GENERATING ANTIBODIES AGAINST SECRETED PROTEINS USING VASCULAR SMOOTH-MUSCLE CELLS TRANSDUCED WITH REPLICATION-DEFECTIVE RETROVIRUS

The traditional method of antibody (Ab) generation requires repeated i njections of antigen (Ag). We have developed an alternative method tha t allows an investigator to generate a polyclonal antiserum with only a cDNA ill hand. We cloned a cDNA encoding the coding frame for baboon tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Fischer rat arterial smooth muscle cells (SMC) transduced, with the baboon TIMP-1 using a replication-defective retrovirus were propagated in culture. T IMP-1 over-expressing rat SMC were seeded into de-endothelialized rat carotid arteries. Three weeks after cell seeding in the rat, the prese nce of Ab to the baboon TIMP-1 was detected by dot blot and enzyme-lin ked immunosorbent assay in 5 of 6 of the animals. The major portion of the Ab generated against baboon TIMP-1 during a 12-month monitoring p eriod after the cell seeding was identified as belonging to the IgG1 s ubtype. More interestingly the titer of the Ab kept rising throughout an 8-month monitoring period. Among the salient features of this Ab ar e its capacity to block TIMP-1 activity and its utility for detecting TIMP-1 by immunohistochemistry. These results demonstrate that Ab agai nst a secreted protein can be obtained in response to continuous expre ssion of the cDNA by vascular SMC. Purified Ag is not required.