RESUMEN
The study describes development of a glycan biosensor for detection of a tumor-associated antibody. The glycan biosensor is built on an electrochemically activated/oxidized graphene screen-printed electrode (GSPE). Oxygen functionalities were subsequently applied for covalent immobilization of human serum albumin (HSA) as a natural nanoscaffold for covalent immobilization of Thomsen-nouvelle (Tn) antigen (GalNAc-O-Ser/Thr) to be fully available for affinity interaction with its analyte-a tumor-associated antibody. The step by step building process of glycan biosensor development was comprehensively characterized using a battery of techniques (scanning electron microscopy, atomic force microscopy, contact angle measurements, secondary ion mass spectrometry, surface plasmon resonance, Raman and energy-dispersive X-ray spectroscopy). Results suggest that electrochemical oxidation of graphene SPE preferentially oxidizes only the surface of graphene flakes within the graphene SPE. Optimization studies revealed the following optimal parameters: activation potential of +1.5 V vs. Ag/AgCl/3 M KCl, activation time of 60 s and concentration of HSA of 0.1 g L-1. Finally, the glycan biosensor was built up able to selectively and sensitively detect its analyte down to low aM concentration. The binding preference of the glycan biosensor was in an agreement with independent surface plasmon resonance analysis.
Asunto(s)
Anticuerpos Antineoplásicos/sangre , Antígenos de Carbohidratos Asociados a Tumores/química , Técnicas Biosensibles/métodos , Grafito/química , Anticuerpos Antineoplásicos/inmunología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Técnicas Electroquímicas , Electrodos , Humanos , Límite de Detección , Albúmina Sérica/químicaRESUMEN
Here, we introduce a novel scFv antibody, G2-D11, specific for two adjacent Tn-antigens (GalNAc-Ser/Thr) binding equally to three dimeric forms of the epitope, Ser-Thr, Thr-Thr and Thr-Ser. Compared to other anti-Tn reagents, the binding of G2-D11 is minimally influenced by the peptide structure, which indicates a high degree of carbohydrate epitope dominance and a low influence from the protein backbone. With a high affinity (KDapp = 1.3 × 10-8 M) and no cross-reactivity to either sialyl-Tn epitope or blood group A antigens, scFv G2-D11 is an excellent candidate for a well-defined anti-Tn-antigen reagent. Detailed immunohistochemical evaluation of tissue sections from a cohort of 80 patients with gastric carcinoma showed in all cases positive tumor cells. The observed staining was localized to the cytoplasm and in some cases to the membrane, whereas the surrounding tissue was completely negative demonstrating the usefulness of the novel Tn-antigen binding antibody.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/inmunología , Carcinoma/metabolismo , Epítopos/química , Anticuerpos de Cadena Única/inmunología , Neoplasias Gástricas/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/química , Carcinoma/patología , Línea Celular Tumoral , Mapeo Epitopo , Epítopos/inmunología , Humanos , Anticuerpos de Cadena Única/química , Neoplasias Gástricas/patologíaRESUMEN
The main aim of the study was to optimize the interfacial presentation of a small antigen-a Tn antigen (N-acetylgalactosamine)-for binding to its analyte anti-Tn antibody. Three different methods for the interfacial display of a small glycan are compared here, including two methods based on the immobilization of the Tn antigen on a mixed self-assembled monolayer (SAM) (2D biosensor) and the third one utilizing a layer of a human serum albumin (HSA) for the immobilization of a glycan forming a 3D interface. Results showed that the 3D interface with the immobilized Tn antigen is the most effective bioreceptive surface for binding its analyte. The 3D impedimetric glycan biosensor exhibited a limit of detection of 1.4 aM, a wide linear range (6 orders of magnitude), and high assay reproducibility with an average relative standard deviation of 4%. The buildup of an interface was optimized using various techniques with the visualization of the glycans on the biosensor surface by atomic force microscopy. The study showed that the 3D biosensor is not only the most sensitive compared to other two biosensor platforms but that the Tn antigen on the 3D biosensor surface is more accessible for antibody binding with better kinetics of binding (t50% = 137 s, t50% = the time needed to attain 50% of a steady-state signal) compared to the 2D biosensor configuration with t50% = 354 s. The 3D glycan biosensor was finally applied for the analysis of a human serum sample spiked with an analyte.
Asunto(s)
Anticuerpos/química , Técnicas Biosensibles , Polisacáridos/química , Cinética , Microscopía de Fuerza AtómicaRESUMEN
Malignant melanoma (MM) patients are being treated with an increasing number of personalized medicine (PM) drugs, several of which are small molecule drugs developed to treat patients with specific disease genotypes and phenotypes. In particular, the clinical application of protein kinase inhibitors has been highly effective for certain subsets of MM patients. Vemurafenib, a protein kinase inhibitor targeting BRAF-mutated protein, has shown significant efficacy in slowing disease progression. In this paper, we provide an overview of this new generation of targeted drugs, and demonstrate the first data on localization of PM drugs within tumor compartments. In this study, we have introduced MALDI-MS imaging to provide new information on one of the drugs currently used in the PM treatment of MM, vemurafenib. In a proof-of-concept in vitro study, MALDI-MS imaging was used to identify vemurafenib applied to metastatic lymph nodes tumors of subjects attending the regional hospital network of Southern Sweden. The paper provides evidence of BRAF overexpression in tumors isolated from MM patients and localization of the specific drug targeting BRAF, vemurafenib, using MS fragment ion signatures. Our ability to determine drug uptake at the target sites of directed therapy provides important opportunity for increasing our understanding about the mode of action of drug activity within the disease environment.
Asunto(s)
Antineoplásicos , Indoles , Melanoma , Imagen Molecular/métodos , Medicina de Precisión/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Sulfonamidas , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Sistemas de Liberación de Medicamentos , Humanos , Indoles/farmacocinética , Indoles/uso terapéutico , Melanoma/química , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/química , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapéutico , VemurafenibRESUMEN
Currently there are no clinically recognized molecular biomarkers for malignant melanoma (MM) for either diagnosing disease stage or measuring response to therapy. The aim of this feasibility study was to develop targeted selected reaction monitoring (SRM) assays for identifying candidate protein biomarkers in metastatic melanoma tissue lysate. In a pilot study applying the SRM assay, the tissue expression of nine selected proteins [complement 3 (C3), T-cell surface glycoprotein CD3 epsilon chain E (CD3E), dermatopontin, minichromosome maintenance complex component (MCM4), premelanosome protein (PMEL), S100 calcium binding protein A8 (S100A8), S100 calcium binding protein A13 (S100A13), transgelin-2 and S100B] was quantified in a small cohort of metastatic malignant melanoma patients. The SRM assay was developed using a TSQ Vantage triple quadrupole mass spectrometer that generated highly accurate peptide quantification. Repeated injection of internal standards spiked into matrix showed relative standard deviation (RSD) from 6% to 15%. All nine target proteins were identified in tumor lysate digests spiked with heavy peptide standards. The multiplex SRM peptide assay panel was then measured and quantified on a set of frozen MM tissue samples obtained from the Malignant Melanoma Biobank collected in Lund, Sweden. All nine proteins could be accurately quantified using the new SRM assay format. This study provides preliminary data on the heterogeneity of biomarker expression within MM patients. The S100B protein, which is clinically used as the pathology identifier of MM, was identified in 9 out of 10 MM tissue lysates. The use of the targeted SRM assay provides potential advancements in the diagnosis of MM that can aid in future assessments of disease in melanoma patients.
Asunto(s)
Biomarcadores de Tumor/análisis , Melanoma/diagnóstico , Proteínas de Neoplasias/análisis , Subunidad beta de la Proteína de Unión al Calcio S100/análisis , Neoplasias Cutáneas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Bancos de Muestras Biológicas , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Estudios de Factibilidad , Femenino , Expresión Génica , Humanos , Metástasis Linfática , Masculino , Melanoma/genética , Melanoma/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteómica , Subunidad beta de la Proteína de Unión al Calcio S100/genética , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Extractos de Tejidos/químicaRESUMEN
Requirements for patient safety and improved efficacy are steadily increasing in modern healthcare and are key drivers in modern drug development. New drug characterization assays are central in providing evidence of the specificity and selectivity of drugs. Meeting this need, matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) is used to study drug localization within microenvironmental tissue compartments. Thin sections of human lung tumor and rat xenograft tissues were exposed to pharmaceutical drugs by either spotting or submerging. These drugs, the epidermal growth factor receptor antagonists, erlotinib (Tarceva) and gefitinib (Iressa), and the acetylcholine receptor antagonist, tiotropium, were characterized by microenvironment localization. Intact tissue blocks were also immersed in drug solution, followed by sectioning. MALDI-MSI was then performed using a Thermo MALDI LTQ Orbitrap XL instrument to localize drug-distribution patterns. We propose three MALDI-MSI models measuring drug disposition that have been used to map the selected compounds within tissue compartments of tumors isolated from lung cancer patients.
Asunto(s)
Adenocarcinoma/química , Antagonistas Colinérgicos/farmacocinética , Neoplasias Pulmonares/química , Inhibidores de Proteínas Quinasas/farmacocinética , Quinazolinas/farmacocinética , Derivados de Escopolamina/farmacocinética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Animales , Transporte Biológico , Antagonistas Colinérgicos/farmacología , Clorhidrato de Erlotinib , Gefitinib , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Microtomía , Neoplasias Experimentales , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Derivados de Escopolamina/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Bromuro de Tiotropio , Técnicas de Cultivo de Tejidos , Microambiente TumoralRESUMEN
BACKGROUND: Low-density lipoprotein (LDL) particles accumulate in the arterial wall and become oxidized during atherogenesis, leading to the formation of atherosclerotic plaques. The major protein of the LDL particle, apolipoprotein B-100 (apoB-100), becomes fragmented during oxidation and a target for the immune system. METHODS: In this study we used in situ ellipsometry to monitor the adsorption of LDL to solid silica surfaces and the effects of oxidation on the structure of the adsorbed LDL layer. We additionally investigated the binding kinetics of two recombinant human antibodies with different specificities recognizing epitopes of apoB-100 in surface-bound native and CuCl2-oxidized LDL (oxLDL). The latter process was studied by adsorbing LDL and then adding the antibody and CuCl2 while continuously monitoring adsorbed amount and the thickness of the film. The molar ratios between the antibodies and surface-bound LDL and oxLDL were calculated from these data. RESULTS: Our results indicate that oxidation of surface-bound LDL induces swelling of the layer, accompanied by a slight desorption. We further found that both antibodies were able to recognize LDL and oxLDL in its adsorbed orientation. Quantitative information was obtained on the number of available binding sites on surface-bound LDL and oxLDL for these two antibodies. GENERAL SIGNIFICANCE: Using ellipsometry for real-time monitoring of adsorption, in situ oxidation of LDL and binding of specific recombinant antibodies to surface-bound LDL, will open up possibilities to map different conformations and orientations of LDL in the adsorbed state.
Asunto(s)
Anticuerpos/química , Lipoproteínas LDL/química , Dióxido de Silicio/química , Adsorción , Anticuerpos/genética , Anticuerpos/metabolismo , Apolipoproteína B-100/inmunología , Cobre/química , Cobre/metabolismo , Epítopos/inmunología , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacocinética , Malondialdehído/química , Malondialdehído/metabolismo , Oxidación-Reducción , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Refractometría/métodos , Dióxido de Silicio/metabolismo , Propiedades de SuperficieRESUMEN
The Tn antigen (GalNAc α-O-Ser/Thr) is heterogeneously synthesized by a variety of tumors and contains an epitope defined by lectins and antibodies as a cluster of αGalNAc carbohydrates synthesized within a peptide sequence, which is rich in serine and/or threonine. The Tn antigen has been utilized as a target in vaccine experiments and also used as a biomarker for prognosis of different cancer forms. In this paper, we present a new monoclonal antibody, GOD3-2C4, with the clear hallmarks of an anti-Tn antibody. It was generated through somatic cell hybridization after immunization of a mouse with a tumor cell line and a Tn carrying mucin. The antibody recognizes synthetic Tn antigen and binds breast, colon, lung, ovarian and pancreas cancer. The GOD3-2C4 antibody has antibody-dependent cellular cytotoxicity activity against Jurkat cells in vitro, and for the first time, it can be shown that an anti-Tn antibody has a significant in vivo effect on a human cancer cell line grown as a xenograft in severe combined immunodeficiency mice.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Antineoplásicos/farmacología , Inmunoglobulina G/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Antineoplásicos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inmunoglobulina G/farmacología , Ratones , Relación Estructura-ActividadRESUMEN
BACKGROUND: Phthalates may pose a risk for perinatal developmental effects. An important question relates to the choice of suitable biological matrices for assessing exposure during this period. OBJECTIVES: This study was designed to measure the concentrations of phthalate diesters or their metabolites in breast milk, blood or serum, and urine and to evaluate their suitability for assessing perinatal exposure to phthalates. METHODS: In 2001, 2-3 weeks after delivery, 42 Swedish primipara provided breast milk, blood, and urine samples at home. Special care was taken to minimize contamination with phthalates (e.g., use of a special breast milk pump, heat treatment of glassware and needles, addition of phosphoric acid). RESULTS: Phthalate diesters and metabolites in milk and blood or serum, if detected, were present at concentrations close to the limit of detection. By contrast, most phthalate metabolites were detectable in urine at concentrations comparable to those from the general population in the United States and in Germany. No correlations existed between urine concentrations and those found in milk or blood/serum for single phthalate metabolites. Our data are at odds with a previous study documenting frequent detection and comparatively high concentrations of phthalate metabolites in Finnish and Danish mothers' milk. CONCLUSIONS: Concentrations of phthalate metabolites in urine are more informative than those in milk or serum. Furthermore, collection of milk or blood may be associated with discomfort and potential technical problems such as contamination (unless oxidative metabolites are measured). Although urine is a suitable matrix for health-related phthalate monitoring, urinary concentrations in nursing mothers cannot be used to estimate exposure to phthalates through milk ingestion by breast-fed infants.
Asunto(s)
Ésteres/análisis , Exposición Materna , Leche Humana/química , Ácidos Ftálicos/análisis , Biomarcadores/análisis , Ésteres/sangre , Ésteres/orina , Estudios de Factibilidad , Femenino , Humanos , Recién Nacido , Ácidos Ftálicos/sangre , Ácidos Ftálicos/orinaRESUMEN
Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Aterosclerosis/metabolismo , Productos Finales de Glicación Avanzada/inmunología , Lisina/análogos & derivados , Neoplasias Pancreáticas/metabolismo , Animales , Mapeo Epitopo , Femenino , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Lisina/inmunología , Lisina/metabolismo , Ratones , Ratones Endogámicos BALB C , Análisis por Matrices de ProteínasRESUMEN
OBJECTIVE: The immune system plays an important regulatory role in the development of atherosclerotic plaques and neointima formation following various types of angioplasty. In the present study we investigated the effect of antibodies against aldehyde-modified apolipoprotein B-100 (apoB-100), a component of oxidized LDL, on atherosclerosis and response to arterial injury in mice. METHODS: The ability of a high affinity human recombinant antibody (2D03), specific for malondialdehyde-modified apoB-100, to influence formation of atherosclerosis as well as remodelling and neointima formation after a collar-induced injury of the carotid artery was studied in LDL receptor(-/-) mice over-expressing human apoB-100. RESULTS: The antibody recognized epitopes present in mouse plasma and reduced the plasma level of oxidized LDL by 34%. Antibody treatment inhibited injury-induced restrictive vascular remodelling but did not influence the size of the neointima. Atherosclerosis in the uninjured contra lateral carotid artery was determined by computerized image analysis and the mean plaque area in animals given control IgG1 was 7608+/-10,336 micro m(2). In contrast, essentially no plaques were present in animals treated with the 2D03 antibody (397+/-235 micro m(2), P<0.01 versus control IgG1). CONCLUSIONS: Treatment with antibodies against aldehyde-modified apoB-100 dramatically reduces atherosclerosis and inhibits restrictive vascular remodelling in mice expressing human apoB-100.
Asunto(s)
Anticuerpos/uso terapéutico , Apolipoproteína B-100/genética , Apolipoproteína B-100/inmunología , Enfermedades de las Arterias Carótidas/terapia , Receptores de LDL/deficiencia , Aldehídos , Animales , Arterias Carótidas/fisiología , Humanos , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de LDL/inmunología , Proteínas Recombinantes/uso terapéuticoRESUMEN
OBJECTIVE: Immune complexes containing oxidatively modified low-density lipoprotein (oxLDL) particles are deposited in human atherosclerotic lesions during atherogenesis. Here we studied whether OxLDL-IgG immune complexes (OxLDL-IgG ICs) affect survival of human monocytes. METHODS AND RESULTS: As demonstrated by light microscopy, and analysis of cell proliferation, caspase-3 activity, and DNA fragmentation, OxLDL-IgG ICs promoted survival of cultured human monocytes by decreasing their spontaneous apoptosis. OxLDL-IgG ICs induced a concentration-dependent production of the major monocyte growth factor, monocyte colony-stimulating factor (M-CSF), by the monocytes, but its inhibition was without effect on OxLDL-IgG IC-induced monocyte survival. Rather, OxLDL-IgG ICs induced rapid phosphorylation of Akt, suggesting a direct anti-apoptotic effect mediated by cross-linking of Fcgamma receptors. Experiments with receptor blocking antibodies revealed that the OxLDL-IgG IC-induced monocyte survival was mediated by Fcgamma receptor I. CONCLUSIONS: The results show that OxLDL-IgG ICs promote survival of monocytes by cross-linking Fcgamma receptor I and activating Akt-dependent survival signaling. The results reveal a novel mechanism by which an immune reaction toward oxLDL can play a role in the accumulation of macrophages in human atherosclerotic lesions.
Asunto(s)
Aterosclerosis/inmunología , Inmunoglobulina G/inmunología , Lipoproteínas LDL/inmunología , Monocitos/citología , Monocitos/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Apoptosis/inmunología , Aterosclerosis/metabolismo , Supervivencia Celular/inmunología , Células Cultivadas , Reactivos de Enlaces Cruzados/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Lipoproteínas LDL/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/inmunología , Monocitos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de IgG/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Biomolecular recognition is often characterised by low affinity where many weak interactions work either alone or in concert, resulting in an inherent dynamic situation. For example the well-studied weak binding of cell-cell interactions is predominantly based on a range of carbohydrates that interact with numerous (protein) ligands. Finding appropriate binders to these carbohydrate structures may pave the way for new analytical strategies based on low affinity, and recombinant antibody technology is a promising approach to the development of such reagents. We have in the present study characterised two low affinity human single chain antibody fragments (scFv) by surface plasmon resonance for use in such applications. The two clones, LeX1 and sLeX10, had been selected from a naive phage display library against Lewis x (Le(x)) and sialyl Le(x) (sLe(x)), respectively. Both LeX1 and sLeX10 showed low affinity, with K(D) values of 3.5+/-0.7 x 10(-5) M for Le(x) and 2.6+/-0.7 x 10(-5) M for sLe(x), respectively. Kinetic studies revealed the scFvs to be associated with fast dissociation rates, with Kd values higher than 0.1 s(-1) for both LeX1 and sLeX10. Apart from the Lewis structures Le(x) and sLe(x), we investigated the conformational isomers Lewis a and sialyl-Lewis a together with the monosaccharide units of the Lewis structures, and both scFvs showed high specificity for their respective carbohydrate. Taking these observations together we have demonstrated that scFv with fast reaction kinetics and low affinity have the necessary characteristics for further development as specific tools in analytical strategies, e.g. differentiation of cells based on the various configurations of carbohydrate epitopes.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Antígeno Lewis X/análisis , Oligosacáridos/análisis , Biblioteca de Péptidos , Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Escherichia coli/genética , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/aislamiento & purificación , Cinética , Antígeno Lewis X/inmunología , Oligosacáridos/inmunología , Antígeno Sialil Lewis X , Resonancia por Plasmón de SuperficieRESUMEN
We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.
Asunto(s)
Biblioteca de Péptidos , Análisis por Matrices de Proteínas , Anticuerpos de Cadena Única/análisis , Secuencia de Aminoácidos , Animales , Reacciones Antígeno-Anticuerpo , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/inmunología , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/aislamiento & purificaciónRESUMEN
A high frequency of IgA1-positive tumour cells was found in tissue micro-arrays of oesophagus, colon, testis, lung, breast, bladder and ovarian cancer. IgA1 was observed in the cytoplasm and the plasma membrane. A correlation was found between intra-tumour IgA1 and poor overall survival in a large cohort of bladder cancer patients (n = 99, p = 0.011, log-rank test). The number of IgA1-positive tumour cells was also found to be higher in female than male bladder cancer patients. The presence of IgA1 was confirmed in formalin-fixed paraffin-embedded ovarian carcinoma samples using LC-MS/MS analysis. Uptake of IgA1 was also observed in breast cancer and melanoma cell lines when cultivated in the presence of serum from healthy individuals, indicating a possible origin of the IgA1 antibodies in cancer cells.
RESUMEN
Cytokeratin 20 (CK20) is a well-established marker for colon epithelium. Herein, we suggest that CK20 is a biomarker for detecting circulating tumor cells (CTCs) in patients with metastatic colorectal cancer. Blood specimens (7.5 mL) were collected during surgery after liver mobilization from 25 patients with colorectal cancer. The FDA approved CellSearch™ system and two panels of antibodies against cytokeratins, cytokeratin 8, 18 and 19 (CK8/18/19) and CK8/18/19/20, were used for the detection of CTCs. All the patients' samples were processed using the anti-CK8/18/19 panel. The number of detected CTCs was low, 52% of the patients lacked CTCs and 40% had ≤ 2 CTCs/7.5 mL blood. Nine of the patients' blood samples were processed with both antibody panels. The detection rate of CTCs was significantly higher using the anti-CK8/18/19/20 panel compared with the anti-CK8/18/19 panel, p-value 0.0078. Our data show that inclusion of CK20 as a biomarker efficiently improves the detection of CTCs in colorectal cancer patients. The finding in our study is of clinical importance since a new prognostic biomarker would provide an important tool in individual clinical decision-making for colorectal cancer patients.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias Colorrectales/sangre , Queratina-20/sangre , Células Neoplásicas Circulantes/metabolismo , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/patologíaRESUMEN
Malignant melanoma has the highest increase of incidence of malignancies in the western world. In early stages, front line therapy is surgical excision of the primary tumor. Metastatic disease has very limited possibilities for cure. Recently, several protein kinase inhibitors and immune modifiers have shown promising clinical results but drug resistance in metastasized melanoma remains a major problem. The need for routine clinical biomarkers to follow disease progression and treatment efficacy is high. The aim of the present study was to build a protein sequence database in metastatic melanoma, searching for novel, relevant biomarkers. Ten lymph node metastases (South-Swedish Malignant Melanoma Biobank) were subjected to global protein expression analysis using two proteomics approaches (with/without orthogonal fractionation). Fractionation produced higher numbers of protein identifications (4284). Combining both methods, 5326 unique proteins were identified (2641 proteins overlapping). Deep mining proteomics may contribute to the discovery of novel biomarkers for metastatic melanoma, for example dividing the samples into two metastatic melanoma "genomic subtypes", ("pigmentation" and "high immune") revealed several proteins showing differential levels of expression. In conclusion, the present study provides an initial version of a metastatic melanoma protein sequence database producing a total of more than 5000 unique protein identifications. The raw data have been deposited to the ProteomeXchange with identifiers PXD001724 and PXD001725.
Asunto(s)
Bases de Datos de Proteínas , Melanoma/metabolismo , Análisis de Secuencia de Proteína/métodos , Neoplasias Cutáneas/metabolismo , Bancos de Muestras Biológicas , Cromatografía Liquida , Biología Computacional , Minería de Datos , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Metástasis de la Neoplasia , Proteómica/métodos , Suecia , Espectrometría de Masas en Tándem , Melanoma Cutáneo MalignoRESUMEN
A method based on gas chromatography electron capture detection (GC-ECD) for the analysis of chlorinated paraffins (CPs) in biological samples has been investigated. The method includes photolytic destruction of halogenated aromatic compounds, such as PCBs, to eliminate some of the interferences in the analysis of CPs in environmental samples. Gel permeation chromatography was used to isolate CPs from the interfering components of Toxaphene and chlordane after the photolysis. GC-ECD gave a detection limit of 20 ng CPs/g fresh muscle tissue. The recovery of CPs from a spiked moose liver sample was estimated to 94%.
Asunto(s)
Cromatografía de Gases/métodos , Hidrocarburos Clorados/análisis , Hidrocarburos Clorados/química , Parafina/análogos & derivados , Animales , Clordano/aislamiento & purificación , Cromatografía en Gel , Ciervos/metabolismo , Hidrocarburos Clorados/efectos de la radiación , Hígado/química , Músculos/química , Parafina/efectos de la radiación , Fotólisis , Sensibilidad y Especificidad , Toxafeno/aislamiento & purificación , Rayos UltravioletaRESUMEN
The aim of this study was to use multi-radionuclide autoradiography to compare the different distributions of three radiolabelled tracers in an atherosclerotic mouse model. This method, along with immunohistochemistry, was applied to investigate the intra-aortic distribution of 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG), (131)I/(125)I labeled anti-oxidized Low Density Lipoprotein (oxLDL), and non-binding control antibodies. Aortas were isolated from a total of 12 apoB-100/LDL receptor deficient mice 73 h post injection of radioiodine-labeled anti-oxLDL and control antibody and 1 h post injection of (18)F-FDG. A solid-state real-time digital autoradiography system was used to image the slide mounted aortas. Contributions from each radionuclide were separated by half-life and emission energy and the aortas were subsequently stained with Oil Red O for plaque to aorta contrast ratios. Immunohistochemical staining was performed to detect anti-oxLDL and control antibody localization. Radiolabeled anti-oxLDL showed increased total activity uptake in the aorta over control antibody and immunohistochemical analysis of plaques indicated increased binding of the specific antibody compared to control. The intra-aortic activity distribution of the anti-oxLDL antibody was however very similar to that of the control antibody although both had higher atherosclerotic plaques to aorta wall ratios than (18)F-FDG. Given the right choice of radionuclides, multi-radionuclide digital autoradiography can be employed to compare several tracers ex vivo in the same animal. The distribution of anti-oxLDL antibodies did not significantly differ from the control antibody but it did appear to have a better plaque to aorta contrast at 73 h post injection than (18)F-FDG at 1 h post injection.
RESUMEN
The aim of this investigation was to assess the Ku70/Ku80 complex as a potential target for antibody imaging of prostate cancer. We evaluated the in vivo and ex vivo tumor targeting and biodistribution of the (111)In-labeled human internalizing antibody, INCA-X ((111)In-DTPA-INCA-X antibody), in NMRI-nude mice bearing human PC-3, PC-3M-Lu2 or DU145 xenografts. DTPA-conjugated, non-labeled antibody was pre-administered at different time-points followed by a single intravenous injection of (111)In-DTPA-INCA-X. At 48, 72 and 96 h post-injection, tissues were harvested, and the antibody distribution was determined by measuring radioactivity. Preclinical SPECT/CT imaging of mice with and without the predose was performed at 48 hours post-injection of labeled DTPA-INCA-X. Biodistribution of the labeled antibody showed enriched activity in tumor, spleen and liver. Animals pre-administered with DTPA-INCA-X showed increased tumor uptake and blood content of (111)In-DTPA-INCA-X with reduced splenic and liver uptake. The in vitro and in vivo data presented show that the (111)In-labeled INCA-X antibody is internalized into prostate cancer cells and by pre-administering non-labeled DTPA-INCA-X, we were able to significantly reduce the off target binding and increase the (111)In-DTPA-INCA-X mAb uptake in PC-3, PC-3M-Lu2 and DU145 xenografts. The results are encouraging and identifying the Ku70/Ku80 antigen as a target is worth further investigation for functional imaging of prostate cancer.