Isolation of functional phage displayed single domain antibody fragments against dichloro-diphenyl-trichloroethane (DDT),chloroquine and plasmodial thiazole kinase.

Abstract

The target of molecular engineers has been to generate smaller antibody fragments from the large complete antibodies that still retain the antigen binding activity. Small fragments equipped with radioisotopes can be used for imaging or therapy and are particularly attractive for use in vivo as they penetrate tissue boundaries more effectively. Such fragments are also cleared faster from serum and tissues and although this may compromise their use as targeting agents, it can aid clearance of toxic drug complexes associated with digoxin from circulation. Small fragments have advantages in fundamental research for high-resolution X-ray crystallography studies of antigen binding fragments. The discovery of naturally occurring antibodies devoid of light chains as well as the first constant heavy domain (CHI) was a great boost to molecular engineers. These antibodies are quite soluble and easy to handle in contrast to other insoluble heavy chain domains that can be prepared from classical antibodies. The aim of the research was to generate functional camel phage displayed antibody fragments with :.: strong binding affinity against Dichloro-diphenyltrichloroethane (DDT), Plasmodial Thiazole kinase and Chloroquine. These were to aid in monitoring environmental pollution in the case of DOT, monitoring chloroquine resistance by Plasmodium parasites and as a potential therapeutic for malaria in the case of plasmodial Thiazole kinase (Thik). Gene fragments encoding the VHHS were amplified from cDNA templates derived from peripheral blood lymphocytes from ten camels using a set of primers able to amplify the entire VHH-D-JHrepertoire present within the peripheral blood lymphocytes sample because all the isotypes (in Llama and dromedary) are members of a single sub-group (sub-group III). The VHH-D-JHrepertoire was ligated into a phagemid vector (pCANT AB 5 E) behind the periplasmic secretion signal and in front of minor coat protein (g3p) of filamentous phage. Expression of cloned VHHleads to a fusion protein that is incorporated at the tip of M13 virions thus generating a genetic linkage between the VHHpeptide expressed at the tip and its gene inside the viral particle. In this way, repertoires of antibody fragments were displayed on surface of filamentous phage, each displaying a single antibody species. To select for phages displaying antibodies specific to the three antigens, panning experiments were carried out. The selected phages not only express the antibody that recognizes the antigen, but also rescues the corresponding VHHgene fragment coding for the antibody. Colony PCR with nested primers that was carried out confirmed this. Sequencing of the. positive clones was also carried out to find out whether the gene fragments were antibody genes. The partial gene obtained for anti-DDT is similar to other antibody genes in the gene data bank e.g. ScFv against the pesticide, parathion. ELISA experiments confirmed the affinity and specificity of the various gene fusions on the phages directed against individual antigens. Binders against pesticide DDT (cAb-DDT), Chloroquine (cAb-Chloro) and plasmodial ThiK{ cAb- ThiK) were retrieved. Thus it is possible to obtain anti-pesticide or anti-drug phage displayed antibodies of camel origin.