Abstract

The suitability o f in vitro immunisation, for monoclonal antibody (Mab) production, was investigated. A panel o f nine Mabs were produced, and analysed for polyreactivity. Three different assays for polyreactivity were utilised, including measurement of the affinity of each Mab for a small panel of antigens. Using these three assays, eight of the nine Mabs were demonstrably polyreactive. It was concluded, that in vitro immunisation may not be suitable for Mab production. A second area of interest was the generation o f intrabody libraries, for functional genomic analyses. A PCR-based strategy was developed to convert any ScFv gene, from the Nissim library, to an intrabody gene. As a model, an anti-NIP ScFv gene was converted to an intrabody gene. Cytochemical studies indicated correct cytoplasmic localisation o f this intrabody, but poor expression levels. The strategy was applied to generate a large intrabody library. A novel PCRcloning methodology was used. This utilised T4 DNA polymerase, and required the creation of a novel vector, referred to as pMK. The cloning strategy proved very successful and a 'one-shot' intrabody library o f 1.3xl07 independent clones was generated. These studies demonstrate the feasibility o f the strategy, but suggest that further studies are required to improve intrabody stability.