Automated monoclonal radioimmunoassays for pirenzepine, a selective muscarinic receptor antagonist, in plasma and urine.

Sensitive and specific monoclonal radioimmunoassays (RIA) for pirenzepine, a muscarinic receptor (M1) antagonist, were developed and validated for rapid automated routine analysis of plasma and urine samples from clinical studies. Three discrete stable hybridoma clones secreting monoclonal antibodies (MAb) to pirenzepine were produced by fusing the myeloma line X63-Ag8.653 to spleen cells of BALB/c mice immunized with pirenzepine-5-N-propionate-protein conjugates. Of three carrier proteins investigated (HSA, BSA and edestin), optimal humoral immune responses and affinity of hybridoma antibody were attained using HSA. All three MAb displayed high affinity to pirenzepine (Ka = 0.6-1.2 X 10(9) l/mol) but showed differing cross-reactivities with its 4'-N-desmethyl metabolite (less than 1%, 6% and 40% respectively). The MAb with essentially zero metabolite cross-reactivity, 58-7/7, was selected for RIA development. In comparison, eight rabbit polyclonal antisera raised against pirenzepine-5-N-propionate-HSA or pirenzepine-5-N-butyrate-HSA possessed a similar range of affinities to the MAbs, but none approached MAb 58-7/7 in specificity. The bridge length had no significant effect on antisera characteristics. Competitive solid-phase RIAs for pirenzepine in human plasma and urine were established using MAb 58-7/7 and [3H]pirenzepine as tracer. All fluid transfers were automated using a programmable sample processing system (Microlab 2,000). Detection limits were 0.25 ng/ml (plasma) and 4 ng/ml (urine) in 0.1 ml sample. The coefficient of within-assay variation was 4% or better in the range 2-100 ng/ml (plasma) or 30-1,000 ng/ml (urine), the between-assay CV was 5.3% or better in the range 5-90 ng/ml (plasma) or 40-660 ng/ml (urine). Excellent correlation was observed between the plasma monoclonal RIA and the hitherto used polyclonal RIA (n = 106, r = 0.994), and between the urine monoclonal RIA and HPLC (n = 82, r = 0.998). We expect that these assays will prove valuable in pharmacokinetic and pharmacological investigations of pirenzepine.