Time-resolved Fluoroimmunoassay Research Articles

Towards Libraries of Luminescent Lanthanide Complexes and Labels from Generic Synthons

A synthetic approach is developed to obtain families of luminescent lanthanide complexes and markers from a generic family of precursors built from nonadentate coordination sites. The syntheses of the precursors, based on a directed regioselective nucleophilic aromatic substitution on polyfluoropyridines, are described. Functionalisation of the synthons on the aromatic moieties allowed the introduction of labelling functions and/or the extension of the electronic delocalisation, with concomitant changes in the spectroscopic properties. The synthesis of two such families of ligands and of some of their complexes of Eu(III) and Tb(III) are described, and the photo-physical properties of the complexes were measured, revealing excellent luminescence quantum yields reaching unity in some cases. For some of these complexes, the emphasis was further put on the preparation of an N-hydroxylsuccinimide (NHS) ester as activated function for labelling. The Tb and La complexes in the NHS activated form were synthesized and fully characterized. The labelling was first demonstrated on amino functionalized polymer beads and characterized by time-resolved luminescence microscopy. In a second step, the activated Tb complex was used for the labelling of GFR44 monoclonal antibody, and was applied to the detection of carcinoembryonic antigene (CEA) within the frame of a time-resolved fluoroimmunoassay. Comparison with a commercially available kit based on a europium cryptate as energy donor confirms the efficiency of Tb to act as an energy donor with an unoptimised 35% increase of the detection efficiency.

Determination of diethylstilbestrol based on biotin–streptavidin‐amplified time‐resolved fluoro‐immunoassay

A rapid and sensitive time-resolved fluoroimmunoassay (TR-FIA) based on the biotin-streptavidin amplification system was developed for the determination of diethylstilbestrol (DES). Europium-labelled streptavidin derivatives combined with europium and anhydride of diethylene triamine penta-acetic acid were used to label streptavidin; biotin was coupled with goat anti-rabbit IgG to form a biotin-goat anti-rabbit IgG bridge between streptavidin-europium and the anti-DES antibody in the immunoassay. The DES assay was carried out by measuring the fluorescence of Eu(3+) -SA at 615 nm. The presented method produced a wide linear range, 0.001-1000.0 ng/mL, and a detection limit up to 0.81 pg/mL for DES. The method was applied to determine DES in serum samples, with recoveries of 97.4-107.8% and RSD 1.32-4.04%. The assay results by the present method showed that biotin-streptavidin amplified TR-FIA for DES detection; it may offer high sensitivity and promising alternative special methods in biological samples.

Lanthanide Complexes in FRET Applications

In this article, the main principles of Forster resonance energy transfer (FRET) are described with special emphasis on luminescent lanthanide complexes (LLCs) in modern fluorescence spectroscopy and microscopy and their use in biomolecular analysis. The theoretical fundamentals of FRET are explained and the unique photophysical properties of LLCs are presented. Their narrow emission bands, long excited-state lifetimes and large “Stokes shifts” make LLCs very promising energy donors for various FRET systems. Numerous recent literature examples as well as commercial applications, demonstrating the particular advantages of LLCs in FRET spectroscopy and microscopy, are reported. Afterwards, the benefits of multi-parametric FRET measurements using LLC donors are emphasized. The multiplexed approach allows the simultaneous detection of several analytes or binding events, which makes it especially important for modern high-throughput-screening as well as low-cost lab-on-a-chip devices. Finally, semiconductor quantum dots (QDs) and the advantages of their use as novel FRET acceptors are presented. A combination of LLCs and QDs in FRET provides extraordinary donor-acceptor pairs with outstanding properties, and long biomolecular distances of up to 20 nm become accessible. Extremely high sensitivity combined with multiplexed detection make the LLC-QD FRET pairs substantial for application in modern time-resolved fluoroimmunoassays (TR-FIA). Luminescent lanthanide complexes have been very efficient tools in various FRET applications and regarding the ever increasing amount of newly arising biomolecular questions and the development of novel fluorescent materials the future of LLC-based FRET is still wide open and highly promising.

Detection of the circulating antigen 14-3-3 protein of Schistosoma japonicum by time-resolved fluoroimmunoassay in rabbits

BackgroundSchistosomiasis remains a major public health concern that afflicts millions of people worldwide. Low levels of Schistosoma infection require more sensitive diagnostic methods. In this study, a time-resolved fluoroimmunoassay (TRFIA) was developed for detecting the signal transduction protein 14-3-3, a circulating antigen of Schistosoma japonicum.ResultsThe detection limit of 14-3-3-TRFIA was 0.78 ng/ml, with a linear measurement range from 0.78 to 800 ng/ml. The average intra-assay and inter-assay variability of this TRFIA was 8.9% and 12.2% respectively, and the mean recovery rate ranged from 92.1% to 115.5%. Within the first 21 days post-infection in rabbits, the positive rates of the 14-3-3-TRFIA were distinctly higher compared to ELISA. All these findings illustrate that 14-3-3-TRFIA has a higher detection efficacy and is a good early diagnostic method for active Schistosoma infection.ConclusionsA sandwich TRFIA for detecting the circulating antigen 14-3-3 of S. japonicum has been developed, and has demonstrated to be a good potential diagnostic method for schistosomiasis.

A competitive dual-label time-resolved fluoroimmunoassay for the simultaneous determination of chloramphenicol and ractopamine in swine tissue

A novel dual-label time-resolved fluoroimmunoassay method was developed for the simultaneous determination of chloramphenicol (CAP) and ractopamine (RAC) residues in 18 swine tissue samples, using anti-CAP and anti-RAC monoclonal antibodies labeled with europium (Eu3+) and samarium (Sm3+), respectively. The detection limits for CAP and RAC were 0.06 and 0.25 ng/g. The recovery from swine muscle samples was 102%–121% for CAP at spiking levels of 0.1–5 ng/g, and 69.8%–85.8% for RAC at spiking levels of 1–10 ng/g. The results obtained from the swine tissue samples using this method showed good agreement with those obtained using ELISA and GC-MS, with correlation coefficients (R) between 0.92–0.98.

More invasive procedures are done to detect each case of Down's syndrome in younger women

To evaluate the performance of first-trimester combined screening in 5-year periods according to maternal age in a low-risk population. A prospective study. Multicenter study in Finland. A total of 76949 voluntary women with singleton pregnancies participated in first-trimester combined screening in public healthcare between 1 May 2002 and 31 December 2008. The serum samples were analyzed using the PerkinElmer AutoDELFIA® time-resolved fluoroimmunoassay kit for the measurement of pregnancy-associated plasma protein-A and free beta-human chorionic gonadotropin. Nuchal translucency was measured by trained personnel (midwives or physicians) in a university or central hospital. Performance, detection rate, false positive rate and the number of invasive procedures needed to detect a single case of Down's syndrome were analyzed. There was a direct connection between maternal age and the prevalence of Down's syndrome with a low prevalence in young women being 1:1 193 in the 25-29 age group and 1:150 in the 35-39 age group. Consequently, for a fixed false positive rate of 5%, the number of invasive procedures needed to detect one case of Down's syndrome is higher in younger women to achieve the same detection rate. In combined first trimester screening the risk for Down's syndrome is individual, varying with maternal age. This should be taken into consideration when counseling women.

Highly Sensitive Immunoassay Based on Immunogold−Silver Amplification and Inductively Coupled Plasma Mass Spectrometric Detection

In this work, we demonstrated a highly sensitive inductively coupled plasma mass spectrometric (ICPMS) method for the determination of human carcinoembryonic antigen (CEA), which combined the inherent high sensitivity of elemental mass spectrometric measurement with the signal amplification of catalytic silver deposition on immunogold tags. The silver amplification procedure was easy to handle and required cheap reagents, and the sensitivity was greatly enhanced to 60-fold after a 15 min silver amplification procedure. The experimental conditions, including detection of gold and silver by ICPMS, immunoassay parameters, silver amplification parameters, analytical performance, and clinical serum samples analysis, were investigated. The ICPMS Ag signal intensity depends linearly on the logarithm of the concentration of human CEA over the range of 0.07-1000 ng mL(-1) with a limit of detection (LOD, 3σ) of 0.03 ng mL(-1) (i.e., 0.15 pM). The LOD of the proposed method is around 2 orders of magnitude lower than that by the widely used enzyme-linked immunosorbent assay (ELISA) and 1 order of magnitude lower than that by clinical routine chemiluminescence immunoassay (CLIA) or time-resolved fluoroimmunoassay (TRFIA) and conventional ICPMS immunoassay. The present strategy was applied to the determination of human CEA in clinical human serum samples, and the results were in good agreement with those obtained by chemiluminescence immunoassay.

Comparisons of insulin related parameters in commercial-type chicks: Evidence for insulin resistance in broiler chicks

The aim of this study is to elucidate whether insulin acts differentially within the central nervous system (CNS) of two types of commercial chicks to control ingestive behavior. Male layer and broiler chicks (4-day-old) were intracerebroventricularly (ICV) injected with saline or insulin under satiated and starved conditions. Feed intake was measured at 30, 60 and 120 min after treatment. Secondly, blood and hypothalamus were collected from both chick types under ad libitum feeding and fasting for 24 h. Plasma insulin concentration was measured by time-resolved fluoro-immunoassay. Hypothalamic insulin receptor mRNA expression levels were measured by quantitative RT-PCR. The ICV injection of insulin significantly inhibited feed consumption in layer chicks when compared with saline (P<0.05), but not broiler chicks (P>0.1). Plasma insulin concentration of both chick types significantly decreased following 24 h of fasting, while insulin concentrations in the broiler chicks were significantly higher compared to the layers fed under ad libitum conditions. Hypothalamic insulin receptor mRNA expression levels were significantly lower (P<0.05) in broiler chicks than in layer ones under ad libitum feeding. Feed deprivation significantly decreased insulin receptor mRNA levels in layer chicks (P<0.01), but not in broiler chicks (P>0.1). Moreover, plasma insulin concentrations correlated negatively with hypothalamic insulin receptor protein expression in the two types of chicks fed ad libitum (P<0.05). These results suggest that insulin resistance exists in the CNS of broiler chicks, possibly due to persistent hyperinsulinemia, which results in a down-regulation of CNS insulin receptor expression compared to that in layer chicks.

Inter-laboratory comparison of methods to measure androstenone in pork fat

Today, different analytical methods are used by different laboratories to quantify androstenone in fat tissue. This study shows the comparison of methods used routinely in different laboratories for androstenone quantification: Time-resolved fluoroimmunoassay in Norwegian School of Veterinary Science (NSVS; Norway), gas chromatography coupled to mass spectrometry in Co-operative Central Laboratory (CCL; The Netherlands) and in Institut de Recerca i Tecnologia Agroalimentàries (IRTA; Spain), and high-pressure liquid chromatography in Agroscope Liebefeld-Posieux Research Station (ALP; Switzerland). In a first trial, a set of adipose tissue (AT) samples from 53 entire males was sent to CCL, IRTA and NSVS for determination of androstenone concentration. The average androstenone concentration (s.d.) was 2.47 (2.10) μg/g at NSVS, 1.31 (0.98) μg/g at CCL and 0.62 (0.52) μg/g at IRTA. Despite the large differences in absolute values, inter-laboratory correlations were high, ranging from 0.82 to 0.92. A closer look showed differences in the preparation step. Indeed, different matrices were used for the analysis: pure fat at NSVS, melted fat at CCL and AT at IRTA. A second trial was organised in order to circumvent the differences in sample preparation. Back fat samples from 10 entire males were lyophilised at the ALP labortary in Switzerland and were sent to the other laboratories for androstenone concentration measurement. The average concentration (s.d.) of androstenone in the freeze-dried AT samples was 0.87 (0.52), 1.03 (0.55), 0.84 (0.46) and 0.99 (0.67) μg/g at NSVS, CCL, IRTA and ALP, respectively, and the pairwise correlations between laboratories ranged from 0.92 to 0.97. Thus, this study shows the influence of the different sample preparation protocols, leading to major differences in the results, although still allowing high inter-laboratory correlations. The results further highlight the need for method standardisation and inter-laboratory ring tests for the determination of androstenone. This standardisation is especially relevant when deriving thresholds of consumer acceptance, whereas the ranking of animals for breeding purposes will be less affected due to the high correlations between methods.

Neuroendocrine control of thyroid‐stimulating hormone and adrenocorticotropic hormone in amphibians

Event Abstract Back to Event Neuroendocrine control of thyroid‐stimulating hormone and adrenocorticotropic hormone in amphibians Reiko Okada1*, Kazutoshi Yamamoto2, Itaru Hasunuma3, Shigeyasu Tanaka1 and Sakae Kikuyama2 1 Shizuoka University, Integrated Bioscience Section, Graduate School of Science and Technology, Japan 2 Waseda University, Department of Biology, Faculty of Education and Integrated Arts and Science, Japan 3 Toho University, Department of Biology, Faculty of Science, Japan In non-mammalian vertebrates, pituitary thyrotropin (TSH) secretion is considered to be regulated by multiple hypothalamic neuropeptides such as TSH-releasing hormone (TRH), corticotropin-releasing factor (CRF), gonadotropin-releasing hormone, pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP) and somatostatin. The main TSH-releasing factor varies according to the animal classes and the developmental stages. By performing in vitro experiments combined with specific radioimmunoassay for bullfrog TSH, we demonstrated that the frog CRF potently stimulates the release of TSH from both adult and larval bullfrog pituitary cells, whereas TRH moderately stimulates the TSH release from adult pituitary cells but not from larval ones and that the action of CRF is mediated by the CRF receptor 2. It was also shown that TSH-releasing activity in the bullfrog hypothalamic extract is profoundly diminished by a CRF receptor antagonist, suggesting that endogenous CRF contributes as a main TSH-releasing factor. On the other hand, both CRF and arginine-vasotocin (AVT)/arginine-vasopressin (AVP) have been reported to stimulate the release of adrenocorticotropic hormone (ACTH) from the pituitary of various vertebrates. In order to compare the potency of both peptides in terms of stimulating the ACTH release, experiments were conducted using dispersed bullfrog pituitary cells. The amount of ACTH released into the culture medium, as measured by a newly developed time-resolved fluoroimmunoassay for frog ACTH, was much higher when the pituitary cells were incubated in the presence of AVT than when they were incubated with CRF. This raised possibility that in amphibians, AVT rather than CRF is the main ACTH-releasing factor. Keywords: ACTH, amphibian, AVT, CRF, TSH Conference: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology, Ann Arbor, United States, 11 Jul - 13 Jul, 2011. Presentation Type: Invited Symposium Topic: Abstracts Citation: Okada R, Yamamoto K, Hasunuma I, Tanaka S and Kikuyama S (2011). Neuroendocrine control of thyroid‐stimulating hormone and adrenocorticotropic hormone in amphibians. Front. Endocrinol. Conference Abstract: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology. doi: 10.3389/conf.fendo.2011.03.00036 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. Reiko Okada, Shizuoka University, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka, 422-8529, Japan, drokada@ipc.shizuoka.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Reiko Okada Kazutoshi Yamamoto Itaru Hasunuma Shigeyasu Tanaka Sakae Kikuyama Google Reiko Okada Kazutoshi Yamamoto Itaru Hasunuma Shigeyasu Tanaka Sakae Kikuyama Google Scholar Reiko Okada Kazutoshi Yamamoto Itaru Hasunuma Shigeyasu Tanaka Sakae Kikuyama PubMed Reiko Okada Kazutoshi Yamamoto Itaru Hasunuma Shigeyasu Tanaka Sakae Kikuyama Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Toward sensitive, quantitative point-of-care testing (POCT) of protein markers: miniaturization of a homogeneous time-resolved fluoroimmunoassay for prostate-specific antigen detection

Point-of-care testing (POCT) systems which allow for a sensitive, quantitative detection of protein markers are extremely useful for the early detection and therapy progress monitoring of cancer. However, currently commercially available POCT devices are mainly limited to the qualitative detection of protein markers. In this study we demonstrate the successive miniaturization of a sensitive and fast assay for the quantitative detection of prostate-specific antigen (PSA) using a well established and clinically approved homogeneous time-resolved fluoroimmunoassay technology (TRACE®) on a commercial plate-reader system (KRYPTOR®). Regarding the initial requirements for the development of POCT devices we applied a 30-fold assay volume reduction (150 µL to 5 µL) to achieve a reasonable lab-on-a-chip volume and a 24-fold and 120-fold excitation pulse energy reduction to achieve reasonable pulse energies for low-cost miniature excitation sources. Due to highly efficient optimization of key POCT parameters our miniaturized PSA assay achieved a 30% increased sensitivity and a 2-fold improved limit of detection compared to the standard plate-reader method. Our results demonstrate the successful implementation of key parameters for a significant miniaturization and for cost reduction in the clinically approved KRYPTOR® platform for protein detection. The technological alterations required are easy-to-implement and can be immediately adapted for more than 30 diagnostic protein markers already available for the KRYPTOR® platform. These features strongly recommend our assay format to be utilized in innovative, sensitive, quantitative POCT of protein markers.

Validation of a direct time-resolved fluoroimmunoassay for progesterone in milk from dairy and beef cows

The aim of this study was to validate a direct time-resolved fluoroimmunoassay (TR-FIA) for quantifying progesterone concentrations in milk during the bovine oestrous cycle. Holstein-Friesian and suckled and non-suckled Japanese Black cows were used to demonstrate the relationship between milk and plasma progesterone concentrations and to monitor progesterone profiles in milk and plasma during the oestrous cycle. The minimum detection level of the assay was 1.53ng/mL. Progesterone concentrations in milk and plasma changed in a similar manner throughout the oestrous cycle in dairy and beef cows, and milk and plasma progesterone profiles were significantly correlated (P<0.001). The study confirmed that a direct TR-FIA can be used to monitor the oestrous cycle in cattle and to quantify progesterone concentrations in whole milk.

Biochemical and Lmmunohistochemical Analyses of GnRH-like Peptides in the Nerve Ganglion of the Chiton,Acanthopleura japonica

We examined whether a gonadotropin-releasing hormone (GnRH)-like peptide is present in the nerve ganglion of the chiton Acanthopleura japonica (Mollusca, Polyplacophora) using reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoas-say (TR-FIA) analysis, and immunohistochemistry. An extract of the chiton head region showed a similar retention time to that of synthetic lamprey GnRH-II on rpHPLC combined with TR-FIA analysis using a rabbit polyclonal antibody raised against chicken GnRH-II (aCII6). Cell bodies immunostained with LRH13 (a mouse monoclonal antibody raised against the common amino acid sequence of vertebrate GnRH) were detected in the cerebrobuccal ring (CBR). Cell bodies immunostained with aCII6 were not only observed in the CBR but also in the lateral nerve cord (LCo). Fibers immunostained with LRH13 and aCII6 were widely distributed throughout the central nervous system in the CBR, subradular ganglion (SubRG), pedal nerve cord (PCo), pedal commissure (P/PCom), lateropedal commissure (L/PCom), and from the LCo to the suprarectal commissure (SupRecCom). The cell bodies and fibers immunostained with these two antisera were distinguishable by dual-label immunohistochemistry. These results suggest that multiple GnRH-like peptides are present in the nerve ganglion of the chiton Acanthopleura japonica.

Simultaneous detection of deoxynivalenol and zearalenone by dual-label time-resolved fluorescence immunoassay

The health risks of deoxynivalenol (DON) and zearalenone (ZEN) necessitate the development of analytical methods for widespread food and feed screening. We sought to establish a rapid, economic and sensitive dual-label time-resolved fluoroimmunoassay (TRFIA) to detect DON and ZEN simultaneously. Eu(3+)- and Sm(3+)-labelled antibodies were used, as lanthanides are more stable and have narrower emission spectra than most fluorescent dyes. The limit of detection was 0.0194 ng mL(-1) for DON (range: 0.0194-100 ng mL(-1)) and 0.37 ng mL(-1) for ZEN (range: 0.37-50 ng mL(-1)). DON recovery in spiked cereal samples was 88-107%, and for ZEN was 83-108%, with both intra- and inter-assay coefficients of variation (CVs) less than 5%. The dual-label TRFIA results correlated well with ELISA results (correlation coefficients: 0.9733 for DON and 0.9784 for ZEN). The dual-label TRFIA is a simple, fast and sensitive method for high-throughput screening of DON and ZEN in food and feedstuff.

A novel bifunctional europium chelate applied in quantitative determination of human immunoglobin G using time-resolved fluoroimmunoassay

The authors demonstrate herein a novel time-resolved fluoroimmunoassay (TRFIA) protocol for quantification of human IgG with the new bifunctional chelate Eu(TTA) 3(5-NH 2-phen) (ETNP) labeling the goat anti-human IgG. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreactions. Goat anti-human IgG was immobilized on aldehyde-modified glass slides. The human IgG analyte was first captured by the primary antibody and then sandwiched by a secondary antibody labeled with the chelate ETNP. The experimental procedure was simple to follow and gave desirable levels of sensitivity and low limits of detection. To the best of our knowledge, this is the first application of the new chelate, ETNP, in an immunoassay. In comparison to typical organic, fluorescent compounds and other lanthanide fluorescent chelates used in immunoassay, the detection sensitivity of our method using ETNP chelate in the solid phase was greatly improved and a concentration of human IgG about 5 μg/L could be detected under optimal conditions. The main result of this work shows that the new chelate ETNP can be applied as a powerful fluorescent labeling material for constructing ultrasensitive TRFIAs. The detection of human IgG, using ETNP as the chelate, is a model example of the effectiveness of this immunoassay. Many other types of antigen–antibody immunoassays should be possible using the protocol described herein.

Sex steroid hormones do not enhance the direct stimulatory effect of kisspetin‐10 on the secretion of growth hormone from bovine anterior pituitary cells

The aims of the present study were to clarify the effect of kisspeptin10 (Kp10) on the secretion of growth hormone (GH) from bovine anterior pituitary (AP) cells, and evaluate the ability of sex steroid hormones to enhance the sensitivity of somatotrophic cells to Kp10. AP cells prepared from 8-11-month-old castrated calves were incubated for 12 h with estradiol (E(2), 10(-8) mol/L),progesterone (P(4), 10(-8) mol/L), testosterone (T, 10(-8) mol/L), or vehicle only (control), and then for 2 h with Kp10. The amount of GH released in the medium was measured by a time-resolved fluoroimmunoassay. Kp10 (10(-6) or 10(-5) mol/L) significantly stimulated the secretion of GH from the AP cells regardless of steroid treatments (P < 0.05), and E(2), P(4), and T had no effect on this response. The GH-releasing response to growth hormone-releasing hormone (GHRH, 10(-8) mol/L) was significantly greater than that to Kp10 (P < 0.05). The present results suggest that Kp10 directly stimulates the release of GH from somatotrophic cells and sex steroid hormones do not enhance the sensitivity of these cells to Kp10. Furthermore, they suggest that the GH-releasing effect of Kp10 is less potent than that of GHRH.

Expanded newborn screening in Greece: 30 months of experience

In Greece, the National Newborn Screening Program was initiated in 1974 and is performed by the Institute of Child Health (ICH). However, there is a complete absence of conditions that have high rates of mortality and a relatively high prevalence listed in the Catalogue of Disorders screened by the ICH. Our laboratory has expanded the existing NBS program to include newborn screening for inborn errors of metabolism, screening for cystic fibrosis (the most common congenital disorder in the Greek population), congenital adrenal hyperplasia, and for biotinidase deficiency. From July 2007 to December 2009, 45,000 dried blood spots (DBS) were collected from infants born in Athens, Greece, and were analyzed. We present a report of our 30-month experience in the newborn screening area. The samples were tested for amino acidopathies, fatty acid oxidation disorders (FAOD), and organic acid metabolic disorders by applying flow injection analysis-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS); for cystic fibrosis by immunoreactive trypsinogen (IRT) measurement (time-resolved fluoroimmunoassay); for congenital adrenal hyperplasia by fluoroimmunoassay to measure the 17 hydroxy-progesterone level; and for biotinidase deficiency using a colorimetric method and a semiquantitative fluoroimmunoassay to determine biotinidase activity. Sample analysis resulted in establishing cutoff values for the respective disease markers for the first time in the Greek population. Four infants were identified with cystic fibrosis, two with congenital adrenal hyperplasia, two with phenylketonuria (PKU), one with medium-chain acyl CoA dehydrogenase deficiency (MCADD), and one with biotinidase deficiency. To the best of our knowledge, this is the first article reporting the status of expanded newborn screening in Greece.

The adaptive significance of crustacean hyperglycaemic hormone (CHH) in daily and seasonal migratory activities of the Christmas Island red crab Gecarcoidea natalis

The Christmas Island red crab Gecarcoidea natalis undergoes extreme changes in metabolic status, ranging from inactivity during the dry season, to a spectacular annual breeding migration at the start of the wet season. The dramatic change in metabolic physiology that this polarisation entails should be reflected in changes in endocrine physiology, particularly that of the crustacean hyperglycaemic hormone (CHH), of which we know relatively little. CHH levels were measured using a novel ultrasensitive time-resolved fluoroimmunoassay (TR-FIA), together with metabolites (glucose, lactate), in the field at several scales of temporal resolution, during migratory activities (wet season) and during the inactive fossorial phase (dry season). Release patterns of CHH were measured during extreme (forced) exercise, showing for the first time an unexpectedly rapid pulsatile release of this hormone. A seasonally dependent glucose-sensitive negative-feedback loop was identified that might be important in energy mobilisation during migration. Haemolymph lactate levels were strongly correlated with CHH levels in both field and experimental animals. During migration, CHH levels were lower than during the dry season and, during migration, daytime CHH levels (when most locomotor activity occurred) increased. However, the intense dawn activity in both dry and wet seasons was not always associated with repeatable hyperglycaemia or CHH release. The results obtained are discussed in relation to the life history and behaviour of G. natalis.

Biochemical and Immunohistochemical Analyses of a GnRH-like Peptide in the Neural Ganglia of the Pacific AbaloneHaliotis Discus Hannai(Gastropoda)

We examined whether gonadotropin-releasing hormone (GnRH)-like peptides are present in the neural ganglia of the gastropod Pacific abalone (Haliotis discus hannai) by reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoassay (TR-FIA) analysis and by immunohistochemistry. Cerebral ganglion extracts showed a similar retention time to lamprey GnRH-II (lGnRH-II) in rpHPLC combined with TR-FIA analysis. GnRH-like-immunoreactive (ir) cell bodies (which reacted with a mouse monoclonal antibody raised against the common amino acid sequence of vertebrate GnRH) were detected in the peripheral region of the cerebral ganglion, and they were observed to send fibers into the neuropil. GnRH-like-ir fibers were also detected in the neuropil of the pedal ganglion, the visceral nerve, and the nerve originating from the pedal ganglion. Chicken GnRH-II (cGnRH-II)-like-ir fibers (which reacted with a rabbit polyclonal antibody raised against cGnRH-II) were also observed in the neuropil of the cerebral ganglion. GnRH-like-ir fibers and cGnRH-II-like-ir fibers were distinguishable in the neuropil of the cerebral ganglion by double-staining immunohistochemistry. These results suggest that multiple GnRH-like peptides exist in the neural ganglia of the Pacific abalone.

Identification and characterization of Schistosoma japonicum Sjp40, a potential antigen candidate for the early diagnosis of schistosomiasis

The pathogenesis of schistosomiasis is mainly caused by egg-induced granuloma formation and subsequent fibrosis. If Schistosoma japonicum infections could be detected in the early stage, especially before the egg deposition in the host tissues, the development of severe pathologic lesions might be prevented efficiently. The present study identified and characterized S. japonicum Sjp40, a potential antigen candidate for the early diagnosis of schistosomiasis. From the S. japonicum cercariae cDNA library, a clone encoding Sjp40 was identified by screening with the pooled rabbit sera collected on day 21 postinfection. Then, the recombinant Sjp40 protein (rSjp40) and monoclonal antibodies (McAbs) anti-rSjp40 were developed. The expression profiles of Sjp40 at 3 stages of S. japonicum, including egg, cercariae, and adult, were also determined at both mRNA and protein level, which displayed that the expression pattern of Sjp40 varied at different stages. Quantification of circulatory anti-Sjp40 IgG in the infected mice sera by time-resolved fluoroimmunoassay showed a statistically significant increase on days 21, 28, 35, and 42 postinfection compared with the mice sera prior to infection and the control mice. It was further confirmed by Western blot that all 8 clones of anti-rSjp40 McAbs could react specifically with the native antigen in S. japonicum cercariae, and rSjp40 could be recognized by the pooled infected mouse sera on days 21, 28, 35, and 42 postinfection as well as the pooled patient sera with acute schistosomiasis japonica. These findings indicated that Sjp40 and its antibodies are detectable from the host at a relatively early phase (day 21 postinfection with S. japonicum) and suggested that Sjp40 is a potential antigen candidate for the early diagnosis of schistosomiasis.