Antibody phage-displayed libraries derived from chicken immunoglobulin genes : a source of highly specific diagnostic antibodies

Abstract

In meeting the high demand for monoclonal antibodies, the chicken immunoglobulin system was exploited to generate recombinant antibodies against multiple target antigens. Following simultaneous immunisation of two chickens with a mixture of Plasmodium falciparum recombinant lactate dehydrogenase (LDH), histidine rich protein II (HRPII) and aldolase (ALDO), recombinant trypanosome variable surface glycoprotein (VSG) and malignant catarrhal fever virus (MCFV) each chicken produced egg yolk antibodies (IgY) against four of the five antigens. Using phage display technology, two single-chain variable fragment (scFv) antibody libraries, one with the immunoglobulin VH and VL chain regions joined by a single amino acid (G) and the other with a 15 amino acid flexible linker [(G4S) 3] were constructed using pooled splenic RNA. The single amino acid-linked scFv repertoire was evaluated as a source of highly specific diagnostic antibodies by panning against each of the five different antigens. After two rounds of panning, polyclonal phage ELISA showed the presence of antigen-specific phage antibodies against three (LDH, HRPII and VSG) of the five antigens. Five different anti-LDH and six different anti-HRPII scFvs were identified by sequence analysis. Evidence of high levels of antigen-driven gene conversion events was found in the framework and complementary determining regions and the VL chain pseudogene donors were identified. Stability of the selected scFvs was determined by incubation at different times and at different temperatures. The specificity and potential use of an LDH-specific scFv as a diagnostic reagent was shown in sandwich and competitive inhibition ELISAs.