Isolation and Purification of Hepatitis B Antigen by Electrochrornatography

Abstract

The electrochromatography column is a unique glass device designed to function both as a chromatography column and as an electrophoresis column; it can also be operated so that both column chromatography and electrophoresis can take place in one simultaneous operation (electrochromatography). When the column is used for electrochromatography, proteins of the same molecular size, but with different electrophoretic mobilities, will be separated by electrophoresis, whereas proteins with the same electrophoretic mobilities but of different molecular size will be separated by molecular sieving. If proteins vary only slightly in these physical properties, separation is greatly enhanced by the action of the electric field on the filtering molecules. Thus, electrochromatography allows proteins to be separated simply, rapidly, and with greater resolution than other systems currently available. Electrochromatography has been used successfully for the purification of hepatitis B antigen, which is not separated from closely related serum immunoglobulins by either chromatography or electrophoresis. This method may become an important tool for the isolation and purification of a large number of proteins and viruses not easily separated by means of current technology.

Mechanistic description and experimental studies of electrochromatography of proteins

“…Electrochromatography (EC) is a form of gradient liquid chromatography in which an electric potential is applied to columns packed with gel-filtration media (Hybarger et al, 1963;Vermeulen et al, 1971;Salak and Roch, 1972;Luzzio, 1975;O'Farrell, 1985;Scott, 1986;Tsuda, 1987;Rudge et al, 1993). The method of Rudge et al (1993) and Ivory (1988) combines gel electrophoresis and liquid chromatography to resolve biomacromolecules on the basis of molecular weights and electrophoretic mobilities.…”

Comparative purification and characterization of hepatitis B virus-like particles produced by recombinant vaccinia viruses in human hepatoma cells and human primary hepatocytes

“…An additional major advantage of that purification method is that it can be carried out in a fast, economical, and simple way. Therefore, this method provides several advantages over other previously described expensive and time-consuming purification strategies, such as precipitation [64,65], affinity chromatography [66,67], and ultracentrifugation [68,69]. Taken together, the combination of different purification methods leads to a maximum in purity, but to a reduced amount of final product.…”

Purification of Hepatitis B Surface Antigen Using Polyethylene Glycol, Pepsin and Tween 80

“…Isolation and purification of hepatitis B surface antigen (HBsAg) has been achieved by many techniques such as isopycnic banding in cesium chloride [11,12,22], by column chromatography [2,19,20], affinity chromatography [5,24], isoelectric focusing [15,28], precipita tion by ammonium sulfate [3], or polyethylene glycol (PEG) [6][7][8]17] and various combinations of these procedures. The most commonly used method is that of isopycnic banding in cesium chloride; however, not many laboratories have the equipment or the resources to be able to use this method effectively for routine production of moderately large batches of immunologically pure HBsAg.…”