RESUMO
OBJECTIVES: In this study, we compared IFA and real-time PCR in bronchoalveolar lavage specimens of HIV infected patients. A total of 66 BALs from 62 HIV patients were included in the study. 30 IFA positive and 36 IFA negative specimens were tested with real-time PCR, targeting the major surface glycoprotein. We performed a retrospective analysis of the patient's medical records, compared the results of the IFA and PCR tests and analyzed costs, expenditure of time and personal expenses. RESULTS: All of the 30 IFA positive samples were PCR positive. 35 of 36 IFA negative probes were also negative in the PCR assay. Considering the PCR results as a binary outcome (positive/negative) sensitivity was 100%, specificity 97.2%. The patient with negative IFA and positive PCR had a clear clinical picture of PCP and responded to PCP treatment. PCR was more than twice as expensive and time-consuming as IFA. Diagnostic accuracy for PCP of PCR and IFA was comparable in HIV-infected patients, but IFA was significantly less expensive and less time-consuming. Therefore, IFA testing can continue to be used as gold standard in the diagnosis of PCP in HIV patients. However, in special cases, IFA may lack sensitivity and PCR should be added to the diagnostic armamentarium.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Técnica Direta de Fluorescência para Anticorpo/normas , Pneumonia por Pneumocystis/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/normas , Adulto , Líquido da Lavagem Broncoalveolar , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We evaluated two currently available rapid antigen detection tests (RADTs) for Respiratory syncytial virus (RSV), Sofia® RSV FIA and BinaxNOW RSV Card (BinaxNOW). Between November 2017 and February 2018, 395 nasopharyngeal swabs were collected from children diagnosed with acute respiratory infections. The swabs were evaluated using the aforementioned RADTs, the reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR), and the direct immunofluorescence assay (DFA). The sensitivity of Sofia® RSV FIA (80.82%) was significantly higher than that of BinaxNOW (53.42%) when RT-qPCR was used as the standard. This was confirmed with DFA. The sensitivities of Sofia® RSV FIA (85.4% [41/48]) and BinaxNOW (58.3% [28/48]) were higher for RSV A than for RSV B (69.6% [16/23] and 43.5% [10/23], respectively). The optimal critical cycle threshold (Ct) values on RT-qPCR that correlated with Sofia® RSV FIA and BinaxNOW were 24 and 22, respectively. The kappa value for Sofia® RSV FIA and RT-qPCR was 0.962 in patients who were two years old or younger, but 0.648 in those who were more than two years old. Thus, Sofia® RSV FIA is more sensitive than BinaxNOW; its results were affected by the RSV viral strain and load. Sofia® RSV FIA is more effective in children who are ≤ 2 years old than in those who are > 2 years old.
Assuntos
Antígenos Virais/isolamento & purificação , Técnica Direta de Fluorescência para Anticorpo/normas , Kit de Reagentes para Diagnóstico/normas , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/isolamento & purificação , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Nasofaringe/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/imunologia , Infecções Respiratórias/diagnóstico , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is potentially fatal if not diagnosed and treated. Accurate and timely diagnosis is considered one of the pillars needed for the reduction in disease-related lethality. Brazil is currently one of the three eco-epidemiological hotspots for this disease. Several serological tests are commercially available in this country for VL diagnosis, although information on the performance of these tests is fragmented and insufficient. The aim of this study was to directly compare the performance of six commercial kits: three enzyme-linked immunosorbent assays (ELISAs), two immunofluorescence antibody tests (IFATs), one immunochromatographic test (ICT), besides one ICT, currently not commercially available in Brazil and one in-house direct agglutination test (DAT-LPC), not yet marketed. METHODOLOGY/PRINCIPAL FINDINGS: A panel of 236 stored samples from patients with clinically suspected VL, including 77 HIV-infected patients, was tested. IT-LEISH and DAT-LPC showed the highest accuracy rate among the non-HIV-infected patients, 96.2% [CI95%: 92.8-99.7%] and 95.6% [CI95%: 91.9-99.3%], respectively. For the ELISA tests evaluated, the maximum accuracy was 91.2%, and in the inter HIV-status group analysis, no significant differences were observed. For both IFATs evaluated, the maximum accuracy was 84.3%, and a lower accuracy rate was observed among the HIV-infected patients (p = 0.039) than among the non-HIV-infected patients. The DAT-LPC was the most accurate test in the HIV-infected patients (p≤0.115). In general, no significant difference in accuracy was observed among the VL-suspected patients stratified by age. CONCLUSIONS/SIGNIFICANCE: In summary, the differences in the performance of the tests available for VL in Brazil confirm the need for local studies before defining the diagnostic strategy.
Assuntos
Anticorpos Antiprotozoários/sangue , Leishmaniose Visceral/diagnóstico , Kit de Reagentes para Diagnóstico/normas , Testes Sorológicos/normas , Adolescente , Adulto , Idoso , Testes de Aglutinação/métodos , Testes de Aglutinação/normas , Antígenos de Protozoários/sangue , Antígenos de Protozoários/imunologia , Brasil , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Técnica Direta de Fluorescência para Anticorpo/normas , Infecções por HIV/complicações , Infecções por HIV/parasitologia , Humanos , Imunoensaio/normas , Lactente , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
Marine bivalve shellfish are of public health interest because they can accumulate pollutants in their tissues. As they are usually consumed raw or lightly cooked, they are considered to be a possible source of foodborne infections, including giardiosis and cryptosporidiosis. Although data indicating contamination of shellfish with Giardia cysts and Cryptosporidium oocysts have been published, comparing results from different studies is difficult, as there is no standardized protocol for the detection and quantification of these parasites in mussels, and different researchers have used different analytical approaches. The aim of this study was to identify and characterize the most sensitive protocol for the detection of Giardia cysts and Cryptosporidium oocysts in shellfish. In an effort to test the sensitivity and the detection limits of the protocol, every step of the process was investigated, from initial preparation of the mussel matrix through detection of the parasites. Comparative studies were conducted, including several methods previously applied by other researchers, on commercial mussels Mytilus galloprovincialis spiked with a known number of (oo)cysts of both parasites. As preparation of the mussel matrix plays an important role in the sensitivity of the method, different techniques were tested. These included: (ia) removal of the coarse particles from the matrix with sieving, (ib) extraction of the lipids with diethyl ether, and (ic) artificial digestion of the matrix with pepsin digestion solution, and (ii) the use or not of immunomagnetic separation (IMS) for the concentration of the (oo)cysts. Pre-treatment of the mussel homogenate with pepsin digestion solution, followed by IMS, then detection with a direct immunofluorescence assay, achieved the highest sensitivity: 32.1% (SD: 21.1) of Giardia cysts and 61.4% (SD: 26.2) Cryptosporidium oocysts were recovered, with a detection limit of 10 (oo)cysts per g of mussel homogenate. The outcome of the current study was the standardization of a protocol, with defined detection limits, for the detection of these two protozoan transmission stages in mussels, in order to be used as a reference technique in future studies. Further advantages of this protocol are that it uses the whole mussel as a starting material and does not require difficult handling procedures. The method has potential to be applied in larger surveys and, potentially, to other species of shellfish for the detection of these parasites. However, the composition (lipid to protein ratio) may be of relevance for detection efficiency for some other species of shellfish.
Assuntos
Cryptosporidium/isolamento & purificação , Técnica Direta de Fluorescência para Anticorpo/normas , Microbiologia de Alimentos/métodos , Giardia/isolamento & purificação , Mytilus/microbiologia , Oocistos/isolamento & purificação , Frutos do Mar/microbiologia , Animais , Criptosporidiose/prevenção & controle , Giardíase/prevenção & controle , Separação Imunomagnética , Mar MediterrâneoRESUMO
BACKGROUND: Bullous pemphigoid (BP) is the most common subepidermal autoimmune disorder characterized by tense bullae. It is associated with circulating autoantibodies against BP antigen-1 and BP antigen-2. Diagnosis is based upon clinical, histopathologic, and immunopathologic examination. Direct immunofluorescence (DIF) of perilesional skin highlights C3 with or without IgG in a linear pattern along the basement membrane. OBJECTIVES: We hypothesized that repeat biopsies may be required for a definitive DIF diagnosis of BP, as initial DIF evaluation may result in a false-negative result. METHODS: A retrospective chart review was conducted on 1143 specimens collected for evaluation for BP. Cases from 2 Vancouver Coastal Health Authority laboratories from 2006 to 2016 were reviewed. Results were interpreted as positive, negative, or indeterminate based on pathologic description and specimen quality. RESULTS: After meeting the inclusion criteria, 739 specimens were further evaluated. There were 289 cases of BP in the 10-year period. Five patients (1.73%; 95% confidence interval [CI], 1.50-1.96) required a second biopsy to support a BP diagnosis, and within this group, 1.04% of the 289 (95% CI, 0.811-1.27) were true successive negative-to-positive DIF results. CONCLUSIONS: DIF is the most reliable test used to diagnose BP; however, a small percentage of patients will initially have a negative result. False-negative or indeterminate results may be due to specimen sampling from lesional skin or due to a subthreshold quantity of immune complexes in the skin. Repeat biopsy is warranted despite an initial negative DIF if BP is clinically suspected.
Assuntos
Técnica Direta de Fluorescência para Anticorpo/normas , Penfigoide Bolhoso/diagnóstico , Penfigoide Bolhoso/patologia , Biópsia , Reações Falso-Negativas , Humanos , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
BACKGROUND: Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. OBJECTIVES: First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. STUDY DESIGN: We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. RESULTS: The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p<0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p>0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2 and 2h for DFA, 31.8 and 1.5h for Simplexa™ and 55 and 3h for TAC testing. CONCLUSIONS: Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.
Assuntos
Coinfecção/diagnóstico , Técnica Direta de Fluorescência para Anticorpo/métodos , Hospedeiro Imunocomprometido , Influenza Humana/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Líquido da Lavagem Broncoalveolar/virologia , Coinfecção/virologia , Feminino , Técnica Direta de Fluorescência para Anticorpo/instrumentação , Técnica Direta de Fluorescência para Anticorpo/normas , Humanos , Influenza Humana/virologia , Masculino , Análise em Microsséries/métodos , Análise em Microsséries/normas , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/normas , Nasofaringe/virologia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Infecções por Vírus Respiratório Sincicial/virologia , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: Within the framework of routine fitness examinations, French Air Force military crew underwent urine testing for 3,4 methylenedioxymetamphetamine (MDMA [ecstasy]). The cross-reactivity of a dyslipidemic drug, fenofibrate, with an MDMA immunoassay was studied and confirmed on a large population sample. METHODS: A 3-year retrospective study was performed on the MDMA DRI Ecstasy Assay on the Unicel DXC 600. In the event of positive test result, a confirmatory testing was carried out by gas chromatography/mass spectrometry (GC/MS) to establish the presence of MDMA. When analysis by GC/MS did not confirm the presence of MDMA, a false-positive result was suspected and the samples were analyzed by high-performance liquid chromatography-mass spectrometry to identify a potential interfering substance. RESULTS: A total of 15,169 urine samples, from 7,803 patients, were tested for 3 years. Of the tested samples, 22 (0.15%) were positive by DRI Ecstasy Assay. None of them were positive by GC/MS. A cross-reactivity of fenofibrate's metabolite with MDMA using this assay was systematically found. CONCLUSION: Fenofibrate's interference with MDMA immunoassay was confirmed. Fenofibrate being widely prescribed, physicians had to be alerted that this treatment could lead to false-positive results.
Assuntos
Avaliação Pré-Clínica de Medicamentos/normas , Reações Falso-Positivas , Fenofibrato/análise , N-Metil-3,4-Metilenodioxianfetamina/urina , Adolescente , Adulto , Idoso , Avaliação Pré-Clínica de Medicamentos/métodos , Fenofibrato/uso terapêutico , Fenofibrato/urina , Técnica Direta de Fluorescência para Anticorpo/métodos , Técnica Direta de Fluorescência para Anticorpo/normas , França , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Militares/estatística & dados numéricos , N-Metil-3,4-Metilenodioxianfetamina/análiseRESUMO
CV2 antibodies (CV2-ab) associate with paraneoplastic neurological syndromes (PNS) and small-cell lung cancer. This study was designed to assess the sensitivity of two widely used anti-CV2 commercial kits. Fifty three sera with CV2-ab identified by immunohistochemistry on paraformaldehyde-perfused rat brain were tested with two commercial immunoblot kits (Euroimmun AG, and Ravo Diagnostika) and 4 (7.5%) of them were negative with the commercial kits. The 4 samples were positive by immunofluorescence on HEK293 cells transfected with CRMP5 and immunoblot of these cells lysate. A few CV2-ab-positive sera may be missed by commercial immunoblots. Negative samples from patients with high suspicion for PNS should be tested by alternative methods.
Assuntos
Autoanticorpos/sangue , Autoanticorpos/imunologia , Proteínas do Tecido Nervoso/sangue , Proteínas do Tecido Nervoso/imunologia , Animais , Técnica Direta de Fluorescência para Anticorpo/métodos , Técnica Direta de Fluorescência para Anticorpo/normas , Células HEK293 , Humanos , Hidrolases , Immunoblotting/métodos , Immunoblotting/normas , Masculino , Proteínas Associadas aos Microtúbulos , Ratos , Ratos Sprague-Dawley , Estudos RetrospectivosAssuntos
Autoanticorpos/sangue , Receptores de N-Metil-D-Aspartato/imunologia , Autoanticorpos/biossíntese , Transtorno da Personalidade Borderline/imunologia , Transtorno Depressivo Maior/imunologia , Técnica Direta de Fluorescência para Anticorpo/normas , Humanos , Prevalência , Ensaios Clínicos Controlados Aleatórios como Assunto/tendências , Receptores de N-Metil-D-Aspartato/sangue , Reprodutibilidade dos Testes , Estudos Retrospectivos , Esquizofrenia/imunologia , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: As quality by design (QbD) for pharmaceutical product development is being expanded to include analytical methods, we applied QbD to the development of antigenicity assays measuring in vitro relative potency of a quadrivalent vaccine candidate. RESULTS: After establishing development targets together with customers, immunoassays were developed to meet objectives. Statistical design of experiments was used to optimize method parameters and establish a design space. Systematic risk analysis enabled identification of potential risks to method performance that were mitigated by investigating the available design space around risk factors and by establishing appropriate control strategies. CONCLUSION: We found QbD-based method development was a more efficient and systematic approach that could also potentially facilitate assay transfers and life cycle management.
Assuntos
Antígenos/análise , Análise Fatorial , Técnica Direta de Fluorescência para Anticorpo/normas , Técnica Indireta de Fluorescência para Anticorpo/normas , Vacinas/análise , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/isolamento & purificação , Coelhos , Reprodutibilidade dos Testes , Medição de Risco , Sensibilidade e Especificidade , Vacinas/imunologia , Estudos de Validação como AssuntoRESUMO
ß-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of ß-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of ß-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by ß-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual ß-lactam was known. The IC50 values of 15 ß-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 µg L(-1). Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected ß-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 µg kg(-1), respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 ß-lactams in edible tissues, among which 11 ß-lactams controlled by European Union could be detected below maximum residue limits.
Assuntos
Antibacterianos/isolamento & purificação , Técnica Direta de Fluorescência para Anticorpo/normas , Proteínas Imobilizadas/química , Carne/análise , Leite/química , Proteínas de Ligação às Penicilinas/química , beta-Lactamas/isolamento & purificação , Ampicilina/química , Animais , Ligação Competitiva , Bovinos , Galinhas , Escherichia coli/genética , Contaminação de Alimentos , Peroxidase do Rábano Silvestre/química , Proteínas Imobilizadas/genética , Limite de Detecção , Proteínas de Ligação às Penicilinas/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reprodutibilidade dos TestesRESUMO
A general and universal analytical strategy for characterization of hapten-BSA conjugates based on complementary optical spectroscopy and molecular mass spectrometry techniques is here described. The proposed procedure provides highly-valuable information about the molecular weight of the conjugate, its stoichiometry and the concentration of the precursors (hapten and BSA) in the conjugate; such information is of great analytical interest for further development of novel quantitative immunoassays. Further, due to great demand of new, simple and robust methodologies for the melamine analysis in milk infant formula, a new immunoprobe melamine-bovine serum albumin-quantum dot was synthetized, characterized and successfully applied in a competitive fluorescent quantum dot-based immunoassay. It should be highlighted that the limit of detection achieved without any sample pretreatment, 0.15 mg kg(-1) for melamine in milk infant formula, is one order of magnitude lower than the maximum concentration level allowed by international legislation in such type of samples. Finally, this simple approach was validated by the use of an alternative technique (HPLC-UV) for the analysis of melamine in contaminated milk infant formula, showing a good agreement between the results obtained by using both analytical methodologies.
Assuntos
Laticínios/análise , Técnica Direta de Fluorescência para Anticorpo/normas , Contaminação de Alimentos/análise , Fórmulas Infantis/química , Pontos Quânticos , Triazinas/análise , Animais , Ligação Competitiva , Bovinos , Haptenos/química , Humanos , Imunoconjugados/química , Lactente , Limite de Detecção , Soroalbumina Bovina/químicaRESUMO
BACKGROUND: Serration pattern analysis of direct immunofluorescence (DIF) allows the differentiation of epidermolysis bullosa acquisita from other subtypes of pemphigoid. In daily practice its use is limited due to lack of experience and unfamiliarity. OBJECTIVES: To test the learnability of DIF serrated-pattern recognition in groups with various a priori levels of competence. METHODS: An online nversusu-test (www.nversusu.umcg.nl) was created, which contained 26 DIF images of the epidermal basement membrane zone, IgG stained and photographed with a magnification of × 40 and × 63. All images represented patients with a form of subepidermal autoimmune bullous disease. Thirteen DIF images were presented before and 13 DIF images after an instruction video about n- and u-serrated patterns. There were three options to choose from: n-serrated, u-serrated or undetermined. The test was completed by three groups of professionals: dermatology residents in training at the University Medical Center Groningen (UMCG), international experts on bullous diseases, and dermatologists and pathologists who had participated in the Groningen blistering course during the past 10 years. RESULTS: The overall number of correct answers of serration patterns was significantly higher after instruction than before instruction (median 9.0 correct answers vs. 11.0 correct answers, P < 0.001). Participants showed a mean improvement after instruction of 15.4% in the UMCG group (66.7% vs. 82.1%), 16.2% in the international expert group (67.2% vs. 83.4%) and 12.1% in the blistering course group (60.7% vs. 72.8%). The u-serrated pattern was better recognized than the n-serrated pattern. CONCLUSIONS: Serration pattern analysis by DIF can be learned irrespective of background of expertise.
Assuntos
Competência Clínica/normas , Dermatologia/normas , Epidermólise Bolhosa Adquirida/patologia , Internato e Residência/normas , Penfigoide Bolhoso/patologia , Adulto , Dermatologia/educação , Diagnóstico Diferencial , Feminino , Técnica Direta de Fluorescência para Anticorpo/normas , Humanos , Países Baixos , EnsinoRESUMO
Studies comparing rabbit monoclonal SP1 antibody with 1D5 for estrogen receptor (ER) immunohistochemical testing show conflicting results. Here we use a standardized quantitative immunofluorescent (QIF) ER assay to determine the level and significance of discordance between the antibodies. Both antibodies were assessed by QIF on our Index TMA of cell lines and case controls, followed by QIF and immunohistochemical analysis on 2 retrospective cohorts from Yale. On the Index TMA, SP1 displayed stronger signal-to-noise ratio compared with 1D5. On the patient cohorts, the range of discrepancy between the 2 antibodies was 8% to 16.9%, with the majority of discrepant cases being SP1 positive/1D5 negative. Kaplan-Meier analysis of the discrepant cases showed outcomes comparable to those of double-positive cases, suggesting that SP1 is more sensitive than 1D5. A series of cases with high levels of ER-ß shows that neither antibody cross-reacts, suggesting equivalent specificity. Future efforts are needed to determine whether response to endocrine therapies show superiority of either antibody as a companion diagnostic test.
Assuntos
Anticorpos Monoclonais/análise , Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Receptor beta de Estrogênio/genética , Técnica Direta de Fluorescência para Anticorpo/normas , Adulto , Animais , Anticorpos Monoclonais/imunologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Reações Falso-Negativas , Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Coelhos , Estudos Retrospectivos , Razão Sinal-Ruído , Análise Serial de TecidosRESUMO
INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Cryptosporidium/imunologia , Água Doce/parasitologia , Animais , Cryptosporidium/isolamento & purificação , Técnica Direta de Fluorescência para Anticorpo/normas , Oocistos/imunologia , Coelhos , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20 percent of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.
INTRODUÇÃO: A produção de anticorpos policlonais anti-Cryptosporidium e sua utilização na imunofluorescência para determinar a presença de Cryptosporidium em água são descritas no presente trabalho. MÉTODOS: Dois coelhos foram imunizados com antígeno solúvel e particulado provenientes de oocistos purificados de Cryptosporidium. O soro produzido foi preparado para a extração de imunoglobulinas G, que foram purificadas e conjugadas com isotiocianato de fluoresceína (FITC). Lâminas contendo quantidades conhecidas de oocistos foram preparadas para determinar a sensibilidade da técnica. Para testar a especificidade foram preparadas lâminas contendo cistos de Giardia duodenalis. RESULTADOS: O conjugado foi usado com sucesso em amostras de água contaminadas experimentalmente com oocistos de Cryptosporidium, sendo capaz de detectar até cinco oocistos/spots que corresponde a uma contaminação de 250 oocistos/mL. CONCLUSÕES: As três imunizações realizadas nos coelhos foram suficientes para produção de anticorpos contra Cryptosporidium; a reação de imunofluorescência direta padronizada permitiu a detecção de cinco oocistos em 20 por cento das amostras; não houve reação cruzada com cistos de Giardia duodenalis.