Instability of chimaeric antibody secretion by anti-carcinoembryonic antigen producing hybridoma cells after gene targeting.
OBJECTIVE
To produce a chimaeric version of the 11-285-14 anti-carcinoembryonic antigen (CEA) monoclonal antibody using a gene targeting approach.


METHODS
A replacement vector was constructed to insert the human constant gamma1 gene within the mouse heavy chain locus of 11-285-14 hybridoma cells. The mouse constant gamma1 gene (1.5 kb) and the mouse mu intron fragment (2.2 kb) were amplified by PCR and cloned into a pKO Scrambler vector. The human constant gamma1 gene fragment (2.2 kb) was cloned next to the intron fragment. Resistant colonies were screened by ELISA for the presence of the human isotype in their supernatants.


RESULTS
Of the 4,370 resistant colonies obtained, 87 colonies showed secretion of the human isotype at levels between 4 and 32 ng/ml. PCR and Southern blot results confirmed the correct integration of the human gene by homologous recombination within the heavy chain locus. Most of the producers ceased to express the human isotype within a few weeks after the initial positive ELISA results. Instability of secretion could not be explained by genetic instability in all the clones, which suggests the presence of other undefined epigenetic or physiologic mechanisms.


CONCLUSIONS
Gene targeting resulted in transformants with unstable and low production rates of chimaeric anti-CEA antibody.
El-Abdallah
Abir A
AA
Department of Pathology, Faculty of Medicine, Kuwait University, Kuwait. abeer.a@hsc.edu.kw
Ford
Christopher H J
CH
D000483:Alleles; D000818:Animals; D000917:Antibody Formation; D015139:Blotting, Southern; D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018390:Gene Targeting; D005820:Genetic Testing; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000276:immunology
D002272:Carcinoembryonic Antigen; D007127:Immunoglobulin Constant Regions; D011993:Recombinant Fusion Proteins