| dc.description.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. | en |
