RESUMO
Salmonella spp. are zoonotic pathogens of substantial public health concern. To enable detection in the field or under instrument-free conditions, we developed a rapid and robust lateral flow fluorescent immunoassay based on strand exchange amplification (SEA-LFIA) for the quantitative detection of Salmonella spp. As far as we know, this work is the first report regarding the use of Bst DNA polymerase-assisted SEA for fluorescence sensing to detect Salmonella spp. The SEA method was further confirmed by enzymatic digestion and Sanger dideoxy sequencing. The specificity of SEA-LFIA assay was verified by 89 Salmonella strains (18 Salmonella reference strains and 71 clinical isolates) and 15 non-Salmonella reference strains (different genera). The sensitivity of SEA-LFIA assay was 6 × 100 CFU mL-1 of Salmonella pure culture or 3 × 104 CFU 25 g-1 of artificially spiked raw chicken meat. Using this assay, it was found that 37 (16%) of the 236 samples collected were positive, which was consistent with the results of conventional PCR. The cutoff value is 15 and SEA-LFIA assay only takes â¼30 min without high equipment and reagent cost. In addition, the proposed strategy can be easily extended by redesigning the corresponding amplification primers to detect target analytes. In conclusion, the optimized SEA-LFIA assay is an efficient and specific method for the detection of Salmonella spp., and can potentially serve as a new on-site diagnostic tool in life sciences.
Assuntos
Fluorimunoensaio/métodos , Aves Domésticas/microbiologia , Infecções por Salmonella/microbiologia , Salmonella/isolamento & purificação , Animais , Galinhas/microbiologia , DNA Bacteriano/análise , DNA Bacteriano/genética , Desenho de Equipamento , Imunofluorescência/economia , Imunofluorescência/métodos , Fluorimunoensaio/economia , Análise de Alimentos/economia , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Humanos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Salmonella/genética , Fatores de TempoRESUMO
BACKGROUND: Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium, Orientia tsutsugamushi. Immunochromatography (ICT) and IgM ELISA are two of the routinely employed antibody based assays for diagnosis of Scrub typhus fever in Nepal, although the recommended gold standard diagnostic test is IgM Immunofluorescence assay (IFA). This study evaluated InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission. METHOD: Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal. Blood sample was collected from the suspected patients of scrub typhus, presenting with acute febrile illness. IgM antibody to Orientia tsusugamushi was detected by using Scrub Typhus Detect™ Kit and an in-house IgM IFA. The IFA assay was performed with the Gilliam, Karp, Kato strains and O. chuto antigens following the ARRL protocol. RESULT: Statistical analysis of IgM ELISA results when compared to reference test, IgM IFA results demonstrated the following characteristics, sensitivity 84.0% (95%CI: 79.73-87.68%), specificity 94.82% (95% CI: 93.43-95.99%), positive likelihood ratio 16.21% (95% CI: 12.71-20.67%), negative likelihood ratio 0.17% (95% CI: 0.13-0.21%), disease prevalence 22.08% (95% CI: 20.06 -24.21%), positive predictive value 82.12% (95% CI: 78.28-85.42%) and negative predictive value 95.44% (95% CI: 94.27-96.38%) respectively. CONCLUSION: Although IgM IFA is considered the gold standard test for the diagnosis of scrub typhus cases, it is relatively expensive, requires trained personal and a microscope with fluorescence filters. Scrub typhus IgM ELISA may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.
Assuntos
Testes Diagnósticos de Rotina/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Imunoglobulina M/sangue , Orientia tsutsugamushi/imunologia , Tifo por Ácaros/diagnóstico , Tifo por Ácaros/epidemiologia , Adulto , Anticorpos Antibacterianos/sangue , Doenças Endêmicas , Feminino , Imunofluorescência/economia , Humanos , Masculino , Nepal/epidemiologia , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: Use of cell culture and conventional in vivo mammalian models to assess nerve regeneration across guidance conduits is resource-intensive. Herein we describe a high-throughput platform utilizing transgenic mice for stain-free axon visualization paired with rapid cryosection techniques for low-cost screening of novel bioengineered nerve guidance conduit performance. METHODS: Interposition repair of sciatic nerve transection in mice expressing yellow fluorescent protein in peripheral neurons (Thy1.2 YFP-16) was performed with various bioengineered neural conduit compositions using a rapid sutureless entubulation technique under isoflurane anesthesia. Axonal ingrowth was assessed at 3 and 6 weeks using epifluorescent microscopy following cryosectioning. RESULTS: Mean procedure time (incision-to-closure) was less than 2½ minutes. Direct operational costs of a 3-week experiment was calculated at $21.47 per animal. Tissue processing steps were minimized to aldehyde fixation, cryoprotection and sectioning, and rapid fluorescent dye staining for conduit visualization. Fluorescent microscopy readily resolved robust axonal sprouting at 3 weeks, with clear elucidation of ingrowth-permissive, semipermissive, or restrictive nerve guidance conduit environments. CONCLUSION: A rapid and cost-efficient in vivo platform for screening of nerve guidance conduit performance has been described. LEVEL OF EVIDENCE: NA. Laryngoscope, E392-E392, 2018.
Assuntos
Imunofluorescência/métodos , Regeneração Tecidual Guiada/métodos , Microscopia de Fluorescência/métodos , Regeneração Nervosa/fisiologia , Nervo Isquiático/lesões , Alicerces Teciduais , Animais , Axônios/fisiologia , Técnicas de Cultura de Células , Feminino , Imunofluorescência/economia , Regeneração Tecidual Guiada/economia , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência/economia , Duração da Cirurgia , Nervo Isquiático/cirurgiaRESUMO
INTRODUCTION: Rabies is an acute zoonotic disease, caused by a rhabdovirus that can affect all mammals, and is commonly transmitted by the bite of a rabid animal. The definitive diagnosis is laboratorial, by the Fluorescent Antibody Test (FAT) as a quick test and Mouse Inoculation Test (MIT) as a confirmatory test (gold standard). Studies conducted over the past three decades indicate that MIT and Virus Isolation in Cell Culture (VICC) can provide the same effectiveness, the latter being considered superior in bioethics and animal welfare. The aim of this study was to compare VICC with MIT, in terms of accuracy, biosafety and occupational health, supply and equipment costs, bioethics and animal welfare, in a Brazilian public health lab. METHODS: We utilized 400 samples of animal neurological tissue to compare the performance of VICC against MIT. The variables analyzed were accuracy, biosafety and occupational health, time spent in performing the tests, supply and equipment costs, bioethics and animal welfare evaluation. RESULTS: Both VICC and MIT had almost the same accuracy (99.8%), although VICC presented fewer risks regarding biosafety and mental health of the technicians, and reduced time between inoculation and obtaining the results (approximately 22 days less). In addition, VICC presented lower supply costs (86.5% less), equipment costs (32.6% less), and the advantage of not using animals. CONCLUSIONS: These results confirm that VICC can replace MIT, offering the same accuracy and better features regarding cost, results, biosafety and occupational health, and bioethics and animal welfare.
Assuntos
Temas Bioéticos , Técnicas de Cultura de Células/métodos , Contenção de Riscos Biológicos , Imunofluorescência/métodos , Saúde Ocupacional , Vírus da Raiva/imunologia , Raiva/diagnóstico , Bem-Estar do Animal , Animais , Técnicas de Cultura de Células/economia , Custos e Análise de Custo , Imunofluorescência/economia , Camundongos , Vírus da Raiva/isolamento & purificação , Reprodutibilidade dos Testes , Fatores de Risco , Sensibilidade e EspecificidadeRESUMO
Abstract INTRODUCTION: Rabies is an acute zoonotic disease, caused by a rhabdovirus that can affect all mammals, and is commonly transmitted by the bite of a rabid animal. The definitive diagnosis is laboratorial, by the Fluorescent Antibody Test (FAT) as a quick test and Mouse Inoculation Test (MIT) as a confirmatory test (gold standard). Studies conducted over the past three decades indicate that MIT and Virus Isolation in Cell Culture (VICC) can provide the same effectiveness, the latter being considered superior in bioethics and animal welfare. The aim of this study was to compare VICC with MIT, in terms of accuracy, biosafety and occupational health, supply and equipment costs, bioethics and animal welfare, in a Brazilian public health lab. METHODS: We utilized 400 samples of animal neurological tissue to compare the performance of VICC against MIT. The variables analyzed were accuracy, biosafety and occupational health, time spent in performing the tests, supply and equipment costs, bioethics and animal welfare evaluation. RESULTS: Both VICC and MIT had almost the same accuracy (99.8%), although VICC presented fewer risks regarding biosafety and mental health of the technicians, and reduced time between inoculation and obtaining the results (approximately 22 days less). In addition, VICC presented lower supply costs (86.5% less), equipment costs (32.6% less), and the advantage of not using animals. CONCLUSIONS: These results confirm that VICC can replace MIT, offering the same accuracy and better features regarding cost, results, biosafety and occupational health, and bioethics and animal welfare.
Assuntos
Animais , Raiva/diagnóstico , Vírus da Raiva/imunologia , Saúde Ocupacional , Imunofluorescência/métodos , Técnicas de Cultura de Células/métodos , Contenção de Riscos Biológicos , Temas Bioéticos , Vírus da Raiva/isolamento & purificação , Bem-Estar do Animal , Reprodutibilidade dos Testes , Fatores de Risco , Imunofluorescência/economia , Sensibilidade e Especificidade , Técnicas de Cultura de Células/economia , Custos e Análise de Custo , CamundongosRESUMO
G protein-coupled receptors (GPCRs) are modulators of almost every physiological process, and therefore, are most favorite therapeutic target for wide spectrum of diseases. Ideally, high-throughput functional assays should be implemented that allow the screening of large compound libraries in cost-effective manner to identify agonist, antagonist, and allosteric modulators in the same assay. Taking advantage of the increased understanding of the GPCR structure and signaling, several commercially available functional assays based on fluorescence or chemiluminescence detection are being used in both academia and industry. In this chapter, we provide step-by-step method and guidelines to perform cAMP measurement using GloSensor assay. Finally, we have also discussed the analysis and interpretation of results obtained using this assay by providing several examples of Gs- and Gi-coupled GPCRs.
Assuntos
Bioensaio/métodos , Técnicas Biossensoriais/métodos , AMP Cíclico/análise , Descoberta de Drogas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Bioensaio/economia , Bioensaio/instrumentação , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/instrumentação , Análise Custo-Benefício , Proteínas Quinases Dependentes de AMP Cíclico/química , Descoberta de Drogas/economia , Descoberta de Drogas/instrumentação , Imunofluorescência/economia , Imunofluorescência/instrumentação , Imunofluorescência/métodos , Células HEK293 , Humanos , Luciferases de Vaga-Lume/química , Medições Luminescentes/economia , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Transdução de Sinais/efeitos dos fármacosRESUMO
The demand for testing to detect celiac disease (CD) autoantibodies has increased, together with the cost per case diagnosed, resulting in the adoption of measures to restrict laboratory testing. We designed this study to determine whether opportunistic screening to detect CD-associated autoantibodies had advantages compared to efforts to restrict testing, and to identify the most cost-effective diagnostic strategy. We compared a group of 1678 patients in which autoantibody testing was restricted to cases in which the test referral was considered appropriate (G1) to a group of 2140 patients in which test referrals were not reviewed or restricted (G2). Two algorithms A (quantifying IgA and Tissue transglutaminase IgA [TG-IgA] in all patients), and B (quantifying only TG-IgA in all patients) were used in each group, and the cost-effectiveness of each strategy was calculated. TG-IgA autoantibodies were positive in 62 G1 patients and 69 G2 patients. Among those positive for tissue transglutaminase IgA and endomysial IgA autoantibodies, the proportion of patients with de novo autoantibodies was lower (p=0.028) in G1 (11/62) than in G2 (24/69). Algorithm B required fewer determinations than algorithm A in both G1 (2310 vs 3493; p<0.001) and G2 (2196 vs 4435; p<0.001). With algorithm B the proportion of patients in whom IgA was tested was lower (p<0.001) in G2 (29/2140) than in G1 (617/1678). The lowest cost per case diagnosed (4.63 euros/patient) was found with algorithm B in G2. We conclude that opportunistic screening has advantages compared to efforts in the laboratory to restrict CD diagnostic testing. The most cost-effective strategy was based on the use of an appropriate algorithm.
Assuntos
Algoritmos , Autoanticorpos/sangue , Doença Celíaca/diagnóstico , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Proteínas de Ligação ao GTP/imunologia , Imunoglobulina A/sangue , Transglutaminases/imunologia , Adolescente , Adulto , Doença Celíaca/imunologia , Criança , Pré-Escolar , Técnicas de Laboratório Clínico/economia , Análise Custo-Benefício , Feminino , Imunofluorescência/economia , Humanos , Técnicas Imunoenzimáticas/economia , Medições Luminescentes/economia , Masculino , Pessoa de Meia-Idade , Proteína 2 Glutamina gama-Glutamiltransferase , Kit de Reagentes para Diagnóstico , Adulto JovemRESUMO
Immunohistochemistry (IHC) is a ubiquitous used technique to identify and analyze protein expression in the context of tissue and cell morphology. In the connexin research field, IHC is applied to identify the subcellular location of connexin proteins, as this can be directly linked to their functionality. The present chapter describes a protocol for fluorescent IHC to detect connexin proteins in tissues slices and cells, with slight modifications depending on the nature of biological sample, histological processing, and/or protein expression level. Basically, fluorescent IHC is a short, simple, and cost-effective technique, which allows the visualization of proteins based on fluorescent-labeled antibody-antigen recognition.
Assuntos
Conexinas/metabolismo , Imunofluorescência/métodos , Junções Comunicantes/metabolismo , Histocitoquímica/métodos , Imuno-Histoquímica/métodos , Animais , Imunofluorescência/economia , Humanos , Imuno-Histoquímica/economia , Fígado/metabolismo , Camundongos , Miocárdio/metabolismoRESUMO
Despite a massive research effort to elucidate Alzheimer's disease (AD) in recent decades, effective treatment remains elusive. This failure may relate to an oversimplification of the pathogenic processes underlying AD and also lack of understanding of AD progression during its long latent stages. Although evidence shows that the two specific neuropathological hallmarks in AD (neuronal loss and protein accumulation), which are opposite in nature, do not progress in parallel, the great majority of studies have focused on only one of these aspects. Furthermore, research focusing on single structures is likely to render an incomplete picture of AD pathogenesis because as AD involves complete brain networks, potential compensatory mechanisms within the network may ameliorate impairment of the system to a certain extent. Here, we describe an approach for enabling integrative analysis of the dual-nature lesions, simultaneously, in all components of one of the brain networks most vulnerable to AD. This approach is based on significant development of methods previously described mainly by our group that were optimized and complemented for this study. It combines unbiased stereology with immunohistochemistry and immunofluorescence, making use of advanced graphics computing for three-dimensional (3D) volume reconstructions. Although this study was performed in human brainstem and focused in AD, it may be applied to the study of any neurological disease characterized by dual-nature lesions, in humans and animal models. This approach does not require a high level of investment in new equipment and a significant number of specimens can be processed and analyzed within a funding cycle.
Assuntos
Doença de Alzheimer/patologia , Tronco Encefálico/patologia , Imunofluorescência/métodos , Imageamento Tridimensional/métodos , Imuno-Histoquímica/métodos , Idoso , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encefalopatias/metabolismo , Encefalopatias/patologia , Tronco Encefálico/metabolismo , Modelos Animais de Doenças , Imunofluorescência/economia , Humanos , Imageamento Tridimensional/economia , Imuno-Histoquímica/economia , Locus Cerúleo/metabolismo , Locus Cerúleo/patologia , Masculino , Pessoa de Meia-Idade , Vias Neurais/metabolismo , Vias Neurais/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Núcleos da Rafe/metabolismo , Núcleos da Rafe/patologia , Fatores de TempoRESUMO
The mainstay of laboratory diagnosis for Lyme disease is two-tiered serological testing, in which a reactive first-tier enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay is supplemented by separate IgM and IgG immunoblots. Recent data suggest that the C6 ELISA can be substituted for immunoblots without a reduction in either sensitivity or specificity. In this study, the costs of 4 different two-tiered testing strategies for Lyme disease were compared using the median charges for these tests at 6 commercial diagnostic laboratories in 2012. The study found that a whole-cell sonicate ELISA followed by the C6 ELISA was the most cost-effective two-tiered testing strategy for Lyme disease with acute-phase serum samples. We conclude that the C6 ELISA can substitute for immunoblots in the two-tiered testing protocol for Lyme disease without a loss of sensitivity or specificity and is less expensive.
Assuntos
Doença de Lyme/diagnóstico , Análise Custo-Benefício , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/economia , Imunofluorescência/métodos , Humanos , Immunoblotting/economia , Immunoblotting/métodos , Sensibilidade e Especificidade , Testes Sorológicos/economia , Testes Sorológicos/métodosRESUMO
Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 µl of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that â¼0.5% HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis.
Assuntos
Citomegalovirus/metabolismo , Imunofluorescência/métodos , Proteínas Virais/metabolismo , Internalização do Vírus , Linhagem Celular Tumoral , Células Cultivadas , Análise Custo-Benefício , Citomegalovirus/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/virologia , Imunofluorescência/economia , Glioblastoma/patologia , Glioblastoma/virologia , Humanos , Proteínas Imediatamente Precoces/metabolismo , Pulmão/citologia , Pulmão/embriologia , Microscopia de Fluorescência , Células-Tronco Neurais/virologia , Fosfoproteínas/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo , Proteínas da Matriz Viral/metabolismoRESUMO
A rapid, accurate tuberculosis diagnostic tool that is compatible with the needs of tuberculosis-endemic settings is a long-sought goal. An immunofluorescence microtip sensor is described that detects Mycobacterium tuberculosis complex cells in sputum in 25 minutes. Concentration mechanisms based on flow circulation and electric field are combined at different scales to concentrate target bacteria in 1 mL samples onto the surfaces of microscale tips. Specificity is conferred by genus-specific antibodies on the microtip surface. Immunofluorescence is then used to detect the captured cells on the microtip. The detection limit in sputum is 200 CFU mL(-1) with a success rate of 96%, which is comparable to PCR.
Assuntos
Imunofluorescência/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose/diagnóstico , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Imunofluorescência/economia , Humanos , Limite de Detecção , Técnicas Analíticas Microfluídicas/economia , Fatores de TempoRESUMO
BACKGROUND & OBJECTIVES: Acute promyelocytic leukaemia (APL) is a distinct subtype of acute myeloid leukaemia (AML) characterized by a reciprocal translocation, t(15;17) and a high incidence of life-threatening coagulopathy. APL diagnosis is considered a medical emergency. As reverse transcription-polymerase chain reaction (RT-PCR) for PML-RAR fusion oncoprotein is time consuming, there is a need for a rapid and accurate diagnostic test for APL. This study evaluates the role of PG-M3 monoclonal antibody using immunofluorescence (IF) in the early diagnosis of APL. MATERIALS AND METHODS: Thirty-six new untreated APL cases diagnosed with RT-PCR for PML-RAR as the gold standard and 38 non-APL controls (28 non-APL AMLs and 10 non-leukaemic samples) were evaluated by routine morphology and cytochemistry, RT-PCR and IF using PG-M3 monoclonal antibody. RESULTS: Using IF, 34 of 36 (94·4%) APL cases showed a microgranular pattern suggestive of APL and two cases (5·6%) showed a speckled pattern typical of wild-type PML protein (False negative). By comparison, two of 28 (7·1%) non-APL AMLs showed microgranular pattern (false positive). Hence, IF as a diagnostic test for APL resulted in a sensitivity of 94·4%, specificity of 92·9% and positive and negative predictive values of 94·4% and 92·9% respectively. All 10 non-leukaemic samples showed a speckled pattern. CONCLUSIONS: IF using PG-M3 antibodies can be used as a rapid (takes 2 h), cheap, sensitive and specific method to identify APL. It can be a useful adjunct for diagnosis of APL especially if facilities for RT-PCR are not available, particularly in resource-limited settings.
Assuntos
Anticorpos Monoclonais , Leucemia Promielocítica Aguda/diagnóstico , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/economia , Estudos de Casos e Controles , Criança , Feminino , Imunofluorescência/economia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fatores de Tempo , Adulto JovemRESUMO
The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5(55-98) protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5(55-98) MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP5(55-98) MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.
Assuntos
Anticorpos Antivirais/análise , Infecções por Arterivirus/veterinária , Equartevirus/imunologia , Imunofluorescência/métodos , Doenças dos Cavalos/imunologia , Doenças dos Cavalos/virologia , Imunoensaio/métodos , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Infecções por Arterivirus/imunologia , Infecções por Arterivirus/virologia , Equartevirus/isolamento & purificação , Imunofluorescência/economia , Cavalos , Imunoensaio/economia , Microesferas , Dados de Sequência Molecular , Testes de Neutralização/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologiaRESUMO
This paper describes simple procedures to process digital images in quantitative immunofluorescence microscopy. Monoclonal antibodies directed against the sarcoplasmic myosin heavy chain isoforms and against laminin, located on the basement membranes, were applied to sections of human skeletal muscle. The localisation and staining intensity of a fluorescent secondary antibody were recorded using an indirect histochemical method. The digitised images were pre-processed and the luminosities of appropriate structures were determined using existing tools in the widely used image processing software Photoshop from Adobe. Procedures to obtain a quantitative measure for the specific fluorescence signal (the background corrected fluorescence in the object) were developed. In addition, antibody binding to individual cells could be quantified whether these cells are well separated or not. The relation between the specific fluorescence signal and the dilution factor of the primary antibody could be measured to determine a suitable concentration of the antibody for incubation of the sections. The potential fading of the fluorescence signal with time and prolonged exposure to light from the microscope was explored and analysed. With the tools described in the present report it is thus possible also to optimize the topical immunohistochemical protocol in order to quantify the fluorescence signal.
Assuntos
Custos e Análise de Custo/economia , Imunofluorescência/métodos , Imuno-Histoquímica/métodos , Software/economia , Adenosina Trifosfatases/metabolismo , Biópsia , Imunofluorescência/economia , Humanos , Imuno-Histoquímica/economia , Técnicas de Diluição do Indicador , Magnetismo , Músculos/metabolismoRESUMO
Calgary Laboratory Services (CLS) in Alberta, Canada, is the regional reference laboratory providing flow cytometry services for southern Alberta and southeastern British Columbia. As a busy reference flow laboratory we provide flow cytometry immunophenotyping for investigation and diagnosis of acute and chronic leukemias, lymphomas, immunodeficiencies, neuroblastoma, platelet disorders, and interstitial lung disease (ILD). Because of increasing workload and the continual effort to improve the service to our health care providers, CLS invested in the new Beckman Coulter Cytomics FC 500 5-color flow cytometer. In addition to time and labor savings due to reduced maintenance and operating system design, this new flow cytometer automates many of the previous manual steps involved in quality control and flow cytometric analysis. It also incorporates 2 lasers and is capable of measuring 5-color antibody combinations in a single tube, enabling us to reduce the number of tubes and overall costs, giving us better gating options for minimal residual disease analysis. We present the first published evaluation, an assessment of the overall productivity and cost impact of the new state-of-the-art Cytomics FC 500 flow cytometer. Implementation of the Cytomics FC 500 has resulted in a 20% reduction in reagent costs and shorter turnaround time for analysis and diagnosis. This instrument has allowed us to reduce our acute leukemia panel from 17 to 13 tubes, our lymphoma panel from 13 to 7 tubes, and our ILD panel from 4 to 2 tubes. The availability of 2 lasers provides more flexibility in choosing antibodies and conjugates to customize immunophenotyping panels. It also allows us to use the DRAQ5 dye and simultaneously analyze the immunophenotype and DNA content of cells with very little compensation. Many of the arduous, time-consuming flow operator tasks often associated with previous generation flow cytometry instruments, such as color compensation, list mode analysis, sample repeats, and interpretations, have been substantially reduced with the Cytomics FC 500 5-color flow cytometer. In conclusion the Cytomics FC 500 5-color flow cytometer is a major advance in flow cytometry instrumentation and has reduced our overall reagent costs by 20%, provided better information and speedier turnaround time to our health care professionals. It is an ideal flow cytometer for any busy clinical or research flow cytometry service.
Assuntos
Eficiência , Citometria de Fluxo/economia , Citometria de Fluxo/instrumentação , Laboratórios/economia , Automação/economia , Automação/instrumentação , Sistemas de Informação em Laboratório Clínico/economia , Cor , Estudos de Avaliação como Assunto , Citometria de Fluxo/normas , Imunofluorescência/economia , Imunofluorescência/instrumentação , Humanos , Síndromes de Imunodeficiência/diagnóstico , Doenças Pulmonares Intersticiais/diagnóstico , Transtornos Linfoproliferativos/diagnóstico , Controle de QualidadeAssuntos
Herpes Simples/diagnóstico , Herpesvirus Humano 1/isolamento & purificação , Herpesvirus Humano 2/isolamento & purificação , Antígenos Virais/análise , Custos e Análise de Custo , Meios de Cultura , Ensaio de Imunoadsorção Enzimática/economia , Imunofluorescência/economia , Genes Virais , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/imunologia , Humanos , Reação em Cadeia da Polimerase/economia , Fatores de Tempo , Cultura de VírusRESUMO
The detection of a large variety of viruses requires a number of different susceptible cell lines in a shell vial method--currently considered to be the most enhanced method of viral detection. However, this method is unsuitable in its standard form for handling a large volume of specimens. We have developed a time and cost saving new method, named rapid enhanced tissue culture immunofluorescence (RETCIF) and incorporates the attributes of the current methods. It is specific, uses the most sensitive cell lines and monoclonal antibodies and does not require the use of cover slips. The RETCIF method, in our hands, is a time saving procedure, with higher isolation ratio than the shell vial method. We recommend the RETCIF method for busy diagnostic virology laboratories.
Assuntos
Imunofluorescência/métodos , Vírus , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Técnicas de Cultura , Imunofluorescência/economia , Humanos , Sensibilidade e Especificidade , Vírus/isolamento & purificaçãoRESUMO
Rapid detection of viruses in clinical samples is important for continuing appropriate antiviral treatment and discontinuing unnecessary antibacterial treatment, as well as for excluding viral pathogens. Yet detection of viral agents may require numerous susceptible cell lines. Even with the shell vial culture method, it is cumbersome for handling large volumes of specimens. A procedure has been developed, which is time and cost-saving and uses specific cell lines in a 96-well microtitre plate and monoclonal antibodies (RETCIF-rapid enhanced tissue culture immunofluorescence). Each clinical sample was inoculated into 12 different wells with five different cell lines. Enhancement was achieved by sonication, centrifugation and hormonal supplementation to the medium used. Cytomegalovirus (CMV), herpes simplex virus (HSV) and respiratory viruses were detected by monoclonal antibodies on day 2, whilst varicella zoster virus (VZV) and enteroviruses were detected on days 5 and 7, respectively. During July-December 1998, 3298 patient specimens were compared by RETCIF and a modified shell vial method. Either or both methods isolated 779 viruses (24% positivity rate), whilst both methods detected 621. Of the 779 viruses, 87% (679) were isolated by the shell vial method in an average time of 4.9 days. For RETCIF the respective rate was 92.5% (721), in an average time of 3.0 days. The RETCIF method is a time-saving procedure, with higher isolation rates than the shell vial method.
Assuntos
Imunofluorescência/métodos , Vírus/imunologia , Vírus/isolamento & purificação , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Células Cultivadas , Centrifugação , Imunofluorescência/economia , Humanos , Sensibilidade e Especificidade , Sonicação , Fatores de TempoRESUMO
Toxoplasmosis infection is commonly asymptomatic, but it may have severe teratogenic consequences. The authors review the literature on serologic screening in the first trimester of pregnancy.