Use of a single-chain antibody library for ovarian cancer biomarker discovery

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.

Fig. 1.

Panning procedure of scFv sublibrary. The Tomlinson I and J human naïve scFv libraries were positively selected with proximal fluid of ovarian cancer patients and used to infect a lawn of E. coli. Phage rescue was performed to obtain virus particles that were used to negatively select (three times) the library against control serum from healthy individuals. Finally, a second round of positive selection with proximal fluid proteins was done. Eluted phage were used to infect E. coli colonies, which now produce individual scFv clones that bind differentially expressed proteins between cases and controls. Shown are the input and output colony-forming units obtained after each step of the panning process.

Fig. 2.

Image of scFv printed on hydrogel slide. Purified scFv were printed in triplicate on a glass slide and incubated with sera from a cancer patient labeled with Cy5 dye (red channel) and a control pool labeled with Cy3 dye (green channel). The array was scanned with a GenePix 4000B microarray scanner.

Fig. 3.

Functional protein arrays. A, three-dimensional representation of the spot volume for USF1 protein detected with scFv 4, ZNF297 detected with scFv 3, BAT4 detected with scFv 10, and TLR2 detected with scFv 17 on the NAPPA. The large peak at the center of each image corresponds to the feature of the protein shown in the inset (i.e. USF1, ZNF297, BAT4, and TLR2). B, plots of signal intensities of all 19 scFv to a particular protein feature, indicating the specificity of the interaction between one antibody and one antigen on the array. Signal intensity was calculated as the spot signal intensity divided by the median signal for all spots on the slide.

Fig. 4.

Dot blots using full-length antibody and scFv to detect antigens. Images of dot blots of purified recombinant full-length USF1 (A) or a cell lysate that overexpresses ZNF297 (B) are shown. The leftmost spot in each blot (1) is the most concentrated sample, and each spot to the right of it is a 2-fold dilution (1/2, 1/4, 1/8, and 1/16). In the USF1 sample, the highest concentration contained 640 ng of USF1 protein; in the case of BSA, it contained 8 μg. ZNF297 protein supplied as a cell lysate was not quantified. The first row of each blot contains the control BSA samples, and the second row has the putative biomarker (USF1 in A and ZNF297 in B). The uppermost blot of each panel uses a full-length antibody to detect the protein spotted; the second blot uses the specific scFv for that protein, and the third uses an scFv specific for BSA. The USF1 antibody used was from Abnova (Abn), clone M02.

Fig. 5.

Proximal fluid and serum dot blots. Column scatter graphs representing the signal intensities of the features on a dot blot of BAT4, TLR2, USF1, and ZNF297 in individual samples of benign cystic fluid, cancer cystic fluid, cancer ascites fluid, healthy serum, cancer serum, and serum from women with benign disease of the ovary are shown. Each measurement is the median of three independent experiments. Horizontal lines represent the mean signal intensity for that group. Signal intensities are in arbitrary units. p values indicated are from a Wilcoxon test ranking the signal intensities and measuring how well each antibody can discriminate disease status. Abc means the antibody was purchased from Abcam, Abn means the antibody was purchased from Abnova, and GW means the antibody was purchased from GenWay. arb, arbitrary.

Fig. 6.

Immunohistochemical staining using full-length antibodies against scFv targets. Formalin-fixed ovarian tumors and normal ovarian tissue were stained with a USF1 antibody and a hematoxylin counterstain. A shows the normal tissue (mostly stroma) stained with hematoxylin and the endothelial cells from the blood vessels stained with the antibody. B shows a higher magnification of the normal stroma and endothelial cells found in the box in A. C shows the cancerous tissue (stage IIIC) with USF1 staining and unstained tissue surrounding the tumor. D shows a higher magnification of the tumor tissue in the box of C indicating that the USF1 staining is mainly perinuclear, possibly Golgi. Bar at low magnification, 100 μm; bar at high magnification, 5 μm.