ABSTRACT
Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.
Subject(s)
Immunoglobulin Variable Region/genetics , Single-Chain Antibodies/genetics , Animals , Enzyme-Linked Immunosorbent Assay , Mice , Mice, Inbred BALB C , Peptide Library , Polymerase Chain ReactionABSTRACT
A 43 year-old male patient presented with small bowel obstruction while being treated for cervical tuberculous lymphadenopathy. Laparotomy revealed multiple adhesions and multiple jejuno-jejunal fistulae. Absence of previous abdominal surgery or other abdominal insult favoured an 'idiopathic' origin of these unusual lesions, although treated tuberculosis may have been the underlying cause. To the best of our knowledge this intestinal condition has never previously been reported in the medical literature.
ABSTRACT
Magnetic resonance angiography (MRA) is a method of non-invasive vascular imaging that has evolved and improved significantly since first described. In this article, the basic principles of MRA are reviewed and MRA compared with the more conventional imaging techniques of Doppler ultrasound and angiography. The ways in which MRA can be used in conjunction with more conventional vascular imaging techniques in investigation and management of carotid disease, aortic disease, and peripheral vascular disease are discussed in this article.
