Bacterial expression and characterization of an anti-desipramine single-chain antibody fragment. 1996

G Lin, and P R Pentel, and W L Shelver, and D E Keyler, and C A Ross, and Y Hieda, and M C Flickinger, and C A Pennell, and M P Murtaugh
Department of Veterinary PathoBiology, University of Minnesota, St Paul 55108, USA.

Tricyclic antidepressant toxicity is a leading cause of death from intentional drug overdose. Monoclonal antibody Fab' fragments specific for the tricyclic antidepressant, desipramine, reverse acute drug toxicity but may themselves have adverse effects at therapeutic doses. To evaluate the characteristics of smaller antibody fragments, we cloned, expressed and characterized a 26 kD single chain Fv fragment (G5-sFv). A DNA sequence encoding VH-linker-V1 was constructed from hybridoma mRNA encoding a high affinity monoclonal desipramine specific IgG1 and expressed in E. coli. G5-sFv was produced at high levels as insoluble inclusion bodies. Single chain Fv was solubilized, folded in a redox buffer and affinity purified on desipramine-Sepharose. The affinity of G5-sFv for desipramine was similar to that of the corresponding monoclonal Fab' as measured by surface plasmon resonance (Fab' 5.5 +/- 0.5 x 10(8) M-1, sFv 2.3 +/- 0.5 x 10(8) M-1). G5-sFv administered to rats after a tracer dose of 3H-desipramine produced rapid and marked redistribution of drug from tissues into serum. G5-sFv was stable at 4 C for greater than 6 months but lost activity at higher temperatures. We conclude that desipramine-specific-single chain Fv expressed in E. coli retains the affinity of the parent antibody for desipramine. The pharmacokinetic effect of G5-sFv on desipramine distribution suggests that it may be useful as an antidote for desipramine overdose.

UI MeSH Term Description Entries
D007128 Immunoglobulin Fragments Partial immunoglobulin molecules resulting from selective cleavage by proteolytic enzymes or generated through PROTEIN ENGINEERING techniques. Antibody Fragment,Antibody Fragments,Ig Fragment,Ig Fragments,Immunoglobulin Fragment,Fragment, Antibody,Fragment, Ig,Fragment, Immunoglobulin,Fragments, Antibody,Fragments, Ig,Fragments, Immunoglobulin
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D002645 Chickens Common name for the species Gallus gallus, the domestic fowl, in the family Phasianidae, order GALLIFORMES. It is descended from the red jungle fowl of SOUTHEAST ASIA. Gallus gallus,Gallus domesticus,Gallus gallus domesticus,Chicken
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D003891 Desipramine A tricyclic dibenzazepine compound that potentiates neurotransmission. Desipramine selectively blocks reuptake of norepinephrine from the neural synapse, and also appears to impair serotonin transport. This compound also possesses minor anticholinergic activity, through its affinity to muscarinic receptors. Desmethylimipramine,Apo-Desipramine,Demethylimipramine,Desipramine Hydrochloride,Norpramin,Novo-Desipramine,Nu-Desipramine,PMS-Desipramine,Pertofran,Pertofrane,Pertrofran,Petylyl,Ratio-Desipramine,Apo Desipramine,Hydrochloride, Desipramine,Novo Desipramine,Nu Desipramine,PMS Desipramine,Ratio Desipramine
D004797 Enzyme-Linked Immunosorbent Assay An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. ELISA,Assay, Enzyme-Linked Immunosorbent,Assays, Enzyme-Linked Immunosorbent,Enzyme Linked Immunosorbent Assay,Enzyme-Linked Immunosorbent Assays,Immunosorbent Assay, Enzyme-Linked,Immunosorbent Assays, Enzyme-Linked
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein

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