Computer‐assisted 2‐D agarose electrophoresis of Haemophilus influenzae type B meningitis vaccines and analysis of polydisperse particle populations in the size range of viruses: A review

Abstract

When protein-polysaccharide conjugated vaccines were first developed for the immunization of small children against meningitis caused by infection with Haemophilus influenzae type b (Hib), the vaccine preparations varied in immunogenicity. Testing for immunogenicity was time-consuming and alternative analytical procedures for determining vaccine quality were unsatisfactory. For example, due to the very high molecular weight of the vaccine particles, immunogens could only be physically characterized as a fraction in the void volume of Sepharose gel filtration. In search of better analytical methods, a computer-assisted electrophoretic technique for analyzing such vaccines was developed in the period from 1983 to 1995. This new approach made it possible to analyze highly negatively charged particles as large as or larger than intact viruses. 2-D gel patterns were generated that varied depending on the conditions of the particular vaccine preparation and were therefore characteristic of each vaccine sample. Thus, vaccine particle populations with a continuous size variation over a wide range (polydisperse) could be characterized according to size and free mobility (related to particle surface net charge density). These advances are reviewed in this article, since the developed methods are still a promising tool for vaccine quality control and for predicting immunogen effectiveness in the production of vaccines. The technique is potentially beneficial for Hib immunogens and other high-molecular-mass vaccines. Additional biomedical applications for this nondenaturing electrophoretic technique are briefly discussed and detailed information about computational and mathematical procedures and theoretical aspects is provided in the Appendices.

DNA Packaging-Associated Hyper-Capsid Expansion of Bacteriophage T3

“…An arc of this shape is expected of particles that all have the same σ, but vary in R E and sometimes have R E large enough to be significantly sieved in the first dimension (i.e., particles with the smaller values of θ) 26. The curved arrow in Figure 3a illustrates an arc of this type; examples of this behavior have been previously described 26,27…”

“…The sample preparation for 2d-AGE was the same as it was for 1d-AGE, except that time in the sample well was increased for enzyme digestions (below) and 100 µg/ml bovine serum albumen was added to reduce adherence to the gel origin. The first dimensional gel was 0.3% agarose (Seakem LE, Lonza, Rockland Maine) that was embedded within a 1.8% second dimensional Seakem LE agarose gel, submerged beneath electrophoresis buffer and cast in an apparatus in which the direction of electrophoresis is changed by 90° without manipulating the gel 26,27. The first dimensional electrophoresis was performed at 2.0 V/cm for 4.0 hr.…”

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

“…where m is the observed gel mobility, m o is the mobility of the polyion in free solution, l' is the length of the gel fibers, R is the effective hydrodynamic radius of the polyion, and T is the gel concentration [16][17][18]. Equation (1) predicts that semilogarithmic plots of the observed mobilities as a function of gel concentration should be linear and should extrapolate to the free solution mobility of the polyion at zero gel concentration.…”

Gels for the Propagation of Bacteriophages and the Characterization of Bacteriophage Assembly Intermediates

“…This aspect was originally demonstrated for vaccine conjugates heterogeneous in R E ) and has been developed in quantitative detail, given that these conjugates are of high utility (Tietz, 2007(Tietz, , 2009. More recently, the use of 2d-AGE with particles heterogeneous in both  and R E has been used to detect ipDNAcapsids of new type.…”