Production of Mouse Monoclonal Antibody against Morphine without Cross Reactivity with Heroin

Immunoassay procedures for detecting and determining opioids in blood and other biologic fluids are based on Monoclonal antibodies. In the present study, monoclonal antibody against Morphine was taken into account. Hybridoma protocol was used in order to produce the monoclonal antibody against morphine in mice. For this purpose, five 6–8-week old female BALB/c mice were immunized with morphine C6- hemisuccinated derivative conjugated to cationized bovine serum albumin (cBSA). The spleens lymphocytes were fused with SP2/0 cells using polyethylene glycol (PEG). Hybridoma clones were subcloned by limiting dilution. Class and subclass of monoclonal antibody were determined using Roche isostrip test. Moreover, antibody was purified by protein G affinity chromatography and affinity was determined according to the method described by Beatty et al. Finally, the cross reaction of monoclonal antibody was determined with some structurally related molecules such as codeine and apomorphine. Among 3 hybridoma clones that reacted with the morphine-BSA, but not with BSA, after thrice limiting dilution, one stable hybridoma monoclon was obtained. The monoclonal antibody (MAb) was found to be of IgG2b class and subclass and containing lambda light chain. The affinity of the MAb to morphine was obtained 2.8 ×109 M-1 by non competitive enzyme immunoassay. The titer of supernatant of cell culture medium was 1/400. The MAb was cross reacted with codeine (100%) and apomorphine (16.5%), though no reaction was observed with heroin, naloxone, naltrexone, and papavrin. The study findings revealed that the produced antibody against morphine was comparable with other antibodies for specificity and affinity; therefore it is usable in design of diagnostic immunoassay in biologic fluids.