Generating antibodies against secreted proteins using vascular smooth muscle cells transduced with replication-defective retrovirus.
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 de-endothelialized 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 the 12-month monitoring period after the cell seeding was identified as belonging to the IgG1 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 capacity 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.
Forough
R
R
University of Washington, Seattle, USA.
Hasenstab
D
D
Koyama
N
N
Lea
H
H
Clowes
M
M
Clowes
A W
AW
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000917:Antibody Formation; D001158:Arteries / Q000737:chemistry; D001483:Base Sequence; D015152:Blotting, Northern; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000737:chemistry* / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000276:immunology; D051381:Rats; D011916:Rats, Inbred F344; D018741:Replication Origin / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D019714:Tissue Inhibitor of Metalloproteinases; D014161:Transduction, Genetic
D000906:Antibodies; D006023:Glycoproteins; D011480:Protease Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases