James G. Files

Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli

An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12. The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge. De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation. A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated. The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides. The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein. The products synthesized in E. coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite. The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form.

Enzymatic properties and processing of bovine prochymosin synthesized in Escherichia coli

Abstract Active chymosin has been produced from calf prochymosin synthesized in E. coli . A preprochymosin cDNA clone was used to construct a plasmid, pWHA43, which expresses methionyl-prochymosin. This zymogen protein is synthesized in the bacteria in a stable but insoluble form localized in cytoplasmic inclusion bodies. Partially purified and solubilized prochymosin from E. coli can be processed by the same apparent mechanism as authentic calf prochymosin upon activation with acid. The chymosin thus derived from E. coli showed the same substrate specificity and the same kinetics of activation as that of chymosin derived from purified calf stomach prochymosin. These results also demonstrate that the bovine prochymosin synthesized in E. coli can be reconstituted and activated to a form having the functional properties necessary for an industrial milk coagulant to be used in cheese manufacturing.

Use of a Standardized MxA Protein Measurement-Based Assay for Validation of Assays for the Assessment of Neutralizing Antibodies Against Interferon-β

Effective monitoring of the development of neutralizing antibodies (NAbs) against IFN-β in multiple sclerosis (MS) patients on IFN-β therapy is important for clinical decision making and disease management. To date, antiviral assays have been the favored approach for NAb determination, but variations in assay conditions between laboratories and the increasing use of novel assays have contributed to the reporting of inconsistent antibody data between laboratories and between products. This study, undertaken at the request of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA), is a joint effort by manufacturers of IFN-β products (approved in Europe) towards harmonization of a NAb assay that facilitates generation of comparable NAb data, which, in conjunction with clinical outcomes, should prove useful for clinicians treating MS patients with IFN-β products. This article describes the standardized cellular myxovirus resistance protein A (MxA) protein measurement-based assay for detection of IFN-β NAbs and its use for the validation of assays used for the quantitative determination of such antibodies. Although titers varied between laboratories and the products used, utilization of IFN-β1a rather than IFN-β1b as the challenge antigen produced more consistent results in the NAb assay. Adoption of the standardized assay improves comparability between laboratories circumventing problems that arise when different, nonstandardized assays are employed for immunogenicity assessment. Based on the data, the EMA recommended for standardization purposes, the use of IFN-β1a in NAb assays, independent of the therapeutic product used for therapy and validation of new NAb procedures against the standardized assay described.

Analytical Assays to Characterise Adenoviral Vectors and Their Applications

Recombinant adenovirus preparations are used for gene delivery in a number of clinical gene therapy approaches. Adenovirus offers short term (transient) gene expression without integration into the host cell genome1. The adenovirus is a complex biological system containing several different structural proteins and a linear double stranded DNA molecule held together by non-covalent interactions2. The classical assays used to characterise the adenovirus preparations include infectivity analysis (typically a plaque assay format3), SDS-PAGE analysis of the adenoviral proteins2,4,5 and the estimation of virus concentration by lysing the virus in SDS and measuring absorption at 260 nm in the lysate2.