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.     

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.     

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.     

Development of time-resolved fluoroimmunoassay with enhanced fluorescence reaction for the quantitation of atezolizumab in plasma

Atezolizumab (ATZ) is a human monoclonal antibody, which has been granted multiple approvals from the US Food and Drug Administration (FDA) for the immunotherapy of different types of cancer. This study describes the prototype of a time-resolved fluoroimmunoassay (TRFIA) for the quantitation of ATZ in plasma. The assay involved the non-competitive binding of ATZ to its specific antigen [programmed death-ligand 1 (PD-L1) protein]. The immune complex formed on the inner surface of the assay plate wells was quantified by anti-human secondary antibody labeled with a chelate of europium-ethylenediaminetetraacetic acid. The enhanced fluorescence signal was generated by an enhanced fluorescence solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The conditions of the TRFIA were refined, and its optimum procedures were established. The assay was validated in accordance with the immunoassay validation guidelines, and all the validation parameters were acceptable. The working range of the assay was 20–1000 pg mL⁻¹, and its limit of quantitation was 20 pg mL⁻¹. The assay was applied to the quantitation of ATZ in plasma samples with satisfactory accuracy and precision. The proposed TRFIA has significant benefits over the existing methodologies for the quantitation of ATZ in clinical settings.     

Simultaneous determination of alpha-fetoprotein and carcinoembryonic antigen in human serum by time-resolved fluoroimmunoassay

A novel simultaneous measurement method for alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera by time-resolved fluoroimmunoassay (TR-FIA) is described. The proposed approach combines the use of europium-labeled anti-AFP antibody for AFP TR-FIA and biotinylated anti-CEA antibody complexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microtiter plate coated with a mixture of anti-AFP and anti-CEA monoclonal antibodies was used for the assay. After it was reacted with a solution containing AFP and CEA, a mixture of anti-AFP antibody labeled with BHHCT-Eu(3+) and biotinylated anti-CEA antibody was added. The AFP concentration was determined by measuring the solid-phase fluorescence of the europium-labeled anti-AFP antibody at 615 nm. Then a BHHCT-Sm(3+)-labeled streptavidin-bovine serum albumin conjugate (SA-BSA) was added to react with the biotinylated anti-CEA antibody. After the reaction, the unreacted SA-BSA was washed out, and a 0.1 M NaOH solution containing 1.0 x 10(-5) M TOPO and 0.05% SDS was added to dissociate the samarium-labeled SA-BSA in the immune complex on the surface of the well into the solution. The CEA concentration was determined by measuring the solution fluorescence of 643 nm from the samarium-labeled SA-BSA. The present method gives detection limits of 0.07 ng/ml for AFP and 0.3 ng/ml for CEA. The coefficient variations of the method are less than 7%, and the recoveries are in the range of 90-110% for serum samples. The AFP and CEA concentrations in 27 human serum samples were determined by the present method as well as by single assay for comparison. A good correlation was obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA.     

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.     

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。     

Development of a sensitive and specific new plasma 4-androstene-3,17- dione time-resolved fluoroimmunoassay (TR-FIA)

We describe, for the first time to our knowledge, the development of a new, non-isotopic time resolved-fluoroimmunoassay of 4-androstene-3,17-dione in plasma or serum. This steroid exhibits a key role in steroid metabolism and is often assayed in the investigation of various pathologic endocrine states. Most of the 4-androstene-3,17-dione immunoassays are performed using a radioactive tracer. We synthesized a biotinylated 4-androstene-3,17-dione tracer from 4-androstene-3,17-dione-3-carboxymethyloxime by acylation of biotinylaminopropylammonium trifluoroacetate. A specific rabbit anti 6-hemisuccinate-4-androstene-3,17-dione/BSA was indirectly bound via an anti-rabbit sheep antibody immobilized on microtiter plate wells. The amount of biotinylated-4-androstene-3,17-dione tracer was then measured by adding streptavidin-europium, and the europium fluorescence was quantified by time resolved-fluorescence (TR-FIA, Delfia System). The plasma 4-androstene-3,17-dione-levels measured with this non-isotopic assay were compared to those measured with a radioimmunoassay previously published. In both cases, the same anti-4-androstene-3,17-dione antibody was used, and the assays were performed after an extraction step and a chromatographic step. The results obtained by the two methods were virtually the same. However, the main advantages of the new plasma 4-androstenedione-3,17-dione time-resolved-fluorescence immunoassay were its greater sensitivity than radioimmunoassay and its higher precision.     

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.     

Time-resolved fluoroimmunoassay of plasma and urine O-desmethylangolensin

We present a method for the determination of the phytoestrogen metabolite O-desmethylangolensin (O-DMA) in plasma (serum) and in urine. O-DMA is a metabolite of daidzein, which occurs in soybeans. It has been suggested that isoflavones may afford protection against breast and prostate cancer and therefore, also the metabolites are of interest. The method is based on time-resolved fluoroimmunoassay (TR–FIA) using a europium chelate as a label. After the synthesis of 4′′-O-carboxymethyl-O-DMA, this compound is coupled to bovine serum albumin, and then used as antigen in immunization of rabbits. The tracers with the europium chelate are synthesized using the same 4′′-O-derivative of the -methyldeoxybenzoin. After enzymatic hydrolysis and ether extraction the immunoassay is carried out by time resolved fluoroimmunoassay (TR–FIA). Cross-reactivity was tested with angolensin, dihydrogenistein, dihydrodaidzein, equol, 6′-OH-angolensin, trans-4-OH-equol, 6′-OH-O-DMA, cis-4-OH-equol and 5-OH-equol. The antiserum cross-reacted only with angolensin. This cross-reactivity seems not to influence the results, which were highly specific. Plasma samples are hydrolyzed and extracted. Urine samples are analyzed directly after hydrolysis without extraction. The correlation coefficient between the plasma TR–FIA results and the GC–MS results was high; r value was 0.985. The correlation coefficient between the urine TR–FIA results and the GC–MS results was high over the entire range of concentrations (0–1500 nmol/l); r value was 0.976, but lower in the low concentration range (0–100 nmol/l), i.e. value was 0.631. The intra-assay coefficients of variation (CVs) for plasma O-DMA concentrations and for urine O-DMA concentrations at three different concentrations varied 2.8–7.7 and 3.0–6.0%, respectively and the inter-assay CVs varied 3.8–8.9 and 4.4–6.6%, respectively. The working range of the plasma and urine O-DMA assays was 0.5–512 nmol/l.     

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.     

Development of a new sensitive and specific time-resolved fluoroimmunoassay (TR-FIA) of chlormadinone acetate in the serum of treated menopausal women

We describe the development of a serum chlormadinone acetate (CMA) time-resolved fluoroimmunoassay (TR-FIA). We prepared haptens (3-CMO-chlormadinone acetate and 6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinate), biotinylated tracers (3(biotinylaminopropylamido) 3-CMO-chlormadinone acetate and 3-(6-chloropregna-4,6-dien-17,20-diol-3-one-20-hemisuccinylamino)1-biotinylaminopropane), and immunogens necessary for eliciting two antibodies (anti-chlormadinone acetate 3-CMO/BSA and anti-chlormadinone 20-hemisuccinate/BSA). The specificity of the assay was rigorously studied to eliminate possible interference by polar metabolites of CMA, particularly 17 alpha-acetoxy-6-chloro-3beta-hydroxypregna-4,6-diene-20-one (3beta-hydroxy metabolite), employing an easy-to-use ethylene glycol chromatographic step prior to immunoassay, so as to separate the polar metabolites, in particular the 3beta-hydroxy-CMA metabolite, from the intact CMA. The choice of the anti-CMA antibody was guided by the high assay sensitivity obtained with the anti-CMA 3-CMO/BSA antibody. The detection limit was 51pg/ml. Interassay reproducibility CVs were between 2.6 and 4.5%. This TR-FIA thus appeared to be a sensitive, specific, precise, and consequently well-suited method for measurement of serum CMA during a pharmacokinetic study in women.     

Time-resolved fluoroimmunoassay of plasma daidzein and genistein

We present a method for the determination of the phytoestrogens daidzein and genistein in plasma (serum). These weakly estrogenic isoflavones occur in soybeans and in smaller amounts in some other beans and plants. It has been suggested that they may afford protection against prostate and breast cancer. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After synthesis of 4'-O-carboxymethyl-daidzein and 4'-O-carboxymethyl-genistein the compounds are coupled to bovine serum albumin (BSA), then used as antigens to immunize rabbits. The tracers with the europium chelate are synthesized using the same 4'-O-derivative of the isoflavones. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). The antisera cross-reacted to some extent with some isoflavonoids but not with flavonoids. The cross-reactivity seems not to influence the results, which were highly specific for both compounds. The correlation coefficients between the TR-FIA methods and the reference method based on isotope dilution gas chromatography-mass spectrometry were high; r-values were about 0.95-0.99 depending on concentration. The intra-assay coefficients of variation (CV%) for daidzein and genistein at three different concentrations vary 3.2-4.5 and 3.2-4.1, respectively. The inter-assay CVs vary 5.0-6.3 and 4.5-5.3, respectively. The working ranges of the daidzein and genistein assays are 1.0-216 and 1.7-370 nmol/l, respectively. The plasma values (n = 80) of daidzein and genistein are very low in Finnish subjects (mean for daidzein, 3.8+/-6.8 and for genistein, 3.2+/-7.6 nmol/l; median value for daidzein 1.5 and for genistein 1.4 nmol/l).     

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.     

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.     

Comparison between radioimmunoassay and a new time-resolved fluoroimmunoassay: Determination of total serum testosterone in male mink (Mustela vison)

A comparison of a solid-phase immunoassay using time-resolved fluorescence (TR-FIA) and a conventional radioimmunoassay (RIA) was performed for the determination of total serum testosterone in peripheral blood samples obtained from 231 mink males (Mustela vison). The correlation between the values obtained with the two methods was good (r=0.85;y=1.52+1.05x). The values obtained with FIA (11.67±6.70 ng/ml) were slightly higher than those obtained with RIA (9.64±5.39 ng/ml). Standards prepared from female mink serum behaved similarly to the bovine serum standards used in the commercial kits. The data obtained show that FIA is a reproducible method and provides a useful tool for measurement of a large number of samples within a short period of time.     

Time-resolved fluoroimmunoassay for equol in plasma and urine

We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4′-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4′-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography–mass spectrometry (GC–MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC–MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC–MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5–6.5 and 3.4–6.9, respectively. The inter-assay CV% varies between 5.4–9.7 and 7.4–7.7, respectively. The working ranges of the plasma and urine assay are 1.27–512 and 1.9–512 nmol/l, respectively.