Abstract
An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv) clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg) but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC50 values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe.
Highlights
Morphine is one of the most medicinally important analgesics and narcotics
This study presents the results for the development of an single-chain Fv (scFv) antibody, M86, with its detailed characteristics for morphine-binding specificity being defined for the first time
M86 was prepared for docking studies in which (i) hydrogen atoms were added to the structure with standard geometry; (ii) the structure was minimized using a MMFF94s force field; (iii) Molecular Operating Environment (MOE) Alpha Site Finder was used for active site searches within M86, and dummy atoms were created from the obtained alpha spheres; and (iv) the obtained M86 structure was used in the ASEDock program (Ryoka Systems Inc., Tokyo, Japan) [30] with a narcotics library in which the conformational analysis data for morphine, codeine, and heroin were determined by the MOE program
Summary
Morphine is one of the most medicinally important analgesics and narcotics. Structurally, it is classified as an alkaloid because of the presence of nitrogen. After a polyclonal anti-morphine antibody was reported in 1971, a number of studies attempted to establish anti-morphine antibodies with higher specificities [3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22] All of these antibodies have shown cross-reactivity among structurally related compounds, with codeine and heroin. We prepared eight types of carrier protein-conjugated narcotics as immunogens or panning antigens to assure specificity to the morphine portion (Figure 1d) These were N conjugates (conjugated through the piperidine ring nitrogen atom of narcotics; Figure 1) and C conjugates (conjugated through the carbon atom at the 2-position of narcotics; Figure 1). MOE analysis based on only one X-ray crystallographic structural data of the morphine-reactive antibody 9B1 (PDB ID: IQ0Y, [20]) suggested the possibility of creating a free morphine-specific antibody; M86 may be useful for developing analytical probes that can detect morphine, codeine, heroin, and therapeutic reagents for opiate addictions
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