Effect of VH and VL consensus sequence-specific primers on the binding and neutralizing potential of a single-chain FV directed towards HuIFN-γ

Froyen, Guy; Hendrix, Diana; Ronsse, Isabelle; Fiten, Pierre; Martens, Erik; Billiau, Alfons

doi:10.1016/0161-5890(95)00010-c

Cited by 8 publications

(1 citation statement)

References 17 publications

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“…In order to retrieve the rearranged variable region genes from genomic DNA, degenerate primers were used. Though changes in the nucleotide sequences of FR1 and FR4 is a major disadvantage of using degenerate primers, sequence-alteration by degenerate primers was considered as a minor influence [41]. On the other hand, the use of family-specific primers assures less change in nucleotide sequences, but a large number of family specific primer pairs will be required for retrieving the Ig variable region genes from genomic DNA.…”

Section: Discussionmentioning

confidence: 99%

Construction and Characterization of Single-Chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes

et al. 2011

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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.

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Section: Discussionmentioning

confidence: 99%