Biomedical subjects

J M Baptista de Sousa

Publications and source records attributed to J M Baptista de Sousa.

3 recordsLinked to original sources

PubMed2001

A fluorimetric method based on changes in membrane potential for screening paralytic shellfish toxins in mussels.

To prevent the consumption of bivalves contaminated with paralytic shellfish poisoning (PSP), toxin levels in seafood products are estimated by using the official mouse bioassay. Because of the limitations of this bioassay other methods of monitoring toxins are clearly needed. We have developed a test to screen for PSP toxins based on its functional activity; the toxins bind to the voltage-gated Na+ channels and block their activity. The method is a fluorimetric assay that allows quantitation of the toxins by detecting changes in the membrane potential of human excitable cells. This assay gives an estimate of toxicity, since each toxin present in the sample binds to sodium channels with an affinity which is proportional to its intrinsic toxic potency. The detection limits for paralytic shellfish toxins were found to be 1 ng saxitoxin equivalents/ml compared to the regulatory limit threshold of 400 ng/ml (equivalent to 80 microg/100 g) used in most countries. Our results indicate that this fluorescent assay is a specific, very sensitive, rapid, and reliable method of monitoring PSP toxin levels in samples from seafood products and toxic algae.

Animals↗

PubMed1999

A fluorescent microplate assay for microcystin-LR.

A fluorescent enzyme inhibition assay for microcystin-LR was developed using a new fluorescent substrate of protein phosphatases 1 (PP1) and 2A (PP2A), 6,8-difluoro-4-methylumbelliferyl phosphate. The PP1 and PP2A inhibition assay for microcystin-LR was performed in a microtiter plate and the fluorescence yielded by the enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The concentration of microcystin-LR causing 50% inhibition of PP1 and PP2A activity (IC50) was 0.01 nM for PP1 and 0.08 nM for PP2A. The measurable range of microcystin-LR was 800 to 0.08 pg/well for both enzymes. The described assay is fast and very sensitive for the detection of microcystin-LR. Furthermore, this assay can be successfully applied to the study of toxins that inhibit PP1 or PP2A.

Cyanobacteria↗

PubMed1997

A fluorescent microplate assay for diarrheic shellfish toxins.

A fluorescent enzyme inhibition assay for okadaic acid using 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for the enzyme phosphatase 2A was developed. In the inhibition assay, performed in a microtiter plate, the PP2A was inhibited by adding okadaic acid and the resulting fluorescence enhancement derived from enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The measurable range of okadaic acid was 3.2 to 3200 pg/ml with an IC50 = 0.1 nM. The detection limit of okadaic acid was 2.56 pg/well in buffer solutions and 12.8 ng/g hepatopancreas in shellfish extracts. The coefficient of variation (CV, n = 22) for each point ranged from 18.80 to 37.90% (mean 28.35%). The proposed method is very convenient, rapid, and sensitive by using the enzyme inhibition assay system and fluorescent reaction as a detection system. This work demonstrates that the fluorescent assay can be used to quantify the amount of okadaic acid in shellfish samples and also is valid for very dilute samples, such as phytoplankton samples.

Animals↗