A highly sensitive immuno-polymerase chain reaction assay for human angiotensinogen using the identical first and second polyclonal antibodies

We describe an immuno-polymerase chain reaction (immuno-PCR) assay for the detection of human angiotensinogen using identical first and second polyclo nal antibodies. The reporter DNA was initially generated by PCR amplificati on using a biotinylated primer, and was bound with streptavidin to biotinyl ated second antibody. Human recombinant angiotensinogen sandwiched by antib odies was detected by amplifying the reporter DNA using PCR. To reduce the effect of nonspecific amplification, the optimal concentrations of streptav idin and DNA label were determined to be 0.1 mg/l and 0.5 ng/l, respectivel y. The detection limit of the immuno-PCR assay was 0.1 ng/l, an approximate ly 2.5 x 10(5)-fold improvement compared with a conventional enzyme-linked immunosorbent assay. These results indicate that a highly sensitive immuno- PCR for human angiotensinogen can be developed even with identical first an d second polyclonal antibodies. (C) 2000 Elsevier Science B.V. All rights r eserved.