curve constructed with each test. The LOD for this test, based on MC-LR, is 4 μg/L. The coefficients of variation (CV%) for the standards are less than 10%; for samples below 15%. | ||
| + | /* > FIXME **To be revised.**\\ //(remove this paragraph once finished)// */ | ||
