Detection of HbA(1c) by boronate affinity immunoassay using bacterial magnetic particles.

We have developed a boronate affinity immunoassay system using m-aminophenylboronic acid (mAPB) coupling to bacterial magnetic particles (BMPs). Homobifunctional crosslinker, Bis-(succcimidyl)suberate (BS3), was employed for preparation of mAPB-BMPs conjugates (mAPB-BMPs). Quantities of HbA_1c on mAPB-BMPs were evaluated based on luminescence from alkaline phosphatase-conjugated anti-Hb antibody (ALP–antibody) binding to HbA_1c on the BMP surface. The binding of HbA_1c to mAPB-BMPs occurred gradually and was almost completed within 10 mm. The coupling reaction is enhanced due to static electric interaction between the positive charges on HbA_1c and negative charges on BMPs. The amount of HbA_1c binding to mAPB-BMPs increased with increasing sodium chloride concentrations in the range of 0–100 mM. However, the amount of Hb binding to mAPB-BMPs also increased in high concentration of sodium chloride. The Hb binding to mAPB-BMPs was detached from mAPB-BMPs when Hb–mAPB-BMPs were washed with low salt buffer. This indicates that Hb is nonspecifically adsorbed onto the surface of mAPB-BMPs in high concentration of sodium chloride. These results suggest that selective separation of HbA_1c using mAPB-BMPs can be achieved with these conditions. A dose–response curve was obtained between luminescence intensity and HbA_1c concentration using a fully automated boronate affinity immunoassay. A linear relationship between luminescence intensity and HbA_1c concentration was obtained in the range of 10–10⁴ ng/ml.     

Detection of HbA1c by boronate affinity immunoassay using bacterial magnetic particles

We have developed a boronate affinity immunoassay system using m-aminophenylboronic acid (mAPB) coupling to bacterial magnetic particles (BMPs). Homobifunctional crosslinker, Bis-(succcimidyl)suberate (BS3), was employed for preparation of mAPB-BMPs conjugates (mAPB-BMPs). Quantities of HbA_1c on mAPB-BMPs were evaluated based on luminescence from alkaline phosphatase-conjugated anti-Hb antibody (ALP–antibody) binding to HbA_1c on the BMP surface. The binding of HbA_1c to mAPB-BMPs occurred gradually and was almost completed within 10 mm. The coupling reaction is enhanced due to static electric interaction between the positive charges on HbA_1c and negative charges on BMPs. The amount of HbA_1c binding to mAPB-BMPs increased with increasing sodium chloride concentrations in the range of 0–100 mM. However, the amount of Hb binding to mAPB-BMPs also increased in high concentration of sodium chloride. The Hb binding to mAPB-BMPs was detached from mAPB-BMPs when Hb–mAPB-BMPs were washed with low salt buffer. This indicates that Hb is nonspecifically adsorbed onto the surface of mAPB-BMPs in high concentration of sodium chloride. These results suggest that selective separation of HbA_1c using mAPB-BMPs can be achieved with these conditions. A dose–response curve was obtained between luminescence intensity and HbA_1c concentration using a fully automated boronate affinity immunoassay. A linear relationship between luminescence intensity and HbA_1c concentration was obtained in the range of 10–10⁴ ng/ml.     

Development of a micro-plate magnetic chemiluminescence enzyme immunoassay (MMCLEIA) for rapid- and high-throughput analysis of 17beta-estradiol in water samples

A novel method of micro-plate magnetic chemiluminescence enzyme immunoassay (MMCLEIA) for the screening of 17beta-estradiol in water samples was proposed. It used the micro-plate magnetic separator designed by ourselves which can achieve the high-throughput analysis without the samples pre-treatment and the sensitive chemiluminescence system of AMPPD-ALP system. The method showed specific recognition of estrogen, without cross-reactions for three other major estrogenic compounds (17beta-estradiol (E2), estriol (E3), ethinyl (E2)) commonly found in water. The MMCLEIA was also especially suitable for the large-scale samples processing. The working range for 17beta-estradiol was 10-3000 pg/ml. The assay sensitivity was 5.4 pg/ml. Both intra- and inter-assay had relative standard deviation of less 15%. The effect of several physico-chemical parameters, such as the ratio of antibody versus antigen, incubation time and the concentration of detergent were studied. This method has been successfully applied to the preliminary detection of the sea-water. Compared with the chemiluminescence enzyme-linked immunosorbed assay, the correlation was good.     

ENUMERATION OF IMMUNOMAGNETICALLY CAPTURED ESCHERICHIA COLI IN WATER SAMPLES USING QUANTUM DOT-LABELED ANTIBODIES

Immunomagnetic separation was coupled with quantum dot (QD) labeling for the rapid, selective and sensitive detection of Escherichia coli in water samples. The target bacteria were recovered from the solution by antibody-coated paramagnetic beads, and sandwich complexes were formed by using secondary antibodies labeled with QDs. The fluorescence intensities, as a result of the capturing of different concentrations of bacteria, were measured, and a linear correlation ( R ²  =  0.976) was obtained between log E. coli concentration ( x ) and the intensity ( y ) with a regression model of y =  26.9x  +  41.1 in a working range of 8.9  ×  10¹ and 1.9  ×  10⁶ cfu/mL. The selectivity of the developed sensor was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The ability of the immunoassay to detect E. coli in real water samples was investigated and the results were compared with the experimental results from plate-counting methods. A good agreement was observed between the QD-enhanced detection and plate counting.

PRACTICAL APPLICATIONS

In this study, a rapid, sensitive and convenient fluorometric assay based on the immunomagnetic separation (IMS) and quantum dot (QD) labeling was employed for the detection of Escherichia coli in water samples. The incorporation of QDs into fluorometric immunoassay techniques has various advantages over labeling with organic dyes and enzymes. In addition, the spectroscopic properties of QDs can allow multiplexed immunoassays coupled with IMS for bacteria detection, which will be investigated in further studies. Here we showed that QD labeling is a promising tool for the detection of E. coli in real water samples containing different components with a lower detection limit.     

Evaluation of measurement method of a bacterial sepsis marker, procalcitonin

Procalcitonin (PCT) is a novel biomarker for diagnosis and severity evaluation of bacterial sepsis. PCT measurement methods provided by Wako Pure Chemical Industries, Ltd. include a fully automated chemiluminescent immunoassay system SphereLight Wako and fully automated immunoanalyzer microTASWako i30 for a quantitative measurement, and immunochromatographic assay method, B R A H M S PCT-Q kit. This time, basic performance of SphereLight Wako and microTASWako i30 was evaluated as quantitative determination methods for PCT. The lower limit of detection for the both methods was 0.02 ng/ml. Correlation coefficients of 0.993 to 0.997 indicated good correlation between the two methods. The both methods allow quick and easy measurement of PCT, therefore they are helpful for diagnosis and severity evaluation of bacterial sepsis.     

Development and validation of a liquid chromatography and tandem mass spectrometry method for determination of roscovitine in plasma and urine samples utilizing on-line sample preparation

Roscovitine, a purine analogue that selectively inhibits cyclin-dependent kinases, has been considered as a potential anti-tumor drug. The determination of roscovitine in plasma and urine was performed using microextraction in packed syringe as on-line sample preparation method with liquid chromatography and tandem mass spectrometry. The sampling sorbent utilized was polystyrene polymer. 2H3-lidocaine was used as internal standard. The limit of detection for roscovitine was as low as 0.5 ng/mL and the lower limit of quantification was 1.0 ng/mL. The accuracy and precision values of quality control samples were between +/-15% and < or =11%, respectively. The calibration curve was obtained within the concentration range 0.5-2000 ng/mL in both plasma and urine. The regression correlation coefficients for plasma and urine samples were > or =0.999 for all runs. The present method is miniaturized and fully automated and can be used for pharmacokinetic and pharmacodynamic studies.     

A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway

We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.     

A screen-printed, amperometric biosensor array incorporated into a novel automated system for the simultaneous determination of organophosphate pesticides

Organophosphate pesticides present serious risks to human and environmental health. A rapid reliable, economical and portable analytical system will be of great benefit in the detection and prevention of contamination. A biosensor array based on six acetylcholinesterase enzymes for use in a novel automated instrument incorporating a neural network program is described. Electrochemical analysis was carried out using chronoamperometry and the measurement was taken 10s after applying a potential of 0 V vs. Ag/AgCl. The total analysis time for the complete assay was less than 6 min. The array was used to produce calibration data with six organophosphate pesticides (OPs) in the concentration range of 10(-5) M to 10(-9) M to train a neural network. The output of the neural network was subsequently evaluated using different sample matrices. There were no detrimental matrix effects observed from water, phosphate buffer, food or vegetable extracts. Furthermore, the sensor system was not detrimentally affected by the contents of water samples taken from each stage of the water treatment process. The biosensor system successfully identified and quantified all samples where an OP was present in water, food and vegetable extracts containing different OPs. There were no false positives or false negatives observed during the evaluation of the analytical system. The biosensor arrays and automated instrument were evaluated in situ in field experiments where the instrument was successfully applied to the analysis of a range of environmental samples. It is envisaged that the analytical system could provide a rapid detection system for the early warning of contamination in water and food.     

Automated method for the determination of fat-soluble vitamins in serum

A new fully automated high-performance liquid chromatography (HPLC) method using 1 ml of serum has been developed for the determination of retinol (Vitamin A), alpha-tocopherol (Vitamin E), 25-hydroxyvitamin D(3) and 24 R,25-hydroxyvitamin D(3). The eluate was monitored with a photodiode-array detector at three wavelengths-namely: 265 nm for Vitamin D(3), 291 nm for Vitamin E and 325 nm for Vitamin A. The detection limits were equal to or lower than 1 ng ml(-1) for all vitamins. The linearity obtained with serum samples (standard addition method) gives correlation coefficients (r(2)) ranging between 0.999 and 0.996 in all cases, with standard deviation of the slope between 3.2 and 1.6%. The repeatability was between 4.0 and 6.0% and the within-laboratory reproducibility was lower than 10% in all cases. The most outstanding features of the present method are its ease of use, its rapidity and fully automation, which enables its use for routine analysis. The time required per sample was 30 min, because the overlapped development of the steps. This method was used for the determination of normality range of these vitamins in healthy people in the 18-80-year-old interval.     

Improved strategy for biosensor-based monitoring of water bodies with diverse organic carbon levels

To protect water resources and to control the water quality it is necessary to develop fast, sensitive, cost-effective, and easy-to-use analytical systems, which are able to measure a variety of contaminants in water. Monitoring water bodies with various matrices can be very difficult. The diverse organic carbon level in water samples (e.g. river water or seawater) causes problems at common analysis and in particular at immunological methods. Here, we demonstrate a new method to overcome the partly occurring matrix problems at quasi-continuous real-world biosensor monitoring. Therefore, we developed an easy matrix referencing method for our fully automated immunoassays that could be adapted to other applications depending on a similar test-format. The method was developed using a synthetic organic carbon standard, and validated using a diluted turf extract. Results for the ultra-sensitive immunoassay for estrone quantification are shown as example. The developed method was verified using immunoassays for testosterone, progesterone, ethinylestradiol, estradiol, and estriol.     

Determination of carbaryl in natural water samples by a surface plasmon resonance flow-through immunosensor

The analysis of carbaryl in natural water samples was accomplished using a portable immunosensor based on surface plasmon resonance (SPR) technology. The assay was based on a binding inhibition immunoassay format with the analyte derivative covalently immobilized on the sensor surface. An alkanethiol self-assembled monolayer (SAM) was formed onto the gold-coated sensor surface to allow the reusability of the same sensing surface during 220 regeneration cycles. Reproducibility was evaluated by performing three independent assays in triplicate on 3 different days. The batch-assay variability was also calculated using three different gold-coated sensor surfaces. The intra- and inter-day relative standard deviation were 8.6 and 15.3%, respectively, whilst a variation of 7.4% in assay sensitivity was obtained by employing different sensor chips. The lowest detection limit, calculated as the concentration providing a 10% decrease of the blank signal, was of 1.38 microg L(-1). Matrix effects were also evaluated in different water types, showing I50 values (carbaryl concentrations that produced a 50% decrease of the blank signal) within the range of carbaryl standard curves in distilled water (2.78-3.55 microg L(-1)). The carbaryl immunoassay performance was validated with respect to conventional high-performance liquid chromatography-mass spectrometry (HPLC-MS). The correlation between methods was in good agreement (r2 = 0.998, 0.999 and 0.999) for the three types of natural water samples tested. A complete assay cycle, including regeneration, is accomplished in 20 min. All measurements were carried out with the SPR sensor system (beta-SPR) commercialised by the company SENSIA, SL (Spain). The small size and low-time of response of the beta-SPR platform would allow its utilization in real contaminated locations.     

血清与血浆中HBsAg 在全自动酶免分析检测结果比较

目的:探讨血浆取代血清检测乙肝标志物的检测结果以及临床意义。方法:选取我科检测乙肝标志物的血样30份,分别放在抗凝剂管和普通干燥试管,采用全自动酶免疫分析仪检测HBsAg,将检测结果进行OD及S/CO值统计处理,并进行比较分析。结果:血清与二种血浆检测结果相关系数均0.99,二种血浆与血清结果相关关系良好(P>0.0 5),无显著性差异。结论:血浆代替血清完全可以用于全自动酶标分析仪进行检测,既可以节省预处理时间,又可以减少标本在分离吸移血清过程中出现差错,值得临床推广和应用。     

Evaluating the Acute Toxicity of Estrogen Hormones and Wastewater Effluents Using Vibrio fischeri

Toxicity evaluation of environmental substances such as those in wastewater and contaminated water bodies has become an important part of environmental monitoring of pollution. The study evaluated the toxicity of estrogen hormones and the removal of toxicity in full-scale wastewater treatment plants (WWTPs) using the marine bacterium, Vibrio fischeri, and to determine if there is a correlation between the hormones and the toxicity in the effluents. Three different types of full-scale WWTPs were investigated and presence of estrogens in the treated wastewater was evaluated by enzyme linked immunoassay (ELISA). The toxicity of individual estrogens (E2, EE2, and a mixture of E1, E2, and E3) was investigated as well as influents and treated wastewater. The results revealed that all estrogen hormones had less than 50% inhibitions and fell in the Class II group that exhibits slight acute toxicity. The toxicity of the individual E2 hormone had higher inhibitions when compared to the individual synthetic EE2 and the mixture of the hormones. The toxicity results of the WWTP revealed that biological treatment can reduce the toxicity of the influent to an extent. The findings suggest that the residual estrogen contents as well as toxicity can be reduced in certain WWTPs.     

Prevalence of Escherichia coli O157:H7 in industrial minced beef

AIMS: The lack of baseline data on the prevalence of Escherichia coli O157:H7 in retail minced beef in France prompted this survey of industrial minced beef production. METHODS AND RESULTS: An automated enzyme-linked fluorescence immunoassay (ELFA), the VIDAS E. coli O157 method, was used to detect E. coli O157 in industrial minced beef samples. Confirmation of samples positive according to the ELFA was performed using an automated immunoconcentration (ICE) system, VIDAS ICE, which allows the selective capture and release of target organisms. The ICE was followed by culture on cefixime tellurite sorbitol MacConkey agar and a chromogenic medium, O157:H7 ID. Of the 3450 minced beef samples tested, 175 samples were positive with the ELFA method and, of these, four were confirmed by the ICE method. They were identified as sorbitol-negative, O157-positive, H7-positive, mobile, verotoxin-producing E. coli. CONCLUSIONS: The prevalence of E. coli O157:H7 in industrial French minced beef was 0.12%, consistent with many other reports. SIGNIFICANCE AND IMPACT OF THE STUDY: The low infective dose of E. coli O157:H7 presents a major threat. The main means of combating this organism are thermal destruction and good food hygiene covering activities on-farm, in the abattoir and in minced beef industries.     

Serum insulin-like growth factor I reference values for an automated chemiluminescence immunoassay system: results from a multicenter study

BACKGROUND: Analysis of insulin-like growth factor I in serum (S-IGF-I) is an integral component in the diagnosis of GH-related disorders and is going to be of interest in the diagnosis and follow-up of many disorders. The objective of the present study was to develop cross-sectional reference values for S-IGF-I measured by an automated chemiluminescence immunoassay (Nichols Advantage). METHODS: The study included samples from 3,961 healthy subjects (2,201 males, 1,760 females) aged 1 month to 88 years. Six laboratories were involved in this study and the samples were analyzed by one of seven automated immunoassay systems run in these laboratories. For data analysis, polynomial age and sex-specific models were fitted after transformation of S-IGF-I values. RESULTS: The results show the well-known age dependency of S-IGF-I levels. At ages <20, higher S-IGF-I levels were seen in girls with an estimated mean peak of 410 microg/l at age 14 and an estimated mean peak of 382 microg/l at age 16 in boys. Thereafter, a rapid decrease was seen to approximately 25 years of age, followed by a slow age-dependent decrease. In adulthood, S-IGF-I in males were slightly, but significantly higher than in females. It could be shown that the mean values of some reference sample subgroups differed significantly from the total mean. However, the multicenter approach used in this study reduces the impact of systematic population, sample handling and laboratory differences on the calculated reference mean. CONCLUSION: The present study establishes age- and sex-specific reference values for a fully automated immunoassay system based on a large population of healthy subjects. The established reference values may be used for this immunoassay system in different laboratories provided that the systematic difference between systems is low.     

Colorimetric enumeration of Escherichia coli based on beta-glucuronidase activity

A medium containing a chromogenic substrate was developed for the enumeration of Escherichia coli on the basis of beta-glucuronidase activity. In this medium there was an inverse linear relationship between the log initial E. coli concentration and the time taken for the color to reach a threshold optical density of 0.05. This relationship applied even when the E. coli population contained 5% beta-glucuronidase-negative cells. Incubation at 44 degrees C reduced the time taken for color development and allowed the procedure to be used in the presence of a competitive microflora that outnumbered the E. coli population by a factor of 10(4). Sodium lauryl sulfate as an additional selective agent gave no significant improvement. In the analysis of environmental water samples, the technique gave a good correlation with a standard cultural method. The procedure shows promise as a simple method for testing the compliance of environmental samples with microbiological criteria for E. coli.     

Flow Immunoassay for Detection of Wheat Allergen Using an Anion Exchange Column and Alkaline Phosphatase Conjugated IgE

A simple and rapid detection system for wheat allergen was developed based on a luminescence immunoassay with a continuous flow system. Wheat allergen and alkaline phosphatase conjugated IgE (ALP-IgE) was separated from free ALP-IgE on the basis of a difference in isoelectric point, using with an anion exchange resin. Luminescence output of the assay correlated linearly with the concentration of allergen in the range of 1-100 µg/ml. Reuse of free ALP-IgE was possible.     

Colorimetric enumeration of Escherichia coli based on beta-glucuronidase activity.

A medium containing a chromogenic substrate was developed for the enumeration of Escherichia coli on the basis of beta-glucuronidase activity. In this medium there was an inverse linear relationship between the log initial E. coli concentration and the time taken for the color to reach a threshold optical density of 0.05. This relationship applied even when the E. coli population contained 5% beta-glucuronidase-negative cells. Incubation at 44 degrees C reduced the time taken for color development and allowed the procedure to be used in the presence of a competitive microflora that outnumbered the E. coli population by a factor of 10(4). Sodium lauryl sulfate as an additional selective agent gave no significant improvement. In the analysis of environmental water samples, the technique gave a good correlation with a standard cultural method. The procedure shows promise as a simple method for testing the compliance of environmental samples with microbiological criteria for E. coli.     

Factors influencing expression of luxCDABE and nah genes in Pseudomonas putida RB1353(NAH7, pUTK9) in dynamic systems.

Bioluminescent reporter organisms have been successfully exploited as analytical tools for in situ determination of bioavailable levels of contaminants in static environmental samples. Continued characterization and development of such reporter systems is needed to extend the application of these bioreporters to in situ monitoring of degradation in dynamic environmental systems. In this study, the naphthalene-degrading, lux bioreporter bacterium Pseudomonas putida RB1353 was used to evaluate the relative influences of cell growth stage, cell density, substrate concentration, oxygen tension, and background carbon substrates on both the magnitude of the light response and the rate of salicylate disappearance. The effect of these variables on the lag time required to obtain maximum luminescence and degradation was also monitored. Strong correlations were observed between the first three factors and both the magnitude and induction time of luminescence and degradation rate. The maximum luminescence response to nonspecific background carbon substrates (soil extract broth or Luria broth) was 50% lower than that generated in response to 1 mg of sodium salicylate liter(-1). Oxygen tension was evaluated over the range of 0.5 to 40 mg liter(-1), with parallel inhibition to luminescence and degradation rate (20 mg of sodium salicylate liter(-1)) observed at 1.5 mg liter(-1) and below and no effect observed above 5 mg liter(-1). Oxygen tensions from 2 to 4 mg liter(-1) influenced the magnitude of luminescence but not the salicylate degradation rate. The results suggest that factors causing parallel shifts in the magnitude of both luminescence and degradation rate were influencing regulation of the nah operon promoters. For factors that cause nonparallel shifts, other regulatory mechanisms are explored. This study demonstrates that lux reporter bacteria can be used to monitor both substrate concentration and metabolic response in dynamic systems. However, each lux reporter system and application will require characterization and calibration.     

Automated methods for multiplexed pathogen detection

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.