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Biomedical subjects

R W Hepler

Publications and source records attributed to R W Hepler.

4 recordsLinked to original sources
PubMed1999

Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae.

Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice.

Amino Acids↗
PubMed1998

One-step fluorescent probe product-enhanced reverse transcriptase assay.

Recently developed PCR-based reverse transcriptase (RT) assays are useful in the detection of retroviruses since they are approximately a millionfold more sensitive than conventional RT assays. However, these assays are both labor- and time-intensive. The previously described product-enhanced reverse transcriptase (PERT) assay involves a two-step RT-PCR followed by detection and quantitation of PCR products by either Southern blot or enzyme-linked immunosorbent assay (ELISA). We have modified the PERT assay to be a one-step, fluorescent probe, PCR-based RT assay that can be completed from sample dilution to final quantitative assay results in approximately 5 h without loss of assay sensitivity or specificity. The assay has a dynamic range of 6 logs, and therefore, extensive sample dilution is not necessary for quantitation. This newly enhanced fluorescent PERT assay can play an important role in the high-throughput detection of retroviral infection and characterization of RT activity.

Animals↗
PubMed1994

Application of capillary ion electrophoresis and ion chromatography for the determination of O-acetate groups in bacterial polysaccharides.

Many bacterial polysaccharides possess O-linked acetate groups as constituents of their repeating units which often can serve as immunological determinants. It is therefore important to develop analytical methods for process monitoring as well as product characterization when such O-acetylated polysaccharides are used as components of vaccines. This is the case in a polysaccharide conjugate vaccine under development for treatment of diseases caused by Streptococcus pneumoniae. An ion chromatographic (IC) method utilizing suppressed conductivity detection (SCD) was developed to quantitatively measure O-acetate groups in the capsular polysaccharides from S. pneumoniae types 18C and 9V following hydrolytic release of O-acetate from the polysaccharide backbones using 2 mM sodium hydroxide. IC was carried out using an OmniPac PAX-500 column and 0.98 mM NaOH in 2% methanol as the mobile phase. Capillary ion electrophoresis (CIE) with indirect photometric detection was evaluated as an alternative method. The CIE method utilized a 72 cm x 75 microns I.D. fused-silica capillary and an electrolyte composed of 5 mM potassium hydrogenphthalate, 0.5 mM tetradecyltrimethylammonium bromide, and 2 mM sodium tetraborate, pH 5.88. A comparison of CIE and IC-SCD in terms of reproducibility, accuracy, linearity, and sensitivity will be presented.

Acetates↗