Preparation and identification of specific and high-affinity monoclonal antibodies against morphine

A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin and ovalbumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol generated twenty-six stable murine monoclonal antibodies (MAbs) producing cell lines to morphine. The donor mouse produced antiserum with a high titer of 1/640,000. Twelve MAbs were selected for further characterization since they showed high sensitivities (53 pg/well to inhibit 50% of the tracer) in improved group-selective immunoassay (IGSI). The assay, which maintains high sensitivity, high precision, and a wide range of optical density (OD) values, was developed using the conjugate M-6-S-OVA to screen and characterize the anti-morphine MAbs. After four successive limiting dilutions, antibodies produced by 12 clones had high affinities ranging from 10(9) to 10(10) M(-1). These clones were found to be of Ig(G) class and IgM class with kappa and lambda light chain. Subclass determination showed that the clones produced IgG1, IgG2a, IgG3, and IgM types of antibody. One clone (2F8B11F2A12) was used to establish the calibration curve with a sensitivity of 400 pg/mL covering up to 25.6 ng/mL in urine.