Abstract
The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers. We report on the use of a single-chain variable fragment antibody library for screening ovarian serum to find novel biomarkers for the detection of cancer. We alternately panned the library with ovarian cancer and disease-free control sera to make a sublibrary of antibodies that bind proteins differentially expressed in cancer. This sublibrary was printed on antibody microarrays that were incubated with labeled serum from multiple sets of cancer patients and controls. The antibodies that performed best at discriminating disease status were selected, and their cognate antigens were identified using a functional protein microarray. Overexpression of some of these antigens was observed in cancer serum, tumor proximal fluid, and cancer tissue via dot blot and immunohistochemical staining. Thus, our use of recombinant antibody microarrays for unbiased discovery found targets for ovarian cancer detection in multiple sample sets, supporting their further study for disease diagnosis.
Highlights
The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers
Our previous results [10, 11] and those of others [12,13,14] using high density, full-length IgG antibody microarrays to validate and discover cancer serum biomarkers demonstrated that this platform is valuable for simultaneously comparing the levels of hundreds of proteins on dozens of serum samples from cancer patients and healthy controls
Phage Library Panning—To find biomarkers for the detection of ovarian cancer, we used a naïve phage-displayed, human scFv library to select a sublibrary of those antibodies that bound proteins that were differentially expressed in tumor proximal fluid and serum of cancer patients with respect to healthy controls
Summary
Selection of scFv Sublibrary—We used the complete library to generate a sublibrary of antibodies that bind proteins differentially expressed in either tumor proximal fluid or serum of cancer patients relative to serum from controls. For the first round of panning (positive selection), we pooled albumin-depleted proximal fluid consisting of seven cyst and nine ascites fluid cancer samples (for epidemiological data on samples used for panning, see Table I). The second positive selection was performed in a MaxiSorp tube coated with 25 l of albumin-depleted, pooled cyst and ascites fluids. Eluate from this selection was used to infect TG1 Escherichia coli. Immunohistochemical staining was performed as reported previously [36] using the USF1 antibody from GenWay at a 1:200 dilution
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