Single-chain variable fragment technology in forensic toxicological analysis: production of an antibody to fluvoxamine

Abstract

Immunoassay techniques are widely used for drug screening in the fields of forensic toxicology and emergency medicine, because of their simple procedures and rapid outcome of results [1, 2]. To create an immunoassay method for a compound, the most laborious step is the production of an antibody that is specific to the compound. Here, we present a new recombinant antibody technology for producing a single-chain variable fragment (scFv). scFv is a small antibody molecule that retains high antigen specificity and binding activity of the original whole immunoglobulin [3]. One advantage of scFv is its simple production using bacterial cell cultures, which also provide variable functionalities by introducing mutations. Furthermore, creating scFv phage libraries, which display various scFvs, might be useful in searching for drug-reactive elements. This seems to be a powerful tool for rapid creation of new antidrug antibodies without immunizing animals. Recently, the frequency of psychotropic drug poisoning has increased in Japan [4]. Among such drugs, fluvoxamine (FLV), one of the selective serotonin reuptake inhibitor (SSRI) antidepressant drugs, is widely used for treatment of depression and obsessive compulsive disorders [5]. Although SSRIs are relatively safe as compared with conventional antidepressant drugs, such as tricyclic antidepressants and monoamine oxidase inhibitors [6], the number of FLV poisoning cases has increased according to the increase in its prescription [7, 8]. In this study, we created an anti-FLV scFv using novel recombinant antibody technology. FLV malate was obtained from Sigma-Aldrich (St. Louis, MO, USA). Mercaptosuccinyl bovine serum albumin (MS-BSA) was a generous gift from Prof. K. Fujiwara, Sojo University (Kumamoto, Japan). All other solvents and chemicals were of analytical grade, and purchased through local suppliers. BALB/c mice (female, 4 weeks old; Kyudo, Kumamoto, Japan) were maintained in the Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan, and were kept in an environmentally controlled room (22 ± 2 C, 50–70 % humidity, illuminated from 0700 to 1900 hours). All procedures were approved by the Kumamoto University Ethics Review Committee for Animal Experimentation. The immunogen (BSA–FLV) for induction of anti-FLV antibodies was prepared as described previously with a slight modification [9]. Briefly, FLV maleate (4.3 mg; approximately 10 lmol) in 1.0 ml of 50 mM phosphate buffer (pH 7.0) was mixed with N-[c-maleimidobutyryloxy] succinimide (GMBS) (0.25 mg; approximately 0.89 lmol), and incubated at room temperature with stirring. MS-BSA, estimated to contain 18 thiol groups per BSA molecule, was diluted with 3.0 ml of 0.1 M phosphate buffer (pH 7.0), and added immediately to the This article is for the special issue TIAFT2012 edited by Osamu Suzuki.