Direct time-resolved fluoroimmunoassay of estriol in serum

A rapid, direct, solid-phase, time-resolved fluoroimmunoassay for free estriol in serum, using Europium-chelate-protein A as a label, is described. The coefficient of correlation with the results of RIA was 0.983.     

Rapid analysis of phytoestrogens in human urine by time-resolved fluoroimmunoassay

A time-resolved fluoroimmunoassay (TR-FIA), with europium labeled phytoestrogens as tracers, was developed for the quantitative determination of enterolactone, genistein and daidzein in human urine. The aim was to create a method for the screening of large populations in order to assess the possible correlations between the urinary levels and the risk of Western diseases.

After the synthesis of the 5′-carboxymethoxy derivative of enterolactone and 4′-O-carboxymethyl derivatives of daidzein and genistein, the respective compound was coupled to bovine serum albumin and then used as an antigen in the immunization of rabbits. The same derivatives of the phytoestrogen were used in preparing the europium tracers. After the enzymatic hydrolysis, the TR-FIA was carried out using the Victor 1420 multilabel counter. The method has sufficient sensitivity to measure the phytoestrogens at concentrations even below 5 nmol/l. The intra- and inter-assay coefficients of variation, at three different concentrations, varied from 1.9 to 5.3 and from 2.4 to 9.7, respectively.

We measured urinary enterolactone, genistein and daidzein in 215 samples from Finnish healthy women and found that more than 50% of the values ranged between 1 and 7, <0.1 and 0.6 and below 0.6 μmol/24 h, respectively. The TR-FIA method including only a hydrolysis step gave higher values than those measured by gas chromatography–mass spectrometry (GC–MS). However, the assay results by the present method showed strong correlation with those obtained by GC–MS. It is concluded that the TR-FIA is suitable for population screening of urinary phytoestrogens.     

Development of a highly sensitive and specific new testosterone time-resolved fluoroimmunoassay in human serum

A new time-resolved fluoroimmunoassay (TR-FIA) of testosterone in serum is described, using a biotinylated testosterone tracer, with a long spacer arm between biotin and testosterone, coupled to the C3 of the testosterone: a biotinylaminodecane carboxymethyloxime testosterone. This tracer affords a great sensitivity of the standard curve, because a amount of 0.3 pg of testosterone can be significantly measured on the testosterone standard curve. The "functional" sensitivity is at least equal to 21 pg/ml of serum. The specificity of the assay is insured by a celite chromatographic step on new minicolumns before immunoassay. The variation coefficient of inter-series reproducibility measured on low and normal testosterone levels in untreated and testosterone treated hypogonadal men were between 2.17 and 5.07%. The accuracy test, (overload and dilution tests) gave satisfying results. Moreover, in a comparison with GCMS, it appeared that the correlation coefficient was 0.992 and no significant difference could be exhibited between the two methods. Consequently, this specific, sensitive reproducible and easy to use method is well suited to the measurement of testosterone in clinical and pharmacological conditions.     

Evaluation of time-resolved fluoroimmunoassay with Eu-labelled protein-A for serum progesterone

Progesterone has been assayed in several serum samples by a time-resolved fluoroimmunoassay (TR-FIA). The solid phase was 11 alpha-hydroxyprogesterone hemisuccinate bound covalently to ovalbumin and adsorbed on wells of polystyrene. The assay was based on competitive reaction of solid phase-bound hormone and samples with specific antibody labelled in situ with protein-A prelabelled with europium. The bound Eu was dissociated from the solid phase by an enhancement solution and measured by time-resolved fluorometry. The coefficient of correlation between TR-FIA and RIA was 0.97.     

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-NH₂-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.     

The fluorometric determination of europium ion concentration as used in time-resolved fluoroimmunoassay

A model of the relationships between optimal fluorescence yield, Eu3+ ion concentration, and the concentration of beta-diketone in the determination of Eu3+ ion concentration in aqueous solutions as used in time-resolved fluoroimmunoassays was developed. The model, in addition to optimizing the determination of the metal ion, also presents a new simple and rapid method for the determination of stability constants for the formation of beta-diketone-Eu3+ complexes. It is shown how the method may be extended to determine the formation constants for Eu3+ with other chelates.     

Determination of bufalin-like immunoreactivity in serum of humans and rats by time-resolved fluoroimmunoassay for using a monoclonal antibody

The existence of a mammalian natriuretic substance or endogenous digitalis-like factor, which inhibits Na+,K+-ATPase and thereby regulates body fluid volume, has been speculated for a long time but has yet to be defined. We established in the present study a simple and highly sensitive procedure to measure bufalin, a constituent of toad venom preparation and a specific inhibitor of Na+,K+-ATPase by time-resolved fluoroimmunoassay (TR-FIA) and using a monoclonal antibody. The antibody was specific to bufalin and resembled bufadienolides but showed no cross-reactivity with digitoxin and ouabain. A bufalin-like immunoreactivity was detectable in serum of humans and rats by the proposed TR-FIA. The levels of bufalin-like immunoreactivity in serum of healthy volunteers were significantly correlated with their systolic blood pressure. Moreover, bufalin-like immunoreactivity in serum of Dahl-S rats increased in parallel with a period of high-salt diet. These results suggest that increased bufalin-like immunoreactivity may be associated with certain types of hypertension.     

时间分辨荧光免疫分析技术研究现状及进展

时间分辨荧光免疫分析是一种新型的超微量的免疫标记分析方法,集酶标记免疫分析和放射免疫分析等优点于一身,且无放射性污染等.本文主要介绍了时间分辨荧光免疫分析的原理、优点,螯合剂的种类,以及各种分析技术及其应用现状和进展.     

Channel-resolved multianalyte immunosensing system for flow-through chemiluminescent detection of alpha-fetoprotein and carcinoembryonic antigen

A novel flow-through immunosensing system for chemiluminescent (CL) multianalyte immunoassay was designed based on channel-resolved technique. Using alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as model analytes, two polyethersulfone membranes modified with the corresponding capture antibodies were set in two channels of a flow cell, and then two mixtures of the sample and corresponding alkaline phosphatase labeled antibodies were introduced into the channels for on-line incubation, respectively. Upon injection of CL substrate the catalyzed CL signals from the two channels were sequentially collected with the aid of an optical shutter for CL detection of two analytes. The antibodies immobilized membranes could be regenerated for reuse. Under optimal conditions AFP and CEA could be assayed in the ranges of 5.0-150 and 0.50-80 ng/ml with detection limits of 1.5 and 0.25 ng/ml, respectively. The assay results of clinical serum samples with the proposed system were in acceptable agreement with those with the reference method in single-analyte test mode. This novel immunosensing system based on the designed channel-resolved technique provided an automated, reusable, simple, sensitive and low-cost approach for multianalyte immunoassay without using of expensive array detector.     

Co-ordinate action of bacterial adhesins and human carcinoembryonic antigen receptors in enhanced cellular invasion by capsulate serum resistant Neisseria meningitidis

Neisseria meningitidis (Nm) is a human specific opportunistic pathogen that occasionally penetrates mucosal barriers via the action of adhesins and invasins and evades host immune mechanisms during further dissemination via capsule expression. From in vitro studies, the primary adhesion of capsulate bacteria is believed to be mediated by polymeric pili, followed by invasion via outer membrane adhesins such as Opa proteins. As the latter requires the surface capsule to be down-modulated, invading bacteria would be serum sensitive and thus avirulent. However, there is recent evidence that capsulate bacteria may interact via Opa proteins when host cells express high levels of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), their target receptors. Such a situation may arise following increased circulation of inflammatory cytokines that upregulate certain adhesion molecules on host cells. In this study, using a tetracycline controlled expression system, we have developed cell lines with inducible CEACAM expression to mimic post-inflammation state of target tissues and analysed the interplay between the three surface components capsule, pili and Opa proteins in cellular interactions. With two distinct cell lines, not only the level but also the rate of adhesion of capsulate Opa-expressing Nm increased concurrently with CEACAM density. Moreover, when threshold levels of receptor were reached, cellular invasion ensued in an Opa-dependent manner. In studies with cell lines intrinsically expressing pilus receptors, notable synergism in cellular interactions between pili and Opa of several meningococcal strains was observed and was independent of capsule type. A number of internalized bacteria were shown to express capsule and when directly isolated from host cells, these bacteria were as serum resistant as the inoculated phenotype. Furthermore, we observed that agents that block Opa-CEACAM binding substantially reduced cellular invasion, while maintaining a low level of cellular adhesion. These studies highlight some of the factors that may determine increased host susceptibility to infection by serum resistant phenotypes; and demonstrate the potential of selective inhibition of key interactions in preventing target tissue penetration while maintaining a level of colonization.     

Comparison of plasma and urinary phytoestrogens in Japanese and Finnish women by time-resolved fluoroimmunoassay

Time-resolved fluoroimmunoassays (TR-FIA), with europium labeled phytoestrogens as tracers, were developed for the quantitative measurement of genistein, daidzein and enterolactone in plasma and urine for the purpose of screening large populations and studies on possible correlation between the values in biological fluids and the risk of western diseases. The mean values of the three phytoestrogens in plasma as determined by TR-FIA were similar to those obtained by gas chromatography-mass spectrometry (GC-MS). The urinary excretion levels of total individual phytoestrogens were higher than those obtained by GC-MS, with the exception of the daidzein values. However, comparing the assay results obtained by the present method and those obtained by GC-MS, a strong correlation was evident (r = 0.87 - 0.99, p < 0.001). We measured plasma levels of genistein, daidzein and enterolactone in 111 healthy Japanese women The mean and median levels of genistein were 406.8 and 306.3 nmol/l, respectively, and those of daidzein were 118.4 and 76.8 nmol/l, respectively. These levels are higher than those reported for Americans and Western Europeans. Isoflavone intake as calculated from dietary records (genistein: mean, 86.5 mircomol/day and daidzein: mean, 57.4 micromol/day) was correlated with the plasma concentrations observed (genistein: r = 0.287, p < 0.01 and daidzein: r = 0.313, p < 0.01). Plasma enterolactone levels were low in Japanese women (mean, about 10 nmol/l). The levels of urinary excretions of genistein, daidzein were also measured and it was found that, in the majority, the levels ranged between 5-25 and 5-50 micromol/24 h, respectively. In contrast, healthy Finnish women showed very low values of isoflavones (below 10 nmol/l in plasma (n = 87) and below 0.6 micromol/24 h in urine (n = 126) for both compounds) and high levels of enterolactone in both plasma and urine (plasma: mean, 25 nmol/l and urine: majority range, 1-7 micromol/24 h).     

Localisation of carcinoembryonic antigen in embryonic and fetal human tissues

Summary Carcinoembryonic antigen (CEA) was localized in various embryonic and fetal human tissues between 8 and 16 weeks of gestation as well as in the colorectal mucosa of older fetuses, newborns and adults. Among the embryonic tissues, CEA was always present in the esophagus, the gastric antrum, the duodenum and the rectum. CEA positive staining of bile cannaliculi of the liver was inconstant. All other embryonic tissues were CEA negative. During early fetal development CEA positive staining of the esophagus, antrum and duodenum was inconstant. However, the whole colon became intensively stained. An inconstant CEA specific staining was found in parts of the midgut and in the bile cannaliculi of the liver. The other organs remained CEA negative. Between the 17th week of gestation and birth, CEA staining pattern of the colorectal mucosa did not change. The staining intensity of late fetal colonic mucosa was similar to that of adult colonic mucosa.Deceased 20th August 1982     

Localisation of carcinoembryonic antigen in embryonic and fetal human tissues

Carcinoembryonic antigen (CEA) was localized in various embryonic and fetal human tissues between 8 and 16 weeks of gestation as well as in the colorectal mucosa of older fetuses, newborns and adults. Among the embryonic tissues, CEA was always present in the esophagus, the gastric antrum, the duodenum and the rectum. CEA positive staining of bile canaliculi of the liver was inconstant. All other embryonic tissues were CEA negative. During early fetal development, CEA positive staining of the esophagus, antrum and duodenum was inconstant. However, the whole colon became intensively stained. An inconstant CEA specific staining was found in parts of the midgut and in the bile canaliculi of the liver. The other organs remained CEA negative. Between the 17th week of gestation and birth, CEA staining pattern of the colorectal mucosa did not change. The staining intensity of late fetal colonic mucosa was similar to that of adult colonic mucosa.     

Rapid time-resolved fluoroimmunoassay for diethylstilbestrol residues in chicken liver

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of diethylstilbestrol (DES) residues in chicken liver. Prior to analysis, the residues were extracted from chicken liver with acetonitrile. The assay could be used in a quantitative or qualitative mode. The limit of detection (LOD) was determined to be 0.05 ngg(-1), and the limit of quantification (LOQ) was less than 0.18 ngg(-1). The intraassay variations were less than 10%, and the interassay variations were from 9.8 to 12.7%. The mean recoveries established at six concentration levels varied from 84.3 to 109.6%, and the coefficients of variation were from 8.3 to 12.4%. The results obtained by the TR-FIA and enzyme-linked immunosorbent assay (ELISA) showed a good correlation. The established TR-FIA was validated for the determination of incurred chicken liver and was confirmed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). This proposed technique could be applied to routine residue analysis.     

Simultaneous determination of prednisolone, prednisolone acetate and hydrocortisone in human serum by high-performance liquid chromatography

A method for the simultaneous determination of prednisolone, prednisolone acetate and hydrocortisone has been established to monitor the serum levels of these three compounds in healthy volunteers following intramuscular administration of prednisolone acetate. Serum samples of 0.75 ml were extracted with ethyl acetate after addition of the internal standard, dexamethasone. The compounds were separated using a LiChrosorb Si 60 column and detected by UV absorbance. Specificity, linearity, as well as the repeatability, intermediate-precision and accuracy of the method were established. The lower limit of quantification was 2.0 ng/ml for prednsolone (C.V. = 14.7%, N=6) and 5.0 ng/ml for prednisolone acetate (C.V. = 13.9%, N= 6 and hydrocortisone (C.V. = 11.7%, N=6). Data on the recovery of the compounds and the internal standard are provided. The results of quality control samples determined during routine analysis (n = 114) are presented. Serum levels of the compounds after intramuscular adminstration of 25 mg of prednisolone acetate are discussed.     

时间分辨荧光免疫分析在兽药残留检测中的应用

近年来,兽药残留引起食物中毒的报道日益增多,兽药残留检测的意义重大。传统的气相色谱法、液相色谱法存在前处理复杂、仪器成本昂贵等缺陷,酶联免疫吸附分析(enzyme-linked immunosorbent assay,ELISA)灵敏度也不高,而时间分辨荧光免疫分析(time-resolved fluoroimmunoassay,TRFIA)操作简便、灵敏度高,已在兽药残留检测领域引起重视。介绍了TRFIA的原理和优势,综述了其在促生长繁殖类、瘦肉增产类和杀菌驱虫类兽药残留检测中的应用,并与传统方法进行了对比,TRFIA有望取代传统的检测方法成为兽药残留检测的常规方法。     

Simultaneous determination of dehydroepiandrosterone and its 7-oxygenated metabolites in human serum by high-resolution gas chromatography--mass spectrometry

A highly sensitive and specific method has been developed for the simultaneous measurement of free (unconjugated) or sulfate-conjugated forms of dehydroepiandrosterone (DHEA), 7alpha-hydroxy-DHEA (7alpha-OH-DHEA), 7beta-hydroxy-DHEA (7beta-OH-DHEA), and 7-oxo-DHEA (7-oxo-DHEA) in human serum. This method is based upon a stable isotope-dilution technique by gas chromatography-selected-ion monitoring mass spectrometry. Free steroids were extracted from serum with an organic solvent and the sulfate-conjugated steroids remained in aqueous phase. Free steroids were purified by solid-phase extraction, while sulfate-conjugated steroids were hydrolyzed by sulfatase and deconjugated steroids were purified by solid-phase extractions. The extracts were treated with O-methylhydroxylamine hydrochloride and were subsequently dimethylisopropylsilylated. The resulting methyloxime-dimethylisopropylsilyl (MO-DMIPS) ether derivatives were quantified by gas chromatography-selected-ion monitoring mass spectrometry in a high-resolution mode. The detection limits of MO-DMIPS ether derivatives of DHEA, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA were 1.0, 0.5, 0.5 and 2.0pg, respectively. Coefficients of variation between samples ranged from 10.6 to 22.9% for free 7-oxygenated DHEA to less than 10% for DHEA and sulfate-conjugated 7-oxygenated DHEA. The concentrations of these steroids were measured in 18 sera samples from healthy volunteers (9 males and 9 females; aged 23-78 years). Free DHEA, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA levels ranged between 0.21-3.55, 0.001-0.194, 0.003-0.481, and 0.000-0.077ng/ml, respectively, and the sulfate-conjugated steroid levels of these metabolites ranged between 253-4681, 0.082-3.001, 0.008-0.903, and 0.107-0.803ng/ml, respectively. The free DHEA-related steroid concentrations were much lower than those previously measured by RIA and low-resolution GC-MS. The present method made it possible to determine simultaneously serum DHEA-related steroid levels with sufficient sensitivity and accuracy.