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Validação, valores normativos e aplicabilidade clínica de um ensaio imunoenzimático para determinação sérica do hormônio anti-Mülleriano; Validation, normative values and clinical applicability of an enzyme immunoassay for the determination of serum anti-Müllerian hormone

Woloszynek, Renata dos Santos Batista Reis
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 09/06/2014 PT
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O hormônio anti-Mülleriano (AMH) é um marcador de reserva ovariana e função testicular. A aplicação clínica desse hormônio requer padronização adequada de valores de referência, de acordo com o imunoensaio utilizado. Os objetivos deste estudo foram: validar o imunoensaio AMH Gen II (Beckman Coulter Company, TX, USA), estabelecer valores normais de AMH em homens e mulheres saudáveis, avaliar a influência do uso de contraceptivos hormonais, tabagismo e índice de massa corpórea (IMC) sobre os valores de AMH e verificar as concentrações séricas desse hormônio em pacientes com síndrome de Turner (ST), síndrome dos ovários policísticos (SOP) e em meninos com criptorquidismo submetidos ao teste de estímulo com rhCG (gonadotrofina coriônica humana recombinante). A validação do ensaio AMH Gen II foi realizada utilizando protocolo simplificado, conforme as recomendações do Clinical and Laboratory Standards Institute. Para estabelecer valores normais de AMH, 133 mulheres e 135 homens saudáveis foram selecionados prospectivamente. Além disso, 66 pacientes com ST, 29 com SOP e 31 com criptorquidismo foram estudados. A sensibilidade analítica e funcional do ensaio AMH Gen II foram 0,02 e 0,2 ng/mL, respectivamente. Os coeficientes de variação intra e interensaio variaram entre 5...

COMPARISON OF A COMMERCIAL ENZYME IMMUNOASSAY WITH PLAQUE REDUCTION NEUTRALIZATION FOR MATERNAL AND INFANT MEASLES ANTIBODY MEASUREMENT

GONÇALVES,Guilherme; CUTTS,Felicity; FORSEY,Timothy; ANDRADE,Helena Rebelo
Fonte: Instituto de Medicina Tropical Publicador: Instituto de Medicina Tropical
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/1999 EN
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The most practicable assay for measurement of measles IgG (mIgG) in large numbers of sera is an enzyme immunoassay (EIA). To assess how EIA results would agree with those by the gold standard method of plaque reduction neutralization (PRN) we compared the results from the two methods in 43 pairs of maternal and umbilical cord sera, and sera from the corresponding infants when aged 11 - 14 months. In maternal-cord sera, the differences between mean antibody levels by EIA or PRN were not statistically significant, though in individual sera, differences could be large. However, agreement was less good for infants sera, in which levels of mIgG were very low. The conclusions of a study of transplacental transport of mIgG would not be affected by the use of either technique. When studying waning immunity in infants, PRN should be the method of choice, while results from studies using EIA should be interpreted with caution.

Vancomycin monitoring in term newborns: comparison of peak and trough serum concentrations determined by high performance liquid chromatography and fluorescence polarization immunoassay

Feferbaum,Rubens; Machado,José Kleber Kobol; Diniz,Edna Maria de Albuquerque; Okay,Thelma S.; Santos,Silvia R. C. J.; Ceccon,Maria Esther J.R.; Krebs,Vera L. J.; Araújo,Maria C. K. de; Vaz,Flávio Adolfo Costa
Fonte: Faculdade de Medicina / Universidade de São Paulo - FM/USP Publicador: Faculdade de Medicina / Universidade de São Paulo - FM/USP
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/10/2001 EN
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INTRODUCTION: Peak and trough serum concentrations of vancomycin were determined in term newborn infants with confirmed or suspected Staphylococcus sp sepsis by high performance liquid chromatography and flourescence polarization immunoassay. OBJECTIVE: To statistically compare the results of the high performance liquid chromatography and flourescence polarization immunoassay techniques for measuring serum vancomycin concentrations. METHODS: Eighteen peak and 20 trough serum samples were assayed for vancomycin concentrations using high performance liquid chromatography and flourescence polarization immunoassay from October 1995 to October 1997. RESULTS: The linear correlation coefficients for high performance liquid chromatography and flourescence polarization immunoassay were 0.27 (peak, P = 0.110) and 0.26 (trough, P = 0.1045) respectively, which were not statistically significant. CONCLUSION: There was wide variation in serum vancomycin concentrations determined by high performance liquid chromatography as compared with those determined by flourescence polarization immunoassay. There was no recognizable pattern in the variability; in an apparently random fashion, the high performance liquid chromatography measurement was sometimes substantially higher than the flourescence polarization immunoassay measurement...

RS virus diagnosis: comparison of isolation, immunofluorescence and enzyme immunoassay

Siqueira,Marilda M.; Ferreira,Vanja; Nascimento,Jussara P.
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/1986 EN
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Two techniques for rapid diagnosis, immunofluorescence (IFAT) and enzyme immunoassay (EIA), have been compared with virus isolaion in tissue culture for the detection of respiratory syncytial virus (RSV) in specimens of nasopharyngeal secretions. The specimens were obtained from children under five years of age suffering from acute respiratory iliness, during a period of six months from January to June 1982. Of 471 specimens examined 54 (11.5%) were positive by virus isolation and 180 (38.2%) were positive by immunofluorescence. The bacterial contamination of inoculated tissue cultures unfortunately prevented the isolation of virus from many samples. Specimens from 216 children were tested to compare enzyme immunoassay and immunofluorescence. Of these 60 (27%) were positive by EIA and 121 (56%) were positive by IFAT. Our results suggest that the EIA technique although highly specific is rather insensitive. This may be because by the time these tests were done the originl nasopharyngeal secretions were considerably diluted and contained more mucus fragments than the call suspension used for IFAT. Of the three techniques, IFAT gives the best results although EIA may be useful where IFAT is not possible.

Dot-dye-immunoassay for the diagnosis of schistosomiasis mansoni

Rabello,Ana Lúcia Teles; Dias Neto,Emmanuel; Garcia,Maria Monica Aguiar; Katz,Naftale
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/1992 EN
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A new serological assay dot-dye-immunoassay (dot-DIA) was evaluated for the diagnosis of schistosomiasis mansoni. This method consist of four steps: (a) biding of antigens to a nitrocellulose membrane (NC); (b) blocking of free sites of the NC; (c) incubation in specific primary antibody; (d) detection of primary antibody reactivity by color development using second antibody coupled to textile dyes. Sera from 82 individuals, 61 with Schistosoma mansoni eggs in the stool and 21 stool negative were tested by ELISA, dot-ELISA, and dotDIA. A high level of agreement between the methods tested was observed for all sera tested: ELISA x dot-ELISA: 95.1%, ELISA x dot-DIA: 92.7% and dot-ELISA x dot-DIA: 97.6%. In this study, dot-DIA proved to be a feasible, sensitive, rapid and practical test for the diagnosis of shcistosomiasis.

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

Lopes,EPA; Granato,CH; Lanzoni,V; Granero,L; Paranhos-Baccala,G; Tomiyama,H; Silva,AEB; Ferraz,ML
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/10/2000 EN
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This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott®), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95%) sera from patients in the study group and negative in 38 of the 39 (97%) sera from those in the control group...

Comparison of a Recombinant-antigen Enzyme Immunoassay with Treponema pallidum Hemagglutination Test for Serological Confirmation of Syphilis

Rodríguez,Islay; Álvarez,Elvio L; Fernández,Carmen; Miranda,Alina
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2002 EN
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A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN TM anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)

Villar,L.M.; Amado,L.A.; Gaspar,A.M.C.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/07/2004 EN
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Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

Validation of immunoassay methods to determine hydrocarbon contamination in estuarine sediments

Fillmann,Gilberto; Bicego,Márcia C.; Zamboni,Ademilson; Fileman,Tim W.; Depledge,Michael H.; Readman,James W.
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2007 EN
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The performance of two commercially available enzyme-linked immunosorbent assay (ELISA) kits (with antibodies attached to magnetic particles) for quantification of hydrocarbons in estuarine sediments is described. The BTEX RaPID Assay® was employed to analyse aliphatic and small aromatic hydrocarbons whilst the c-PAH RaPID Assay® was used to analyse the carcinogenic (> 4 aromatic rings) polycyclic aromatic hydrocarbons. Results were validated by comparison with analyses by gas chromatography (GC)- Flame Ionisation Detection (FID) (with GC-MS confirmation). Correlations between the techniques were good with r² values ranging between 0.68 and 0.97. Disparity between immunoassay and GC techniques were related to differences in the relative compositions of the complex mixtures of hydrocarbons, which alter ELISA responses. Overall, results from the ELISA techniques are shown to compare well with those obtained by GC, confirming ELISA as a useful screening protocol to focus use of the more expensive and time consuming high resolution analytical techniques.

NON-RADIOMETRIC IMMUNOASSAYS [FLUOROIMMUNOASSAY (FIA) AND FLUOROMETRIC ENZYME IMMUNOASSAY (FEIA)] WITH RADIOIMMUNOASSAY (RIA) FOR EVALUATION OF ADRENAL FUNCTION IN NORMAL AND HYPERCORTISOLEMIC DOGS

Jericó,Márcia Marques; Mendonça,Berenice Bilharino de; Otsuka,Mary; Maganin Jr,Aristides; Larsson,Carlos Eduardo
Fonte: Universidade Federal de Santa Maria Publicador: Universidade Federal de Santa Maria
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2002 EN
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Non-radiometric immunoassays offer many advantages over radiometric assays, such as higher stability of kit compounds and absence of potential hazardous effects for users and environment. The comparison of cortisol measurements by fluoroimmunoassay (FIA) and fluorometric enzyme immunoassay (FEIA) with radioimmunoassay (RIA) in adrenal function evaluation of normal (n=50) and hypercortisolemic dogs (n=12) was proposed. Serum concentrations of cortisol were measured in basal conditions and 8 hours after dexamethasone (DEX) suppression (0.01mg/kg/IV). All our reference values were based on the 5th and 95th percentile. The values for basal cortisol of healthy dogs were 0.20 to 2.35mug/d for FIA, 0.30 to 5.39mug/d for FEIA, and 0.65 to 4.64mug/d for RIA. After DEX suppression the values were <0.87mug/d, <0.30mug/d and < 0.80mug/d for FIA, FEIA and RIA, respectively. In hypercortisolemic dogs, the values of cortisol (mean ± SD) in basal and post-DEX conditions were 2.71 + 0.41mug/d and 1.73 + 1.15mug/d for FIA...

Development of an enzyme immunoassay for poliovirus antigens

Hashimoto,Newton; Benati,Fabrício José; Lauretti,Flávio; Linhares,Rosa Elisa Carvalho; Nozawa,Carlos Mitihiko
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2007 EN
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An indirect solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Virus antigen was obtained in LLC-MK2 cell cultures and used to prepare antibodies in rabbit and guinea pig. Antibodies were evaluated by double immunodiffusion and neutralization test. Optimal concentrations of guinea pig and rabbit immunoglobulins were determined by checkerboard titration. Microtitre plates were coated with 15.0 µg/ml guinea pig anti-polio immunoglobulin and rabbit anti-polio immunoglobulin at the concentration of 7.94 µg/ml was used as detecting antibody. The standard curve with eight different antigen concentrations in eight replicates resulted in a coefficient of variation (CV) between 2.1% to 7.8%. The dose-response relationship was determined by simple linear regression with a coefficient of correlation (R²) equal to 96.4%. The assay detected a minimum of 2.3 µg/ml poliovirus antigen.

A comparison between enzyme immunoassay and HPLC for ochratoxin A detection in green, roasted and instant coffee

Fujii,Simone; Ono,Elisabete Yurie Sataque; Ribeiro,Ricardo Marcelo Reche; Assunção,Fernanda Garcia Algarte; Takabayashi,Cássia Reika; Oliveira,Tereza Cristina Rocha Moreira de; Itano,Eiko Nakagawa; Ueno,Yoshio; Kawamura,Osamu; Hirooka,Elisa Yoko
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2007 EN
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An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for ochratoxin A (OTA) detection in green, roasted and instant coffees was developed using anti-OTA monoclonal antibody. Immunological reagents prepared were OTA-BSA (4.76 mg/mL), anti-OTA.7 MAb (2x10³-fold dilution) and HRP-anti IgG (10³-fold dilution). The detection limit was 3.73 ng OTA/g and correlation coefficients (r) between this immunoassay and high performance liquid chromatography were 0.98 for green coffee, 0.98 for roasted and 0.86 for instant. OTA levels detected by ic-ELISA were higher than by HPLC, with ELISA/HPLC ratio of 0.66 - 1.46 (green coffee), 0.96 - 1.11 (roasted) and 0.93 - 1.82 (instant). ELISA recoveries for OTA added to coffee (5 - 70 ng/g) were 81.53 % for green coffee, 46.73 % for roasted and 64.35 % for instant, while recoveries by HPLC were 80.54 %, 45.91 % and 55.15 %, respectively. Matrices interferences were minimized by samples dilution before carrying out the ELISA assay. The results indicate that MAb-based ic-ELISA could be a simple, sensitive and specific screening tool for OTA detection, contributing to quality and safety of coffee products.

Diagnostic performance of an immunoassay for simultaneous detection of HCV core antigen and antibodies among haemodialysis patients

El-Emshaty,Wafaa M; Raafat,Douaa; Elghannam,Doaa M; Saudy,Niveen; Eltoraby,Ehab E; Metwalli,Abd Elhameed A
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2011 EN
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Nosocomial transmission of HCV is a concern in haemodialysis (HD) units worldwide. Diagnosis of HCV infection among dialysis patients is currently based on the detection of anti HCV antibodies by ELISA, and is confirmed by HCV RNA. The average window period between HCV infection and seroconversion with new generations of HCV antibody tests remains approximately 70 days with more prolonged period among dialysis patients. In this study we assessed the diagnostic performance of an immunoassay designed for simultaneous detection of anti HCV antibodies and core antigen in one step in comparison to qualitative RT-PCR and anti HCV antibodies detection test among Egyptian haemodialysis patients. The studied patients were 39 chronic renal failure patients on maintenance haemodialysis. The results obtained in the present study revealed HCV infection of 56.4%. Combined Ag/Ab test detected 3 out of the 4 anti-HCV negative viraemic patients who were in the window period. The sensitivity, specificity and accuracy of the test were higher than that of anti HCV antibodies detection test (95.45%, 94.1% and 94.87% versus 81.8%, 88.23% and 84.6%) and they were raised to 100% on combining its positivity with liver enzymes elevation results. Therefore, this simple combined Ag/Ab test can be applied for early detection of HCV infection during window period among HD patients as an alternative to HCV RNA detection.

Seradyn quantitative microsphere system lamotrigine immunoassay on a Hitachi 911 analyzer compared with HPLC-UV

Westley, I.; Morris, R.
Fonte: Lippincott Williams & Wilkins Publicador: Lippincott Williams & Wilkins
Tipo: Artigo de Revista Científica
Publicado em //2008 EN
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Lamotrigine (LTG) is used currently as monotherapy or, more frequently, as add-on therapy with other antiepileptic drugs. It demonstrates efficacy against partial seizures, primary and secondary tonic clonic seizures, absence seizures, and drop attacks. LTG pharmacokinetics is complicated by coadministration with other antiepileptic drugs such as valproic acid, phenytoin, or carbamazepine. The wide interpatient variability in LTG dosage required to attain therapeutic plasma LTG concentrations for seizure control suggests that LTG is a good candidate for therapeutic drug monitoring (TDM). In this study, we compared the quantitative microsphere system (QMS) LTG immunoassay with the LTG high-performance liquid chromatography-ultra violet (HPLC-UV) assay routinely employed for TDM in our laboratory. Samples tested by these methods were patient samples presented for TDM and from a quality assurance program. Quality control material demonstrated within- and between-run (n = 6) coefficient of variation and biases of less than 10%. Patient samples demonstrated a Deming regression of QMS = 1.09 HPLC-UV - 0.17 and quality assurance program samples had a Deming regression of QMS = 1.03 HPLC-UV - 0.11. Patient samples demonstrated a mean bias of 6.1% and quality assurance program samples had a mean bias of 0.2%. The QMS LTG assay had a clinically small but significant overestimation of plasma LTG concentrations. It may be useful as a convenient alternative method that would provide TDM guidance if a chromatographic assay was not available.; Westley...

Thermodynamic and kinetic characterisation of antibody / hapten pairs and optimisation of an immunoassay of fluorescence in homogeneous phase; Thermodynamische und kinetische Charakterisierung von Antikörper / Hapten Paaren und Optimierung von einem Fluoreszenzimmunoassay in homogener Phase

Coille, Ingrid
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
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Zuerst wurde ein monoklonaler Anti-Estradiol-Antikörper mit Hilfe der SPR (Oberflächenplasmonenresonanz) vollständig charakterisiert. Die aktive Konzentration des Antikörpers wurde bestimmt. Die Gleichgewichtskonstanten, die kinetischen Konstanten und die thermodynamischen Variablen wurden gemessen. Die Bestimmung der Aktivität, der Affinität und der Kinetik von drei polyklonalen Antikörpern wurde mit SPR durchgeführt. Die Affinitätskonstanten wurden parallel dazu mit Reflektometrische Interferenzspektroskopie bestimmt. Die verwendeten Antikörper waren Anti-Östron, Anti-Östradiol und Anti-Östrogen. Die Analyte waren natürliche und synthetische Östrogene. Die auf der Oberfläche und in der Lösung bestimmten Affinitätskonstanten sowie die mit den zwei Methoden bestimmten Affinitätskonstanten wurden mit statistischen Tests verglichen. Für den Einsatz dieser Antikörper/Hapten-Systeme in einem Fluoroimmunoassay wurden außerdem die aktiven Konzentrationen und die Affinitätskonstanten vor und nach dem Markieren mit dem Fluorophor Cy5 gemessen. Simulierungen des Fluoroimmunoassays zeigten den Einfluss von wichtigen Parametern auf einen kompetitiven Immunoassay. Die mit SPR untersuchten Antikörper/Analyte-Systeme wurden in einem ETIA (Energietransfer Immunoassay) in Lösung eingesetzt. Der Assay wurde in Mikrotiterplatten entwickelt. Die gemessenen Kalibrierkurven wurden mit den gemessenen Affinitätskonstanten und den Simulationen verglichen. Der ETIA wurde bei verschiedenen Messungen von Östrogenen in synthetischem Abwasser validiert. Der letzte Schritt bestand in der Miniaturisierung des ETIA. Der Assay wurde erfolgreich in einer Nanotiterplatte mit einem Wellsvolumen von 70 nl gemessen. Der Fluoroimmunoassay wurde auf ein Antikörper/Protein-Paar (Vitellogenin/Anti-vitellogenin-Antikörper) angewendet. Die Fähigkeit zur Bindung von markiertem Antikörper und Protein wurde mit einem Fluoroimmunoassay in heterogener Phase geprüft.; A part of this study consisted of the characterisation of different systems of antibodies and analytes and analyte derivatives. A monoclonal anti-estradiol antibody (15H11) was fully characterised by surface plasmon resonance (SPR). The active concentration decreased with increasing dilution. The kinetic rate constants at several temperatures allowed to determine the affinity constants and thermodynamic constants. The antibody showed affinity for estradiol and ethinylestradiol and according to the thermodynamic results the binding was entropy driven. SPR was also used to characterise three polyclonal anti-estrogen antibodies. The average of active concentration of each polyclonal antibody was measured...

Der Weg vom Heterogenen Immunoassay zum Immunosensor: Prinzipien und Applikationen im Bereich der Klinischen Chemie

Luppa, Peter B.
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Sonstiges
DE_DE
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Immunosensoren sind ligandenaffinitäts-messende Sensoren, bei denen immunochemische Reaktionen über eine Transducerschaltung aufgezeichnet werden. Das fundamentale Merkmal aller Immunosensoren ist hierbei die Spezifität von Antikörpern zu geeigneten Analyten unter Ausbildung eines stabilen Immunkomplexes. Dies ist auch die Basis der Immunoassay-Methodologie. Im Vergleich beider Methoden ermöglicht die Transducer-Tech-nologie eine markierungsfreie Detektion und zugleich eine Quantifizierung des gebildeten Immunkomplexes. In den letzten Jahren wurden Immunoassays auch verstärkt zur Spurenanalyse von toxischen Substanzen in der pharmazeutischen und der Nahrungsmittelindustrie sowie in der Umweltanalytik eingesetzt. Entsprechende Mess-ungen können hierbei jedoch nur diskontinuierlich erfolgen. Um eine kontinuierliche Analytik zu ermöglichen, er-scheint der Einsatz von Immunosensoren besonders vielversprechend. Auch im Bereich der klinischen Diagnostik gibt es viele Anwendungsgebiete, für die ein kontinuierliches „Monitoring“ von verschiedenen Analyten von großem Nutzen wäre. Deshalb sollten gerade Labormediziner die Vorteile des Einsatzes von Immunosensoren in der klinische Diagnostik und Laboratoriumsmedizin bedenken. Dieser Vortrag wendet sich somit vor allem an Klinische Chemiker...

Serodiagnosis of Trypanosoma cruzi Infection Using the New Particle Gel Immunoassay - ID-PaGIA Chagas

Rabello,Ana; Luquetti,Alejandro O; Moreira,Eliana F; Gadelha,Maria de Fátima; Santos,José Alisson dos; Melo,Laércio de; Schwind,Peter
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/1999 EN
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The ID-Chagas test is a particle gel immunoassay (PaGIA). Red coloured particles are sensitised with three different synthetic peptides representing antigen sequences of Trypanosoma cruzi: Ag2, TcD and TcE. When these particles are mixed with serum containing specific antibodies, they agglutinate. The reaction mixture is centrifuged through a gel filtration matrix allowing free agglutinated particles to remain trapped on the top or distributed within the gel. The result can be read visually. In order to investigate the ability of the ID-PaGIA to discriminate negative and positive sera, 111 negative and 119 positive, collected in four different Brazilian institutions, were tested by each of the participants. All sera were previously classified as positive or negative according to results obtained with three conventional tests (indirect immunofluorescence, indirect hemaglutination, and enzime linked immunosorbent assay). Sensitivity rates of ID-PaGIA varied from 95.7% to 97.4% with mean sensitivity of 96.8% and specificity rates varied from 93.8 to 98.8% with mean specificity of 94.6%. The overall Kappa test was 0.94. The assay presents as advantages the simplicity of operation and the reaction time of 20 min. In this study, ID-PaGIA showed to be highly sensitive and specific.

A flow immunoassay for alkylphenol ethoxylate surfactants and their metabolites—questions associated with cross-reactivity, matrix effects, and validation by chromatographic techniques

Badea, Mihaela; Nistor, Catalin; Goda, Yasuhiro; Fujimoto, Shigeru; Dosho, Shin; Danet, Andrei; Barceló, Damià; Ventura, Francesc; Emnéus, Jenny
Fonte: Royal Society of Chemistry (Great Britain) Publicador: Royal Society of Chemistry (Great Britain)
Tipo: Artículo Formato: 117498 bytes; application/pdf
ENG
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8 pages, 5 figures, 5 tables.-- Printed version published Jul 2003.-- Supporting iniformation available at: http://www.rsc.org/suppdata/an/b3/b302110f/; This paper describes the application and evaluation of a competitive enzyme flow injection immunoassay (EFIIA) for screening of alkylphenol ethoxylate (APEO) surfactants in different water samples based on a generic immunoassay system previously developed (see E. Burestedt, C Nistor, U. Schagerlöf and J. Emnéus, Anal. Chem., 2000, 72, 4171–4177). The detection limits for octylphenol ethoxylates (OPEOs), nonylphenol ethoxylates (NPEOs), and nonylphenol (NP) were 0.5 µg l–1, between 2 and 3 µg l–1, and 50 µg l–1, respectively, with a sample throughput of 6 h–1 (i.e., for triplicate analysis of each sample). Different OPEOs and NPEOs were highly cross-reactive within the assay, with sensitivities in the same order of magnitude for all the ethoxylates tested, thus the result obtained by the EFIIA method could be used as an alkylphenol ethoxylate index. No or minor matrix effects with recoveries between 70–120% for the reference analyte NPEO10 in tap, and surface water, and acceptable for rainwater, were observed. Influent and effluent surfactant containing wastewater samples were analysed by EFIIA...

Sensitivity and specificity of a competitive enzyme immunoassay in the serodiagnosis of bovine brucellosis; Sensibilidade e especificidade de um teste imunoenzimático competitivo no diagnóstico sorológico da brucelose bovina

Mathias, Luis Antonio; Macmillan, Allastair Paul; Greiser-Wilke, I.; Moennig, V.
Fonte: Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia Publicador: Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; ; ; Formato: application/pdf
Publicado em 03/01/1993 ENG
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The purpose of this work was to evaluate the sensitivity and the specificity of a competitive enzyme immunoassay, using as conjugate the monoclonal antibodies BM-38 and BM-40, in the serodiagnosis of bovine brucellosis. Seventy-four sera from culture-positive cattle and 2,118 cattle sera from herds free from brucellosis and negative to the Rose Bengal plate test were examined. The competitive enzyme immunoassay, using any of the two conjugates, was able to reveal the presence of antibodies to Brucella lipopolysaccharide in all of the 74 sera of the infected cattle, resulting in a sensitivity of 100%. The specificity of the test using the conjugate BM-38 was 98.82% and using the conjugate BM-40 was 99.95%. These results indicated that the competitive enzyme immunoassay, mainly when using the conjugate BM-40, consists in a technique very useful in the confirmation of the serological diagnosis of bovine brucellosis.; O trabalho teve por objetivo avaliar a sensibilidade e a especificidade de um teste imunoenzimático competitivo, empregando como conjugado os anticorpos monoclonais BM-38 e BM-40, no diagnóstico sorológico da brucelose bovina. Foram examinados 74 soros de bovinos dos quais havia sido isolada Brucella abortus e 2.118 soros de bovinos procedentes de rebanhos livres de brucelose e que apresentaram resultado negativo quando submetidos ao teste Rosa Bengala. O teste imunoenzimático competitivo...

Monitorização do vale e pico sérico de vancomicina em recém-nascidos de termo: comparação entre as técnicas de cromatografia líquida de alta eficácia e imunoensaio por fluorescência polarizada; Vancomycin monitoring in term newborns: comparison of peak and trough serum concentrations determined by high performance liquid chromatography and fluorescence polarization immunoassay

Feferbaum, Rubens; Machado, José Kleber Kobol; Diniz, Edna Maria de Albuquerque; Okay, Thelma S.; Santos, Silvia R. C. J.; Ceccon, Maria Esther J.R.; Krebs, Vera L. J.; Araújo, Maria C. K. de; Vaz, Flávio Adolfo Costa
Fonte: Universidade de São Paulo. Faculdade de Medicina Publicador: Universidade de São Paulo. Faculdade de Medicina
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; ; ; ; Formato: application/pdf
Publicado em 01/10/2001 ENG
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INTRODUÇÃO: Foi realizada monitorização dos níveis séricos de vancomicina em recém-nascidos de termo com sepse ou suspeita de sepse Staphylococcus sp., através da cromatografia líquida de alta eficácia (HPLC) e imunoensaio por fluorescência polarizada (FPIA). OBJETIVO: Verificar a existência de correlação estatística entre os resultados obtidos pelas duas técnicas. MÉTODO E CASUÍSTICA: Foram obtidas dezoito e vinte concentrações séricas de vancomicina no pico e vale respectivamente, em recém-nascidos de termo, no período de outubro de 1995 a outubro de 1997. RESULTADO: O coeficiente de correlação linear para pico sérico foi de 0,27, p=0,110 e para vale sérico 0,26, p= 0,1045 não sendo estatisticamente significativo, não sendo estatisticamente significativo. CONCLUSÃO: Apesar da pequena casuística, não houve correlação estatisticamente significante entre os resultados obtidos pelos duas técnicas.; INTRODUCTION: Peak and trough serum concentrations of vancomycin were determined in term newborn infants with confirmed or suspected Staphylococcus sp sepsis by high performance liquid chromatography and flourescence polarization immunoassay. OBJECTIVE: To statistically compare the results of the high performance liquid chromatography and flourescence polarization immunoassay techniques for measuring serum vancomycin concentrations. METHODS: Eighteen peak and 20 trough serum samples were assayed for vancomycin concentrations using high performance liquid chromatography and flourescence polarization immunoassay from October 1995 to October 1997. RESULTS: The linear correlation coefficients for high performance liquid chromatography and flourescence polarization immunoassay were 0.27 (peak...