The distribution of particles characterized by size and free mobility within polydisperse populations of protein-polysaccharide conjugates, determined from two-dimensional agarose electropherograms

New approaches for the characterization of polydisperse particle populations are presented*. The investigated samples contain virus-sized protein-polysaccharide conjugates which had previously been prepared as immunogens against bacterial meningitis (Hib). The analysis is based on two-dimensional agarose electrophoresis (Serwer-type). This method, like the one of O'Farrell, achieves a separation according to size and charge. It relies on a different principle, however, and is applicable to nondenatured particles which are 100 to more than 1000 times larger in mass than regular uncrosslinked proteins. Data from stained gel patterns are evaluated by the computer program ELPHOFIT, which makes it possible to standardize the gel and to construct a nomogram which defines every position on the gel in terms of particle size and free mobility (related to surface net charge density). The output of ELPHOFIT, consisting of nomogram parameters, is transferred to the image processing program GELFIT. This software is used to evaluate the computer images obtained by digitizing the stained gel patterns: (i) The nomogram is electronically superimposed on the computer image. (ii) The gel pattern is transformed from a curvilinear to a rectangular coordinate system of particle size and free mobility. The center of gravity as well as density maxima are given in coordinates of particle size and free mobility. Ranges of grey levels can be accentuated by adding 16 pseudocolors. (iii) Using surface-stripping techniques, GELFIT provides an estimate for the number of major subpopulations within each preparation. (iv) Numerical values for the distribution of particle size and free mobility are determined. Using program IMAGE, the quantitative physical assessment of a given conjugate preparation is presented in the form of a computer-generated three-dimensional plot, the shape of which serves to identify and characterize the preparation visually. The data analysis based on digitized two-dimensional gel patterns is automated to an extent that a technician can perform routine evaluations. It uses the Macintosh II personal computer.