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1.
Int J Mol Sci ; 25(4)2024 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-38396869

RESUMEN

Overhydration (OH) is a prevalent medical problem that occurs in patients with kidney failure, but a specific marker has still not been found. Patients requiring kidney replacement therapy suffer from a water imbalance, which is correlated with mortality rates in this population. Currently, clinicians employ techniques such as bioimpedance spectroscopy (BIS) and ultrasound (USG) markers of overhydration or markers of heart and kidney function, namely NT-pro-BNP, GFR, or creatinine levels. New serum markers, including but not limited to Ca-125, galectin-3 (Gal-3), adrenomedullin (AMD), and urocortin-2 (UCN-2), are presently under research and have displayed promising results. Ca-125, which is a protein mainly used in ovarian cancer diagnoses, holds great potential to become an OH marker. It is currently being investigated by cardiologists as it corresponds to the volume status in heart failure (HF) and ventricular hypertrophy, which are also associated with OH. The need to ascertain a more precise marker of overhydration is urgent mainly because physical examinations are exceptionally inaccurate. The signs and symptoms of overhydration, such as edema or a gradual increase in body mass, are not always present, notably in patients with chronic kidney disease. Metabolic disruptions and cachexia can give a false picture of the hydration status. This review paper summarizes the existing knowledge on the assessment of a patient's hydration status, focusing specifically on kidney diseases and the role of Ca-125.


Asunto(s)
Antígeno Ca-125 , Fallo Renal Crónico , Insuficiencia Renal Crónica , Intoxicación por Agua , Humanos , Biomarcadores , Fallo Renal Crónico/complicaciones , Diálisis Renal/efectos adversos , Insuficiencia Renal Crónica/complicaciones , Intoxicación por Agua/diagnóstico , Antígeno Ca-125/sangre , Antígeno Ca-125/química
2.
Proteins ; 90(5): 1210-1218, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35037700

RESUMEN

MUC16 is a membrane bound glycoprotein involved in the progression and metastasis of pancreatic and ovarian cancer. The protein is shed into the serum and the resulting cancer antigen 125 (CA125) can be detected by immunoassays. The CA125 epitope is used for monitoring ovarian cancer treatment progression, and has emerged as a potential target for antibody mediated immunotherapy. The extracellular tandem repeat domain of the protein is composed of repeating segments of heavily glycosylated sequence intermixed with homologous SEA (Sperm protein, Enterokinase and Agrin) domains. Here we report the purification and the first X-ray structure of a human MUC16 SEA domain. The structure was solved by molecular replacement using a Rosetta generated structure as a search model. The SEA domain reacted with three different MUC16 therapeutic antibodies, confirming that the CA125 epitope is localized to the SEA domain. The structure revealed a canonical ferredoxin-like fold, and contained a conserved disulfide bond. Analysis of the relative solvent accessibility of side chains within the SEA domain clarified the assignment of N-linked and O-linked glycosylation sites within the domain. A model of the glycosylated SEA domain revealed two major accessible faces, which likely represent the binding sites of CA125 specific antibodies. The results presented here will serve to accelerate future work to understand the functional role of MUC16 SEA domains and antibody recognition of the CA125 epitope.


Asunto(s)
Antígeno Ca-125 , Proteínas de la Membrana , Neoplasias Ováricas , Anticuerpos , Biomarcadores de Tumor , Antígeno Ca-125/química , Antígeno Ca-125/metabolismo , Epítopos , Femenino , Humanos , Proteínas de la Membrana/química , Neoplasias Ováricas/metabolismo
3.
Anal Biochem ; 609: 113964, 2020 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-32979366

RESUMEN

Carcinoembryonic antigen (CEA) is one of the biomarkers most commonly used to determine tumor activity. In this work, a Surface Plasmon Resonance imaging (SPRi) immunosensor was developed. The immunosensor consists of a cysteamine linker attached to a gold chip and mouse monoclonal anti-CEA antibody bonded by the "EDC/NHS protocol". The formation of successive immunosensor layers was confirmed by AFM measurements. The concentration of the antibody was optimized. The linear response range of the developed immunosensor is between 0.40 and 20 ng mL-1, and it is suitable for CEA measurement in both blood cancer patients and healthy individuals. Only 3 µL of serum or plasma sample is required, and no preconcentration is used. The method has a precision of 2-16%, a recovery of 101-104% depending on CEA concentration, a detection limit of 0.12 ng mL-1 and a quantification limit of 0.40 ng mL-1. The method is selective (with respect to albumin, leptin, interleukin 6, metalloproteinase-1, metallopeptidase inhibitor 1 and CA 125/MUC16) and it was validated by comparison with the standard electrochemiluminescence method on a series of colorectal cancer blood samples.


Asunto(s)
Antígeno Carcinoembrionario/sangre , Resonancia por Plasmón de Superficie/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Biomarcadores de Tumor/sangre , Antígeno Ca-125/química , Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/diagnóstico , Humanos , Inmunoensayo , Leptina/química , Límite de Detección , Proteínas de la Membrana/química , Inhibidor Tisular de Metaloproteinasa-1/química
4.
Anal Chim Acta ; 1125: 41-49, 2020 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-32674779

RESUMEN

In clinical diagnosis of cancer, the monitoring of single tumor marker may result in many false and missed results, while simultaneous detection of multiple tumor markers should be more accuracy and effective. Here, we report a new strategy that salt-induced gold nanoparticles (AuNPs) aggregation lights up fluorescence of dual-color DNA-silver nanoclusters-aptamer (DNA-AgNCs-apta) for the simultaneous monitoring of carcinoembryonic antigen (CEA) and carbohydrate antigen 125 (CA125). The dual-color aptasensor system is composed of green-emitting DNA-AgNCs with CEA aptamer (gDNA1-AgNCs-apta1) and red-emitting DNA-AgNCs with CA125 aptamer (rDNA2-AgNCs-apta2) in the ratio of 1:1 in volume. Upon addition of AuNPs, gDNA1-AgNCs-apta1 and/or rDNA2-AgNCs-apta2 are flexibly adsorbed onto the surface of AuNPs by terminal aptamer(s), which prevents salt-induced AuNPs aggregation under high salt condition and results in fluorescence quenching based on surface plasmon enhanced energy transfer (SPEET). With the addition of CEA and/or CA125, the target(s) and corresponding aptamer(s) coordinate to form the complex, keeping DNA-AgNCs-apta(s) far away from the surface of AuNPs and making AuNPs aggregated in high salt medium. The AuNPs aggregation leads to the recovery of fluorescence signals of DNA-AgNCs-apta(s) due to weakened SPEET. Utilizing the fluorescence aptasensor system, the limit of detection of CEA and CA125 are as low as 7.5 pg·mL-1 and 0.015 U·mL-1, respectively. The proposed method can be applied to the selective and simultaneous determination of CEA and CA125 in human serum.


Asunto(s)
Biomarcadores de Tumor/sangre , Antígeno Ca-125/sangre , Antígeno Carcinoembrionario/sangre , ADN/química , Proteínas de la Membrana/sangre , Nanopartículas del Metal/química , Aptámeros de Nucleótidos/química , Biomarcadores de Tumor/química , Técnicas Biosensibles/métodos , Antígeno Ca-125/química , Antígeno Carcinoembrionario/química , Femenino , Fluorescencia , Oro/química , Humanos , Límite de Detección , Proteínas de la Membrana/química , Neoplasias Ováricas/sangre , Plata/química , Espectrometría de Fluorescencia
5.
Nucl Med Biol ; 86-87: 9-19, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32403071

RESUMEN

INTRODUCTION: Despite its limitations, CA125 remains the most widely used biomarker for the diagnosis and treatment monitoring of ovarian cancer. Targeting the unshed portion of serum biomarkers such as CA125/MUC16 may afford more specific imaging and targeting of MUC16-positive tumors in High Grade Serous Ovarian Cancer (HGSOC) patients. METHODS: Six monoclonal antibodies raised against the 58 amino acid sequence between the extracellular cleavage site and the transmembrane region of MUC16 were radiolabeled with [89Zr]Zr4+. The radioimmunoconjugates were evaluated in vitro for molar activities, target binding affinity, cellular internalization and serum stability. In vivo characterization was performed via longitudinal positron emission tomography (PET) imaging and ex vivo biodistribution studies in mice bearing subcutaneous xenografts of SKOV3 cells transfected with the proximal 114 amino-acids of MUC16 carboxy-terminus (SKOV3+). RESULTS: In vitro screening identified 9C9 and 4H11 as the lead antibody candidates based on their comparable binding affinities, serum stability and cellular internalization profiles. Despite an identical molecular footprint for binding to MUC16, [89Zr]Zr-DFO-4H11 yielded a more favorable in vivo radiopharmacologic profile. Furthermore, a humanized variant of 4H11 capable of binding MUC16 in vitro also yielded excellent in vivo profile in subcutaneous xenograft models of SKOV3+, OVCAR3 tumors and a patient-derived xenograft model representative of HGSOC. CONCLUSION: Radiopharmacologic screening of antibodies early during their development can provide crucial information pertinent to the in vitro characterization and in vivo pharmacokinetics. The favorable in vivo profile demonstrated by humanized 4H11 combined with the use of its murine predecessor for immunohistochemical staining of biopsied tumor tissues from HGSOC patients makes a unique pair of antibodies that is poised for clinical translation.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígeno Ca-125/química , Antígeno Ca-125/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Neoplasias Ováricas/inmunología , Investigación Biomédica Traslacional , Línea Celular Tumoral , Femenino , Humanos , Dominios Proteicos , Distribución Tisular
6.
Anal Chim Acta ; 1082: 126-135, 2019 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-31472701

RESUMEN

Considering the high incidence level and mortality rate of ovarian cancer, particularly among the European female population, the carbohydrate antigen 125 (CA-125) was selected as the protein target for this study for the development of a MIP-based biosensor. This work presents the development of molecular imprinting polymers (MIPs) on gold electrode surface for CA-125 biomarker recognition. The preparation of the CA-125 imprinting was obtained by electropolymerization of pyrrole (Py) monomer in a gold electrode using cyclic voltammetry (CV) in order to obtain highly selective materials with great molecular recognition capability. The quantification of CA-125 biomarker was made through the comparison of two methods: electrochemical (square wave voltammetry -SWV) and optical transduction (surface plasmon resonance -SPR). SWV has been widely used in biological molecules analysis since it is a fast and sensitive technique. In turn, SPR is a non-destructive optical technique that provides high-quality analytical data of CA-125 biomarker interactions with MIP. Several analytical parameters, such as sensitivity, linear response interval, and detection limit were determined to proceed to the performance evaluation of the electrochemical and optical transduction used in the development of the CA-125 biosensor. The biosensor based in the electrochemical transduction was the one that presented the best analytical parameters, yielding a good selectivity and a detection limit (LOD) of 0.01 U/mL, providing a linear concentration range between 0.01 and 500 U/mL. This electrochemical biosensor was selected for the study and it was successfully applied in the CA-125 analysis in artificial serum samples with recovery rates ranging from 91 to 105% with an average relative error of 5.8%.


Asunto(s)
Antígeno Ca-125/sangre , Técnicas Electroquímicas/métodos , Proteínas de la Membrana/sangre , Impresión Molecular , Resonancia por Plasmón de Superficie/métodos , Antígeno Ca-125/química , Técnicas Electroquímicas/instrumentación , Electrodos , Oro/química , Humanos , Límite de Detección , Proteínas de la Membrana/química , Polímeros/química , Pirroles/química
7.
Biosens Bioelectron ; 137: 72-81, 2019 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-31082647

RESUMEN

This work describes further developments into the self-powered and self-signalled biosensing system that merges photovoltaic cells, plastic antibodies and electrochromic cells into a single target. Herein, the plasmonic effect is introduced to improve the photoanode features of the photovoltaic cell, a dye sensitized solar cell (DSSC), and better electrocatalytic features are introduced in the electrode containing the sensing element. In brief, the DSSC had a counter-electrode of poly(3,4-ethylenedioxythiophene) on an FTO glass modified by a plastic antibody of 3,4-ethylenedioxythiophene and pyrrol. The photoanode had dye sensitized TiO2 modified with gold nanoparticles (AuNPs) to increase the cell efficiency, aiming to improve the sensitivity of the response of hybrid device for the target biomarker. The target biomarker was carcinoembryonic antigen (CEA). The response of the hybrid device evidenced a linear trend from 0.1 ng/mL to 10 µg/mL, with an anionic slope of 0.1431 per decade concentration. The response of the plastic antibody for CEA revealed great selectivity against other tumour markers (CA 15-3 or CA 125). The colour response of the electrochromic cell was also CEA concentration dependent and more sensitive when the hybrid device was set-up with a photoanode with AuNPs. A more intense blue colour was obtained when higher concentrations of CEA were present. Overall, this improved version of the self-powered and self-signalled set-up has zero-requirements and is particularly suitable for point-of-care analysis (POC). It is capable of screening CEA in real samples and differentiating clinical levels of interest. This concept opens new horizons into the current cancer screening approaches.


Asunto(s)
Técnicas Biosensibles , Antígeno Ca-125/aislamiento & purificación , Antígeno Carcinoembrionario/aislamiento & purificación , Mucina-1/aislamiento & purificación , Anticuerpos/química , Anticuerpos/inmunología , Antígeno Ca-125/química , Antígeno Carcinoembrionario/química , Técnicas Electroquímicas , Oro , Humanos , Límite de Detección , Nanopartículas del Metal/química , Mucina-1/química , Plásticos/química , Titanio/química
8.
Biosens Bioelectron ; 126: 301-307, 2019 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-30445305

RESUMEN

In this work, we developed benchtop and handheld Giant Magnetoresistive (GMR) biosensing systems that serve as platforms for detecting a wide variety of protein biomarkers for human diseases. System development included spintronic and nanomagnetic materials, biomolecular chemistry, electronic circuitry, analog and digital signal processing, firmware programming, user interface programming on both PC and Android smartphone, communications over both USB and Bluetooth, and mechanical integration. In this work, we demonstrated the benchtop GMR biosensing system in the context of ovarian cancer assay development. The prototype system delivered the required performance in terms of high-sensitivity multiplex assays in a portable format with enough flexibility to serve as a platform for ovarian cancer and many other diseases. We achieved multiplex detection of cancer antigen 125 (CA125 II), human epididymis protein 4 (HE4), and interleukin 6 (IL6), with limits of detection (LOD) as low as 3.7 U/mL, 7.4 pg/mL, and 7.4 pg/mL, respectively.


Asunto(s)
Biomarcadores de Tumor/aislamiento & purificación , Técnicas Biosensibles , Neoplasias Ováricas/diagnóstico , Teléfono Inteligente , Biomarcadores de Tumor/química , Antígeno Ca-125/química , Antígeno Ca-125/aislamiento & purificación , Femenino , Humanos , Interleucina-6/química , Interleucina-6/aislamiento & purificación , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Proteínas/química , Proteínas/aislamiento & purificación , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP
9.
Biosens Bioelectron ; 115: 77-82, 2018 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-29803103

RESUMEN

A dual-wavebands-resolved electrochemiluminescence (ECL) multiplexing immunoassay (MIA) was developed for simultaneously detecting alpha fetoprotein antigen (AFP) in greenish waveband with CdSe550 (λmax = 550 nm) nanocrystals (NCs) and carbohydrate antigen 125 (CA125) in near-infrared waveband with CdTe790 (λmax = 790 nm) NCs via one-pot ECL reaction, in which dichroic mirror works as a key part to reflect ECL from CdSe550 to one photomultiplier-tube (PMT) and transmit ECL from CdTe790 to the other PMT for dual-wavebands-resolved assay. The proposed ECL-MIA strategy was capable of simultaneously determining AFP with linearly response from 5 pg/mL to 5 ng/mL and limit of detection at 1 pg/mL, and CA125 with linearly response from 5 mU/mL to 1 U/mL and limit of detection at 1 mU/mL, with desired specificity and without obvious energy-transfer between ECL tags. The dichroic mirror assistant ECL setup is easy-to-assemble and convenient for the popularization of color-resolved multiplexing ECL assay.


Asunto(s)
Técnicas Biosensibles , Antígeno Ca-125/aislamiento & purificación , Nanopartículas/química , alfa-Fetoproteínas/aislamiento & purificación , Antígeno Ca-125/química , Compuestos de Cadmio/química , Técnicas Electroquímicas , Humanos , Límite de Detección , Mediciones Luminiscentes , Compuestos de Selenio/química , alfa-Fetoproteínas/química
10.
Methods Enzymol ; 598: 169-196, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29306434

RESUMEN

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges associated with the analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and the speed of lectin microarrays. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, the CA125 protein purified from ovarian cancer cell lines, and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated "sample-to-answer" microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glycobiomarker development.


Asunto(s)
Glicómica/métodos , Lectinas/química , Microfluídica/métodos , Polisacáridos/química , Análisis por Matrices de Proteínas/métodos , Antígeno Ca-125/análisis , Antígeno Ca-125/química , Estudios de Factibilidad , Glicómica/instrumentación , Glicosilación , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Microfluídica/instrumentación , Análisis por Matrices de Proteínas/instrumentación
11.
Anal Chim Acta ; 997: 60-66, 2018 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-29149995

RESUMEN

Sensitivity enhancement and proteins adsorption are the common challenges faced in protein immunoassays. In this work, an ultrasensitive and protein-resistant label-free amperometric immunosening platform for carcinoma antigen-125 (CA125) based on redox polyaniline-polythionine hydrogel (PANI-PThi gel) was developed. The as-prepared hydrogel, which was facilely synthesized by electropolymerization, exhibited good conductivity and strong hydrophilicity while the sensitivity and specificity of the immunosensor can be enhanced. Furthermore, the as-prepared AuNPs functionalized PANI-PThi gel exhibited strong current signal and H2O2 electrocatalytic ability, which guaranteed a large current variable range. Based on these, the prepared immunosensor revealed a wide linear range from 0.0001 U mL-1 to 1 kU mL-1, a limit of detection of 0.00125 U mL-1 and its sensitivity was at least three-fold higher than previous works. More importantly, the prepared immunosensor exhibited excellent specificity, making it capable of assaying CA125 in human serum.


Asunto(s)
Biomarcadores de Tumor/análisis , Técnicas Biosensibles/métodos , Antígeno Ca-125/análisis , Técnicas para Inmunoenzimas/métodos , Compuestos de Anilina/química , Biomarcadores de Tumor/química , Antígeno Ca-125/química , Técnicas Electroquímicas , Oro/química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/síntesis química , Peróxido de Hidrógeno/química , Interacciones Hidrofóbicas e Hidrofílicas , Límite de Detección , Nanopartículas del Metal/química , Oxidación-Reducción , Fenotiazinas/química
12.
Glycobiology ; 27(10): 920-926, 2017 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-28673046

RESUMEN

MUC16 is a large transmembrane mucin expressed on the apical surfaces of the epithelium covering the ocular surface, respiratory system and female reproductive tract. The transmembrane mucin is overexpressed by ovarian carcinomas, it is one of the most frequently used diagnostic markers for the disease and it is considered a promising target for immunotherapeutic intervention. Immunodetection of the mucin has to date been through antibodies that recognize its exceptionally large ectodomain. Similar to other membrane anchored mucins, MUC16 has a short cytoplasmic tail (CT), but studies of the biological relevance of the C-terminal domain of MUC16 has been limited by lack of availability of monoclonal antibodies that recognize the native CT. Here, we report the development of a novel monoclonal antibody to the CT region of the molecule that recognizes native MUC16 and its enzymatically released CT region. The antibody is useful for immunoprecipitation of the released CT domain as demonstrated with the OVCAR3 ovarian cancer cell line and can be used for detailed cytolocalization in cells as well as in frozen sections of ocular surface and uterine epithelium.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Biomarcadores de Tumor/inmunología , Antígeno Ca-125/inmunología , Proteínas de la Membrana/inmunología , Animales , Antígeno Ca-125/química , Femenino , Células HeLa , Humanos , Proteínas de la Membrana/química , Ratones , Ratones Endogámicos BALB C , Dominios Proteicos
13.
ACS Chem Biol ; 12(8): 2085-2096, 2017 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-28617578

RESUMEN

Expression of the retained C-terminal extracellular portion of the ovarian cancer glycoprotein MUC16 induces transformation and tumor growth. However, the mechanisms of MUC16 oncogenesis related to glycosylation are not clearly defined. We establish that MUC16 oncogenic effects are mediated through MGAT5-dependent N-glycosylation of two specific asparagine sites within its 58 amino acid ectodomain. Oncogenic signaling from the C-terminal portion of MUC16 requires the presence of Galectin-3 and growth factor receptors colocalized on lipid rafts. These effects are blocked upon loss of either Galectin-3 expression or activity MGAT5. Using synthetic MUC16 glycopeptides, we developed novel N-glycosylation site directed monoclonal antibodies that block Galectin-3-mediated MUC16 interactions with cell surface signaling molecules. These antibodies inhibit invasion of ovarian cancer cells, directly blocking the in vivo growth of MUC16-bearing ovarian cancer xenografts, elucidating new therapeutic modalities.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno Ca-125/química , Carcinogénesis/efectos de los fármacos , Proteínas de la Membrana/química , Animales , Sitios de Unión , Antígeno Ca-125/genética , Antígeno Ca-125/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicosilación/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Neoplasias Ováricas/fisiopatología , Transducción de Señal
14.
Clin Chem ; 62(10): 1390-400, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27540033

RESUMEN

BACKGROUND: Measurement of serum cancer antigen 125 (CA125) is the standard approach for epithelial ovarian cancer (EOC) diagnostics and follow-up. However, the clinical specificity is not optimal because increased values are also detected in healthy controls and in benign diseases. CA125 is known to be differentially glycosylated in EOC, potentially offering a way to construct CA125 assays with improved cancer specificity. Our goal was to identify carbohydrate-reactive lectins for discriminating between CA125 originating from EOC and noncancerous sources. METHODS: CA125 from the OVCAR-3 cancer cell line, placental homogenate, and ascites fluid from patients with cirrhosis were captured on anti-CA125 antibody immobilized on microtitration wells. A panel of lectins, each coated onto fluorescent europium-chelate-doped 97-nm nanoparticles (Eu(+3)-NPs), was tested for detection of the immobilized CA125. Serum samples from high-grade serous EOC or patients with endometriosis and healthy controls were analyzed. RESULTS: By using macrophage galactose-type lectin (MGL)-coated Eu(+3)-NPs, an analytically sensitive CA125 assay (CA125(MGL)) was achieved that specifically recognized the CA125 isoform produced by EOC, whereas the recognition of CA125 from nonmalignant conditions was reduced. Serum CA125(MGL) measurement better discriminated patients with EOC from endometriosis compared to conventional immunoassay. The discrimination was particularly improved for marginally increased CA125 values and for earlier detection of EOC progression. CONCLUSIONS: The new CA125(MGL) assay concept could help reduce the false-positive rates of conventional CA125 immunoassays. The improved analytical specificity of this test approach is dependent on a discriminating lectin immobilized in large numbers on Eu(+3)-NPs, providing both an avidity effect and signal amplification.


Asunto(s)
Biomarcadores de Tumor/sangre , Antígeno Ca-125/sangre , Inmunoensayo/métodos , Lectinas/química , Nanopartículas/química , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Ováricas/sangre , Adulto , Biomarcadores de Tumor/química , Antígeno Ca-125/química , Carcinoma Epitelial de Ovario , Femenino , Humanos , Persona de Mediana Edad , Adulto Joven
15.
Methods ; 97: 58-68, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26542762

RESUMEN

Early identification of neoplastic diseases is essential to achieve timely therapeutic interventions and significantly reduce the mortality of patients. A well-known biomarker is the Cancer Antigen 125 (CA125) or 16 mucin (MUC 16), a glycoprotein of the human family of mucins, already used for the diagnostic and prognostic evaluation of ovarian cancer. Therefore, the detection of CA125 to now remains a promising tool in the early diagnosis of this tumor. In this paper, we describe the development of RNA aptamers that bind with high affinity the tumor antigen CA125. We performed eight cycles of selection against CA125 purified protein. The selected aptamers were cloned and sequenced and the binding properties of the most promising sequences were studied by Real Time PCR and Surface Plasmon Resonance (SPR) to evaluate their ability in targeting CA125 protein with perspective applications in aptamer-based bioassays.


Asunto(s)
Aptámeros de Nucleótidos/química , Antígeno Ca-125/química , Proteínas de la Membrana/química , Neoplasias Ováricas/diagnóstico , Técnicas Biosensibles , Detección Precoz del Cáncer , Femenino , Humanos , Proteínas Inmovilizadas/química , Secuencias Invertidas Repetidas , Unión Proteica , Técnica SELEX de Producción de Aptámeros
16.
Cancer Immunol Res ; 4(2): 157-64, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26589767

RESUMEN

High-grade epithelial ovarian cancer kills more women than any other gynecologic cancer and is rarely diagnosed at an early stage. We sought to identify tumor-associated antigens (TAA) as candidate diagnostic and/or immunotherapeutic targets by taking advantage of tumor autoantibody responses in individuals with ovarian cancer. Plasma-derived IgG from a pool of five patients with advanced ovarian cancer was subjected to iterative biopanning using a library of bacteriophage MS2 virus-like particles (MS2-VLPs) displaying diverse short random peptides. After two rounds of biopanning, we analyzed the selectant population of MS2-VLPs by Ion Torrent deep sequencing. One of the top 25 most abundant peptides identified (DISGTNTSRA) had sequence similarity to cancer antigen 125 (CA125/MUC16), a well-known ovarian cancer-associated antigen. Mice immunized with MS2-DISGTNTSRA generated antibodies that cross-reacted with purified soluble CA125 from ovarian cancer cells but not membrane-bound CA125, indicating that the DISGTNTSRA peptide was a CA125/MUC16 peptide mimic of soluble CA125. Preoperative ovarian cancer patient plasma (n = 100) was assessed for anti-DISGTNTSRA, anti-CA125, and CA125. Patients with normal CA125 (<35 IU/mL) at the time of diagnosis had significantly more antibodies to DISGTNTSRA and to CA125 than those patients who had high CA125 (>35 IU/mL). A statistically significant survival advantage was observed for patients who had either normal CA125 and/or higher concentrations of antibodies to CA125 at the time of diagnosis. These data show the feasibility of using deep sequence-coupled biopanning to identify TAA autoantibody responses from cancer patient plasma and suggest a possible antibody-mediated mechanism for low CA125 plasma concentrations in some ovarian cancer patients.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Formación de Anticuerpos/inmunología , Antígeno Ca-125/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunología , Animales , Autoanticuerpos/química , Autoanticuerpos/genética , Autoanticuerpos/inmunología , Antígeno Ca-125/química , Carcinoma Epitelial de Ovario , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Femenino , Humanos , Inmunoglobulina G/inmunología , Ratones , Estadificación de Neoplasias , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/inmunología , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Péptidos/química , Péptidos/genética , Modelos de Riesgos Proporcionales
17.
Biosens Bioelectron ; 78: 396-403, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26655179

RESUMEN

This paper addresses the question - Is EDC-NHS activated gold nanoparticles modified electrode surface the best available option for antibody immobilization for immunosensor fabrication? Is there any other alternative covalent immobilization strategy for orthogonal orientation of antibody, ensuring enhanced sensitivity of immunosensors? Does EDC-NHS activation of carboxyl functionalized nanoparticles surface really leads to orthogonal or directed immobilization of antibody? Gold nanoparticles synthesized using L-Asparagine as reducing and stabilization agent were employed for orthogonal immobilization of antibody for immunosensor fabrication. Anti-CA125 antibody was used as a model system for immunosensor fabrication. A comparative evaluation of immunosensors fabricated using L-Asparagine stabilized gold nanoparticles and citrate stabilized gold nanoparticles via different immobilization strategies/chemistries was done. The three strategies involved immobilization of Anti-CA125 antibody - (1) after EDC-NHS activation of citrate stabilized gold nanoparticles, (2) directly onto citrate stabilized gold nanoparticles and (3) directly onto L-Asparagine stabilized gold nanoparticles modified electrode surfaces. Comparative evaluation of Impedimetric response characteristics showed 2.5 times increase in sensitivity (349.36 Ω/(IU/mL)/cm(2)) in case of third strategy as compared to first (147.53 Ω/(IU/mL)/cm(2)) and twice that of second strategy (166.24 Ω/(IU/mL)/cm(2)). Additionally, an extended dynamic range of 0-750 IU/mL was observed while for others it was up to 500 IU/mL. Amino acid coated gold nanoparticles ensured orthogonal immobilization, lesser randomization, with 88% of active antibody available for antigen binding as opposed to other two strategies with less than 30% active antibody.


Asunto(s)
Anticuerpos Inmovilizados/química , Asparagina/química , Técnicas Biosensibles , Antígeno Ca-125/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Antígeno Ca-125/química , Ácido Cítrico/química , Oro/química , Proteínas de la Membrana/química , Nanopartículas del Metal/química
18.
Glycobiology ; 25(11): 1172-82, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26201951

RESUMEN

The MUC16 mucin is overexpressed and aberrantly glycosylated in ovarian carcinomas. Immunodetection of circulating MUC16 is one of the most used cancer biomarker assays, but existing antibodies to MUC16 fail to distinguish normal and aberrant cancer glycoforms. Although all antibodies react with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent MUC16 immunity. Here, we developed a MUC16 vaccine based on a 1.7TR (264 aa) expressed in Escherichia coli and in vitro enzymatically glycosylated to generate the aberrant cancer-associated glycoform Tn. This vaccine elicited a potent serum IgG response in mice and we identified two major immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry of ovarian benign and cancer lesions, 5E11 showed similar reactivity as traditional MUC16 antibodies, suggesting that the epitope is not efficiently glycosylated. The study provides a vaccine design and immunodominant MUC16 TR epitopes.


Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Antígeno Ca-125/inmunología , Epítopos/inmunología , Proteínas de la Membrana/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales de Origen Murino/química , Antígeno Ca-125/química , Células CHO , Cricetinae , Cricetulus , Epítopos/química , Femenino , Humanos , Proteínas de la Membrana/química , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular
19.
Sci Rep ; 5: 9759, 2015 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-26044153

RESUMEN

MUC16, precursor of the most widely used ovarian cancer biomarker CA125, is up regulated in multiple malignancies and is associated with poor prognosis. While the pro-tumorigenic and metastatic roles of MUC16 are ascribed to the cell-associated carboxyl-terminal MUC16 (MUC16-Cter), the exact biochemical nature of MUC16 cleavage generating MUC16-Cter has remained unknown. Using different lengths of dual-epitope (N-terminal FLAG- and C-terminal HA-Tag) tagged C-terminal MUC16 fragments, we demonstrate that MUC16 cleavage takes place in the juxta-membrane ectodomain stretch of twelve amino acids that generates a ~17 kDa cleaved product and is distinct from the predicted sites. This was further corroborated by domain swapping experiment. Further, the cleavage of MUC16 was found to take place in the Golgi/post-Golgi compartments and is dependent on the acidic pH in the secretory pathway. A similar pattern of ~17 kDa cleaved MUC16 was observed in multiple cell types eliminating the possibility of cell type specific phenomenon. MUC16-Cter translocates to the nucleus in a cleavage dependent manner and binds to the chromatin suggesting its involvement in regulation of gene expression. Taken together, we demonstrate for the first time the oft-predicted cleavage of MUC16 that is critical in designing successful therapeutic interventions based on MUC16.


Asunto(s)
Antígeno Ca-125/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Dominios y Motivos de Interacción de Proteínas , Animales , Antígeno Ca-125/química , Antígeno Ca-125/genética , Membrana Celular/metabolismo , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Espacio Intracelular/metabolismo , Elastasa de Leucocito/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Estabilidad Proteica , Transporte de Proteínas , Proteolisis , Transducción de Señal , Ubiquitinación
20.
PLoS One ; 10(5): e0126633, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25965947

RESUMEN

The CA125 antigen is found in the serum of many patients with serous ovarian cancer and has been widely used as a disease marker. CA125 has been shown to be an independent factor for clinical outcome in this disease. In The Cancer Genome Atlas ovarian cancer project, MUC16 expression levels are frequently increased, and the highest levels of MUC16 expression are linked to a significantly worse survival. To examine the biologic effect of the proximal portion of MUC16/CA125, NIH/3T3 (3T3) fibroblast cell lines were stably transfected with the carboxy elements of MUC16. As few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the AKT and ERK pathways. MUC16 transformation was associated with up-regulation of a number of metastases and invasion gene transcripts, including IL-1ß, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular "ectodomain" of MUC16. The biologic impact of MUC16 was also explored through the creation of a transgenic mouse model expressing 354 amino acids of the carboxy-terminal portion of MUC16 (MUC16c354). Under a CMV, early enhancer plus chicken ß actin promoter (CAG) MUC16c354 was well expressed in many organs, including the brain, colon, heart, kidney, liver, lung, ovary, and spleen. MUC16c354 transgenic animals appear to be viable, fertile, and have a normal lifespan. However, when crossed with p53-deficient mice, the MUC16c354:p53+/- progeny displayed a higher frequency of spontaneous tumor development compared to p53+/- mice alone. We conclude that the carboxy-terminal portion of the MUC16/CA125 protein is oncogenic in NIH/3T3 cells, increases invasive tumor properties, activates the AKT and ERK pathways, and contributes to the biologic properties of ovarian cancer.


Asunto(s)
Antígeno Ca-125/genética , Antígeno Ca-125/metabolismo , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Animales , Antígeno Ca-125/química , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/química , Ratones , Ratones Desnudos , Células 3T3 NIH , Invasividad Neoplásica , Neoplasias Experimentales , Neoplasias Ováricas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo
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