title: Real-time immuno-polymerase chain reaction in a 384-well format: detection of vascular endothelial growth factor and epidermal growth factor-like domain 7.
abstract: Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.
authors.affiliation: Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA.
authors.forename: Jianhuan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Vernes
authors.affiliation: Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Ni
authors.affiliation: Protein Chemistry, Genentech, South San Francisco, CA 94080, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Nelson
authors.affiliation: Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Wong
authors.affiliation: Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Chen
authors.affiliation: Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA.
authors.forename: Aarati
authors.initials: A
authors.lastname: Asundi
authors.affiliation: Protein Chemistry, Genentech, South San Francisco, CA 94080, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Vandlen
authors.affiliation: Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA 94080, USA. Electronic address: meng.gloria@gene.com.
authors.forename: Y Gloria
authors.initials: YG
authors.lastname: Meng
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D014408:Biomarkers, Tumor / Q000032:analysis / Q000097:blood; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D066255:EGF Family of Proteins; D016228:Endothelial Growth Factors / Q000032:analysis* / Q000097:blood / Q000276:immunology; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D060888:Real-Time Polymerase Chain Reaction; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis* / Q000097:blood / Q000276:immunology
chemical_list: D000906:Antibodies; D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D066255:EGF Family of Proteins; C527877:EGFL7 protein, human; D016228:Endothelial Growth Factors; D042461:Vascular Endothelial Growth Factor A; D001710:Biotin; D004247:DNA; D019809:Streptavidin
keywords: 384-Well; Biomarker assay; Epidermal growth factor-like domain 7; Hepatocellular carcinoma; Real-time immuno-PCR; Vascular endothelial growth factor