Isolation and characterization of a novel PDGF-induced human gene.

Using a differential display RT-PCR strategy to identify novel growth-factor-induced transcripts, we cloned and characterized the human homolog of yeast NOP5/NOP58, whose gene product has been implicated in the execution of early pre-rRNA processing steps. Human NOP5 cDNA was isolated from an M426 fibroblast cDNA library. Determination of the cDNA nucleotide sequence revealed an open reading frame of 1587 nucleotides encoding a predicted gene product of 529 amino acids and mass of 59554Da. The yeast and human NOP5 gene products were found to share 63% homology and 46% identity. NOP5 mRNA was induced within 2h of platelet-derived growth factor (PDGF) treatment of human M426 fibroblasts. Pretreatment with cycloheximide enhanced, while actinomycin blocked induction of the NOP5 transcript. In vitro translational analysis of the cDNA revealed a 60kDa species, consistent with the predicted molecular weight of the gene product. Ubiquitous, but differential NOP5 mRNA expression was revealed after Northern blot analysis of total RNA from several human tissues. Moreover, NOP5 mRNA expression was also demonstrated in cell lines of fibroblast, epithelial, and myeloid origin. A highly charged carboxy terminal domain and consensus phosphorylation sites were identified. The presence of potential regulatory elements, together with growth factor induction and widespread expression is consistent with the hypothesis that the NOP5 gene product may play a role in fundamental cellular growth processes.

Avidin- or streptavidin-biotin as a highly sensitive method to stain total protein on membranes.

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate alpha-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5 ng of transferred protein in a single band and is thus 5-10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.

Recombinant polyclonal antibody libraries.

We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen). Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen 'escape variants', and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations. The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries. This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies. We review here our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.

Generation of a polyclonal fab phage display library to the human breast carcinoma cell line BT-20.

We have previously described a vector system for generating recombinant polyclonal antibody libraries. This system uses bidirectional phagemid and mammalian expression vectors to facilitate mass transfer of selected variable light and variable heavy (VL-VH) region gene pairs from the phagemid to the mammalian vector, to express polyclonal libraries of whole IgG antibodies. We report here the first stage of generating a polyclonal antibody library to the human breast carcinoma cell line BT-20, using this vector system. VL and VH region gene pairs were obtained from a mouse immunized with BT-20 cells, and cloned, in opposite transcriptional orientations, in the bidirectional phagemid vector, to produce an Fab phage display library. This library was selected by panning on BT-20 cells and shown to bind specifically to BT-20 cells. Such libraries, after suitable negative selection to eliminate major reactivities against normal tissue, could be transferred in mass to our bidirectional mammalian expression vector for production of libraries of chimeric antibodies with mouse V regions and human constant (C) regions. These polyclonal antibody libraries will mediate effector functions and are expected to be useful for breast cancer therapy, as well as diagnosis.

Generation of a polyclonal Fab phage display library to the protozoan parasite Cryptosporidium parvum.

We had developed a technology for creation of recombinant polyclonal antibody libraries, standardized perpetual mixtures of polyclonal whole antibodies for which the genes are available and can be altered as desired. We report here the first phase of generating a polyclonal antibody library to Cryptosporidium parvum, a protozoan parasite that causes severe disease in AIDS patients, for which there is no effective treatment. BALB/c mice, immunized by neonatal oral infection with oocysts followed by intraperitoneal immunization with a sporozoite/oocyst preparation of C. parvum, were used for construction of a Fab phage display library in a specially-designed bidirectional vector. This library was selected for reactivity to an oocyst/sporozoite preparation, and was shown to be antigen-specific and diverse. Following mass transfer of the selected variable region gene pairs to appropriate mammalian expression vectors, such anti-C. parvum Fab phage display libraries could be used to develop chimeric polyclonal antibody libraries, with mouse variable regions and human constant regions, for passive immunotherapy of C. parvum infection.