Time-resolved Fluoroimmunoassay Research Articles

Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L−1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates.Graphical We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody.

A New Method for Determination of Alfatoxin M1 in Milk by Ultrasensitive Time-Resolved Fluoroimmunoassay

A competitive indirect time-resolved fluoroimmunoassay (TRFIA) for detecting aflatoxin M1(AFM1) contamination in milk was developed, by using aflatoxin M1-bovineserum albumin conjugate, anti-AFM1 antibody, and Eu-labeled goat anti-rabbit antibody. To improve the sensitivity of the assay, the concentrations of the coating antigen and anti-AFM1 antibody were varied to optimize the condition of the immunological assay. The limit of detection values, limit of quantification values, and dynamic working range were 0.006, 0.022, and 0.022–1.334 μg/kg, respectively. Values of recovery within and between assays were 88.0–116.0% and 92.69–108.63%. The method was applicable for the full-cream, semi-skimmed, skimmed, and raw milk as well. Values of repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) were 1.2–4.5% and 0.8–5.0%, respectively. The results of using AFM1-TRFIA to analyze samples of 23 brands of milk that were purchased in Wuxi revealed that AFM1 was absent from all studied samples. This study suggests that the novel method is a simple, sensitive, specific, reproducible, economic, and adequate method for screening large quantities of samples and has good prospects of application.

Effects of methyl-beta-cyclodextrin on the release of luteiniz-ing hormone, follicle stimulating hormone, prolactin and growth hormone from cultured bovine anterior pituitary cells

The aims of the present study are to clarify the effect of methyl-beta-cyclo-dextrin (MβCD) on the secretions of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL) and growth hormone (GH) from the anterior pituitary (AP) cells of prepubertal male cattle and to compare the characteristics of secretion to those of gonadotrophic-releasing hormone (GnRH), thyrotropin- releasing hormone (TRH) and growth hormone-releasing hormone (GHRH), respectively. The AP cells prepared from six castrated Holstein calves (age: 8-11 months) were incubated for 2 h with MβCD (10-3 M, 10-2 M), GnRH (10-8 M), TRH (10-8 M) and GHRH (10-8 M) or vehicle only as a control (CTL). The amount of LH, PRL and GH released in the medium was measured by a time-resolved fluoroimmunoassay (TRFIA). The amount of FSH released in the medium was measured by the radioimmunoassay (RIA). MβCD significantly (P<0.05) stimulated the secretion of LH, FSH, PRL and GH from the AP cells. Furthermore, GnRH significantly (P<0.05) stimulated the secretion of LH and FSH. Also, TRH and GHRH significantly (P<0.05) stimulated the secretion of PRL and GH, respectively. However, the potency of the MβCD was less compared to each respective hormone secretions in response to GnRH, TRH and GHRH (P<0.05).

Direct competitive fluoroimmunoassays for detection of imidaclothiz in environmental and agricultural samples using quantum dots and europium as labels.

A direct quantum dots-based fluoroimmunoassay (QDFIA) and a time-resolved fluoroimmunoassay (TRFIA) for imidaclothiz (IMI) were developed by using the quantum dots (QDs)-labeled antibody and the europium (Eu3+)-labeled antibody, respectively. After optimization, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC10) are 20.41 and 0.52μgL-1 for the QDFIA, while 6.91 and 0.018μgL-1 for the TRFIA, respectively. The cross-reactivities (CRs) with the analogues are negligible except for imidacloprid with CRs of 29.0% for the QDFIA and 26.6% for the TRFIA. The spiked recoveries of IMI in paddy water, soil, pear, tomato, rice, apple, cabbage and cucumber are 72.7-117.6% with a standard deviation (RSD) of 2.4-13.5% for the QDFIA, and 81.3-117.7% with a RSD of 1.6-7.5% for TRFIA. The detection results of the analyses for the real paddy water and pear samples are markedly correlated with these of high-performance liquid chromatography (HPLC).

Simultaneous determination of free and total prostate‐specific antigen by a magnetic particle‐based time‐resolved fluoroimmunoassay

In this paper, a novel, sensitive and rapid method to simultaneously determine the free and total prostate-specific antigen (fPSA and tPSA) in serum by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation was described. The new approach uses magnetic particles as an immobilization matrix and means of separation, whereas the luminescent europium (Eu) and samarium (Sm) chelates are used as probes. The proposed method was evaluated via a single-step, sandwich-type TRFIA immunoassay of tPSA and fPSA as model analytes in serum. A new one-step method to simultaneously detect fPSA and tPSA in less than 15minutes was built up with the detection limits 0.006ng/mL for fPSA and 0.05ng/mL for tPSA. The assay ranges for fPSA and tPSA were both 0.5-100ng/mL, whereas the average recovery of those two obtained by our original mode were 95.9% and 95.3%, respectively. The present new TRFIA method carrying larger reproducibility, higher recovery, and sensitive specificity was demonstrated to be widely acceptable. This simultaneous determination method containing a fast and sensitive approach can be put into an important position to screening large quantities of specimen, and be commonly applied to the clinical determination of fPSA and tPSA in human serum.

A New Method Based on Time-Resolved Fluoroimmunoassay for the Detection of Streptomycin in Milk

Streptomycin (STR), used extensively in the treatment of bovine mastitis, may cause damage such as ototoxicity, allergic reaction, and increasing bacterial resistance to consumers on account of remnant in milk. A time-resolved fluoroimmunoassay (TRFIA) was developed to quantify STR for the first time to ensure food safety. Using secondary antibody labeled with europium (Eu3+) chelate as a tracer, the proposed TRFIA showed that the linear working range was 0.32–5.0 ng/mL under the optimal conditions. Milk samples were deproteinized by trichloroacetic acid and the limit of detection of STR in milk was 1.8 μg/kg. The recoveries of milk samples fortified with 4.0, 20, and 40 μg/kg of STR ranged from 86.2 to 96.3% with relative standard deviations less than 11%. Results of TRFIA for the authentic samples were coincided with those of UHPLC-MS/MS analyses. This study confirmed that the established TRFIA was sensitive as well as reliable and could be an alternative method to monitor STR residue in milk.

Development of a Dual-Label Time-Resolved Fluorescence Immunoassay (TRFIA) for Screening of Bladder Cancer based on Simultaneous Detection of BLCA-4 and NMP52 in Urine

Bladder cancer is a heterogeneous disease and occupies highest incidence in developed country. Time-resolved fluoroimmunoassays (TRFIA) is a new detection technique with a feature of sensitive, simple and inexpensive. The aim of this study is to establish a dual-label TRFIA for the simultaneous detection of BLCA-4 and NMP52 in urine in a single run. The sandwich immunoassay was used to detect the concentration of BLCA-4 and NMP52 in urine. BLCA- 4 and NMP52 in urine were captured by anti-BLCA-4 and anti-NMP52 antibodies immobilized on microtiter wells, and then banded together with another anti-BLCA-4 and anti-NMP52 labeled with europium(III) Sm3+ and samarium(III) Eu3+ chelates, followed by fluorescence measurement using time resolved fluorometry. To assess the performance of this assay, clinical urine samples were used, and then commercialized kits were compared. The sensitivity for BLCA-4 and NMP52 detection of this assay were 2 U/mL (dynamic range, 5-300 U/L) and 1 μg/ml (dynamic range, 2-150 μg/ml) respectively. The correlation coefficients (R) between the present dual-label TRFIA and commercially available kits were high. R-values were 0.99 for BLCA-4 and NMP52. The cross-reactivity seems not to influence the results. The present dual-label TRFIA, allowing the simultaneous detection ofBLCA-4 and NMP52, has high sensitivity, specificity, and accuracy in clinical sample analysis. Therefore, it has good prospects of application.

Alteration of thyroid hormone concentrations in juvenile Chinook salmon (Oncorhynchus tshawytscha) exposed to polybrominated diphenyl ethers, BDE-47 and BDE-99

Polybrominated diphenyl ethers (PBDEs) have been used as flame-retardants in consumer products and are currently detected in salmon globally. The two most predominant PBDE congeners found in salmon are BDE-47 (2,2′,4,4’-tetrabromodiphenyl ether) and BDE-99 (2,2′,4,4′,5-pentabromodiphenyl ether). In the present study, groups of juvenile Pacific Chinook salmon were fed five environmentally relevant concentrations of either BDE-47 (0.3–552 ng total PBDEs/g food), BDE-99 (0.3–580 ng total PBDEs/g food), or nearly equal mixtures of both congeners (0.7–690 ng total PBDEs/g food) for 39–40 days. The concentrations of circulating total thyroid hormones, thyroxine (T4) and 3,5,3’-triiodothyronine (T3), were measured using a hormone-specific time-resolved fluoroimmunoassay to determine if PBDE exposure disrupts the hypothalamic-pituitary-thyroid endocrine axis. The concentrations of both circulating T4 and T3 were altered in juvenile salmon by dietary uptake of BDE-99. Exposure to BDE-47 did not alter either T3 or T4 circulating hormone concentrations. However, exposure to a mixture of BDE-47 and BDE-99 reduced T3 in fish with lower concentrations of total whole body PBDEs than with either congener alone at equivalent PBDE whole body concentrations. Accordingly, the disruption of PBDEs on circulating thyroid hormone concentrations has the potential to impact a number of critical functions in juvenile salmon including growth, parr-smolt transformation, and immunological processes.

Development and effectiveness evaluation of time-resolved fluoroimmunoassay kit for detection of Schistosoma japonicum

To develop a kit of time-resolved fluoroimmunoassay (TRFIA) for detection of Schistosoma japonicum protein SjP38, and evaluate its effectiveness. The anti 9G7 SjP38 monoclonal antibody was used as the capture anti-body coated with 96-hole plate, and the Eu3+ labeled 1A6 monoclonal antibody was used as the detection antibody to establish the TRFIA SjP38 kit. In addition, the accuracy, sensitivity, precision, stability and coincidence rate to pathogenic diagnosis of the kit were evaluated. This established kit possessed high accuracy, wide linear range from 2 to 1 250 ng/ml, high sensitivity with the minimum detectable concentration of 0.14 ng/ml, and good precision (the coefficient variation of the intra- and inter-assay were 3.6% to 4.6% and 5.1% to 6.7%, respectively). The stability tests showed that the reagents could be stable for six months at 4 ℃, 7 d at 37 ℃. The positive and negative corresponding rates to the pathogen detection method were 95% and 100% respectively. All the performance and detection indicators of the kit have reached the requirements of clinical test, but its clinical application still needs further validation.

Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08–6.44 ng mL−1 and the medium inhibition of control (IC50) was 0.73 ng mL−1. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%–96.6% and with a coefficient of variation (CV) among 2.0%–8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples.

Simultaneous determination of β2-agonists clenbuterol and salbutamol in water and swine feed samples by dual-labeled time-resolved fluoroimmunoassay

Lanthanide ions, the excellent fluorescence markers, have the potential to simplify the performance of multi-analytes analysis. In this work, a powerful time-resolved fluoroimmunoassay (TRFIA) based on dual-labeled format were performed for detecting clenbuterol and salbutamol simultaneously in water and swine feed samples. Europium (Eu3+)-labeled goat anti-mouse IgG and Samarium (Sm3+)-labeled goat anti-rabbit IgG were prepared and used as fluorescent probes. Under optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC10) of the TRFIA were 0.263 and 0.007 ng/mL for clenbuterol, and 0.179 and 0.014 ng/mL for salbutamol, respectively. The cross-reactivities of the TRFIA between these two β2-agonists and with their analogues were negligible, thus indicated high specificity and promoted the simultaneous determination. The spiked recoveries were 72.2%–102.2% with relative standard deviations (RSDs) of 3.4–13.8% for clenbuterol and salbutamol in water and swine feed samples. Furthermore, the results of TRFIA correlated well with those obtained by HPLC. The proposed dual-labeled TRFIA was a satisfactory tool for simultaneous, sensitive, efficient and labour-saving detection of clenbuterol and salbutamol in water and swine feed samples.

A Pilot Study on the Physiological Effects of Three Essential Oils in Humans

In the present study, the physiological effects on 32 humans exposed to experimental stress provoked by inhalation of the essential oils of East Indian sandalwood (Santalum album L.), Western Australian sandalwood (Santalum spicatum R.Br.) and lavender (Lavandula angustifolia MILL.) were investigated. During individual testing sessions, several saliva samples were collected, blood pressure was regularly measured and parameters of the autonomic nervous system (heart rate, skin conductance response) were continuously monitored. Salivary cortisol, as an endocrine stress indicator, was determined by time- resolved fluoroimmunoassay. Statistical analyses evidenced that the tested sandalwood essential oils significantly reduced systolic blood pressure, especially during the recreation phase. This finding corresponds with a distinct reduction of salivary cortisol levels during recreation in the Western Australian sandalwood oil compared with the control. In conclusion, the results demonstrate that essential oils can alleviate the physiological reactions to psychological stress and facilitate recovery after exposition to stress.

Simultaneous Quantification of Anticardiolipin IgG and IgM by Time Resolved Fluoroimmunoassay.

The autoimmune disease antiphospholipid syndrome (APS) is characterized by the presence of anticardiolipin antibodies (aCL), along with anti-β2-glycoprotein I (β2GPI) antibodies and lupus anticoagulant (LA). In this study, we developed a time-resolved fluoroimmunoassay (TRFIA) system for simultaneous quantification of aCL IgG and IgM. A 96-well microtiter plate precoated with the complex of cardiolipin from bovine heart and bovine β2GPI was incubated with the anticardiolipin IgG and IgM standard substance or serum, and the conjugate of Eu3+-labeled anti-human IgG and Sm3+-labeled anti-human IgM was pipetted to the wells to form a tipical double-antibody-sandwich immunoreactions; finally the fluorescent intensity of Eu3+ and Sm3+ was detected to reflect the quantity of anticardiolipin IgG and IgM. This assay showed a good relationship between fluorescence intensities and the concentration of anticardiolipin antibody(aCL) IgG and IgM, with a low-end sensitivity of 0.1 U/ml for IgG and 0.1 U/ml for IgM, respectively. The intra- and inter-assay coefficients of variation (CV) of the calibrators was 3.0% and 4.51% for IgG, and 2.76% and 4.45% for IgM. The average recovery was 100.38% for aCL IgG and 100.45% for aCL IgM. For serum samples, the results of our method showed a good correlation with those obtained with ELISA kit. Simultaneous detection of aCL-IgG and aCL-IgM in the same reaction well can optimize assay performance by avoiding potential influence of different reaction conditions-timing, and well-to-well difference in concentration and characteristics of cardiolipin antigen. The results of a combo aCL-IgG and aCL-IgM assay for the same sample are more consistent and more reliable. This dual-label time-resolved fluoroimmunoassay is sensitive for detecting aCL IgG and IgM across a wide concentration range with stable reagents and may assist in the clinical diagnosis of antiphospholipid syndrome.

Application of Time-resolved Fluroimmunoassay for Determination of Furaltadone Metabolite 3-Amino-5-morpholinomethyl-2-oxazolidinone

Abstract To detect furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in fish sample, a Eu3+ labeling time-resolved fluoroimmunoassay (TRFIA) was developed. The experimental conditions were optimized as follows: the AMOZA-OVA concentration was 0.25 μg mL−1; the antibody was diluted by 50000 folds, and the competitive reaction time was 50 min. Under the optimal conditions, the method showed a detection limit of 0.01 ng mL−1, an IC50 of 0.26 ng mL−1 and a linear range (IC20−IC80) of 0.025−2.83 ng mL−1. The spiked recoveries of AMOZ in fish at three spiked levels ranged from 78.0% to 86.0%, and the relative standard deviations were less than 15%. Good correlation between ic-TRFIA and high performance liquid chromatography-tandem mass spectrometry was obtained for spiked food samples. The proposed ic-TRFIA method was suitable for the determination of AMOZ residue in food samples.

Immunohistochemical detection of corticotropin-releasing hormone (CRH) in the brain and pituitary of the hagfish, Eptatretus burgeri

The distribution of corticotropin-releasing hormone (CRH) in the brain and pituitary of the hagfish Eptatretus burgeri, representing the earliest branch of vertebrates, was examined by immunohistochemistry to better understand the neuroendocrine system of hagfish. CRH-immunoreactive (ir) cell bodies were detected in the preoptic nucleus, periventricular preoptic nucleus, infundibular nucleus of the hypothalamus, and in the nucleus “A” of Kusunoki et al. (1982) in the medulla oblongata. In the brain, CRH-ir fibers were detected in almost all areas except for the olfactory bulb and telencephalon. Bundles of CRH-ir fibers were detected in the dorsal wall of the neurohypophysis. However, CRH-ir fibers were distant from adrenocorticotropic hormone (ACTH) cells in the adenohypophysis, as studied by dual-label immunohistochemistry. Cortisol and corticosterone were detected in the plasma by a combination of reverse-phase high performance liquid chromatography and a time-resolved fluoroimmunoassay. These results suggest that in the hagfish, CRH, ACTH, and corticosteroids exist and that CRH released in the neurohypophysis likely reaches the adenohypophysis via diffusion.

Abstract 4293: Combination of hepatocyte growth factor, genistein and daidzein concentrations as a biomarker for gastric cancer risk: A nested case-control study

Abstract Backgrounds: Hepatocyte growth factor (HGF) induces ERK pathway which is associated with carcinogenesis by promoting cell proliferation. Inhibition of ERK signal transduction pathways by genistein, one of the isoflavones, has been reported by several in vitro studies. Therefore, gastric cancer risk by HGF and isoflavone concentrations was evaluated in this study. Methods: Nested case-control study with 93 gastric cancer cases and 1:1 matched controls was conducted within the Korean Multi-center Cancer Cohort (KMCC). HGF and ISF (genistein, daidzein, equol) concentrations in plasma were measured with enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay, respectively. Odds ratios (ORs) and 95% confidence intervals (95% CIs) on gastric cancer risk according to HGF and ISF level was calculated using conditional logistic regression models. Results: Compared to the group with low level of HGF (&amp;lt;315pg/mL) and high level of genistein (≥212.5nmol/L), the group having high level of HGF and lower level of genistein was associated with increased gastric cancer risk (OR = 5.08, 95% CI = 1.39-18.57). Elevated gastric cancer risk in the subjects having high HGF level and low daidzein level than subjects with low HGF and high daidzein levels was also shown (OR = 3.67, 95% CI = 1.18-11.45). There was significant interaction between HGF and genistein levels on gastric cancer was found (p for interaction&amp;lt;0.01). Continual increase in gastric cancer risk according to the counts of risky status by HGF, genistein, and daidzein levels was found (p for trend = 0.03). Conclusion: Our study suggests that combination of HGF, genistein and daidzein concentrations has potential as a biomarker for gastric cancer risk. Citation Format: Jieun Jang, Yunji Hwang, Choonghyun Ahn, Kwang-Pil Ko, Keun-Young Yoo, Sue K. Park. Combination of hepatocyte growth factor, genistein and daidzein concentrations as a biomarker for gastric cancer risk: A nested case-control study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4293.

Highly efficient detection of paclobutrazol in environmental water and soil samples by time-resolved fluoroimmunoassay

A fast and ultrasensitive indirect competitive time-resolved fluoroimmunoassay (TRFIA) was developed for the analysis of paclobutrazol in environmental water and soil samples. Paclobutrazol hapten was synthesized and conjugated to bovine serum albumin (BSA) for producing polyclonal antibodies. Under optimal conditions, the 50% inhibitory concentration (IC50 value) and limit of detection (LOD, IC20 value) were 1.09μgL−1 and 0.067μgL−1, respectively. The LOD of TRFIA was improved 30-fold compared to the already reported ELISA. There was almost no cross-reactivity of the antibody with the other structural analogues of triazole compounds, indicating that the antibody had high specificity. The average recoveries from spiked samples were in the range from 80.2% to 104.7% with a relative standard deviation of 1.0–9.5%. The TRFIA results for the real samples were in good agreement with that obtained by high-performance liquid chromatography analyses. The results indicate that the established TRFIA has potential application for screening paclobutrazol in environmental samples.

Polystyrene latex particles containing europium complexes prepared by miniemulsion polymerization using bovine serum albumin as a surfactant for biochemical diagnosis

Luminescent particles have been attracting significant attention because they can be used in biochemical applications, such as detecting and imaging biomolecules. In this study, luminescent polystyrene latex particles were prepared through miniemulsion polymerization of styrene with dissolved europium complexes in the presence of bovine serum albumin (BSA) and poly(ethylene glycol) monomethoxy methacrylate as surfactants. The solubility of the europium complex in styrene has a strong effect on the yield of the particle. Europium tris(2-thenoyl trifluoroacetonate) di(tri-n-octyl phosphine oxide), which has a high solubility in styrene, was sufficiently incorporated into the polystyrene particles compared to europium tris(2-thenoyl trifluoroacetonate), which has a low solubility in styrene. The luminescence property of the europium complex could remain intact even after its incorporation through the miniemulsion polymerization. In the aqueous dispersion, the resulting particles could emit strong luminescence, which is a characteristic of the europium complex. The antibody fragments were covalently attached to BSA-covered particles after a reaction with a bifunctional linker, N-(6-maleimidocaproyloxy)succinimide. The time-resolved fluoroimmunoassay technique showed that 3.3pg/mL of human α-fetoproteins (AFP) can be detected by using the resulting luminescent particles. An immunochromatographic assay using the resulting particles was also performed as a convenient method to qualitatively detect biomolecules. The detection limit of AFP measured by the immunochromatographic assay was determined to be 2000pg/mL. These results revealed that the luminescent particles obtained in this study can be utilized for the highly sensitive detection of biomolecules and in vitro biochemical diagnosis.

Three GnRH forms in brain and pituitary of Asian seabass, Lates calcarifer (Bloch, 1790) analysed by high performance liquid chromatography combined with time-resolved flouroimmunoassay and immunohistochemistry

Three forms of gonadotropin-releasing hormone (GnRH) namely, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH) were identified in the brain of Asian seabass Lates calcarifer using reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoassay (TR-FIA) using rabbit polyclonal antibody raised against sGnRH, cGnRH-II and sbGnRH. An extract of L. calcarifer brain showed a similar retention time to that of synthetic sGnRH, cGnRH-II and sbGnRH on rpHPLC. In the brain, cGnRH-II and sGnRH levels were high, whereas sbGnRH was absolutely dominant in the pituitary. In addition, cGnRH-II, sGnRH and sbGnRH immunoreactive (ir) cell bodies and fibers in the brain and pituitary were also examined by immunohistochemistry. sGnRH-ir cell bodies were localised in the ventromedial part of the rostral olfactory bulb, terminal nerve ganglion region, and the transitional area between the olfactory bulb and telencephalon. cGnRH-II-ir cell bodies were located only in the midbrain tegmentum and sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were found in many parts of the brain, being more dense in the midbrain, whereas sbGnRH-ir fibers were localised in the preoptic-hypothalamic area, innervating the pituitary. In the pituitary, sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis. These results confirm the existence of multiple GnRH forms with different physiological roles in L. calcarifer. Among them, sbGnRH is considered to be the best candidate for pituitary regulation while sGnRH and cGnRH-II can function as neurotransmtters or as neuromodulators in this species.

The Establishment of an HE4-CLIA Method and the Combined Analysis of HE4 and CA125 in Ovarian Cancer.

The human epididymal secretory protein 4 (HE4) is a novel, verified biomarker for the early diagnosis of ovarian cancer. Magnetic beads were coated with capture antibodies and were used with acridinium ester labeled detection antibodies in a sandwich-type immunoassay. The patient's HE4 serum levels were measured simultaneously with the chemiluminescence immunoassay (CLIA) kit we developed and electrochemiluminescence immunoassay (ECLIA) kit from Roche (Mannheim, Germany). CA125 was also detected by time-resolved fluoroimmunoassay. The diagnostic value was analyzed. The assay demonstrated a linear range from 2.5 to 2,000 pmol/l, with an analytical sensitivity of 2.5 pmol/l. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. Compared with the ECLIA kit from Roche in 124 serum samples (40 patients with ovarian cancer, 35 patients with benign gynecological diseases, and 49 health controls), there is a satisfied correlation coefficient of 0.875. The area under the receiver-operating curve (ROC-AUC) was 0.903 (95% CI was 0.839-0.966, P < 0.001) for HE4, 0.787 (95% CI was 0.694-0.879, P < 0.001) for CA125, and 0.914 (95% CI was 0.866-0.962, P < 0.001) for combined analysis of HE4 and CA125. A quantitative method (HE4-CLIA) for detecting HE4 in serum was successfully established. Preliminary clinical sample analysis showed HE4-CLIA has a certain clinical value in the screening and diagnosis of ovarian cancer. Moreover, in distinguishing benign from malignant ovarian lesions, HE4 has higher demonstrated accuracy than CA125.