Sahar Sibani

Protein Microarray Signature of Autoantibody Biomarkers for the Early Detection of Breast Cancer

Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a prescreen was performed to eliminate uninformative antigens. Sera from stage I-III IBC (n = 53) and healthy women (n = 53) were screened for AAb to all 4988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p < 0.05) and if the number of cases with signals above the 95th percentile of controls was significant (p < 0.05). These 761 antigens were screened using an independent set of IBC sera (n = 51) and sera from women with benign breast disease (BBD) (n = 39). From these, 119 antigens had a partial area under the ROC curve (p < 0.05), with sensitivities ranging from 9-40% at >91% specificity. Twenty-eight of these antigens were confirmed using an independent serum cohort (n = 51 cases/38 controls, p < 0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC = 0.756). These are potential biomarkers for the early detection of breast cancer.

Detection of Multiple Autoantibodies in Patients with Ankylosing Spondylitis Using Nucleic Acid Programmable Protein Arrays

Ankylosing spondylitis (AS) is a common, inflammatory rheumatic disease that primarily affects the axial skeleton and is associated with sacroiliitis, uveitis, and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine whether plasma from patients with AS contained autoantibodies and, if so, characterize and quantify this response in comparison to patients with rheumatoid arthritis (RA) and healthy controls. Two high density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA, and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis to determine the patterns of signaling cascades or tissue origin. 44% of patients with ankylosing spondylitis demonstrated a broad autoantibody response, as compared with 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The autoantibody responses in the AS patients were targeted toward connective, skeletal, and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic acid programmable protein arrays constitute a powerful tool to study autoimmune diseases.

Rapid detection of antibodies in sera using multiplexed self-assembling bead arrays

Rapid detection of antibody immunity in serum or plasma, whether to pathogenic antigens, tumor antigens, or autoimmune antigens, is critical for diagnosis, monitoring, and biomarker assessment of the immune response. Individual or multiplexed ELISAs that use purified recombinant proteins are dependent on a priori protein purification, a labor-intensive process that may take months to obtain proteins of sufficient purity and stability for serologic assays. We developed a programmable multiplexed immunoassay for the rapid monitoring of humoral immunity using the Luminex suspension bead array platform. In this approach, epitope-tagged antigens (GST- or FLAG-tagged) are expressed using in vitro transcription and translation, and captured onto anti-epitope-coupled Luminex SeroMap beads. The antigen-loaded beads are mixed, serum is added, and human IgG is detected with standard secondary detection reagents. By coupling high-throughput DNA preparation of cDNA ORFs with antigen expression/capture, we demonstrate that 71/72 (98.6%) of GST-tagged proteins can be expressed and specifically detected on the bead ELISA. Detection of antibodies to the test viral antigen EBNA-1 in human sera is highly reproducible, with intra-assay variation of 3-8%, inter-assay variation of 5%, and with stability over 11 months. The specificity and limits of detection of the bead ELISAs for the tumor antigen p53 are comparable to both standard protein ELISAs and plate-based programmable (RAPID) ELISAs, and are also comparable to the detection of directly-conjugated p53 protein. Multiplexing a panel of analytes does not impair the sensitivity of antibody detection. Immunity to a panel of EBV-derived antigens (EBNA-1, EBNA-3A, EBNA-3B, and LMP-2) is specifically and differentially detected within healthy donor sera. This method allows for rapid conversion of ORFeome-derived cDNAs to a multiplexed bead ELISA to detect antibody immunity to both infectious and tumor antigens.

Systematic analysis of the IgG antibody immune response against varicella zoster virus (VZV) using a self-assembled protein microarray

Varicella zoster virus (VZV) is a human herpesvirus encoding at least 69 distinct viral proteins which causes chickenpox after primary infection and shingles during reactivation and which is particularly important in pregnancy and immunocompromised patients. Current serodiagnostic tests are either based on whole cell lysates or glycoprotein preparations. In order to investigate the humoral immune response to VZV infection or vaccination in more detail, and to improve the currently available diagnostic assays, we developed a nucleic acid programmable protein array (NAPPA) containing all 69 VZV proteins and performed a detailed analysis of 68 sera from individuals with either no, a previous or an acute VZV infection. In addition to the known reactive glycoprotein antigens (ORF 5, ORF 14, ORF 31, ORF 37, ORF 68), we discovered IgG antibodies against a variety of other membrane (ORF 2, ORF 24), capsid (ORF 20, ORF 23, ORF 43) and tegument (ORF 53, ORF 9, ORF 11) proteins, as well as other proteins involved in virus replication and assembly (ORF 25, ORF 26, ORF 28) and the transactivator proteins ORF 12, ORF 62 and ORF 63. All of these antigens were only reactive in a subset of VZV-positive individuals. A subset of the newly identified VZV antigens was validated by western blot analysis. Using these seroreactive new VZV antigens, more sensitive assays and tests distinguishing between different clinical entities may be developed.

Tracking humoral responses using self assembling protein microarrays

The humoral immune response is a highly specific and adaptive sensor for changes in the bodys protein milieu, which responds to novel structures of both foreign and self antigens. Although Igs represent a major component of human serum and are vital to survival, little is known about the response specificity and determinants that govern the human immunome. Historically, antigen‐specific humoral immunity has been investigated using individually produced and purified target proteins, a labor‐intensive process that has limited the number of antigens that have been studied. Here, we present the development of methods for applying self‐assembling protein microarrays and a related method for producing 96‐well formatted macroarrays for monitoring the humoral response at the proteome scale. Using plasmids encoding full‐length cDNAs for over 850 human proteins and 1700 pathogen proteins, we demonstrate that these microarrays are highly sensitive, specific, reproducible, and can simultaneously measure immunity to thousands of proteins without a priori protein purification. Using this approach, we demonstrate the detection of humoral immunity to known and novel self‐antigens, cancer antigens, autoimmune antigens, as well as pathogen‐derived antigens. This represents a powerful and versatile tool for monitoring the immunome in health and disease.

Autoantibody signature for the serologic detection of ovarian cancer.

Sera from patients with ovarian cancer contain autoantibodies (AAb) to tumor-derived proteins that are potential biomarkers for early detection. To detect AAb, we probed high-density programmable protein microarrays (NAPPA) expressing 5177 candidate tumor antigens with sera from patients with serous ovarian cancer (n = 34 cases/30 controls) and measured bound IgG. Of these, 741 antigens were selected and probed with an independent set of ovarian cancer sera (n = 60 cases/60 controls). Twelve potential autoantigens were identified with sensitivities ranging from 13 to 22% at >93% specificity. These were retested using a Luminex bead array using 60 cases and 60 controls, with sensitivities ranging from 0 to 31.7% at 95% specificity. Three AAb (p53, PTPRA, and PTGFR) had area under the curve (AUC) levels >60% (p < 0.01), with the partial AUC (SPAUC) over 5 times greater than for a nondiscriminating test (p < 0.01). Using a panel of the top three AAb (p53, PTPRA, and PTGFR), if at least two AAb were positive, then the sensitivity was 23.3% at 98.3% specificity. AAb to at least one of these top three antigens were also detected in 7/20 sera (35%) of patients with low CA 125 levels and 0/15 controls. AAb to p53, PTPRA, and PTGFR are potential biomarkers for the early detection of ovarian cancer.

Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery

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.

Immunoprofiling using NAPPA protein microarrays.

Protein microarrays provide an efficient method to immunoprofile patients in an effort to rapidly identify disease immunosignatures. The validity of using autoantibodies in diagnosis has been demonstrated in type 1 diabetes, rheumatoid arthritis, and systemic lupus, and is now being strongly considered in cancer. Several types of protein microarrays exist including antibody and antigen arrays. In this chapter, we describe the immunoprofiling application for one type of antigen array called NAPPA (nucleic acids programmable protein array). We provide a guideline for setting up the screening study and designing protein arrays to maximize the likelihood of obtaining quality data.

Optimierung von Western Blot-Signalen mit Chemilumineszenz

ZusammenfassungWestern Blot zum Nachweis von Proteinen in Zelllysaten ist eine wertvolle Ergänzung zu immunzytochemischen Methoden. Erfolgreiche Western Blots erfordern die Optimierung mehrerer Variablen. Diese Studie zeigt die Optimierung der Chemilumineszenz-Detektion.AbstractUsing Western blotting to detect proteins in a cell lysate is a powerful, complementary approach to immunocytochemistry. Successful Western blotting is dependent on the optimization of multiple variables. In this study, we focused on optimization of chemiluminescence detection