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

A C6-hemisuccinate derivative of morphine was prepared and conjugated to bo vine serum albumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. A C3-hemisuccinate derivativ e of morphine was prepared and conjugated to enzyme penicillinase used as a tracer molecule. A novel enzyme-linked immunoadsorbent assay was developed using this conjugate to screen and characterize the monoclonal antibody pr oduced in these experiments. After two successive limiting dilutions, antib odies produced by 5 clones with good affinities ranging from 10(8) to 10(12 ) M-1 and less cross-reaction (least for codeine and other structurally rel ated molecules) were selected. These clones were found to be of IgG class w ith kappa light chain. Subclass determination showed that two of the clones produced IgG2b and three of them produced IgG1 type of antibody. Affinity purifications were performed for the selected clone (MOR-I). Purified antib ody was coated onto the wells of microtiter plate. The standard curve was c onstructed with a sensitivity of 100 pg/mL covering up to 10 ng/mL in buffe r and urine. The slope of the standard curve for selected clone in buffer a nd urine was calculated to be -0.7 and -0.64, respectively.