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 polyclonal antibodies. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound with streptavidin to biotinylated second antibody. Human recombinant angiotensinogen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. To reduce the effect of nonspecific amplification, the optimal concentrations of streptavidin and DNA label were determined to be 0.1 mg/l and 0.5 ng/l, respectively. The detection limit of the immuno-PCR assay was 0.1 ng/l, an approximately 2.5x10(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 and second polyclonal antibodies.
Sugawara
K
K
Department of Laboratory Diagnosis, Sapporo Medical University, School of Medicine, South-1, West-16, 060-0061, Sapporo, Japan.
Kobayashi
D
D
Saito
K
K
Furuya
D
D
Araake
H
H
Yagihashi
A
A
Yajima
T
T
Hosoda
K
K
Kamimura
T
T
Watanabe
N
N
D000808:Angiotensinogen / Q000032:analysis*; D000906:Antibodies / Q000032:analysis / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007120:Immunochemistry / Q000379:methods*; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction / Q000379:methods*; D019809:Streptavidin
D000906:Antibodies; D007202:Indicators and Reagents; D000808:Angiotensinogen; D001710:Biotin; D019809:Streptavidin