Naslov
Electrophoretic analyses of clotting factor VIII concentrates

Autori
Pock, Katharina ; Jungbauer, Alois ; Kannicht, Christoph ; Löster, Klemens ; Buchacher, Andrea ; Josic, Djuro

Izvornik
Analytica Chimica Acta (0003-2670) 372 (1998), 1-2; 219-229

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Plasma proteins ; Clotting factor VIII ; 2D-PAGE (two-dimensional electrophoresis) ; Proteomics

Sažetak
Preparations of clotting factor VIII (FVIII) are complex protein mixtures, regardless of whether they are made by genetically engineered cells or isolated from human plasma. Commercially available FVIII preparations contain either von Willebrand factor (vWF) or human serum albumin (HSA), both of which function as stabilizers. Apart from these, the preparations contain other plasma proteins as impurities. Such protein mixtures were analyzed with SDS–PAGE and 2D-electrophoresis. Single bands and spots were identified by immunoblot using monoclonal and polyclonal antibodies. The aim was to detect every preparation which has an increased level of FVIII cleavage products. Chromatographic methods and SDS–PAGE under reducing and non-reducing conditions usually do not show any differences between single preparations. Only by a combination of separations under non-reducing and increasingly reducing conditions cleavage products of FVIII can in some cases be detected in immunoblot. In 2D electrophoresis the detection of proteins with high molecular masses was achieved by applying a mixture of thiourea and urea. The spots in 2D-electrophoresis of plasma-derived FVIII preparations have been identified as FVIII and vWF, but also as impurities, chiefly from fibrinogen. It was shown that especially 2D-electrophoresis and immunoblot can be used for better characterization of FVIII concentrates. The 2D protein maps of FVIII concentrates will be an efficient tool for quality control of such products.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)

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