A comparison of chemiluminescence immunoassay and radioimmunoassay for the detection of nortestosterone residues in meat samples

A chemiluminescence immunoassay, with a nantiserum raised against 19-nortestorone-3- carboxymethyloxime/bovine serum albumin and the N-(4-aminobutyl)-N-ethylisoluminol conjugate of 19-nortestosterone as tracer is compared to a radioimmunoassay, with an antiserum against 19-nortestosterone-17-hemisuccinate/bovine serum albumin and 19-(6,7-³H) nor testosterone as the radioactive label. Both methods have a similar sensitivity but the limit of quantification is much lower for radioimmunoassay (0.08 μg kg^-1) than for chemiluminescence immunoassay (0.6 μg kg^-1). A more practical appraoch, the limit of decision, is defined which is determined by the analytical results obtained from certified blank reference samples. This concept gives a good qualitative agreement between the two immunoassay techniques and the number of false positive and negative results, as ascertaind by GC/MS, is minimized; this is shown for assays of 38 meat samples.     

Séparation des alcalino-terreux par chromatographie sur colonne de cellulose

A good separation of tracer amounts of. Ca- Sr- Ba- Ra could be obtained by chromatography on a cellulose powder column on using successively the following two eluants: 1. Methanol-ether-HCl 12N (v/v: 75-25-5) (Ca, Sr). 2. Methanol - HCI 12N (v/v: 100-5) (Ba, Ra). Eluant 1, which has already been proposed for the separation of Ba and Sr in tracer amounts. is well suited for the separation of Ca Sr (carrier free) A further test of this eluant for the separation of ⁸⁹Sr-¹⁴⁰Ba, obtained carrier-free, from an irradiated uranium nitrate sample, proved to be successful.Eluant 2, gives a satisfactory Ba- Ra separation (Ba either carries free or not) and may be used for a rapid preparation of mesothorium 1 -sources (²²⁸Ra), free from barium; it is also recommended for the purification of small quantities of radium.     

Séparation Rb-Cs par chromatographie sur cellulose extraction sélective DU 137Cs de fission

A search for a good method of separating Rb-Cs in micro-quantities by chromatography on cellulose has revealed that only with a phenolic eluant (equilibrated with H₂O) or 2N HC1) can a specially good separation be easily obtained.This separation remains quite effective from quantities of a few milligrams to “tracer” amounts, and we were able to obtain a selective separation of fission ¹³⁷Cs “carrier free”, directly from a nitric solution of irradiated uranium.     

Determination of clenbuterol by capillary electrophoresis immunoassay with chemiluminescence detection

A competitive immunoassay for clenbuterol (CLB) based on capillary electrophoresis with chemiluminescence (CL) detection was established. The method was based on the competitive reaction of horseradish peroxidase (HRP)-labeled CLB (CLB-HRP) and free CLB with anti-CLB antiserum. The factors affecting the electrophoresis and CL detection were systematically investigated with HRP as a model sample. Under the optimal conditions, the tracer CLB-HRP and the immunoassay complex were separated, and the linear range and the detection limit (S/N = 3) for CLB were 5.0-40 nmol l⁻¹ and 1.2 nmol l⁻¹, respectively. The proposed method has been applied satisfactorily in the analysis of urine sample.     

NMR study of phase separation in D2O/ethanol solutions of poly(N-isopropylmethacrylamide) induced by solvent composition and temperature

¹H and ¹³C NMR spectroscopies were applied to investigate phase separation in solutions of poly(N-isopropylmethacrylamide) (PIPMAm) in D₂O/ethanol (EtOH) mixtures induced by solvent composition (cononsolvency) and temperature. Effects of EtOH content in D₂O/EtOH mixtures and temperature on the appearance and extent of the phase separation were characterized. Differences in mesoglobules formed during the phase separation induced by cononsolvency and temperature were found. For temperature-induced phase separation, ¹³C spin-spin relaxation times showed that besides the free EtOH expelled from the PIPMAm mesoglobules, there are also EtOH molecules bound in these mesoglobules. On the other hand, virtually no bound EtOH molecules were detected for mesoglobules formed as a consequence of the cononsolvency. For PIPMAm random copolymers containing negatively charged methacrylate units the phase separation induced by solvent composition was not observed.     

Procedure for quality control of chemiluminogenic labels

A method is presented to monitor the chemical, chemiluminogenic and immunochemical properties of steroid N-(4-aminobutyl)-N-ethylsoluminol (ABEI) labels. The label preparation was applied to an isocratic reversed-phase HPLC system (with on-line ultraviolet detection) and fractionated into seventy 12-s fractions. In the fractions, the immunochemicla response was determined by radioimmunoassay and the chemiluminescence was determined in a luminometer or with photplate detection. From the results of the three detection techniques, conclusions can be drawn about the quality and purity of the label. The presence and identity of immunochemically or chemiluminogenically reactive decomposition products cn also be monitored. As an example, a preparation of 17β-testoterone-3-carboxymethyloxime/aminobutylethylisoluminol is analyzed. Possibilities for quality control, stability control and preparative purification are discussed.     

Development of a chemiluminescence immunoassay for salivary progesterone using microtitre plates as a solid phase

Progesterone is determined in whole saliva by a direct solid-phase chemiluminescence immunoassay (CIA), which uses a monoclonal antibody raised against progesterone-11α-hemisuccinate bovine serum albumin (BSA) and the homologous marker conjugate progesterone-11α-hemisuccinate aminobutylethylisoluminol. Rabbit anti-mouse immunoglobulins (second antibody) are coated on the wells of Nunc Maxisorb microtitre plates during an overnight incubation. Then monoclonal anti-estradiol antibody is bound overnight to the second antibody and residual “binding” places are blocked with BSA. Saliva is frozen, thawed and the clear supernate obtained after centrifugation is used. The calibration graph is linear over the range 3–200 pg of progesterone and the detection limit is 1.6 pg per well (0.096 nmol l^?1). The mean recovery of added progesterone is 97%. The within-assay relative standard deviation (RSD) is 2.7–9.7% for 0.3–5.4 nmol l^?1 progesterone and the between-assay RSD is 5.7–10.6% for 0.79–3.7 nmol l^?1 progesterone. In pregnancy salivary progesterone rises from 1 nmol l^?1 (6–15 weeks) to 4 nmol l^?1 (36–40 weeks).     

Determination of Pyridinium Ionic Liquid Cations by Reversed Phase Ion-Pair Chromatography Using Gradient Elution

Four pyridinium ionic liquid cations (N-ethyl-pyridinium, N-butyl-pyridinium, N-butyl-4-methyl-pyridinium, N-hexyl-pyridinium) were separated and determined by reversed phase ion-pair chromatography with ultraviolet-visible detection. The effects of ion-pair reagent, acetonitrile concentration and column temperature on the retention and separation of the cations were evaluated. Then the four pyridinium cations could be separated at baseline within 13 min. The detection limits (S/N = 3) were 0.30–0.70 mg L^?1, and relative standard deviations (n = 5) for peak areas were 0.18–0.58%. The method was applied to analyze surface water with recoveries of 99.5–104.0%, which is accurate, reliable and practical.     

Solid-phase chemiluminescence immunoassay for screening of anabolic agents in application sites in slaughtered cattle

A solid-phase chemiluminescence immunoassay for 19-nortestosterone (NT) is presented; NT-3-carboxymethyloxime/N-(4-aminobutyl)-N-ethylisoluminol serves as the label with an antiserum raised against NT-3-carboxymethyloxime/bovine serum albumin. Some other anabolic compounds (e.g., testosterone and trenbolone) showed substantial cross-reactivity. The assay can be used generally for the detection of anabolic agents in application sites. Because of the high sensitivity (0.1 pg NT/tube at 90% relative binding), only 250 μg of muscle tissue is needed for the assay. Apparent NT contents of 0.4 to 16 000 μg kg^?1 tissue can be measured. With a simplified isolation method, about 40 samples can be screened in a working day.     

Direct radioimmunoassay of testosterone in human saliva

A simple direct radioimmunoassay for testosterone in 400 μl of whole human saliva is reported. Neither extraction nor chromatography is needed. The direct assay involves a commercially available, highly selective antiserum raised against testosterone-19-(O-carboxymethyl)-ether-BSA, an iodinated tracer, a serum added to give parallel inhibition of analyte and tracer binding to serum proteins and a double antibody/polyethylene glycol separation technique. The lower limit of determination is about 3 pM salivary testosterone and the calibration curve covers the range of clinical interest both for males and females (0–868 pM). The assay is specific as judged from recovery and dilution tests, from the cross-reactivity of the antiserum used and from comparisons with an extraction procedure. Within-assay and between-assay relative standard deviations are 4.1, 1.9, 10.5% and 6.3, 2.8, 15.3% for saliva samples with testosterone concentrations of 310, 117 and 52.7 pM, respectively.     

Effects of side-chains ofN-acetylamino acids on the structures of their triphenyltin(IV) complexes

Triphenyltin(IV) complexes ofN-acetylglycine,N-acetyl-L-leucine,N-acetyl-L-asparagine andN-acetyl-L-tyrosine were prepared by two methods and characterized by means of different spectroscopic methods (FTIR, multinuclear,¹H,¹³C and¹¹⁹Sn NMR and¹¹⁹Sn Mössbauer). The spectroscopic data indicated that theN-acetylglycine complex adopts a trigonal-bipyramidal structure in which the monodentate carboxylate and the amide-C=O group are bound to the same organotin(IV) moiety. The other three complexes are linear oligomers in which the planar Ph₃Sn(IV) is coordinated axially by a monodentate carboxylate and an amide-C=O from two different ligands. At theC-terminal end of the oligomer chain there is a tetracoordinated tin(IV) with a monodentate carboxylate as donor group.     

Studies on Am(III) separation from simulated high-level waste using cobalt bis(dicarbollide) (1? ) ion derivative covalently bound to N,N′-di-n-octyl diglycol diamide as extractant and DTPA as stripping agent

The separation of minor actinides from high level liquid waste (HLLW) belongs to the principal challenges in current nuclear treatment. A derivative based on two cobalt bis(dicarbollide) (1^?) ions covalently bound to the N,N??-di-n-octyl diglycolyl amide platform via diethyleneglycol chain with the formula {[(N,N??-(8-(OCH₂ CH₂)₂-1,2-C₂B₉H₁₀)(1??,2??-C₂B₉H₁₁)-3,3??-Co)(N,N??-n-C₈H₁₇)NCOCH₂]₂O}Na₂ (TODGA-COSAN), dissolved in low polar mixture of hexyl methyl ketone and n-dodecane, was used as an extractant for efficient Am(III)/Eu(III) separation from PUREX HLLW. Am(III) could be selectively stripped from loaded organic phase by using a stripping agent composed from 0.05?M DTPA and 1?M citric acid as a buffer and 1?M NaNO₃ at pH?3.0. Separation factor between europium and americium of 13 was achieved. The europium remaining in the organic phase could be consecutively effectively stripped by using solution of ammonium citrate or ammonium citrate with ammonium DTPA at pH~7.     

Flow-injection spectrophotometry of vanadium by catalysis of the bromate oxidation of N,N'-bis(2-hydroxyl-3-sulfopropyl)-tolidine

A highly sensitive flow-injection method is proposed for the catalytic determination of vanadium(V) at sub-nanogram per milliliter levels using a new indicator reaction. The method is based on the catalytic effect of vanadium(V) on the bromate oxidation of N,N′-bis(2-hydroxyl-3-sulfopropyl)-tolidine. 1,2-Dihydroxybenzene-3,5-disulfonate was used as an activator in the vanadium(V)-catalyzed reaction and significantly enhanced the sensitivity of the method. Vanadium(V) in the range 0.01-3.0 ng ml⁻¹ was easily determined with sampling rate of about 30 h⁻¹. Vanadium(IV) could be also determined. The limit of detection (S/N=3) was 0.008 ng ml⁻¹ and the relative standard deviations were 1.4 and 1.6% for ten determinations of 0.2 ng ml⁻¹ vanadium(IV) and vanadium(V), respectively. Interferences from metal ions could be suppressed by the addition of ethylenediamine-N,N,N′,N′-tetrakis(methylenephosphonic acid) as a masking agent. The proposed method was successfully applied to the determination of vanadium in water samples.     

Séparation baryum-strontium par chromatographie sur cellulose : Chromatographie sur poudre de cellulose

The systematic investigation of a good Ba⁺²- Sr⁺² separation using a. cellulose powder column has given us the choice of two eluants: (a) Methanol 1.2N en HCl (gazeux) + water ($\text{v}\text{v}$: $\text{100}\text{5}$) (b) Methanol-ether-HCl 12N ($\text{v}\text{v}$:75-25-5). The second one gave excellent separations, both in quantities of the order of 200 mg and in tracer amounts: Sr ? 0.001 μg-Ba ? 0.8 μg If we increase the eluting power of the solvent after the complete elution of Sr⁺² (suppression of ether), we considerably reduce the time and volume needed for the Ba⁺² elution. This work was done with the help of radioactive isotopes ¹⁴⁰Ba (12.8 days) and ⁹⁰Sr (25 years). At the same time, we found that the eluant used is also efficient for the separation of ¹⁴⁰Ba and ⁹⁰Sr from their respective decay products: ¹⁴⁰La and ⁹⁰Y (40h and 65h).     

Development of an ELISA procedure to study sorption of atrazine onto a sewage sludge-amended luvisol soil

Pesticides may contaminate ground and surface waters and one of the major factors governing this property is soil sorption. Sorption can be assessed by batch equilibrium technique which produces lots of extracts with high dissolved organic carbon concentration in which the pesticide concentration has to be determined. We developed an ELISA procedure to analyse atrazine based on polyclonal antibodies (C193) for which tracer structure and dilutions of immunochemical reagents were adapted to fit the purpose. After a 1000-fold dilution (or after an SPE clean-up procedure) extracts of a sewage-sludge amended luvisol (used as an example application of the methodology developed) could be reliably analysed. The Freundlich model is able to describe adsorption for this system (r² = 0.977) delivering a distribution coefficient K_F of 1.6 ± 0.2 (mg kg⁻¹) (mg L⁻¹)^−N and an isotherm nonlinearity factor N of 0.70 ± 0.09.     

Homogeneous immunoassay for the detection of trinitrotoluene (TNT) based on the reactivation of apoglucose oxidase using a novel FAD-trinitrotoluene conjugate

A flavin adenine dinucleotide-trinitrotoluene derivative (FAD-TNT) was synthesized by coupling N ⁶-(2-aminoethyl)-FAD covalently to the N-hydroxysuccinimidyl ester of trinitrophenyl-γ-aminobutyric acid and characterized by negative-ion electrospray-ionization mass spectrometry (ESI-MS) after purification by reversed-phase HPLC. Free FAD-TNT can be detected at very low levels by recombination with apoglucose oxidase, since the FAD-TNT-glucose oxidase complex is enzymatically active. On the contrary, if FAD-TNT has been bound by an anti-TNT antibody, the conjugate cannot recombine with apoglucose oxidase any more. Based on these two phenomena, a homogeneous apoenzyme reactivation immunoassay system (ARIS) was developed for the detection of TNT. No separation step is needed in this assay. Proportionality between the TNT concentration and enzyme activity was demonstrated with a detection limit of 5 μg/L TNT.     

Click novel glycosyl amino acid hydrophilic interaction chromatography stationary phase and its application in enrichment of glycopeptides

A novel glycosyl amino acid hydrophilic interaction chromatography (HILIC) stationary phase was prepared via click chemistry. The key intermediate N₃-glycosyl d-phenylglycine was prepared by a three steps procedure, including selective condensation of amino glucose with N-succinimidyl ester of Boc-d-phenylglycine, deprotection and transformation of amino group to azido group. The structure of all the intermediates and functionalized silica beads were confirmed by ¹H NMR, IR, elemental analysis and ¹³C CP-MAS. The chromatography test showed that this new type of separation material possessed good HILIC properties and glycopeptide enrichment characteristics. Nucleosides and bases could be separated in a simple eluent composition (only acetonitrile in combined with water), and with the same condition, these model compounds could not be separated on the commercial HILIC column (Atlantis). Click glycosyl amino acid thus prepared also showed longer retention and better separation ability in the separation of polar organic acids.     

Photogeneration and transport studies on differently polymerized n-vinylcarbazole polymers

Photogeneration and transport studies were carried out on carefully purified, poly(N-vinylcarbazole) (PVK) samples prepared with different initiators. The results were compared both with those obtained from commercial samples and those reported in the literature. The experimental mobility values obtained [μ] = [cm²/Vsec] were low compared with the “trap free” mobility value, recently reported (μ = 10⁻³cm²/Vsec). The presence of persistent impurities, formed during the polymerization procedure and bound to the polymer backbone, together with the presence of specific structural faults, could explain the rather low observed mobility values. The photoelectric yield for all the samples, except those prepared from EtAlCl₂ (PVK-IV), is about 3 × 10⁻³. The higher value shown by the PVK-IV samples (10⁻²) could be ascribed to a lower concentration of traps for excitons (viz the so-called “second excimer site”).     

A novel P-doped and NCDs loaded g-C3N4 with enhanced charges separation for photocatalytic hydrogen evolution

Graphite carbon nitride(g-C₃N₄) is a promising non-metal photocatalyst for photocatalytic hydrogen production, but its performance is still limited due to sluggish charges separation and low utilization of light.In this work, P-doped and N-doped carbon dots(NCDs) supported g-C₃N₄were successfully prepared via hydrothermal and polymerization reactions. The sub-bandgap formed by P-doping enhances the utilization of visible light, and the high electron de...     

Fluorescein labeling of estrogen for application of fluorescence polarization binding assays for antibody and receptor

The fluorescence polarization binding assay (FPBA) using fluorescein-labeled estrogen tracer is a homogeneous assay applicable to both estrogen antibody and estrogen receptor-binding assays. Two estrogen-ethylendiamine fluoresceinthiobamyl (E-EDF) tracers were synthesized; estrogen-6-EDF (E-6-F) derived from 6-ketoestradiol 6-(o-carboxymethyl) oxime and estrogen-17-EDF (E-17-F) was from 17β-estradiol 17-hemisuccinate. In both FPBAs using antibody and receptor, E-6-F tracer (Rf_365nm=0.58) showed a better binding response than E-17-F (Rf_365nm=0.70) indicating that the 17-position of estrogen seems to play an essential role as a binding site for antibody or receptor. In the optimized conditions of FPBA for E₂ using E-6-F tracer, antibody binding (K_d=9.4×10⁻⁹ M) is 50 times sensitive than receptor binding (K_d=4.6×10⁻⁸ M). Binding responses of estrogen and its related chemicals by FPBA indicate that antibody binding assay is able to screen the structural similarity of estrogen showing some response with methyltestosterone (K_i=2.1×10⁻⁵ M). On the other hand, the receptor assay is able to screen for estrogenic chemicals such as tamoxifen (K_i=4.5×10⁻⁹ M) and diethylstilbesterol (K_i=8.1×10⁻⁷ M). Therefore, E-6-F tracer is useful as a tracer for FPBA that is able to screen for chemicals structurally similar to estrogen using antibody, and that is able to screen for chemicals functionally similar to estrogen using receptor binding assay.