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Extensive knowledge about protein-protein interactions is fundamental to fully understand signaling pathways and for the development of new drugs. Rho GTPases are key molecules in cellular signaling processes and their deregulation is implicated in the development of a variety of diseases such as neurofibromatosis type 2 and cancer. Here, we describe a bead-based protein-protein interaction assay for overexpressed HA-tagged Rho GTPases to study the GTPγS-dependent interaction with the regulatory protein RhoGDIα. This assay provides a useful tool for the analysis of both macromolecular and small molecule activators and inhibitors of the protein-protein interactions of Rho GTPases with their regulatory proteins in a multiplexed miniaturized format.

Extensive knowledge about protein-protein interactions is fundamental to fully understand signaling pathways and for the development of new drugs. Rho GTPases are key molecules in cellular signaling processes and their deregulation is implicated in the development of a variety of diseases such as neurofibromatosis type 2 and cancer. Here, we describe a bead-based protein-protein interaction assay for overexpressed HA-tagged Rho GTPases to study the GTPγS-dependent interaction with the regulatory protein RhoGDIα. This assay provides a useful tool for the analysis of both macromolecular and small molecule activators and inhibitors of the protein-protein interactions of Rho GTPases with their regulatory proteins in a multiplexed miniaturized format.

Receptor tyrosine kinases (RTK) are important targets in drug discovery processes. Studying the phosphorylation pattern of RTKs enables the determination of their activation and inactivation states. Multiplex bead-based sandwich immunoassays are powerful tools for measuring the phosphorylation state of key regulators within cellular signalling networks. Here, we describe the analysis of the phosphorylation state of receptor tyrosine kinases using the epidermal growth factor receptor (EGFR) as an example. We provide a protocol for a bead-based sandwich immunoassay that enables a relative quantification of the EGFR and its generic tyrosine phosphorylation. We also present data from a kinase inhibitor experiment using 96-well cell-culture plates and a commercially available kit for the analysis of seven receptor tyrosine kinases.

Receptor tyrosine kinases (RTK) are important targets in drug discovery processes. Studying the phosphorylation pattern of RTKs enables the determination of their activation and inactivation states. Multiplex bead-based sandwich immunoassays are powerful tools for measuring the phosphorylation state of key regulators within cellular signalling networks. Here, we describe the analysis of the phosphorylation state of receptor tyrosine kinases using the epidermal growth factor receptor (EGFR) as an example. We provide a protocol for a bead-based sandwich immunoassay that enables a relative quantification of the EGFR and its generic tyrosine phosphorylation. We also present data from a kinase inhibitor experiment using 96-well cell-culture plates and a commercially available kit for the analysis of seven receptor tyrosine kinases.

Over the last 10 years, DNA microarrays have achieved a robust analytical performance, enabling their use for analyzing the whole transcriptome or for screening thousands of single-nucleotide polymorphisms in a single experiment. DNA microarrays allow scientists to correlate gene expression signatures with disease progression, to screen for disease-specific mutations, and to treat patients according to their individual genetic profiles; however, the real key is proteins and their manifold functions. It is necessary to achieve a greater understanding of not only protein function and abundance but also their role in the development of diseases. Protein concentrations have been shown to reflect the physiological and pathologic state of an organ, tissue, or cells far more directly than DNA, and proteins can be profiled effectively with protein microarrays, which require only a small amount of sample material. Protein microarrays have become wellestablished tools in basic and applied research, and the first products have already entered the in vitro diagnostics market. This review focuses on protein microarray applications for biomarker discovery and validation, disease diagnosis, and use within the area of personalized medicine. Protein microarrays have proved to be reliable research tools in screening for a multitude of parameters with only a minimal quantity of sample and have enormous potential in applications for diagnostic and personalized medicine.

Purpose of this study was to determine the accuracy of prediction of non-sentinel lymph node (NSLN) involvement in sentinel node (SLN)-positive breast cancer patients based on protein concentrations measured in lysates from initially taken breast biopsies. Data on protein expression, previously generated by multiplexed bead-based immunoassays, were analysed by multivariate logistic regression to define parameter sets of value to predict NSLN involvement. Receiver-operator characteristics (ROCs) were calculated as indicators of diagnostic significance. Analyses of data from all patients (n = 99) resulted in parameter sets that allowed direct prediction of the NSLN status with a ROC area under the curve (AUC) of 0.83. The clinically most relevant prediction of NSLN status in SLN-positive patients (n = 37) based on only seven parameters (including TIMP-2 as the most relevant single value) was possible with high accuracy indicated by an AUC of 0.89. Parallel assessment of protein concentrations in breast biopsies is a highly promising approach to predict nodal involvement and even the NSLN status in SLN-negative breast cancer patients. Such diagnostic information could substantially reduce the number of completion axillary lymph node dissections in clinical practice.

Triple-negative breast cancers (TNBC), characterized by absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor 2 (HER2), are typically associated with poor prognosis, due to aggressive tumor phenotype(s), only partial response to chemotherapy and present lack of clinically established targeted therapies. Advances in the design of individualized strategies for treatment of TNBC patients require further elucidation, by combined ‘omics’ approaches, of the molecular mechanisms underlying TNBC phenotypic heterogeneity, and the still poorly understood association of TNBC with BRCA1 mutations. An overview is here presented on TNBC profiling in terms of expression signatures, within the functional genomic breast tumor classification, and ongoing efforts toward identification of new therapy targets and bioimaging markers. Due to the complexity of aberrant molecular patterns involved in expression, pathological progression and biological/clinical heterogeneity, the search for novel TNBC biomarkers and therapy targets requires collection of multi-dimensional data sets, use of robust multivariate data analysis techniques and development of innovative systems biology approaches.

BACKGROUND: Over the last ten years, miniaturized multiplexed immunoassays have become robust, reliable research tools that enable researchers to simultaneously determine a multitude of parameters. Among the numerous analytical protein arrays available, bead-based assay systems have evolved into a key technology that enables the quantitative protein profiling of biological samples whilst requiring only a minimal amount of sample material. METHODOLOGY/PRINCIPAL FINDINGS: A microfluidic bead-based immunoassay, µFBI, was developed to perform bead-based multiplexed sandwich immunoassays in a capillary. This setup allows the simultaneous detection of several parameters and only requires 200 ng of tissue lysate in a 1 µL assay volume. In addition, only 1 µL of detection antibodies and 1 µL of the reporter molecule Streptavidin-Phycoerythrin were required. The µFBI was used to compare the expression of seven receptor tyrosine kinases and their degree of tyrosine phosphorylation in breast cancer tissue and in normal tissue lysates. The total amount of HER-2, as well the degree of tyrosine phosphorylation was much higher in breast cancer tissue than in normal tissue. µFBI and a standard bead-based assay led to identical protein expression data. Moreover, it was possible to reduce the quantity of sample material required by a factor of 100 and the quantity of reagents by a factor of 30. CONCLUSIONS/SIGNIFICANCE: The µFBI, microfluidic bead-based immunoassay, allows the analysis of multiple parameters from a very small amount of sample material, such as tumor biopsies or tissue sections.

Within the last decade, protein microarray technology has been successfully used for the simultaneous quantification of target proteins from minimal amounts of samples in basic and applied proteome research. The robustness and appropriate sensitivity of these miniaturized assays have been demonstrated and thus the transfer to routine and high-throughput applications is now possible. In this study, multiplexed bead-based sandwich immunoassays were used to determine the concentrations of 54 protein analytes, including HER 2 and the estrogen receptor, from ultrasound-guided large-core needle biopsies (LCNBs) from breast cancer patients. Expression levels for HER 2, estrogen receptors and progesterone receptors were also assessed by immunohistochemical routine staining, performed in the clinic on corresponding biopsy samples. The high concordance of the data sets generated with the bead-based protein arrays and by conventional immunohistochemical assessment of HER 2 and the estrogen receptor expressed by breast cancer cells present in the biopsies was demonstrated.

Background: Over the last 10 years, DNA microarrays have achieved a robust analytical performance, enabling their use for analyzing the whole transcriptome or for screening thousands of single-nucleotide polymorphisms in a single experiment. DNA microarrays allow scientists to correlate gene expression signatures with disease progression, to screen for disease-specific mutations, and to treat patients according to their individual genetic profiles; however, the real key is proteins and their manifold functions. It is necessary to achieve a greater understanding of not only protein function and abundance but also their role in the development of diseases. Protein concentrations have been shown to reflect the physiological and pathologic state of an organ, tissue, or cells far more directly than DNA, and proteins can be profiled effectively with protein microarrays, which require only a small amount of sample material. Content: Protein microarrays have become well-established tools in basic and applied research, and the first products have already entered the in vitro diagnostics market. This review focuses on protein microarray applications for biomarker discovery and validation, disease diagnosis, and use within the area of personalized medicine. Summary: Protein microarrays have proved to be reliable research tools in screening for a multitude of parameters with only a minimal quantity of sample and have enormous potential in applications for diagnostic and personalized medicine.

Inflammation is a defense reaction of an organism against harmful stimuli such as tissue injury or infectious agents. The relationship between the infecting microorganism and the immune, inflammatory, and coagulation responses of the host is intricately intertwined. Due to its complex nature, the molecular mechanisms of inflammation are not yet understood in detail and additional diagnostic tools are required to clarify further aspects. In recent years, protein microarray-based research has moved from being technology-based to application-oriented. Protein microarrays are perfect tools for studying inflammatory diseases. High-density protein arrays enable new classes of autoantibodies, which cause autoimmune diseases, to be discovered. Protein arrays consisting of miniaturized multiplexed sandwich immunoassays allow the simultaneous expression analysis of dozens of signaling molecules such as the cytokines and chemokines involved in the regulation of the immune system. The data enable statements to be made on the status of the disease and its progression as well as support for the clinicians in choosing patient-specific treatment. This chapter reviews the technology and the applications of protein microarrays in diagnosing and monitoring inflammatory diseases.

Inflammation is a defense reaction of an organism against harmful stimuli such as tissue injury or infectious agents. The relationship between the infecting microorganism and the immune, inflammatory, and coagulation responses of the host is intricately intertwined. Due to its complex nature, the molecular mechanisms of inflammation are not yet understood in detail and additional diagnostic tools are required to clarify further aspects. In recent years, protein microarray-based research has moved from being technology-based to application-oriented. Protein microarrays are perfect tools for studying inflammatory diseases. High-density protein arrays enable new classes of autoantibodies, which cause autoimmune diseases, to be discovered. Protein arrays consisting of miniaturized multiplexed sandwich immunoassays allow the simultaneous expression analysis of dozens of signaling molecules such as the cytokines and chemokines involved in the regulation of the immune system. The data enable statements to be made on the status of the disease and its progression as well as support for the clinicians in choosing patient-specific treatment. This chapter reviews the technology and the applications of protein microarrays in diagnosing and monitoring inflammatory diseases.

Vascular endothelial growth factor (VEGF) is involved in various physiologic processes, such as angiogenesis or wound healing, but is also crucial in pathologic events, such as tumor growth. Thus, clinical anti-VEGF treatments have been developed that could already show beneficial effects for cancer patients. In this article, we describe the first VEGF-derived CD8+ T-cell epitope. The natural HLA ligand SRFGGAVVR was identified by differential mass spectrometry in two primary renal cell carcinomas (RCC) and was significantly overpresented on both tumor tissues. SRFGGAVVR is derived from a cryptic translated region of VEGF presumably by initiation of translation at the nonclassic start codon CUG499. SRFGGAVVR-specific T cells were generated in vitro using peptide-loaded dendritic cells or artificial antigen-presenting cells. SRFGGAVVR-specific CD8+ T cells, identified by HLA tetramer analysis after in vitro stimulation, were fully functional T effector cells, which were able to secrete IFN-γ on stimulation and killed tumor cells in vitro. Additionally, we have quantitatively analyzed VEGF mRNA and protein levels in RCC tumor and normal tissue samples by gene chip analysis, quantitative reverse transcription-PCR, in situ hybridization, and bead-based immunoassay. In the future, T cells directed against VEGF as a tumor-associated antigen may represent a possible way of combining peptide-based anti-VEGF immunotherapy with already existent anti-VEGF cancer therapies. [Cancer Res 2008;68(7):2447–54]

Within recent years, protein microarrays have been developed to quantify a large number of parameters present in a given sample simultaneously. Such miniaturised and parallelised sandwich immunoassays are of general interest for all proteomic and diagnostic approaches in which several parameters have to be determined from small samples, e.g. biopsy material. In addition to planar microarray-based approaches, bead-based flow cytometry is quite suitable for the multiplex detection of target molecules, especially when a limited number of parameters are to be analysed. Appropriate sensitivity, reproducibility and robustness have to be demonstrated before protein microarray technology can be used to characterise clinical samples and generate reliable data sets. As a model system to analyse these issues, a set of multiplexed sandwich immunoassays based on Luminex beads were developed to screen clinical samples for the presence or absence of marker proteins indicative of prognosis or response to therapeutic options.

To investigate the vitreous levels of bevacizumab and vascular endothelial growth factor-A (VEGF-A) after intravitreal injection of the drug in patients with choroidal neovascularization (CNV).Interventional case series.Eleven eyes of 11 patients with submacular hemorrhage and CNV due to age-related macular degeneration (n = 10) or angioid streaks (n = 1).All patients were treatment naïve except for a single dose of intravitreal injection of bevacizumab (1.25 mg/50 muL dose) and subsequent vitrectomy after various intervals (1-101 days) because of active and progressive lesion. Intravitreal free bevacizumab and VEGF-A levels were measured using enzyme-linked immunosorbent assay and microsphere-based immunoassay, respectively. Vitreous VEGF-A isoforms were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.Intravitreal bevacizumab and VEGF-A levels were measured and pharmacokinetic parameters were calculated.Pharmacokinetics of intravitreal bevacizumab followed a 2-compartment model with initial and terminal half-lives of 0.5 and 6.7 days, respectively. Bevacizumab could be detected in all cases, ranging from 2.63 ng/ml to 165 microg/ml. The peak concentration was observed on the second day after intravitreal bevacizumab injection. Vitreous free VEGF-A levels ranged from 0.2 to 33.9 pg/ml and showed a negative correlation with the bevacizumab concentration (P0.001; r = -0.955) and a positive correlation with time (P0.001; r = 0.964). However, the percentage expression of VEGF-A(165) exhibited a positive correlation with the bevacizumab concentration (P = 0.032, r = 0.645) and a negative correlation with time (P = 0.007, r = -0.755). A time-dependent increase was found for the percentage expression of VEGF-A(189) (P = 0.023, r = 0.673). Neither bevacizumab- nor time-related alterations were found for VEGF-A(121).Based on pharmacokinetics, the interval of 6-7 weeks would be appropriate for efficacy, although clinical trials should guide dosing recommendations. Vitreous levels of free VEGF-A showed a negative correlation with the bevacizumab concentration, which confirmed the in vivo binding affinity of bevacizumab to VEGF-A. The analysis of the VEGF-A isoforms suggests differences of interaction between bevacizumab and individual VEGF-A isoforms.

Background & Aims We report a novel experimental immunotherapeutic approach in a patient with metastatic intrahepatic cholangiocarcinoma. In the 5year course of the disease, the initial tumor mass, two local recurrences and a lung metastasis were surgically removed. Lacking alternative treatment options, aiming at the induction of anti-tumor T cells responses, we initiated a personalized multi-peptide vaccination, based on in-depth analysis of tumor antigens (immunopeptidome) and sequencing. Methods Tumors were characterized by immunohistochemistry, next-generation sequencing and mass spectrometry of HLA ligands. Results Although several tumor-specific neo-epitopes were predicted in silico , none could be validated by mass spectrometry. Instead, a personalized multi-peptide vaccine containing non-mutated tumor-associated epitopes was designed and applied. Immunomonitoring showed vaccine-induced T cell responses to three out of seven peptides administered. The pulmonary metastasis resected after start of vaccination showed strong immune cell infiltration and perforin positivity, in contrast to the previous lesions. The patient remains clinically healthy, without any radiologically detectable tumors since March 2013 and the vaccination is continued. Conclusions This remarkable clinical course encourages formal clinical studies on adjuvant personalized peptide vaccination in cholangiocarcinoma. Lay Summary Metastatic cholangiocarcinomas, cancers that originate from the liver bile ducts, have very limited treatment options and a fatal prognosis. We describe a novel therapeutic approach in such a patient using a personalized multi-peptide vaccine. This vaccine, developed based on the characterization of the patient's tumor, evoked detectable anti-tumor immune responses, associating with long-term tumor-free survival.

Background The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated individuals showed a substantial reduction. Methods We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals. Results While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to Omicron only improved after administration of a third dose. Conclusions Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies.

The ongoing COVID-19 pandemic and the emergence of new SARS-CoV-2 variants of concern (VOCs) requires continued development of effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nbs) specific for the receptor-binding domain (RBD) of SARS-CoV-2. Taking advantage of detailed epitope mapping, we generate two biparatopic Nbs (bipNbs) targeting a conserved epitope outside and two different epitopes inside the RBD:ACE2 interface. Both bipNbs bind all currently circulating VOCs with high affinities and are capable to neutralize cellular infection with VOC B.1.351 (Beta) and B.1.617.2 (Delta) in vitro. To assess if the bipNbs NM1267 and NM1268 confer protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice are treated intranasally before infection with a lethal dose of SARS-CoV-2 B.1, B.1.351 (Beta) or B.1.617.2 (Delta). Nb-treated mice show significantly reduced disease progression and increased survival rates. Histopathological analyses further reveal a drastically reduced viral load and inflammatory response in lungs. These data suggest that both bipNbs are broadly active against a variety of emerging SARS-CoV-2 VOCs and represent easily applicable drug candidates.