Cloning, expression, and purification of an anti-desipramine single chain antibody in NS/O myeloma cells.
Drug-specific monoclonal antibodies and their antigen-binding Fab fragments reverse acute desipramine toxicity in a rat experimental model by inducing a redistribution of drug from cardiac tissue into serum and extracellular fluid. In order to investigate the use of smaller recombinant antibody fragments such as single chain Fv (sFv) as an antidote, an efficient murine NS/O myeloma expression system was developed. The variable light (VL) and variable heavy (VH) domains of a murine anti-desipramine monoclonal antibody were cloned and sequenced. A 270 amino acid VH-(Gly4Ser)3-VL sFv was prepared by overlapping polymerase chain reaction (PCR) amplification of VH with heavy chain leader peptide, VL, and the linker. This construct was subcloned into a mammalian expression vector which utilizes the SR alpha promoter, a hybrid promoter consisting of the SV40 early promoter with portions of the human T-cell leukemia virus type I long terminal repeat and also containing the Escherichia cloi xanthine-guanine phosphoribosyltransferase gene for selection. NS/O myeloma cells were transfected by electroporation. Stable recombinant NS/O clones were screened for expression of sFv using reverse transcriptase-PCR to detect mRNA and an enzyme-linked immunosorbent assay (ELISA) to detect sFv. Secreted sFv from clones capable of growth to a cell density of 2-4 x 10(6) viable cells/mL was purified in a single step using a desipramine affinity column resulting in 12-39 mg/L of purified sFv. Affinity-purified sFv had comparable desipramine binding activity to Fab when evaluated by competitive ELISA.
Kitchin
K
K
Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul 55108, USA.
Lin
G
G
Shelver
W L
WL
Murtaugh
M P
MP
Pentel
P R
PR
Pond
S M
SM
Oberst
J C
JC
Humphrey
J E
JE
Smith
J M
JM
Flickinger
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D000931:Antidotes / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003891:Desipramine / Q000276:immunology* / Q000378:metabolism; D006825:Hybridomas / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000931:Antidotes; D007128:Immunoglobulin Fragments; D003891:Desipramine