2-SITE IMMUNOASSAY OF RECOMBINANT HIRUDIN BASED ON 2 MONOCLONAL-ANTIBODIES

Two monoclonal antibodies (mAbs), 10-2 and 10-5, both directed against recombinant hirudin variant 2-Lys47 (rHV2), were selected for their h igh affinity and epitopic specificities to develop a two-site immunoas say of rHV2. The mAb concentrations, incubation time, and temperature were optimized. The immunoassay has a detection limit for rHV2 of 45 n g/L in plasma and 30 ng/L in urine. The reactivity of the mAbs was tes ted against rHV2 and several forms of this protein truncated in the ca rboxyl terminus. The capture mAb 10-2 was found to be mainly directed against rHV2, whereas tracer mAb 10-5 was independent of the carboxyl- terminal region of the protein. This explains the high specificity of the immunoassay for the 65-amino acid form of hirudin.