RESUMO
Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.
Assuntos
Imunidade Inata , Células-Tronco Pluripotentes/imunologia , Viroses/imunologia , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Interferons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Pluripotentes/virologia , Especificidade da EspécieRESUMO
BACKGROUND: To compare the analgesic efficacy and safety of preoperative, single-shot ultrasound-guided thoracic paravertebral block (TPVB), erector spinae plane block (ESB), and serratus anterior plane block (SAPB) in thoracotomy pain. DESIGN: A prospective, randomized study. SETTING: The cardiothoracic operating room and intensive care unit of a tertiary-care hospital in India. PARTICIPANTS: Ninety adult patients scheduled to undergo posterolateral thoracotomy for lung surgery under general anesthesia were recruited and randomized into 3 equal groups. INTERVENTIONS: Preoperatively, the patients received ultrasound-guided, single-shot nerve blocks within their respective groups, as follows: Erector spinae plane block in the ESB group, Thoracic paravertebral block in the TPVB group, and Serratus anterior plane block in the SAPB group. MEASUREMENTS AND RESULTS: The primary outcome measure, the visual analog scale (VAS) score, was recorded postoperatively in the intensive care unit at 0, 3, 6, 12, and 24 hours. The secondary outcome measures were the time to first rescue analgesic, total rescue opioid dose used, patient satisfaction at 24 hours, success of one-time attempt, and occurrence of adverse events. Data were statistically analyzed and a significant difference was found in the VAS score at all time points, the time to rescue analgesic and total opioid dosage, and patient satisfaction level (p < 0.05) among the groups with only 1 incidence of hypotension in the TPVB group. From post hoc analysis, ESB was found to have better analgesic efficacy compared with TPVB and SAPB. Serratus anterior plane block was found to be least efficacious and shortest acting among the three. CONCLUSION: The nerve blocks in decreasing order of analgesic efficacy in relieving post-thoracotomy pain would be ESB, TPVB, and SAPB.
Assuntos
Analgesia , Bloqueio Nervoso , Adulto , Humanos , Toracotomia/efeitos adversos , Analgésicos Opioides , Dor Pós-Operatória/diagnóstico , Dor Pós-Operatória/etiologia , Dor Pós-Operatória/prevenção & controle , Estudos Prospectivos , Ultrassonografia de Intervenção , Medição da Dor , Bloqueio Nervoso/efeitos adversos , PulmãoRESUMO
Induction of vast transcriptional programs is a central event of innate host responses to viral infections. Here we report a transcriptional program with potent antiviral activity, driven by E74-like ETS transcription factor 1 (ELF1). Using microscopy to quantify viral infection over time, we found that ELF1 inhibits eight diverse RNA and DNA viruses after multi-cycle replication. Elf1 deficiency results in enhanced susceptibility to influenza A virus infections in mice. ELF1 does not feed-forward to induce interferons, and ELF1's antiviral effect is not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNA-seq revealed that the ELF1 transcriptional program is distinct from interferon signatures. Thus, ELF1 provides an additional layer of the innate host response, independent from the action of type I interferons.
Assuntos
Antivirais/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Vírus da Influenza A/imunologia , Interferon Tipo I/farmacologia , Proteínas Nucleares/metabolismo , Infecções por Orthomyxoviridae/imunologia , Fatores de Transcrição/metabolismo , Replicação Viral/imunologia , Células A549 , Animais , Feminino , Humanos , Imunidade Inata , Vírus da Influenza A/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Fosforilação , Fator de Transcrição STAT1 , Transdução de Sinais , Fatores de Transcrição/genética , Replicação Viral/efeitos dos fármacosRESUMO
SEE STEPHAN ET AL DOI101093/AWW120 FOR A SCIENTIFIC COMMENTARY ON THIS WORK: Real world information is often abstract, dynamic and imprecise. Deciding if changes represent random fluctuations, or alterations in underlying contexts involve challenging probability estimations. Dysfunction may contribute to erroneous beliefs, such as delusions. Here we examined brain function during inferences about context change from noisy information. We examined cortical-subcortical circuitry engaging anterior and dorsolateral prefrontal cortex, and midbrain. We hypothesized that schizophrenia-related deficits in prefrontal function might overestimate context change probabilities, and that this more chaotic worldview may subsequently gain familiarity and be over-reinforced, with implications for delusions. We then examined these opposing information processing biases against less expected versus familiar information patterns in relation to genetic risk for schizophrenia in unaffected siblings. In one experiment, 17 patients with schizophrenia and 24 normal control subjects were presented in 3 T magnetic resonance imaging with numerical information varying noisily about a context integer, which occasionally shifted up or down. Subjects were to indicate when the inferred numerical context had changed. We fitted Bayesian models to estimate probabilities associated with change inferences. Dynamic causal models examined cortical-subcortical circuitry interactions at context change inference, and at subsequent reduced uncertainty. In a second experiment, genetic risk for schizophrenia associated with similar cortical-subcortical findings were explored in an independent sample of 36 normal control subjects and 35 unaffected siblings during processing of intuitive number sequences along the number line, or during the inverse, less familiar, sequence. In the first experiment, reduced Bayesian models fitting subject behaviour suggest that patients with schizophrenia overestimated context change probabilities. Here, patients engaged anterior prefrontal cortex relatively less than healthy controls, in part driven by reduced effective connectivity from dorsolateral prefrontal cortex to anterior prefrontal cortex. In processing subsequent information indicating reduced uncertainty of their predictions, patients engaged relatively increased mid-brain activation, driven in part by increased dorsolateral prefrontal cortex to midbrain connectivity. These dissociable reduced and exaggerated prefrontal and subcortical circuit functions were accentuated in patients with delusions. In the second experiment, analogous dissociable reduced anterior prefrontal cortex and exaggerated midbrain engagement occurred in unaffected siblings when processing less expected versus more familiar number sequences. In conclusion, patients overestimated ambiguous context change probabilities with relatively reduced anterior frontal engagement. Subsequent reduced uncertainty about contextual state appeared over-reinforced, potentially contributing to confirmation bias and a cascade of aberrant belief processing about a more chaotic world relevant to delusions. These opposing cortical-subcortical effects relate in part to genetic risk for schizophrenia, with analogous imbalances in neural processing of less expected versus familiar information patterns.
Assuntos
Conectoma/métodos , Delusões/fisiopatologia , Mesencéfalo/fisiopatologia , Rede Nervosa/fisiopatologia , Córtex Pré-Frontal/fisiopatologia , Esquizofrenia/fisiopatologia , Adulto , Antecipação Psicológica/fisiologia , Delusões/diagnóstico por imagem , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Mesencéfalo/diagnóstico por imagem , Esquizofrenia/diagnóstico por imagem , Esquizofrenia/genética , Irmãos , Pensamento/fisiologia , Incerteza , Adulto JovemRESUMO
Patients with estrogen receptor-positive breast cancer receive adjuvant endocrine therapies (ET) that delay relapse by targeting clinically undetectable micrometastatic deposits. Yet, up to 50% of patients relapse even decades after surgery through unknown mechanisms likely involving dormancy. To investigate genetic and transcriptional changes underlying tumor awakening, we analyzed late relapse patients and longitudinally profiled a rare cohort treated with long-term neoadjuvant ETs until progression. Next, we developed an in vitro evolutionary study to record the adaptive strategies of individual lineages in unperturbed parallel experiments. Our data demonstrate that ETs induce nongenetic cell state transitions into dormancy in a stochastic subset of cells via epigenetic reprogramming. Single lineages with divergent phenotypes awaken unpredictably in the absence of recurrent genetic alterations. Targeting the dormant epigenome shows promising activity against adapting cancer cells. Overall, this study uncovers the contribution of epigenetic adaptation to the evolution of resistance to ETs. SIGNIFICANCE: This study advances the understanding of therapy-induced dormancy with potential clinical implications for breast cancer. Estrogen receptor-positive breast cancer cells adapt to endocrine treatment by entering a dormant state characterized by strong heterochromatinization with no recurrent genetic changes. Targeting the epigenetic rewiring impairs the adaptation of cancer cells to ETs. See related commentary by Llinas-Bertran et al., p. 704. This article is featured in Selected Articles from This Issue, p. 695.
Assuntos
Neoplasias da Mama , Epigênese Genética , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Feminino , Recidiva Local de Neoplasia/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a â¼25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.
Assuntos
Precursores de RNA/genética , Splicing de RNA , RNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Biocatálise , Éxons/genética , Peso Molecular , Ligação Proteica , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Spliceossomos/genética , TemperaturaRESUMO
Neoadjuvant endocrine treatment (NET) associates to satisfactory rates of breast conservative surgery and conversions from inoperable to operable hormone receptor-positive (HR+)/HER2-negative breast cancer (BC), with less toxicities than neoadjuvant chemotherapy (NACT) and similar outcomes. Hence, it has been proposed as a logical alternative to NACT in patients with HR+/HER2- BC candidate to a neoadjuvant approach. Nevertheless, potential barriers to the widespread use of NET include the heterogeneous nature of patient response coupled with the long duration needed to achieve a clinical response. However, interest in NET has significantly increased in the last decade, owing to more in-depth investigation of several biomarkers for a more adequate patient selection and on-treatment benefit monitoring, such as PEPI score, Ki67 and genomic assays. This review is intended to describe the state-of-the-art regarding NET, its future perspectives and potential integration with molecular biomarkers for the optimal selection of patients, regimen and duration of (neo)adjuvant treatments.
Assuntos
Neoplasias da Mama , Terapia Neoadjuvante , Humanos , Feminino , Neoplasias da Mama/genética , Mastectomia , Quimioterapia Adjuvante , Receptor ErbB-2 , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
The yeast Bud31 protein, a Prp19 complex (NTC) member, aids spliceosome assembly and thus promotes efficient pre-mRNA splicing. The bud31 null cells show mild budding abnormalities at optimal growth temperatures and, at higher temperatures, have growth defects with aberrant budding. Here we have assessed cell cycle transitions which require Bud31. We find Bud31 facilitates passage through G1-S regulatory point (Start) but is not needed for G2-M transition or for exit from mitosis. To co-relate Bud31 functions in cell division with splicing, we studied the splicing status of transcripts that encode proteins involved in budding. We find Bud31 promotes efficient splicing of only some of these pre-mRNAs, for example, ARP2 and SRC1. Wild type cells have a long and a short isoform of SRC1 mRNA and protein, out of which the shorter mRNA splice variant is predominant. bud31Δ cells show inefficient SRC1 splicing and entirely lack the shorter SRC1 spliced mRNA isoform. Yeast PRP17, another NTC sub-complex member, is also required for G1-S and G2-M cell cycle transitions. We examined genetic interactions between BUD31 and PRP17. While both factors were needed for efficient cell cycle dependent gene expression, our data indicate that distinct pre-mRNAs depend on each of these non-essential splicing factors.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Fatores de Processamento de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
A filtration-based staining method has been developed for sensitive estimation of proteins in a batch of samples. It is based on focused absorption of an applied protein standard (or sample) and dye solution on nitrocellulose membrane through an aqueous network of capillary channels formed between the membrane and a wetted filter paper. The method does not require any equipment for creating vacuum. Compared with the conventional pouring methods, the new approach reduces the consumption of staining solution and lowers the staining and destaining times (from 1.5 h to approximately 10 min) without compromising the sensitivity.
Assuntos
Membranas Artificiais , Proteínas/química , Coloração e Rotulagem/métodos , Adsorção , Colódio/química , Corantes/química , Filtração/métodos , Reprodutibilidade dos TestesRESUMO
A strategy for rapid in situ elimination of interfering substances that are present in extracts of food samples during assay is described in this article. The novel feature of this method is that the sample purification is carried out as a part of the assay, and a separate sample cleanup step is not required. The assay procedure involves the sequential addition of standard or sample, cleaning solutions, and aflatoxin B1-horseradish peroxidase conjugate (AFB1-HRP) over antibody-spotted zones of a membrane, and 3,3'-diaminobenzidine was used as the substrate for visualization. We have determined that trifluoroacetic acid and propionic acids at concentrations of 100 mM are highly effective for cleaning groundnut, wheat, corn, and poultry feed samples and that NaHCO3 (100 mM) is successful in cleaning processed soybean. In all cases, subsequent washing was performed with phosphate-buffered saline solution to facilitate the removal of traces of adhering interfering substances. A batch of 12 samples can be analyzed within 8 min either by visual comparison of the color intensity (inversely related to the analyte concentration) of a sample spot with those of reference standards or, more precisely, by densitometry. The method was tested for the analysis of AFB1 in groundnut, wheat, corn, processed soybean, chili, and poultry feed. The detection limit obtained was 5 microg/kg, except for chili, where it was 10 microg/kg. The average recoveries from different noninfected food samples spiked with AFB1 at concentrations of 5 to 100 microg/kg were between 99 and 105%. The values obtained for infected corn and groundnut samples correlated well with the estimates obtained by high-pressure liquid chromatography. The absence of a sample extraction step reduces the cost and labor involved in the assay. The method may be potentially applicable to the assay of other mycotoxins and environmental pollutants.
Assuntos
Aflatoxina B1/isolamento & purificação , Contaminação de Alimentos/análise , Imunoensaio/métodos , Aflatoxina B1/análise , Ração Animal/análise , Arachis/química , Capsicum/química , Carbonatos/farmacologia , Qualidade de Produtos para o Consumidor , Microbiologia de Alimentos , Indicadores e Reagentes , Propionatos/farmacologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Glycine max/química , Fatores de Tempo , Ácido Trifluoracético/farmacologia , Triticum/química , Zea mays/químicaRESUMO
AIM: The present study was carried out to compare the associated role of micro minerals and hormones in repeat breeding animals with the normal crossbred cows. MATERIALS AND METHODS: Blood samples were collected from 10 normal cycling and 10 repeat breeding crossbred cows of Ramakrishna Mission Ashram, Narendrapur to study the plasma mineral profile and hormonal activities. RESULTS: Zn was found to be highly significant (p<0.01) between the two groups. Follicle stimulating hormone (FSH) and progesterone showed significant (p<0.05) difference in repeat breeding animal from the normal cyclic animal, whereas no significant differences were observed in Ca, P, Cu, Se, Co, luteinizing hormone and estradiol level. CONCLUSION: It may conclude that repeat breeding condition of crossbred cows in farm condition is mainly due to the low level of progesterone, FSH and zinc.
RESUMO
A new approach for quantitative determination of AFB1 based on the separation of pre-immune complexes in the same immunoassay system has been developed. No additional step for the separation of pre-immune complexes is required. The method uses a test device for separation of pre-immune complexes from the free AFB1-enzyme conjugate by filtration through the membrane strips spotted with anti-AFB1 antibody. The bound enzyme conjugate was visualized by super-catalyzed reporter deposition (Super-CARD) signal amplification method. The measured signal intensity is directly proportional to the amount of AFB1 present in the sample. The detection limit obtained by the present method was 15 pg/ml. The data on the analytical parameters indicate that the new format of AFB1 detection in foodstuffs is reproducible, accurate and specific. The method is user friendly and does not require any costly equipment or a well-equipped laboratory.
Assuntos
Aflatoxina B1/química , Complexo Antígeno-Anticorpo/química , Imunoensaio/métodos , Anticorpos/química , Reações Cruzadas , Filtração/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Triticum/química , Zea mays/químicaRESUMO
Alternaria alternata produces more than 60 secondary metabolites, among which alternariol (AOH) and alternariol-9-methyl ether (AME) are important mycotoxins. Whereas the toxicology of these two polyketide-based compounds has been studied, nothing is known about the genetics of their biosynthesis. One of the postulated core enzymes in the biosynthesis of AOH and AME is polyketide synthase (PKS). In a draft genome sequence of A. alternata we identified 10 putative PKS-encoding genes. The timing of the expression of two PKS genes, pksJ and pksH, correlated with the production of AOH and AME. The PksJ and PksH proteins are predicted to be 2222 and 2821 amino acids in length, respectively. They are both iterative type I reducing polyketide synthases. PksJ harbors a peroxisomal targeting sequence at the C-terminus, suggesting that the biosynthesis occurs at least partly in these organelles. In the vicinity of pksJ we found a transcriptional regulator, altR, involved in pksJ induction and a putative methyl transferase, possibly responsible for AME formation. Downregulation of pksJ and altR caused a large decrease of alternariol formation, suggesting that PksJ is the polyketide synthase required for the postulated Claisen condensations during the biosynthesis. No other enzymes appeared to be required. PksH downregulation affected pksJ expression and thus caused an indirect effect on AOH production.
Assuntos
Alternaria/enzimologia , Alternaria/genética , Lactonas/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Alternaria/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Lactonas/química , Metaboloma , Família Multigênica/genética , Policetídeo Sintases/química , Interferência de RNARESUMO
The catalyzed reporter deposition (CARD) method of signal amplification, also called "tyramide signal amplification", has been used in immunoassays not only to increase sensitivity but also to reduce assay time. The current approach to tyramide amplification in immunoassays involves slow incubation with agitation. In this paper we describe new filtration-based tyramide amplification and substrate visualization techniques. Compared with the standard method, this new approach greatly enhances spot intensities in membrane immunoassay and reduces biotinylated tyramide (B-T) and substrate consumption approximately fiftyfold, without loss of specificity. An improved test device and a cost-effective method for preparation of membranes for Super-CARD amplification have also been developed. The techniques have been used for rapid detection of aflatoxin B(1) (AFB(1)) in a variety of foodstuffs with a detection limit of 12.5 microg kg(-1). The assay procedure involves sequential addition of standards or sample, AFB(1)-horseradish peroxidase (HRP) conjugate, B-T, avidin-HRP, and substrate solution over anti-AFB(1) antibody-spotted zones of the membrane surface. The method saves time, improves reproducibility, eliminates many washing steps and avoids manipulation of the membranes between the different steps, while maintaining the sensitivity of the standard method. Average recoveries from different non-infected food samples spiked with AFB(1) at concentrations from 25 to 100 mg kg(-1) were between 95 and 105%. AFB(1) results obtained on different days for Aspergillus parasiticus infection of corn and groundnut samples correlated well with estimates obtained by HPLC.
Assuntos
Aflatoxina B1/análise , Filtração/métodos , Contaminação de Alimentos/análise , Imunoensaio/métodos , Tiramina/análogos & derivados , Aflatoxina B1/toxicidade , Anticorpos Monoclonais/imunologia , Avidina/imunologia , Avidina/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Peroxidase do Rábano Silvestre/imunologia , Peroxidase do Rábano Silvestre/metabolismo , Membranas/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Membrane-based immunoassay has been developed for simultaneous estimation of aflatoxin B(1) (AFB(1)) and ochratoxin A (OA) in chili samples. The combined estimation of both the mycotoxins is more economical in respect of time, work and materials than two separate assays. The method uses a low cost test device consisting of a membrane with immobilized anti-AFB(1) and anti-OA antibodies and a filter paper attached to a polyethylene card below the membrane. It allows direct analysis of sample extracts containing substantial amount (40%) of methanol. This permits the use of two-fold diluted sample extracts resulting in minimum dilution error. The limit of quantitation obtained was 2 and 10 microg kg(-1) for AFB(1) and OA, respectively. The tolerance of 40% methanol was found to be due to the application of small size (0.8 mm diameter) spots on membranes, as the tolerance decreases to 20% with gradual increase in spot size. The combined method is capable of producing acceptable results to analyze AFB(1) and OA in chili with accuracy and precision. The AFB(1) and OA values obtained for spiked and naturally contaminated chili samples by the simultaneous method were in good correlation with those measured by individual ELISA. The method offers a simple, rapid and cost-effective screening tool to meet the requirements of the rapidly evolving EU legislation.
Assuntos
Aflatoxina B1/análise , Capsicum/química , Contaminação de Alimentos/análise , Ocratoxinas/análise , Aflatoxina B1/imunologia , Anticorpos/análise , Técnicas Imunoenzimáticas , Ocratoxinas/imunologia , Extratos Vegetais/químicaRESUMO
Membrane-based dot immunoassays are now widely used in almost every branch of biology and medicine. However, the quality of the immobilized antigen or antibody spots on the membranes was found to be highly operator-dependent and spotting by conventional methods often leads to heterogeneous spot morphologies and deposition inconsistencies. To circumvent these problems, a spotting method has been developed which is based on focussed absorption of an applied antibody solution through an aqueous network of capillary channels formed between the membrane and a wetted absorbent body. The method does not require any equipment for creating vacuum and according to assay requirements highly homogeneous spots of uniform size, in the range of 0.8- to 9-mm diameter, can be obtained by varying the volume of the applied antibody solution. Spot intensities were sufficiently high even at high antibody dilutions. Immobilization of anti-ochratoxin A (anti-OA) antibody by this method gave 2-fold increased sensitivity in a competitive assay of the toxin compared to conventional spotting methods. The calculated CV of the colour intensity for spots of different sizes (0.8 to 9 mm) was between 4.5 and 1%. Application of this spotting technique has been demonstrated for detection of OA in wine and coffee samples with the elimination of matrix interferences in the same immunoassay system. This was achieved by selective removal of nonspecific interfering substances from the sample extract during the assay. The detection limit of OA in wine (1 microg L(-1)) and coffee (2.5 microg kg(-1)) obtained by the present new method is superior to values reported recently. Thus, the present new method will be highly useful for improved performance of membrane-based immunoassays in almost every branch of biology and medicine.
Assuntos
Anticorpos/análise , Anticorpos/imunologia , Imunoensaio/métodos , Ocratoxinas/análise , Ocratoxinas/imunologia , Adsorção , Café , Ensaio de Imunoadsorção Enzimática , Sensibilidade e Especificidade , Soluções , Vinho/análiseRESUMO
An improved analytical device capable of performing simultaneous immunofiltration-based immunoassay on 30 samples in the presence of reference standards has been developed. The device consists of a rectangular membrane with 36 antibody spotted zones, one end of which was attached to a semirigid polyethylene card. A piece of wetted filter paper between the membrane and the polyethylene card absorbs the added reagent. The assay is a competitive one using T-2 toxin-horseradish peroxidase (T-2 toxin-HRP) as the labeled analyte and 4-chloro-1 naphthol (4CN) as the substrate. Signal amplification was done by the Super-CARD signal amplification method. Semiquantitative results were obtained by visual comparison of the color intensity of a sample spot with those of reference standards. Densitometric analysis was used for quantitation. The method allows rapid and easy determination of T-2 toxin in wheat and poultry feed with detection limits of 12.5 and 25 microg x kg(-)(1), respectively, with accuracy and precision. Matrix interference was eliminated by appropriate dilution of sample extracts with assay buffer. The detection sensitivity in ELISA was 10-fold higher than that in the membrane-based method. Noninfected samples were spiked with T-2 toxin at several concentrations and analyzed by the present method and rapid ELISA. Mean recoveries by both methods were between 80 and 108%. The correlation between the two methods was excellent (R(2) = 0.99).