Protein Macroarrays Research Articles

Protein array processing software for automated semiquantitative analysis of serum antibody repertoires.

Effective immunotherapies activate natural antitumor immune responses in patients undergoing treatment. The ability to monitor immune activation in response to immunotherapy is critical in measuring treatment efficacy over time and across patient cohorts. Protein arrays are systematically arranged, large collections of annotated proteins on planar surfaces, which can be used for the characterization of disease-specific and treatment-induced antibody repertoires in individuals undergoing immunotherapy. However, the absence of appropriate image analysis and data processing software presents a substantial hurdle, limiting the uptake of this approach in immunotherapy research. We developed a first, automated semiquantitative open-source software package for the analysis of widely used protein macroarrays. The software allows accurate single array and inter-array comparative studies through the tackling of intra-array inconsistencies arising from experimental disparities. The innovative and automated image analysis process includes adaptive positioning, background identification and subtraction, removal of null signals, robust statistical analysis, and protein pair validation. The normalized values allow a convenient semiquantitative data analysis of different samples or timepoints. Enabling accurate characterization of sample series to identify disease-specific immune profiles or their relative changes in response to treatment may serve as a diagnostic or predictive tool of disease.

Hyper N-Glycosylated SEL1L3 As B-Cell Receptor Autoantigen of Primary Vitreoretinal Lymphoma

Chronic antigenic stimulation of B lymphocytes through their B-cell receptor (BCR) is thought to play an important role in the genesis of B-cell lymphomas. SAMD14/neurabin-I has been described as auto-antigenic target of primary central nervous system lymphoma (PCNSL) BCRs. Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress to PCNSL. Besides manifestation in an immunologically privileged site, PVRL and PCNSL share frequent recurrent genetic alterations like MYD88 and CD79B mutations. Both PVRL and PCNSL are considered being related to the MCD subtype of DLBCLs and may depend on chronic BCR signaling through antigenic stimulation. To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed BCRs from PVRL patients as antibody fragments (Fabs) and tested for specific binding against common antigens. All cases with corresponding BCR sequences were previously reported by Belhouachi et al. (Blood Adv. 2020;4(7):1357-1366). For 20 of 55 PVRL BCR sequences both immunoglobulin heavy and light chain variable region (V) genes were available. DNA clones comprising an ApaLI restriction site, the complete light chain V gene, a κ- or λ-constant region gene, a ribosome binding site, a signal sequence, the heavy chain V gene and a BstEII restriction site were ordered from GeneCust® (GeneCust, 5690 Ellange, Luxembourg) in a pUC57 vector. DNA fragments were cloned in front of a γ-1 constant region gene and a Histidine-tag into a modified pCES-1 vector for expression as Fabs. PVRL-BCRs were screened on protein macroarrays representing clones of UniPEx human cDNA expression libraries expressed in E. coli (Bioscience, Dublin, Ireland). SEL1L3 was identified as the auto-antigenic target of the B-cell receptors in 3 out of 20 PVRL cases. None of the PVRL Fabs bound to LRPAP1 (common BCR antigen of mantle cell lymphomas), RpoC (Moraxella catarrhalis bacterial antigen and target of nodular lymphocyte-predominant Hodgkin lymphoma cells) or galectin-3 (member of the lectin family). SEL1L3 is a 1132 amino acid long protein with multiple glycosylation and phosphorylation sites (uniprot.org). This is especially important since previously identified BCR antigens from different lymphoma entities showed post-translational modifications such as hyper-N-glycosylation or hypo-phosphorylation. Furthermore, SEL1L3 is highly expressed in the pigmented layer of the retina. Western Blot analysis of the blood of a PVRL patient with serum antibodies against SEL1L3 showed an increased molecular weight of SEL1L3 as compared to SEL1L3 of controls. After treatment with N-acetyl-β-d-glucosaminidase or PNGase F, this difference in molecular weight disappeared, indicating a hyper N-glycosylation of SEL1L3 in PVRL patients with SEL1L3-reactive BCRs as trigger for its' immunogenicity. SEL1L3 induced proliferation of aggressive lymphoma cell lines transfected with SEL1L3-reactive BCRs. Moreover, the BCR binding epitope of SEL1L3 (aa 560 - 585) conjugated to a truncated form of Pseudomonasaeruginosa exotoxin A, killed exclusively lymphoma cells expressing BCRs with the respective reactivity. Additionally, BCRs of a locally treated PVRL patient with serum antibodies against SEL1L3 were cloned from biopsies of the vitreous body at initial diagnosis and of a lymphoma manifestation in the breast at relapse. Both BCRs showed binding to SEL1L3 suggesting a continued dependence of the BCR on antigen stimulation. These results suggest an important role of chronic antigenic stimulation by the post-translationally modified auto-antigen SEL1L3 in the genesis of a subgroup of patients with PVRL, might give an explanation for the selective tropism of PVRLs and provide the basis for a new treatment approach with high specificity.

Autoantibodies against SUMO1-DHX35 in long-COVID

Long COVID is a collection of symptoms as a late sequelae of SARS-CoV-2 infection. It often includes mental symptoms such as cognitive symptoms, persisting loss of smell and taste, in addition to exertional dyspnea. A role of various autoantibodies (autoAbs) has been postulated in long-COVID and is being further investigated. With the goal of identifying potentially unknown autoAbs, we screened plasma of patients with long COVID on in-house post-translationally modified protein macroarrays including citrullinated, SUMOylated and acetylated membranes. SUMO1ylated isoform DEAD/H (Asp-Glu-Ala-Asp/His) box helicase 35 (SUMO1-DHX35) was identified as only candidate antigen. In adult patients with long COVID, IgG autoAbs against SUMO1-DHX35 of IgG class were found in seven of 71 (9.8%) plasma samples, of IgM and IgG class in one of 69 (1.4%) samples, not in 200 healthy adult controls, not in 442 healthy children, and 146 children after SARS-CoV-2 infection. All autoAb-positive seven patients were female. AutoAb titers ranged between 200 to up to 400 By point mutagenesis and expression of FLAG-tagged mutants of DHX35 in HEK293 cells, and subsequent SUMOylation of purified constructs, lysine 53 was identified as a unique, never yet identified, SUMOylation site. The autoAbs had no reactivity against the non-SUMO1ylated mutant (K53R) of DHX35. To summarize, autoAbs against SUMO1-DHX35 were identified in adult female patients with long-COVID. Further studies are needed to verify the frequency of occurrence. The function of DHX35 has not yet been determined and there is no available information in relation to disease implication. The molecular mechanism causing the SUMOylation, the potential functional consequences of this post-translational modification on DHX35, and a potential pathogenicity of the autoAbs against SUMO1-DHX35 in COVID-19 and other possible contexts remain to be elucidated.

Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection—a missing link for ACPA production

ObjectivesPorphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007...

Identification of Posttranvslationally Modified Neoantigens As Targets of B Cell Receptors of Burkitt Lymphoma

Introduction:Burkitt lymphoma represents the most aggressive neoplasm of mature sIg+ B cells. Besides the characterisitc translocation of the MYC gene with an immunoglobulin heavy or light chain gene locus, activating mutations in the TCF3 gene and inactivating mutations in the ID3 gene represent the key events in the pathogenesis of Burkitt lymphoma. These TCF3/ID3 mutations result in tonic and antigen-independent B cell receptor (BCR) pathway activation. Additionally, chronic BCR activation by antigens might play a role in Burkitt lymphoma pathogenesis and we set out to identify such potential target antigens of BCRs of Burkitt lymphoma.Methods:BCRs were expressed as recombinant Fabs in TG1 E.coli based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen sporadic Burkitt lymphoma specimens. Additionally, natural Fabs and recombinant Fabs were produced of 8 established Burkitt lymphoma cell lines by Papain digestion and BCR expression cloning. The purified pooled Fabs were screened for reactivities against non-modified and differently posttranslationally modified human protein macroarrays. Reactivities were verified by ELISA with coated N-terminally FLAG-tagged candidate antigens, each separately for the posttranslationally modified and non-modified isoforms. Recombinant Fabs derived of mantle cell lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma served as controls. Moreover the functional effects on proliferation and BCR pathway activation after addition of the identified target antigens to Burkitt lymphoma cell lines with and without reactive BCRs were analyzed by proliferation assays and western blots. Finally, mutation status, methylation status and expression level of identified target antigens were analyzed in ICGC MMML-Seq lymphoma databases for differences in Burkitt lymphoma versus distinct lymphoma entities.Results:The Burkitt lymphoma derived Fabs were tested on posttranslationally modified protein arrays. The BCR of CA46 line showed specific reactivity against sumoylated Bystin, the Fab of the BL41 line reacted specifically against acetylated HSP40. Recombinant Fabs of diffuse large B cell lymphoma, primary central nervous system lymphoma and mantle cell lymphoma did neither bind sumoylated Bystin nor acetylated HSP40. Addition of the posttranslationally modified cognate antigens to respective Burkitt lymphoma cell line with the reactive BCR induced proliferaton. The analysis of the ICGC MMML-Seq lymphoma databases (representing different cohorts) showed a higher expression of Bystin in Burkitt lymphoma compared to other aggressive B-cell lymphomas. However, the mutation and methylation status of Bystin and HSP40 in the ICGC MMML-seq cohort did not provide any direct indication of the origin of their immunogenic post-translational modifications.Conclusions:A subgroup of sporadic Burkitt lymphoma has autoreactive BCRs with specific affinity against posttranslationally modified self antigens, demonstrating a new aspect in the pathogenesis of Burkitt lymphoma. Specific secondary modifications, as sumolytation of Bystin or acetylation of HSP40 appear to evoke the immunogenicity. Future studies will focus on the functional consequences of the antigen/BCR interaction on Burkitt cells. Furthermore, the causes of these posttranslationally modified neoantigens will be investigated in more detail. DisclosuresStilgenbauer:Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding.

Sumoylated HSP90 is a dominantly inherited plasma cell dyscrasias risk factor.

Posttranslationally modified proteins serve as autoimmunogenic targets in a wide spectrum of autoimmune diseases. Here, we identified a posttranslationally modified paraprotein target (paratargs) in monoclonal gammopathies of undetermined significance (MGUS), multiple myelomas (MM), and Waldenstrom's macroglobulinemias (WM) using protein macroarrays that were sumoylated and screened for reactivity with paraproteins from MGUS, MM, and WM patients. We found that paraproteins from a proportion of European, African-American, and Japanese patients specifically reacted with the sumoylated heat-shock protein 90 β isoform-α (HSP90-SUMO1, where SUMO indicates small ubiquitin-like modifier), while no reactivity with HSP90-SUMO1 was detected in over 800 controls. HSP90-SUMO1 was present in blood cells from all patients with HSP90-SUMO1-binding paraproteins. We determined that the HSP90-SUMO1 carrier state is autosomal-dominantly inherited and caused by the inability of SUMO peptidase sentrin/SUMO-specific protease 2 (SENP2) to desumoylate HSP90-SUMO1. HSP90-SUMO1 was detected in a small percentage of healthy individuals from all backgrounds; however, only MGUS, MM, and WM patients who were HSP90-SUMO1 carriers produced HSP90-SUMO1-specific paraproteins, suggesting that sumoylated HSP90 promotes pathogenesis of these diseases through chronic antigenic stimulation. This study demonstrates that harboring HSP90-SUMO1 identifies healthy individuals at risk for plasma cell dyscrasias and that dominant inheritance of posttranslationally modified autoantigenic paratargs is one of the strongest molecular defined risk factors for MGUS, MM, and WM.

Identification of the Antigenic Targets of the Major CLL-Derived Stereotyped BCRs and First Demonstration of Sub-Subsets

Background: The B-cell receptor (BCR) is the hallmark of mature B cells and a clonal BCR is expressed on the neoplastic cells of B-cell malignancies. The BCR mediated signalling provides proliferative and anti-apoptotic signals for the neoplastic B cells and represents an attractive therapeutic target. The BCRs of about 1/3 of CLL cases display highly homologous complementary determining regions 3 (CDR3), on the basis of which stereotyped subsets have been defined. The homologies of the BCR of a given subset suggest selection by shared antigen(s), which might play an important role in the pathogenesis of the respective B-cell neoplasm by chronic antigenic stimulation.Methods: Tumor cells from CLL patients belonging to subsets 2, 3, 4, 5, 6, 8 were studied. Natural Fabs (nFab) were obtained by papain digestion of membrane-bound Igs. Subset-derived nFabs were used to screen for reactivity with proteins represented in two high-density protein macroarrays, one derived from human fetal brain cDNA and a second derived from a mixture of expression libraries derived from activated T-cells, lung and colon. Identified antigens were analysed by standard techniques. All methods used (nFab generation by papain digestion, BCR cloning, membrane screening, epitope mapping etc.) are described in detail in Zwick C et al., Blood 2013;121(23):4708-4717.Results: Our ongoing systematic search for CLL subset-specific antigens together with our previously published data (Zwick et al) led to the identification and epitope characterization of the antigenic targets for the 6 most common CLL subsets: MARK3 as antigen for subset 2, NCOR2 as antigen for subset 3, CaCyBP as antigen for subset 4, FAM32A protein as antigen for subset 5 and GLDC as antigen for subset 8. In addition we could confirm MYHIIA as antigen for subset 6 (in accordance with Chu CC et al, Blood 2010;115(19):3907-3915). The analysis of the BCR binding revealed (except for oxLDL) in minimum 2 different epitopes for each subset-specific antigen, thus defining sub-subsets with BCRs binding to distinct epitopes. Sequence analysis of the respective BCR suggests that this epitope-spreading is due to antigen-driven mutation/maturation of BCRs with a highly homologous CDR3.Conclusions: The antigenic targets (and stimulus) for the clonal BCRs of the major sterotyped subsets have been identified providing for the first time experimental evidence for the hypothesis that BCRs of a given subset indeed target the identical antigen. Moreover, based on the BCR-binding epitopes, sub-subsets of stereotypes have been defined by BCRs obviously originating from the same germline, but having undergone different antigen-driven mutations.Supported by Deutsche Forschungsgemeinschaft DFG, Deutsche José Carreras Leukämie Stiftung, Wilhelm-Sander-Stiftung, Deutsche Krebshilfe e.V. DisclosuresStilgenbauer:Pharmacyclics, Janssen: Honoraria, Research Funding.

Autoantigenic targets of B-cell receptors derived from chronic lymphocytic leukemias bind to and induce proliferation of leukemic cells

AbstractAntigenic targets of the B-cell receptor (BCR) derived from malignant cells in chronic lymphocytic leukemia (CLL) might play a role in the pathogenesis of this neoplasm. We screened human tissue–derived protein macroarrays with antigen-binding fragments derived from 47 consecutive cases of CLL. An autoantigenic target was identified for 12/47 (25.5%) of the cases, with 3 autoantigens being the target of the BCRs from 2 patients each. Recombinantly expressed autoantigens bound specifically to the CLL cells from which the BCR used for the identification of the respective autoantigen was derived. Moreover, binding of the autoantigen to the respective leukemic cells induced a specific activation and proliferation of these cells. In conclusion, autoantigens are frequent targets of CLL-BCRs. Their specific binding to and induction of proliferation in the respective leukemic cells provide the most convincing evidence to date for the long-time hypothesized role of autoantigens in the pathogenesis of CLL.

Myasthenia gravis: analysis of serum autoantibody reactivities to 1827 potential human autoantigens by protein macroarrays.

BackgroundMyasthenia gravis is a disorder of neuromuscular transmission associated with autoantibodies against the nicotinic acetylcholine receptor. We have previously developed a customized protein macroarray comprising 1827 potential human autoantigens, which permitted to discriminate sera of patients with different cancers from sera of healthy controls, but has not yet been evaluated in antibody-mediated autoimmune diseases.ObjectiveTo determine whether autoantibody signatures obtained by protein macroarray separate sera of patients with myasthenia gravis from healthy controls.MethodsSera of patients with acetylcholine receptor antibody-positive myasthenia gravis (n = 25) and healthy controls (n = 32) were analyzed by protein macroarrays comprising 1827 peptide clones.ResultsAutoantibody signatures did not separate patients with myasthenia gravis from controls with sufficient sensitivity, specificity, and accuracy. Intensity values of one antigen (poly A binding protein cytoplasmic 1, p = 0.0045) were higher in patients with myasthenia gravis, but the relevance of this and two further antigens, 40S ribosomal protein S13 (20.8% vs. 0%, p = 0.011) and proteasome subunit alpha type 1 (25% vs. 3.1%, p = 0.035), which were detected more frequently by myasthenia gravis than by control sera, currently remains uncertain.ConclusionSeroreactivity profiles of patients with myasthenia gravis detected by a customized protein macroarray did not allow discrimination from healthy controls, compatible with the notion that the autoantibody response in myasthenia gravis is highly focussed against the acetylcholine receptor.

Autoantigenic Targets of B-Cell Receptors (BCR) Derived From Chronic Lymphocytic Leukemias Bind to and Induce Proliferation of Leukemic Cells.

AbstractAbstract 2884 BackgroundAuto-antigenic targets of the B-cell receptor (BCR) derived from malignant cells in chronic lymphocytic leukemia (CLL) might play a role in the pathogenesis of this neoplasm. Patients and MethodsIn order to identify autoantigenic targets of CLL-derived BCR we screened human tissue-derived protein macroarrays with Fab fragments obtained by papain treatment of CLL cells derived from 50 consecutive cases. Antigens were biochemically and molecularly characterized and recombinantly expressed. ResultsAn autoantigenic target was identified for 12/50 (24%) of the cases, with 3 autoantigens being the target of the BCR from two patients each. CLL-BCR derived from the same stereotype subset recognized the same antigen, but differed epitopes. By flow cytometry using flag-tagged recombinantly expressed autoantigens binding of antigen to the surface of CLL was demonstrated, which was specific for the CLL cells from which the BCR used for the identification of the respective autoantigen was derived. Moreover, binding of the autoantigen to the respective leukemic cells induced specific activation as shown by increased cytoplasmic calcium concentration, induced MYC expression and proliferation of leukemic CLL cells as demonstrated by a proliferation assay (EZ4U). ConclusionsAutoantigens are frequent targets of CLL-derived BCR. Their specific binding to and induction of proliferation in respective leukemic cells, which has been demonstrated for the first time, provide the most convincing evidence to date for the long-time hypothesized role of autoantigens in the pathogenesis of chronic lymphocyte leukemia. Supported by Sander-Stiftung (Munich, Germany) Disclosures:No relevant conflicts of interest to declare.

Progranulin antibodies in autoimmune diseases

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg–Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis.

F08 The autoimmune profile of Huntington's disease patients: a pilot study

Huntington9s disease (HD) is a progressive neurodegenerative disorder, which is caused by expansion of a polyglutamine tract in the first exon of the huntingtin gene. Genetic testing allows the unambiguous diagnose of HD and the identification of individuals carrying the gene defect before onset of clinical symptoms. However, molecular markers that are suited to predict disease onset, monitor disease progression and asses the response to therapy are still not available. To identify HD-specific molecular markers we performed a comparative analysis of the autoimmune profile of symptomatic patients, presymptomatic gene carriers and a representative control cohort. Serum samples were tested for antigen/autoantibody interactions using a sequential arrangement of protein macroarrays and microarrays. The macroarrays contained E coli clones expressing approximately 10 000 human proteins and were used to characterise the autoimmune profile of 20 HD patients. Hereby, 273 human proteins were selected to generate a HD-specific protein microarray. Serum samples of 100 HD patients, 29 presymptomatic gene carriers and 100 healthy individuals were analysed using the HD-specific microarray. Thereby, several autoantigens were detected that represent putative molecular markers for HD. Further validation experiments are necessary to confirm the relevance of the identified molecular markers. The presence of HD-specific autoantibodies in serum samples supports the idea that progression of HD is associated with alterations of the immune system.

Protein array screening reveals IgA autoantigenicity patterns predicting anti-TNFα therapy response in rheumatoid arthritis patients

BackgroundOne third of rheumatoid arthritis (RA) patients treated with biologicals targeting tumour necrosis factor α (TNFα) are therapy non-responders. The authors investigated the differences in seroreactivity of patients responding and...

Protein Macroarray Profiling of Serum Autoantibodies in Pseudoexfoliation Glaucoma

Complex repertoires of IgG autoantibodies have been detected against ocular antigens in patients with glaucoma. The goal was to identify and characterize the IgG autoantibody repertoires in sera of patients with pseudoexfoliation glaucoma (PXFG) with protein macroarrays. Serum samples of 21 patients with PXFG and 19 age- and sex-matched control subjects were profiled on high-density colony protein macroarrays expressing His-tagged recombinant human proteins derived from a human fetal brain cDNA library. Statistically prevalent expression clones in the PXFG group were sequenced. mRNA expression of identified antigens was examined in the rat ganglion cell line RGC-5 and in human brain and optic nerve cDNA. The IgG immunoreactivity of the sera of 20 control and 26 PXFG patients to purified C6orf129 was analyzed in a reverse enzyme-linked immunosorbent assay. An increased prevalence was detected among the PXFG patients of serum antibodies to seven proteins: C6orf129; stathmin-like 4; transmembrane protein 9 domain family, member B; fibroblast growth factor receptor 3; cleft lip and palate transmembrane protein 1; EH-domain-containing protein 1; and eukaryotic translation elongation factor 2. All antigens were expressed in the RGC-5 cells and in cDNA from human brain and optic nerve, with the exception of stathmin-like 4, which was not expressed in the RGC-5 cells. The patients with PXFG had increased anti-C6orf129 IgG immunoreactivity compared with that in the control subjects (P < 0.05). Screening high-density protein arrays identifies unique antibody profiles that may discriminate between patients with and without PXFG. Characterization of the autoantibody repertoire may provide new insights into the pathophysiology of PXFG.

A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies

AbstractThe restricted immunoglobulin (Ig) repertoire found in B-cell chronic lymphocytic leukemia (CLL) implies a role for antigen(s) in the leukemogenesis. The nature of the antigens has, however, not been characterized, although examples of autoantigens have been demonstrated. We have analyzed a panel of 28 CLL cell lines and primary cultures, producing monoclonal Ig with different Ig heavy-chain variable region gene usage and mutational status, including several complementarity determining region 3 homology subset members. Using mass-spectrometry, immunoassays, or protein macroarrays, we have discovered novel antigens binding to CLL Igs. These antigens included cytoskeletal proteins vimentin, filamin B, and cofilin-1, but also phosphorylcholine-containing antigens (eg, Streptococcus pneumoniae polysaccharides and oxidized low-density lipoprotein [oxLDL]). Additional new antigens identified were cardiolipin and proline-rich acidic protein-1. Remarkably, these antigens represent molecular motifs exposed on apoptotic cells/blebs and bacteria, and several CLL Igs bound to apoptotic Jurkat cells. In conclusion, these intriguing data, showing a limited target structure recognition, indicate that CD5+ CLL B cells are derived from a cell compartment that produces “natural antibodies,” which may be instrumental in elimination and scavenging of apoptotic cells and pathogenic bacteria.

Diagnostic Markers of Ovarian Cancer by High-Throughput Antigen Cloning and Detection on Arrays

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.

Articles selected by Faculty of 1000: bacterial ORFans: ORFs with no known homologs; fluorescent marker of histone phosphorylation; genome-wide chromatin analysis; protein macroarrays; clinically relevant Trichomonas variation

Articles selected by Faculty of 1000: bacterial ORFans: ORFs with no known homologs; fluorescent marker of histone phosphorylation; genome-wide chromatin analysis; protein macroarrays; clinically relevant Trichomonas variation

High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies.

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.