RESUMO
BACKGROUND: Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence. METHODS: We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival. RESULTS: The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003). CONCLUSIONS: Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.
Assuntos
Carcinoma Ductal de Mama/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Receptores ErbB/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , PrognósticoRESUMO
Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. Metabotropic glutamate receptors are dimeric GPCRs that can control synaptic activity in a multitude of ways. Here we identify llama nanobodies that specifically recognize mGlu2 receptors, among the eight subtypes of mGluR subunits. Among these nanobodies, DN10 and 13 are positive allosteric modulators (PAM) on homodimeric mGlu2, while DN10 displays also a significant partial agonist activity. DN10 and DN13 have no effect on mGlu2-3 and mGlu2-4 heterodimers. These PAMs enhance the inhibitory action of the orthosteric mGlu2/mGlu3 agonist, DCG-IV, at mossy fiber terminals in the CA3 region of hippocampal slices. DN13 also impairs contextual fear memory when injected in the CA3 region of hippocampal region. These data highlight the potential of developing antibodies with allosteric actions on GPCRs to better define their roles in vivo.
Assuntos
Medo/fisiologia , Hipocampo/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Sítios de Ligação , Camelídeos Americanos , AMP Cíclico/metabolismo , Ciclopropanos , Ácido Glutâmico/sangue , Ácido Glutâmico/metabolismo , Glicina/análogos & derivados , Células HEK293 , Hipocampo/efeitos dos fármacos , Humanos , Fosfatos de Inositol/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neurônios/fisiologia , Receptores OpioidesRESUMO
Exploratory clinical trials using therapeutic anti-HER3 antibodies strongly suggest that neuregulin (NRG1; HER3 ligand) expression at tumor sites is a predictive biomarker of anti-HER3 antibody efficacy in cancer. We hypothesized that in NRG1-expressing tumors, where the ligand is present before antibody treatment, anti-HER3 antibodies that do not compete with NRG1 for receptor binding have a higher receptor-neutralizing action than antibodies competing with the ligand for binding to HER3. Using time-resolved-fluorescence energy transfer (TR-FRET), we demonstrated that in the presence of recombinant NRG1, binding of 9F7-F11 (a nonligand-competing anti-HER3 antibody) to HER3 is increased, whereas that of ligand-competing anti-HER3 antibodies (H4B-121, U3-1287, Ab#6, Mab205.10.2, and MOR09825) is decreased. Moreover, 9F7-F11 showed higher efficacy than antibodies that compete with the ligand for binding to HER3. Specifically, 9F7-F11 inhibition of cell proliferation and of HER3/AKT/ERK1/2 phosphorylation as well as 9F7-F11-dependent cell-mediated cytotoxicity were higher in cancer cells preincubated with recombinant NRG1 compared with cells directly exposed to the anti-HER3 antibody. This translated in vivo into enhanced growth inhibition of NRG1-expressing BxPC3 pancreatic, A549 lung, and HCC-1806 breast cell tumor xenografts in mice treated with 9F7-F11 compared with H4B-121. Conversely, both antibodies had similar antitumor effect in NRG1-negative HPAC pancreatic carcinoma cells. In conclusion, the allosteric modulator 9F7-F11 shows increased anticancer effectiveness in the presence of NRG1 and thus represents a novel treatment strategy for NRG1-addicted tumors. Mol Cancer Ther; 16(7); 1312-23. ©2017 AACR.
Assuntos
Anticorpos Monoclonais Murinos/administração & dosagem , Biomarcadores Tumorais/imunologia , Neoplasias/tratamento farmacológico , Neuregulina-1/genética , Receptor ErbB-3/imunologia , Células A549 , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais Murinos/imunologia , Biomarcadores Tumorais/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Transferência Ressonante de Energia de Fluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neuregulina-1/imunologia , Fosforilação , Ligação Proteica , Receptor ErbB-3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The epidermal growth factor receptor (EGFR) is a cell-surface receptor with a single transmembrane domain and tyrosine kinase activity carried by the intracellular domain. This receptor is one of the four members of the ErbB family including ErbB2, ErbB3, and ErbB4. Ligand binding, like EGF binding, induces a conformational rearrangement of the receptor and induces a homo/hetero dimerization essentially with ErbB family receptors that leads to the phosphorylation of the kinase domain, triggering a signaling cascade. EGFR can also form inactive dimers in a ligand-independent way through interactions between cytoplasmic domains. To date, the conformation of EGFR extracellular domain engaged in these inactive dimers remains unclear. In this study, we describe the successful selection and characterization of llama anti-EGFR nanobodies and their use as innovative conformational sensors. We isolated three different specific anti-EGFR clones binding to three distinct epitopes. Interestingly, the binding of all three nanobodies was found highly sensitive to ligand stimulation. Two nanobodies, D10 and E10, can only bind the ligand-free EGFR conformation characterized by an intramolecular tether between domains II and IV, whereas nanobody G10 binds both ligand-free and ligand activated EGFR, with an 8-fold higher affinity for the extended conformation in the presence of ligand. Here we took advantage of these conformational probes to reveal the existence of tethered EGFR in EGFR/ErbB2 predimers. These biosensors represent important tools allowing the determination of EGFR conformations and should help the design of relevant inhibitors.
Assuntos
Técnicas Biossensoriais , Receptores ErbB/química , Receptores ErbB/imunologia , Multimerização Proteica , Receptor ErbB-2/química , Anticorpos de Domínio Único/imunologia , Animais , Especificidade de Anticorpos , Sítios de Ligação , Camelídeos Americanos , Linhagem Celular , Epitopos/imunologia , Humanos , Camundongos , Estrutura Quaternária de ProteínaRESUMO
Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water-soluble, and negatively charged sulfonic- or carboxylic acid derivatives of para-substituted aryl-alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell-surface proteins with time-resolved Förster resonance energy transfer (TR-FRET) assays in plate-based format and using TR-FRET microscopy.
Assuntos
Compostos Aza/análise , Complexos de Coordenação/análise , Európio/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Substâncias Luminescentes/análise , Microscopia/métodos , Piperidinas/análise , Receptores Acoplados a Proteínas G/metabolismo , Compostos Aza/metabolismo , Complexos de Coordenação/metabolismo , Európio/metabolismo , Células HEK293 , Humanos , Ligantes , Substâncias Luminescentes/metabolismo , Piperidinas/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/análise , Solubilidade , Água/químicaRESUMO
The design and application of luminescent lanthanide cryptates for sensing biological interactions is highlighted through the review of the work performed in our laboratory and with academic collaborations. The path from the initial applications probing biochemical interaction in vitro to "state-of-the-art" cellular assays toward clinical applications using homogeneous time-resolved fluorescence technology is described. An overview of the luminescent lanthanide macrocyclic compounds developed at Cisbio in the recent past is given with an emphasis on specific constraints required by specific applications. Recent assays for drug-discovery and diagnostic purposes using both antibody-based and suicide-enzyme-based technology are illustrated. New perspectives in the field of molecular medicine and time-resolved microscopy are discussed.
Assuntos
Éteres de Coroa/química , Descoberta de Drogas , Elementos da Série dos Lantanídeos/química , Substâncias Luminescentes/química , Medicina Molecular/tendências , HumanosRESUMO
Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 breast tumors and the results were compared with those obtained by using reference methods. The EGFR number per cell determined by TR-FRET was significantly correlated with EGFR mRNA copy number (P<0.0001). Moreover, our method detected HER2 overexpression with 100% specificity and sensibility, as confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 dimerization was then assessed, using as controls xenograft tumors from cell lines with known dimer expression profiles. Our results show that quantification of HER dimerization provides information about receptor activation that cannot be obtained by quantification of single receptors. Quantifying HER expression and dimerization by TR-FRET assays might help identifying novel clinical markers for optimizing patients' treatment in oncology.
Assuntos
Neoplasias da Mama/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Animais , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Humanos , Técnicas In Vitro , Camundongos , Multimerização ProteicaRESUMO
In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Neoplasias/tratamento farmacológico , Multimerização Proteica/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Cloridrato de Erlotinib , Humanos , Lapatinib , Camundongos , Células NIH 3T3 , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genéticaRESUMO
Probing the interactions of the DNA mismatch repair protein MutS with altered and damaged DNA is of great interest both for the understanding of the mismatch repair system function and for the development of tools to detect mutations. Here we describe a homogeneous time-resolved fluorescence (HTRF) assay to study the interactions of Escherichia coli MutS protein with various DNA substrates. First, we designed an indirect HTRF assay on a microtiter plate format and demonstrated its general applicability through the analysis of the interactions between MutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin. Then we directly labeled MutS with the long-lived fluorescent donor molecule europium tris-bipyridine cryptate ([TBP(Eu(3+))]) and demonstrated by electrophoretic mobility shift assay that this chemically labeled protein retained DNA mismatch binding property. Consequently, we used [TBP(Eu(3+))]-MutS to develop a faster and simpler semidirect HTRF assay.
Assuntos
DNA/metabolismo , Transferência de Energia , Proteínas de Escherichia coli/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , Escherichia coli , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Ligação Proteica , Fatores de TempoRESUMO
Synthesis, absorption spectra and luminescebce properties of a series of lanthanide trisbipyridine cryptates Ln within R-Bpy x R-Bpy x R-Bpy, where Ln = Eu, Gd and R = H, COOH, COOCH3, CONH(CH2)2NH2 are described. Comparison of the unsubstituted parent compound with the substituted compounds shows that bipyridine substitution doesn't alter significantly the photophysical properties of the lanthanide cryptate. The absorption maximum is slightly red-shifted when three bipyridines are substituted, whereas substituting one bipyridines has a negligible effect on the absorption spectra. The experimental triplet state energy is between 21600 and 22 100 cm(-1) for the series of compounds and the luminescence lifetimes at 77 K are between 0.5 and 0.8 ms in HO2 and equal to 1.7 ms in D2O. The experimental characterizations are completed by DFT and TD-DFT calculations to assess the ability of these approaches to predict absorption maxima, triplet state energies and structural parameters of lanthanide cryptates and to characterize the electronic structure of the excited states. The calculations on the unsubstituted parent and substituted compounds show that absorption maxima and lowest 3pipi* triplet state energies can be accurately determined from density functional theory (DFT) and time-dependent (TD) DFT calculations.
Assuntos
2,2'-Dipiridil/análogos & derivados , Substâncias Luminescentes/química , Compostos Organometálicos/química , 2,2'-Dipiridil/química , Éteres de Coroa/química , Cristalização , Elementos da Série dos Lantanídeos/química , Modelos Moleculares , Estrutura Molecular , Nitrogênio/química , Espectrofotometria , Água/químicaRESUMO
Phospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade. We show here that IP1 can be used as a surrogate of IP3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP-One assay was validated on various GPCR models. It has the advantage over the traditional Ca2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-beta activity in any nontransfected primary cultures. Finally, the high assay specificity for D-myo-inositol 1 monophosphate (IP1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.
Assuntos
Inositol 1,4,5-Trifosfato , Fosfatos de Inositol , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células CHO , Cricetinae , Humanos , Inositol 1,4,5-Trifosfato/química , Fosfatos de Inositol/química , Fosfolipases Tipo C/metabolismoAssuntos
Técnicas de Química Combinatória/métodos , Fluorescência , Biblioteca de Peptídeos , Desenho de Fármacos , Ativação Enzimática , Imunoensaio de Fluorescência por Polarização/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Fluorimunoensaio/métodos , Elementos da Série dos Lantanídeos/farmacologia , Medições Luminescentes , Proteínas Luminescentes , Modelos Biológicos , Fragmentos de Peptídeos , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodosRESUMO
Single nucleotide primer extension reaction has been widely used in DNA testing, and several detection methods based on this core allelic discrimination have been developed. Most of the reported formats are based on a two step protocol involving first, a liquid phase extension reaction, then a physical separation process (chromatography, electrophoresis, capture on solid support, mass spectrometry). Here we describe a new strategy based on homogeneous time-resolved fluorescence (HTRF), which does not involve any separation process and which allows a simple "mix and measure" protocol. In this approach, a 5'-(europium) cryptate-labeled primer is elongated by a biotinylated dideoxynucleoside-triphosphate, followed by the addition of a streptavidin-acceptor conjugate, which gives rise to a long-life fluorescence resonance energy transfer (FRET) signal between the cryptate donor and the acceptor. We present the development of HTRF technology as applied to the diagnosis of tumor suppressor gene p53 (TP53) mutations, and its application to the analysis of genomic DNA from human tumoral samples. The sensitivity of the reported method is compared to the corresponding fluorescent polarization assay.
Assuntos
Análise Mutacional de DNA/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Linhagem Celular Tumoral , Polarização de Fluorescência/métodos , Genes p53/genética , Humanos , Mutação , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The homogeneous time-resolved fluorescence (HTRF) technology is an assay developed to study the interaction between biomolecules. This detection system is based on a fluorescence resonance energy transfer (FRET) between a Tris-bipyridine europium cryptate used as a long-lived fluorescent donor and a chemically modified allophycocyanine as acceptor. This technology is characterized by both a spectral selectivity and a temporal selectivity (due to the time-resolved mode), ensuring a highly specific signal. Here a europium-cryptate-labeled deoxyuridine triphosphate analogue (K-11-dUTP) was used to monitor the extension reaction on a biotinylated oligonucleotide used as substrate for telomerase in a telomeric repeat amplification protocol (TRAP). After the addition of an allophycocyanine-streptavidin conjugate, the extension products give rise to a FRET between the incorporated cryptate moieties and the allophycocyanine acceptor that then displays a specific long-lived emission. The TRAP-HTRF format was validated as a screening tool by using a 2,6-diaminoanthraquinone analogue, a known inhibitor of telomerase activity. The IC(50) measured was consistent with the reported values, showing the convenience of the HTRF technology for the study of telomerase activity and inhibitors.
Assuntos
Desoxiuridina/análogos & derivados , Transferência Ressonante de Energia de Fluorescência/métodos , Telomerase/metabolismo , Antraquinonas/farmacologia , Desoxiuridina/química , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Humanos , Células K562 , Oligodesoxirribonucleotídeos/química , Compostos Organometálicos/química , Ficocianina/química , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Telomerase/antagonistas & inibidores , Telomerase/químicaRESUMO
Direct or indirect interactions between membrane proteins at the cell surface play a central role in numerous cell processes, including possible synergistic effects between different types of receptors. Here we describe a method and tools to analyze membrane protein-protein interaction at the surface of living cells. This technology is based on the use of specific antibodies directed against each partner and labeled either with europium cryptate or with Alexa Fluor 647. This allows the measurement of a fluorescence resonance energy transfer (FRET) signal in a time-resolved manner if both antibodies are in close proximity. This approach is here validated using the heterodimeric gamma-aminobutyrate B receptor as a model. We show that after washing out the unbound antibodies, the time-resolved FRET signal can be measured together with the expression level of both partners via the quantification of the donor and the acceptor fluorophores bound to the cells. Thanks to the high sensitivity of this method and to the low concentration of antibodies required, we show that the signal can also be measured directly after the incubation period without washing out the unbound antibody (homogeneous time-resolved FRET). As such, this method is highly sensitive, reproducible, and compatible with the development of high-throughput screening protocols.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Mapeamento de Interação de Proteínas/métodos , Receptores de GABA-B/metabolismo , Anticorpos/imunologia , Humanos , Receptores de GABA-B/imunologia , Fatores de TempoRESUMO
The p53 protein is a tumor suppressor that protects the organism against malignant consequences of DNA damage. Interaction of p53 with numerous cellular or viral proteins regulates its functional activity either positively or negatively. An approach leading to identification of such protein interactions directly in a cell extract could be of help in the development of screening assays to search for drugs acting on p53 in its cellular environment, either by disrupting its association with inhibitory proteins or by increasing its affinity for activating proteins. We show that the homogeneous time-resolved fluorescence (HTRF) assay based on the time-resolved amplified cryptate emission (TRACE) technology allows identification of such an interaction by simply adding a mixture of two labeled monoclonal antibodies, directly in a cellular extract. We validate this assay by studying p53/SV40-LTAg interactions. The antibodies directed against genuine p53 and SV40-LTAg epitopes were labeled with europium cryptate (donor) and XL665, a crosslinked allophycocyanin (acceptor), respectively. We demonstrated that a nonradiative energy transfer occurs between labeled antibodies only when p53 interacts with SV40-LTag, which opens up the possibility of extending this approach to other p53 partners to search for drugs that restore p53 tumor-suppressor activity.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Fluorimunoensaio/métodos , Proteína Supressora de Tumor p53/metabolismo , Animais , Anticorpos Monoclonais , Antígenos Transformantes de Poliomavirus/genética , Baculoviridae/genética , Células COS , Ensaio de Imunoadsorção Enzimática , Haplorrinos , Humanos , Neoplasias Pulmonares/metabolismo , Mutação , Plasmídeos , Ligação Proteica , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
A miniaturized immunoassay for human interleukin-13 (IL-13) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IL-13 which was secreted from NK3.3 cells stimulated with interleukin-2 (IL-2) was detected by measuring the time-resolved fluorescence after adding a mixture of three reagents, biotinylated anti-IL-13 monoclonal antibody, europium cryptate (fluorescence donor)-labeled different anti-IL-13 monoclonal antibody and crosslinked allophycocyanin (fluorescence acceptor)-conjugated with streptavidin in a 384-well assay plate. The detection limit of IL-13 using this immunoassay was estimated to be less than 600 pg/ml and IL-13 levels measured by this method were very close to those measured by enzyme linked immunosorbent assay (ELISA; the correlation coefficient was 0.9535). The proposed assay requires only a fourth of the quantities of all reagents compared with the assay using a conventional 96-well microtiter plate. Furthermore, there is no need to transfer the culture supernatant to another assay plate and wash the plate. Therefore, this miniaturized immunoassay is economical and efficient and is particularly suitable for high-throughput drug screening.