One-step competitive immunoassay for cadmium ions: development and validation for environmental water samples

A rapid, simple, and reliable competitive immunoassay was developed and validated for measurement of Cd(II) in environmental water samples. This assay employed a monoclonal antibody that recognizes Cd(II)-EDTA complexes as capture reagent and a Cd(II)-EDTA conjugate of horseradish peroxidase as an enzyme label. The assay depended on a competitive binding reaction between the enzyme conjugate and Cd(II)-EDTA complexes, derived from the environmental water sample, for the binding sites of the immobilized antibody. The concentration of Cd(II) in the sample was quantified by the ability of its EDTA complexes to inhibit the binding of the enzyme conjugate to the antibody and, subsequently, color formation in the assay. The assay was specific to Cd(II), with a limit of detection of 0.3 ppb. Ca(II), Mg(II), and Fe(III), the metal ions commonly found in ambient water at relatively high concentrations, did not interfere with the assay. Mean analytical recovery of added Cd(II) was 100.29 +/- 3.60. The precision of the assay was satisfactory; coefficients of variation were 3.6-10.9 and 4.81-10.21% for intra- and interassay precision, respectively. The assay compared favorably with graphite furnace atomic absorption spectroscopy in its ability to accurately measure Cd(II) spiked into water samples from a Louisiana bayou.     

Development and validation of a rapid HPLC method for the determination of ketotifen in pharmaceuticals

In the present study, a simple, sensitive, rapid, and stability-indicating high performance liquid chromatographic (HPLC) method with ultraviolet detection for the analysis of ketotifen was developed and validated. The method was applied to the determination of ketotifen in pharmaceutical formulations (tablets and syrups). The HPLC method utilized isocratic elution technique with a reversed phase C8 column, detection at 297 nm and a mixture of methanol, triethylamine phosphate buffer (pH 2.8; 0.04 M), and tetrahydrofuran (43: 55: 2, v/v/v) as mobile phase at a flow rate of 1.2 mL/min. Total analysis time was about 7 min with typical retention time of ketotifen of about 5 min. The method was validated for selectivity, linearity, accuracy, and precision following International Conference of Harmonization, 1996 (ICH) recommendations. Due to its simplicity and accuracy, the method can be used for routine quality control analysis.     

Dual-color fluorescence and homogeneous immunoassay for the determination of human enterovirus 71

We have developed a new fluorescent immune ensemble probe comprised of a conjugated lower toxic water-soluble CdTe:Zn(2+) quantum dots (QDs) and Ru(bpy)(2)(mcbpy-O-Su-ester)(PF(6))(2)- antibody complex (Ru-Ab) for the dual-color determination of human enterovirus 71 (EV71) in homogeneous solution. EV71 monoantibody was easily covalently conjugated with Ru(bpy)(2)(mcbpy-O-Su-ester)(PF(6))(2) to form a stable complex Ru-Ab, which acted both as an effective quencher of QDs fluorescence and the capture probe of virus antigen EV71. Herein, the target EV71 can break up the low fluorescent ionic ensemble by antigen-antibody combination to set free the fluorescent QDs and restore the fluorescence of QDs whereas the fluorescence intensity of Ru-Ab remains the same. Thus, the determination of EV71 by the complex Ru-Ab and QDs can be realized via the restoration of QDs fluorescence upon addition of EV71 and even can be directly evaluated by the ratio of green-colored QDs fluorescence intensity to Ru-Ab red-colored fluorescence intensity. The green-colored fluorescence of QDs was very sensitive to the change of EV71 concentration, and its fluorescence intensity increased with the increase of EV71 concentration between 1.8 ng/mL and 12 μg/mL. With this method, EV71 was detected at subnanogram per milliliter concentration in the presence of 160 μg/mL bovine serum albumin. More importantly, this strategy can be used as a universal method for any protein or virus by changing conjugated antibodies in disease early diagnosis providing a fast and promising clinical approach for virus determination. In a word, a simple, fast, sensitive, and highly selective assay for EV71 has been described. It could be applied in real sample analysis with a satisfactory result. It was notable that the sensor could not only achieve rapid and precise quantitative determination of protein/virus by fluorescent intensity but also could be applied in semiquantitative protein/virus determination by digital visualization.     

Latex piezoelectric immunoassay: analysis of C-reactive protein in human serum

We constructed an integrated immunosensor system to combine a quartz crystal microbalance with the agglutination reaction of immunized latex beads. C-reactive protein (CRP) induced an immunoreaction due to the latex bends with anti-CRP antibody. We succeeded in determining the concentration of CRP necessary in human serum for disease diagnosis using a CRP Sensor I.     

Development of a method for the determination of human skin moisture using a portable near-infrared system.

In this study, a portable near-infrared (NIR) system was newly integrated with a photodiode array detector that has no moving parts, and this system has been successfully applied for the evaluation of human skin moisture. The good correlation between NIR absorbance and the absolute water content of separated hairless mouse skin, in vitro, was showed, depending on the water content (7.4-84.9%) using this portable NIR system. Partial least squares (PLS) regression was used for calibration with the 1150-1650-nm wavelength range. For practical use for the evaluation of human skin moisture, the PLS model for human skin moisture was developed in vivo using the portable NIR system on the basis of the relative water content values of stratum corneum from the conventional capacitance method. The PLS model showed a good correlation. This study indicated that the portable NIR system, as compared to conventional methods, could be a powerful tool for human skin moisture, which may be much more stable to environmental conditions, such as temperature and humidity. Furthermore, to confirm the performance of the newly integrated portable NIR system, a scanning-type conventional NIR spectrometer was used in the same experiments, and the results were compared.     

Development and evaluation of a reference measurement procedure for the determination of estradiol-17beta in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry

Estradiol is the most potent natural estrogen and is derived from the ovaries. Its concentration in blood is measured to determine ovarian function. A reference measurement procedure for estradiol in serum involving isotope-dilution coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. A deuterated estradiol (estradiol-d3) was used as an internal standard. The estradiol and its internal standard were extracted from serum matrix using solid-phase extractions and derivatized with dansyl chloride prior to reversed-phase LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this reference method on lyophilized human serum reference materials for estradiol [Certified Reference Materials (CRMs) 576, 577, and 578] with the certified values determined by gas chromatography/mass spectrometry (GC/MS) reference methods and by a recovery study for the added estradiol. The results of this method for estradiol agreed with the certified values within the uncertainty of the measurements for the three CRMs. The recovery of the added estradiol ranged from 100.7 to 101.8%. This method was applied to the determination of estradiol in frozen serum samples from three individual female donors. Excellent reproducibility was obtained with within-set coefficient of variations (CVs) ranging from 0.6 to 2.2% and between-set CVs ranging from 0.2 to 0.4%. Excellent linearity was also obtained, with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.998 to 1.000. The detection limit at a signal-to-noise ratio of approximately 3 was 0.6 pg of estradiol (or 1 ng/L, as expressed as a concentration). This well-characterized LC/MS/MS method for serum estradiol, which demonstrates good accuracy and precision, low susceptibility to interferences, and comparability with GC/MS reference methods, qualifies as a reference measurement procedure that can be used to provide an accuracy base to which routine methods for estradiol can be compared and that will serve as a standard of higher order for measurement traceability.     

Development of a "membrane cloaking" method for amperometric enzyme immunoassay and surface plasmon resonance analysis of proteins in serum samples

Detection of trace amounts of target proteins in the presence of high concentrations of matrix proteins (e.g., serum samples) without separation steps is of great significance to biomedical research but remains technically challenging. Here we report a "membrane cloaking" method to overcome nonspecific protein adsorption and fouling problems for label-free surface plasmon resonance detection and heterogeneous immunosensing. A thin, hybrid, self-assembled monolayer on gold was formed with 70 mol % mercaptopropanol and 30 mol % cysteamine/propanedithiol to facilitate membrane fusion and covalent attachment of antibodies. After antibody immobilization, the surface was incubated with lipid vesicles, which fused to form a supported membrane. The analyte spiked in serum was introduced for binding, and the membrane and nonspecifically adsorbed proteins on the membrane were subsequently removed using a nonionic surfactant before the final measurement was carried out. Selection of a suitable surfactant can preserve antibody/antigen binding and selectively remove the membrane, allowing accurate measurement of the captured proteins without interference from nonspecifically adsorbed species. Surface plasmon resonance (SPR) quantification of IgG spiked in undiluted serum ( approximately 75 mg/mL protein) was achieved with the membrane cloaking method, whereas direct measurement without membrane removal resulted in a significantly large error. The cloaking method was also used to develop an enzyme amplified amperometric assay using HRP-conjugated IgG. Detection of concentrations as low as 5 fM proteins was obtained. Finally, a membrane cloaking assay combining SPR and in situ electrochemical measurement was demonstrated on a gold substrate. Similar sensitivity was observed using a continuous flow injection measurement. The method opens new avenues to develop direct assay methods with ultrahigh sensitivity for protein samples using SPR and enzyme-linked amplification mechanisms.     

Development and certification of a standard reference material for vitamin D metabolites in human serum

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health's Office of Dietary Supplements (NIH-ODS), has developed a Standard Reference Material (SRM) for the determination of 25-hydroxyvitamin D [25(OH)D] in serum. SRM 972 Vitamin D in Human Serum consists of four serum pools with different levels of vitamin D metabolites and has certified and reference values for 25(OH)D(2), 25(OH)D(3), and 3-epi-25(OH)D(3). Value assignment of this SRM was accomplished using a combination of three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). Chromatographic resolution of the 3-epimer of 25(OH)D(3) proved to be essential for accurate determination of the metabolites.     

Development and validation of a technique for the determination of 226Ra and 228Ra by liquid scintillation in liquid samples

Radium isotopes are dispersed in the environment according to their physicochemical characteristics. Considering their long half-lives and radiological effects, (226)Ra and (228)Ra are very important issues in radiological protection. In Brazil, radium isotopes represent an exposure problem both in nuclear fuel cycle installations and in high natural radiation background areas. The experimental part of this work includes the development of a technique for the determination of (226)Ra and (228)Ra by liquid scintillation with potential application in biological samples. Radium was concentrated and then separated from other constituents of the sample by co-precipitation/precipitation with Ba(Ra)SO(4). The precipitate was filtered and weighted to calculate the chemical yield. The filter containing the precipitate of Ba(Ra)SO(4) was transferred to a scintillation vial. Two methods were used to prepare the sources. The first one consisted of the addition of water (8 ml), Instagel XF (8 ml) and UltimaGold (4 ml) in the vial containing the filter and the precipitate, forming a gel suspension. In the second method, the precipitate was dissolved with 0.2 M ethylene-diamine-tetra-acetic-acid solution (9 ml) and 11 ml of scintillation solution (Optiphase Hisafe 3) was added to the vial, forming an aqueous and an organic phase. The solutions obtained were counted in a low background scintillation spectrometry system (Quantulus) suitable for the detection and identification of both alpha and beta particles for the determination of (226)Ra and (228)Ra. The activity values of (226)Ra and (228)Ra calculated by the two methods are in good agreement with the reference values, indicating that both methods are suitable for the determination of (226)Ra and (228)Ra.     

Development and validation of an analytical method for tensile strength determination of fibrous bulk solids

The increasing significance of products consisting of elongated particles or fibres along with a lack of understanding flow properties of fibrous bulk solids in processes urge for appropriate test procedures.Therefore, a tensile tester was designed with respect to the special needs in terms of test techniques of those bulk solids. The procedure of tensile strength determination was tested with regard to several possible influencing factors. Following from that, the manner of filling and filling height were identified to have the greatest influence on results. Furthermore, it is shown that the developed load system is capable of improving the repeatability of test results for fibrous bulk solids.Based on the derived standard procedure, systematic tests were carried out on beech and spruce chips in three different fractions each as well as model materials such as polypropylene fibres and differently sized bunches of glass fibres. The biomass materials have been characterised by dynamic image analysis prior to and after experiments resulting in particle size and shape factor distributions. It is found that tensile strength is affected by particle shape, size, roughness and interparticle contact area.     

Development and evaluation of a candidate reference measurement procedure for the determination of progesterone in human serum using isotope-dilution liquid chromatography/tandem mass spectrometry

Progesterone is a steroid hormone that is involved in regulating female reproductive processes. Its concentration in blood is measured to determine ovarian function. A candidate reference measurement procedure for progesterone in human serum involving isotope dilution coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. The progesterone along with its internal standard (progesterone-13C2) was extracted from the serum matrix using liquid-liquid extraction prior to reversed-phase LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this method on a lyophilized human serum reference material for progesterone [Certified Reference Material (CRM) 347] with the certified values determined by gas chromatography/mass spectrometry (GC/MS) reference methods and by a recovery study for the added progesterone. The results of this method for progesterone agreed with the certified value within the uncertainty of the measurements for the CRM 347. The recovery of the added progesterone ranged from 100.1 to 100.9%. This method was applied to the determination of progesterone in frozen serum samples from three individual female donors with the progesterone concentrations ranging from 0.151 to 24.42 ng/g. Excellent reproducibility was obtained with within-set coefficients of variation (CVs) ranging from 0.1 to 1.4%, and between-set CVs ranging from 0.3 to 0.5%. Excellent linearity was also obtained with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.9998 to 1.0000. The detection limit at a signal-to-noise ratio of approximately 3 was 1.8 pg of progesterone. This well-characterized LC/MS/MS method for serum progesterone, which demonstrates good accuracy and precision, low susceptibility to interferences, and comparability with GC/MS reference methods, qualifies as a reference measurement procedure that can be used to provide an accuracy base to which routine methods for progesterone can be compared and that will serve as a standard of higher order for measurement traceability.     

Development and validation of a high-performance liquid chromatography-mass spectrometry assay for determination of amphetamine, methamphetamine, and methylenedioxy derivatives in meconium

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of amphetamine, methamphetamine, and methylendioxy derivatives in meconium, using 3,4-methylendioxypropylamphetamine as internal standard. The analytes were initially extracted from the matrix by 17 mM methanolic HCl. Subsequently, a solid-phase extraction with Bondelut Certify columns was applied. Chromatography was performed on a C(18) reversed-phase column using a linear gradient of 10 mM ammonium bicarbonate, pH 9.0-methanol as a mobile phase. Analytes were determined in LC-MS single ion monitoring mode with an atmospheric pressure ionization-electrospray interface. The method was validated in the range 0.005-1.00 microg/g using 1 g of meconium per assay. Mean recoveries ranged between 61.1 and 87.2% for different analytes. The quantification limits were 0.005 microg/g meconium for amphetamine, methamphetamine, and 4-hydroxy-3-methoxymethamphetamine and 0.004 microg/g meconium for 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine. The method was applied to analysis of meconium in newborns to assess eventual fetal exposure to amphetamine derivatives.     

Surface-enhanced Raman scattering based approach for quantitative determination of creatinine in human serum

A novel surface-enhanced Raman scattering (SERS) based approach for the quantitative determination of creatinine in human serum is described. Using isotopically labeled (2-13C, 2,3-15N2) creatinine as internal standard, SERS acquires the character of a ratio method that works similar to the well-established isotope dilution techniques. In conjunction with multivariate data analysis, the method was successfully applied for quantifying creatinine at clinically relevant levels and below. A partial least-squares regression model was generated from a set of 87 calibration spectra covering the full range of mole fractions of neat creatinine. The prediction performance of the model was thereafter validated with independent reference samples giving a standard deviation of less than 2%. Finally, a conditioning procedure to prepare real serum samples for SERS-based creatinine analysis was worked out and validated. Measured serum creatinine concentrations are within 3% of the values obtained from gas chromatography/isotope dilution mass spectrometry on the same serum starting material.     

Direct construction of an open-sandwich enzyme immunoassay for one-step noncompetitive detection of thyroid hormone T4

To establish a sensitive noncompetitive immunoassay for thyroxine (T4), we attempted to isolate anti-T4 antibodies from a phage display library based on a phagemid pDong1 ( Dong et al. Anal. Biochem.2009, 36, 386 ), which was designed to enable open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) after selection on immobilized antigen. After the Fab-displaying phage library made from the splenocytes of T4-KLH immunized mice was subjected to biopanning on T4-BSA, two T4-specific clones were obtained. When they were assayed by indirect competitive ELISA, both clones showed low IC(50) (5-13 ng/mL), indicating their high affinity to T4. When they were used for OS-ELISA that detects antigen-dependency of the interaction between variable domains V(H) and V(L), a clone successfully detected 1 ng/mL of T4 with a working range superior to that of competitive IA. OS-ELISA was also performed with maltose binding protein (MBP)-fused V(H)/V(L) of this clone, which showed a detection limit less than 0.1 ng/mL T4. Moreover, the assay showed cross-reactivity with T3 similar to that of competitive ELISA, and also gave a reasonable total serum T4 concentration (90 ng/mL) from ethanol-extracted sample serum using the recombinant proteins. This is the first direct construction of an OS-ELISA system bypassing hybridoma, which will be applicable to the detection of many other small molecule antigens.     

Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1beta and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1beta and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal-Wallis analysis of variance with Dunn's multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences ( P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1beta determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.     

Development and evaluation of a reference measurement procedure for the determination of total 3,3',5-triiodothyronine in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry

3,3',5-Triiodothyronine (T3) is an important diagnostic marker for thyroid function. A reference measurement procedure (RMP) for total T3 in serum involving isotope dilution coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. The method uses solid-phase extraction with mixed-mode retention mechanisms of reversed phase and ion exchange prior to reversed-phase LC/MS/MS. In addition to a labeled T3 internal standard (T3-13C9), labeled thyroxine (T4-d5) is also added to serum samples in order to monitor the degradation of T4 to T3. The accuracy of the measurement was evaluated by a recovery study for added T3 and was supported by a comparison study with the other RMP. The recovery of the added T3 ranged from 98.9% to 99.4%. The results of this method and the other RMP agreed to within 1%. Samples of frozen serum pools were prepared and measured in three separate sets. Excellent reproducibility was obtained with within-set coefficients of variation (CVs) ranging from 0.8% to 1.6% and between-set CVs ranging from 1.9% to 2.6%. Excellent linearity was also obtained with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.9995 to 0.9996. The detection limit at a signal-to-noise ratio of approximately 3 was 1 pg of T3. The T4 degradation during sample preparation was minimized to a small percentage (no more than 3% of the T3 values) by use of antioxidants (ascorbic acid, dithiothreitol, citric acid) and can be accounted for in the T3 measurement process. This well-characterized LC/MS/MS method for total serum T3, which demonstrates good accuracy and precision, low susceptibility to interferences, accountability of the conversion of T4 to T3, and comparability with the other RMP, qualifies as a reference measurement procedure and can be used to provide an accuracy base to which routine methods for T3 can be compared.