Porphyrin detection in denatured cnidarian tissue extracts

Porphyrin metabolic disruption from exposure to xenobiotic contaminants such as heavy metals, dioxins, and aromatic hydrocarbons can elicit overproduction of porphyrins. Measurement of porphyrin levels, when used in conjunction with other diagnostic assays, can help elucidate an organism’s physiological condition and provide evidence for exposure to certain toxicants. A sensitive microplate fluorometric assay has been optimized for detecting total porphyrin levels in detergent solubilized protein extracts from symbiotic, dinoflagellate containing cnidarian tissues. The denaturing buffer used in this modified assay contains a number of potentially interfering components (e.g., sodium dodecyl sulfate (SDS), dithiothreitol (DTT), protease inhibitors, and chlorophyll from the symbiotic zooxanthellae), which required examination and validation. Examination of buffer components were validated for use in this porphyrin assay; while the use of a specific spectrofluorometric filter (excitation 400 ± 15 nm; emission 600 ± 20 nm) minimized chlorophyll interference. The detection limit for this assay is 10 fmol of total porphyrin per μg of total soluble protein and linearity is maintained up to 5000 fmol. The ability to measure total porphyrins in a SDS protein extract now allows a single extract to be used in multiple assays. This is an advantage over classical methods, particularly when tissue samples are limiting, as is often the case with coral due to availability and collection permit restrictions.

Rapid detection of white spot syndrome virus (WSSV) of Penaeus monodon by latex agglutination test using monoclonal antibodies

Latex beads were sensitized with monoclonal antibodies (MAb) rose against VP28 of WSSV. The optimum concentration of MAb required to sensitize the latex beads was 125 µg/ml. The sensitized latex beads were used to detect WSSV from PCR-positive stomach tissue homogenates obtained from infected shrimp. Stomach tissue homogenates from WSSV-infected shrimp agglutinated the sensitized latex beads within 10 minutes, while uninfected samples did not produce any agglutination, although non-specific agglutinations were observed in some samples. The analytical sensitivity, analytical specificity, diagnostic sensitivity and diagnostic specificity of the (LAT) agglutination test were assessed. The analytical sensitivity of the test was 40 ng of purified WSSV (2 µg/ml). The sensitized latex beads did not agglutinate with normal shrimp tissue or MBV-infected tissue homogenate. The test has a diagnostic sensitivity of 70 and 45%, respectively, compared to single-step and nested PCR. The diagnostic specificity of the test was 82%. This test is a simple and rapid on-farm test which can be used to corroborate clinical signs for the detection of WSSV in grow-out ponds.