RESUMO
Monitoring anti-drug antibodies (ADAs) to infliximab and adalimumab is critical to treatment management in various autoimmune disorders. The growing need for proactive therapeutic monitoring further requires the detection of ADAs in the presence of measurable concentrations of infliximab or adalimumab. To provide robust analytical assays for clinical application, we evaluated two automated immunoassays developed using ImmunoCAP™ technology and based on the bridging format to measure serum ADAs to infliximab and adalimumab respectively. Without an acid-dissociation step, these research prototype assays can detect a positive control monoclonal ADA towards infliximab and adalimumab, ranging from < 25 ng/ml to 10,000 ng/mL. Both assays exhibit imprecision less than 20% at different ADA titer levels and can distinguish ADAs towards different drug targets. In method comparison using authentic patient samples, the quantitative results of the ADA assays are not directly comparable to two existing clinical immunoassays for ADAs (correlation coefficient rs = 0.673 for infliximab ADAs; rs = 0.510 for adalimumab ADAs), presumably due to the lack of commutable ADA standards and the polyclonal nature of ADAs. Nevertheless, there is qualitative agreement between the methods when evaluating putative positive and negative patient samples (overall agreement 0.83 for infliximab ADAs; 0.76 for adalimumab ADAs). Biotin and high levels of rheumatoid factors may interfere with the performance of the automated assays due to competitive binding with the biotinylated drug and non-specific formation of bridging complexes. The two ImmunoCAP assays can provide new analytical methods for proactive therapeutic monitoring of adalimumab and infliximab.
Assuntos
Adalimumab , Monitoramento de Medicamentos , Infliximab , Adalimumab/imunologia , Adalimumab/sangue , Infliximab/imunologia , Infliximab/sangue , Humanos , Imunoensaio/métodos , Monitoramento de Medicamentos/métodos , Anticorpos/imunologia , Anticorpos/sangueRESUMO
We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes -UnFold probes - where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic "unfolding" step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.
Assuntos
Anticorpos Imobilizados/química , Oligonucleotídeos/química , Proteínas/análise , Anticorpos Imobilizados/imunologia , Sequência de Bases , Caderinas/química , Caderinas/metabolismo , Linhagem Celular , DNA Circular/química , DNA Circular/metabolismo , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência , Conformação de Ácido Nucleico , Mapeamento de Interação de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Proteínas/imunologia , Pele/metabolismo , Pele/patologia , beta Catenina/química , beta Catenina/metabolismoRESUMO
The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-ß signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-ß signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-ß signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.
Assuntos
Anticorpos de Cadeia Única/análise , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Biblioteca de Peptídeos , Fosforilação , Transdução de Sinais/efeitos dos fármacosRESUMO
Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.
Assuntos
DNA Circular/química , DNA Circular/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de DNA/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Razão Sinal-RuídoRESUMO
Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.
Assuntos
Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Fator de Crescimento Epidérmico/química , Receptores ErbB/química , Citometria de Fluxo , Corantes Fluorescentes/química , Ligação ProteicaRESUMO
The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGFß. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGFß (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGFß stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGFß induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ-Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP-Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGFß signaling pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias do Colo/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/análise , Linhagem Celular Tumoral , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/patologia , Via de Sinalização Hippo , Humanos , Fosfoproteínas/análise , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/análise , Proteínas Smad Reguladas por Receptor/análise , Proteína Smad2/análise , Proteína Smad2/metabolismo , Proteína Smad3/análise , Proteína Smad3/metabolismo , Fatores de Transcrição/análise , Fator de Crescimento Transformador beta/análise , Proteínas de Sinalização YAPRESUMO
The fate of the cell is governed by interactions among proteins, nucleic acids, and other biomolecules. It is vital to look at these interactions in a cellular environment if we want to increase our understanding of cellular processes. Herein we will describe how the in situ proximity ligation assay (in situ PLA) can be used to visualize protein interactions in fixed cells and tissues. In situ PLA is a novel technique that uses DNA, together with DNA modifying processes such as ligation, cleavage, and polymerization, as tools to create surrogate markers for protein interactions of interest. Different in situ PLA designs make it possible not only to detect protein-protein interactions but also post-translational modifications and interactions of proteins with nucleic acids. Flexibility in DNA probe design and the multitude of different DNA modifying enzymes provide the basis for modifications of the method to make it suitable to use in many applications. Furthermore, examples of how in situ PLA can be combined with other methods for a comprehensive view of the cellular activity status are discussed.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Animais , DNA/genética , DNA/metabolismo , Humanos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas/genéticaRESUMO
The emergence of targeted therapies for cancer has created a need for the development of companion diagnostic tests. Assays developed in recent years are aimed at determining both the effectiveness and safety of specific drugs for a defined group of patients, thus, enabling the more efficient design of clinical trials and also supporting physicians when making treatment-related decisions. Immunohistochemistry (IHC) is a widely accepted method for protein expression analyses in human tissues. Immunohistochemical assays, used to localize and quantitate relative protein expression levels within a morphological context, are frequently used as companion diagnostics during clinical trials and also following drug approval. Herein, we describe established immunochemistry-based methods and their application in routine diagnostics. We also explore the possibility of using IHC to detect specific protein mutations in addition to DNA-based tests. Finally, we review alternative protein binders and proximity ligation assays and discuss their potential to facilitate the development of novel, targeted therapies against cancer.
RESUMO
Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/ultraestrutura , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Imagem Molecular/métodos , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Animais , Biópsia , Neoplasias da Mama/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Receptores ErbB/metabolismo , Feminino , Humanos , Imunoconjugados/química , Sondas Moleculares/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais , Análise de Célula Única , SuínosRESUMO
In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, ß-catenin, PIPKIγ and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein.
Assuntos
Caderinas/genética , Caderinas/metabolismo , Mutação de Sentido Incorreto , Neoplasias Gástricas/genética , Animais , Antígenos CD , Sítios de Ligação , Células CHO , Cateninas/metabolismo , Cricetinae , Citoplasma/genética , Citoplasma/metabolismo , Exocitose/genética , Humanos , Biologia Molecular/métodos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Neoplasias Gástricas/patologia , Ubiquitina-Proteína Ligases/metabolismo , beta Catenina/metabolismo , delta CateninaRESUMO
The heterogeneous nature of cancer results in highly variable therapeutic responses even among patients with identical stages and grades of a malignancy. The move towards personalised medicine in cancer therapy has therefore been motivated by a need to customise therapy according to molecular features of individual tumours. Companion diagnostics serves to support early drug development, it can provide surrogate markers in clinical trials, and also guide selection of individual therapies and monitoring of responses in routine clinical care. The era of companion diagnostics can be said to have begun with the introduction of the HercepTest - a first-of-a-kind diagnostic tool developed by DakoCytomation in 1998 to select patients for therapy with the anticancer drug Herceptin (trastuzumab). Herceptin and the paired test proved that companion diagnostics can help guide patient-tailored therapies. We will discuss herein technologies to analyse companion diagnostics markers at the level of DNA, RNA or protein, focusing on a series of methods developed in our laboratory that can facilitate drug development and help stratify patients for therapy.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Neoplasias/diagnóstico , Técnicas de Genotipagem , Humanos , Proteínas de Neoplasias/sangue , Neoplasias/sangue , Neoplasias/genética , Células Neoplásicas Circulantes/patologia , Análise de Sequência de DNARESUMO
The family Chlamydiaceae contains several bacterial pathogens of important human and veterinary medical concern, such as Chlamydia trachomatis and Chlamydophila psittaci. Within the order Chlamydiales there are also an increasing number of chlamydia-like bacteria whose biodiversity, host range and environmental spread seem to have been largely underestimated, and which are currently being investigated for their potential medical relevance. In this study we present 16S rRNA, rnpB and ompA gene sequence data congruently indicating a novel chlamydia-like bacterium found in faecal specimens from opportunistic fish-eating sea birds, belonging to the Laridae and Alcidae families, from the Bering Sea. This novel bacterium appears to be closer to the Chlamydiaceae than other chlamydia-like bacteria and is most likely a novel genus within the Chlamydiaceae family.