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1.
Front Immunol ; 10: 1820, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31428101

RESUMEN

Antibodies are well-known protein mediators of immunity. IgM is the primordial member and the neglected sibling of the later-evolved and more proficient IgG in regard to their therapeutic and diagnostic use. Serendipitously, however, we found a paradox: While murine IgM antibodies specific for guanosine triphosphate (GTP) were able to recognize native guanylyl antigens found in primate or rat muscle tissues by immunofluorescence assays (which mimicked the auto-antibodies from autoimmune patients to skeletal or smooth muscle), the murine and human IgG counterparts failed. The results were replicated in cell-free direct binding assays using small latex microspheres decorated densely with GTP. The IgG antibodies could bind, however, if GTP was presented more spaciously on larger particles or as a univalent hapten. Accordingly, oligomerization of GTP (30-mer) destroyed the binding of the IgG antibodies but enhanced that of the IgMs in inhibition ELISA. We reason that, contrary to current belief, IgM does not bind in a lock-and-key manner like IgG. We hypothesize that whereas the intact and rigid antigen-binding site of IgG hinders the antibody from docking with antigens that are obstructed, in IgM, the two component polypeptides of the antigen-binding site can dissociate from each other and navigate individually through obstacles like the ancestral single-polypeptide antibodies found in sharks and camelids, both components eventually re-grouping around the antigen. We further speculate that polyreactive IgMs, which enigmatically bind to more than one type of antigen, use the same modus operandi. These findings call for a re-look at the clinical potential of IgM antibodies particularly in specific areas of cancer therapy, tissue pathology and vaccine design, where IgG antibodies have failed due to target inaccessibility.


Asunto(s)
Anticuerpos/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Animales , Especificidad de Anticuerpos/inmunología , Línea Celular , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Ratones , Ratones Endogámicos BALB C , Vacunas/inmunología
2.
PLoS One ; 11(8): e0160412, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27494141

RESUMEN

We investigated whether circulating osteopontin (OPN) could be used as a biomarker for cervical cancer. We employed a monoclonal antibody (mAb 659) specific for the unique and intact thrombin-sensitive site in OPN using an inhibition ELISA. We found significantly higher levels of OPN in 33 cervical cancer patients in both the plasma (mean +/- SD, 612 +/- 106 ng/mL) and serum (424 +/- 121 ng/mL) compared to healthy subjects [409 +/- 56 ng/mL, from 31 plasma samples (P < 0.0001), and 314 +/- 98 ng/mL, from 32 serum samples (P = 0.0002), respectively]. Similar results were obtained when the plasma from a bigger group (147 individuals) of cervical cancer patients (560 +/- 211 ng/mL) were compared with the same plasma samples of the healthy individuals (P = 0.0014). More significantly, the OPN level was highest in stage III-IV disease (614 +/- 210 ng/mL, from 52 individuals; P = 0.0001) and least and non-discriminatory in stage I (473 +/- 110 ng/mL, from 40 individuals; P = 0.5318). No such discrimination was found when a mAb of a different specificity (mAb 446) was used in a similar inhibition ELISA to compare the two groups in the first study; a commercial capture ELISA also failed. The possibility that the target epitope recognized by the antibody probe in these assays was absent from the circulating OPN due to protein truncation was supported by gel fractionation of the OPN found in patients' plasma: 60-64 kDa fragments were found instead of the presumably full-length OPN (68 kDa) seen in healthy people. How these fragments are generated and what possible role they play in cancer biology remain interesting questions.


Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Osteopontina/metabolismo , Neoplasias del Cuello Uterino/sangre , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales/metabolismo , Biomarcadores de Tumor/sangre , Femenino , Humanos , Ratones , Persona de Mediana Edad , Osteopontina/genética , Osteopontina/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Trombina/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología
3.
PLoS One ; 7(11): e49586, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23166719

RESUMEN

Definitive diagnosis of infectious diseases, including food poisoning, requires culture and identification of the infectious agent. We described how antibodies could be used to shorten this cumbersome process. Specifically, we employed an anti-Salmonella lipopolysaccharide O12 monoclonal antibody in an epitope-inhibition 10-min test (TUBEX TP) to detect O12⁺Salmonella organisms directly from routine blood culture broths. The aim is to obviate the need to subculture the broth and subsequently identify the colonies. Thus, blood from 78 young outpatients suspected of having enteric fever was incubated in an enrichment broth, and after 2 or 4 days, broth samplings were examined by TUBEX TP as well as by conventional agar culture and identification. TUBEX TP was performed before the culture results. Eighteen isolates of S. Typhi (15 after 2 days) and 10 isolates of S. Paratyphi A (4 after 2 days) were obtained by conventional culture. Both these Salmonella serotypes, the main causes of enteric fever, share the O12 antigen. In all instances where either of these organisms was present (cultured), TUBEX TP was positive (score 4 [light blue]--to--score 10 [dark blue]; negative is 0 [pink-colored]) i.e. 100% sensitive. Identification of the specific Salmonella serotype in TUBEX-positive cases was achieved subsequently by conventional slide agglutination using appropriate polyclonal antisera against the various serotypes. Twelve Escherichia coli, 1 Alcaligenes spp. and 1 Enterobacter spp. were isolated. All of these cases, including all the 36 culture-negative broths, were TUBEX-negative i.e. TUBEX TP was 100% specific. In a separate study using known laboratory strains, TUBEX TF, which detects S. Typhi but not S. Paratyphi A via the O9 antigen, was found to efficiently complement TUBEX TP as a differential test. Thus, TUBEX TP and TUBEX TF are useful adjuncts to conventional culture because they can save considerable time (>2 days), costs and manpower.


Asunto(s)
Anticuerpos Monoclonales , Pruebas Inmunológicas/métodos , Antígenos O/inmunología , Salmonella enterica/inmunología , Fiebre Tifoidea/diagnóstico , Adolescente , Anticuerpos Monoclonales/inmunología , Niño , Preescolar , Humanos , Juego de Reactivos para Diagnóstico , Salmonella enterica/clasificación , Sensibilidad y Especificidad , Fiebre Tifoidea/inmunología , Fiebre Tifoidea/microbiología
4.
PLoS One ; 6(9): e24743, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21935450

RESUMEN

Rapid diagnostics can be accurate but, often, those based on antibody detection for infectious diseases are unwittingly underrated for various reasons. Herein, we described the development of a combined rapid test for two clinically-indistinguishable bacterial diseases, typhoid and paratyphoid A fever, the latter fast emerging as a global threat. By using monoclonal antibodies (mAbs) to bacterial antigens of known chemical structures as probes, we were able to dissect the antibody response in patients at the level of monosaccharides. Thus, a mAb specific for a common lipopolysaccharide antigen (O12) found in both the causative organisms was employed to semi-quantify the amounts of anti-O12 antibodies present in both types of patients in an epitope-inhibition particle-based (TUBEX) immunoassay. This colorimetric assay detected not only anti-O12 antibodies that were abundantly produced, but also, by steric hindrance, antibodies to an adjoining epitope (O9 or O2 in the typhoid or paratyphoid bacillus, respectively). Sensitivity and, particularly, reaction intensities, were significantly better than those obtained using an anti-O9 or anti-O2 mAb-probe in the examination of paired sera from 22 culture-confirmed typhoid patients (sensitivity, 81.8% vs 75.0%) or single sera from 36 culture-confirmed paratyphoid patients (52.8% vs 28.6), respectively. Importantly, sensitivity was better (97.1% for typhoid, 75.0% for paratyphoid) if allowance was made for the absence of relevant antibodies in certain specimens as determined by an independent, objective assay (ELISA)--such specimens might have been storage-denatured (especially the older paratyphoid samples) or procured from non-responders. Benchmarking against ELISA, which revealed high concordance between the two tests, was useful and more appropriate than comparing with culture methods as traditionally done, since antibody tests and culture target slightly different stages of these diseases. Paired sera analysis was insightful, revealing 64% of typhoid patients who had no change in antibody titer over 4-16 days, and 14% with no IgM-IgG class-switching.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos O/inmunología , Fiebre Paratifoidea/diagnóstico , Juego de Reactivos para Diagnóstico , Fiebre Tifoidea/diagnóstico , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Sensibilidad y Especificidad , Adulto Joven
5.
J Med Microbiol ; 57(Pt 11): 1349-1353, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18927411

RESUMEN

The TUBEX test for typhoid fever detects serum antibodies in a simple and rapid assay system based on the inhibition of binding between two types of reagent particles - magnetic particles coated with an antigen (Salmonella O9 LPS) and coloured indicator particles coated with an anti-O9 mAb. A magnet is used to separate the colour indicator particles bound to the magnetic particles from the unbound indicator particles. Specific colour changes following magnetic separation are indicative of antibodies in the patient's serum; however, because results are interpreted based on changes in the colour red, haemolytic or icteric specimens cannot be used. This study describes a simple modification of the protocol to accommodate such specimens, including whole blood. This involves the addition of a quick and simple washing step after mixing the specimen with the antigen-bound magnetic particles. This modification has the advantage of allowing larger sample volumes to be used, thus enhancing the assay sensitivity, and also enables cases considered to be borderline positive by the original method to be re-assessed.


Asunto(s)
Anticuerpos Antibacterianos/sangre , Salmonella typhi/inmunología , Fiebre Tifoidea/diagnóstico , Animales , Hemólisis , Humanos , Sueros Inmunes/inmunología , Ratones , Ratones Endogámicos BALB C
6.
Diagn Microbiol Infect Dis ; 62(2): 142-50, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18715736

RESUMEN

We described a 5-min colorimetric test for paratyphoid A fever, which detects anti-Salmonella O2 antibodies by inhibiting the binding between 2 types of reagent particles. This test (TUBEX-PA) is based on that (TUBEX-TF) used for typhoid fever, which detects anti-O9 antibodies. TUBEX-PA showed a sensitivity of 81.0% (47/58 culture-confirmed patients) to 93.3% (14/15) and was 98.1% (52/53) specific for healthy subjects. However, TUBEX-PA also detected 50% (7/14) to 81.8% (9/11) of typhoid patients, and conversely, TUBEX-TF detected 46.7% (7/15) to 73.3% (11/15) of paratyphoid A cases. This cross-detection could be abrogated in both tests by adding a blocker (heterologous antigen) to remove the antibodies responsible, which presumably bind to a common antigen (O12) located close to O2 and O9. The presence of anti-O12 antibodies in typhoid (9/12 or 75.0% sensitive) and paratyphoid A (22/33 or 66.7%) patients was demonstrated directly using a prototypic TUBEX test designed specifically to detect these antibodies. Thus, using TUBEX-PA and TUBEX-TF together can increase the diagnostic accuracy of detecting both typhoid and paratyphoid A fever, while the further use of differential tests allows possible immediate discrimination between these diseases.


Asunto(s)
Anticuerpos Antibacterianos/sangre , Colorimetría/métodos , Antígenos O/inmunología , Fiebre Paratifoidea/diagnóstico , Juego de Reactivos para Diagnóstico , Salmonella paratyphi A/inmunología , Adolescente , Adulto , Niño , Preescolar , Humanos , Inmunoensayo , Persona de Mediana Edad , Fiebre Paratifoidea/microbiología , Salmonella paratyphi A/clasificación , Serotipificación , Factores de Tiempo
7.
J Immunol ; 181(3): 2246-57, 2008 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-18641365

RESUMEN

It is puzzling how autoreactive B cells that escape self-tolerance mechanisms manage to produce Abs that target vital cellular processes without succumbing themselves to the potentially deleterious effects of these proteins. We report that censorship indeed exists at this level: when the Ab synthesis in the cell is up-regulated in IL-6-enriched environments (e.g., adjuvant-primed mouse peritoneum), the cell dies of the increased intracellular binding between the Ab and the cellular autoantigen. In the case in which telomerase is the autoantigen, mouse hybridoma cells synthesizing such an autoantibody, which appeared to grow well in culture, could not grow in syngeneic BALB/c mice to form ascites, but grew nevertheless in athymic siblings. Culture experiments demonstrated that peritoneal cell-derived IL-6 (and accessory factors) affected the growth and functions of the hybridoma cells, including the induction of mitochondria-based apoptosis. Electron microscopy revealed an abundance of Abs in the nuclear chromatin of IL-6-stimulated cells, presumably piggy-backed there by telomerase from the cytosol. This nuclear presence was confirmed by light microscopy analysis of isolated nuclei. In two other cases, hybridoma cells synthesizing an autoantibody to GTP or osteopontin also showed similar growth inhibition in vivo. In all cases, Ab function was crucial to the demise of the cells. Thus, autoreactive cells, which synthesize autoantibodies to certain intracellular Ags, live delicately between life and death depending on the cytokine microenvironment. Paradoxically, IL-6, which is normally growth-potentiating for B cells, is proapoptotic for these cells. The findings reveal potential strategies and targets for immunotherapy.


Asunto(s)
Apoptosis/inmunología , Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Animales , Especificidad de Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Ascitis/genética , Ascitis/inmunología , Autoanticuerpos/genética , Autoanticuerpos/metabolismo , Secuencia de Bases , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Hibridomas , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Interleucina-6/farmacología , Ratones , Microscopía Inmunoelectrónica , Mitocondrias/inmunología , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Telomerasa/inmunología , Telomerasa/metabolismo
8.
J Med Microbiol ; 57(Pt 3): 316-323, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18287294

RESUMEN

TUBEX (IDL Biotech) is a 5 min semiquantitative colorimetric test for typhoid fever, a widely endemic disease. TUBEX detects anti-Salmonella O9 antibodies from a patient's serum by the ability of these antibodies to inhibit the binding between an indicator antibody-bound particle and a magnetic antigen-bound particle. Herein, we report that TUBEX could also be used to specifically detect soluble O9 lipopolysaccharide in antigen-spiked buffer by the ability of the antigen to inhibit the same binding between the particles. Sensitivity of antigen detection was improved (8-31 mug ml(-1)) by using a modified protocol in which the test sample was mixed with the indicator particles first, rather than with the magnetic particles as for antibody detection. The antigen was also detectable in spiked serum and urine samples, albeit less well (2-4-fold) than in buffer generally. However, no antigen was detected from six typhoid sera examined, all of which had anti-O9 antibodies. In addition, whole organisms of Salmonella Typhi (15 strains) and Salmonella Enteritidis (6 strains) (both O9(+) Salmonella), grown in simulated blood broths or on MacConkey agar, were also detectable by TUBEX when suspended at >9 x 10(8) organisms ml(-1). Expectedly, Salmonella Paratyphi A (7 strains), Salmonella Typhimurium (1 strain) and Escherichia coli (2 strains) were negative in the test. Thus, the same TUBEX kit may be used in several ways both serologically and microbiologically for the rapid diagnosis of typhoid fever. However, validation of the newer applications will require the systematic examination of real patient and laboratory materials.


Asunto(s)
Anticuerpos Antibacterianos/sangre , Lipopolisacáridos/análisis , Antígenos O/análisis , Juego de Reactivos para Diagnóstico , Salmonella typhi/inmunología , Salmonella typhi/aislamiento & purificación , Especificidad de Anticuerpos , Humanos , Lipopolisacáridos/sangre , Lipopolisacáridos/inmunología , Lipopolisacáridos/orina , Antígenos O/sangre , Antígenos O/inmunología , Antígenos O/orina , Salmonella enteritidis/inmunología , Salmonella enteritidis/aislamiento & purificación , Sensibilidad y Especificidad , Fiebre Tifoidea/diagnóstico , Fiebre Tifoidea/microbiología , Orina/microbiología
9.
Diagn Microbiol Infect Dis ; 58(3): 275-81, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17350203

RESUMEN

Typhoid remains a global public health problem, and quick accurate immunodiagnosis is needed. Here, we examined the performance of the 5-min TUBEX O9-antibody detection kit in 243 outpatients (mostly children and infants) in their first week of fever and 57 healthy subjects in the Bangladesh community. Based on culture results, TUBEX was 91.2% (31/34) sensitive and 82.3% (172/209) specific in febrile subjects. However, specificity was better in nonfebrile healthy subjects (89.5%, 51/57) or in febrile individuals who serologically had dengue fever (90.5%, 57/63), suggesting that some culture-negative febrile individuals could be truly typhoidal. These individuals were also positive in an anti-crude O9 enzyme-linked immunosorbent assay (ELISA) and the Widal test. Regression analysis of the TUBEX and ELISA results showed good concordance between them, better with the combined IgM-IgG ELISA than with IgM alone, suggesting that TUBEX detects IgM antibodies not necessarily by themselves, as previously reported, but with the help of IgG antibodies.


Asunto(s)
Anticuerpos Antibacterianos/sangre , Fiebre Tifoidea/diagnóstico , Adolescente , Adulto , Bangladesh , Niño , Preescolar , Dengue/complicaciones , Enfermedades Endémicas , Ensayo de Inmunoadsorción Enzimática , Humanos , Análisis de Regresión , Sensibilidad y Especificidad , Pruebas Serológicas/métodos , Estadística como Asunto , Fiebre Tifoidea/inmunología
10.
J Immunol Methods ; 321(1-2): 152-63, 2007 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-17331532

RESUMEN

Bacterially-produced antibody fragments, such as single-chain Fv (scFv) which comprises the variable regions of the light (VL) and heavy (VH) chains joined together by a short flexible linker, are useful as diagnostic and therapeutic agents. We previously constructed a scFv fragment from a hybridoma antibody (Mab2) but it unexpectedly lacked the unique carrier specificity of the native antibody. Thus, it bound indiscriminately to various phosphorylcholine (PC)-associated antigens, whereas the hybridoma antibody recognized the PC epitope only in the context of the immunizing antigen. Here, we investigated whether the problem was linker-related by changing the linker composition or by deleting it, but these attempts proved futile. Instead, we have constructed a recombinant Fab fragment of the antibody in bacteria that was carrier-specific. This suggests that constant regions are required for the carrier specificity, which presumably helps to mould the fine structure of the antibody combining site or in stabilizing such a structure. Consistent with this global effect is the finding that replacing specific residues in VH with germ-line residues, namely, VH49 glycine and VH30 threonine, both thought previously to be important for the carrier specificity, had no effect on the carrier specificity of the recombinant Fab.


Asunto(s)
Anticuerpos Antihelmínticos/química , Antígenos Helmínticos/inmunología , Haptenos/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fosforilcolina/inmunología , Trichinella spiralis/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/genética , Anticuerpos Antihelmínticos/metabolismo , Especificidad de Anticuerpos , Antígenos Helmínticos/metabolismo , Sitios de Unión de Anticuerpos , Glicina/química , Haptenos/metabolismo , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/química , Datos de Secuencia Molecular , Mutación , Fosforilcolina/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Treonina/química , Trichinella spiralis/metabolismo
11.
Immunol Lett ; 103(1): 17-26, 2006 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-16325269

RESUMEN

Accumulating evidence is emerging that B lymphocytes and autoantibodies are critical in the development of autoimmune disease. Even in certain disorders initially thought to be T cell-mediated, these immune components are now considered key players in the disease pathogenesis, and new autoantibody specificities have been added to the growing list of targets including cell surface receptors and ion channels that may be involved in a variety of neuropsychiatric and cardiovascular disorders. Studies of autoantibodies penetrating living cells suggest a dosage effect in generating a biological outcome in vivo. Some autoantibodies, such as those directed to double-stranded DNA, can bind to a variety of surrogate antigens located in different cellular compartments, and this may have different biological consequences. This polyreactive behavior could be related to their conformational diversity, or to the fact that the epitope recognized is distributed among other macromolecular antigens. In addition, recent studies revealed unsuspected mechanisms of pathogenesis, wherein autoantibodies have been described that can activate neuronal, endothelial cells or B lymphocytes. Other autoantibodies inactivate the target antigens, or exhibit a catalytic activity, releasing toxic oxygen products that may be linked to arthritic or atherosclerotic injury.


Asunto(s)
Autoanticuerpos , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/patología , Linfocitos B/inmunología , Humanos , Inmunidad Innata
12.
J Infect Dis ; 192(1): 166-9, 2005 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-15942907

RESUMEN

The maturation of virus-specific immunoglobulin G avidity during severe acute respiratory syndrome-associated coronavirus infection was examined. The avidity indices were low (mean +/- SD, 30.8% +/- 11.6%) among serum samples collected < or =50 days after fever onset, intermediate (mean +/- SD, 52.1% +/- 14.1%) among samples collected between days 51 and 90, and high (mean +/- SD, 78.1% +/- 8.0%) among samples collected after day 90. Avidity indices of 40% and 55% could be considered as cutoff values for determination of recent (< or =50 days) and past (>65 days) infection, respectively. Measurement of antibody avidity can be used to differentiate primary infection from reexposure and to assess humoral responses to candidate vaccines.


Asunto(s)
Anticuerpos Antivirales/fisiología , Afinidad de Anticuerpos/fisiología , Inmunoglobulina G/fisiología , Síndrome Respiratorio Agudo Grave/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Factores de Tiempo
13.
Histochem Cell Biol ; 123(1): 105-12, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15538612

RESUMEN

Telomerase is an important tumor marker but few antibodies to the enzyme have been described or used without difficulty in histochemical detection. Here we report specific detection of the enzyme in cell and tissue preparations using a new monoclonal antibody (mAb 476) and a new antigen-retrieval buffer (Enhancing buffer). When used to detect telomerase under normal immunostaining conditions in HL-60 cells or tissue sections of hepatocellular carcinoma or metastatic choriocarcinoma, unexpectedly, the antibody stained the cytoplasm rather than the nucleus. Nuclear staining, however, was revealed using the Enhancing buffer. Since other nuclear antigens in the HL-60 cell could be stained both ordinarily and in the Enhancing buffer, nuclear telomerase appears to be shrouded by the nuclear matrix or blocked by accessory proteins. The cytoplasmic activity seen in normal buffer but absent largely from the Enhancing buffer may be an artifact or the nascent, "naked" enzyme. With a known cytoplasmic antigen (proteinase-3) chosen arbitrarily for comparison, the antigenicity was found enhanced, instead, by the Enhancing buffer. The mode of action of the Enhancing buffer differs from that of microwave irradiation or the signal amplification (CSA) used by some investigators. The latter was found to enhance the cytoplasmic reactivity rather than the nuclear reactivity of mAb 476.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Biomarcadores de Tumor/inmunología , Núcleo Celular/enzimología , Telomerasa/inmunología , Animales , Especificidad de Anticuerpos , Tampones (Química) , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Núcleo Celular/inmunología , Coriocarcinoma/enzimología , Coriocarcinoma/secundario , Citoplasma/enzimología , Citoplasma/inmunología , Epítopos , Células HL-60 , Humanos , Inmunohistoquímica , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/secundario , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , ARN Mensajero , Telomerasa/genética
14.
J Med Virol ; 75(2): 181-4, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15602743

RESUMEN

A high throughput accurate assay for anti-SARS-CoV IgG detection is needed for large-scale epidemiological studies. The evaluation of a commercial recombinant nucleocapsid protein-based microtitre plate enzyme immunoassay, ELISARS is described. The results on 150 sera from SARS patients and 450 sera from non-SARS controls showed that this assay had a high level of sensitivity (96.2% for late serum samples) and specificity (97.8%). The performance and setup of this assay fulfills the requirement as a screening test for large-scale studies. A vast majority of SARS patients developed antibodies against the nucleocapsid protein. In some patients (10/45), a high level of anti-nucleocapsid antibody appeared very early in the course of the illness. In contrast, a minority (4 of 105 patients) never developed these antibodies. The implication of differences in antibody response to the nucleocapsid protein deserves further investigation.


Asunto(s)
Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/sangre , Proteínas de la Nucleocápside/inmunología , Proteínas Recombinantes/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Sensibilidad y Especificidad , Síndrome Respiratorio Agudo Grave/inmunología
15.
Biochem Biophys Res Commun ; 326(2): 268-73, 2005 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-15582573

RESUMEN

Human BRE, a death receptor-associating intracellular protein, attenuates apoptotic response of human and mouse tumor cell lines to death receptor stimuli in vitro. In this report, we addressed whether the in vitro antiapoptotic effect of BRE could impact on tumor growth in vivo. We have shown that the mouse Lewis lung carcinoma D122 stable transfectants of human BRE expression vector developed into local tumor significantly faster than the stable transfectants of empty vector and parental D122, in both the syngeneic C57BL/6 host and nude mice. In vitro growth of the BRE stable transfectants was, however, not accelerated. No significant difference in metastasis between the transfectants and the parental D122 was detected. Thus, overexpression of BRE promotes local tumor growth but not metastasis. We conclude that the enhanced tumor growth is more likely due to the antiapoptotic activity of BRE than any direct effect of the protein on cell proliferation.


Asunto(s)
Neoplasias/metabolismo , Neoplasias/patología , Proteínas del Tejido Nervioso/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Expresión Génica , Masculino , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias/genética , Proteínas del Tejido Nervioso/genética
16.
J Biol Chem ; 279(50): 52106-16, 2004 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-15465831

RESUMEN

BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-alpha-induced activation of NF-kappaB. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-alpha, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-alpha, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.


Asunto(s)
Apoptosis/fisiología , Proteínas del Tejido Nervioso/fisiología , Apoptosis/efectos de los fármacos , Secuencia de Bases , Núcleo Celular/metabolismo , Cicloheximida/farmacología , Citosol/metabolismo , Etopósido/farmacología , Expresión Génica , Células HeLa , Humanos , Células Jurkat , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Interferente Pequeño/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/metabolismo
17.
J Infect Dis ; 190(2): 379-86, 2004 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-15216476

RESUMEN

The recent outbreak of severe acute respiratory syndrome (SARS) provided an opportunity to study the antibody response of infected individuals to the causative virus, SARS coronavirus. We examined serum samples obtained from 46 patients with SARS, 40 patients with non-SARS pneumonia, and 38 healthy individuals, by use of Western blotting (WB), enzyme-linked immunoassay (ELISA), and immunofluorescence assay, using both native and bacterially produced antigens of the virus. We found a highly restricted, immunoglobulin G-dominated antibody response in patients with SARS, directed most frequently (89% by ELISA) and predominantly at the nucleocapsid. Almost all of the subjects without SARS had no antinucleocapsid antibodies. The spike protein was the next most frequently targeted, but only 63% of the patients (by ELISA) responded. Other targets of the response identified by use of WB included antigens of 80 and 60 kDa. Several nonstructural proteins cloned were not antigenic, and the culture-derived nucleocapsid appeared to be specifically degraded.


Asunto(s)
Anticuerpos Antivirales/sangre , Nucleocápside/inmunología , Síndrome Respiratorio Agudo Grave/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Adolescente , Adulto , Anciano , Antígenos Virales/inmunología , Western Blotting , Niño , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoglobulina G/sangre , Masculino , Glicoproteínas de Membrana/inmunología , Persona de Mediana Edad , Peso Molecular , Síndrome Respiratorio Agudo Grave/virología , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/inmunología
18.
Arthritis Rheum ; 50(5): 1533-8, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15146423

RESUMEN

OBJECTIVE: To investigate why the serum of a pediatric patient with systemic lupus erythematosus was persistently (>30 months) and strongly positive for antibodies to double-stranded DNA (dsDNA) as revealed by enzyme-linked immunosorbent assay (ELISA), but yielded negative results on the antinuclear antibody test (HEp-2 immunofluorescence [IF]). METHODS: The patient's antibodies were isolated on dsDNA and single-stranded DNA (ssDNA) supports, which were then examined by dsDNA ELISA and HEp-2 IF. Tests included the use of various inhibitors to determine the fine specificity of the antibodies. Other tests performed included immunoblotting, immunoprecipitation, Crithidia luciliae IF, and neutrophil IF. RESULTS: The antibodies isolated from the dsDNA and ssDNA supports were similar, in that they were of the IgG type, bound well in the dsDNA ELISA, and recognized a normally hidden nucleolar RNA antigen in HEp-2 cells. With both the dsDNA ELISA and nucleolar antigens, inhibition studies revealed that the epitope recognized was guanosine 5'-triphosphate (GTP). Binding of the antibodies was better to GTP than to guanosine 5'-monophosphate or cytidylyl (3'-5') guanosine, and, in turn, was better than to guanosine, while N7-methylated GTP was unreactive. The antibodies did not bind to dsDNA present in solution or in HEp-2 or Crithidia cells, but bound transfer RNA well and recognized a cytoplasmic RNA antigen in neutrophils. CONCLUSION: A new problem in dsDNA ELISA is revealed in the occurrence of a hitherto-unknown and unusual buckling of the insolubilized DNA molecule, which, absent in dsDNA found in solution or in whole cells, presumably creates gaps of single-strandedness in the molecule. A new antibody specific for GTP is described in this patient, which may be clinically important.


Asunto(s)
Anticuerpos Antinucleares/sangre , ADN/inmunología , Ensayo de Inmunoadsorción Enzimática/normas , Guanosina Trifosfato/inmunología , Lupus Eritematoso Sistémico/inmunología , Especificidad de Anticuerpos , Niño , ADN/metabolismo , Reacciones Falso Positivas , Femenino , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Solubilidad
19.
J Bone Miner Metab ; 22(2): 148-52, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-14999526

RESUMEN

Transforming growth factor-beta-1 (TGF-Beta(1)) has been implicated in bone mineral density (BMD) determination. We investigated the relationship between the TGF polymorphism, BMD, and vertebral fractures in 588 Chinese men and women. No association between TGF polymorphism and BMD was observed in postmenopausal women (aged 55-59 years), elderly men (aged 70-79 years), or elderly women (aged 70-79 years) at the hip, spine, or total body ( P >> 0.05 by two-way ANOVA). In all study groups, there was no effect of an interaction between TGF polymorphism and calcium intake on BMD ( P >> 0.05 for the interaction effects by two-way ANOVA). No statistical significant association was observed between TGF polymorphism and vertebral fracture in elderly men or women ( P >> 0.05 by the chi-square test), even though men of the TT and TC genotypes seem to have more vertebral fractures. Contrary to previous studies that found an association between BMD and TGF polymorphism in the Japanese, we found no association between TGF polymorphism and BMD of elderly Chinese men or women. This finding could result from different sampling methods between the previous and current studies and environmental factors and ethnic differences between the two populations.


Asunto(s)
Densidad Ósea , Osteoporosis/genética , Polimorfismo Genético , Factor de Crecimiento Transformador beta/genética , Anciano , Calcio de la Dieta , China , Femenino , Genotipo , Cadera/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/metabolismo , Radiografía , Fracturas de la Columna Vertebral , Columna Vertebral/diagnóstico por imagen , Estadística como Asunto , Factor de Crecimiento Transformador beta1
20.
J Immunol Methods ; 282(1-2): 83-91, 2003 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-14604543

RESUMEN

A serological test kit (TUBEX, IDL Biotech, Sweden) developed recently for the diagnosis of typhoid fever detects antibodies to the Salmonella enterica serovar Typhi lipopolysaccharide (LPS) O9 antigen. The antibodies are detected by their ability to inhibit the interaction between two types of reagent particles: (a). indicator latex microspheres sensitized with an anti-O9 monoclonal antibody, and (b). magnetic microspheres sensitized with S. typhi LPS. Following rapid mixing of the serum with these reagents and sedimentation of the magnetic particles by magnetic force, the concentration of indicator particles left in suspension provides a measure of the inhibition. Whereas it was previously assumed that both IgM and IgG antibodies could inhibit in the system, the present study reveals, surprisingly, that only the IgM antibodies do. It is not clear why IgG anti-O9 antibodies, both of mouse and human origin, do not inhibit, although these can bind to the LPS-sensitized magnetic particles as efficiently as the IgM antibodies. In addition, they can also inhibit very well in another detection system (ELISA) which uses a similar assay format and the same antibody and antigen reagents. Increasing the size of the LPS-sensitized microspheres made no difference; microscopic analysis of the TUBEX reaction mixture revealed that while the indicator particles bound abundantly to the IgG-aggregated LPS-sensitized particles, forming large clumps, these only formed a very light decoration on the IgM-aggregated particles. Thus, the TUBEX system is ideally suited for use in the diagnosis of infections as it allows IgM antibodies to be detected easily and rapidly from whole sera.


Asunto(s)
Anticuerpos Antibacterianos/análisis , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Antígenos O/inmunología , Juego de Reactivos para Diagnóstico , Salmonella typhi/inmunología , Fiebre Tifoidea/diagnóstico , Animales , Humanos , Inmunoensayo , Ratones
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