Immunoreattività e carcinoma renale

V. Proserpio*, M. Beretta, C. Chinello, M. Soldi, N. Tiberti, F. Raimondo,
C. Bianchi, S. Ferrero, S. Casellato, F. Magni, C. Sarto, P. Mocarelli, P. Brambilla

*Servizio Universitario di Medicina di Laboratorio, Ospedale di Desio (MI)
Biochimica Clinica: 2009; 33(3): 191-195 [Article in italian]

ABSTRACT
Immunoreactive proteins in renal cell carcinoma. Renal cell carcinoma (RCC) represents the most common tumor affecting the adult kidney and is characterised by a variety of histological subtypes. Several molecules, such as proteins involved in the cell cycle, apoptosis, and cell-cell interaction, have been investigated, but unfortunately none of these has been validated as an ideal biomarker. During spontaneous remissions it has been shown that the immune system of cancer patients is activated against specific tumor antigens that might represent new potential biomarkers. In this study we applied a serological proteomic-based approach (SERPA) to identify tumor antigens that induce a humoral immune response in RCC. Protein patterns derived from RCC cases were separated by two-dimensional electrophoresis and western blot. Tumor antigens were immnunodetected using autologous and healthy donor sera as primary antibodies. The analysis of immunoreactive spots from normal and RCC tissues pointed out 14 spots that immunoreacted with sera of at least three patients. None of these protein spots were immunoreactive with the healthy donor sera. Among these proteins, identified by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry, vimentin and lamin A/C were already found immunoreactive in RCC and in other cancer. They might be useful as diagnostic and therapeutic targets for RCC and could represent new tools for clinical use.

Applicazioni dell'elettroforesi capillare all'analisi delle proteine della membrana eritrocitaria

L. Mosca*, R. Paleari, A. Mosca

*Centro Interdipartimentale per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi, Milano

Biochimica Clinica: 2009; 33(3): 196-201 [Article in italian]

ABSTRACT
Application of capillary electrophoresis to the analysis of red cell membrane proteins. Several hereditary hemolytic anemias associated with abnormal red cell shapes, such as hereditary spherocytosis and elliptocytosis, are caused by defects of red cell membrane proteins. The detection of these abnormalities is usually performed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), a time-consuming and laboratory intensive technique. The aim of this study was to set up a method based on SDS-capillary gel electrophoresis (SDS-CGE) for the separation and quantification of major erythrocyte membrane proteins. Twenty blood samples were collected in EDTA and processed within 2 days. Erythrocytes were separated from platelets and leukocytes and then subjected to a hypotonic treatment to obtain membrane ghosts. The analyses of red cell membrane proteins were carried out with a Beckman Coulter ProteomeLab PA800 capillary electrophoresis apparatus equipped with a diode array detector set at 220 nm. The SDS-MW analysis kit (Beckman Coulter Inc.) was used. Seven major erythrocyte membrane proteins were separated and identified: α- and β-spectrin, and bands 3, 4.1, 4.2, 5 and 6. Their MW values were in good agreement with those reported in the literature. The reproducibility (expressed as CV) of the migration times was between 0.1% and 0.3%. The reproducibility of the relative abundances of these main proteins, expressed in relation to band 5, was between 3.6% (band 6/5) and 7.6 % (band 4.1/5). Our preliminary results prove that SDS-CGE may be an alternative approach to standard SDS-PAGE for the identification of red cell membrane disorders.

Sviluppo e validazione di un metodo hplc per il dosaggio del levetiracetam nel siero

C. Corsaro*, M.A. Mangano, A. Signorelli

*Laboratorio di Chimica e Clinica Tossicologica, Azienda USL 3, Catania

Biochimica Clinica: 2009; 33(3): 202-204 [Article in italian]

ABSTRACT
Development and validation of a hplc method for the determination of levetiracetam in human serum. A sensitive and specific HPLC method has been developed and validated for determination of levetiracetam in human serum. The sample preparation involves a clean-up procedure using a SPE C18 column. The separation was carried out on a Zorbax Esclipse XDB-C18 4.6 x 150 mm with Alltima C18 4.6 x 7.5 mm precolumn using phosphate-acetonitril buffer solution. Excellent linearity was found between 2.5 and 40.0 mg/L, with a limit of detection of 0.625 mg/L. Assay imprecision (% CV) was <6,8%. This method could be helpful for monitoring subjects with epileptic pathology to better optimize their therapy.

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