Generating Antibodies Against Secreted Proteins Using Vascular Smooth Muscle Cells Transduced with Replication-Defective Retrovirus

Abstract

The traditional method of antibody (Ab) generation requires repeated injections of antigen (Ag). We have developed an alternative method that allows an investigator to generate a polyclonal antiserum with only a cDNA in 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. TIMP-1 overexpressing rat SMC were seeded into deendothelialized rat carotid arteries. Three weeks after cell seeding in the rat, the presence of Ab to the baboon TIMP-1 was detected by dot blot and enzyme-linked 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 period afer the cell seeding was identified as belonging to the IgGl subtype. More interestingly, the titer of the Ab kept rising throughout an 8-month monitoring period. Among the salient features of this Ab are its capacily to block TIMP-1 activity and its utility for detecting TIMP-1 by immunohistochemistry. These results demonstrate that Ab against a secreted protein can be obtained in response to continuous expression of the cDNA by vascular SMC. Purified Ag is not required.