Diagnostic markers of ovarian cancer by high-throughput antigen cloning and detection on arrays

Abstract

A noninvasive screening test would significantly facilitate early detection of epithelial ovarian cancer. This study used a combination of high-throughput selection and array-based serologic detection of many antigens indicative of the presence of cancer, thereby using the immune system as a biosensor. This high-throughput selection involved biopanning of an ovarian cancer phage display library using serum immunoglobulins from an ovarian cancer patient as bait. Protein macroarrays containing 480 of these selected antigen clones revealed 65 clones that interacted with immunoglobulins in sera from 32 ovarian cancer patients but not with sera from 25 healthy women or 14 patients having other benign or malignant gynecologic diseases. Sequence analysis data of these 65 clones revealed 62 different antigens. Among the markers, we identified some known antigens, including RCAS1, signal recognition protein-19, AHNAK-related sequence, nuclear autoantogenic sperm protein, Nijmegen breakage syndrome 1 (Nibrin), ribosomal protein L4, Homo sapiens KIAA0419 gene product, eukaryotic initiation factor 5A, and casein kinase II, as well as many previously uncharacterized antigenic gene products. Using these 65 antigens on protein microarrays, we trained neural networks on two-color fluorescent detection of serum IgG binding and found an average sensitivity and specificity of 55% and 98%, respectively. In addition, the top 6 of the most specific clones resulted in an average sensitivity and specificity of 32% and 94%, respectively. This global approach to antigenic profiling, epitomics, has applications to cancer and autoimmune diseases for diagnostic and therapeutic studies. Further work with larger panels of antigens should provide a comprehensive set of markers with sufficient sensitivity and specificity suitable for clinical testing in high-risk populations.

Figure 1

Filter macroarray. Five plates of amplified 96 phage clones from BP4 were spotted on nitrocellulose filters using Biomek robot with 96-pin print head. Filters were treated as a Western blot using patient sera or control sera as the primary antibody and antibody binding was detected using HRP-conjugated anti-human IgG antibody. A, filter was processed with PM2280 serum. The pattern consisted of 96 (4 × 4) patches. Each of the plates was spotted in triplicate and indicated by the same number 1, 2, 3, 4, or 5 (refer to box diagram at the right ). The outer four corners of the 96 sets of spots (A1, A12, H1, and H12) were spotted with a 1:10,000 dilution of human serum in the last spot of the 16 for orientation purposes (arrows). Note: In the diagram of the patch indicated by the box, clones from plates 1, 3, and 4 bound to the IgG in the patient’s serum; in this case, it is PM2280. B and C, filters were processed with late-stage patients’ sera, PM0044 and PM2314. D and E, filters showing antigen binding with IgG in the serum of stage I ovarian cancer patients, PM2133 and PM2126. F and G, filters labeled with Con PM0217 and Con PM0136 were treated with healthy control serum. Refer to Table 1A for tumor histology and stage of patients’ sera used.

Figure 2

Antigen microarrays on biochip. Sixty-five clones in quintuplicate were robotically arrayed on biochip (FAST slides). Binding of antigens first with serum IgG from ovarian cancer patient and normal healthy individual and next with Cy3-labeled T7 anti-capsid antibody and Cy5-labeled anti-human IgG was done as described in Materials and Methods. The arrays were scanned at 532 and 635 nm lasers in an Axon Laboratories 4100A scanner. A, microarrays processed with serum IgG from control individual PM0574. A small section of the entire biochip. Arrows, five replicates of a particular clone; the location of the five replicates has been designated as r1_c2, r5_c2, r9_c2, r13_c2, and r17_c2 (r, row; c, column). B, microarrays processed with serum IgG from ovarian cancer patient PM0175. Arrows, five replicates of the same clone as shown in (A).

Figure 3

Determination of a titerable antigen-antibody binding in ELISA macroarray analysis. The clones were spotted on a set of four different nitrocellulose membranes that were later processed with four different dilutions (1:1,000, 1:3,000, 1:10,000, and 1:30,000) of either healthy control serum or patients serum (both stage I and III). Refer to Table 1A for tumor histology and stage of patients’ sera used. A set of four filters was also processed with T7 antibody at 1:10,000 dilution. Phage binding to serum IgG was detected with HRP-conjugated anti-human IgG, and intensity of each spot corresponding to a particular phage clone was determined as described in Materials and Methods. The intensity ratio of the four clones 4H4 (A), 5B12 (B), 2F7 (C), and 2A3 (D) were plotted against dilutions of serum obtained from healthy controls and patients. Intensity ratio = (mean signal intensity of a phage clone reacting with patient’s serum) / (mean signal intensity of that phage clone reacting with T7 antibody) – (mean signal intensity of blank phage clone reacting with patient’s serum) / (mean signal intensity of that blank phage clone reacting with T7 antibody). The intensity ratio versus serum concentration was plotted for each antigen clone.