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1.
Gen Comp Endocrinol ; 293: 113494, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32333913

RESUMO

Integrative behavioral ecology requires accurate and non-invasive measures of hormone mediators for the study of wild animal populations. Biologically sensitive assay systems for the measurement of hormones and their metabolites need to be validated for the species and sample medium (e.g. urine, feces, saliva) of interest. Where more than one assay is available for hormone (metabolite) measurement, antibody selection is useful in identifying the assay that tracks changes in an individuals endocrine activity best, i.e., the most biologically sensitive assay. This is particularly important when measuring how glucocorticoids (GCs) respond to the subtle, additive effects of acute stressors during a predictable metabolic challenge, such as gestation. Here, we validate a group-specific enzyme immunoassay, measuring immunoreactive 11ß-hydroxyetiocholanolone, for use in a wild primate, geladas (Theropithecus gelada). This group-specific assay produced values correlated with those from a previously validated double-antibody, corticosterone 125I radioimmunoassay. However, the results with the group-specific assay showed a stronger response to an ACTH challenge and identified greater variation in gelada immunoreactive fecal glucocorticoid metabolites (iGCMs) compared with the corticosterone assay, indicating a higher biological sensitivity for assessing adrenocortical activity. We then used the group-specific assay to: (1) determine the normative pattern of iGCM levels across gelada gestation, and (2) identify the ecological, social, and individual factors that influence GC output for pregnant females. Using a general additive mixed model, we found that higher iGCM levels were associated with low rank (compared to high rank) and first time mothers (compared to multiparous mothers). This study highlights the importance of assay selection and the efficacy of group-specific assays for hormonal research in non-invasively collected samples. Additionally, in geladas, our results identify some of the factors that increase GC output over and above the already-elevated GC concentrations associated with gestation. In the burgeoning field of maternal stress, these factors can be examined to identify the effects that GC elevations may have on offspring development.


Assuntos
Fezes/química , Glucocorticoides/metabolismo , Metaboloma , Paridade , Theropithecus/metabolismo , Animais , Animais Selvagens/metabolismo , Corticosterona/metabolismo , Feminino , Técnicas Imunoenzimáticas , Masculino , Modelos Biológicos , Gravidez , Radioimunoensaio , Reprodutibilidade dos Testes
2.
PLoS One ; 15(4): e0231257, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32255800

RESUMO

Endogenous oxytocin has been associated with different aspects of social cognition in healthy subjects and patients with schizophrenia. In this pilot study, we investigated the relationship between plasma oxytocin and oxytocin level changes induced by empathy-eliciting, attachment-related movie scenes with correlates of cognitive and emotional empathy in patients and healthy controls. The Multifaceted Empathy Test (MET) and the Interpersonal Reactivity Index (IRI) were administered to patients with schizophrenia (N = 35, 12 females) and healthy controls (N = 35, 12 females) to estimate dimensions of cognitive and emotional empathy. Peripheral basal oxytocin concentrations and oxytocin responses to movie-based emotional stimuli were assessed using radioimmunoassay with sample extraction. In patients, induced oxytocin level changes were inversely correlated with MET cognitive empathy regarding negative emotional states. Controlling for non-social cognition and age revealed a significant negative association between basal oxytocin levels and MET cognitive empathy for positive emotions. In healthy subjects, oxytocin reactivity was inversely correlated with the IRI subscale "fantasy". Oxytocin was not related to any measure of emotional empathy. A hyper-reactive oxytocin system might be linked to impaired cognitive empathy as a part of a dysfunctional regulative circuit of attachment-related emotions and interpersonal stressors or threats by attribution of meaning. Healthy adults with a disposition to identify with fictional characters showed lower oxytocin reactivity, possibly indicating familiarity with movie-based stimuli. The oxytocinergic system may be involved in maladaptive coping mechanisms in the framework of impaired mentalizing and associated dysfunctional responses to interpersonal challenges in schizophrenia.


Assuntos
Cognição , Empatia , Ocitocina/fisiologia , Psicologia do Esquizofrênico , Adaptação Psicológica , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ocitocina/sangue , Projetos Piloto , Radioimunoensaio
3.
Clin Chem ; 66(3): 483-492, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32068832

RESUMO

BACKGROUND: Many medications (including most antihypertensives) and physiological factors affect the aldosterone/renin ratio (ARR) when screening for primary aldosteronism (PA). We sought to validate a novel equilibrium angiotensin II (eqAngII) assay and compare correlations between the aldosterone/angiotensin II ratio (AA2R) and the current ARR under conditions affecting the renin-angiotensin system. METHODS: Among 78 patients recruited, PA was excluded in 22 and confirmed in 56 by fludrocortisone suppression testing (FST). Peripheral levels of eqAngII, plasma renin activity (PRA) and direct renin concentration (DRC) were measured. RESULTS: EqAngII showed good consistency with DRC and PRA independent of PA diagnosis, posture, and fludrocortisone administration. EqAngII showed close (P < 0.01) correlations with DRC (r = 0.691) and PRA (r = 0.754) during FST. DRC and PRA were below their assays' functional sensitivity in 43.9% and 15.1%, respectively, of the total 312 samples compared with only 7.4% for eqAngII (P < 0.01). Bland-Altman analysis revealed an overestimation of PRA and DRC compared with eqAngII in a subset of samples with low renin levels. The AA2R showed not only consistent changes with the ARR but also close (P < 0.01) correlations with the ARR, whether renin was measured by DRC (r = 0.878) or PRA (r = 0.880). CONCLUSIONS: Dynamic changes of eqAngII and the AA2R show good consistency and close correlations with renin and the ARR. The eqAngII assay shows better sensitivity than DRC and PRA assays, especially at low concentrations. Whether the AA2R can reduce the impact of some factors that influence the diagnostic power of the ARR warrants further study.


Assuntos
Angiotensina II/sangue , Hiperaldosteronismo/diagnóstico , Espectrometria de Massas em Tandem/métodos , Adolescente , Adulto , Idoso , Aldosterona/sangue , Cromatografia Líquida de Alta Pressão , Feminino , Fludrocortisona/química , Humanos , Imunoensaio , Masculino , Pessoa de Meia-Idade , Radioimunoensaio , Renina/sangue , Adulto Jovem
4.
Endocrinol. diabetes nutr. (Ed. impr.) ; 67(1): 43-52, ene. 2020. tab
Artigo em Espanhol | IBECS | ID: ibc-186146

RESUMO

ANTECEDENTES Y OBJETIVOS: El alelo de riesgo (G) de la variante rs10830963 en el gen del receptor de melatonina 1 B (MTNR1B) se relaciona con la obesidad. En este trabajo evaluamos el efecto de este SNP sobre los factores de riesgo cardiovascular y la pérdida de peso secundaria a 2 dietas hipocalóricas. MÉTODOS: Trescientos sesenta y un sujetos obesos fueron asignados aleatoriamente durante 3 meses (dieta M: dieta hipocalórica alta en grasas monoinsaturadas vs. dieta P: dieta hipocalórica alta en grasas poliinsaturadas). Se midieron los parámetros antropométricos, glucemia en ayunas, proteína C reactiva, concentración de insulina, resistencia a la insulina (HOMA-IR), perfil de lípidos y los niveles de adipocitoquinas. Se evaluó el genotipo del polimorfismo del gen MTNR1B (rs10830963). RESULTADOS: Todos los parámetros antropométricos, la presión arterial sistólica y los niveles de leptina disminuyeron en todos los sujetos después de ambas dietas. Esta mejora de los parámetros antropométricos fue mayor en los no portadores del alelo G que en los portadores del alelo G. Tras la intervención con dieta M (CC vs. CG + GG), el colesterol total (delta: -10,4 ± 2,1 mg/dl vs. -6,4 ± 1,2 mg/dl: p < 0,05), colesterol LDL (delta: -7,1 ± 0,9 mg/dl vs. -2,8 ± 0,8 mg/dl: p < 0,05), insulina (delta: -3,0 ± 0.8 UI/l vs. -2,0 ± 1,0 UI/l: p < 0,05) y HOMA IR (delta: -3,4 ± 1,0 unidades vs. -2,9 ± 0,9 unidades: p < 0,05) mejoraron en los no portadores del alelo G. Tras la dieta P, en el grupo de sujetos sin alelo G, los niveles de insulina (delta: -2,9 ± 1,0 UI/l vs. -0,6 ± 0,2 UI/l: p < 0,05) y HOMA-IR (delta [CC vs. CG + GG]: -0,8 ± 0,2 unidades vs. -0,4 ± 0,3 unidades: p < 0,05) también disminuyeron. CONCLUSIONES: Nuestro estudio detectó una relación de la variante rs10830963 de MTNR1B con la pérdida de peso corporal y la modificación de la resistencia a la insulina inducida por 2 dietas hipocalóricas diferentes. Solo la dieta hipocalórica enriquecida en grasa monoinsaturada y los no portadores del alelo G mostraron un efecto significativo sobre las lipoproteínas


BACKGROUND & AIMS: The risk allele (G) of rs10830963 in the melatonin receptor 1 B (MTNR1B) gene presents an association with obesity. We study the effect of this SNP on cardiovascular risk factors and weight loss secondary to 2hypocaloric diets. Methods: 361 obese subjects were randomly allocated during 3 months (Diet M - high monounsaturated fat hypocaloric diet vs. Diet P - high polyunsaturated fat hypocaloric diet). Anthropometric parameters, fasting blood glucose, C-reactive protein (CRP), insulin concentration, insulin resistance (HOMA-IR), lipid profile and adipocytokines levels were measured. Genotype of MTNR1B gene polymorphism (rs10830963) was evaluated. Results: All anthropometric parameters, systolic blood pressure and leptin levels decreased in all subjects after both diets. This improvement of anthropometric parameters was higher in non G allele carriers than G allele carriers. After dietary intervention with Diet M, (CC vs. CG + GG); total cholesterol (delta: -10.4 ± 2.1 mg/dl vs. -6.4 ± 1.2 mg/dl: P <.05), LDL-cholesterol (delta:-7.1 ± 0.9 mg/dl vs. -2.8 ± 0.8 mg/dl: P <.05), insulin (delta:-3.0 ± 0.8 UI/L vs. -2.0 ± 1.0 UI/L: P<.05) and HOMA-IR (delta:-3.4 ± 1.0 units vs. -2.9 ± 0.9 units: P<.05) improved in no G allele carriers. After Diet P, in the group of subjects without G allele CC, insulin levels (delta: -2.9 ± 1.0 UI/L vs. -0.6 ± 0.2 UI/L: P <.05) and HOMA-IR (delta (CC vs. CG + GG): -0.8 ± 0.2 units vs. -0.4 ± 0.3 units: P <.05) decreased, too. Conclusions: Our study detected a relationship of rs10830963 MTNR1B SNP with body weight loss and insulin resistance modification induced by 2 different hypocaloric. Only monounsaturated enriched hypocaloric diet and in no-G allele carriers showed a significant effect on lipoproteins


Assuntos
Humanos , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Polimorfismo Genético , Gorduras na Dieta/uso terapêutico , Resistência à Insulina , Perda de Peso/efeitos dos fármacos , Obesidade/dietoterapia , Fatores de Risco , Gorduras na Dieta/metabolismo , Doenças Cardiovasculares , Antropometria , Glicemia , Composição Corporal , Relação Cintura-Quadril , Radioimunoensaio/métodos
5.
Horm Behav ; 118: 104682, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31927020

RESUMO

The first issue of Hormones and Behavior was published 50 years ago in 1969, a time when most of the techniques we currently use in Behavioral Endocrinology were not available. Researchers have during the last 5 decades developed techniques that allow measuring hormones in small volumes of biological samples, identify the sites where steroids act in the brain to activate sexual behavior, characterize and quantify gene expression correlated with behavior expression, modify this expression in a specific manner, and manipulate the activity of selected neuronal populations by chemogenetic and optogenetic techniques. This technical progress has considerably transformed the field and has been very beneficial for our understanding of the endocrine controls of behavior in general, but it did also come with some caveats. The facilitation of scientific investigations came with some relaxation of methodological exigency. Some critical controls are no longer performed on a regular basis and complex techniques supplied as ready to use kits are implemented without precise knowledge of their limitations. We present here a selective review of the most important of these new techniques, their potential problems and how they changed our view of the hormonal control of behavior. Fortunately, the scientific endeavor is a self-correcting process. The problems have been identified and corrections have been proposed. The next decades will obviously be filled with exciting discoveries in behavioral neuroendocrinology.


Assuntos
Comportamento/fisiologia , Invenções/história , Invenções/tendências , Neuroendocrinologia/história , Neuroendocrinologia/tendências , Animais , Comportamento Animal/fisiologia , Técnicas de Silenciamento de Genes/história , Técnicas de Silenciamento de Genes/métodos , Técnicas de Silenciamento de Genes/tendências , História do Século XX , História do Século XXI , Humanos , Hibridização In Situ/história , Hibridização In Situ/métodos , Hibridização In Situ/tendências , Neuroendocrinologia/métodos , Optogenética/história , Optogenética/métodos , Optogenética/tendências , Radioimunoensaio/história , Radioimunoensaio/métodos , Radioimunoensaio/tendências , Técnicas Estereotáxicas/história , Técnicas Estereotáxicas/tendências
6.
J Diabetes Investig ; 11(2): 356-362, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31267698

RESUMO

AIMS/INTRODUCTION: The aim of the present study was to compare the clinical and genetic characteristics between people with type 1 diabetes who were positive and negative for autoantibodies against glutamic acid decarboxylase (GADA) measured by enzyme-linked immunosorbent assay (ELISA) with low-titer GADA measured by radioimmunoassay. MATERIALS AND METHODS: Among Japanese people with type 1 diabetes in whom GADA were measured by both ELISA and radioimmunoassay, those who had low titers of GADA measured by radioimmunoassay (1.5-10 U/mL), regardless of positivity for GADA measured by ELISA, were studied. There were 65 participants with acute-onset type 1 diabetes and 30 participants with slowly progressive insulin-dependent diabetes mellitus. Clinical characteristics and human leukocyte antigen types were compared in ELISA-positive (≥5 U/mL) and ELISA-negative participants. Endogenous insulin secretion was evaluated by C-peptide index. RESULTS: Among participants with slowly progressive insulin-dependent diabetes mellitus, postprandial C-peptide index was significantly higher in ELISA-negative participants than in ELISA-positive participants (r = 0.619, P = 0.002). Among 52 participants whose human leukocyte antigen typing was carried out, all of the participants with slowly progressive insulin-dependent diabetes mellitus who had DRB1*09:01 were positive by GADA-ELISA (P = 0.021). In acute-onset type 1 diabetes participants, there were no significant differences for the C-peptide index and human leukocyte antigen genotypes. CONCLUSIONS: The difference in the positivity for GADA-ELISA might reflect cytotoxicity toward pancreatic ß-cells and preservation of endogenous insulin secretion in people with slowly progressive insulin-dependent diabetes mellitus. We also suggest that the difference in the GADA-ELISA-specific epitope depends on the human leukocyte antigen genotype.


Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Adulto , Estudos Transversais , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/enzimologia , Ensaio de Imunoadsorção Enzimática , Feminino , Glutamato Descarboxilase/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Radioimunoensaio
7.
J Immunoassay Immunochem ; 41(2): 195-207, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31885329

RESUMO

A radioimmunoassay (RIA) method for progesterone has been developed. It has been validated for the determination of progesterone in cow skim milk. The main reagents used in the development work were prepared and characterized in-house. The assay uses a radioiodinated tracer purified by gel filtration on Sephadex G-25, standards prepared in skim milk and coated tubes with specific antibodies as the solid phase (separation system). The radiochemical purity of the tracer was greater than 95%, the maximum binding using solid phase reached 43% and the nonspecific binding didn't exceed 5%. Series of progesterone standards using milk matrix with concentration ranging from 0 to 40ng/mL were prepared. Detection limit of the assay was 0.13ng/mL and the precision evaluation gives an intra and inter-assay coefficient variations between 4.94% and 12.66%. The recovery obtained with skim milk samples was 84% to 119% and the parallelism test indicated good linearity (R2 > 0.99). The clinical tests give high correlation coefficient (r = 0.998) between progesterone concentrations of cow's skim milk assayed using developed progesterone RIA kit and commercial progesterone RIA kit.


Assuntos
Leite/química , Progesterona/análise , Radioimunoensaio , Animais , Bovinos , Progesterona/imunologia , Controle de Qualidade
8.
Gen Comp Endocrinol ; 285: 113226, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374286

RESUMO

A "reproducibility crisis" is widespread across scientific disciplines, where results and conclusions of studies are not supported by subsequent investigation. Here we provide a steroid immunoassay example where human errors generated unreproducible results and conclusions. Our study was triggered by a scientific report citing abnormally high concentrations (means of 4-79 ng L-1) of three natural sex steroids [11-ketotestosterone (11-KT), testosterone (T) and oestradiol (E2)] in water samples collected from two UK rivers over 4 years (2002-6). Furthermore, the data suggested that trout farms were a major source because reported steroid concentrations were 1.3-6 times higher downstream than upstream. We hypothesised that the reported levels were erroneous due to substances co-extracted from the water causing matrix effects (i.e. "false positives") during measurement by enzyme-linked immunoassay (EIA). Thus, in collaboration with three other groups (including the one that had conducted the 2002-6 study), we carried out field sampling and assaying to examine this hypothesis. Water samples were collected in 2010 from the same sites and prepared for assay using an analogous method [C18 solid phase extraction (SPE) followed by extract clean-up with aminopropyl SPE]. Additional quality control ("spiked" and "blank") samples were processed. Water extracts were assayed for steroids using radioimmunoassay (RIA) as well as EIA. Although there were statistically significant differences between EIA and RIA (and laboratories), there was no indication of matrix effects in the EIAs. Both the EIAs and RIAs (uncorrected for recovery) measured all three natural steroids at <0.6 ng L-1 in all river water samples, indicating that the trout farms were not a significant source of natural steroids. The differences between the two studies were considerable: E2 and T concentrations were ca. 100-fold lower and 11-KT ca. 1000-fold lower than those reported in the 2002-6 study. In the absence of evidence for any marked changes in husbandry practice (e.g. stock, diet) or environmental conditions (e.g. water flow rate) between the study periods, we concluded that calculation errors were probably made in the first (2002-6) study associated with confusion between extract and water sample concentrations. The second (2010) study also had several identified examples of calculation error (use of an incorrect standard curve; extrapolation below the minimum standard; confusion of assay dilutions during result work-up; failure to correct for loss during extraction) and an example of sample contamination. Similar and further errors have been noted in other studies. It must be recognised that assays do not provide absolute measurements and are prone to a variety of errors, so published steroid levels should be viewed with caution until independently confirmed.


Assuntos
Aquicultura , Água Doce , Imunoensaio/métodos , Esteroides/análise , Truta/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Radioimunoensaio , Padrões de Referência , Reprodutibilidade dos Testes , Rios , Água/química
9.
Pak J Pharm Sci ; 32(5): 1987-1993, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31813862

RESUMO

Imposex is a genital disorder characterized by imposition of male sexual characteristics in female gastropods due to exposure to tributyltin (TBT). TBT is used as biocidal agent in antifouling paints, applied on the ship hulls and marine submerged structures such as fishing gears and buoys. In the present study bioassay experiment was carried out to determine imposex inductive and endocrine disruptive effect of TBT in two species of gastropods of genus Thais. In this experiment normal specimens of T. bufo and T. rudolphi were exposed to three different concentrations (100, 500 and 1000ngl-1) of TBTCl for four weeks in laboratory and at the end of experiment level of free testosterone and TBT body burden was estimated by radioimmunoassay and gas chromatograph coupled with a flame photometric detector respectively. In both tested species exposed to 500 and 1000ngl-1 of TBT imposex stages developed, while in 100ng l-1 and control groups showed no imposex condition. Elevation of free testosterone level in imposex females has also been observed. These observations indicate that the TBT act as potential imposex inducer and endocrine disruptor in the targeted gastropod species and these species can be used as sensitive biomonitoring tool for TBT contamination.


Assuntos
Gastrópodes/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Compostos de Trialquitina/efeitos adversos , Animais , Bioensaio/métodos , Monitoramento Ambiental/métodos , Feminino , Gastrópodes/metabolismo , Masculino , Radioimunoensaio/métodos , Testosterona/metabolismo , Poluentes Químicos da Água/efeitos adversos
10.
Pol J Vet Sci ; 22(4): 695-701, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31867920

RESUMO

Progesterone (P4) is responsible for the main reproduction processes. Concentration of P4 varies widely among different determination methods, and interpretation of these values may be difficult. The objective of the current study was to assess the agreement of three different enzyme immunoassays (ELISA) in relation to radioimmunoassay (RIA) of P4 concentration assessment of beef cow serum samples. Samples were collected randomly considering high (pregnant cows) and low (non-pregnant cows) P4 concentrations. Depending on the P4 assessment method, four groups were created as follows: Group 1 - direct samples assessed by ELISA, Group 2 - extracted samples assessed by ELISA, Group 3 - samples assessed by automated ELISA, and Group 4 - samples assessed by RIA. The mean progesterone concentration was 4.50 ng/mL, 1.24 ng/mL, 4.07 ng/mL and 4.39 ng/mL from Group 1 to Group 4, respectively. The mean difference (MD) between Group 1, Group 2 and Group 3 individually compared with Group 4 was -0.10 ± 1.24 ng/mL, 3.15 ± 3.58 ng/mL and 0.33 ± 1.42 ng/mL, and the 95% confidence interval (CI) for the differences (s) was from -0.99 to 0.78 ng/mL, from 0.59 to 5.71 ng/mL, and from -0.69 to 1.34 ng/mL, respectively. The confidence interval for the lower and upper limit of the agreement ranged from -4.12 to -1.05 ng/mL and from 0.84 to 3.91 ng/mL between Group 1 and Group 4, from -8.45 to 0.42 ng/ mL and from 5.88 to 14.75 ng/mL between Group 2 and Group 4, from -4.29 to -0.76 ng/mL, and from 1.41 to 4.94 ng/mL between Group 3 and Group 4. Our findings show that the best agreement with RIA was observed for Group 1 and Group 3, while the agreement in the extraction method was least accurate.


Assuntos
Análise Química do Sangue/veterinária , Bovinos/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Progesterona/sangue , Radioimunoensaio/veterinária , Animais , Análise Química do Sangue/métodos , Feminino
11.
Clin Lab ; 65(11)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710438

RESUMO

BACKGROUND: Radioimmunoassays, which are often not automated and time-consuming, are gradually being re-placed in medical laboratories by non-radioactive methods that need to be evaluated. The purpose was to compare the measurement of thyroid-stimulating hormone receptor antibodies (TRAb) by the new Brahms' kit using Kryptor TRACE technology and the Brahms' radioimmunoassay. METHODS: We prospectively collected all samples from patients who received thyroid-stimulating hormone receptor antibodies testing in July 2018 at the University Hospital of Brest. The radioimmunoassay used was the Dynotest TRAK human by BRAHMS Diagnostica (Berlin, Germany). The Kryptor method used the BRAHMS TRAK human Kryptor kit performed with the Kryptor Compact Plus system. RESULTS: The inter-assay coefficient variations for the radioimmunological and Kryptor methods were 11.07% and 8.36%, respectively, with the low level quality control and 8.36% and 4.38%, respectively, with the high level quality control. Forty-four patients were included in the study including thirty-two Graves' disease patients in follow-up. The sensitivity of the radioimmunological method for the detection of Graves' disease was 0.94 and the specificity was 0.73. The sensitivity of the Kryptor method was 0.91 and the specificity was 0.91. A non-proportional systematic bias in favor of higher values of TRAb concentrations with the radioimmunological method was observed: slope of 0.93 (0.74 - 1.07, 95% confidence interval) and an intercept of -0.69 IU/L (-1.58 to -0.30, 95% confidence interval). Compared to the Kryptor method, the radioimmunological method tends to overestimate TRAb concentrations by up to 120%. CONCLUSIONS: The fully automated Brahms Kryptor kit using TRACE technology to measure TRAb reduces sampling time and intra- as well as inter-assay variations. The Kryptor kit underestimates the results of TRAb leading to a lower sensitivity and higher specificity compared to the radioimmunoassay. Thus, the new Brahms Kryptor kit has good laboratory performances but the interpretation of the results must still be performed with caution.


Assuntos
Doença de Graves/diagnóstico , Hipotireoidismo/diagnóstico , Imunoglobulinas Glândula Tireoide-Estimulantes/sangue , Radioimunoensaio , Receptores da Tireotropina/imunologia , Tireoidite/diagnóstico , Adulto , Automação Laboratorial , Feminino , Doença de Graves/sangue , Doença de Graves/imunologia , Humanos , Hipotireoidismo/sangue , Hipotireoidismo/imunologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Radioimunoensaio/normas , Reprodutibilidade dos Testes , Tireoidite/sangue , Tireoidite/imunologia , Fluxo de Trabalho
12.
J S Afr Vet Assoc ; 90(0): e1-e6, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31714111

RESUMO

The Coat-A-Count® radioimmunoassay has been long and widely used to determine the concentration of progesterone in serum or plasma of bitches (progRIA), but was discontinued in 2014. The Immulite® 1000 LKPG1 chemiluminescence immunoassay has gained prominence since 2003 to determine the concentration of progesterone in serum of bitches, but the assay changed in 2012 (Immulite® 1000 LKPW1). This study assessed the feasibility of using Immulite® 1000 LKPW1 (progImm) to estimate the time of clinically relevant events during oestrus and compared progRIA and progImm 2 and 3 days after the first or only day of the luteinizing hormone surge (LH1). ProgImm first exceeded 5.1 nmol/L on the same day that progRIA first exceeded 6 nmol/L, a proxy for the occurrence of the LH surge, or the day before in 28 of 31 (90%) of oestrous periods. ProgImm first exceeded 13.6 nmol/L on the same day that progRIA first exceeded 16 nmol/L (a proxy for the day of ovulation) or the day before in 34 of 35 (97%) oestrous periods. ProgImm first exceeded 5.4 nmol/L on LH1 or the day before in 24 of 25 (95%) of oestrous periods. The median of progImm 2 days after LH1 was 1.2 nmol/L lower than the 10.7 nmol/L of progRIA (p = 0.001). The mean of progImm 3 days after LH1 was 2.2 nmol/L lower than the 19.0 nmol/L of progRIA (p 0.001). In conclusion, the days on which progImm first exceeded 5.1 nmol/L, 13.6 nmol/L and 5.4 nmol/L effectively estimate the days on which progRIA reached 6 nmol/L or 16 nmol/L or LH1.


Assuntos
Cães/sangue , Medições Luminescentes/veterinária , Detecção da Ovulação/veterinária , Progesterona/sangue , Radioimunoensaio/veterinária , Animais , Tomada de Decisão Clínica , Estro/sangue , Feminino , Medições Luminescentes/métodos , Detecção da Ovulação/métodos , Radioimunoensaio/métodos , Reprodução/fisiologia
13.
Artigo em Alemão | MEDLINE | ID: mdl-31634940

RESUMO

OBJECTIVE: To assess testicular endocrine function in the male donkey (Equus asinus) during the course of the year. MATERIAL AND METHODS: In 5 miniature and 4 standard donkey stallions, peripheral blood concentrations of testosterone (T), estrone (E1) and estrone sulfate (E1S) were determined using radioimmunoassay. RESULTS: There was a highly significant influence of the season (p < 0.0001) on the course of all 3 steroids. Values were low in November until January and high in April, May and June. As delineated by the measurement of E1 the breed also had an effect on the expression of seasonality. Mean T concentration (X̅g × SF ±â€…1) was 1.58 × 1.20 ±â€…1 ng/ml, values ranged between 0.39 and 5.95 ng/ml, which is approximately double the plasma T concentrations observed in horse stallions. As in horse stallions, E1 levels were only slightly above the detection limit of the assay (0.10-0.17 ng/ml). Mean E1S concentration amounted to 0.91 ±â€…0.23 ng/ml, values ranged between 0.34-1.36 ng/ml and taking peak levels into account measured approximately 300-fold lower than in the horse stallion. CONCLUSIONS: The data obtained confirm that the donkey belongs to the group of long day breeders. Irrespective of the close phylogenetical relationship the course of E1S concentrations reveals distinct differences between horse and donkey. CLINICAL RELEVANCE: Even between closely related species established reference values for sex steroids cannot be transferred without verification.


Assuntos
Equidae/sangue , Estrona/análogos & derivados , Estrona/sangue , Testosterona/sangue , Animais , Equidae/fisiologia , Masculino , Radioimunoensaio/veterinária , Estações do Ano
14.
Poult Sci ; 98(12): 6611-6617, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504910

RESUMO

Stocking density is an important environment factor that affects the development of poultry farming, which has caused widespread concern. This study was carried out to determine the effects of stocking density on growth performance, growth regulatory factors, and endocrine hormones in broilers under appropriate environments. A total of 144 Arbor Acres male broilers (BW 1000 ± 70 g) were randomly divided into low stocking density (LSD; 6.25 birds/m2), medium stocking density (MSD; 12.50 birds/m2), and high stocking density (HSD; 18.75 birds/m2) groups, with 6 replicates in each group, and raised in 3 environmental chambers (same size) from 29-day-old to 42-day-old, respectively. The trial period lasted for 14 D with 21 ± 1°C and 60 ± 7% relative humidity, wind speed < 0.5 m/s, ammonia level<5 ppm. The results indicated that average daily food intake and average daily gain in HSD group showed significantly lower than other 2 groups (P < 0.05). Besides, the HSD group significantly reduced breast muscle yield, tibial length, tibial width, and tibial weight of broilers (P < 0.05). The HSD group increased the mRNA expression level of myostatin, and reduced the mRNA expression levels of insulin-like growth factor 1 (IGF-1) and myogenic determination factor 1 (P < 0.05). The HSD group significantly reduced the expression of parathyroid hormone-related protein in tibial growth plate (P < 0.05). The HSD group increased the serum corticosterone levels of broilers (P < 0.05), and decreased the serum IGF-1 and thyroxine (T4) levels of broiler chickens (P < 0.05) than other stocking density groups. Moreover, the serum alkaline phosphatase levels were decreased (P < 0.05) with increasing stocking density, whereas there were no significant effects on the serum 3,5,3'-triiodothyronine (T3) concentrations in 3 groups (P > 0.05). In conclusion, under appropriate environments HSD reduced the growth performance of broilers and this negative effect was likely associated with decreased growth of muscle and bone.


Assuntos
Galinhas/fisiologia , Hidrocortisona/sangue , Fator de Crescimento Insulin-Like I/farmacologia , Tiroxina/sangue , Tri-Iodotironina/sangue , Criação de Animais Domésticos , Animais , Galinhas/crescimento & desenvolvimento , Masculino , Densidade Demográfica , Radioimunoensaio/veterinária , Distribuição Aleatória
15.
J Thromb Haemost ; 17(12): 2123-2130, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31420903

RESUMO

BACKGROUND: Accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential to ensure timely treatment and prevent complications. Current diagnostic assays include enzyme-linked immunosorbent assays (ELISAs) and rapid immunoassays (RIs). RIs offer fast turnaround times but were not significantly represented in previous external proficiency testing challenges. OBJECTIVES: To use external proficiency testing to assess qualitative concordance for heparin/PF4 antibody detection. METHODS: From 2013 to 2017, the External Quality Control for Assays and Tests (ECAT) Foundation distributed 10 samples internationally. RESULTS: In total, 437 laboratories submitted 3149 results. ELISAs accounted for 1484 (47%) responses with RIs accounting for 1665 (53%) responses. RI use increased over the 5-year period. ELISAs classified 96% of both consensus positive and consensus negative samples concordantly. The coefficient of variation (CV) for positive sample optical densities (ODs) ranged from 35% to 50% when combining ELISA assay methods together. Quantitative RIs classified 97% of consensus-positive and 98% of consensus-negative samples concordantly. Qualitative RIs had a higher proportion of discordant responses and classified 88% of consensus-positive samples and 73% of consensus-negative samples concordantly. Of RIs only latex immunoassays and IgG specific chemiluminescent assays identified > 95% of samples concordantly with consensus. CONCLUSION: Quantitative RIs and ELISAs classify > 95% of samples concordantly. The ODs from different ELISA methods vary considerably and are not interchangeable. Qualitative RI use is increasing despite a greater proportion of discordant classifications. This includes a higher than expected number of negative classifications for consensus-positive samples among many RIs, challenging their use as "rule out" tests.


Assuntos
Anticorpos/sangue , Anticoagulantes/efeitos adversos , Ensaio de Imunoadsorção Enzimática/normas , Heparina/efeitos adversos , Ensaio de Proficiência Laboratorial , Fator Plaquetário 4/imunologia , Radioimunoensaio/normas , Testes Sorológicos/normas , Trombocitopenia/diagnóstico , Anticoagulantes/imunologia , Austrália , Biomarcadores/sangue , Europa (Continente) , Heparina/imunologia , Humanos , Israel , América do Norte , Variações Dependentes do Observador , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Trombocitopenia/sangue , Trombocitopenia/induzido quimicamente , Trombocitopenia/imunologia
16.
Vet Immunol Immunopathol ; 215: 109904, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31420068

RESUMO

During immune activation, CD25 is expressed by T cells, and its soluble form (sCD25) is released into the extracellular matrix and the bloodstream. In humans, serum sCD25 concentrations are used as a surrogate marker for autoimmune diseases, malignancies, and transplant rejection. However, a canine-specific assay for the measurement of sCD25 in dog serum has not previously been described. Therefore, the aims of this study were to develop and analytically validate a radioimmunoassay to measure sCD25 in canine serum, to establish a reference interval for canine sCD25, and to test the clinical utility of this assay with serum samples for dogs with various diseases. A competitive radioimmunoassay (RIA) was developed and analytically validated. Analytical validation consisted of lower limit of detection (LLOD), dilutional parallelism, spiking recovery, and intra- and inter-assay variability using pooled surplus canine serum samples. A reference interval was established in healthy dogs and serum samples from dogs with various types of neoplasia, IBD, liver disease, suspected pancreatitis, or suspected small intestinal disease and serum samples with an increased C-reactive protein concentration (CRP) were analyzed to test the clinical utility of the assay. LLOD was calculated to be 0.5 ng/mL. The mean (±SD) observed-to-expected ratio (O/E) for serial dilutions was 101.7 ±â€¯14.0%, and the mean (± SD) O/E for spiking recovery was 93.2 ±â€¯4.2%. Coefficients of variation (CVs) for intra-assay variability were ≤12.5% (mean ±â€¯SD: 7.5 ±â€¯4.2%), and inter-assay CVs were ≤15.7% (mean ±â€¯SD: 11 ±â€¯4.4%). A reference interval (RI) for canine sCD25 of 1.2-4.2 ng/mL was established from a population of 112 clinically healthy dogs. Dogs with neoplasia and dogs with suspected small intestinal disease had decreased concentrations of serum sCD25 when compared to healthy dogs (p < 0.0001, respectively). However, the majority of clinical samples used in this study were within the reference interval. Median concentrations of serum sCD25 were 1.9 ng/mL for healthy dogs. Dogs with cancer, IBD, liver disease, suspected pancreatitis, or suspected small intestinal disease, as well as sera with an increased serum CRP concentration, had median serum sCD25 concentrations of 1.6 ng/mL, 2.1 ng/mL, 2.2 ng/mL, 1.7 ng/mL, 1.5 ng/mL, and 1.8 ng/mL, respectively. Thus, the RIA described here is linear, accurate, precise, and reproducible for measuring sCD25 in canine serum. However, this assay shows little clinical utility of sCD25 as a biomarker for dogs with inflammatory, autoimmune, and/or neoplastic conditions.


Assuntos
Doenças do Cão/sangue , Cães/sangue , Subunidade alfa de Receptor de Interleucina-2/sangue , Radioimunoensaio/veterinária , Animais , Doenças do Cão/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Humanos , Radioimunoensaio/métodos , Valores de Referência , Sensibilidade e Especificidade
17.
Int Forum Allergy Rhinol ; 9(9): 1077-1082, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31369207

RESUMO

BACKGROUND: The integration of the immune and endocrine systems through a network of signaling molecules-including cytokines and hormones-produces a consequent modulatory effect on immune function. In this study we aimed to comparatively evaluate the serum concentrations of dehydroepiandrosterone sulfate (DHEA-S) and cortisol in patients with allergic rhinitis and healthy control subjects. METHODS: Morning fasting blood samples were obtained from 96 participants, including 66 patients with allergic rhinitis (mean age, 28 ± 10.7 years; range, 11-59 years) and 30 healthy control subjects (mean age, 32.4 ± 11.1 years; range, 14-57 years). Total immunoglobulin E (IgE), DHEA-S, and cortisol concentrations were measured by radioimmunoassay. RESULTS: Female patients with allergic rhinitis exhibited lower DHEA-S levels than male patients and control subjects (p < 0.001). Although no intergroup differences were observed in the morning:evening cortisol ratio, the DHEA-S:cortisol ratio was the highest among female patients with allergic rhinitis (1:11). Total IgE levels were significantly higher among patients of both sexes (p < 0.05) than among the control subjects. There was a positive correlation between body mass index and age among all groups of participants (male and female patients: r = 0.6 and 0.7, respectively; male and female controls: r = 0.6 and 0.4, respectively) and a negative correlation between DHEA-S level and age among females (patients and controls: r = -0.4 and -0.5, respectively) and healthy male subjects (r = -0.7). CONCLUSION: In this study, female patients with allergic rhinitis exhibited significantly lower DHEA-S levels than their healthy counterparts and male patients. These results should be confirmed in a larger group of participants.


Assuntos
Sulfato de Desidroepiandrosterona/sangue , Hidrocortisona/sangue , Rinite Alérgica/diagnóstico , Fatores Sexuais , Adolescente , Adulto , Albânia/epidemiologia , Criança , Regulação para Baixo , Feminino , Humanos , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Radioimunoensaio , Rinite Alérgica/epidemiologia , Adulto Jovem
18.
Methods Mol Biol ; 1934: 247-264, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256384

RESUMO

α-Amidation is a terminal modification in peptide biosynthesis that can itself be rate limiting in the overall production of bioactive α-amidated peptides. More than half of the known neural and endocrine peptides are α-amidated and in most cases this structural feature is essential for receptor recognition, signal transduction, and thus biologic function. This chapter describes methods for developing and using analytical tools to study the biology of α-amidated peptides. The principal analytical method used to quantify α-amidated peptides is the radioimmunoassay (RIA). Detailed protocols are provided for (1) primary antibody production and characterization; (2) radiolabeling of RIA peptides; (3) sample preparation; and (4) performance of the RIA itself. Techniques are also described for the identification and verification of α-amidated peptides. Lastly, in vivo models used for studying the biology of α-amidation are discussed.


Assuntos
Peptídeos/química , Peptídeos/metabolismo , Amidas/química , Radioimunoensaio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
19.
Anal Chem ; 91(14): 9323-9329, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31260261

RESUMO

Low levels of hypocretin-1 (Hcrt1) in cerebrospinal fluid (CSF) are associated with narcolepsy type 1 (NT1). Although immunoassays are prone to antibody batch differences, detection methods and variation between laboratories, the standard method for Hcrt1 measurement is a radioimmunoassay (RIA). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is an antibody- and radioactive free alternative for precise measurement of Hcrt1. We developed an LC-MS/MS method for measurement of Hcrt1 in CSF with automated sample preparation by solid-phase extraction (SPE). The LC-MS/MS method was compared with the RIA method for Hcrt1 detection. CSF samples from healthy subjects and NT1 patients was obtained by lumbar puncture. NT1 patients were diagnosed according to the minimal criteria by the International Classification of Sleep Disorders (ICSD). The LC-MS/MS method showed linearity across the range of calibrators and had a limit of detection (LOD) of 2.5 pg/mL and a limit of quantitation (LOQ) of 3.6 pg/mL. Comparison of the LC-MS/MS method with RIA revealed a 19 times lower level in healthy controls and 22 times lower level in NT1 patients with the LC-MS/MS method than with RIA. Bland-Altman analysis demonstrated agreement between the methods. These results question what is detected by RIA and strongly suggest that the physiological concentrations of the peptide are much lower than previously believed. LC-MS/MS proves to be an alternative for detection of Hcrt1 for diagnosis of narcolepsy.


Assuntos
Cromatografia Líquida/métodos , Orexinas/líquido cefalorraquidiano , Espectrometria de Massas em Tandem/métodos , Adulto , Sequência de Aminoácidos , Humanos , Limite de Detecção , Narcolepsia/líquido cefalorraquidiano , Narcolepsia/diagnóstico , Radioimunoensaio , Reprodutibilidade dos Testes , Extração em Fase Sólida
20.
Am J Reprod Immunol ; 82(2): e13146, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31206871

RESUMO

PROBLEM: Current methods to quantify kisspeptin (KP) are limited. To this end, a radioimmunoassay (RIA) specific for KP was developed and validated. We hypothesized that use of a RIA would reveal multiple hypothalamic regions as targets of negative seasonal feedback of estradiol on KP production in sheep. METHOD OF STUDY: Ovariectomized (OVX) ewes bearing a subcutaneous implant of estradiol were euthanized during the breeding season (BS) (n = 4) and non-breeding season (NBS) (n = 3). Coronal sections of preoptic area (POA), anterior hypothalamic area (AHA), and mediobasal hypothalamus (MBH) were collected, as well as the median eminence (ME), cortex, brain stem, and cerebellum. Amounts of KP and gonadotropin-releasing hormone (GnRH) in individual hypothalamic nuclei were quantified by radioimmunoassay. RESULTS: Concentration and content of KP were lower during the NBS than the BS in the MBH (P < 0.01) and POA (P < 0.01). Levels of KP in tissue adjacent to the POA and MBH were much lower, and neither concentration nor content of KP differed between the BS and NBS. Kisspeptin was also detected in the cortex, brain stem, and cerebellum, but concentrations were not affected by season. In addition, concentration and content of GnRH in the POA, AHA, MBH, and ME were similar between seasons. CONCLUSION: Our RIA results indicate that in addition to the MBH, the POA and AHA appear to be involved in the seasonal negative feedback of estradiol on KP expression.


Assuntos
Córtex Cerebelar/metabolismo , Estradiol/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo Médio/metabolismo , Kisspeptinas/metabolismo , Área Pré-Óptica/metabolismo , Ovinos/metabolismo , Animais , Cruzamento , Retroalimentação Fisiológica/fisiologia , Feminino , Radioimunoensaio , Estações do Ano
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