title: Cloning and expression in E. coli of a functional Fab fragment obtained from single human lymphocyte against anthrax toxin.
abstract: Human lymphocytes derived from the blood of a donor immunized with anthrax vaccine were isolated and enriched for B-cells by Nycoprep density centrifugation. Individual anti-anthrax protective antigen (PA) B-cells were isolated by fluorescence activated cell sorting with fluorescence-labeled recombinant PA (rPA). The RNA from sorted single B-cells was extracted using plant total RNA as the carrier prior to purification by Nanoprep RNA isolation columns and then cDNA was prepared. Donor specific human Fab primer sets were developed based on rapid amplification of 5'-complementary DNA end results. Heavy chain and light chain of human Fab were amplified from the donor single B-cells by PCR. The amplified heavy and light chain were then cloned into the expression vector pASK-IBA2 and expressed in Escherichia coli (E. coli). The chains combined in vivo to form a functional Fab which was then purified as one protein. The human Fab antibodies produced by this technique were functional when tested in Western blots where the rPA was the target as well as in ELISA. This approach allowed us to obtain human Fab that retained the natural heavy and light chain pairing, which is supposed to have a high antigen-binding affinity.
authors.affiliation: Canadian Food Inspection Agency, Centre for Plant Health, 8801, East Saanich Road, Sidney, British Columbia, Canada. msris@inspection.gc.ca
authors.forename: Saad A
authors.initials: SA
authors.lastname: Masri
authors.forename: Heidi
authors.initials: H
authors.lastname: Rast
authors.forename: Wei-Gang
authors.initials: WG
authors.lastname: Hu
authors.forename: Les P
authors.initials: LP
authors.lastname: Nagata
authors.forename: Damon
authors.initials: D
authors.lastname: Chau
authors.forename: Scott
authors.initials: S
authors.lastname: Jager
authors.forename: David
authors.initials: D
authors.lastname: Mah
mesh_terms: D022122:Anthrax Vaccines / Q000008:administration & dosage / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology*; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D022122:Anthrax Vaccines; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; C030325:anthrax toxin