Biomedical subjects

M Vecchiati

Publications and source records attributed to M Vecchiati.

3 recordsLinked to original sources

PubMed1996

A polyclonal antiserum against a recombinant viral protein combines specificity with versatility.

A polyclonal rabbit antiserum was obtained to the nucleoprotein of tomato spotted wilt tospovirus expressed as a recombinant fusion protein in E. coli. In indirect plate trapping ELISA, the antiserum gave similar titres against purified TSWV nucleocapsids in native form, the fusion protein and the carrier protein. The crude antiserum was also tested by Western blotting, indirect plate trapping ELISA and immunogold electron microscopy of thin sections: purified immunoglobulins were tested by DAS-ELISA. In all cases, with both glasshouse and field material, the antibodies had good detectability and specificity. By ELISA and Western blots against other tospoviruses, impatiens necrotic spot and groundnut bud necrosis viruses did not react but there was a reaction with groundnut ringspot virus, reflecting the nucleoprotein amino acid sequence similarity. These antibodies combine specificity to the target protein and versatility with regard to all the more important serological techniques. There were no undesired reactions resulting from immunization using a complex virus purified from infected host material.

Animals↗

PubMed1995

Purification of tomato yellow leaf curl geminivirus.

Attempts were made to find a good purification procedure for tomato yellow leaf curl virus (TYLCV), a dangerous and continuously spreading whitefly-transmitted germinivirus, up to now only partially purified. Electron microscopy, serology and spectrophotometry were used to evaluate different procedures. The scheme finally adopted was the following: collect leaves and stems from Nicotiana benthamiana graft-infected 45-60 days previously (5-10 g/plant); homogenize with 0.5 M phosphate buffer pH 6 containing 2.5 mM NaEDTA, 10 mM Na2SO3, 0.1% 2-mercaptoethanol, 1% Triton X-100 and 0.1% Driselase (3-4 ml of buffer for each g of material); incubate overnight on ice with gentle agitation; filter; emulsify with 15% cold chloroform; centrifuge at low speed; ultracentrifuge supernatant; resuspend pellets in 0.5 M phosphate buffer pH 7 containing 2.5 mM NaEDTA; centrifuge at low speed; repeat resuspension of the pellets and low-speed centrifugation; ultracentrifuge the pooled supernatant on a Cs2SO4 gradient (e.g. for 5 h at 41,000 rpm); collect the virus band and dialyse or ultracentrifuge the virus. The virus yield was 5-10 mg per kg of tissue.

Animals↗

PubMedDate not supplied

Using non-radioactive probes on plants: a few examples.

Due to costs in using and disposing of radiochemicals and to health considerations, we have been developing applications which include non-isotopic detection of DNA and proteins using chemiluminescence. Our major interests are in the detection of viral nucleic acids and in the analysis of transgenic plants. Generally, probes were labelled with digoxigenin, either by the random priming method or by PCR, and then detected with CSPD or CDP-Star. We routinely use a tissue blotting protocol for diagnosing TYLCV, a plant virus becoming a post in the Mediterranean region. Test results were comparable with those using the same radiolabelled probe. When total nucleic acids are extracted from the plant samples and used in dot-blot or Southern blot assays, viral DNAs are promptly detected by chemiluminescence. In transgenic plants, chemiluminescence was used to detect the transgene on genomic Southern blots, the transgenic mRNAs on Northern blots, and the transgenic protein on Western blots. In Southern and Northern blots, the quality of the results obtained was usually satisfactory, but not as good as with a radiolabelled probe, the main problem being the signal-to-background ratio. Our goal is now to improve the quality of results in demanding applications such as genomic Southern blots, by reducing the background on membranes.

Adamantane↗