Sample records for solid-phase enzyme immunoassay

  1. The use of ferromagnetic dacron as solid-phase in enzyme immunoassays

    Ana Maria dos A. Carneiro Leão


    Full Text Available Ferromagnetic dacron is proposed as an alternative solid-phase for magnetic enzyme immunoassays. Human serum albumin (HSA was covalentlyimmobilized onto ferromagnetic dacron and as enzyme immunoassay was developed using anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD and hydrogen peroxide were used anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD and hydrogen peroxide were used as the enzymatic label and substrates, respectively. Best results were observed when particles of 63-100 µm (diameter and 10 µg of immobilized antigen were used. Positive reactions were detected until dilutions of1:51200 of immune sera. Its reproducibility was similar to standard ELISA. Disruption of the immunocomplexes formed and recuperation of the immobilized antigen in other immunoassays also proved to be reliable.

  2. Performance of a solid phase enzyme immunoassay for detection of group A streptococci in a pediatric office laboratory as refereed by a hospital laboratory.

    Yuckienuz, S A; Thorne, G M; Macone, A B; Goldmann, D A; St Pierre, J; Marcus, E P


    We evaluated the performance of a new rapid solid phase enzyme immunoassay, SUDS Group A Strep (MUREX Corp., Norcross, GA) for the detection of Group A beta-hemolytic streptococci in a pediatric office practice. Duplicate throat swabs were obtained from 341 children with pharyngitis. One swab was used in the SUDS test and the other was cultured in the office laboratory. Office SUDS and culture (sheep blood agar plate, aerobic 24-hour incubation) were compared with culture using reference techniques (sheep blood agar plate, anaerobic 48-hour incubation) in a hospital laboratory. Compared with hospital laboratory culture, the sensitivity of office SUDS (73.8%) was superior to that of office culture (66.6%) at P = 0.05. Specificities were 93.1 and 98.6%, respectively; positive predictive values were 86.1 and 96.6%; and negative predictive values were 85.9 and 83.5%. The sensitivity and specificity of SUDS compared with office culture were 88.5 and 87.8%, respectively, but would have been 93 and 94% had hemolyzed media not been used on several occasions in the office culture procedure. We conclude that SUDS Group A Strep was significantly more sensitive than throat cultures as performed in a typical pediatric practice although the performance of office cultures could have been improved by standard quality control techniques.

  3. Enzyme immunoassay

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M


    An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from...

  4. Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays.

    IJsselmuiden, O E; Herbrink, P; Meddens, M J; Tank, B; Stolz, E; Van Eijk, R V


    The technical variables of the solid-phase immunofiltration assay (SPIA) for the detection of antibodies bound to antigens on a solid-phase filter have been investigated. The binding to solid-phase filters of 125I-labelled axial filament proteins derived from Treponema phagedenis and the optimal conditions for blocking non-specific protein binding were analysed. Axial filament was applied to nitrocellulose, Hybond Nylon and Zeta Probe. After extensive rinsing, the highest amount (68%) of axial filament was observed bound to Zeta Probe. However, blocking non-specific protein binding by pre-wetting the filter with rinsing buffer containing 0.5% Tween 20, prevented the binding of protein to the filter only when nitrocellulose was used as solid phase. Tween 20 (0.5%) in the rinsing and incubation solutions was found to be necessary for the reduction of non-specific binding of contaminants in turbid sera. However, the use of such solutions resulted in a substantial leakage of antigen (47%) during rinsing procedures. Binding of antigen-specific antibody was analysed using 125I-labelled protein A. The maximal possible binding of the antibody occurred within 5 min when the antibody solution was filtered. For optimal binding of 125I-labelled protein A an incubation time of 1 h was needed. It is suggested that solid-phase immunofiltration may provide a rapid alternative for radioimmunoassays or enzyme immunoassays for the detection of specific antibodies.

  5. Solid-Phase Immunoassay of Polystyrene-Encapsulated Semiconductor Coreshells for Cardiac Marker Detection

    Sanghee Kim


    Full Text Available A solid-phase immunoassay of polystyrene-encapsulated semiconductor nanoparticles was demonstrated for cardiac troponin I (cTnI detection. CdSe/ZnS coreshells were encapsulated with a carboxyl-functionalized polystyrene nanoparticle to capture the target antibody through a covalent bonding and to eliminate the photoblinking and toxicity of semiconductor luminescent immunosensor. The polystyrene-encapsulated CdSe/ZnS fluorophores on surface-modified glass chip identified cTnI antigens at the level of ~ng/mL. It was an initial demonstration of diagnostic chip for monitoring a cardiovascular disease.

  6. Rapid solid-phase immunoassay for 6-keto prostaglandin F1 alpha on microplates

    Schramm, W.; Smith, R.H.; Jackson, T.M.; Craig, P.A.; Grates, H.E.; Minton, L.L. (BioQuant of Ann Arbor, Inc., MI (USA))


    We describe, for the measurement of 6-keto prostaglandin F1 alpha in biological media, a solid-phase immunoassay with immobilized antibodies that requires a total processing time of less than 2 h with hands-on time less than 30 min for 40 samples. The method combines the convenience of the microplate format with the sensitivity of radiolabeled prostaglandin derivatives as tracers in a competitive immunoassay. The intra- and interassay variations at 50% displacement of the radiolabeled prostaglandin derivative as tracer were 9.0% and 11.8%, respectively. At 50% displacement of the radiolabeled tracer, the sensitivity is about 20 pg per well. Optimal incubation time is between 60 and 90 min. Nonspecific binding was less than 1% if about 8 pg of tracer (approximately 25,000 counts/min per well) was used. Inhibition curves of samples in different dilutions were parallel to standard curves. The variation of bound radiolabeled prostaglandin derivative within the wells of one microplate (n = 96) was less than 3%. Human plasma samples and medium from tissue culture assayed for 6-keto prostaglandin F1 alpha correlated well with results obtained with a solid-phase assay based on use of magnetic particles (r = 0.99, n = 24) for culture-medium samples; r = 0.99; n = 26 for plasma samples.

  7. Non-specific binding in solid phase immunoassays for autoantibodies correlates with inflammation markers.

    Güven, Esin; Duus, Karen; Lydolph, Magnus Christian; Jørgensen, Charlotte Sværke; Laursen, Inga; Houen, Gunnar


    Enzyme-linked immunosorbent assay (ELISA) is a validated and sensitive method for detection of human autoantibodies, but may have problems with specificity. Non-specific binding is a well-known problem often observed in tests for autoantibodies, when sera are incubated on plastic surfaces, e.g. an ELISA plate. To understand the mechanisms underlying non-specific immunoglobulin deposition, we here analyse the phenomenon in detail and we propose means of reducing false positive test results caused by non-specific binding. The level of non-specific binding, in sera with suspected autoreactivity, was analysed in non-coated and autoantigen-coated ELISA wells and 4-32% of sera showed a high level of non-specific binding depending on the assay conditions and serum properties. Non-specifically binding sera were found to contain increased concentrations of IgG and other inflammatory mediators. Moreover, non-specific binding could be induced in serum by increasing the concentration of IgG and incubating the serum at 40 °C. This suggests that non-specific binding immunoglobulins can be formed during inflammation with high immunoglobulin levels and elevated temperature. We show that the level of non-specific binding correlates with the IgG concentration and therefore propose that non-specific binding may be interpreted as an informative finding indicative of elevated IgG and inflammation.

  8. [Enzyme immunoassay of usnic acid in lichens].

    Burkin, A A; Kononenko, G P; Tolpysheva, T Iu


    An enzyme immunoassay for usnic acid in lichens was developed, the sensitivity of which was 0.1 microg/g of air-dried material (0.00001%). Polyclonal rabbit antibodies against bovine serum albumin conjugated to (+)-usnic acid under the conditions of formaldehyde condensation made it possible to determine the analyzed substance in solutions at concentrations from 1 ng/mL when it interacts with an immobilized gelatin conjugate homologous in the binding mode. Usnic acid in 2-26600 microg/g (0.0002-2.6%) amounts was found in all 236 studied samples of lichens belonging to 53 species and 8 families.

  9. Fast Diagnosis of Gonorrhea Witth Enhanced Luminescence Enzyme Immunoassay

    ZHENG Heyi(郑和义); CAO Jingjiang(曹经江); SHAO Yanglin(邵燕玲)


    Objective:To evaluate the value of enhanced luminescence enzyme immunoassay in the diagnosis of Neisseria gonorrhea(NG) infection.Methods: Anti-catalase antibody for Neisseria gonorrheae combined with enhanced luminescence enzyme immunoassay were used to test for N. Gonorrhea.Results: A minimum of 1x104/CFU of GC in genital tract secretions or urine could be detected with the technique of luminescence enzyme immunoassay.Conclusion : The enhanced luninescence enzyme immunoassay has the advantage of high sensitivity and specificity for diagnosing NG from genitourinary tract secretion and urine.

  10. Solid-Phase Synthesis of Modified Peptides as Putative Inhibitors of Histone Modifying Enzymes

    Cohrt, Anders Emil O'Hanlon

    and purities. Libraries of histone H2B tail pieces were synthesised using both parallel and split-pool synthesis protocols. Changes in the acetylation pattern of the individual library members upon treatment with HDAC3 enzyme were measured using LCMS-MS techniques. An MSMS deconvolution strategy was employed......,2,3-triazoles were cleanly deprotected by treatment with TFA (CH2Cl2). Four different libraries of histone demethylase inhibitor candidates have been synthesised based on metal chelation, cofactor mimicking and radial stabilising inhibition strategies. The libraries have all been synthesised on solid......-phase using various handle strategies for the clean release of products. Two cofactor mimicking inhibitor candidates, which were synthesised using a safety-catch benzyl hydrazide handle, were found to inhibit the histone demethylase JMJD2C with IC50-values of 23.5µM and 24µM. Two mild and selective methods...

  11. Competitive enzyme immunoassay for urinary vanillylmandelic acid.

    Taran, F; Bernard, H; Valleix, A; Créminon, C; Grassi, J; Olichon, D; Deverre, J R; Pradelles, P


    An enzyme immunoassay for urinary vanillylmandelic acid (VMA) using polyclonal antiserum and VMA-acetylcholinesterase conjugate as enzymatic tracer is described. Two different strategies for immunogen preparation were developed and enantioselectivity was demonstrated. Selected EIA allowed direct measurement of urinary VMA using D(-)-VMA as standard with good sensitivity (MDC = 0.1 mumol/l) and precision (CV less than 7% in 0.2-2.25 mumol/l range). Cross-reactivity with homovanillic acid (HVA) was 0.8% and less than 0.4% with other structurally related catecholamine metabolites. Intra- and inter-assay repeatability were less than 10% and recovery was 97.3% +/- 3%. Good correlation was obtained for EIA and HPLC analysis with normal and pathologic human urine samples (EIA = 0.895 HPLC-7.085, r2 = 0.98, n = 47).

  12. An enzyme immunoassay for rat growth hormone - Applications to the study of growth hormone variants

    Farrington, Marianne A.; Hymer, W. C.


    A sensitive and specific competitive enzyme immunoassay for rat growth hormone (GH) is described and its use in the detection of GH variants is demonstrated. In the present assay, soluble GH and GH adsorbed to a solid-phase support compete for monkey anti-GH antibody binding sites. The immobilized antibody-GH complex is detected and quantified using goat antimonkey immunoglobin G covalently conjugated to horseradish peroxidase. It is noted that the assay can be performed in 27 hours and that sensitivities in the range of 0.19 to 25 ng can be obtained in the region of 10 to 90 percent binding.


    The occurrence of triclosan in the water environment around a Mediterranean region was investigated. Triclosan and methyl-triclosan content of ninety five environmental samples were screened using a magnetic particle enzyme immunoassay. Positive samples were confirmed by solid phase extraction (SPE...

  14. Confirmation of amphetamine, methamphetamine, MDA and MDMA in urine samples using disk solid-phase extraction and gas chromatography-mass spectrometry after immunoassay screening.

    Huang, Zengping; Zhang, Shaoyu


    A method using mixed phase disk solid-phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) was developed for confirmation of amphetamine (AMP), methamphetamine (MET), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples after immunoassay screening. Disk SPE provided hydrophobic (C(18)) and strong cation-exchange (SCX) interactions. The analytes were retained on SCX functional groups in the disk and eluted with ammoniated ethyl acetate after washed with methanol. Confirmation and quantitation was exercised by selected ion monitoring using nikethamide as chromatographic standard. Recoveries of the amphetamines were between 73.0 and 104.6% with RSDs in range of 2.1-6.4% (n=3). The limits of detection were 2 ng/ml for AMP, MET and MDMA, and 4 ng/ml for MDA. Five real urine samples were tested with the method after immunoassay screening, and the results were comparable to those of traditional liquid-liquid extraction (LLE). The method was solvent-saved, simple, rapid and reliable, and the extract was cleaner than that of LLE.

  15. Development of an enzyme immunoassay for poliovirus antigens

    Newton Hashimoto


    Full Text Available An indirect solid-phase enzyme immunoassay (EIA was developed for the detection of poliovirus antigen. Virus antigen was obtained in LLC-MK2 cell cultures and used to prepare antibodies in rabbit and guinea pig. Antibodies were evaluated by double immunodiffusion and neutralization test. Optimal concentrations of guinea pig and rabbit immunoglobulins were determined by checkerboard titration. Microtitre plates were coated with 15.0 µg/ml guinea pig anti-polio immunoglobulin and rabbit anti-polio immunoglobulin at the concentration of 7.94 µg/ml was used as detecting antibody. The standard curve with eight different antigen concentrations in eight replicates resulted in a coefficient of variation (CV between 2.1% to 7.8%. The dose-response relationship was determined by simple linear regression with a coefficient of correlation (R² equal to 96.4%. The assay detected a minimum of 2.3 µg/ml poliovirus antigen.O trabalho apresenta o desenvolvimento de um ensaio imunoenzimático indireto para a detecção de antígeno de poliovírus. O antígeno viral foi obtido em cultura de células LLC-MK2 e usado para imunização de coelho e cobaia. Os soros hiperimunes foram avaliados por imunodifusão dupla e teste de neutralização. Após padronização, o soro de captura, produzido em cobaia, foi usado na concentração protéica de 15.0 µg/ml para sensibilizar microplacas de poliestireno e o soro de coelho (detector foi usado na concentração de 7.94 µg/ml. A curva padrão resultante da utilização de oito diferentes concentrações do antígeno padrão definiu um coeficiente de variação de 2.1% a 7.8%. A relação dose-resposta foi determinada por regressão linear simples com o estabelecimento do coeficiente de correlação (R² igual a 96.4%. O ensaio possibilitou a detecção mínima de 2.3 µg/ml de antígeno de poliovírus.

  16. Determination of alachlor and its sulfonic acid metabolite in water by solid-phase extraction and enzyme-linked immunosorbent assay

    Aga, D.S.; Thurman, E.M.; Pomes, M.L.


    Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were combined for the trace analysis of the herbicide alachlor and its major soil metabolite, ethanesulfonic acid (ESA). The anti-alachlor antibody cross-reacted with ESA, which produced false-positive detections of alachlor in water samples by immunoassay screens. Alachlor and ESA were isolated from water by SPE on a C18 resin and eluted sequentially with ethyl acetate and methanol. Alachlor is soluble in ethyl acetate while the anionic ESA is not. Thus ESA remained adsorbed on the C18 resin and was eluted later with methanol. The combination of SPE with ELISA effectivety separated and quantified both alachlor and ESA using the same antibody for two ELISA methods. The general method may have applicability for the separation of other herbicides and their ionic metabolites. The SPE-ELISA method has a, detection limit of 0.01 ??g/L for alachlor and 0.05 ??g/L for ESA, with a precision of ?? 10%. Analyses of surface and ground water samples were confirmed by gas chromatography/mass spectrometry and high-performance liquid chromatography with photodiode-array detection. Results showed widespread occurrence of ESA in surface and ground water of the midwestern United States, with concentrations ranging from 10 ??g/L.

  17. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko


    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  18. Coupling solid-phase extraction and enzyme-linked immunosorbent assay for ultratrace determination of herbicides in pristine water

    Aga, D.S.; Thurman, E.M.


    Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were coupled for automated trace analysis of pristine water samples containing 2-chloro-4-ethylamino-6-isopropylamine-s-triazine (atrazine) and 2-chloro-2???,6???-diethyl-N-(methoxymethyl)acetanilide (alachlor). The isolation of the two herbicides on a C18-resin involved the selection of an elution solvent that both removes interfering substances and is compatible with ELISA. Ethyl acetate was selected as the elution solvent followed by a solvent exchange with methanol/water (20/80, % v/v). The SPE-ELISA method has a detection limit of 5.0 ng/L (5 ppt), >90% recovery, and a relative standard deviation of ??10%. The performance of a microtiter plate-based ELISA and a magnetic particle-based ELISA coupled to SPE was also evaluated. Although the sensitivity of the two ELISA methods was comparable, the precision using magnetic particles was improved considerably (??10% versus ??20%) because of the faster reaction kinetics provided by the magnetic particles. Finally, SPE-ELISA and isotope dilution gas chromatography/ mass spectrometry correlated well (correlation coefficient of 0.96) for lake-water samples. The SPE-ELISA method is simple and may have broader applications for the inexpensive automated analysis of other contaminants in water at trace levels.

  19. Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

    Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi


    We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

  20. Immunoassays

    Hsieh, Y.-H. Peggy

    Immunochemistry is a relatively new science that has developed rapidly in the last few decades. One of the most useful analytical developments associated with this new science is immunoassay. Originally immunoassays were developed in medical settings to facilitate the study of immunology, particularly the antibody-antigen interaction. Immunoassays now are finding widespread applications outside the clinical field because they are appropriate for a wide range of analytes ranging from proteins to small organic molecules. In the food analysis area, immunoassays are widely used for chemical residue analysis, identification of bacteria and viruses, and detection of proteins in food and agricultural products. Protein detection is important for determination of allergens and meat species content, seafood species identification, and detection of genetically modified plant tissues. While immunoassays of all formats are too numerous to cover completely in this chapter, there are several procedures that have become standard for food analysis because of their specificity, sensitivity, and simplicity.

  1. Comparison of an enzyme immunoassay to an indirect fluorescent immunoassay for the detection of antinuclear antibodies.

    Reisner, B S; DiBlasi, J; Goel, N


    The standard method for detecting antinuclear antibodies (ANAs) is by immunofluorescence assay (IFA), a method that is labor intensive and subjective. In an attempt to overcome these limitations, several commercial enzyme immunoassays (EIAs) have been developed. We report the results of our evaluation of the ANA Microplate EIA (Sanofi Diagnostics Pasteur, Chaska, MN). For the evaluation, 808 serum samples were tested by EIA and IFA; 52 specimens were positive by both assays, 561 were negative by both assays, 91 were positive by EIA only, and 3 were positive by IFA only. Borderline results (not positive or negative) were obtained for 101 specimens, which were excluded when calculating the sensitivity, specificity, and positive and negative predictive values of this assay, which were 94.6%, 86.0%, 36.4%, and 99.5%, respectively. Because of its high negative predictive value, this assay can be used reliably to detect ANA-negative samples; however, the low positive predictive value indicates that EIA-positive specimens should be retested by an IFA to determine the final result.

  2. Immunoassays

    Pas, Hendrikus; Jonkman, Marcel


    Immunoassays are helpful serological techniques for the laboratory diagnosis of autoimmune blistering diseases (AIBD) and for monitoring disease activity of individual patients. The three main immunoassaysare immunoblotting, immunoprecipitation, and ELISA. All three make use of the ability of the au

  3. [State of Fungal Lipases of Rhizopus microsporus, Penicillium sp. and Oospora lactis in Border Layers Water-Solid Phase and Factors Affecting Catalytic Properties of Enzymes].

    Khasanov, Kh T; Davranov, K; Rakhimov, M M


    We demonstrated that a change in the catalytic activity of fungal lipases synthesized by Rhizopus microsporus, Penicillium sp. and Oospora lactis and their ability to absorb on different sorbents depended on the nature of groups on the solid phase surface in the model systems water: lipid and water: solid phase. Thus, the stability of Penicillium sp. lipases increased 85% in the presence ofsorsilen or DEAE-cellulose, and 55% of their initial activity respectively was preserved. In the presence of silica gel and CM-cellulose, a decreased rate of lipid hydrolysis by Pseudomonas sp. enzymes was observed in water medium, and the hydrolysis rate increased by 2.4 and 1.5 times respectively in the presence of aminoaerosil and polykefamid. In an aqueous-alcohol medium, aminoaerosil and polykefamid decreased the rate of substrate hydrolysis by more than 30 times. The addition of aerosil to aqueous and aqueous-alcohol media resulted in an increase in the hydrolysis rate by 1.2-1.3 times. Sorsilen stabilized Penicillium sp. lipase activity at 40, 45, 50 and 55 degrees C. Either stabilization or inactivation of lipases was observed depending on the pH of the medium and the nature of chemical groups localized on the surface of solid phase. The synthetizing activity of lipases also changed depending on the conditions.

  4. Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

    Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J;


    We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA...

  5. Development of a synchronous enzyme-reaction system for a highly sensitive enzyme immunoassay.

    Inouye, Kuniyo; Ueno, Iori; Yokoyama, Shin-ichi; Sakaki, Toshiyuki


    A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.

  6. Competitive Enzyme Immunoassay for Diagnosis of Human Brucellosis

    Lucero, Nidia E.; Foglia, Luis; Ayala, Sandra M.; Gall, David; Nielsen, Klaus


    The methods commonly used for human brucellosis serological testing are agglutination tests and the complement fixation test (CFT). Among the newer serological tests, primary binding assays were developed to improve sensitivity and specificity. The competitive enzyme immunoassay (CELISA) for the detection of serum antibody to Brucella is a multispecies assay which appears to be capable of differentiating vaccinal and cross-reacting antibodies from antibodies elicited by field infection in cattle. The competing monoclonal antibody used in this assay is specific for a common epitope of smooth lipopolysaccharide (S-LPS). In this study, we compared the CELISA to the classical tests for the diagnosis of human brucellosis. The CELISA cutoff value was determined to calculate its diagnostic specificity and sensitivity. A survey was performed with 911 sera. Of the sera, 341 were from an asymptomatic population that tested negative with conventional serological tests (screening and confirmatory). Based on these samples, the CELISA specificities were determined to be 99.7 and 100% with cutoff values of 28 and 30% inhibition (%I), respectively. In a further study with 393 additional sera from an asymptomatic population found negative by the conventional screening tests, the CELISA specificities were calculated to be 96.5 and 98.8% with cutoff values of 28 and 30%I. The CELISA sensitivities were determined to be 98.3 and 94.8% with cutoff values of 28 and 30%I, respectively, for sera from 116 individuals found positive by the classical tests. For the 51 culture-positive patients, CELISA was positive for 100%, the CFT was positive for 92%, and the standard tube agglutination test (TAT) was positive for 100%. The CELISA specificity was 100% for 31 sera from patients found negative by conventional serological tests but with brucellosis-like symptoms. The CELISA is fairly rapid to perform, somewhat faster than TAT, and cross-reacts less with other antigens (or antibodies) than the

  7. Enzyme-catalyzed reaction of voltammetric enzyme-linked immunoassay system based on OAP as substrate

    张书圣; 陈洪渊; 焦奎


    The o-aminophenol (OAP)-H2O2-horseradish peroxidase (HRP) voltammetric enzyme-linked immunoassay new system has extremely high sensitivity. HRP can be measured with a detection limit of 6.0×10-(10) g/L and a linear range of 1.0×10-9—4.0×10-6 g/L. The pure product of H2O2 oxidizing OAP catalyzed by HRP was prepared with chemical method. The enzyme-catalyzed reaction has been investigated with electroanalytical chemistry, UV/Vis spectrum, IR spectrum, 13C NMR, 1H NMR, mass spectrum, elemental analysis, etc. Under the selected enzyme-catalyzed reaction conditions, the oxidation product of OAP with H2O2 catalyzed by HRP is 2-aminophe-noxazine-3-one. The processes of the enzyme-catalyzed reaction and the electroreduction of the product of the enzymecatalyzed reaction have been described.

  8. A solid phase enzyme-linked immunosorbent assay for the antigenic detection of Legionella pneumophila (serogroup 1): A compliment for the space station diagnostic capability

    Hejtmancik, Kelly E.


    It is necessary that an adequate microbiology capability be provided as part of the Health Maintenance Facility (HMF) to support expected microbial disease events and environmental monitoring during long periods of space flight. The application of morphological and biochemical studies to confirm the presence of certain bacterial and fungal disease agents are currently available and under consideration. This confirmation would be facilitated through employment of serological methods to aid in the identification of bacterial, fungal, and viral agents. A number of serological approaches are currently being considered, including the use of Enzyme Linked Immunosorbent Assay (ELISA) technology, which could be utilized during microgravity conditions. A solid phase, membrane supported ELISA for the detection of Legionella pneumophila, an expected disease agent, was developed to show a potential model system that would meet the HMF requirements and specifications for the future space station. These studies demonstrate the capability of membrane supported ELISA systems for identification of expected microbial disease agents as part of the HMF.

  9. 多功能灵敏固相时间分辨荧光免疫分析仪设计%Design of a new solid-phase time-resolved fluorescence immunoassay analyzer

    宋克非; 张佩杰


    A new solid-phase time-resolved fluorescence immunoassay analysis system has been designed for solid-phase time-resolved fluorescence spectroscopy measurements. The device uses nitrogen molecular laser as the excitation light source and employs integrating sphere combined with grating monochromator as the fluorescence collecting subsystem to minimize the influences of stray light on the sample fluorescence. Photomultiplier tube is used to convert the sampled optic signal to electronic signal and high resolution measurements of fluorescence spectrum in the range of 500 ~ 700 nm are implemented. The converted electrical signals are sampled and integrated digitally with a single chip micro-computer, which improves the signal to noise ratio of the received signal. The instrument can complete fluorescence lifetime measurement, time-resolved fluorescence spectroscopy measurement and substance concentration measurement. Experimental results show that the measurement sensitivity is up to 10 ~12 mol/L, linearity range is 10 ~'2 ~ 10 "9 mol/L, relative stability error is lass than 3% and the measurement resolution is 0. 5 nm.%针对固相时间分辨荧光光谱的测量,设计出一种全新的固相时间分辨荧光免疫分析系统.使用氮分子激光器作为激发光源,采用积分球和单色仪相结合的荧光收集结构,使杂散光对样品荧光的影响降到最低;用光电倍增管进行光电转换,在500~700 nm范围实现了高分辨荧光光谱测量;利用数字方式实现取样积分功能,提高了系统的信噪比.系统可实现荧光寿命、时间分辨荧光光谱、物质浓度的自动测量,仪器的检测灵敏度可达10 - 12 moL/L,线性范围为10-12~10-9 mol/L,稳定性相对误差小于3%,荧光光谱分辨为0.5nm.

  10. Detection of antibodies against Actinobacillus pleuropneumoniae serotype 5 using an inhibition enzyme immunoassay

    Stenbæk, Eva I.; DeLaSalle, F.; Gottschalk, M.


    An inhibition enzyme immunoassay (EIA) for detection of antibodies against A. pleuropneumoniae serotype 5 (App-5) in pig sera, based on the inhibition of the binding of an App-5 specific monoclonal antibody was established. The monoclonal antibody (MAb 210-F11) was found to be directed against...

  11. Electrochemical Enzyme Immunoassay of Tumor Marker CA15-3 with Capillary Electrophoresis


    Tumor marker CA15-3 was determined by using capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED). The method can be used to detect CA15-3 with a limit of 0.024 U/mL.

  12. Thermophilic Campylobacter spp. in turkey samples: evaluation of two automated enzyme immunoassays and conventional microbiological techniques

    Borck, Birgitte; Stryhn, H.; Ersboll, A.K.


    Aims: To determine the sensitivity and specificity of two automated enzyme immunoassays (EIA), EiaFoss and Minividas, and a conventional microbiological culture technique for detecting thermophilic Campylobacter spp. in turkey samples. Methods and Results: A total of 286 samples (faecal, meat...

  13. Analytical accuracy of determinations of aminoglycoside concentrations by enzyme multiplied immunoassay, fluorescence polarization immunoassay, and radioimmunoassay in the presence of heparin.

    O?Connell, M. E.; Heim, K L; Halstenson, C E; Matzke, G R


    The accuracy of gentamicin, netilmicin, and tobramycin concentration determinations by enzyme multiplied immunoassay technique (EMIT; Syva Corp., Palo Alto, Calif.), fluorescence polarization immunoassay (TDx; Abbott Diagnostics, Irving, Tex.), and radioimmunoassay were compared in the presence of 0 to 3,000 USP units of porcine heparin per ml. Gentamicin, netilmicin, and tobramycin concentrations determined by EMIT decreased by 10 and 50% in the presence of 75 and 1,000 USP units/ml, 2 and 5...

  14. Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

    Haraya, Shiomi; Karasawa, Koji; Sano, Yoshihiro; Ozawa, Kimiko; Kato, Nobumasa; Arakawa, Hidetoshi


    Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

  15. Analysis of Benzo[a]pyrene in Vegetable Oils Using Molecularly Imprinted Solid Phase Extraction (MISPE Coupled with Enzyme-Linked Immunosorbent Assay (ELISA

    Michael Pschenitza


    Full Text Available This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE method coupled with enzyme-linked immunosorbent assay (ELISA for determination of the PAH benzo[a]pyrene (B[a]P in vegetable oils. Different molecularly imprinted polymers (MIPs were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene yielding a broad-specific polymer for PAHs with a maximum binding capacity (Q of ~32 μg B[a]P per 50 mg of polymer. The vegetable oil/n-hexane mixture (1:1, (v/v was pre-extracted with acetonitrile, the solvent evaporated, the residue reconstituted in n-hexane and subjected to MISPE. The successive washing with n-hexane and isopropanol revealed most suitable to remove lipid matrix constituents. After elution of bound PAHs from MISPE column with dichloromethane, the solvent was evaporated, the residue reconstituted with dimethyl sulfoxide and diluted 100-fold with methanol/water (10:90, (v/v for analysis of B[a]P equivalents with an ELISA. The B[a]P recovery rates in spiked vegetable oil samples of different fatty acid composition were determined between 63% and 114%. The presence of multiple PAHs in the oil sample, because of MIP selectivity and cross-reactivity of the ELISA, could yield overestimated B[a]P values.

  16. Detection of abnormally high amygdalin content in food by an enzyme immunoassay.

    Cho, A-Yeon; Yi, Kye Sook; Rhim, Jung-Hyo; Kim, Kyu-Il; Park, Jae-Young; Keum, Eun-Hee; Chung, Junho; Oh, Sangsuk


    Amygdalin is a cyanogenic glycoside compound which is commonly found in the pits of many fruits and raw nuts. Although amygdalin itself is not toxic, it can release cyanide (CN) after hydrolysis when the pits and nuts are crushed, moistened and incubated, possibly within the gastrointestinal tract. CN reversibly inhibits cellular oxidizing enzymes and cyanide poisoning generates a range of clinical symptoms. As some pits and nuts may contain unusually high levels of amygdalin such that there is a sufficient amount to induce critical CN poisoning in humans, the detection of abnormal content of amygdalin in those pits and nuts can be a life-saving measure. Although there are various methods to detect amygdalin in food extracts, an enzyme immunoassay has not been developed for this purpose. In this study we immunized New Zealand White rabbits with an amygdalin-KLH (keyhole limpet hemocyanin) conjugate and succeeded in raising anti-sera reactive to amygdalin, proving that amygdalin can behave as a hapten in rabbits. Using this polyclonal antibody, we developed a competition enzyme immunoassay for determination of amygdalin concentration in aqueous solutions. This technique was able to effectively detect abnormally high amygdalin content in various seeds and nuts. In conclusion, we proved that enzyme immunoassay can be used to determine the amount of amygdalin in food extracts, which will allow automated analysis with high throughput.

  17. Detection of Escherichia coli in wastewater based on enzyme immunoassay

    XI Haiyan; CAI Qiang; HE Miao; SHI Hanchang


    This research describes a fast detection method on the basis of enzyme-linked immunosorbent assay (ELISA)for Escherichia coli in drainage of wastewater treatment plants.Optimized conditions such as the reaction format(sandwich or direct),the concentrations of diluted horseradish peroxidase (HRP)-E.coli conjugate,and anti-HPR antibody and pretreatment of E.coli were studied.Those results showed that the linear range of detection for E.coli was 10 cfu/mL-6×104 cfu/mL.Compared with conventional methods,it is a convenient and sensitive detection method with low cost.

  18. Development and application of an enzyme immunoassay for the detection of the mycotoxin Fumigaclavine A


    The present study describes the development of specific polyclonal antibodies against fumigaclavine A (FuA) in rabbits, and the development of a highly sensitive enzyme immunoassay (EIA) for this mycotoxin. The Mannich condensation reaction with formaldehyde was used to conjugate FuA to keyhole limpet hemocyanin (KLH) as the immunogen, and to bovine serum albumine (BSA) for as the coating antigen. Conjugation of FuA to KLH with formaldehyde proved to be an effective approach...

  19. Utility of salivary enzyme immunoassays for measuring estradiol and testosterone in adolescents: a pilot study.

    Amatoury, Mazen; Lee, Jennifer W; Maguire, Ann M; Ambler, Geoffrey R; Steinbeck, Katharine S


    We investigated the utility of enzyme immunoassay kits for measuring low levels of salivary estradiol and testosterone in adolescents and objectively assessed prevalence of blood contamination. Endocrine patients provided plasma and saliva for estradiol (females) or testosterone (males) assay. Saliva samples were also tested with a blood contamination kit. Picomolar levels of salivary estradiol in females failed to show any significant correlation with plasma values (r=0.20, p=0.37). The nanomolar levels of salivary testosterone in males showed a strong correlation (r=0.78, p<0.001). A significant number of saliva samples had blood contamination. After exclusion, correlations remained non-significant for estradiol, but strengthened for testosterone (r=0.88, p<0.001). The salivary estradiol enzyme immunoassay is not clinically informative at low levels. Users should interpret clinical saliva with caution due to potential blood contamination. Our data supports the utility of the salivary testosterone enzyme immunoassay for monitoring adolescent boys on hormone developmental therapy.

  20. Synthetic glycosylation of proteins using N-(beta-saccharide) iodoacetamides: applications in site-specific glycosylation and solid-phase enzymic oligosaccharide synthesis.

    Wong, S Y; Guile, G R; Dwek, R A; Arsequell, G


    A simple and efficient synthetic glycosylation method suitable for use in solid-phase enzymic oligosaccharide synthesis and site-specific glycosylation of recombinant proteins to produce defined glycoforms is described. This strategy utilizes N-(beta-saccharide) haloacetamides for attaching oligosaccharides specifically to cysteine residues of proteins in solution to form neoglycoproteins. The alkylation reaction was tested using N-(beta-chitotriose) bromoacetamide and an unprotected synthetic hexapeptide containing a single cysteine residue. The glycosylated product was confirmed by amino acid and hexosamine analyses as well as laser desorption mass spectrometry. Similarly N-(beta-chitotriose) iodoacetamide was covalently linked to non-reduced BSA to produce a defined glycoform of this protein. The specific attachment of chitotriose at the single cysteine residue in non-reduced serum albumin was suggested by Ellman's assay for free thiols. This was verified by amino acid sequencing of tryptic glycopeptide derived from this neoglycoprotein. Multiple sugar attachment was accomplished using fully reduced serum albumin as demonstrated by the formation of two neoglycoproteins using iodoacetamide derivatives of galactose beta 1-3-N-acetylgalactosamine (Gal beta 1-3GalNAc) and the major xylose/fucose-class plant-type oligosaccharide of horseradish peroxidase. These two neoglycoproteins with an average of 18-21 sugar residues attached were assayed positively for binding to peanut agglutinin and a sugar-specific anti-(horseradish peroxidase) monoclonal antibody YZ1/2.23 respectively. Sialylation of the neoglycoprotein containing Gal beta 1-3GalNAc was accomplished using alpha-2,3-sialyltransferase and radiolabelled CMP-N-acetylneuraminic acid. Significantly, glycan attachment using this conjugation method is reversible as demonstrated by the release of oligosaccharides from these two neoglycoproteins using hydrazinolysis. Therefore this method could provide invaluable

  1. Optimization of headspace solid-phase microextraction for the analysis of specific flavors in enzyme modified and natural Cheddar cheese using factorial design and response surface methodology.

    Januszkiewicz, Julien; Sabik, Hassan; Azarnia, Sorayya; Lee, Byong


    A headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC/MS) method was developed using experimental designs to quantify the flavor of commercial Cheddar cheese and enzyme-modified Cheddar cheese (EMCC). Seven target compounds (dimethyl disulfide, hexanal, hexanol, 2-heptanone, ethyl hexanoate, heptanoic acid, delta-decalactone) representative of different chemical families frequently present in Cheddar cheese were selected for this study. Three types of SPME fibres were tested: Carboxen/polydimethylsiloxane (CAR/PDMS), polyacrylate (PA) and Carbowax/divinylbenzene (CW/DVB). NaCl concentration and temperature, as well as extraction time were tested for their effect on the HS-SPME process. Two series of two-level full factorial designs were carried out for each fibre to determine the factors which best support the extraction of target flavors. Therefore, central composite designs (CCDs) were performed and response surface models were derived. Optimal extraction conditions for all selected compounds, including internal standards, were: 50 min at 55 degrees C in 3M NaCl for CAR/PDMS, 64 min at 62 degrees C in 6M NaCl for PA, and 37 min at 67 degrees C in 6M NaCl for CW/DVB. Given its superior sensitivity, CAR/PDMS fibre was selected to evaluate the target analytes in commercial Cheddar cheese and EMCC. With this fibre, calibration curves were linear for all targeted compounds (from 0.5 to 6 microg g(-1)), except for heptanoic acid which only showed a linear response with PA fibres. Detection limits ranged from 0.3 to 1.6 microg g(-1) and quantification limits from 0.8 to 3.6 microg g(-1). The mean repeatability value for all flavor compounds was 8.8%. The method accuracy is satisfactory with recoveries ranging from 97 to 109%. Six of the targeted flavors were detected in commercial Cheddar cheese and EMCC.

  2. Quantification of polynuclear aromatic hydrocarbons in transformer oils by enzyme immunoassay.

    Kim, I S; Ritchie, L; Setford, S; Allen, M; Wilson, G; Heywood, R; Pahlavanpour, B; Saini, S


    Many polynuclear aromatic hydrocarbons (PAHs) are either known or suspected carcinogens and are a common constituent of mineral oils. Due to the large number of possible PAH structures, standard quantification methods fail since they either lack specificity or are too complex, requiring individual fractionation, identification, and quantification. A rapid, low-cost, novel analytical screening method, incorporating a silica-based solid-phase extraction (SPE) method linked to co-solvent dilution and quantification of total and carcinogenic PAH levels by immunoassay, is reported here. The method yielded high extraction efficiencies and minimal matrix effects. This novel approach yielded total and carcinogenic PAH levels x 5.7 and x 126, respectively, lower than that recorded by the industry-recognised BS2000 Pt. 346 (IP346) method which estimates the polyaromatic carbon (PAC) content of oils by gravimetry. The method is expected to be of benefit where an indication of PAH levels in oils is important for purchasing, management or disposal purposes and also for risk assessment and for appropriate labelling of oils in line with current legislation.

  3. Sugar additives improve signal fidelity for implementing two-phase resorufin-based enzyme immunoassays.

    Sandoz, Patrick A; Chung, Aram J; Weaver, Westbrook M; Di Carlo, Dino


    Enzymatic signal amplification based on fluorogenic substrates is commonly used for immunoassays; however, when transitioning these assays to a digital format in water-in-mineral oil emulsions, such amplification methods have been limited by the leakage of small reporting fluorescent probes. In the present study, we used a microfluidic system to study leakage from aqueous droplets in a controlled manner and confirmed that the leakage of fluorescent resorufin derivatives is mostly due to the presence of the lipophilic surfactant Span80, which is commonly used to preserve emulsion stability. This leakage can be overcome by the addition of specific sugars that most strongly interfered with the surfactants ability to form micelles in water. The application of the microfluidic system to the quantitative analysis of droplets and the implementation of the described sugar additives would allow for alternatives to fluorinated surfactant-based platforms and improve the signal fidelity in enzyme immunoassays implemented through multiphase microfluidics.

  4. Solid-phase microextraction

    Nilsson, Torben

    The objective of this study has been to develop new analytical methods using the rapid, simple and solvent-free extraction technique solid-phase microextraction (SPME) for the quantitative analysis of organic pollutants at trace level in drinking water and environmental samples. The dynamics...

  5. Study of two different enzyme immunoassays for the detection of Mayaro virus antibodies

    Figueiredo, Luiz Tadeu Moraes; Nogueira, Rita Maria Ribeiro; Cavalcanti,Silvia Maria Baêta; Schatzmayr, Hermann; Rosa,Amélia Travassos da


    This paper presents the evaluation of an enzyme immunoassay in which Mayaro virus-infected cultured cells ara used as antigen (EIA-ICC) and an IgM antibody capture ELISA (MAC-ELISA) for Mayaro serologic diagnosis using 114 human sera obtained during a Mayaro outbreak occurred in Bolivia, in 1987. Results were compared with those obtained by haemagglutination-inhibition test (HAI). MAC-ELISA was the most sensitive technique for anti-Mayaro IgM detection. MAC-ELISA was twice sensitive as IgM EI...

  6. A New Voltammetric Enzyme Immunoassay System for the Detection of Alkaline Phosphatase

    KuiJIAO; WeiSUN; 等


    A new voltammetric enzyme immunoassay system was invesigated based on p-nitrophenyl phosphate (PNPP) as the subsrate for alkaline phosphatase (ALP). PNPP is enzymatically hydrolyzed and the product p-nitrophenol (PNP) is detected by differential pulse voltammetry (DPV), which can be oxidized at +1.02 V(vs.Ag/AgCl) on bare glass carbon electrode (GCE). The conditions for enzymatic reaction and electrochemical detection were studied. According to this method,ALP can be detected with a detection limit of 2.8×102 mU/L and a linear range of 4.0×102-1.0×106mU/L.

  7. A New Voltammetric Enzyme Immunoassay System for the Detection of Alkaline Phosphatase


    A new voltammetric enzyme immunoassay system was investigated based on p-nitrophenyl phosphate (PNPP) as the substrate for alkaline phosphatase (ALP). PNPP is enzymatically hydrolyzed and the product p-nitrophenol (PNP) is detected by differential pulse voltammetry (DPV), which can be oxidized at +1.02 V (vs. Ag/AgCl) on bare glass carbon electrode (GCE). The conditions for enzymatic reaction and electrochemical detection were studied. According to this method, ALP can be detected with a detection limit of 2.8′102 mU/L and a linear range of 4.0′102 ~ 1.0′106 mU/L.

  8. Enzyme immunoassay of oestrogen receptors in needle biopsies from human liver

    Becker, U; Andersen, J; Poulsen, H S;


    For quantitative assessments of sex hormone receptors in liver tissue, ligand binding assays are inconvenient, as they require large biopsies (0.5-1.0 g). The present study shows that it is possible to measure oestrogen receptors (ER) quantitatively in needle biopsy specimens as small as 10 mg...... by modifications of a commercial enzyme immunoassay employing monoclonal antibodies. Sucrose gradient centrifugation and the dextran charcoal method served as reference methods. A consecutive series of needle biopsies from patients suspected of liver disease were investigated. The biopsies (n = 37) had a median...... is a convenient tool for further studies of ER in routine needle biopsies from the liver....

  9. A sensitive enzyme immunoassay for amygdalin in food extracts using a recombinant antibody.

    Cho, A-Yeon; Shin, Kum-Joo; Chung, Junho; Oh, Sangsuk


    Amygdalin (laterile) is a cyanogenic glycoside commonly found in the pits of many fruits and raw nuts. When amygdalin-containing seeds are crushed and moistened, free cyanide is formed. Pits and nuts containing unusually high levels of amygdalin can therefore cause cyanide poisoning, and detection of amygdalin in food extracts can be a life-saving measure. In this study, we generated recombinant antibodies against amygdalin from a phage display of a combinatorial rabbit/human chimeric antibody library and used it in a sensitive competition enzyme immunoassay system to detect amygdalin in extracts of pits and nuts. The detection limit was determined to be 1 x 10(-9) M.

  10. An enzyme immunoassay for detection of Japanese encephalitis virus-induced chemotactic cytokine

    Aditi Singh; Rajesh Kulshreshtha; Asha Mathur


    Japanese encephalitis virus (JEV) induces human peripheral blood monocytes to secrete a chemotactic cytokine [human macrophage-derived factor (hMDF)] which causes chemotaxis of neutrophils. The only known assay for hMDF cannot quantify its level in samples, so an enzyme immunoassay has been standardized for detection of hMDF and hMDF-specific antibodies in test samples. The reported enzyme linked immunosorbent assay (ELISA) was found to be sensitive (89%), specific (91%), accurate (92·2%) and reproducible and was able to detect a minimum concentration of 23 ng hMDF/ml in test samples. The chemotactic factor could be detected in JEV inoculated mouse sera and JEV infected culture fluids. Significant finding of the test was the detection of hMDF in sera of human cases of JE.

  11. Solid phase transformations II

    Čermák, J


    This topical volume includes ten invited papers that cover selected areas of the field of solid phase transformations. The first two contributions represent a burgeoning branch; that of the computer simulation of physical phenomena. The following three articles deal with the thermodynamics of phase transformations as a basic theory for describing the phenomenology of phase changes in matter. The next paper describes the interconnections between structural stability and the electronic structure of phases. Two further articles are devoted to displacive transformations; a field where there are ma

  12. Solid phase transformations

    Čermák, J


    This special-topic book, devoted to ""Solid Phase Transformations"" , covers a broad range of phenomena which are of importance in a number of technological processes. Most commercial alloys undergo thermal treatment after casting, with the aim of imparting desired compositions and/or optimal morphologies to the component phases. In spite of the fact that the topic has lain at the center of physical metallurgy for a long time, there are numerous aspects which are wide open to potential investigative breakthroughs. Materials with new structures also stimulate research in the field, as well as n

  13. Enzyme immunoassay of benzyl penicilloyl (BPO) groups using acetylcholinesterase as label. Application to the study of the BPO-binding sites on albumin.

    Wal, J M; Yvon, M; Pradelles, P; Grassi, J


    Benzyl penicilloyl groups (BPO) derive from penicillin G by cleavage of the beta lactam ring; they covalently bind to proteins to give conjugates which have lost all antibiotic properties but are considered as the major allergenic determinants in penicillin allergy. A solid-phase Enzyme Immuno Assay (EIA) of BPO groups in different biological fluids is described. It is a competitive immunoassay using acetylcholinesterase as label. In all biological fluids, very low non-specific binding values are observed. The sensitivity and the precision of the assay are good since ca. 0.5 ng/ml can be measured with a coefficient of variation less than 10%. Cross reactions between BPO and penicillin or penicillin derivatives are nil or very low. This assay is more sensitive, much more rapid and easier to handle than the other methods available and is thus suitable for routine determinations. In association with reversed-phase high performance liquid chromatography this EIA has allowed an initial investigation of the location of BPO-binding sites on micro quantities of serum albumin (ca. 1 mg) from penicillin treated patients.

  14. Electrochemical studies of 1,5-dihydroxynaphthalene as substrate for voltammetric enzyme immunoassay

    JIAO Kui; YAO Hong; XU Jin; ZHANG Shusheng


    A sensitive voltammetric enzyme immunoanalytical method is described for assay of horseradish peroxidase (HRP), labelled-HRP and antigen:antibody:labelled-HRP conjugate (Ag︰AB︰IgG-HRP) using 1,5-dihydroxynaphthalene (1,5-DHN) as substrate. 1,5-DHN is an electrochemical inactive substance, but it may be oxidized by O2 in air to 5-hydroxy-1,4-naphtho- quinone in basic solution, which produces a well-defined voltammetric reduction peak. After adding H2O2 and HRP, HRP catalyzes H2O2 oxidizing 5-hydroxy-1,4-naphthoquinone to 3-hydroxyphthalic acid, leading to the decrease of the voltammetric reduction peak, with which the activity of HRP and the labelled-HRP, further the antigen and antibody may be determined. The mechanism of this voltammetric enzyme-linked immunoassay system is different from the previous reports, in which there are no voltammetric reduction peaks before the addition of H2O2 and HRP, and the reduction peaks appear after the addition of H2O2 and HRP. The height of the voltammetric peak is directly proportional to the concentration of HRP in the previous systems, while the decrease of the voltammetric peak is linear with the concentration of HRP in this system. This new electrochemical method is combined with an indirect enzyme immunoassay using direct antigen-coating format for the detection of the purified tobacco mosaic virus (TMV) with a linear range from 25.0 to 340.0 ng/mL, and a detection limit 25.0 ng/mL.

  15. The clinical value of enzyme-multiplied immunoassay technique monitoring the plasma concentrations of cyclosporine A a%The clinical value of enzyme-multiplied immunoassay technique monitoring the plasma concentrations of cyclosporine A aft

    Xiao-Hui Luo; wu-Jun Xue; Pu-Xun Tian; Xiao-Ming Ding; Hang Yan; He-Li Xiang; Yang Li


    The feasibility and the clinical value of the enzyme-multiplied immunoassay technique (EMIT) monitoring of blood concentrations of cyclosporine A (CsA) in patients treated with CsA were investigated after kidney transplantation. The validation method was

  16. Development of a sensitive enzyme immunoassay for human epidermal growth factor (urogastrone).

    Kurobe, M; Tokida, N; Furukawa, S; Ishikawa, E; Hayashi, K


    A sensitive two-site enzyme immunoassay (EIA) for human epidermal growth factor (hEGF) was developed, based on the sandwiching of an antigen between anti-hEGF IgG-coated polystyrene beads and anti-hEGF Fab'-linked peroxidase complex (horseradish peroxidase, EC. This method has four advantages: the anti-hEGF Fab'-linked peroxidase complex is more stable than 125I-labelled antibody; the procedure is simple and rapid compared to bioassay; its discriminatory sensitivity is as low as 0.1 pg/assay tube; and serial dilution curves of unextracted human serum and urine samples all paralleled that of standard hEGF. The validity of the measurement of hEGF-like immunoreactivity in human serum and plasma is discussed.

  17. Detection of hidden hazelnut protein in food by IgY-based indirect competitive enzyme-immunoassay

    Baumgartner, S.; Bremer, M.G.E.G.; Kemmers - Voncken, A.E.M.; Smits, N.G.E.; Haasnoot, W.; Banks, J.; Reece, P.; Danks, C.; Tomkies, V.; Immer, U.; Schmitt, K.; Krska, R.


    The development of an indirect competitive enzyme-immunoassay for the detection of hidden hazelnut protein in complex food matrices is described. A sensitive and selective polyclonal antibody was raised by immunisation of laying hens with protein extracts from roasted hazelnuts. In contrast to tradi

  18. Evaluation of two automated enzyme-immunoassays for detection of thermophilic campylobacters in faecal samples from cattle and swine

    Hoorfar, Jeffrey; Nielsen, E.M.; Stryhn, H.


    We evaluated the performance of two enzyme-immunoassays (EIA) for the detection of naturally occurring, thermophilic Campylobacter spp. found in faecal samples from cattle (n = 21 and n = 26) and swine (n = 43) relative to the standard culture method, and also assuming that none of the tests was ...

  19. Role of Triton X-100 in chemiluminescent enzyme immunoassays capable of diagnosing genetic disorders.

    Chong, Richard; Rho, Jee-Eun R; Yoon, Hye-Joo; Park, Paul S; Rho, Tae-Ho D; Park, Jee Y; Park, Lucienne; Kim, Young-Hwan; Lee, Ji Hoon


    The use of Triton X surfactants in developing 1,1'-oxalylimidazole chemiluminescent enzyme immunoassays (ODI CEIs) with extended linear response range for the quantification of unconjugated estriol (uE3), alpha-fetoprotein (AFP), and human chorionic gonadotropin (hCG) is reported for the first time. The wider linear dynamic range in ODI CLEIA results from Triton X series (e.g., Triton X-100, -114, -405, -705) acting as an inhibitor in the interaction between Amplex Red (hydrophobic substrate) and horseradish peroxidase (hydrophilic enzyme) to produce resorufin (hydrophobic fluorescent dye). Triton X-100 acts as the appropriate inhibitor in ODI CLEIA. The maximum concentrations of AFP and hCG quantified with sandwich ODI CLEIA in the presence of Triton X-100 were 8 times higher than when analyzed with the same system in the absence of Triton X-100. In addition, the lowest concentration of uE3 determined using competitive ODI CLEIA in the presence of Triton X-100 was 20 times lower than that measured with competitive ODI CLEIA in the absence of Triton X-100. These results indicate that rapid quantification of AFP, uE3, and hCG using cost effective and highly sensitive ODI CLEIAs in the presence of Triton X-100 can be applied as an accurate, precise, and reproducible method to diagnose genetic disorders (e.g., trisomy 18 and trisomy 21) in fetuses. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Simultaneous Photoelectrochemical Immunoassay of Dual Cardiac Markers Using Specific Enzyme Tags: A Proof of Principle for Multiplexed Bioanalysis.

    Zhang, Nan; Ma, Zheng-Yuan; Ruan, Yi-Fan; Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan


    In this Letter, on the basis of the CdS quantum dots functionalized TiO2 nanotubes electrode, we proposed a simultaneous photoelectrochemical (PEC) immunoassay of dual cardiac markers using specific enzyme tags of alkaline phosphatase (ALP) and acetylcholine esterase (AChE). ALP and AChE were integrated into the PEC system through the sandwich immunobinding and could specifically catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) or the acetylthiocholine (ATC) to in situ generate ascorbic acid (AA) or thiocholine (TC) for sacrificial electron donating. These two enzymes were thus used to differentiate the signals of two cardiac targets in connection with the sandwich immunorecognition and PEC responses to the corresponding electron donors. This strategy demonstrates a proof of principle for the successful integration of dual enzyme tags with PEC immunoassay that can potentially provide a general format for multiplexed PEC bioanalysis.

  1. Investigation of voltammetric enzyme-linked immunoassay based on new system of ODA-H2O2-HRP

    焦奎; 张书圣; 韦璐


    A voltammetric enzyme-linked immunoassay based on a new system of ODA-H2O2-HRP has first been developed and used in the detection of HRP and labelled HRP. By this method, the enzyme-catalyzing reaction of H2O2 oxidizing odianisidine (ODA) couples the electrode-reduction reaction of the oxidizing product of odianisidine, which produces a sensitive polarographic wave at potential of -0.56V (SCE) in Britton-Robinson buffer solution. In using this polarographic wave, a detection limit to HRP is 3.7×10-12g/mL and a linear range 1.0×10-11-2.0×10-9g/mL. And the mechanisms of the coupling reaction and the process of electro-reduction in the ODA-H2O2-HRP voltammetric enzyme-linked immunoassay system have also been carefully studied.

  2. On-chip enzyme quantification of single Escherichia coli bacteria by immunoassay-based analysis.

    Stratz, Simone; Eyer, Klaus; Kurth, Felix; Dittrich, Petra S


    Individual bacteria of an isogenic population can differ significantly in their phenotypic characteristics. This cellular heterogeneity is thought to increase the adaptivity to environmental changes on a population level. Analytical methods for single-bacteria analyses are essential to reveal the different factors that may contribute to this cellular heterogeneity, among them the stochastic gene expression, cell cycle stages and cell aging. Although promising concepts for the analysis of single mammalian cells based on microsystems technology were recently developed, platforms suitable for proteomic analyses of microbial cells are by far more challenging. Here, we present a microfluidic device optimized for the analysis of single Escherichia coli bacteria. Individual bacteria are captured in a trap and isolated in a volume of only 155 pL. In combination with an immunoassay-based analysis of the cell lysate, the platform allowed the selective and sensitive analysis of intracellular enzymes. The limit of detection of the developed protocol was found to be 200 enzymes. Using this platform, we could investigate the levels of β-galactosidase in cells grown under different nutrient conditions. We successfully determined the enzyme copy numbers in cells cultured in defined medium (3517 ± 1578) and in complex medium (4710 ± 2643), and verified the down-regulation of expression in medium that contained only glucose as carbon source. The strong variations we found for individual bacteria confirm the phenotype heterogeneity. The capability to quantify proteins and other molecules in single bacterial lysates is encouraging to use the new analysis platform in future proteomics studies of isogenic bacteria populations.

  3. Direct appraisal of latex agglutination testing, a convenient alternative to enzyme immunoassay for the detection of rotavirus in childhood gastroenteritis, by comparison of two enzyme immunoassays and two latex tests.

    Sambourg, M; Goudeau, A; Courant, C; Pinon, G; Denis, F


    During February and March 1984, 207 fecal samples from infants and children with gastroenteritis were tested for rotavirus with four techniques: two enzyme immunoassays (Rotazyme; Abbott Laboratories, North Chicago, Ill., and Enzygnost-Rotavirus; Calbiochem-Behring, La Jolla, Calif.) and two latex agglutination tests (Rotalex; Orion Research, Inc., Cambridge, Mass., and Slidex Rota-Kit; Biomérieux). All stool samples were also tested for yeasts and bacterial pathogens. Electron microscopy was used to investigate discrepant results. We found 47% positive samples with Enzygnost-Rotavirus, 38% with Rotazyme, 37% with Slidex Rota-Kit, and 34% with Rotalex. No specimen was found positive by Rotazyme only or Slidex Rota-Kit only. On the contrary, 12 samples which were positive with Enzygnost-Rotavirus only and 3 which were positive with Rotalex only were not confirmed as positive by electron microscopy. Both enzyme immunoassays gave 6% equivocal results; Slidex Rota-Kit gave significantly fewer equivocal results than did Rotalex: 2.9% versus 9.7% (P less than 0.01). The sensitivity and specificity of latex tests compared favorably with that of enzyme immunoassays. Latex agglutination tests can be performed by unskilled personnel and are rapid and relatively cheap. They appear to be very suitable for routine laboratory work and may prove useful for large-scale screening in developing countries.

  4. Detection and identification of platelet-associated alloantibodies by a solid-phase modified antigen capture enzyme-linked immunosorbent assay method and its correlation to platelet refractoriness in multiplatelet concentrate-transfused patients.

    Jain, Neelesh; Sarkar, Shankar; Philip, Joseph


    Platelets express a variety of polymorphic glycoproteins (GPs), such as GPIIb/IIIa, GPib/IX, GPla/Ila, GPIV, and class I human leukocyte antigen. In the platelet transfusion setting, alloimmunization involves the production of antibodies against these glycoproteins. Patients transfused with multiple units of platelet concentrates for longer periods are the main individuals with platelet alloimmunization. This study was performed to detect the development of platelet antibodies in patients who are transfused with multiple units of leukodepleted platelet concentrates, such as those with hemato-oncologic diseases and bone marrow failure syndromes. The method used was solid phase modified antigen capture enzyme-linked immunosorbent assay. Platelet refractoriness was assessed by measuring the corrected count increment at 1 and 24 hours after transfusion.

  5. High sensitivity chemiluminescence enzyme immunoassay for detecting staphylococcal enterotoxin A in multi-matrices.

    Zhang, Chunmei; Liu, Zhijia; Li, Yongming; Li, Qi; Song, Chaojun; Xu, Zhuwei; Zhang, Yun; Zhang, Yusi; Ma, Ying; Sun, Yuanjie; Chen, Lihua; Fang, Liang; Yang, Angang; Yang, Kun; Jin, Boquan


    In this study, detection of staphylococcal enterotoxin A (SEA) in multi-matrices using a highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) has been established. A pair of monoclonal antibodies (mAbs) was selected from 37 anti-SEA mAbs by pairwise analysis, and the experimental conditions of the CLEIA were optimized. This CLEIA exhibited high performance with a wide dynamic range from 6.4 pg mL(-1) to 1600 pg mL(-1), and the measured low limit of detection (LOD) was 3.2 pg mL(-1). No cross-reactivity was observed when this method was applied to test SEB, SEC1, and SED. It has also been successfully applied for analyzing SEA in a variety of environmental, biological, and clinical matrices, such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, human urine, and serum. Thus, the highly sensitive and SEA-specific CLEIA should make it attractive for quantifying SEA in public health and diagnosis in near future.

  6. Study of two different enzyme immunoassays for the detection of Mayaro virus antibodies.

    Figueiredo, L T; Nogueira, R M; Cavalcanti, S M; Schatzmayr, H; da Rosa, A T


    This paper presents the evaluation of an enzyme immunoassay in which Mayaro virus-infected cultured cells are used as antigen (EIA-ICC) and an IgM antibody capture ELISA (MAC-ELISA) for Mayaro serologic diagnosis using 114 human sera obtained during a Mayaro outbreak occurred in Bolivia, in 1987. Results were compared with those obtained by haemagglutination-inhibition test (HAI). MAC-ELISA was the most sensitive technique for anti-Mayaro IgM detection. MAC-ELISA was twice as sensitive as IgM EIA-ICC. The data shows that MAC-ELISA is a practical and valid technique for diagnosis of recent Mayaro infection. IgG EIA-ICC showed high sensitivity and high specificity compared to HAI. The combination of anti-Mayaro IgG and IgM EIA-ICC results presented the highest sensitivity of the study. Anti-Mayaro IgG and IgM simultaneous detection by EIA-ICC can be used for recent infection diagnosis (in spite of a less sensitive IgM detection than by MAC-ELISA), for surveillance and sero-epidemiologic studies, and for studies of IgG and IgM responses to Mayaro infection.

  7. Evaluation of enzyme immunoassay techniques for diagnosis of the most common intestinal protozoa in fecal samples.

    Gaafar, Maha R


    This study was designed to evaluate the antigen capture enzyme immunoassays (EIAs) Triage parasite panel and TechLab Entamoeba histolytica II in detecting Giardia intestinalis, Cryptosporidium sp, and Entamoeba histolytica in fecal samples in comparison to microscopy, and in differentiating Entamoeba histolytica from Entamoeba dispar. The Triage EIA was evaluated using 100 stool specimens that were tested by standard ova and parasite examination, including staining with both trichrome and modified acid-fast stains. Differentiation between E. histolytica and E. dispar was performed using TechLab. Microscopic examination revealed that 19% of the samples were positive for Giardia, 4% for Cryptosporidium, and 1% for E. histolytica/E. dispar, and other parasites were found in 5%. By Triage, 23% of the samples were infected with Giardia, 5% with Cryptosporidium, and 2% with E. histolytica/E. dispar. Triage showed a sensitivity of 100% and specificity of 91.5%. The TechLab assay was negative for both samples diagnosed as E. histolytica/E. dispar by Triage, which suggested that they were E. dispar. Both tests showed no cross-reactivity with other intestinal protozoa. These results indicate that antigen detection by EIA has the potential to become a valuable tool, capable of making stool diagnostics more effective. Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  8. Competitive chemiluminescent enzyme immunoassay for vitamin B12 analysis in human milk.

    Hampel, Daniela; Shahab-Ferdows, Setareh; Domek, Joseph M; Siddiqua, Towfida; Raqib, Rubhana; Allen, Lindsay H


    Recent discoveries of matrix interferences by haptocorrin (HC) in human milk and serum show that past analyses of vitamin B12 in samples with high HC content might have been inaccurate (Lildballe et al., 2009; Carmel & Agrawal, 2012). We evaluated two competitive enzyme-binding immunoassays for serum/plasma (IMMULITE and SimulTRAC-SNB) for B12 analysis in human milk. B12-recovery rates (United States Environmental Protection Agency, 2007) were determined to be 78.9 ± 9.1% with IMMULITE and 225 ± 108% (range 116-553%) using SimulTRAC-SNB, most likely due to the presence of excess HC. HC-interferences were not observed with the IMMULITE assay, rendering previously reported mandatory HC-removal (Lildballe et al., 2009) unnecessary. Linearity continued at low B12-concentrations (24-193 pM; r(2)>0.985). Milk B12 concentrations from Bangladeshi women (72-959 pM) were significantly lower than those from California (154-933 pM; pmilk matrix and its ability to measure low milk B12 concentrations.

  9. Evaluation of an enzyme immunoassay for estrogen receptors in human breast cancers.

    Nicholson, R I; Colin, P; Francis, A B; Keshra, R; Finlay, P; Williams, M; Elston, C W; Blamey, R W; Griffiths, K


    An estrogen receptor enzyme immunoassay kit (ER-EIA) has been evaluated in 70 human breast carcinomas against a routine cytoplasmic [3H]estradiol binding assay (ERU). A linear correlation between the ER-EIA and the ERU was observed for binding values up to 400 fmol/mg of cytosol protein. Above this value, the ERU underestimates the concentration of receptor. The ERU gave a lower number of estrogen receptor-positive tumors (50 of 70) than did the ER-EIA assay (59 of 70). In the ERU-negative ER-EIA-positive tumors, receptor values as determined by the ER-EIA assay all fell below 50 fmol/mg of protein (mean, 19.9 +/- 4.2 fmol/mg of protein). Application of an exchange procedure which estimates the total steroid binding capacity of the cytosol gave positive results in 7 of 9 ERU-negative ER-EIA-positive tumors (mean, 16.9 +/- 2.95 fmol/mg of protein). Subdivision of the binding data according to the menopausal status of the patient indicates low receptor values in premenopausal women by each assay. A correlation between the ER-EIA assay and the histological grade of tumors was observed; Grade I well-differentiated tumors were all positive, while Grade II and III tumors were 86% and 75% positive, respectively. No correlation between the ER-EIA assay and tumor lymph node stage or tumor size was observed.

  10. Comparison of Salivary and Serum Enzyme Immunoassays for the Diagnosis of Helicobacter pylori Infection

    John M Embil


    Full Text Available Infection with Helicobacter pylori has been established as an important risk factor for the development of peptic ulcer disease, gastritis and gastric cancer. The diagnosis of H pylori infection can be established by invasive or noninvasive techniques. Two noninvasive enzyme immunoassays (EIAs for antibody detection – HeliSal and Pylori Stat – were compared with histology. Both assays detect immunoglobulin (Ig G directed against purified H pylori antigen. The test populations consisted of 104 consecutive patients scheduled for upper gastrointestinal endoscopy. Of these patients, 97 (93% had symptoms compatible with peptic ulcer disease. Saliva and serum were collected simultaneously at the time of endoscopy. Salivary EIA had a sensitivity of 66%, specificity of 67%, positive predictive value of 67% and negative predictive value of 66% compared with the serum EIA, where the results were 98%, 48%, 64% and 96%, respectively. Although the salivary EIA is an appealing noninvasive test, it was not a sensitive and specific assay. The serum EIA also lacked specificity, but was highly sensitive with a good negative predictive value. Although a negative serum EIA rules out H pylori infection, a positive result must be interpreted in the clinical context and confirmed with a more specific measure.

  11. A sensitive two-site enzyme immunoassay for human epidermal growth factor (urogastrone).

    Hayashi, K; Nomoto, H; Kurobe, M; Nishimuro, S; Hiratani, H; Furukawa, S


    A sensitive enzyme immunoassay (EIA) was developed for human epidermal growth factor (hEGF) or urogastrone, which was isolated from human urine. Our EIA system is based on the sandwiching of an antigen between anti-hEGF IgG coated on a polystyrene tube and anti-hEGF antibody Fab'-linked beta-D-galactosidase (beta-D-galactosidase, EC This method has the advantages that the procedures are simple and rapid and that the antibody Fab'-beta-D-galactosidase complex is more stable than radioisotope-labeled IgG. Purified hEGF is detectable at as low as 100 pg/ml, which is very sensitive compared to the radioimmuno-assays or radioreceptor assays already reported. Using this new EIA system, hEGF levels in human urine were examined. The values for normal males and females were 48.4 and 83.5 ng/mg creatinine, respectively, which shows that females excrete 1.7 times more hEGF than males.

  12. Evaluation of an enzyme immunoassay for verotoxin detection in Escherichia coli.

    Frias, C; Majò, M; Margall, N; Llobet, T; Mirelis, B; Prats, G


    Verotoxin-producing Escherichia coli strains (VTEC) cause hemorrhagic colitis and hemolytic-uremic syndrome in humans. Laboratory diagnosis by conventional methods is slow and cumbersome. The results of a new rapid enzyme immunoassay (EIA Premier EHEC) for verotoxin detection both in isolated strains and in clinical samples are presented, and they are compared with cell culture (CC) and polymerase chain reaction (PCR) techniques. Fifty-four strains have been analyzed by both EIA and PCR, and 33 by all three methods. The kit has also been evaluated for experimentally infected stool samples directly and after their enrichment on MacConkey broth. Nineteen, out of the 54 strains, were positive by EIA and 20 by PCR. The results of the 33 strains evaluated by the three techniques were coincident with one exception. The latter was uninterpretable by CC, negative by EIA and positive by PCR. The sensitivity of the kit for experimentally infected stool samples was approximately 5 x 10(7) bacteria/ml in the direct test, and 5 x 10(4) bacteria/ml after broth enrichment. EIA sensitivity and specificity were similar to those of CC and PCR. The diagnostic times were 18h for EIA, 3 days for PCR and 5 days for CC. Sensitivity, rapidity and ease of performance make this technique especially valuable for clinical diagnosis.

  13. Laboratory and epidemiologic evaluation of an enzyme immunoassay for antibodies to HTLV-III

    Ward, J.W.; Grindon, A.J.; Feorino, P.M.; Schable, C.; Parvin, M.; Allen, J.R.


    The enzyme immunoassays (EIAs) for antibody to human T-cell lymphotropic virus type III (HTLV-III) were rapidly adopted for screening donated blood and plasma. To evaluate the significance of a positive EIA reaction, test performance was examined in a blood bank screening program. Specimens were tested by EIA, Western blot assay, and HTLV-III/lymphadenopathy-associated virus (LAV) culture. The EIA was positive in 0.25% of 67 190 blood donations. Specimens were categorized and 57.3% had low (weak) reactivity, 12.7% had moderate reactivity, and 30.0% had high reactivity. Highly reactive specimens were strongly associated with a positive Western blot or culture (86.7%) in contrast to moderately and weekly reactive specimens (1.9%). Twenty-five of 29 donors interviewed with a highly reactive EIA had risk factors for HTLV-III/LAV infection. Risk factors were not identified for 74 of 75 interviewed donors with specimens of lower reactivity. The minimum calculated specificity was 99.82%. The use of the HTLV-III EIA has virtually eliminated the use of blood and plasma for HTLV-III/LAV infected donors.

  14. Direct radio-immunoassay of renin substrate: effect of converting enzyme inhibition

    Metsaerinne, Kaj; Rosenloef, Katarina; Groenhagen-Riska, Carola; Fyhrquist, Frej


    A direct radio-immunoassay (RIA) for renin substrate (RS) was compared to enzymatic (indirect) assay. In normal subjects, a significant, albeit weak, correlation between the methods was seen. In hypertensive patients with different levels of plasma renin activity (PRA), RS concentration measured by both assays increased with increasing PRA, and for patients with PRA > 10 AI/l/h, direct assay gave significantly higher RS values (55%), compared to the enzymatic assay, indicating consumption of RS by increasing plasma renin and production rate of RS with increasing PRA. In 11 patients with renovascular hypertension, treatment with angiotensin-converting enzyme (ACE) inhibitor, lisinopril, resulted in a significant increase in PRA, accompanied by a decrease in RS measured by enzymatic assay. No change in RS measured by direct RIA was noticed. The results suggest that ACE inhibition may not have an effect upon RS production and that its effect on plasma RS is limited to a reduction of intact RS measured by the enzymatic assay.

  15. An ultrasensitive and universal photoelectrochemical immunoassay based on enzyme mimetics enhanced signal amplification.

    Wang, Guang-Li; Shu, Jun-Xian; Dong, Yu-Ming; Wu, Xiu-Ming; Li, Zai-Jun


    An ultrasensitive photoelectrochemical (PEC) immunoassay based on signal amplification by enzyme mimetics was fabricated for the detection of mouse IgG (as a model protein). The PEC immunosensor was constructed by a layer-by-layer assembly of poly (diallyldimethylammonium chloride) (PDDA), CdS quantum dots (QDs), primary antibody (Ab1, polyclonal goat antimouse IgG), and the antigen (Ag, mouse IgG) on an indium-tin oxide (ITO) electrode. Then, the secondary antibody (Ab2, polyclonal goat antimouse IgG) combined to a bio-bar-coded Pt nanoparticle(NP)-G-quadruplex/hemin probe was used for signal amplification. The bio-bar-coded Pt NP-G-quadruplex/hemin probe could catalyze the oxidation of hydroquinone (HQ) using H2O2 as an oxidant, demonstrating its intrinsic enzyme-like activity. High sensitivity for the target Ag was achieved by using the bio-bar-coded probe as signal amplifier due to its high catalytic activity, a competitive nonproductive absorption of hemin and the steric hindrance caused by the polymeric oxidation products of HQ. For most important, the oxidation product of HQ acted as an efficient electron acceptor of the illuminated CdS QDs. The target Ag could be detected from 0.01pg/mL to 1.0ng/mL with a low detection limit of 6.0fg/mL. The as-obtained immunosensor exhibited high sensitivity, good stability and acceptable reproducibility. This method might be attractive for clinical and biomedical applications.

  16. Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA).

    Léo, P; Ucelli, P; Augusto, E F; Oliveira, M S; Tamashiro, W M


    The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.

  17. Heat identification by 17β-estradiol and progesterone quantification in individual raw milk samples by enzyme immunoassay

    Domènech, Anna; Pich,Sara; Arís,Anna; Plasencia,Carmen; Bach, Alex; Serrano,Alicia


    Background: There is a substantial decline in first-service-pregnancy-rate in dairy cows. In this regard, future prospects are to measure milk hormones on-farm and progesterone levels in milk are not enough to precise ovulation unless connected to other data. The objectives of this study were to investigate whether 17β-estradiol could be measured from individual cow milk samples using a commercially available non-radiolabelled enzyme immunoassay kit (EIA) with no previously reported milk...

  18. Evaluation of rapid commercial enzyme immunoassay for detection of Giardia lamblia in formalin-preserved stool specimens.

    E. H. Scheffler; Van Etta, L L


    Two hundred twenty-three formalin-preserved stool specimens were evaluated by using ProSpecT Giardia Rapid Assay (membrane bound) (Alexon, Inc., Sunnyvale, Calif.). Enzyme immunoassay (EIA) results were compared with those by conventional microscopic examination. Two hundred four specimens were negative by both methods, and 13 (6.3%) were positive. Five specimens were negative by initial microscopic exam and positive by EIA; three of these specimens were found to be positive upon extensive mi...

  19. An efficient sample preparation method for high-throughput analysis of 15(S)-8-iso-PGF2α in plasma and urine by enzyme immunoassay.

    Bielecki, A; Saravanabhavan, G; Blais, E; Vincent, R; Kumarathasan, P


    Although several methods have been reported on the analysis of the oxidative stress marker 15(S)-8-iso-prostaglandin-F2alpha (8-iso-PGF2α) in biological fluids, they either involve extensive sample preparation and costly technology or require high sample volume. This study presents a sample preparation method that utilizes low sample volume for 8-iso-PGF2α analysis in plasma and urine by an enzyme immunoassay (EIA). In brief, 8-iso-PGF2α in deproteinized plasma or native urine sample is complexed with an antibody and then captured by molecular weight cut-off filtration. This method was compared with two other sample preparation methods that are typically used in the analysis of 8-iso-PGF2α by EIA: Cayman's affinity column purification method and solid-phase extraction on C-18. The immunoaffinity purification method described here was superior to the other two sample preparation methods and yielded recovery values of 99.8 and 54.1% for 8-iso-PGF2α in plasma and urine, respectively. Analytical precision (relative standard deviation) was ±5% for plasma and ±15% for urine. The analysis of healthy human plasma and urine resulted in basal 8-iso-PGF2α levels of 31.8 ± 5.5 pg/mL and 2.9 ± 2.0 ng/mg creatinine, respectively. The robustness and analytical performance of this method makes it a promising tool for high-throughput screening of biological samples for 8-iso-PGF2α.

  20. Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

    Lopes EPA


    Full Text Available This study was undertaken to evaluate an enzyme immunoassay (EIA for hepatitis C virus antibody detection (anti-HCV, using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm. Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott®, specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95% sera from patients in the study group and negative in 38 of the 39 (97% sera from those in the control group, showing an accuracy of 96%. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

  1. Solid-Phase Random Glycosylation

    Agoston, K.; Kröger, Lars; Dekany, Gyula


    Two different approaches were employed to study solid phase random glycosylations to obtain oligosaccharide libraries. In approach I, Wang resin esters were attached to the acceptors structures. Following their glycosylation and resin cleavage, the peracetylated components of the oligosaccharide ...

  2. Which one of the two common reporter systems is more suitable for chemiluminescent enzyme immunoassay: alkaline phosphatase or horseradish peroxidase?

    Yu, Songcheng; Yu, Fei; Liu, Lie; Zhang, Hongquan; Zhang, Zhenzhong; Qu, Lingbo; Wu, Yongjun


    Alkaline phosphatase and horseradish peroxidase are the most commonly used reporter systems in chemiluminescent enzyme immunoassay (CLEIA). Which one, therefore, would be better when establishing a CLEIA method for a new target substance? There was no standard answer. In this study, both reporters were compared systematically including luminescence kinetics, conjugation methods, optimal condition and detection performance, using two common drugs, SD-methoxy-pyrimidine and enrofloxacin, as determination objects. The results revealed that there was much difference between the luminescence kinetics of the two systems. However, there was little difference between these systems when detecting the same substance, including in optimal conditions and determination of performance. Both reporters were suitable for establishing chemiluminescent enzyme immunoassays. Therefore, the choice of alkaline phosphatase or horseradish peroxidase as the reporter system in chemiluminescent enzyme immunoassays depends on availability. Conversely, these two report systems could be applied in simultaneous analysis of multicomponents due to their different optical behaviors and similar performances. But attention should be paid to conjugation method and coating buffer, which affected the luminescent intensity of different determination targets.

  3. Particle counting assay for anti-toxoplasma IgG antibodies. Comparison with four automated commercial enzyme-linked immunoassays.

    Galanti, L M; Dell'Omo, J; Wanet, B; Guarin, J L; Jamart, J; Garrino, M G; Masson, P L; Cambiaso, C L


    An assay for anti-toxoplasma IgG antibodies based on agglutination of latex particles was set up and compared with commercial immunoassays. The reaction was measured by instrumental counting of particles remaining unagglutinated. The running time was 45 min. This test (PaC) was compared using 243 serum samples with four automated commercial immunoassays: the Enzymum test Toxo IgG (ES300, Boehringer), the Vidas Toxo IgG (Biomérieux), the IMX Toxo IgG (Abbott), the Magia Toxoplasma gondii IgG (Merck). The mean values (+/- SD) obtained by IMX (25 IU +/- 68) and ES300 (45 IU +/- 142) were significantly lower than the values obtained by Vidas (73 IU +/- 237, p Magia (80 IU +/- 300, p < 10(-4) and p = 0.0005) and by PaC (70 IU +/- 260, p < 10(-4) and p = 0.0126). The correlations between PaC and Toxo IgG Boehringer, Biomérieux, Abbott, Merck were r = 0.97, r = 0.98, r = 0.94, r = 0.98, respectively. The correlation coefficients between the enzyme-immunoassays ranged from 0.96 to 0.99. All positive samples by PaC were found to be positive by enzyme-immunoassays except for eight sera which were doubtful positives by the Enzymum test ToxoIgG from Boehringer. No negative sample by PaC was found positive by any of the enzyme-immunoassays. In PaC, when two latex preparations coated with different antigen were compared, the correlation was rather weak (r = 0.93) suggesting that the selection of the antigen can be critical. In conclusion, the four automated commercial immunoassays now available gave similar results. However, the discrepancies observed in this study underlined the importance of clinical and biological follow-up of the patients and the necessity to confirm the result. The introduction of a new technique such as PaC, which is now available for a large variety of assays in Clinical Chemistry and Microbiology, is justified by its intrinsic advantage of homogeneity. Therefore, automation is easy as well as the control of possible interference.

  4. Evaluation of Correlation between Pretest Probability for Clostridium difficile Infection and Clostridium difficile Enzyme Immunoassay Results.

    Kwon, Jennie H; Reske, Kimberly A; Hink, Tiffany; Burnham, C A; Dubberke, Erik R


    The objective of this study was to evaluate the clinical characteristics and outcomes of hospitalized patients tested for Clostridium difficile and determine the correlation between pretest probability for C. difficile infection (CDI) and assay results. Patients with testing ordered for C. difficile were enrolled and assigned a high, medium, or low pretest probability of CDI based on clinical evaluation, laboratory, and imaging results. Stool was tested for C. difficile by toxin enzyme immunoassay (EIA) and toxigenic culture (TC). Chi-square analyses and the log rank test were utilized. Among the 111 patients enrolled, stool samples from nine were TC positive and four were EIA positive. Sixty-one (55%) patients had clinically significant diarrhea, 19 (17%) patients did not, and clinically significant diarrhea could not be determined for 31 (28%) patients. Seventy-two (65%) patients were assessed as having a low pretest probability of having CDI, 34 (31%) as having a medium probability, and 5 (5%) as having a high probability. None of the patients with low pretest probabilities had a positive EIA, but four were TC positive. None of the seven patients with a positive TC but a negative index EIA developed CDI within 30 days after the index test or died within 90 days after the index toxin EIA date. Pretest probability for CDI should be considered prior to ordering C. difficile testing and must be taken into account when interpreting test results. CDI is a clinical diagnosis supported by laboratory data, and the detection of toxigenic C. difficile in stool does not necessarily confirm the diagnosis of CDI. Copyright © 2017 American Society for Microbiology.

  5. Magnetic-particle-based, ultrasensitive chemiluminescence enzyme immunoassay for free prostate-specific antigen.

    Liu, Ruping; Wang, Cheng; Jiang, Quan; Zhang, Wei; Yue, Zhao; Liu, Guohua


    We report a magnetic-particle (MMP)-based chemiluminescence enzyme immunoassay (CLEIA) for free prostate-specific antigen (f-PSA) in human serum. In this method, the f-PSA is sandwiched between the anti-PSA antibody coated MMPs and alkaline phosphatase (ALP)-labeled anti-f-PSA antibody. The signal produced by the emitted photons from the chemiluminescent substrate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2'-adamantane)) is directly proportional to the amount of f-PSA in a sample. The present MMP-based assay can detect f-PSA in the range of 0.1-30 ng mL(-1) with the detection limit of 0.1 ng mL(-1). The linear detection range could match the concentration range within the "diagnostic gray zone" of serum f-PSA levels (4-10 ng mL(-1)). The detection limit was sufficient for measuring clinically relevant f-PSA levels (>4 ng mL(-1)). Furthermore, the method was highly selective; it was unaffected by cross-reaction with human glandular kallikrein-2, a kallikrein-like serine protease that is 80% similar to f-PSA. The proposed method was finally applied to determine f-PSA in 40 samples of human sera. Results obtained using the method showed high correlation with those obtained using a commercially available microplate CLEIA kit (correlation coefficient, 0.9821). This strategy shows great potential application in the fabrication of diagnostic kits for determining f-PSA in serum.

  6. Power-free chip enzyme immunoassay for detection of prostate specific antigen (PSA) in serum.

    Adel Ahmed, Heba; Azzazy, Hassan M E


    A power-free, portable "Chip EIA" was designed to render the popular Enzyme Linked Immunosorbent Assay (ELISA) more suitable for point-of-care testing. A number of microfluidic platforms have enabled miniaturization of the conventional microtitre plate ELISA, however, they require external pumping systems, valves, and electric power supply. The Chip EIA platform has eliminated the need for pumps and valves through utilizing a simple permanent magnet and magnetic nanoparticles. The magnetic nanoparticles act as solid support to capture the target and are then moved through chambers harboring different reagents necessary to perform a sandwich ELISA. The use of magnetic nanoparticles increases the volume-to-surface ratio reducing the assay time to 30 min. Changing the color of horseradish peroxidase (HRP) substrate to green indicates a positive result. In addition, a quantitative read-out was obtained through the use of cellphone camera imaging and analyzing the images using Matlab®. Cell phones, including smart ones, are readily available almost everywhere. The Chip EIA device was used to assay total prostate specific antigen (tPSA) in 19 serum samples. The PSA Chip EIA was tested for accuracy, precision, repeatability, and the results were correlated to the commercial Beckman Colter, Hybritech immunoassay® for determination of tPSA in serum samples with a Pearson correlation coefficient (R(2)=0.96). The lower detection limit of the PSA Chip EIA was 3.2 ng/mL. The assay has 88.9% recovery and good reproducibility (% CV of 6.5). We conclude that the developed Chip EIA can be used for detection of protein biomarkers in biological specimens.

  7. Detection of virus-specific intrathecally synthesised immunoglobulin G with a fully automated enzyme immunoassay system

    Weissbrich Benedikt


    Full Text Available Abstract Background The determination of virus-specific immunoglobulin G (IgG antibodies in cerebrospinal fluid (CSF is useful for the diagnosis of virus associated diseases of the central nervous system (CNS and for the detection of a polyspecific intrathecal immune response in patients with multiple sclerosis. Quantification of virus-specific IgG in the CSF is frequently performed by calculation of a virus-specific antibody index (AI. Determination of the AI is a demanding and labour-intensive technique and therefore automation is desirable. We evaluated the precision and the diagnostic value of a fully automated enzyme immunoassay for the detection of virus-specific IgG in serum and CSF using the analyser BEP2000 (Dade Behring. Methods The AI for measles, rubella, varicella-zoster, and herpes simplex virus IgG was determined from pairs of serum and CSF samples of patients with viral CNS infections, multiple sclerosis and of control patients. CSF and serum samples were tested simultaneously with reference to a standard curve. Starting dilutions were 1:6 and 1:36 for CSF and 1:1386 and 1:8316 for serum samples. Results The interassay coefficient of variation was below 10% for all parameters tested. There was good agreement between AIs obtained with the BEP2000 and AIs derived from the semi-automated reference method. Conclusion Determination of virus-specific IgG in serum-CSF-pairs for calculation of AI has been successfully automated on the BEP2000. Current limitations of the assay layout imposed by the analyser software should be solved in future versions to offer more convenience in comparison to manual or semi-automated methods.


    鄢盛恺; 林其燧; 宋耀虹; 王树琴


    Objective. To evaluate the clinical utility of a new non-invasive enzyme immunoassay(EIA) for the diagnosis of Helicobacter pylori (H.pylori) infection. Methods. Stool specimens of 63 patients were collected and tested by using a commercial kit for detecting Helicobacter pylori stool antigen (HpSA), of which 61 patients also underwent 13C-Urea breath test (13C-UBT). The tissue samples of 31 patients were obtained endoscopically and were examined with histologic technique (Warthin-Starry silver stain).Regarded 13C-UBT as a golden standard, HpSA test and histologic techniques were evaluated. Using this method,we also investigated the positive rate of H.Pylori infection in children in Beijing.Results.The sensitivity and specificity of HpSA test were 94.7% and 95.1% respectively; the positive and negative predictive values were 97.3% and 91.7% respectively; and the accuracy was 95.1%.The results showed the prevalence of H.pylori infection was 26.0% in children (3~18 years) of district of Xicheng in Beijing. After treatment, HpSA seems to disappear rapidly(3~5 days) from the feces. Conclusion. The detection of HpSA in stool samples by HpSA test is a rapid noninvasive test for detecting H.pylori infection, and has both high sensitivity and high specificity. It is suitable for screening and diagnosis of H.pylori infection, monitoring the treatment efficacy in routine in all hospitals.

  9. Retrospective review of dot enzyme immunoassay test for typhoid fever in an endemic area.

    Jackson, A A; Ismail, A; Ibrahim, T A; Kader, Z S; Nawi, N M


    Typhoid fever remains a common problem in Malaysia, but for its diagnosis both blood culture and the Widal test have drawbacks. A dot enzyme immunoassay (EIA) has been developed which detects IgM and IgG antibodies to a specific 50 kDa outer membrane protein on Salmonella typhi. This study was performed among outpatients attending the university hospital in Kelantan, a state on the east coast of Peninsular Malaysia where typhoid is endemic. The dot EIA was done on 149 outpatients of all ages in whom typhoid was suspected. Of these, 60 were not analysable due to insufficient data. The other 89 were retrospectively classed as typhoid (total = 21), or not typhoid (total = 68). The criteria for diagnosis of typhoid was either, blood culture was positive, or with blood culture negative, temperature was at least 38 degrees C and Widal O and/or H titer greater than or equal to 1/160. We then compared the diagnosis with the EIA result. For the result where either IgM or IgG was positive, sensitivity was 90%, specificity 91% and negative predictive value 97%. For IgM positive, specificity was 100%. But the specificity of IgG positive alone was reduced by six false positives, which were probably due to persistence of IgG after acute infection. Other cases were found where IgG positive alone appeared in the first week of typhoid fever, probably due to rapid response in a second or subsequent infection. We also found that IgM-producing patients were significantly younger than those showing IgG alone positive.

  10. Comparison of murex single-use diagnostic system with traditional enzyme immunoassay for detection of exposure to human immunodeficiency virus.

    Martin, Christin A; Keren, David F


    Because a retrospective study detected 13 negative Western blots out of 38 single-use diagnostic system (SUDS)-positive cases over a 1-year period, we performed a prospective study to compare the performance of the SUDS test with that of enzyme immunoassay (EIA). Of 888 SUDS-tested sera, 875 (98.4%) were both SUDS and EIA negative and 5 (0.6%) were SUDS, EIA, and Western blot positive. The rate of SUDS-positive samples decreased from 3.16/month in the retrospective study to 1.33/month in the prospective study. The immunoassays had sensitivities and specificities of 100 and 99.7 (SUDS) and 100 and 99.4% (traditional EIA), respectively. In laboratories with experienced personnel, the SUDS test performs as well as the EIA as a screen for infection with the human immunodeficiency virus.

  11. Solid-phase peptide synthesis

    Jensen, Knud Jørgen


    This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective.......This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective....

  12. Evaluation of a homogenous enzyme immunoassay for the detection of synthetic cannabinoids in urine

    Barnes, Allan J.; Young, Sheena; Spinelli, Eliani; Martin, Thomas M.; Klette, Kevin L.; Huestis, Marilyn A.


    Introduction The recent emergence and widespread availability of many new synthetic cannabinoids support the need for an accurate and high-throughput urine screen for these new designer drugs. We evaluated performance of the immunalysis homogeneous enzyme immunoassay (HEIA) to sensitively, selectively, and rapidly identify urinary synthetic cannabinoids. Methods 2443 authentic urine samples were analyzed with the HEIA that targets JWH-018 N-pentanoic acid, and a validated LC-MS/MS method for 29 synthetic cannabinoids and metabolites. Semiquantitative HEIA results were obtained, permitting performance evaluation at and around three cutoffs (5, 10 and 20 μg/L), and diagnostic sensitivity, specificity and efficiency determination. Performance challenges at ±25 and ±50% of each cutoff level, cross-reactivity and interferences also were evaluated. Results Sensitivity, specificity, and efficiency of the immunalysis HEIA K2 Spice kit with the manufacturer's recommended 10 μg/L cutoff were 75.6%, 99.6% and 96.8%, respectively, as compared to the reference LC-MS/MS method with limits of detection of 0.1 -10 μg/L. Performance at 5 μg/L was 92.2%, 98.1% and 97.4%, and for the 20 μg/L cutoff were 62.9%, 99.7% and 95.4%. Semi-quantitative results for in-house prepared standards were obtained from 2.5-30 μg/L, and documented acceptable linearity from 5-25 μg/L, with inter-day imprecision <30% (n = 17). Thirteen of 74 synthetic cannabinoids evaluated were classified as highly cross-reactive (≥50% at 10 μg/L); 4 showed moderate cross-reactivity (10–50% at 10 μg/L), 30 low cross-reactivity (<10% at 500 μg/L), and 27 <1% cross-reactivity at 500 μg/L. There was no interference from 102 investigated compounds. Only a mixture containing 1000 μg/L each of buprenorphine/norbuprenorphine produced a positive result above our proposed cutoff (5 μg/L) but below the manufacturer's recommended cutoff concentration (10 μg/L). Conclusion The Immunalysis HEIA K2 Spice kit



    The solid-phase synthesis of isoxazolines on 2-polystyrylsulfonamidoethanol resin isreported. 2-Polystyrylsuifonamidoethanol resin 1 was reacted with acryloyl chloride to afford2-polystyrylsulfonylamidoethyl acrylate resin 2, which was further reacted with brominatedaldoximes by [3+2] cycioaddition to give isoxazoline resin 4. Resin 4 was treated with aqueous 6mol/L HCI solution to obtain isoxazolines in good yield and purity.

  14. Multiple solid-phase microextraction

    Koster, EHM; de Jong, GJ


    Theoretical aspects of multiple solid-phase microextraction are described and the principle is illustrated with the extraction of lidocaine from aqueous solutions. With multiple extraction under non-equilibrium conditions considerably less time is required in order to obtain an extraction yield that


    Talat Mokhtari Azad; Anahid Ehteda; Parvin Yavari; R Hamkar; Zahra Safar Pour; M. Essalat Rakhsheh Nategh


    Laboratory diagnosis of acute measles is usually achieved by serology assays for measle-specific IgM antibody. For comparison of measle-specific IgM antibody in saliva and serum, 95 paired blood and saliva samples were collected 1-14 days after the onset of rash. The specimens were tested for specific IgM antibody by an IgM antibody-capture Enzyme Immunoassay (EIA). Measles IgM antibody was detected in 89 (93.7%) of serum samples and in 85(89.5%) of saliva specimens. Of the 6(6.3%) serum samp...

  16. The evolution of pretransfusion testing: from agglutination to solid-phase red cell adherence tests.

    Plapp, F V; Sinor, L T; Rachel, J M


    Hospital transfusion services and blood centers still use manual hemagglutination tests for most of their serological procedures. Automation of hemagglutination reactions has proven to be difficult, primarily because hemagglutination lacks an objective endpoint which can be easily interpreted by inexpensive instruments. Alternatively, solid-phase red cell adherence assays for ABO cell and serum grouping, Rh typing, red cell and platelet antibody screening, red cell and platelet crossmatching, IgA deficiency screening, hepatitis B surface antigen, and HIV antibody screening have been developed. The performance of these assays compares favorably with current hemagglutination and enzyme immunoassay methods. All of these tests share a common objective endpoint of adherence or nonadherence of indicator red cells. This uniformity allows easy interpretation of results visually, spectrophotometrically, or by image analysis. The latter technique has the potential to revolutionize the reading and interpretation of all agglutination tests. Solid-phase red cell adherence tests in microplates are ideal for batch processing large numbers of specimens. However, adherence tests are not restricted to this format. Therefore, blood grouping dipsticks have been produced, which permit testing of individual blood samples even outside of the laboratory.

  17. Triclosan and methyl-triclosan monitoring study in the northeast of Spain using a magnetic particle enzyme immunoassay and confirmatory analysis by gas chromatography mass spectrometry

    Kantiani, Lina; Farré, Marinella; Asperger, Danijela; Rubio, Fernando; González, Susana; López de Alda, Maria J.; Petrović, Mira; Shelver, Weilin L.; Barceló, Damià


    SummaryFor the first time, the occurrence of triclosan and its metabolite methyl-triclosan was investigated in a typical Mediterranean area using a two-step methodology based on screening using a magnetic particle immunoassay (IA) and confirmatory analysis by solid phase extraction (SPE) followed by gas chromatography-mass spectrometry (GC-MS). In this study, 95 environmental samples were analyzed. A commercial immunoassay was assessed for use in the different types of water selected for this study. A large monitoring study was performed on the influent and the effluent of eight wastewater treatment plants (WWTPs), water samples from Ebro and Llobregat rivers, and drinking water. All wastewater samples tested in this study (influents and effluents) showed the presence of triclosan, with concentrations for raw influents being high (10 μg/L as average value). The percentages of triclosan removal for the WWTPs were evaluated (30-70%) along the different treatment processes showing that the best removal rates were obtained by the processes equipped with membrane bioreactors (MBRs). However, important concentrations of triclosan were detected even after treatment by MBRs. The presence of this biocide was confirmed in 50% of the river samples analyzed. Twenty two drinking water samples from the Barcelona city area were investigated, and in this case no triclosan was detected. Due to its properties and the widespread usage of triclosan, there is a need for monitoring and controlling the amounts present in wastewater effluents, river water, drinking water catchments areas, and drinking water. To this end, we present a feasible methodology using a magnetic particle-based immunoassay as a screening, followed by confirmatory analysis using solid phase extraction-gas chromatography-mass spectrometry (SPE-GC-MS).

  18. Multiplexed Electrochemical Immunoassay of Phosphorylated Proteins Based on Enzyme-Functionalized Gold Nanorod Labels and Electric Field-Driven Acceleration

    Du, Dan; Wang, Jun; Lu, Donglai; Dohnalkova, Alice; Lin, Yuehe


    A multiplexed electrochemical immunoassay integrating enzyme amplification and electric field-driven strategy was developed for fast and sensitive quantification of phosphorylated p53 at Ser392 (phospho-p53 392), Ser15 (phospho-p53 15), Ser46 (phospho-p53 46) and total p53 simultaneously. The disposable sensor array has four spatially separated working electrodes and each of them is modified with different capture antibody, which enables simultaneous immunoassay to be conducted without cross-talk between adjacent electrodes. The enhanced sensitivity was achieved by multi-enzymes amplification strategy using gold nanorods (AuNRs) as nanocarrier for co-immobilization of horseradish peroxidase (HRP) and detection antibody (Ab2) at high ratio of HRP/Ab2, which produced an amplified electrocatalytic response by the reduction of HRP oxidized thionine in the presence of hydrogen peroxide. The immunoreaction processes were accelerated by applying +0.4 V for 3 min and then -0.2 V for 1.5 min, thus the whole sandwich immunoreactions could be completed in less than 5 min. The disposable immunosensor array shows excellent promise for clinical screening of phosphorylated proteins and convenient point-of-care diagnostics.

  19. Combined liquid and solid-phase extraction improves quantification of brain estrogen content

    Andrew eChao


    Full Text Available Accuracy in quantifying brain-derived steroid hormones (‘neurosteroids’ has become increasingly important for understanding the modulation of neuronal activity, development, and physiology. Relative to other neuroactive compounds and classical neurotransmitters, steroids pose particular challenges with regard to isolation and analysis, owing to their lipid solubility. Consequently, anatomical studies of the distribution of neurosteroids have relied primarily on the expression of neurosteroid synthesis enzymes. To evaluate the distribution of synthesis enzymes vis-à-vis the actual steroids themselves, traditional steroid quantification assays, including radioimmunoassays (RIA, have successfully employed liquid extraction methods (e.g., ether, dichloromethane or methanol to isolate steroids from microdissected brain tissue. Due to their sensitivity, safety and reliability, the use of commercial enzyme immunoassays (EIA for laboratory quantification of steroids in plasma and brain has become increasingly widespread. However, EIAs rely on enzymatic reactions in vitro, making them sensitive to interfering substances in brain tissue and thus producing unreliable results. Here, we evaluate the effectiveness of a protocol for combined, two-stage liquid/solid phase extraction as compared to conventional liquid extraction alone for the isolation of estradiol (E2 from brain tissue. We employ the songbird model system, in which brain steroid production is pronounced and linked to neural mechanisms of learning and plasticity. This study outlines a combined liquid-solid phase extraction protocol that improves the performance of a commercial EIA for the quantification of brain E2 content. We demonstrate the effectiveness of our optimized method for evaluating the region specificity of brain E2 content, compare these results to established anatomy of the estrogen synthesis enzyme and estrogen receptor, and discuss the nature of potential EIA interfering

  20. Microfluidic Platform for Enzyme-Linked and Magnetic Particle-Based Immunoassay

    Dorota G. Pijanowska


    Full Text Available This article presents design and testing of a microfluidic platform for immunoassay. The method is based on sandwiched ELISA, whereby the primary antibody is immobilized on nitrocelluose and, subsequently, magnetic beads are used as a label to detect the analyte. The chip takes approximately 2 h and 15 min to complete the assay. A Hall Effect sensor using 0.35-μm BioMEMS TSMC technology (Taiwan Semiconductor Manufacturing Company Bio-Micro-Electro-Mechanical Systems was fabricated to sense the magnetic field from the beads. Furthermore, florescence detection and absorbance measurements from the chip demonstrate successful immunoassay on the chip. In addition, investigation also covers the Hall Effect simulations, mechanical modeling of the bead–protein complex, testing of the microfluidic platform with magnetic beads averaging 10 nm, and measurements with an inductor-based system.


    SUNWeimin; LUOJuntao; 等


    The solid-phase synthesis of isoxazolines on 2-polystyrylsulfonamidoethanol resin is reported.2-Polystyrylsulfonamidoethanol resin 1 was reacted with acryloyl chloride to afford 2-polystyrylsulfonylamidoethyl acrylate resin 2,which was further reacted with brominated aldoximes by [3+2] cycloaddition to give isoxazoline resin 4.Resin 4 was treated with aqueous 6 mol/L HCl solution to obtain isoxazolines in good yield and purity.

  2. A single-step enzyme immunoassay capillary sensor composed of functional multilayer coatings for the diagnosis of marker proteins.

    Funano, Shun-ichi; Sugahara, Masato; Henares, Terence G; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki


    A single-step, easy-to-use enzyme immunoassay capillary sensor, composed of functional multilayer coatings, was developed in this study. The coatings were composed of substrate-immobilized hydrophobic coating, hydrogel coating, and soluble coating containing an enzyme-labeled antibody. The response mechanism involved a spontaneous immunoreaction triggered by capillary action-mediated introduction of a sample antigen solution and subsequent separation of unreacted enzyme-labeled antibodies and antigen-enzyme-labeled antibody complexes by the molecular sieving effect of the hydrogel. An enzyme reaction at the substrate-immobilized hydrophobic coating/hydrogel coating interface resulted in a protein-selective fluorescence response. An antigen concentration-dependent response was obtained for diagnostic marker protein samples (hemoglobin A1c (HbA1c), 7.14-16.7 mg mL(-1); alpha-fetoprotein (AFP), 1.4-140 ng mL(-1); C-reactive protein (CRP), 0.5-10 μg mL(-1)) that cover a clinically important concentration range. The successful measurement of CRP in diluted serum samples demonstrated the application of this capillary sensor.

  3. Improved methods for urinary atrazine mercapturate analysis-Assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE)

    Koivunen, Marja E. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Dettmer, Katja [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Vermeulen, Roel [Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Rockville, MD (United States); Bakke, Berit [Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Rockville, MD (United States); National Institute of Occupational Health, Oslo (Norway); Gee, Shirley J. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Hammock, Bruce D. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States)]. E-mail:


    Elimination of interfering substances in urine by solid phase extraction (SPE) prior to analysis resulted in 10-fold improvement in the sensitivity of atrazine mercapturate (AM) enzyme-linked immunosorbent assay (ELISA) compared to previous reports. Of the two tested SPE systems, Oasis[reg] HLB and MCX, the mixed-mode MCX gave good recoveries (82%) of AM in spiked samples measured by ELISA, whereas the reverse-phase HLB phase was not compatible with the immunochemical method. At relatively high concentrations of urinary AM (>20 ng mL{sup -1}), sample dilution was effective enough for the elimination of interfering substances. The new liquid chromatography-mass spectrometry (LC-MS) method developed for AM utilizes online-SPE with Oasis[reg] HLB, column switching and a stable-isotope internal standard. The limit of quantification (0.05 ng mL{sup -1}) indicates improved sensitivity compared with most previously published LC-MS methods for AM. Validation of all three methods, LC-MS, ELISA + SPE and ELISA + dilution with spiked urine samples showed good correlation between the known and measured concentrations with R {sup 2} values of 0.996, 0.957 and 0.961, respectively. When a set (n = 70 plus 12 blind duplicates) of urine samples from farmers exposed to atrazine was analyzed, there was a good agreement (R {sup 2} = 0.917) between the log normalized data obtained by ELISA + SPE and LC-MS. High correlation among the data obtained by the two tested methods and the LC-MS method by the Center of Disease Control and Prevention (CDC), together with low variability among the blind duplicates, suggests that both methods reported here would be suitable for the analysis of urinary AM as a biomarker for human exposure of atrazine.

  4. Comparison of automated chemiluminescence immunoassays with capture enzyme immunoassays for the detection of measles and mumps IgM antibodies in serum.

    Haywood, Becky; Patel, Mauli; Hurday, Samantha; Copping, Ruth; Webster, Daniel; Irish, Dianne; Haque, Tanzina


    Outbreaks of measles and mumps occur regularly in the UK. Rapid diagnosis of acute infection is important for both infection control and epidemiological purposes. The objective of this study was to compare the performance of an automated platform (DiaSorin Liaison(®), Saluggia, Italy) with a manual capture enzyme immunoassay (EIA; Microimmune, Hounslow, UK) for the detection of measles and mumps IgM antibodies in serum from symptomatic individuals. Ninety sera tested previously for measles (n=50) and mumps (n=40) IgM using the manual EIA were tested retrospectively using the DiaSorin Liaison(®) and the results compared. Sensitivity, specificity, inter-assay variability and intra-assay variability of the Liaison(®) assays were calculated. Sensitivity and specificity of the Liaison(®) assay for measles IgM were 92% and 100% respectively, with inter-assay variation of 14.1% and intra-assay variation of 12.5%. The sensitivity and specificity of the mumps IgM Liaison(®) assay were 88% and 95% respectively, with an inter-assay and intra-assay variation of 13.9% and 5.3% respectively. Both the measles and mumps IgM Liaison(®) assays gave fewer equivocal results than the EIA. Neither Liaison(®) IgM assay showed any cross-reactivity with sera positive against other viruses, however the measles IgM EIA cross-reacted with parvovirus IgM. The automated Liaison(®) assays are more specific, cheaper and less labour-intensive compared to the manual EIA. The Liaison(®) assays benefit from reduced number of equivocal results compared to the EIA for both measles and mumps IgM. This allows clinical decisions to be made accurately and in a timely manner.

  5. Chemiluminescence enzyme immunoassay based on magnetic nanoparticles for detection of hepatoceUular carcinoma marker glypican-3

    Qian-Yun Zhang; Hui Chen; Zhen Lin; Jin-Ming Lin


    Glypican-3 (GPC3) is reported as a great promising tumor marker for hepatocellular carcinoma (HCC) diagnosis. Highly sensitive and accurate analysis of serum GPC3 (sGPC3), in combination with or instead of traditional HCC marker alpha-fetoprotein (AFP), is essential for early diagnosis of I-ICC. Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay. Here, the magnetic nanoparticles (MnPs) and magnetic microparticles (MmPs) with carboxyl groups were further modified with streptavidin, and applied for the development of chemiluminescence enzyme immunoassay (CLEIA). After comparing between MnPs- and MmPs-based CLEIA, MnPs-based CLEIA was proved to be a better method with less assay time, greater sensitivity, better linearity and longer chemiluminescence platform. MnPs-based CLEIA was applied for detection of sGPC3 in normal liver, hepatocirrhosis, secondary liver cancer and HCC serum samples. The results indicated that sGPC3 was effective in diagnosis of HCC with high performance.

  6. Development and characterization of an enzyme-immunoassay with polyclonal antisera for benzene, toluene, and xylenes (BTX); Entwicklung und Charakterisierung eines Enzym-Immunoassays mit polyklonalen Antikoerpern fuer Benzol, Toluol und Xylole (BTX)

    Beyer, K.; Knopp, D.; Niessner, R. [Technische Univ. Muenchen (Germany). Lehrstuhl fuer Hydrogeologie, Hydrochemie und Umweltanalytik


    An indirect competitive enzyme immunoassay for the detection of the volatile organic compounds benzene, toluene, and xylenes has been developed with polyclonal antibodies. The limit of determination for the equal amount of the five analytes in water was 210 {mu}g/l with a center point value of 1.7 mg/l. The addition of 10% of dimethyl sulfoxide (DMSO) to the sample decreased the limit of determination of 80 {mu}g/l and the center point value to 540 {mu}g/l. The specificity of the polyclonal antibodies was investigated based on its cross-reactivity. The influence of increasing concentrations of organic solvents and humic acid on the sensitivity of the antibodies was studied. Water samples were analysed both with GC-FID and by an immunochemical method in order to evaluate the suitability of the assay for environmental analysis. (orig.) [Deutsch] Zur Bestimmung von Benzol, Toluol und den drei Xylol-Isomeren (BTX) wird ein indirekter kompetitiver Enzym-Immunoassay auf der Basis von polyklonalen Antikoerpern beschrieben. Die Bestimmungsgrenze in Wasser liegt bei 210 {mu}g/l und der Testmittelpunkt der sigmoiden Kalibrierkurve bei 1,7 mg/l fuer ein aequivalentes Volumengemisch der fuenf Einzelverbindungen. Durch Loesemittelzusatz, zum Beispiel 10% Dimethylsulfoxid (DMSO), laesst sich die Bestimmungsgrenze auf 80 {mu}g/l und der Testmittelpunkt auf 540 {mu}g/l verringern. Die Spezifitaet der Antikoerper wurde durch Bestimmung von Kreuzreaktionen ermittelt. Weiterhin wurde der Einfluss unterschiedlicher Loesemittelgehalte und von Huminsaeure auf die Affinitaet der Antikoerper im Hinblick auf die Anwendung fuer Realproben untersucht. Um die Eignung des Tests fuer die Routineanalytik zu pruefen, wurde parallel zur immunochemischen Methode eine gaschromatographische Bestimmung BTX in Wasserproben durchgefuehrt. (orig.)

  7. Preparation of peanut butter suspension for determination of peanuts using enzyme-linked immunoassay kits.

    Trucksess, Mary W; Brewer, Vickery A; Williams, Kristina M; Westphal, Carmen D; Heeres, James T


    Peanuts are one of the 8 most common allergenic foods and a large proportion of peanut-allergic individuals have severe reactions, some to minimal exposure. Specific protein constituents in the peanuts are the cause of the allergic reactions in sensitized individuals who ingest the peanuts. To avoid accidental ingestion of peanut-contaminated food, methods of analysis for the determination of the allergenic proteins in foods are important tools. Such methods could help identify foods inadvertently contaminated with peanuts, thereby reducing the incidence of allergic reactions to peanuts. Commercial immunoassay kits are available but need study for method performance, which requires reference materials for within- and between-laboratory validations. In this study, National Institute of Standards and Technology Standard Reference Material 2387 peanut butter was used. A polytron homogenizer was used to prepare a homogenous aqueous Peanut Butter suspension for the evaluation of method performance of some commercially available immunoassay kits such as Veratox for Peanut Allergen Test (Neogen Corp.), Ridascreen Peanut (R-Biopharm GmbH), and Bio-Kit Peanut Protein Assay Kit (Tepnel). Each gram of the aqueous peanut butter suspension contained 20 mg carboxymethylcellulose sodium salt, 643 microg peanut, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. The suspension was homogenous, stable, reproducible, and applicable for adding to ice cream, cookies, breakfast cereals, and chocolate for recovery studies at spike levels ranging from 12 to 90 microg/g.

  8. Solid phase syntheses of oligoureas

    Burgess, K.; Linthicum, D.S.; Russell, D.H.; Shin, H.; Shitangkoon, A.; Totani, R.; Zhang, A.J.; Ibarzo, J. [Texas A& M Univ., College Station, TX (United States)


    Isocyanates 7 were formed from monoprotected diamines 3 or 6, which in turn can be easily prepared from commercially available N-BOC- or N-FMOC-protected amino acid derivatives. Isocyanates 7, formed in situ, could be coupled directly to a solid support functionalized with amine groups or to amino acids anchored on resins using CH{sub 2}Cl{sub 2} as solvent and an 11 h coupling time at 25 {degree}C. Such couplings afforded peptidomimetics with an N-phthaloyl group at the N-terminus. The optimal conditions identified for removal of the N-phthaloyl group were to use 60% hydrazine in DMF for 1-3 h. Several sequences of amino acids coupled to ureas (`peptidic ureas`) and of sequential urea units (`oligoureas`) were prepared via solid phase syntheses and isolated by HPLC. Partition coefficients were measured for two of these peptidomimetics, and their water solubilities were found to be similar to the corresponding peptides. A small library of 160 analogues of the YGGFL-amide sequence was prepared via Houghten`s tea bag methodology. This library was tested for binding to the anti-{beta}-endorphin monoclonal antibody. Overall, this paper describes methodology for solid phase syntheses of oligourea derivatives with side chains corresponding to some of the protein amino acids. The chemistry involved is ideal for high-throughput syntheses and screening operations. 51 refs., 3 figs., 2 tabs.

  9. Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies

    Tiel, F.H. van; Kraaijeveld, C.A.; Baller, J.; Harmsen, T.; Oosterlaken, T.A.M.; Snippe, H.


    Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates. This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for

  10. An inhibition enzyme immunoassay, using a human monoclonal antibody (K14) reactive with gp41 of HIV-1, for the serology of HIV-1 infections.

    V.J.P. Teeuwsen; J.J. Schalken; G. van der Groen (Guido); R. van den Akker (Ruud); J. Goudsmit (Jaap); A.D.M.E. Osterhaus (Albert)


    textabstractAn inhibition enzyme immunoassay (IEIA), using a human monoclonal antibody (K14) reactive with gp41 of HIV-1, was evaluated for its applicability to the serology of HIV-1 infections. Using panels of serum samples from seronegative and confirmed HIV-1-seropositive individuals, it was show

  11. Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies

    Tiel, F.H. van; Kraaijeveld, C.A.; Baller, J.; Harmsen, T.; Oosterlaken, T.A.M.; Snippe, H.


    Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates. This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for det

  12. A Comparison of Anti-Nuclear Antibody Quantification Using Automated Enzyme Immunoassays and Immunofluorescence Assays

    Baronaite, Renata; Engelhart, Merete; Mørk Hansen, Troels;


    Anti-nuclear antibodies (ANA) have traditionally been evaluated using indirect fluorescence assays (IFA) with HEp-2 cells. Quantitative immunoassays (EIA) have replaced the use of HEp-2 cells in some laboratories. Here, we evaluated ANA in 400 consecutive and unselected routinely referred patients...... patients were compared. The majority of the results were the same between the two methods (n = 325, 84%); however, 8% (n = 30) yielded equivocal results (equivocal-negative and equivocal-positive) and 8% (n = 31) yielded divergent results (positive-negative). The results showed fairly good agreement......, with Cohen's kappa value of 0.30 (95% confidence interval (CI) = 0.14-0.46), which decreased to 0.23 (95% CI = 0.06-0.40) when the results for dsDNA were omitted. The EIA method was less reliable for assessing nuclear and speckled reactivity patterns, whereas the IFA method presented difficulties detecting...

  13. Evaluation of a Newly Formulated Enzyme Immunoassay for the Detection of Hydrocodone and Hydromorphone in Pain Management Compliance Testing.

    Nascimento, Renata; Poklis, Alphonse; Wolf, Carl E


    A new Hydrocodone Enzyme Immunoassay (HEIA; Lin-Zhi International, Inc.) was evaluated for the detection of hydrocodone and its main metabolite, hydromorphone. All specimens were tested with two different cutoff calibrators, 100 and 300 ng/mL, on an ARCHITECT Plus c4000 Clinical Chemistry Analyzer. Controls containing -25% (negative control) and +25% (positive control) of the cutoff calibrators and a drug-free control were analyzed with each batch. All 1,025 urine specimens were previously analyzed by ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS-MS) for opiates. Approximately, 33% (337/1,019) of the specimens yielded positive results by the HEIA assay at a cutoff concentration of 100 ng/mL and 19% (190/1,025) yielded positive results at the 300 ng/mL cutoff concentration. Of these presumptive positive specimens, UPLC-MS-MS confirmed the presence of hydrocodone and/or hydromorphone >100 ng/mL in 241 specimens and >300 ng/mL in 162 specimens, for each respective cutoff. With the 100 ng/mL cutoff, the HEIA demonstrated a sensitivity of 0.959, a specificity of 0.846 and an overall agreement with the UPLC-MS-MS of 87%. At 300 ng/mL cutoff, the HEIA demonstrated a sensitivity of 0.880, a specificity of 0.966 and an overall agreement of UPLC-MS-MS results of 95%. The Lin-Zhi HEIA 100 ng/mL cutoff assay demonstrated sensitivity for the detection of hydrocodone and hydromorphone in urine. The 300 ng/mL cutoff was less sensitive, but more selective, and should be part of an initial immunoassay screen, particularly in pain management compliance testing.

  14. Sensitive enzyme immunoassay for hepatitis B virus core-related antigens and their correlation to virus load.

    Kimura, Tatsuji; Rokuhara, Akinori; Sakamoto, Yoko; Yagi, Shintaro; Tanaka, Eiji; Kiyosawa, Kendo; Maki, Noboru


    A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 10(3) U/ml, all healthy control (HBsAg/HBV-DNA negative; n = 108) and anti-HCV antibody-positive (n = 59) sera were identified as negative. The assay showed a detection limit of 4 x 10(2) U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification (TMA) or HBeAg radio immunoassay (RIA) in the dilution test. HBcrAg concentrations correlated well with HBV-DNA TMA (r = 0.91, n = 29) and in-house real-time detection-PCR (r = 0.93, n = 47) in hepatitis B patients. On HBeAg/anti-HBe antibody seroconversion panels, the HBcrAg concentration changed in accordance with HBV-DNA levels. HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients.

  15. Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B{sub 1} detection in cereal

    Shu, Mei [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Xu, Yang, E-mail: [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Liu, Xing [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); College of Food Science and Technology, Hainan University, No. 58 Renmin Avenue, Haikou 570228 (China); Li, Yanping; He, Qinghua [Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Tu, Zhui [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Fu, Jinheng [Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Gee, Shirley J.; Hammock, Bruce D. [Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States)


    A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB{sub 1} was developed. The anti-idiotypic nanobody–alkaline phosphatase (Ab2β−Nb−AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB{sub 1} were 2.69 and 0.35 ng mL{sup −1}, respectively, with a linear range of 0.93–7.73 ng mL{sup −1}. The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL{sup −1}, and the IC{sub 50} was 0.89 ± 0.09 ng mL{sup −1} with a linear range of 0.29–2.68 ng mL{sup −1}. Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β−Nb−AP fusion protein based one-step competitive immunoassay for monitoring FB{sub 1} contamination in cereals. The Ab2β−Nb−AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems. - Highlights: • Ab2β−Nb−AP has the potential to replace chemically-coupled probes. • Ab2β−Nb−AP is homogeneous enzyme-labelled antigen can be prepared reproducibly. • We developed a green and rapid one-step competitive enzyme immunoassay. • The sensitivity of one-step CLIA was 9-folds higher than two-step ELISA.

  16. Sensitive and high-fidelity electrochemical immunoassay using carbon nanotubes coated with enzymes and magnetic nanoparticles.

    Piao, Yunxian; Jin, Zongwen; Lee, Dohoon; Lee, Hye-Jin; Na, Hyon-Bin; Hyeon, Taeghwan; Oh, Min-Kyu; Kim, Jungbae; Kim, Hak-Sung


    We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng(-1) mL(-1) and a detection limit of 0.19 ng mL(-1) (1.2 pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months.

  17. Non-radiometric immunoassays fluoroimmunoassay (FIA) and fluorometric enzyme immunoassay (FEIA) and radioimmunoassay (RIA) for evaluation of thyroid function in normal and hypothyroid dogs

    Jerico, M.M.; Larsson, C.E. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina Veterinaria e Zootecnia. Dept. de Clinica Medica]. E-mail:; Mendonca, B.B. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina . Lab. de Hormonios e Genetica Molecular; Otsuka, M. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina Veterinaria e Zootecnia. Hospital Veterinario; Maganin Junior, A. [Canil da Policia Militar do Estado de Sao Paulo, SP (Brazil)


    We proposed the comparison of thyroxine (T4) and free thyroxine (FT4) measurements by fluoroimmunoassay (FIA) and fluorometric enzyme immunoassay (FEIA) with radioimmunoassay (RIA) in thyroid function evaluation of normal (n=50) and hypothyroid dogs (n=9). T4 and FT4 serum concentrations were measured in basal conditions and 6 h after TRH stimulation (200 mug/IV). All our reference values are based on the 5th and 95th percentile. The reference values for basal T4 in healthy dogs were 0.50 to 2.35 mug/dL (FIA), 0.50 to 2.51 mug/dL (FEIA) and 0.35 to 0.74 mug/dL (RIA). After TRH, the values were >= 1.37 mug/dL (FIA), >= 0,26 mug/dL (FEIA) and >= 0.40 mug/dL (RIA). Basal FT4 values in healthy dogs were 0.65 to 2.20 ng/dL (FIA), 0.38 to 1.43 ng/dL (FEIA) and 0.10 to 1.24 ng/dL (RIA). After TRH, the values were >= 1.30 ng/dL (FIA), >= 0.77 ng/dL (FEIA) and >=0.50 ng/dL (RIA). In hypothyroid dogs, the mean +- SD for T4 in basal conditions and after TRH were 0.24 +- 0.20 mug/dL and 0.26 +- 0.20 mug/dL (FIA), 0.27 +- 0.12 mug/dL and 0.32 +- 0.51 mug/dL (FEIA) and 0.19 +- 0.30 mug/dL and 0,24 +- 0.09 mug/dL (RIA), respectively. In the same group the mean +- SD basal FT4 values and after TRH were 0.28 +- 0.33 ng/dL and 0.28 +- 0.39 ng/dL (FIA), 0,12 +- 0.26 ng/dL and 0.23 +- 0.56 ng/dL (FEIA) and 0,15 +- 0,15 ng/dL and 0,17 +- 0,28 ng/dL (RIA), respectively. Significant differences (p<0.05) between the normal and hypothyroid groups (Kruskall-Wallis test) were observed in the three methods and between basal and stimulated values in normal dogs (Wilcoxon test), by the three methods. The best sensitivity for diagnosing hypothyroidism was obtained through T4 values (100%), and the best specificity through FT4 values (100%), both determined by FIA after TRH stimulation. We conclude that T4 and FT4 measured by fluoroimmunoassay after TRH stimulation can be an excellent alternative. (author)

  18. Competitive chemiluminescent enzyme immunoassay for vitamin B12 analysis in human milk

    BACKGROUND Few accurate data exist on the concentration of vitamin B12 in human milk. Binding of the vitamin to haptocorrin (HC) can interfere with the assay if not removed by pretreatment, and very low values can occur in women with poor B12 status. This study evaluated two competitive enzyme bind...

  19. Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy.

    Capobiango, Jaqueline Dario; Pagliari, Sthefany; Pasquali, Aline Kuhn Sbruzzi; Nino, Beatriz; Ferreira, Fernanda Pinto; Monica, Thaís Cabral; Tschurtschenthaler, Nely Norder; Navarro, Italmar Teodorico; Garcia, João Luis; Mitsuka-Breganó, Regina; Reiche, Edna Maria Vissoci


    The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detect Toxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.

  20. Evaluation of two new enzyme immunoassay reagents for diagnosis of histoplasmosis in a cohort of clinically characterized patients.

    Zhang, Chen; Lei, Guang-Sheng; Lee, Chao-Hung; Hage, Chadi A


    The performance characteristics of the recently available analyte-specific reagent based enzyme immunoassay (ASR-EIA) and in vitro diagnostic (IVD) kit for urine Histoplasma antigen detection were evaluated in a cohort of 50 clinically characterized patients with histoplasmosis and 50 control patients. Overall sensitivity and specificity of the ASR-EIA were significantly improved compared with those of the IVD kit (sensitivity 72% vs. 22%, Phistoplasmosis (five with pulmonary histoplasmosis and nine with progressive disseminated histoplasmosis) were falsely negative by ASR-EIA. All 10 specimens from patients with severe symptoms of progressive disseminated histoplasmosis were positive by ASR-EIA, although the average reading value of these 10 specimens was not significantly different from that of others with positive results. Compared to the MiraVista antigen assay, both the IVD kit and the ASR-EIA were significantly less sensitive in detecting Histoplasma antigen in the urine of patients with histoplasmosis. The ASR-EIA and MiraVista assay had comparable specificity. In conclusion, the ASR-EIA has improved performance compared with the IVD kit in the detection of Histoplasma antigen in the urine. However, users should be aware of the potential for false negative results using the currently recommended cutoff value.

  1. Comparison of a recombinant-antigen enzyme immunoassay with Treponema pallidum hemagglutination test for serological confirmation of syphilis.

    Rodríguez, Islay; Alvarez, Elvio L; Fernández, Carmen; Miranda, Alina


    A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

  2. Comparison of a Recombinant-antigen Enzyme Immunoassay with Treponema pallidum Hemagglutination Test for Serological Confirmation of Syphilis

    Islay Rodríguez


    Full Text Available A recombinant-antigen enzyme immunoassay (EIA, BioSCREEN TM anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38, secondary (n = 10, early latent (n = 28 and congenital syphilis (n = 2, patients with leptospirosis ( n= 8, infectious mononucleosis (n = 7, hepatitis (n = 9, diabetes mellitus (n = 11, rheumatoid arthritis (n = 13, leprosy (n = 11, tuberculosis (n = 9, HIV/Aids ( n= 12, systemic lupus erythematosus (n = 4, rheumatic fever (n = 3, old-persons (n = 9, pregnant women (n = 29 and blood donors (n = 164. The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

  3. Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA)

    Leo, P; P. Ucelli; Augusto, EFP; Oliveira,MS; Tamashiro, WMSC


    The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatant...

  4. Development of ultrasensitive direct chemiluminescent enzyme immunoassay for determination of aflatoxin M1 in milk.

    Vdovenko, Marina M; Lu, Chuan-Chen; Yu, Feng-Yih; Sakharov, Ivan Yu


    A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detecting aflatoxin M1 (AFM1) was developed. To improve the sensitivity of the assay, a mixture of 3-(10'-phenothiazinyl)-propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) was used to enhance peroxidase-induced CL. The concentrations of the coating anti-AFM1 antibody and the conjugate of AFB1 with horseradish peroxidase the conditions of the chemiluminescent assay were varied to optimise the condition of the chemiluminescent assay. The lower detection limit values and dynamic working range of CL-ELISA of AFM1 were 0.001 ng mL(-1) and 0.002-0.0075 ng mL(-1), respectively. A 20-fold dilution of milk samples prevented a matrix effect of the milk and allowed measurement of AFM1 at concentrations below than the maximum acceptable limit. Values of recovery within and between assays were 81.5-117.6% and 86-110.6%, respectively. The results of using the developed CL-ELISA to analyse samples of six brands of milk that were purchased in Taiwan revealed that AFM1 was absent from all studied samples.

  5. [Development and application of indirect competitive enzyme immunoassay for detection of neomycin in milk].

    Burkin, M A; Gal'vidis, I A


    As a result of immunization of rabbits with neomycin B (N M) conjugated to periodate-oxidized transferrin, polyclonal antibodies were generated and used to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) of NM. Several heterologous conjugates, namely, glutaraldehyde (GA)-polymerized NM, gelatin-ribostamycin (sp), and gelatin-NM (ga) were used as coating antigens in different ELISA variants for quantification of NM in milk. These variants were characterized by different dynamic ranges and detection limits of 1.0, 0.1, and 0.01 ng/ml, respectively. Maximum residue level (MRL) of this antibiotic in milk accepted in the EU can be detected without any special pretreatment at a 100-fold sample dilution in the least sensitive assay variant. The mean recovery rate from NM-spiked milk containing 1.5-10% fat was 111.7% and ranged from 84 to 125.2%. We found that 57 of 106 tested milk samples contained NM at concentrations higher than 100 ng/ml. In ten percent of cases (11/1 06), the residual level of this antibiotic was greater than 500 ng/ml. In one case, the M RL was exceeded (1690 ng/ml). The assay developed in this study is specific shows no cross-reactivity with other veterinary aminoglycosides, has a good sensitivity reserve, and can serve as an effective tool to monitor the NM content in milk stuff.

  6. Development of an enzyme immunoassay using a monoclonal antibody against the psychoactive diterpenoid salvinorin A.

    Paudel, Madan Kumar; Shirota, Osamu; Sasaki-Tabata, Kaori; Tanaka, Hiroyuki; Sekita, Setsuko; Morimoto, Satoshi


    Salvinorin A (1), the main active constituent in Salvia divinorum, is a highly selective kappa-opioid receptor agonist with hallucinogenic effects, which is regulated in several countries. In the present study, a monoclonal antibody (mAb) against 1 was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) system was developed for the detection of salvinorins. To raise mAbs against 1, salvinorin B (2) hemisuccinate was synthesized and used to prepare the immunogen 2-bovine serum albumin conjugate. This technique was used to prepare a hybridoma cell line, 3D5, which secreted a mAb that recognized 1. The mAb was shown to have specificity for 1 and other salvinorins in cross-reactivity tests. The intra-assay calibration range by icELISA using the mAb against 1 was 0.0195-0.625 μg/mL. After validating the icELISA using intra- and interassays, a recovery experiment and analysis of several plants in the family Lamiaceae, including S. divinorum, confirmed that the analytical method based on ELISA is not only simple but also precise, accurate, sensitive, and sufficiently reliable. The results indicate that icELISA is a useful tool in the identification of S. divinorum.

  7. Development of enzyme immunoassay for captan and its degradation product tetrahydrophthalimide in foods.

    Newsome, W H; Yeung, J M; Collins, P G


    A simple, sensitive, and precise enzyme-linked immunosorbent assay (ELISA) is described for the quantitation of captan as its degradation product tetrahydrophthalimide (THPI) in foods using polyclonal antibodies. Three hapten analogues of THPI with different alkyl spacer arm lengths were synthesized. Immunogens and coating proteins were prepared by coupling these haptens to human serum albumin and ovalbumin, respectively. A 5-carbon spacer arm appeared to be optimum for the production of antibodies. Heterologous coating proteins did not improve the sensitivity, but reduction of homologous coating protein concentration did improve the sensitivity, resulting in a concentration of test compound required to inhibit binding by 50% of 15.5 ng/mL. The antiserum is specific for captan, captafol, and THPI, but not other structurally related compounds. The minimum detection limit was 1 ng/mL; the linearity was 1-200 ng/mL. The overall recoveries of captan and THPI from 11 commodities spiked at 4 levels were 92 and 100%, respectively. The intra-assay and interassay coefficients of variation were 9.1 and 16.8% for apple blanks and 5.9 and 4.2% for apple spiked with 3 ppm THPI, respectively. The ELISA described is suitable for measuring captan and THPI at levels comparable to those typically found in fruit.

  8. Chronic Chagas Disease Diagnosis: A Comparative Performance of Commercial Enzyme Immunoassay Tests

    Santos, Fred Luciano Neves; de Souza, Wayner Vieira; da Silva Barros, Michelle; Nakazawa, Mineo; Krieger, Marco Aurélio; de Miranda Gomes, Yara


    There is a significant heterogeneity in reported performance of serological assays for Chagas disease diagnosis. The conventional serology testing in laboratory diagnosis and in blood banks is unsatisfactory because of a high number of inconclusive and misclassified results. We aimed to assess the quality of four commercially available enzyme-linked immunosorbent assay tests for their ability to detect Trypanosoma cruzi antibodies in 685 sera samples. Cross-reactivity was assessed by using 748 sera from patients with unrelated diseases. Initially, we found that the reactivity index against T. cruzi antigen was statistically higher in sera from Chagas disease patients compared with those from non-chagasic patients, supporting the notion that all evaluated tests have a good discriminatory ability toward the diagnosis of T. cruzi infection in patients in the chronic phase of the disease. Although all tests were similarly sensitive for diagnosing T. cruzi infection, there were significant variations in terms of specificity and cross-reactivity among them. Indeed, we obtained divergent results when testing sera from patient with unrelated diseases, particularly leishmaniasis, with the levels of cross-reactivity being higher in tests using whole T. cruzi extracts compared with those using recombinant proteins. Our data suggest that all four tests may be used for the laboratory diagnosis and routine blood screening diagnose for Chagas disease. We also emphasize that, despite their general good performance, caution is needed when analyzing the results when these tests are performed in areas where other diseases, particularly leishmaniasis, are endemic. PMID:26976886

  9. Development of a sensitive enzyme immunoassay (ELISA for specific identification of Lachesis acrochorda venom

    V Núñez Rangel


    Full Text Available The snake genus Lachesis provokes 2 to 3% of snakebites in Colombia every year. Two Lachesis species, L. acrochorda and L. muta, share habitats with snakes from another genus, namely Bothrops asper and B. atrox. Lachesis venom causes systemic and local effects such as swelling, hemorrhaging, myonecrosis, hemostatic disorders and nephrotoxic symptoms similar to those induced by Bothrops, Portidium and Bothriechis bites. Bothrops antivenoms neutralize a variety of Lachesis venom toxins. However, these products are unable to avoid coagulation problems provoked by Lachesis snakebites. Thus, it is important to ascertain whether the envenomation was caused by a Bothrops or Lachesis snake. The present study found enzyme linked immunosorbent assay (ELISA efficient for detecting Lachesis acrochorda venom in a concentration range of 3.9 to 1000 ng/mL, which did not show a cross-reaction with Bothrops, Portidium, Botriechis and Crotalus venoms. Furthermore, one fraction of L. acrochorda venom that did not show crossreactivity with B. asper venom was isolated using the same ELISA antibodies; some of its proteins were identified including one Gal-specific lectin and one metalloproteinase. This test may be useful to physicians, since it could be applicable for tracking the kinetic distribution of antigens in patients or experimentally envenomed animals.

  10. Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

    Jun Seo Oh


    Full Text Available Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland to detect antibodies to Plasmodium vivax (the indigenous malaria in the blood samples in the Republic of Korea (ROK. Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2% were reactive and 28 cases (8.6% were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.

  11. Validating a commercially available enzyme immunoassay for the determination of 17beta-estradiol and progestogens in the feces of cheetahs (Acinonyx jubatus): a case report.

    Borque, C; Perez-Garnelo, S S; Lopez, M; Talavera, C; Delclaux, M; de la Fuente, J


    Fecal 17beta-estradiol and progestogens excretion was monitored in adult, female cheetahs (Acinonyx jubatus; n = 2), ZGG-12301 (born 3 April 1993), gonadotrophin treated and ZGT-3301, (born 19 August 1993), nontreated, for 120 days using commercially available plate enzyme immunoassay kits prepared for human serum or plasma. There were significant differences (P 0.05) between baseline and gestation 17beta-estradiol values; fecal 17beta-estradiol excretion during pregnancy was statistically different (P cheetah (ZGT-3301), basal and increased progestogen concentrations were statistically different (P cheetahs and could be a practical alternative to other enzyme-linked immunosorbent assays which require more complex procedures.

  12. Development of enzyme immunoassays (ELISA and Western blot) for the serological diagnosis of dermatophytosis in symptomatic and asymptomatic cats.

    Santana, Aline Elisa; Taborda, Carlos Pelleschi; Severo, Julia So; Rittner, Glauce Mary Gomes; Muñoz, Julian Esteban; Larsson, Carlos Eduardo; Larsson, Carlos Eduardo


    Dermatophytosis is the most common fungal infection in cats worldwide and plays an important role in both animal and human health due to their high zoonotic potential. Effective screening is a strong preventive measure and the fungal culture is quite useful but requires full laboratorial experience and it takes a long time to obtain the result. A rapid and accurate screening test for dermatophytosis in cats is crucial for the effective control of disease outbreaks. The aim of this study was to develop and evaluate the diagnostic efficacy of enzyme immunoassays (ELISA and Western blot [WB]) for the rapid and precise diagnosis of dermatophytosis in cats. Seventy cats of various ages were divided into three groups: S (symptomatic, n = 20), AS (asymptomatic, n = 30), and N (negative, n = 20). All animals were submitted to fungal culture and blood samples for carrying out the serological tests. A significant difference (P < 0.05) was found between IgG-specific levels of sera of Microsporum canis positive and negative animals. There was no statistic difference between groups symptomatic and asymptomatic. The ELISA test showed sensitivity of 94% and specificity of 75%. Receiver operating characteristic (ROC) analysis also showed higher diagnostic accuracy (AUC 0.925). The WB technique detected 13 bands, and the 50 kDa protein was considered the most immunogenic protein, observing reactivity in 83.3% in the symptomatic group and 66.6% in the asymptomatic group. The study concluded that ELISA and WB were useful tools to reliably detect cats that have been exposed to M. canis. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail:

  13. Microplate chemiluminescence enzyme immunoassay for the quantitative evaluation of carbohydrate antigen 72-4 in human serum

    JIN Hui; WANG Xu; XIN TianBing; GAO Peng; LIN JinMing; LIANG ShuXuan


    A highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) was de-veloped for the quantitative evaluation of carbohydrate antigen 72-4 (CA72-4) in human serum, using luminol-H2O2 catalyzed by horseradish peroxidase (HRP) as the chemiluminescence system. The sim-ple and quick determination was accomplished through a sandwich reaction mode. Several physico-chemical parameters of the immunoreaction, including incubation conditions, antibody coating condi-tions, dilution ratio of anti-CA72-4-HRP conjugate, and chemiluminescence reaction time, were studied and optimized. The proposed method exhibited a linear range of 0-200 U/mL with correlation coeffi-cient and detection limit of 0.9995 and 0.18 U/mL, respectively. The inter-assay and intra-assay coeffi-cients of variation (CV) were both less than 10%. The average recovery of two clinical sera with low and high concentration CA72-4 was 99.3% and 98.7%, respectively. Normal tumor markers, including AFP, CEA, CA2.4-2, CA19-9 and CA15-3, did not cross-react with each other. The method's stability was evaluated by assessing its analytical performance after storing the immunoreagents at 4℃ and 37℃ for 7 days. Little difference was found, indicating satisfactory stability of the method. The present method has been successfully applied to the detection of CA72-4 human serum, and showed a good correlation with the commercially available ELISA kit (r2=0.9383). This method showed great potential in the fabrication of diagnostic kit for CA72-4, and could be well used in diagnosis of cancer in clinical practice.

  14. Detection of Campylobacter in Stool and Determination of Significance by Culture, Enzyme Immunoassay, and PCR in Developing Countries

    Platts-Mills, James A.; Liu, Jie; Gratz, Jean; Mduma, Esto; Amour, Caroline; Swai, Ndealilia; Taniuchi, Mami; Begum, Sharmin; Peñataro Yori, Pablo; Tilley, Drake H.; Lee, Gwenyth; Shen, Zeli; Whary, Mark T.; Fox, James G.; McGrath, Monica; Kosek, Margaret; Haque, Rashidul


    Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level. PMID:24452175

  15. Evaluation of the C6 Lyme Enzyme Immunoassay for the Diagnosis of Lyme Disease in Children and Adolescents.

    Lipsett, Susan C; Branda, John A; McAdam, Alexander J; Vernacchio, Louis; Gordon, Caroline D; Gordon, Catherine R; Nigrovic, Lise E


    The commercially-available C6 Lyme enzyme immunoassay (EIA) has been approved to replace the standard whole-cell sonicate EIA as a first-tier test for the diagnosis of Lyme disease and has been suggested as a stand-alone diagnostic. However, the C6 EIA has not been extensively studied in pediatric patients undergoing evaluation for Lyme disease. We collected discarded serum samples from children and adolescents (aged ≤21 years) undergoing conventional 2-tiered testing for Lyme disease at a single hospital-based clinical laboratory located in an area endemic for Lyme disease. We performed a C6 EIA on all collected specimens, followed by a supplemental immunoblot if the C6 EIA result was positive but the whole-cell sonicate EIA result was negative. We defined a case of Lyme disease as either a clinician-diagnosed erythema migrans lesion or a positive standard 2-tiered serologic result in a patient with symptoms compatible with Lyme disease. We then compared the performance of the C6 EIA alone and as a first-tier test followed by immunoblot, with that of standard 2-tiered serology for the diagnosis of Lyme disease. Of the 944 specimens collected, 114 (12%) were from patients with Lyme disease. The C6 EIA alone had sensitivity similar to that of standard 2-tiered testing (79.8% vs 81.6% for standard 2-tiered testing; P = .71) with slightly lower specificity (94.2% vs 98.8% 2; P Lyme disease, the C6 EIA could guide initial clinical decision making, although a supplemental immunoblot should still be performed. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail

  16. [Evaluation of infection by Helicobacter pylori in HIV positive patients trough enzyme immunoassay and specific amplification of DNA].

    Gutiérrez, Sandra; Chacón-Petrola, María; Flores, María; Pinto, Angela; Pacheco, Mariela


    The objective of this work was to assess the effectiveness of detection of specific antibodies anti-Helicobacter pylori (H. pylori) by ELISA and amplification of specific DNA by polimerase chain reaction (PCR) as diagnostic methods of infection of H. pylori in HIV positive patients. Twenty two patients with HIV infection were studied, with ages between 26 to 35 years, 17 masculine, 55% with gastrointestinal symptoms, controlled in the Unit of Immunology, CHET. Inclusion approaches: older than 18 years, with confirmed diagnosis of HIV infection (ELISA and WB), lymphocyte subpopulation and good general conditions. Consent in writing was obtained. Exclusion approaches: previous diagnosis of H. pylori infection or treatment with antibiotics in the three previous months to their inclusion. The quantification of IgG anti H. pylori was carried out by Enzyme Immunoassay methods (ELISA). Biopsy of gastric mucosa was obtained by superior endoscopic study. The amplification of DNA for H. pylori was performed by PCR (Wizard SV Genomik and PCR Ready-Promega). In the statistical analysis was used the test of Fisher, with a level of significance of 5% (0.05). In 15 patients of the total group, antibodies anti H. pylori were confirmed, without statistical association with the presence or not of digestives symptoms, neither with the number of lymphocytes CD4 + in peripheral blood. Also 15 patients were positives by PCR for H. pylori DNA, 73.3% of them presented levels of CD4+ above 200 cells. There was not statistical association between the positivity of this method and levels of lymphocytes CD4+. In 12 of the 15 patients with positive results by PCR, antibodies anti H. pylori were evidenced, and among the 7 patients with negative serology to H. pylori, PCR was positive in three of them. In conclusion, serology is an effective method for the diagnose of H. pylori infection in VIH+ patients, but its negativity doesn't discard the infection for this bacillus.

  17. The utility of repeat enzyme immunoassay testing for the diagnosis of Clostridium difficile infection: A systematic review of the literature

    P S Garimella


    Full Text Available Over the last 20 years, the prevalence of healthcare-associated Clostridium difficile (C. diff disease has increased. While multiple tests are available for the diagnosis of C. diff infection, enzyme immunoassay (EIA testing for toxin is the most used. Repeat EIA testing, although of limited utility, is common in medical practice. To assess the utility of repeat EIA testing to diagnose C. diff infections. Systematic literature review. Eligible studies performed >1 EIA test for C. diff toxin and were published in English. Electronic searches of MEDLINE and EMBASE were performed and bibliographies of review articles and conference abstracts were hand searched. Of 805 citations identified, 32 were reviewed in detail and nine were included in the final review. All studies except one were retrospective chart reviews. Seven studies had data on number of participants (32,526, and the overall reporting of test setting and patient characteristics was poor. The prevalence of C. diff infection ranged from 9.1% to 18.5%. The yield of the first EIA test ranged from 8.4% to 16.6%, dropping to 1.5-4.7% with a second test. The utility of repeat testing was evident in outbreak settings, where the yield of repeat testing was 5%. Repeat C. diff testing for hospitalized patients has low clinical utility and may be considered in outbreak settings or when the pre-test probability of disease is high. Future studies should aim to identify patients with a likelihood of disease and determine the utility of repeat testing compared with empiric treatment.

  18. The utility of repeat enzyme immunoassay testing for the diagnosis of Clostridium difficile infection: a systematic review of the literature.

    Garimella, P S; Agarwal, R; Katz, A


    Over the last 20 years, the prevalence of healthcare-associated Clostridium difficile (C. diff) disease has increased. While multiple tests are available for the diagnosis of C. diff infection, enzyme immunoassay (EIA) testing for toxin is the most used. Repeat EIA testing, although of limited utility, is common in medical practice. To assess the utility of repeat EIA testing to diagnose C. diff infections. Systematic literature review. Eligible studies performed >1 EIA test for C. diff toxin and were published in English. Electronic searches of MEDLINE and EMBASE were performed and bibliographies of review articles and conference abstracts were hand searched. Of 805 citations identified, 32 were reviewed in detail and nine were included in the final review. All studies except one were retrospective chart reviews. Seven studies had data on number of participants (32,526), and the overall reporting of test setting and patient characteristics was poor. The prevalence of C. diff infection ranged from 9.1% to 18.5%. The yield of the first EIA test ranged from 8.4% to 16.6%, dropping to 1.5-4.7% with a second test. The utility of repeat testing was evident in outbreak settings, where the yield of repeat testing was 5%. Repeat C. diff testing for hospitalized patients has low clinical utility and may be considered in outbreak settings or when the pre-test probability of disease is high. Future studies should aim to identify patients with a likelihood of disease and determine the utility of repeat testing compared with empiric treatment.

  19. Usefulness of IgM-specific enzyme immunoassays for serodiagnosis of syphilis: comparative evaluation of three different assays.

    Bosshard, Philipp P


    IgM antibodies are usually the first to be produced during treponemal infection. Three commercially available enzyme immunoassays (EIA) for detection of IgM antibodies against Treponema pallidum were evaluated. Results of the Anti-Treponema-pallidum-ELISA (IgM; Euroimmun), Pathozyme Syphilis M Capture (Omega Diagnostics) and recomWell Treponema IgM (Mikrogen) were compared with those of the T. pallidum particle agglutination (TPPA) and the Venereal Disease Research Laboratory (VDRL) tests for 307 serum samples. The overall sensitivity (95% confidence interval [CI]) of the TPPA was 100% (97.7-100%) compared to 83.3% (76.5-88.8%) of the VDRL, 88.5% (82.4-93.0%) of the Pathozyme, 84.6% (78.0-89.9) of the Euroimmun, and 73.6% (66.1-80.4%) of a modified recomWell test procedure. Specificities were in the range of 91.4-100%. In primary syphilis, sensitivities of the Pathozyme (89.8%; 95% CI, 79.2-96.2%) and Euroimmun tests (81.4%; 95% CI, 69.1-90.3%) were significantly higher (p VDRL test (61%; 95% CI, 47.4-73.5%). IgM EIAs even were positive in some cases of suspected very early infection where the VDRL was non-reactive and the TPPA was indeterminate. In cases of suspected early infection specific IgM EIAs should be used in addition to other screening tests. The VDRL is not recommended for screening. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  20. Use of a nested PCR-enzyme immunoassay with an internal control to detect Chlamydophila psittaci in turkeys

    Goddeeris Bruno M


    Full Text Available Abstract Background Laboratory diagnosis of Chlamydophila psittaci, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay (PCR-EIA was developed to detect the Cp. psittaci outer membrane protein A (ompA gene in pharyngeal swabs. Methods The fluorescein-biotin labelled PCR products were immobilized on streptavidin-coated microtiter plates and detected with anti-fluorescein peroxidase conjugate and a colorimetric substrate. An internal inhibition control was included to rule out the presence of inhibitors of DNA amplification. The diagnostic value of the ompA nested PCR-EIA in comparison to cell culture and a 16S-rRNA based nested PCR was assessed in pharyngeal turkey swabs from 10 different farms experiencing respiratory disease. Results The sensitivity of the nested PCR-EIA was established at 0.1 infection forming units (IFU. Specificity was 100%. The ompA nested PCR-EIA was more sensitive than the 16S-rRNA based nested PCR and isolation, revealing 105 out of 200 (52.5% positives against 13 and 74 for the latter two tests, respectively. Twenty-nine (23.8% out of 122 ompA PCR-EIA negatives showed the presence of inhibitors of DNA amplification, although 27 of them became positive after diluting (1/10 the specimens in PCR buffer or after phenol-chloroform extraction and subsequent ethanol precipitation. Conclusion The present study stresses the need for an internal control to confirm PCR true-negatives and demonstrates the high prevalence of chlamydiosis in Belgian turkeys and its potential zoonotic risk. The ompA nested PCR-EIA described here is a rapid, highly sensitive and specific diagnostic assay and will help to facilitate the diagnosis of Cp. psittaci infections in both poultry and man.

  1. Nanogold-penetrated poly(amidoamine) dendrimer for enzyme-free electrochemical immunoassay of cardiac biomarker using cathodic stripping voltammetric method.

    Zhang, Bo; Zhang, Yi; Liang, Wenbin; Cui, Bin; Li, Jiabei; Yu, Xuejun; Huang, Lan


    Methods based on immunoassays have been developed for cardiac biomarkers, but most involve the low sensitivity and are unsuitable for early disease diagnosis. Herein we design an electrochemical immunoassay for sensitive detection of myoglobin (a cardiac biomarker for acute myocardial infarction) by using nanogold-penetrated poly(amidoamine) dendrimer (AuNP-PAMAM) for signal amplification without the need of natural enzymes. The assay was carried out on the monoclonal mouse anti-myoglobin (capture) antibody-anchored glassy carbon electrode using polyclonal rabbit anti-myoglobin (detection) antibody-labeled AuNP-PAMAM as the signal tag. In the presence of target myoglobin, the sandwiched immunocomplex could be formed between capture antibody and detection antibody. Accompanying AuNP-PAMAM, the carried gold nanoparticles could be directly determined via stripping voltammetric method under acidic conditions. Under optimal conditions, the detectable electrochemical signal increased with the increasing target myoglobin in the sample within a dynamic working range from 0.01 to 500 ng mL(-1) with a detection limit of 3.8 pg mL(-1). The electrochemical immunoassay also exhibited high specificity and good precision toward target myoglobin. Importantly, our strategy could be applied for quantitative monitoring of myoglobin in human serum specimens, giving well matched results with those obtained from commercialized enzyme-linked immunosorbent assay (ELISA) method.

  2. Evaluation of the effect of TM208 on the activity of five cytochrome P450 enzymes using on-line solid-phase extraction HPLC-DAD: a cocktail approach.

    Lin, Wensi; Zhang, Jianmei; Ling, Xiaomei; Yu, Ning; Li, Jing; Yang, Haisong; Li, Runtao; Cui, Jingrong


    A rapid, simple, and sensitive on-line solid-phase extraction HPLC-DAD method for simultaneous evaluation of the activity of five CYP450 isoforms (CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in vivo has been developed and validated. The five specific probe substrates include caffeine (1A2), metoprolol (2D6), dapsone (3A4), omeprazole (2C19) and chlorzoxazone (2E1). Automated pre-purification of plasma and enrichment of analytes were performed using a C18 on-line solid-phase extraction cartridge. After being eluted from the cartridge, the analytes and the internal standard antipyrine were separated on a C18 RP analytical column and analyzed by DAD. The method was validated to quantify the concentration ranges of 0.05-50.0 μg/ml for dapsone and omeprazole, 0.1-50.0 μg/ml for caffeine and 0.2-50.0 μg/ml for metoprolol and chlorzoxazone. The linearity (R(2)) for all analytes tested was exceeded 0.99. The intra-day precision ranged from 0.29 to 13% and the inter-day precision ranged from 5.0 to 15%, respectively. The intra-day and inter-day accuracy were between 86.7% and 113.6%. The extraction recoveries were in the range 82.8-109.9% for all the analytes and internal standard antipyrine. This method was successfully applied to evaluate the effects of TM208 on rat five CYP450 isoforms.

  3. Filter paper blood spot enzyme linked immunoassay for adiponectin and application in the evaluation of determinants of child insulin sensitivity.

    Richard M Martin

    Full Text Available BACKGROUND: Adiponectin is an adipocyte-derived hormone that acts as a marker of insulin sensitivity. Bloodspot sampling by fingerstick onto filter paper may increase the feasibility of large-scale studies of the determinants of insulin sensitivity. We first describe the validation of an enzyme-linked immunoassay (ELISA for quantifying adiponectin from dried blood spots and then demonstrate its application in a large trial (PROBIT. METHODS: We quantified adiponectin from 3-mm diameter discs (≈3 µL of blood punched from dried blood spots obtained from: i whole blood standards (validation; and ii PROBIT trial samples (application in which paediatricians collected blood spots from 13,879 children aged 11.5 years from 31 sites across Belarus. We examined the distribution of bloodspot adiponectin by demographic and anthropometric factors, fasting insulin and glucose. RESULTS: In the validation study, mean intra-assay coefficients of variation (n=162 were 15%, 13% and 10% for 'low' (6.78 µg/ml, 'medium' (18.18 µg/ml and 'high' (33.13 µg/ml internal quality control (IQC samples, respectively; the respective inter-assay values (n=40 were 23%, 21% and 14%. The correlation coefficient between 50 paired whole bloodspot versus plasma samples, collected simultaneously, was 0.87 (95% CI: 0.78 to 0.93. Recovery of known quantities of adiponectin (between 4.5 to 36 µg/ml was 100.3-133%. Bloodspot adiponectin was stable for at least 30 months at -80°C. In PROBIT, we successfully quantified fasting adiponectin from dried blood spots in 13,329 of 13,879 (96% children. Mean adiponectin (standard deviation concentrations were 17.34 µg/ml (7.54 in boys and 18.41 µg/ml (7.92 in girls and were inversely associated with body mass index, fat mass, triceps and subscapular skin-fold thickness, waist circumference, height and fasting glucose. CONCLUSIONS: Bloodspot ELISA is suitable for measuring adiponectin in very small volumes of blood collected on filter paper

  4. Comparison of a Monoclonal Antibody-Blocking Enzyme-Linked Immunoassay and a Strip Immunoblot Assay for Identifying Type-Specific Herpes Simplex Virus Type 2 Serological Responses

    van Doornum, G. J. J.; Slomka, M.J.; Buimer, M; Groen, J.; van den Hoek, J.A.R.; Cairo, I.; Vyse, A.; Brown, D. W G


    Detection of herpes simplex virus type 2 (HSV-2)-specific antibodies by a monoclonal antibody (MAb)-blocking enzyme-linked immunoassay (EIA) was compared with detection by a strip immunoblot assay (SIA) in a sexually transmitted disease (STD) clinic population. The study population consisted of 1,683 genitourinary medicine clinic attendees (582 women and 1,101 men). Sera were tested for the presence of HSV-2 antibody by use of the blocking EIA, in which binding of the MAb AP-1 to HSV-2 glycop...

  5. Application of a Newly Developed High-Sensitivity HBsAg Chemiluminescent Enzyme Immunoassay for Hepatitis B Patients with HBsAg Seroclearance


    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstr...

  6. An enzyme immunoassay of phaseolinone and its application in estimation of the amount of toxin in Macrophomina phaseolina-infected seeds.

    Bhattacharya, D; Dhar, T K; Ali, E


    A microtiter plate-based enzyme immunoassay has been developed for phaseolinone, a phytotoxin isolated from the culture filtrate of the plant-pathogenic fungus Macrophomina phaseolina (Tassi) Goid. The smallest amount of phaseolinone detectable by the method is 5 pg per well. The method is validated by comparison with high-performance liquid chromatography and used to confirm and estimate phaseolinone production in seeds infected with the fungus. The degree of seed inhibition correlated well with the amount of toxin produced in infected seeds, 50% inhibition being observed at a toxin concentration of 0.60 micrograms/g of wet tissue. PMID:1622272


    Bondarenko, N P; Lakatosh, V P; Lakatosh, P V; Malanchuk, O B; Poladich, I V


    The combined method of diagnosis parvovirus infection during pregnancy by maternal serum enzyme immunoassay and deoxyribonucleic acid isolation parvovirus B19 polymerase chain reaction in amnniotic fluid and fetal cord blood newborns, can diagnose vertical transmission and anticipate a negative effect on the fetus parvovirus. Lack of maternal IgM antibodies in serum due to parvovirus seroconversion during pregnancy does not exclude the persistence of the virus in the fetus. To analyze the diagnostic value of the method for determining the LHP parvovirus B19 DNA in the amniotic fluid, umbilical cord blood of newborns to determine vertical transmission of parvovirus infection when infected mothers B19 during pregnancy.

  8. Determination of the concentrations of the steroids estradiol, progesterone and testosterone in bovine sera: comparison of commercial dissociation enhanced lanthanide fluorescence immunoassay kits with conventional radio and enzyme immunoassays.

    Elliott, C T; Francis, K S; Shortt, H D; McCaughey, W J


    The performance of three conventional enzyme and radioimmunoassays routinely used to detect residues of anabolic steroids in cattle sera were compared with dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) kits designed for the hospital market. Slight modifications to the kit reagents were required for the analysis of bovine sera. Owing to the large sample volumes used in conventional assays, detection limits were generally better than those obtained with DELFIA kits, however, assay reproducibility was enhanced using the DELFIA technology. Comparison of sera obtained from cattle implanted with anabolic steroids revealed a good correlation between alternate methods (r2 from 0.91 to 0.97). The DELFIA kits offer a faster method for measuring estradiol, progesterone and testosterone with adequate sensitivity and in a safer environment than that encountered using radioimmunoassays.

  9. Multiplexed Colorimetric Solid-Phase Extraction

    Gazda, Daniel B.; Fritz, James S.; Porter, Marc D.


    Multiplexed colorimetric solid-phase extraction (MC-SPE) is an extension of colorimetric solid-phase extraction (C-SPE) an analytical platform that combines colorimetric reagents, solid phase extraction, and diffuse reflectance spectroscopy to quantify trace analytes in water. In CSPE, analytes are extracted and complexed on the surface of an extraction membrane impregnated with a colorimetric reagent. The analytes are then quantified directly on the membrane surface using a handheld diffuse reflectance spectrophotometer. Importantly, the use of solid-phase extraction membranes as the matrix for impregnation of the colorimetric reagents creates a concentration factor that enables the detection of low concentrations of analytes in small sample volumes. In extending C-SPE to a multiplexed format, a filter holder that incorporates discrete analysis channels and a jig that facilitates the concurrent operation of multiple sample syringes have been designed, enabling the simultaneous determination of multiple analytes. Separate, single analyte membranes, placed in a readout cartridge create unique, analyte-specific addresses at the exit of each channel. Following sample exposure, the diffuse reflectance spectrum of each address is collected serially and the Kubelka-Munk function is used to quantify each water quality parameter via calibration curves. In a demonstration, MC-SPE was used to measure the pH of a sample and quantitate Ag(I) and Ni(II).

  10. Enhanced Antibody Detection and Diagnosis of Coccidioidomycosis with the MiraVista IgG and IgM Detection Enzyme Immunoassay.

    Malo, Joshua; Holbrook, Eric; Zangeneh, Tirdad; Strawter, Chris; Oren, Eyal; Robey, Ian; Erickson, Heidi; Chahal, Racquel; Durkin, Michelle; Thompson, Cindy; Hoover, Susan E; Ampel, Neil M; Wheat, L Joseph; Knox, Kenneth S


    Coccidioidomycosis is a common cause of community-acquired pneumonia in areas of the southwestern United States in which the disease is endemic. Clinical presentations range from self-limited disease to severe disseminated disease. Therefore, early and accurate diagnosis is essential to ensure appropriate treatment and monitoring. Currently available diagnostic tests have variable accuracy, particularly in certain patient populations, and new tests may offer improved accuracy for the diagnosis of coccidioidomycosis. Serum samples from 103 cases of coccidioidomycosis and 373 controls were tested for IgG and IgM antibodies using the MVista anti-Coccidioides antibody enzyme immunoassay. Serum specimens from 170 controls from areas in which the disease is endemic and 44 cases were tested by immunodiffusion at MiraVista Diagnostics. The sensitivity of the MVista antibody assay was 88.3%, and the specificity was 90%. The sensitivity was maintained in the presence of immunocompromising conditions or immunosuppressive therapies. The sensitivity of immunodiffusion was 60.2%, and the specificity was 98.8%. The sensitivity of complement fixation (62 cases) was 66.1%, but the specificity could not be determined. The MVista anti-Coccidioides antibody enzyme immunoassay offers improved sensitivity, compared with immunodiffusion and complement fixation, is not impaired in immunocompromised patients, and permits highly reproducible semiquantification. Copyright © 2017 American Society for Microbiology.

  11. A comparison between enzyme immunoassay and HPLC for ochratoxin A detection in green, roasted and instant coffee

    Simone Fujii


    Full Text Available An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA for ochratoxin A (OTA detection in green, roasted and instant coffees was developed using anti-OTA monoclonal antibody. Immunological reagents prepared were OTA-BSA (4.76 mg/mL, anti-OTA.7 MAb (2x10³-fold dilution and HRP-anti IgG (10³-fold dilution. The detection limit was 3.73 ng OTA/g and correlation coefficients (r between this immunoassay and high performance liquid chromatography were 0.98 for green coffee, 0.98 for roasted and 0.86 for instant. OTA levels detected by ic-ELISA were higher than by HPLC, with ELISA/HPLC ratio of 0.66 - 1.46 (green coffee, 0.96 - 1.11 (roasted and 0.93 - 1.82 (instant. ELISA recoveries for OTA added to coffee (5 - 70 ng/g were 81.53 % for green coffee, 46.73 % for roasted and 64.35 % for instant, while recoveries by HPLC were 80.54 %, 45.91 % and 55.15 %, respectively. Matrices interferences were minimized by samples dilution before carrying out the ELISA assay. The results indicate that MAb-based ic-ELISA could be a simple, sensitive and specific screening tool for OTA detection, contributing to quality and safety of coffee products.ELISA competitivo indireto (ic-ELISA baseado em anticorpos monoclonais foi desenvolvido para a detecção de ocratoxina A (OTA em café verde, torrado e instantâneo. Os reagentes imunológicos necessários à reação consistiram de OTA-BSA (4,76 mg/mL, anti-OTA.7 MAb (diluído 2x10³ e anti IgG-HRP (diluído 10³, apresentando limite de detecção de 3,73 ng OTA/g. Os coeficientes de correlação (r entre o imunoensaio e cromatografia líquida de alta eficiência (CLAE foram de 0,98 (café verde, 0,98 (torrado e 0,86 (instantâneo. Ic-ELISA detectou valores superestimados de OTA em relação a CLAE, com valor ELISA/CLAE variando de 0,66 - 1,46 (café verde, 0,96 - 1,11 (torrado e 0,93 - 1,82 (instantâneo. As recuperações médias de OTA adicionada em café (5 - 70 ng/g foram de 81,53 % (café verde, 46

  12. Development of a highly sensitive chemiluminescent assay for hydrogen peroxide under neutral conditions using acridinium ester and its application to an enzyme immunoassay.

    Arakawa, Hidetoshi; Tsuruoka, Keiko; Ohno, Ken-ichi; Tajima, Noriko; Nagano, Hiromi


    We developed a highly sensitive chemiluminescent (CL) assay for hydrogen peroxide using 10-methyl-9-(phenoxycarbonyl) acridinium fluorosulfonate (PMAC) that produced chemiluminescence under neutral conditions and applied it to an enzyme immunoassay (EIA). One picomole of hydrogen peroxide could be detected using the optimized PMAC-CL method and 6.2 × 10(-20) mol β-D-galactosidase (β-gal) could be detected by combining an indoxyl derivative substrate and the proposed PMAC-CL method. This highly sensitive CL β-gal assay was applied to an EIA for thyroid-stimulating hormone (TSH) using β-gal as a label enzyme; 0.02-100.0 μU/mL TSH in human serum could be assayed directly and with high reproducibility.

  13. Measurements in international units of antibody to hepatitis B surface antigen(anti-HBs) after immunization with a yeast-derived, subtype adr hepatitis B vaccine are considerably different between chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA).

    Ogata, Norio


    The worldwide consensus of the minimum protective anti-HBs level against HBV infection is 10 mIU/mL on assays standardized by the World Health Organization (WHO) reference preparations. To investigate whether this value could be applied to recipients of yeast-derived recombinant HB vaccine containing the major surface protein of subtype adr (Bimmugen, Astellas Pharmaceutical, Tokyo), we compared anti-HBs measurements between chemiluminescent immunoassay (CLIA) (Architect Ausab, Abbott Japan, Tokyo) and chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse Forte, Fujirebio, Tokyo) in given serum samples obtained from the vaccinees. The vaccine and the two assay methods are currently in a wide use in Japan. The study included 300 medical students who completed a standard vaccination course (0, 1 and 6 months). Serum samples obtained 1 month or 13 months after completing the vaccination were simultaneously tested for anti-HBs by CLIA and CLEIA. In 147 samples with quantifiable values on both CLIA and CLEIA (10 to 1000 mIU/mL) the geometric mean titer on CLEIA (225.0 mIU/mL) was significantly higher than that on CLIA (94.5 mIU/mL) (p anti-HBs measurements compared to CLIA and discordance in determining critical anti-HBs level of 10 mIU/mL was observed in more than half the samples. This suggests that the minimum HBV-protective anti HBs titer of 10 mIU/mL is difficult to be introduced to Japan where subtype adr-HB vaccines or -HBV infection are prevalent, unless characteristics of assay methods are carefully evaluated.

  14. Solid phase synthesis of bifunctional antibodies.

    DeSilva, B S; Wilson, G S


    Bifunctional antibodies were prepared using the principle of solid-phase synthesis. The two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')2 fragments from intact IgG were prepared using an immobilized pepsin column. Goat, mouse and human antibodies were digested completely within 4 h. The F(ab')2 fragments thus produced did not contain any IgG impurities. The Fab' fragments were produced by reducing the inter-heavy chain disulfide bonds using 2-mercaptoethylamine. The use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of the bifunctional antibody preparation and the rapid optimization of reaction conditions.

  15. A New Enzyme Immunoassay for the Quantitative Determination of Classical Autotaxins (ATXα, ATXβ, and ATXγ and Novel Autotaxins (ATXδ and ATXε.

    Yasunori Tokuhara

    Full Text Available Autotaxin (ATX is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid, a potent bioactive lipid mediator, through its lysophospholipase D activity. Although five alternative splicing isoforms of ATX have been identified as ATXα, ATXβ, ATXγ, ATXδ, and ATXε and the expression patterns of each isoform differ among several tissues, the clinical significance of each isoform remains to be elucidated.Anti-ATXβ and anti-ATXδ monoclonal antibodies were produced by immunization with recombinant human ATXβ and ATXδ expressed using a baculovirus system, respectively. We then developed enzyme immunoassays to measure the serum concentrations of "classical ATX" (ATXα, ATXβ, and ATXγ and "novel ATX" (ATXδ and ATXε antigens and evaluated the usefulness of these assays using human serum samples.The with-run and between-run precision, interference, detection limit, and linearity studies for the present assay were well validated. In healthy subjects, the serum concentrations of classical ATX and novel ATX were significantly (P < 0.01 higher in women than in men, while the ratios of classical ATX or novel ATX to total ATX were not different between women and men. The concentrations of both classical ATX and novel ATX in normal pregnant subjects and patients with chronic liver diseases or follicular lymphoma were significantly higher than those in healthy subjects, while the ratio of both ATX isoforms to total ATX did not vary among these groups.We have developed a new enzyme immunoassay to determine the concentrations of classical ATX and novel ATX in human serum. These assays may be helpful for elucidating the distinct functional roles of each ATX isoform, which are largely unknown at present.

  16. [Solid phase techniques in blood group serology].

    Uthemann, H; Sturmfels, L; Lenhard, V


    As alternatives to hemagglutination, solid-phase red blood cell adherence assays are of increasing importance. The adaptation of the new techniques to microplates offers several advantages over hemagglutination. Using microplates the assays may be processed semiautomatically, and the results can be read spectrophotometrically and interpreted by a personal computer. In this paper, different red blood cell adherence assays for AB0 grouping, Rh typing, Rh phenotyping, antibody screening and identification, as well as crossmatching will be described.

  17. Recent advances in solid phase peptide synthesis

    White, P.D.


    Since its introduction by Merrifield half a century ago, solid phase peptide synthesis has evolved to become the enabling technology for the development of peptide therapeutics. Using modern methods, 100 - 1000s of peptides can be routinely synthesised in parallel for screening as leads for drug development and peptide APIs are produced in ton scale. In this talk I consider the state of art and report on recent advances to overcome remaining issues such as aspartimide formation, racemisation ...

  18. Development of headspace solid-phase microextraction method for ...

    ... solid-phase microextraction method for the analysis of pesticide residues in fruit and ... Journal of Applied Sciences and Environmental Management ... interface temperature) and solid phase microextraction parameters (fiber coating type, ...

  19. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile☆☆☆★

    Strachan, Alastair J.; Evans, Natalie E.; Williams, O. Martin; Spencer, Robert C.; Greenwood, Rosemary; Probert, Chris J.


    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting. PMID:23107315

  20. Galactomannan enzyme immunoassay and quantitative Real Time PCR as tools to evaluate the exposure and response in a rat model of aspergillosis after posaconazole prophylaxis.

    Cendejas-Bueno, Emilio; Forastiero, Agustina; Ruiz, Isabel; Mellado, Emilia; Buitrago, María José; Gavaldà, Joan; Gomez-Lopez, Alicia


    A steroid-immunosuppressed rat model of invasive pulmonary aspergillosis was use to examine the usefulness of galactomannan enzyme immunoassay (GM) and quantitative real time PCR (RT-PCR) in evaluating the association between response and exposure after a high dose of prophylactic posaconazole. Two different strains of Aspergillus fumigatus with different in vitro posaconazole susceptibility were used. Serum concentrations demonstrated similar posaconazole exposure for all treated animals. However, response to posaconazole relied on the in vitro susceptibility of the infecting strain. After prophylaxis, galactomannan index and fungal burden only decreased in those animals infected with the most susceptible strain. This study demonstrated that both biomarkers may be useful tools for predicting efficacy of antifungal compounds in prophylaxis. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  1. Detection of pregnancy and fertility status in big cats using an enzyme immunoassay based on 5α-pregnan-3α-ol-20-one.

    Umapathy, Govindhaswamy; Kumar, Vinod; Wasimuddin; Kabra, Meha; Shivaji, S


    Development of non-invasive steroid hormone assays using fecal samples is crucial for detection of pregnancy and monitoring of fertility status in big cats and thus facilitates conservation and management of wild animals. Due to changes in metabolism and excretory pattern, animals excrete different steroid metabolites in feces and urine. The present study is an attempt to develop a common enzyme immunoassay for 5α-pregnan-3α-ol-20-one one of the predominant progestogen metabolites in the feces samples of big cats. The developed ELISA showed a high sensitivity and low cross reactivity to other hormones compared to commercially available RIA kits based on progesterone antibody. It could be used in a wide range of animals for monitoring fertility status and pregnancy detection by measuring fecal steroid metabolites.

  2. [Development and testing of an enzyme immunoassay-based monoclonal test system for the detection of the Yersinia pestis V antigen].

    Ivashchenko, T A; Belova, E V; Dentovskaia, S V; Bel'kova, S A; Balakhonov, S V; Ignatov, S G; Shemiakin, I G


    An enzyme immunoassay-based test system for Y. pestis V antigen detection was developed. The specificity and sensitivity of this system met the requirements for medical immunobiological preparations for the identification of causative agents of highly fatal diseases. The sensitivity of the test system was assessed, and its high specificity was also demonstrated: the test system did not detect bacterial cells of closely related (four Y. pseudotuberculosis strains) and heterologous microorganism strains. The test system developed was able to detect the V antigen at concentrations as low as 2.0 ng/mL in cells of nine experimental Y. pestis cultures. The obtained preparation can be recommended for use in laboratory diagnostics of plaque.

  3. Enzyme-amplified protein micorarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

    Varnum, Susan M.; Warner, Marvin G.; Dockendorff, Brian P.; Anheier, Norman C.; Lou, Jianlong; Marks, James D.; Smith, Leonard A.; Feldhaus, Michael J.; Grate, Jay W.; Bruckner-Lea, Cindy J.


    With the use of high-affinity recombinant monoclonal antibodies against the receptor binding domain of botulinum neurotoxin A (BoNT/A), two separate immunoassay platforms were developed for either the sensitive or the rapid detection of BoNT/A. An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of BoNT in buffer and clinical fluids. This assay has the sensitivity to detect BoNT in diverse samples down to 14 fM (1.4 pg/mL). Using the recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. While the ELISA microarray assay, because of its sensitivity, offers an alternative to the mouse bioassay, the renewable surface assay has potential as a rapid screening assay for the analysis of complex environmental samples.

  4. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile.

    Strachan, Alastair J; Evans, Natalie E; Williams, O Martin; Spencer, Robert C; Greenwood, Rosemary; Probert, Chris J


    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting.

  5. A solid-phase dot assay using silica/gold nanoshells

    Zharov Vladimir; Khlebtsov Boris; Dykman Lev; Bogatyrev Vladimir; Khlebtsov Nikolai


    AbstractWe report on the first application of silica-gold nanoshells to a solid-phase dot immunoassay. The assay principle is based on staining of a drop (1 µl) analyte on a nitrocellulose membrane strip by using silica/gold nanoshells conjugated with biospecific probing molecules. Experimental example is human IgG (hIgG, target molecules) and protein A (probing molecules). For usual 15-nm colloidal gold conjugates, the minimal detectable amount of hIgG is about 4 ng. By contrast, for na...

  6. Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

    Nguyen, Duc Doan; Busetti, Francesco; Johnson, Stuart Keith; Solah, Vicky Ann


    This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linkedimmunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

  7. Indirect solid-phase immunosorbent assay for detection of arenavirus antigens and antibodies

    Ivanov, A.P.; Rezapkin, G.V.; Dzagurova, T.K.; Tkachenko, E.A. (Institute of Poliomyelitis anU Viral Encephalities of the U.S.S.R. Academy of Medical Sciences, Moscow)


    Indirect enzyme-linked immunosorbent assay (ELISA) and solid phase radioimmunoassay (SPRIA) using either enti-human or anti-mouse IgG labelled with horseradish peroxidase and /sup 125/I, respectively, were developed for the detection of Junin, Machupo, Tacaribe, Amapari, Tamiami, Lassa and LCM arenaviruses. Both methods allow high sensitivity detection of arenavirus antigens and antibodies.

  8. Parallel solid-phase synthesis of diaryltriazoles

    Matthias Wrobel


    Full Text Available A series of substituted diaryltriazoles was prepared by a solid-phase-synthesis protocol using a modified Wang resin. The copper(I- or ruthenium(II-catalyzed 1,3-cycloaddition on the polymer bead allowed a rapid synthesis of the target compounds in a parallel fashion with in many cases good to excellent yields. Substituted diaryltriazoles resemble a molecular structure similar to established terphenyl-alpha-helix peptide mimics and have therefore the potential to act as selective inhibitors for protein–protein interactions.

  9. Evaluation of urinary metanephrine and normetanephrine enzyme immunoassay (ELISA) kits by comparison with isotope dilution mass spectrometry

    Wolthers, BG; Kema, IP; Volmer, M; Wesemann, R; Westermann, J; Manz, B


    Determination of urinary 3-O-methylated catecholamines (metanephrines) is generally considered a principal test for the clinical chemical diagnosis of pheochromocytoma and is currently performed predominantly with chromatographic techniques such as gas-liquid chromatography and HPLC. Enzyme immunoas

  10. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou, E-mail:


    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL{sup −1} with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO{sub 2} particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors.

  11. EQCM Immunoassay for Phosphorylated Acetylcholinesterase as a Biomarker for Organophosphate Exposures Based on Selective Zirconia Adsorption and Enzyme-Catalytic Precipitation

    Wang, Hua; Wang, Jun; Choi, Daiwon; Tang, Zhiwen; Wu, Hong; Lin, Yuehe


    A zirconia (ZrO2) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (AChE) as a potential biomarker for bio-monitoring exposures to organophosphate (OP) pesticides and chemical warfare agents. Hydroxyl-derivatized monolayer was preferably chosen to modify the crystal serving as the template for directing the electro-deposition of ZrO2 film with uniform nanostructures. The resulting ZrO2 film was utilized to selectively capture phosphorylated AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated protein. Enzyme-catalytic oxidation of the benzidine substrate resulted in the accumulation of insoluble product on the functionalized crystal. Ultrasensitive EQCM quantification by mass-amplified frequency responses as well as rapid qualification by visual color changes of product could be thus achieved. Moreover, 4-chloro-1-naphthol (CN) was comparably studied as an ideal chromogenic substrate for the enzyme-catalytic precipitation. Experimental results show that the developed EQCM technique can allow for the detection of phosphorylated AChE in human plasma. Such an EQCM immunosensing format opens a new door towards the development of simple, sensitive, and field-applicable biosensor for biologically monitoring low-level OP exposures.

  12. Pregnancy does not affect HIV incidence test results obtained using the BED capture enzyme immunoassay or an antibody avidity assay.

    Oliver Laeyendecker

    Full Text Available BACKGROUND: Accurate incidence estimates are needed for surveillance of the HIV epidemic. HIV surveillance occurs at maternal-child health clinics, but it is not known if pregnancy affects HIV incidence testing. METHODS: We used the BED capture immunoassay (BED and an antibody avidity assay to test longitudinal samples from 51 HIV-infected Ugandan women infected with subtype A, C, D and intersubtype recombinant HIV who were enrolled in the HIVNET 012 trial (37 baseline samples collected near the time of delivery and 135 follow-up samples collected 3, 4 or 5 years later. Nineteen of 51 women were also pregnant at the time of one or more of the follow-up visits. The BED assay was performed according to the manufacturer's instructions. The avidity assay was performed using a Genetic Systems HIV-1/HIV-2 + O EIA using 0.1M diethylamine as the chaotropic agent. RESULTS: During the HIVNET 012 follow-up study, there was no difference in normalized optical density values (OD-n obtained with the BED assay or in the avidity test results (% when women were pregnant (n = 20 results compared to those obtained when women were not pregnant (n = 115; for BED: p = 0.9, generalized estimating equations model; for avidity: p = 0.7, Wilcoxon rank sum. In addition, BED and avidity results were almost exactly the same in longitudinal samples from the 18 women who were pregnant at only one study visit during the follow-up study (p = 0.6, paired t-test. CONCLUSIONS: These results from 51 Ugandan women suggest that any changes in the antibody response to HIV infection that occur during pregnancy are not sufficient to alter results obtained with the BED and avidity assays. Confirmation with larger studies and with other HIV subtypes is needed.

  13. Solid phase microextraction device using aerogel

    Miller, Fred S.; Andresen, Brian D.


    A sample collection substrate of aerogel and/or xerogel materials bound to a support structure is used as a solid phase microextraction (SPME) device. The xerogels and aerogels may be organic or inorganic and doped with metals or other compounds to target specific chemical analytes. The support structure is typically formed of a glass fiber or a metal wire (stainless steel or kovar). The devices are made by applying gel solution to the support structures and drying the solution to form aerogel or xerogel. Aerogel particles may be attached to the wet layer before drying to increase sample collection surface area. These devices are robust, stable in fields of high radiation, and highly effective at collecting gas and liquid samples while maintaining superior mechanical and thermal stability during routine use. Aerogel SPME devices are advantageous for use in GC/MS analyses due to their lack of interfering background and tolerance of GC thermal cycling.

  14. Solid Phase Synthesis of Polymacromer and Copolymacromer Brushes


    REPORT Solid Phase Synthesis of Polymacromer and Copolymacromer Brushes 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: We report a novel solid phase...form poly-macromer brushes wherein macromonomers are linked via triazole groups. After each addition step, the terminal alkyne group can be deprotected...Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS Solid Phase Synthesis , polymers and copolymers Hernán R. Rengifo, Cristian Grigoras, Benjamin I

  15. Evaluation of recombinant outer membrane protein C based indirect enzyme-linked immunoassay for the detection of Salmonella antibodies in poultry

    Jinu Manoj


    Full Text Available Aim: To evaluate the efficacy of recombinant outer membrane proteinC (rOmpC based enzyme-linked immunoassay (ELISA for the diagnosis of salmonellosis in poultry. Materials and Methods: Three antigens were prepared, and the indirect ELISA was standardized using the antigens and the antiserum raised in chicken against Omp and rOmpC. Sera were collected from a total of 255 apparently healthy field chickens and screened for the presence of Salmonella antibodies by this ELISA. Results: The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Omp revealed major polypeptides at 36, 42 and 52 kDa, and the rOmpC was evident by a single protein band of 43 kDa. The Omp and rOmpC antigen revealed an optimum concentration of 78 and 156 ng, respectively, in the assay, while the whole cell antigen gave an optimum reaction at a concentration of 106 organisms/ml. The test was found to be specific as it did not react with any of the antisera of seven other organisms. The developed ELISA detected Salmonella antibodies from 22 (8.62% samples with rOmpC antigen, while 24 (9.41% samples gave a positive reaction with both Omp and whole cell antigens. Conclusion: We suggest rOmpC based indirect ELISA as a suitable screening tool for serological monitoring of poultry flocks.

  16. Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

    Rodrigo Otávio Silveira Silva


    Full Text Available Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs and one nucleic acid amplification test (NAAT were evaluated against a cytotoxicity assay (CTA and toxigenic culture (TC. Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2% of 92 samples were positive according to the CTA, and 23 (25% were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

  17. Rapid confirmation of enzyme multiplied immunoassay technique (EMIT) cocaine positive urine samples by capillary gas-liquid chromatography/nitrogen phosphorus detection (GLC/NPD).

    Verebey, K; DePace, A


    A rapid gas-liquid chromatographic (GLC) method was developed for the confirmation of benzoylecgonine (BE) positive urine samples screened by the enzyme multiplied immunoassay technique (EMIT) assay. The procedure is performed by solvent extraction of BE from 0.1 or 0.2 mL of urine, followed by an aqueous wash of the solvent and evaporation. The dried residue was derivatized with 50 microL of pentafluoropropionic anhydride and 25 microL of pentafluoropropropanol at 90 degrees C for 15 min. The derivatizing reagents were evaporated to dryness, and the derivatized BE, and cocaine if present, were reconstituted and injected into the gas chromatograph. The column was a 15-m by 0.2-mm fused silica capillary column, coated with 0.25 micron of DB-1, terminating in a nitrogen phosphorus detector (NPD). Cocaine and the pentafluoro BE derivatives retention times were 3.2 and 2.6 min, respectively. Nalorphine was used as reference or internal standard with a retention time of 4.78 min. The complete procedure can be performed in approximately 1.5 h. The EMIT cutoff between positive and negative urine samples is 300 ng/mL of BE. The lower limit of sensitivity of this method is 25 ng of BE extracted from urine. Validation studies resulted in confirmation of 101 out of 121 EMIT cocaine positive urine samples that could not be confirmed by thin-layer chromatography (TLC). This represents 84% confirmation efficiency.

  18. A sensitive enzyme immunoassay for human epidermal growth factor. Determination of hEGF in human serum and urine and pharmacokinetics in mouse.

    Hayashi, T; Hashimoto, K; Sakamoto, S


    A sensitive enzyme immunoassay for human epidermal growth factor (hEGF) is described. The anti-hEGF antibody was prepared by immunizing rabbits with hEGF, which was synthesized by Escherichia coli using the genetic engineering technique. The present assay system was based on the sandwiching of an antigen between anti-hEGF F(ab')2 precoated on a 96-well polystyrene plate and beta-D-galactosidase-labeled anti-hEGF Fab'. The range of measurable hEGF by this assay was 0.1-100 pg/well. Recoveries of hEGF added to serum and urine ranged between 94 and 108%. The intra- and inter-assay coefficients of variation were less than 6 and 8%, respectively. The results obtained by this assay method correlated well with those obtained by the radioimmunoassay method. By using this assay, the time course of serum hEGF levels in mice after the various administrations were also examined.

  19. Evaluation and Comparison of Enzyme Immunoassay (Eia and Acid Fast Staining with Confirmation by Immunofluorescent Antibody Assay for Detection of Cryptosporidium Species in Infants and Young Children.

    D Dorostcar Moghaddam


    Full Text Available Introduction: Cryptosporidiosis is prevalent world wide, causing a variety of problems ranging from acute, self-limiting diarrhea to fatal cases in immunocompromised persons, particulary those with acquired immunodeficiency (AIDS. Diagnosis of Cryptosporidium is made by identification of oocysts in stool specimens. The detection is most commonly made by the acid-fast staining method followed by microscopic examination which has low specificity and sensitivity. Material and Methods: In the present study, we evaluated diagnostic utility of a commercially available enzyme immunoassay (EIA, which detects Cryptosporidium-Specific antigen (CSA in 204 unprocessed stool specimens obtained from patients less than 3 years of age. Results: When compared with the routine screening procedure applied in this field study (screening by acid-fast staining and microscopy after concentration of positive results by IFA, both sensitivity and specificity were 98%. Of the 139 specimens negative by microscopy, 13 (9.3% were positive by EIA, 11 of which were confirmed by inhibition with antibody to Cryptosporidia-specific antigen. Conclusion: The EIA is an important tool for identifying Cryptosporidium in fecal specimens in field studies since it is sensitive, specific, simple to use and unaffected by the presence of a preservative.

  20. Clostridium difficile infection diagnostics - evaluation of the C. DIFF Quik Chek Complete assay, a rapid enzyme immunoassay for detection of toxigenic C. difficile in clinical stool samples.

    Johansson, Karin; Karlsson, Hanna; Norén, Torbjörn


    Diagnostic testing for Clostridium difficile infection (CDI) has, in recent years, seen the introduction of rapid dual-EIA (enzyme immunoassay) tests combining species-specific glutamate dehydrogenase (GDH) with toxin A/B. In a prospective study, we compared the C. DIFF Quik Chek Complete test to a combination of selective culture (SC) and loop-mediated isothermal amplification (LAMP) of the toxin A gene. Of 419 specimens, 68 were positive in SC including 62 positive in LAMP (14.7%). The combined EIA yielded 82 GDH positives of which 47 were confirmed toxin A/B positive (11%) corresponding to a sensitivity and specificity of 94% for GDH EIA compared to SC and for toxin A/B EIA a sensitivity of 71% and a specificity of 99% compared to LAMP. Twenty different PCR ribotypes were evenly distributed except for UK 081 where only 25% were toxin A/B positive compared to LAMP. We propose a primary use of a combined GDH toxin A/B EIA permitting a sensitive 1-h result of 379 of 419 (90%, all negatives plus GDH and toxin EIA positives) referred specimens. The remaining 10% being GDH positive should be tested for toxin A/B gene on the same day and positive results left to a final decision by the physician. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  1. Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus).

    Pribbenow, Susanne; Wachter, Bettina; Ludwig, Carsten; Weigold, Annika; Dehnhard, Martin


    In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs.

  2. Correlation between Clostridium difficile bacterial load, commercial real-time PCR cycle thresholds, and results of diagnostic tests based on enzyme immunoassay and cell culture cytotoxicity assay.

    Dionne, Léa-Laurence; Raymond, Frédéric; Corbeil, Jacques; Longtin, Jean; Gervais, Philippe; Longtin, Yves


    The impact of Clostridium difficile fecal loads on diagnostic test results is poorly understood, but it may have clinical importance. In this study, we investigated the relationship between C. difficile fecal load and the results of four assays: a glutamate dehydrogenase (GDH) enzyme immunoassay (EIA), a toxin A/B antigen EIA (ToxAB), a cell culture cytotoxicity assay (CCA), and PCR targeting the tcdB gene. We also compared the PCR cycle threshold (CT) with the results of quantitative culture using Spearman's rank correlation coefficient. Finally, we sequenced the genomes of 24 strains with different detection profiles. A total of 203 clinical samples harboring toxigenic C. difficile were analyzed and sorted into one of four groups: 17 PCR(+) (group 1), 37 PCR(+) GDH(+) (group 2), 24 PCR(+) GDH(+) CCA(+) (group 3), and 125 PCR(+) GDH(+) ToxAB(+) (group 4). The overall median fecal load in log10 CFU/g was 6.67 (interquartile range [IQR], 5.57 to 7.54). The median fecal bacterial load of groups 1, 2, 3, and 4 were 4.15 (IQR, 3.00 to 4.98), 5.74 (IQR, 4.75 to 6.16), 6.20 (IQR, 5.23 to 6.80), and 7.08 (IQR, 6.35 to 7.83), respectively. Group 1 samples had lower fecal loads than those from each of the other groups (P tests.

  3. Reactivity in the Venereal Diseases Research Laboratory test and the Mercia IgM enzyme immunoassay after treatment of early syphilis.

    McMillan, A; Young, H


    The aim of the study was to compare reactivity in the Mercia immunoglobulin M enzyme immunoassay (IgM EIA) and the Venereal Disease Research Laboratory (VDRL) after treatment of 229 previously untreated patients with early syphilis. At three months, the VDRL and the IgM EIA were negative in 41 (38%) and 71 (62%) cases, respectively; a four-fold or greater decrease in VDRL titre occurred in 106 (99%). At six months, the VDRL and the IgM EIA were negative in 45 (48%) and 69 (71%) patients, respectively; a four-fold or greater decrease in VDRL titre occurred in 88 (95%) and an eight-fold or greater decrease in 80 (86%). At 12 months, the VDRL and the IgM EIA were negative in 35 (70%) and 55 (92%) patients, respectively; a four-fold or greater decrease in VDRL titre occurred in 49 (98%) and an eight-fold or greater decrease in 47 (94%). The Mercia IgM EIA is as sensitive as the VDRL in monitoring treatment of primary syphilis but not as sensitive as the finding of a four-fold or eight-fold decrease in VDRL titre in patients treated for secondary or early latent infection.

  4. Identification of specific antinuclear antibodies in dogs using a line immunoassay and enzyme-linked immunosorbent assay.

    Bremer, Hanna D; Lattwein, Erik; Renneker, Stefanie; Lilliehöök, Inger; Rönnelid, Johan; Hansson-Hamlin, Helene


    Circulating antinuclear antibodies (ANA) are commonly present in the systemic autoimmune disease Systemic Lupus Erythematosus (SLE) and in other systemic rheumatic diseases, in humans as well as in dogs. The indirect immunofluorescence (IIF)-ANA test is the standard method for detecting ANA. Further testing for specific ANA with immunoblot techniques or ELISAs is routinely performed in humans to aid in the diagnosis and monitoring of disease. Several specific ANA identified in humans have been identified also in suspected canine SLE but, in contrast to humans, investigation of autoantibodies in canine SLE is mainly restricted to the IIF-ANA test. Our aim was to identify both known and novel specific ANA in dogs and to investigate if different IIF-ANA patterns are associated with different specific ANA in dogs. Sera from 240 dogs with suspicion of autoimmune disease (210 IIF-ANA positive (ANA(pos)) and 30 IIF-ANA negative (ANA(neg))) as well as sera from 27 healthy controls were included. The samples were analysed with a line immunoassay, LIA (Euroline ANA Profile 5, Euroimmun, Lübeck, Germany) and four different ELISAs (Euroimmun). The ANA(pos) dogs were divided in two groups depending on the type of IIF-ANA pattern. Of the 210 ANA(pos) samples 68 were classified as ANA homogenous (ANA(H)) and 141 as ANA speckled (ANA(S)), one sample was not possible to classify. Dogs in the ANA(H) group had, compared to the other groups, most frequently high levels of anti-double stranded deoxyribonucleic acid (dsDNA) and anti-nucleosome ANA. Anti-dsDNA antibodies were confirmed in some dogs with the Crithidia luciliae indirect immunofluorescence test (CLIFT). The frequency of ANA(H) dogs with values above those observed in the healthy group was significantly higher compared to ANA(S) dogs for anti-dsDNA, anti-nucleosome, and anti-histone reactivity. Dogs in the ANA(S) group had, compared to the other groups, most frequently high levels of anti-ribonucleoproteins (RNP) and

  5. Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

    Tang, Jin-Bao [School of Pharmacy, Weifang Medical University, Weifang 261053 (China); Tang, Ying [Affiliated Hospital of Weifang Medical University, Weifang 261041 (China); Yang, Hong-Ming, E-mail: [School of Pharmacy, Weifang Medical University, Weifang 261053 (China)


    Highlights: • An efficient signal amplification strategy for label-free EIA is proposed. • Divalent biotinylated AP and monovalent biotinylated ZZ were prepared via Avitag–BirA system. • The above site-specific biotinylated fusion proteins form complex via SA–biotin interaction. • The mechanism relies on the ZZ–Avi-B/SA/AP–(Avi-B){sub 2} complex. • The analytical signals are enhanced (32-fold) by the proposed strategy. - Abstract: Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable

  6. Performance of a limiting-antigen avidity enzyme immunoassay for cross-sectional estimation of HIV incidence in the United States.

    Jacob Konikoff

    Full Text Available A limiting antigen avidity enzyme immunoassay (HIV-1 LAg-Avidity assay was recently developed for cross-sectional HIV incidence estimation. We evaluated the performance of the LAg-Avidity assay alone and in multi-assay algorithms (MAAs that included other biomarkers.Performance of testing algorithms was evaluated using 2,282 samples from individuals in the United States collected 1 month to >8 years after HIV seroconversion. The capacity of selected testing algorithms to accurately estimate incidence was evaluated in three longitudinal cohorts. When used in a single-assay format, the LAg-Avidity assay classified some individuals infected >5 years as assay positive and failed to provide reliable incidence estimates in cohorts that included individuals with long-term infections. We evaluated >500,000 testing algorithms, that included the LAg-Avidity assay alone and MAAs with other biomarkers (BED capture immunoassay [BED-CEIA], BioRad-Avidity assay, HIV viral load, CD4 cell count, varying the assays and assay cutoffs. We identified an optimized 2-assay MAA that included the LAg-Avidity and BioRad-Avidity assays, and an optimized 4-assay MAA that included those assays, as well as HIV viral load and CD4 cell count. The two optimized MAAs classified all 845 samples from individuals infected >5 years as MAA negative and estimated incidence within a year of sample collection. These two MAAs produced incidence estimates that were consistent with those from longitudinal follow-up of cohorts. A comparison of the laboratory assay costs of the MAAs was also performed, and we found that the costs associated with the optimal two assay MAA were substantially less than with the four assay MAA.The LAg-Avidity assay did not perform well in a single-assay format, regardless of the assay cutoff. MAAs that include the LAg-Avidity and BioRad-Avidity assays, with or without viral load and CD4 cell count, provide accurate incidence estimates.

  7. Stable solid-phase Rh antigen.

    Yared, M A; Moise, K J; Rodkey, L S


    Numerous investigators have attempted to isolate the Rh antigens in a stable, immunologically reactive form since the discovery of the Rh system over 56 years ago. We report here a successful and reproducible approach to solubilizing and adsorbing the human Rh antigen(s) to a solid-phase matrix in an antigenically active form. Similar results were obtained with rabbit A/D/F red blood cell antigens. The antigen preparation was made by dissolution of the red blood cell membrane lipid followed by fragmentation of the residual cytoskeleton in an EDTA solution at low ionic strength. The antigenic activity of the soluble preparations was labile in standard buffers but was stable in zwitterionic buffers for extended periods of time. Further studies showed that the antigenic activity of these preparations was enhanced, as was their affinity for plastic surfaces, in the presence of acidic zwitterionic buffers. Adherence to plastic surfaces at low pH maintained antigenic reactivity and specificity for antibody was retained. The data show that this approach yields a stable form of antigenically active human Rh D antigen that could be used in a red blood cell-free assay for quantitative analysis of Rh D antibody and for Rh D antibody immunoadsorption and purification.

  8. A New Hapten for Immunoassay of Aldicarb

    Yan Feng ZHANG; Zhi Xian GAO; Qing Min ZHANG; Shu Gui DAI


    A new hapten, aldicarb oxime succinic ester (AOSE), was synthesized for immunoassay of aldicarb. It was conjugated to proteins by active ester method. Polyclonal antibody was raised against AOSE-BSA (bovine serum albumin) conjugate. Enzyme-linked immunosorbent assays (ELISAs) showed that this antiserum had high affinity to aldicarb and can be used for sensitive and selective immunoassay of aldicarb.

  9. Design of indirect solid-phase immunosorbent methods for detecting arenavirus antigens and antibodies

    Ivanov, A.P.; Rezapkin, G.V.; Dzagurova, T.K.; Tkachenko, E.A.


    Specifications have been elaborated for formulating indirect solid-phase enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (SPRIA) methods that employ anti-human and anti-mice G class immunoglobulin (IgG), conjugated with horseradish peroxidase and /sup 125/I for detecting the arenaviruses Junin, Machupo, Tacaribe, Amalpari, Tamiami, Lassa, and LCM (lymphocytic choriomeningitis). These methods make it possible to identify with a high degree of sensitivity arenavirus antigens and antibodies in various kinds of material.

  10. Effect of Microwave Radiation on Enzymatic and Chemical Peptide Bond Synthesis on Solid Phase

    Alessandra Basso


    Full Text Available Peptide bond synthesis was performed on PEGA beads under microwave radiations. Classical chemical coupling as well as thermolysin catalyzed synthesis was studied, and the effect of microwave radiations on reaction kinetics, beads' integrity, and enzyme activity was assessed. Results demonstrate that microwave radiations can be profitably exploited to improve reaction kinetics in solid phase peptide synthesis when both chemical and biocatalytic strategies are used.

  11. Assessment of the stress response in Columbian ground squirrels: laboratory and field validation of an enzyme immunoassay for fecal cortisol metabolites.

    Bosson, Curtis O; Palme, Rupert; Boonstra, Rudy


    Stress responses play a critical role in the ecology and demography of wild animals, and the analysis of fecal hormone metabolites is a powerful noninvasive method to assess the role of stress. We characterized the metabolites of injected radiolabeled cortisol in the urine and feces of Columbian ground squirrels and validated an enzyme immunoassay for measuring fecal cortisol metabolites (FCM) with a 5 alpha-3beta,11 beta-diol structure by stimulation and suppression of adrenocortical activity and by evaluation of the circadian pattern of FCM excretion. In addition, we also evaluated the impact of capture, handling, and acclimation to the laboratory on FCM. Cortisol is highly metabolized, with virtually none being excreted, and of the radiolabeled cortisol injected, 31% was recovered in urine and 6.5% in feces. The lag time between cortisol injection and its appearance in urine and feces was 4.5 +/- 0.82 (SE) h and 7.0 +/- 0.53 (SE) h, respectively. FCM levels varied over the day, reflecting circadian variation in endogenous cortisol. Dexamethasone decreased FCM levels by 33%, and ACTH increased them by 255%. Trapping and housing initially increased FCM levels and decreased body mass, but these reversed within 3-7 d, indicating acclimation. Finally, FCM levels were modestly repeatable over time (r=0.57) in wild, live trapped, nonbreeding animals, indicating that FCMs provide a measure of the squirrel's stress-axis state. This assay provides a robust noninvasive assessment of the stress response of the Columbian ground squirrel and will facilitate an integration of its life history and physiology.

  12. Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study.

    Schwettmann, Lutz; Külpmann, Wolf-Rüdiger; Vidal, Christian


    Two commercially available drug-screening assays were evaluated: the Roche kinetic interaction of microparticles in solution (KIMS) assay and the Microgenics cloned enzyme donor immunoassay (CEDIA). Urine samples from known drug-abuse patients were analyzed for amphetamines, barbiturates, benzodiazepines, benzoylecgonine, cannabinoids, LSD, methadone and opiates. Samples with discordant findings for the two assays were analyzed by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/electron capture detection (GC/ECD). Amphetamines showed 96.0% concordant results, with two false positive findings by CEDIA, three by KIMS and a further two false negatives by KIMS. Barbiturates showed 99.4% concordant results, with one false negative by KIMS. Benzodiazepines showed 97.4% concordant results, with two false negatives by KIMS (cutoff 100 microg/L, CEDIA cutoff 300 microg/L). Benzoylecgonine showed 17.8% concordant positive and 82.2% concordant negative results and no false finding by either assay. Cannabinoids showed 99.3% concordant results, with one sample negative by KIMS at a cutoff of 50 microg/L and positive by CEDIA (cutoff 25 microg/L). For LSD, 6.7% of findings were not in agreement. Methadone showed 97.5% concordant results, with two false positives by CEDIA, and one false positive and one false negative by KIMS. Opiates showed 96.9% concordant results, with no false KIMS results, but four false positives by CEDIA. The results indicate that the agreement of the CEDIA and KIMS results for the eight drugs is rather good (93.3-100%).

  13. Variation in the limit-of-detection of the ProSpecT Campylobacter microplate enzyme immunoassay in stools spiked with emerging Campylobacter species.

    Bojanić, Krunoslav; Midwinter, Anne Camilla; Marshall, Jonathan Craig; Rogers, Lynn Elizabeth; Biggs, Patrick Jon; Acke, Els


    Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. The impact of other Campylobacter spp. is likely to be underestimated due to the bias of culture methods towards Campylobacter jejuni/coli diagnosis. Stool antigen tests are becoming increasingly popular and appear generally less species-specific. A review of independent studies of the ProSpecT® Campylobacter Microplate enzyme immunoassay (EIA) developed for C. jejuni/coli showed comparable diagnostic results to culture methods but the examination of non-jejuni/coli Campylobacter spp. was limited and the limit-of-detection (LOD), where reported, varied between studies. This study investigated LOD of EIA for Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter helveticus spiked in human stools. Multiple stools and Campylobacter isolates were used in three different concentrations (10(4)-10(9)CFU/ml) to reflect sample heterogeneity. All Campylobacter species evaluated were detectable by EIA. Multivariate analysis showed LOD varied between Campylobacter spp. and faecal consistency as fixed effects and individual faecal samples as random effects. EIA showed excellent performance in replicate testing for both within and between batches of reagents, in agreement between visual and spectrophotometric reading of results, and returned no discordance between the bacterial concentrations within independent dilution test runs (positive results with lower but not higher concentrations). This study shows how limitations in experimental procedures lead to an overestimation of consistency and uniformity of LOD for EIA that may not hold under routine use in diagnostic laboratories. Benefits and limitations for clinical practice and the influence on estimates of performance characteristics from detection of multiple Campylobacter spp. by EIA are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Assessment of pregnancy status of Asian elephants (Elephas maximus) by measurement of progestagen and glucocorticoid and their metabolite concentrations in serum and feces, using enzyme immunoassay (EIA).

    Kajaysri, Jatuporn; Nokkaew, Weerapun


    The study was to find patterns of progestagen (progesterone and its metabolite) and glucocorticoid and their metabolite concentrations in serum and feces of pregnant Asian elephants (Elephas maximus). The 5 female Asian domestic elephants were naturally mated until pregnancy. After that, blood and feces samples were collected monthly during pregnancy for progestagen, glucocorticoid and their metabolites analysis by enzyme immunoassay (EIA). The results showed the serum progestagen concentration during gestation was 2.11 ± 0.60 to 18.44 ± 2.28 ng/ml. Overall, serum progestagen concentration rose from the 1st month to reach peak in the 11th month, after which it declined to its lowest level in the 22nd month of pregnancy. Fecal progestagen concentration varied from 1.18 ± 0.54 to 3.35 ± 0.45 µg/g during pregnancy. In general, fecal progestagen concentration increased from the 1st month to its highest level in the 12th month. After this, it declined reaching its lowest point in the 22nd month of pregnancy. Glucocorticoid hormones and their metabolite concentrations both in serum and feces fluctuated from low to medium throughout almost the entire pregnancy period and then rapidly increased around the last week before calving. Our study suggests that this profile of progestagen and glucocorticoid hormones and their metabolite concentration levels in serum and feces can be used to assess the pregnancy status of Asian elephants. If serum and fecal progestagen concentrations were found in very low levels and glucocorticoid and their metabolite concentrations were found in very high levels, it was indicated that the cow elephant would calve within 7 days.

  15. Clinical evaluation of a new enzyme immunoassay for hepatitis B virus core-related antigen; a marker distinct from viral DNA for monitoring lamivudine treatment.

    Rokuhara, A; Tanaka, E; Matsumoto, A; Kimura, T; Yamaura, T; Orii, K; Sun, X; Yagi, S; Maki, N; Kiyosawa, K


    We aimed to assess the clinical performance of a newly developed chemiluminescence enzyme immunoassay (CLEIA) for the detection of hepatitis B virus (HBV) core-related antigen (HBcrAg) in patients with chronic HBV infection. A total of 82 patients with chronic HBV infection and 167 HBV-negative controls were studied. HBcrAg was measured by CLEIA with monoclonal antibodies to hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBcAg), and HBV DNA was measured by transcription-mediated amplification assay (TMA) and in-house real-time detection polymerase chain reaction (RTD-PCR). The HBcrAg assay detected viremia in 189 of 216 samples (88%) collected from 72 patients whilst the TMA assay detected viremia in 178 of the 216 samples (82%) (P = 0.019). The HBcrAg concentration correlated linearly with the HBV DNA concentration (P HBcrAg assay was not affected by the absence of hepatitis B e antigen from the serum or the presence of precore mutations in the HBV genome. In patients without anti-viral drugs, changes in their serum HBcrAg concentration over time corresponded to their HBV DNA concentration. In six additional patients who were later treated with lamivudine, HBV DNA concentration declined more rapidly than their HBcrAg concentration. Three months after treatment commenced, the ratio of HBcrAg: HBV DNA had increased in all six patients (P = 0.031). The HBcrAg assay is a sensitive and useful test for the assessment of a patient's HBV load. When monitoring the anti-viral effect of lamivudine, HBcrAg provides a viral marker which is independent of HBV DNA.

  16. Characterisation and validation of an enzyme-immunoassay for the non-invasive assessment of faecal glucocorticoid metabolites in cheetahs (Acinonyx jubatus).

    Ludwig, C; Wachter, B; Silinski-Mehr, S; Ganswindt, A; Bertschinger, H; Hofer, H; Dehnhard, M


    The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0-4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at -20°C is not feasible. The two cheetahs in Germany also received an injection of [(3)H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs.

  17. Preparation of Ion Exchange Films for Solid-Phase Spectrophotometry and Solid-Phase Fluorometry

    Hill, Carol M.; Street, Kenneth W.; Tanner, Stephen P.; Philipp, Warren H.


    Atomic spectroscopy has dominated the field of trace inorganic analysis because of its high sensitivity and selectivity. The advantages gained by the atomic spectroscopies come with the disadvantage of expensive and often complicated instrumentation. Solid-phase spectroscopy, in which the analyte is preconcentrated on a solid medium followed by conventional spectrophotometry or fluorometry, requires less expensive instrumentation and has considerable sensitivity and selectivity. The sensitivity gains come from preconcentration and the use of chromophore (or fluorophore) developers and the selectivity is achieved by use of ion exchange conditions that favor the analyte in combination with speciative chromophores. Little work has been done to optimize the ion exchange medium (IEM) associated with these techniques. In this report we present a method for making ion exchange polymer films, which considerably simplify the solid-phase spectroscopic techniques. The polymer consists of formaldehyde-crosslinked polyvinyl alcohol with polyacrylic acid entrapped therein. The films are a carboxylate weak cation exchanger in the calcium form. They are mechanically sturdy and optically transparent in the ultraviolet and visible portion of the spectrum, which makes them suitable for spectrophotometry and fluorometry.

  18. Binding of properdin to solid-phase immune complexes

    Junker, A; Baatrup, G; Svehag, S E


    The capacity of serum to support deposition of C3, properdin and factor B was studied by enzyme-linked immunosorbent assay using solid-phase immune complexes (IC) for activation of complement. Deposition of C3 and properdin occurred in fairly dilute normal human serum (NHS), but factor B uptake...... was hardly detectable. Alternative pathway-mediated deposition of C3 with slow kinetics was demonstrated in C2-deficient serum and in NHS depleted of C1q, factor D and properdin (C1qDP-depleted serum) after reconstitution with factor D and properdin. Efficient uptake of properdin required a functional...... classical pathway, in the presence of which C3 and properdin were rapidly deposited onto the IC. Judging from findings in C3-deficient serum, factor I-deficient serum, and C1qDPB-depleted serum, the uptake of properdin was strictly C3-dependent, and did not require the presence of factors B and D. Thus, C3b...

  19. Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork

    Dong Jiexian; Li Zhenfeng; Lei Hongtao; Sun Yuanming [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Ducancel, Frederic [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Xu Zhenlin [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Boulain, Jean-Claude [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Yang Jinyi; Shen Yudong [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Wang Hong, E-mail: [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China)


    Graphical abstract: Detection model of dc-CLEIA based on anti-RAC scFv-AP fusion protein. Highlights: Black-Right-Pointing-Pointer The scFv-AP fusion protein against ractopamine (RAC) was produced. Black-Right-Pointing-Pointer A dc-CLEIA for RAC was developed based on the purified scFv-AP fusion protein. Black-Right-Pointing-Pointer The sensitivity of dc-CLEIA was 10 times as sensitive as dc-ELISA for RAC. Black-Right-Pointing-Pointer Recovery tests from pork samples were studied. Black-Right-Pointing-Pointer Good accuracy was obtained. - Abstract: A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V{sub H} and V{sub L}) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V{sub H} and V{sub L} genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 {+-} 0.03 and 0.02 {+-} 0.004 ng mL{sup -1}, respectively, and the linear response range extended from 0.05 to 1.45 ng mL{sup -1}. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between

  20. A solid-phase dot assay using silica/gold nanoshells

    Zharov Vladimir


    Full Text Available AbstractWe report on the first application of silica-gold nanoshells to a solid-phase dot immunoassay. The assay principle is based on staining of a drop (1 µl analyte on a nitrocellulose membrane strip by using silica/gold nanoshells conjugated with biospecific probing molecules. Experimental example is human IgG (hIgG, target molecules and protein A (probing molecules. For usual 15-nm colloidal gold conjugates, the minimal detectable amount of hIgG is about 4 ng. By contrast, for nanoshell conjugates (silica core diameter of 70 nm and gold outer diameter of 100 nm we have found significant increase in detection sensitivity and the minimal detectable amount of hIgG is about 0.5 ng. This finding is explained by the difference in the monolayer particle extinction.

  1. Paper-based enzyme-free immunoassay for rapid detection and subtyping of influenza A H1N1 and H3N2 viruses.

    Lei, Kin Fong; Huang, Chia-Hao; Kuo, Rei-Lin; Chang, Cheng-Kai; Chen, Kuan-Fu; Tsao, Kuo-Chien; Tsang, Ngan-Ming


    Development of rapid screening in the ambulatory environment is the most pressing needs for the control of spread of infectious disease. Despite there are many methods to detect the immunoassay results, quantitative measurement in rapid disease screening is still a great challenge for point-of-care applications. In this work, based on the internal structural protein, i.e., nucleoprotein (NP), and outer surface glycoproteins, i.e., H1 and H3, of the influenza viruses, specific and sensitive immunoassay on paper-based platform was evaluated and confirmed. Detection and subtyping of influenza A H1N1 and H3N2 viruses found in people were demonstrated by colorimetric paper-based sandwich immunoassay. Concentration-dependent response to influenza viruses was shown and the detection limits could achieve 2.7×10(3) pfu/assay for H1 detection and 2.7×10(4) pfu/assay for H3 detection, which are within the clinical relevant level. Moreover, detection of influenza virus from infected cell lysate and clinical samples was demonstrated to further confirm the reliability of the paper-based immunoassay. The use of paper for the development of diagnostic devices has the advantages of lightweight, ease-of-use, and low cost and paper-based immunoassay is appropriate to apply for rapid screening in point-of-care applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Automated solid-phase synthesis of oligosaccharides containing sialic acids

    Chian-Hui Lai


    Full Text Available A sialic acid glycosyl phosphate building block was designed and synthesized. This building block was used to prepare α-sialylated oligosaccharides by automated solid-phase synthesis selectively.


    B. S. Chandravanshi

    cation exchange-solid phase extraction (SCX-SPE) was investigated as an .... Stock solutions, with a concentration of 1.00 mg/mL were prepared ... Johannesburg, South Africa) connected to a vacuum pump (Vacuubrand, GMBH, Germany).

  4. Combinatorial Solid-Phase Synthesis of Balanol Analogues

    Nielsen, John; Lyngsø, Lars Ole


    The natural product balanol has served as a template for the design and synthesis of a combinatorial library using solid-phase chemistry. Using a retrosynthetic analysis, the structural analogues have been assembled from three relatively accessible building blocks. The solid-phase chemistry inclu...... including MSNT-mediated esterification of both support-bound alcohols and carboxylic acids has been implemented successfully. Copyright (C) 1996 Elsevier Science Ltd....

  5. Combinatorial Solid-Phase Synthesis of Balanol Analogues

    Nielsen, John; Lyngsø, Lars Ole


    The natural product balanol has served as a template for the design and synthesis of a combinatorial library using solid-phase chemistry. Using a retrosynthetic analysis, the structural analogues have been assembled from three relatively accessible building blocks. The solid-phase chemistry inclu...... including MSNT-mediated esterification of both support-bound alcohols and carboxylic acids has been implemented successfully. Copyright (C) 1996 Elsevier Science Ltd....

  6. Fuel spill identification using solid-phase extraction and solid-phase microextraction. 1. Aviation turbine fuels.

    Lavine, B K; Brzozowski, D M; Ritter, J; Moores, A J; Mayfield, H T


    The water-soluble fraction of aviation jet fuels is examined using solid-phase extraction and solid-phase microextraction. Gas chromatographic profiles of solid-phase extracts and solid-phase microextracts of the water-soluble fraction of kerosene- and nonkerosene-based jet fuels reveal that each jet fuel possesses a unique profile. Pattern recognition analysis reveals fingerprint patterns within the data characteristic of fuel type. By using a novel genetic algorithm (GA) that emulates human pattern recognition through machine learning, it is possible to identify features characteristic of the chromatographic profile of each fuel class. The pattern recognition GA identifies a set of features that optimize the separation of the fuel classes in a plot of the two largest principal components of the data. Because principal components maximize variance, the bulk of the information encoded by the selected features is primarily about the differences between the fuel classes.

  7. Comparasion of polyacrylamide gel electrophoresis (PAGE, immuno-electron microscopy (IEM and enzyme immunoassay (EIA for the rapid diagnosis of rotavirus infection in children

    H. G. Pereira


    Full Text Available Detection of rotavirus RNA by polyacrylamide gel electrophoresis (PAGE proved to be a highly sensitive and rapid diagnostic test. A comparison of this assay with immuno-electron microscopy (IEM and enzyme immunoassay (EIA in 245 faeces from children with gastroenteritis revealed complete agreement between the three assays in 238 (97.14% samples. Among 75 samples positive in at least one of the three assays, negative results were observed in 5 (6.48% by PAGE, in 6 (6.76% by EIA and in none by IEM. Silver staining greatly increased the sensitivity of the PAGE assay. We conclude that although IEM remains the most sensitive and rapid rotavirus diagnostic assay, the PAGE technique has many advantages in its favour, including the non-requirement of expensive equipment, the use of only chemically defined reagents and the capacity to distinguish virus subgroup and variants and to detect non-crossreactive rotaviruses which are missed in serological assays.A evidenciação da presença de ácido ribonucleico (ARN viral por eletroforese em gel de policrilamida (EGPA foi comprovada como um método altamente sensível e rápido para o diagnóstico de infecções por rotavirus. Uma comparação desta prova com a imunomicroscopia eletrônica (IEM e com o ensaio imunoenzimático (EIE no exame de 245 fezes de crianças com gastroenterite revelou completa concordancia entre os três ensaios em 238 (97.14% amostras. Entre 75 amostras positivas pelo menos em um dos três ensaios, resultados negativos foram observados em 5 (6.48% por EGPA, em 6 (6.76% por EIE e em nenhum por IEM. Coloração pela prata aumentou consideravelmente a sensibilidade do ensaio por EGPA. Concluímos que embora a IEM ainda seja a prova mais sensível e rápida para o diagnóstico de infecções por rotavirus, o ensaio por EGPA tem muitas vantagens em seu favor, sendo as principais as de não necessitar equipamentos caros, de empregar exclusivamente reagentes quimicamente definidos, de

  8. Application of a newly developed high-sensitivity HBsAg chemiluminescent enzyme immunoassay for hepatitis B patients with HBsAg seroclearance.

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi; Tanaka, Yasuhito


    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring.

  9. Development of a biotin-streptavidin amplified enzyme immunoassay for oxytocin and its application during milk ejection and the reproductive cycle in the mithun (Bos frontalis).

    Mondal, Mohan; Rajkhowa, Chandan; Prakash, Bukkaraya Samudram


    Oxytocin is a key hormone involved in milk ejection. It plays a key role in regulation of reproductive cyclicity in female mammals by taking part in the process of luteolysis. Determination of oxytocin is, therefore, important for studying the control of its secretion and its role in reproduction of the mithun. A simple and sufficiently sensitive enzyme immunoassay (EIA) for oxytocin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique was therefore developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in a competitive assay. The EIA was conducted directly in 200 microl of unknown mithun plasma. Standards prepared in hormone-free plasma were used. The lowest detection limit was 0.5 pg/ml plasma. Plasma volumes for the EIA (50, 100, and 200 microl) did not influence the shape of standard curve, even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare endogenous mithun oxytocin with a bovine oxytocin standard. The former showed good parallelism with the bovine standard curve. For biological validation of the assay, plasma oxytocin was measured in the blood samples collected before, during, and after milking in three mithun cows and in six non-lactating cyclic mithuns during the entire estrous cycle. A sharp release of oxytocin shortly after udder stimulation was observed. A high level of oxytocin was maintained during milking, falling sharply thereafter. The mean plasma oxytocin concentration was different on different days of the estrous cycle (P oxytocin were recorded, one at day 6 and another at day 18 of the estrous cycle. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine plasma oxytocin levels in mithuns. Apart from being non-radioactive, the EIA procedure described here also utilizes a highly

  10. Enzyme-linked immunosorbent assay and colloidal gold immunoassay for sulphamethazine residues in edible animal foods: investigation of the effects of the analytical conditions and the sample matrix on assay performance.

    Wang, Lei; Wang, Shuo; Zhang, Jiayi; Liu, Junwei; Zhang, Yan


    To determine sulphamethazine (SMZ) residues in edible animal foods (pig muscle, chicken muscle, egg, fish, milk and liver), a competitive direct enzyme-linked immunosorbent assay (ELISA) and a colloidal gold immunoassay were established. The limits of detection of the ELISA and the colloidal gold immunoassay were 0.02 and 0.5 microg kg(-1), respectively. The specificity of the ELISA developed to the SMZ was high according to the results of cross-reactivity testing with 14 kinds of sulphonamides. To obtain a more sensitive immunoassay, buffer solution (30 mmol L(-1) phosphate-buffered saline with 0.05% Tween 20, pH 8.5) was optimized through the whole test procedure. A simple and efficient extraction method for the rapid detection of SMZ residues in foods was developed, with recoveries between 74 and 117.5%. Matrix effects can be avoided by 1:10 dilution of pig muscle, chicken muscle, egg, fish, milk and liver with optimal buffer. The detection limit of SMZ was 5 microg kg(-1) in liver and 2 microg kg(-1) in the other five samples. For the validation of the ELISA tests, sample extracts were analysed by ELISA and high-performance liquid chromatography. The results obtained by these two methods showed a good correlation (r(2)) which was greater than 0.9. The colloidal gold immunoassay presented in this assay was successfully applied to determine SMZ in pig muscle, milk and fish below or equal to the maximum residue level (20 microg kg(-1)).

  11. Immunoassay of paralytic shellfish toxins by moving magnetic particles in a stationary liquid-phase lab-on-a-chip.

    Kim, Myoung-Ho; Choi, Suk-Jung


    In this study, we devised a stationary liquid-phase lab-on-a-chip (SLP LOC), which was operated by moving solid-phase magnetic particles in the stationary liquid phase. The SLP LOC consisted of a sample chamber to which a sample and reactants were added, a detection chamber containing enzyme substrate solution, and a narrow channel connecting the two chambers and filled with buffer. As a model system, competitive immunoassays of saxitoxin (STX), a paralytic shellfish toxin, were conducted in the SLP LOC using protein G-coupled magnetic particles (G-MPs) as the solid phase. Anti-STX antibodies, STX-horseradish peroxidase conjugate, G-MPs, and a STX sample were added to the sample chamber and reacted by shaking. While liquids were in the stationary state, G-MPs were transported from the sample chamber to the detection chamber by moving a magnet below the LOC. After incubation to allow the enzymatic reaction to occur, the absorbance of the detection chamber solution was found to be reciprocally related to the STX concentration of the sample. Thus, the SLP LOC may represent a novel, simple format for point-of-care testing applications of enzyme-linked immunosorbent assays by eliminating complicated liquid handling steps.

  12. Performance of the TechLab C. DIFF CHEK-60 enzyme immunoassay (EIA) in combination with the C. difficile Tox A/B II EIA kit, the Triage C. difficile panel immunoassay, and a cytotoxin assay for diagnosis of Clostridium difficile-associated diarrhea.

    Snell, Heather; Ramos, Meredith; Longo, Sue; John, Michael; Hussain, Zafar


    We compared a recently marketed enzyme immunoassay for glutamate dehydrogenase (GDH), TechLab's C. DIFF CHEK-60 (TL-GDH), in combination with the C. difficile Tox A/B II enzyme immunoassay (Tox-A/B) with (i) the Triage C. difficile test, which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); (ii) an in-house cytotoxin assay (C-Tox); and (iii) stool cultures for C. difficile. All C. difficile isolates were tested for the presence of the toxin genes by PCR. If a toxin gene-positive strain of Clostridium difficile was recovered and a toxin was detected by any method, the result was considered to be truly positive. Eighty-seven of 93 and 79 of 93 C. difficile culture-positive samples were also TL-GDH and TR-GDH positive, respectively. No test was able to detect toxin in all samples with true-positive results. Tox-A/B and TR-Tox-A in combination with the GDH detection tests and C-Tox were able to identify 52 and 50 samples with true-positive results. Tox-A/B and TR-Tox-A would have missed 15 and 31% of cases of C. difficile-associated diarrhea, respectively, if used alone.

  13. Solid-phase techniques in blood transfusion serology.

    Beck, M L; Plapp, F V; Sinor, L T; Rachel, J M


    For nearly a century, erythrocyte agglutination has persisted as the most widely used method for the demonstration of antigen-antibody reaction in immunohematology. So far, no other system has been developed which can match its simplicity, versatility, and general reliability. The major disadvantage of agglutination reactions is the lack of an objective endpoint, which has severely hindered attempts to automate routine pretransfusion tests. To overcome this problem, we have designed a series of solid-phase assays for ABO and Rh grouping, antibody screening, compatibility, and hepatitis tests. Each of these solid-phase assays shares a common endpoint of red cell adherence, which is easily interpreted visually or spectrophotometrically. Computer interface permits the automatic interpretation and recording of results. We believe this solid-phase system should finally bring the blood bank laboratory into the age of automation.

  14. 乙型肝炎病毒的液相杂交技术在聚合酶链反应酶联免疫检测的应用探讨%The exploration on application of liquid hybridization of hepatitis B virus in polymerase chain reaction enzyme-linked immunoassay



    OBJECTIVE To take the use of liquid hybridization, to simplify nucleic acid hybridization techniques and establish the method of liquid hybridization of hepatitis B virus (HBV) polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). METHODS Took HBV for the purpose of target DNA, primere of P15 side marked the Bio-PCR amplification of 37 cycles. PCR products of the 5' end tag digoxin probe was liquid mixture, 85℃ 2.5min, 60℃ 1min were taken for hybridization. The hybridization products were fixed by streptomycin and prime microtiter plates, anti-digoxigenin-labeled antibody was recod-ed, combined and colored by microplate. RESULTS The optimization of the liquid hybridization ELISA conditions: probe concentration of digoxin/time was 2.4pmol, liquid hybridization time was 2.5min, and solid-phase hybridization time was 100min, the test results had no significant differences {P> 0.05). The detection rate of reagent for 87 " of HBsAg +, HBeAg +, anti-HBc +" specimens positive were 91.95%. The positive detection rate for 72 cases " of HBsAg +, anti-Hbe +, anti-HBc +" specimens was 68.06%. The combination of detection index for other immune specimen was 16 cases. CONCLUSION The HBV liquid hybridization technology is used in the polymerase chain reaction enzyme-linked immunoassay (PCR-ELJSA). The experimental operation of the hybrid technology is simple, fast, and it is suitable for clinical laboratory testing.%目的 利用液相杂交技术,将核酸杂交技术进行简化,建立乙型肝炎病毒(HBV)的液相杂交的聚合酶链反应酶联免疫检测(PCR-ELISA)方法.方法 以HBV为目的靶DNA,引物P15'端标记Bio-PCR扩增37个循环.PCR扩增产物与5’端标记地高辛的探针呈液相混合,85cC 2.5 min,60C1 min进行杂交,杂交后产物均经过链霉素亲和素酶标板进行固定,经过酶标仪记录抗地高辛标记抗体,结合、显色.结果 液相杂交酶联免疫检测条件的优化:地高辛的探针浓度为2.4 pmol

  15. Magnetic Solid Phase Extraction Applied to Food Analysis

    Israel S. Ibarra


    Full Text Available Magnetic solid phase extraction has been used as pretreatment technique for the analysis of several compounds because of its advantages when it is compared with classic methods. This methodology is based on the use of magnetic solids as adsorbents for preconcentration of different analytes from complex matrices. Magnetic solid phase extraction minimizes the use of additional steps such as precipitation, centrifugation, and filtration which decreases the manipulation of the sample. In this review, we describe the main procedures used for synthesis, characterization, and application of this pretreatment technique which were applied in food analysis.


    Yu-ying Li; Jia-song He


    Solid phase transition of the a form crystals to the β form crystals in syndiotactic polystyrene (sPS) samples has occurred in supercritical CO2. This transformation is different from those detected under other conditions. The effects of some factors (e.g. time, temperature, and pressure) on the solid phase transformation of sPS in supercritical CO2 were analyzed in detail. Experimental results show that longer time, higher temperature or higher pressure favors the transformation of the α form crystals to the β form crystals.

  17. Rapid detection of fungal alpha-amylase in the work environment with a lateral flow immunoassay

    Bogdanovic, J.; Koets, M.; Sander, I.; Wouters, I.; Meijster, T.; Heederik, D.J.J.; Amerongen, van A.; Doekes, G.


    Background Occupational allergen exposure assessment usually requires airborne dust sampling at the worksite followed by dust extraction and enzyme immunoassay (EIA) analysis at the laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with

  18. Solid-phase oligosaccharide and glycopeptide synthesis using glycosynthases

    Tolborg, Jakob Fjord; Petersen, Lars; Jensen, Knud Jørgen;


    Enzymatic approaches for the preparation of oligosaccharides are interesting alternatives to traditional chemical synthesis, the main advantage being the regio- and stereoselectivity offered without the need for protecting groups. The use of solid-phase techniques offers easy workup procedures an...

  19. N-Acyliminium Intermediates in Solid-Phase Synthesis

    Quement, Sebastian Thordal le; Petersen, Rico; Meldal, M.


    N-Acyliminium ions are powerful intermediates in synthetic organic chemistry. Examples of their use are numerous in solution-phase synthesis, but there are unmerited few reports on these highly reactive electrophiles in solid-phase synthesis. The present review covers the literature to date and i...

  20. Solid Phase Synthesis of Ethyl β-Substituted Indolepropionates

    刘占祥; 阮秀秀; 黄宪


    A facile solid phase synthesis of ethyl β-substituted indolepropionates is reported. Condensation between indole, polymer-supported cyclic malonic acid ester and aldehyde yielded the trimolecular adducts, which was cleaved by pyridine/EtOH to release the final products in good yield with high purity.

  1. Recent Approaches Toward Solid Phase Synthesis of β-Lactams

    Mandal, Bablee; Ghosh, Pranab; Basu, Basudeb

    Since the discovery of penicillin in 1929, β-lactam antibiotics have been recognized as potentially chemotherapeutic drugs of incomparable effectiveness, conjugating a broad spectrum of activity with very low toxicity. The primary motif azetidin-2-one ring (β-lactam) has been considered as specific pharmacophores and scaffolds. With the advent of combinatorial chemistry and automated parallel synthesis coupled with ample interests from the pharmaceutical industries, recent trends have been driven mostly by adopting solid phase techniques and polymer-supported synthesis of β-lactams. The present survey will present an overview of the developments on the polymer-supported and solid phase techniques for the preparation of β-lactam ring or β-lactam containing antibiotics published over the last decade. Both unsubstituted and substitutions with different functional groups at various positions of β-lactams have been synthesized using solid phase technology. However, Wang resin and application of Staudinger [2+2] cycloaddition reaction have remained hitherto the major choice. It may be expected that other solid phase approaches involving different resins would be developed in the coming years.

  2. Solid phase extraction method for determination of mitragynine in ...

    mitragynine in urine and its application to mitragynine excretion ... Purpose: To develop a solid phase extraction (SPE) method that utilizes reverse-phase high performance .... solution of MG (1 mg/mL) which was further ... Facility, Prince of Songkla University and carried ..... d), which permit unrestricted use, distribution,.

  3. Solid-phase synthesis of 3-amino-2-pyrazolines

    Lyngsø, Lars O.; Nielsen, John


    The development of a solid-phase synthesis of 3-amino-2-pyrazolines is described. Conjugate addition of hydrazines to α,β-unsaturated nitriles followed by cyclization yields 3-amino-2-pyrazolines. Acylation or sulfonation of the free amino-group yields a 24 member library of 3-amino-2- pyrazolines....

  4. Solid-phase synthesis of 3-amino-2-pyrazolines

    Nielsen, John


    The development of a solid-phase synthesis of 3-amino-2-pyrazolines is described. Conjugate addition of hydrazines to alpha,beta-unsaturated nitriles followed by cyclization yields 3-amino-2-pyrazolines. Acylation or sulfonation of the free amino-group yields a 24 member library of 3-amino-2...

  5. Solid-phase synthesis of complex and pharmacologically interesting heterocycles

    Nielsen, Thomas Eiland


    Efficient routes for the creation of heterocycles continue to be one of the primary goals for solid-phase synthesis. Recent advances in this field rely most notably on transition-metal-catalysis and N-acyliminium chemistry to mediate a range of cyclization processes for the generation of compounds...

  6. Solid-phase synthesis of 3-amino-2-pyrazolines

    Lyngsø, Lars O.; Nielsen, John


    The development of a solid-phase synthesis of 3-amino-2-pyrazolines is described. Conjugate addition of hydrazines to α,β-unsaturated nitriles followed by cyclization yields 3-amino-2-pyrazolines. Acylation or sulfonation of the free amino-group yields a 24 member library of 3-amino-2- pyrazolines....

  7. Solid-phase synthesis of complex and pharmacologically interesting heterocycles

    Nielsen, Thomas Eiland


    Efficient routes for the creation of heterocycles continue to be one of the primary goals for solid-phase synthesis. Recent advances in this field rely most notably on transition-metal-catalysis and N-acyliminium chemistry to mediate a range of cyclization processes for the generation of compounds...

  8. Solid Phase Characterization of Solids Recovered from Failed Sluicer Arm

    Cooke, Gary A. [Hanford Site (HNF), Richland, WA (United States)


    The Enclosure to this memo discusses the solid phase characterization of a solid sample that was retrieved from the single-shell Tank 241-C-111 extended reach sluicer #2. This sluicer, removed from riser #3 on September 25, 2014, was found to have approximately 0.4 gallons of solid tank waste adhering to the nozzle area.

  9. Solid-phase microextraction for the analysis of biological samples

    Theodoridis, G; Koster, EHM; de Jong, GJ


    Solid-phase microextraction (SPME) has been introduced for the extraction of organic compounds from environmental samples. This relatively new extraction technique has now also gained a lot of interest in a broad field of analysis including food, biological and pharmaceutical samples. SPME has a num

  10. Investigation of binary solid phases by calorimetry and kinetic modelling

    Matovic, M.


    The traditional methods for the determination of liquid-solid phase diagrams are based on the assumption that the overall equilibrium is established between the phases. However, the result of the crystallization of a liquid mixture will typically be a non-equilibrium or metastable state of the solid

  11. Wax Precipitation Modeled with Many Mixed Solid Phases

    Heidemann, Robert A.; Madsen, Jesper; Stenby, Erling Halfdan


    The behavior of the Coutinho UNIQUAC model for solid wax phases has been examined. The model can produce as many mixed solid phases as the number of waxy components. In binary mixtures, the solid rich in the lighter component contains little of the heavier component but the second phase shows sub...

  12. Solid Phase Characterization of Tank 241-C-105 Grab Samples

    Ely, T. M. [Washington River Protection Solutions LLC, Richland, WA (United States); LaMothe, M. E. [Washington River Protection Solutions LLC, Richland, WA (United States); Lachut, J. S. [Washington River Protection Solutions LLC, Richland, WA (United States)


    The solid phase characterization (SPC) of three grab samples from single-shell Tank 241-C-105 (C-105) that were received at the laboratory the week of October 26, 2015, has been completed. The three samples were received and broken down in the 11A hot cells.

  13. Sensitive and fast mutation detection by solid phase chemical cleavage

    Hansen, Lise Lotte; Justesen, Just; Kruse, Torben A


    We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly...

  14. Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes

    Liu, Jianbo; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Wei; Wang, Dong


    The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies.The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to

  15. Solid-Phase Extraction Combined with High Performance Liquid ...

    including natural estrone (E1), estradiol (E2), estriol (E3) (see Figure 1). ... include immunoassay [11], gas chromatography- ... HPLC grade acetonitrile and methanol were obtained .... diethyl ether and petroleum ether, acetonitrile, acetic acid ...

  16. Dot enzyme-linked immunosorbent assay (dot-ELISA for schistosomiasis diagnosis using dacron as solid-phase Dot-ELISA (dot enzyme-linked immunosorbent assay, utilizando o dacron como suporte sólido para o diagnóstico da esquistossomose

    Silvia Maria Lucena Montenegro


    Full Text Available Dacron and nitrocellulose were evaluated as matrices for the dot enzyme linked immunosorbent assay (dot-ELISA for schistosomiasis and compared to indirect immunofluorescence (IMF. Titration of sera from 18 schistosomiasis patients against soluble worm antigen preparation (SWAP was carried out and sera from healthy individuals from non-endemic areas were used as controls. The IMF was less sensitive than the dot-ELISAs, although the difference was not statistically significant (p > 0.05. The dot-ELISA based on nitrocellulose was as sensitive as that using dacron. Stability did not differ between nitrocellulose and dacron. Specificity was lower when dacron was used than when nitrocellulose was used, although the difference was not statistically significant (p > 0.05. In conclusion, this work showed that nitrocellulose and dacron performed similarly in dot-ELISA, suggesting that they may be used alternatively in population surveillance in endemic areas.O dacron e a nitrocelulose foram utilizados como matrizes para realização do dot-ELISA na esquistossomose e comparadas com a imunofluorescência indireta (IMF. A titulação dos soros de 18 pacientes esquistossomóticos foi feita, utilizando o antígeno solúvel de verme adulto (SWAP e soro de pessoas normais não endêmicas foram usadas como controle. A IMF foi menos sensível do que os dot-ELISAs, apesar da diferença não ter sido estatisticamente significativa (p > 0,05. O dot-ELISA, utilizando a nitrocelulose foi tão sensível do que aquele utilizando o dacron como suporte. Não houve diferenças significativas entre os suportes em relação à estabilidade do antígeno. Entretanto, a especificidade, utilizando o dacron como suporte foi menor do que a nitrocelulose, apesar da diferença não ter sido estatisticamente significativa (p > 0,05. Em resumo, este trabalho mostrou que os resultados dos suportes utilizados em dot-ELISA para o diagnóstico da esquistossomose mansônica foram

  17. Enzyme

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  18. Studies in Solid Phase Peptide Synthesis: A Personal Perspective

    Mitchell, A R


    By the early 1970s it had became apparent that the solid phase synthesis of ribonuclease A could not be generalized. Consequently, virtually every aspect of solid phase peptide synthesis (SPPS) was reexamined and improved during the decade of the 1970s. The sensitive detection and elimination of possible side reactions (amino acid insertion, N{sup {alpha}}-trifluoroacetylation, N{sup {alpha}{var_epsilon}}-alkylation) was examined. The quantitation of coupling efficiency in SPPS as a function of chain length was studied. A new and improved support for SPPS, the 'PAM-resin', was prepared and evaluated. These and many other studies from the Merrifield laboratory and elsewhere increased the general acceptance of SPPS leading to the 1984 Nobel Prize in Chemistry for Bruce Merrifield.

  19. Entransy dissipation minimization for liquid-solid phase change processes


    The liquid-solid phase change process of a simple one-dimensional slab is studied in this paper.By taking entransy dissipation minimization as optimization objective,the optimal external reservoir temperature profiles are derived by using optimal control theory under the condition of a fixed freezing or melting time.The entransy dissipation corresponding to the optimal heat exchange strategies of minimum entransy dissipation is 8/9 of that corresponding to constant reservoir temperature operations,which is independent of all system parameters.The obtained results for entransy dissipation minimization are also compared with those obtained for the optimal heat exchange strategies of minimum entropy generation and constant reservoir temperature operations by numerical examples.The obtained results can provide some theoretical guidelines for the choice of optimal cooling or heating strategy in practical liquid-solid phase change processes.

  20. Semi-automated microwave assisted solid-phase peptide synthesis

    Pedersen, Søren Ljungberg

    with microwaves for SPPS has gained in popularity as it for many syntheses has provided significant improvement in terms of speed, purity, and yields, maybe especially in the synthesis of long and "difficult" peptides. Thus, precise microwave heating has emerged as one new parameter for SPPS, in addition...... to coupling reagents, resins, solvents etc. We have previously reported on microwave heating to promote a range of solid-phase reactions in SPPS. Here we present a new, flexible semi-automated instrument for the application of precise microwave heating in solid-phase synthesis. It combines a slightly modified...... Biotage Initiator microwave instrument, which is available in many laboratories, with a modified semi-automated peptide synthesizer from MultiSynTech. A custom-made reaction vessel is placed permanently in the microwave oven, thus the reactor does not have to be moved between steps. Mixing is achieved...

  1. Chromatography, solid-phase extraction, and capillary electrochromatography with MIPs.

    Tóth, Blanka; Horvai, George


    Most analytical applications of molecularly imprinted polymers are based on their selective adsorption properties towards the template or its analogs. In chromatography, solid phase extraction and electrochromatography this adsorption is a dynamic process. The dynamic process combined with the nonlinear adsorption isotherm of the polymers and other factors results in complications which have limited the success of imprinted polymers. This chapter explains these problems and shows many examples of successful applications overcoming or avoiding the problems.

  2. Effects of solid-phase extraction of plasma in measuring gut metabolic hormones in fasted and fed blood of lean and diet-induced obese rats.

    Reidelberger, Roger; Haver, Alvin; Anders, Krista; Apenteng, Bettye; Lanio, Craig


    Glucagon-like peptide-1 (GLP-1), peptide YY (3-36) [PYY(3-36)], amylin, ghrelin, insulin, and leptin are thought to act as hormonal signals from periphery to brain to control food intake. Here, we determined the effects of solid-phase extraction of plasma in measuring these hormones in blood of lean and diet-induced obese rats. Individual enzyme-linked immunoassays and a multiplex assay were used to measure active GLP-1, total PYY, active amylin, active ghrelin, insulin, leptin, and total GIP in response to (1) addition of known amounts of the peptides to lean and obese plasma, (2) a large meal in lean and obese rats, and (3) intravenous infusions of anorexigenic doses of GLP-1, PYY(3-36), amylin, and leptin in lean rats. Extraction of lean and obese plasma prior to assays produced consistent recoveries across assays for GLP-1, PYY, amylin, ghrelin, and insulin, reflecting losses inherent to the extraction procedure. Plasma extraction prior to assays generally revealed larger meal-induced changes in plasma GLP-1, PYY, amylin, ghrelin, and insulin in lean and obese rats. Plasma extraction and the multiplex assay were used to compare plasma levels of GLP-1, PYY, and amylin after a large meal with plasma levels produced by IV infusions of anorexigenic doses of GLP-1, PYY(3-36), and amylin. Infusions produced dose-dependent increases in plasma peptide levels, which were well above their postprandial levels. These results do not support the hypothesis that postprandial plasma levels of GLP-1, PYY(3-36), and amylin are sufficient to decrease food intake by an endocrine mechanism.

  3. Solid-phase synthesis of molecularly imprinted nanoparticles.

    Canfarotta, Francesco; Poma, Alessandro; Guerreiro, Antonio; Piletsky, Sergey


    Molecularly imprinted polymers (MIPs) are synthetic materials, generally based on acrylic or methacrylic monomers, that are polymerized in the presence of a specific target molecule called the 'template' and capable of rebinding selectively to this target molecule. They have the potential to be low-cost and robust alternatives to biomolecules such as antibodies and receptors. When prepared by traditional synthetic methods (i.e., with free template in solution), their usefulness has been limited by high binding site heterogeneity, the presence of residual template and the fact that the production methods are complex and difficult to standardize. To overcome some of these limitations, we developed a method for the synthesis of MIP nanoparticles (nanoMIPs) using an innovative solid-phase approach, which relies on the covalent immobilization of the template molecules onto the surface of a solid support (glass beads). The obtained nanoMIPs are virtually free of template and demonstrate high affinity for the target molecule (e.g., melamine and trypsin in our published work). Because of an affinity separation step performed on the solid phase after polymerization, poor binders and unproductive polymer are removed, so the final product has more uniform binding characteristics. The overall protocol, starting from the immobilization of the template onto the solid phase and including the purification and characterization of the nanoparticles, takes up to 1 week.

  4. Prebeta-migrating high density lipoprotein: quantitation in normal and hyperlipidemic plasma by solid phase radioimmunoassay following electrophoretic transfer

    Ishida, B.Y.; Frolich, J.; Fielding, C.J.


    A quantitative solid phase immunoassay has been developed for the determination of the mass of electrophoretically separated prebeta apolipoprotein A-I (apoA-I) in human plasma. Conditions have been identified for the quantitative transfer and immunoblotting of the apolipoprotein in the absence of organic solvents or detergents. In normolipidemic plasma, the prebeta-migrating fraction of apoA-I represented 4.2 +/- 1.8% of total apoA-I (61 +/- 26 micrograms of apoA-I per ml of plasma). Significantly higher levels were found in hypercholesterolemia of genetic origin, in primary and secondary hypertriglyceridemia, and in congenital lecithin:cholesterol acyltransferase deficiency. In all cases prebeta-migrating apoA-I consisted in large part of low molecular weight lipoprotein species, compared to the size of the major, alpha-migrating apoA-I fraction.

  5. Padronização de um teste imunoenzimático para detecção de Salmonella em alimentos Standardization of an enzyme immunoassay for detection of Salmonella in foods

    Regina Baptista dos Reis


    Full Text Available A metodologia convencional utilizada para detecção de Salmonella em alimentos é trabalhosa, apresenta custo elevado e os resultados definitivos somente estão disponíveis após 96 horas. Vários métodos rápidos têm sido propostos, sendo os testes imunoenzimáticos os mais empregados. Este estudo relata o desenvolvimento de um teste imunoenzimático para detecção de Salmonella em alimentos, empregando-se um anti-soro policlonal monovalente contendo aglutininas f,g,s, não absorvido, e um anti-soro polivalente absorvido contendo as aglutininas e,h; 1,6; i; 1,2; f,g,s e m,t. A eficiência foi comparada com a da metodologia de cultivo tradicional. O teste imunoenzimático foi empregado para a detecção de Salmonella em amostras de alimentos infantis experimentalmente inoculadas com este patógeno e com outras enterobactérias, em diferentes proporções. O teste imunoenzimático revelou-se significativamente mais sensível que o método de cultivo. Esse mesmo teste, utilizando-se o anti-soro f,g,s não absorvido com antígenos heterólogos revelou concordância de 89,6% com o método de cultivo e sensibilidade de 100,0%. Por outro lado, empregando-se o anti-soro polivalente absorvido, a concordância com o método de cultivo foi de 81,3% embora a sensibilidade tenha se mantido no mesmo nível. O desempenho do teste imunoenzimático empregando-se um desses dois anti-soros indica um grande potencial de aplicação como método de triagem na detecção de Salmonella em alimentos.The conventional method for detection of Salmonella in foods is cumbersome, it is not cost-effective and results are available only after 96h. Many alternative rapid methods have been already proposed and enzyme immunoassays are the most common. This study reports the standardization of a new enzyme immunoassay for detection of Salmonella in foods, based on a policlonal non-absorbed antiserum containing f,g,s aglutinins and a pool of policlonal absorbed antisera

  6. A comparison of observables for solid-solid phase transitions

    Smilowitz, Laura B [Los Alamos National Laboratory; Henson, Bryan F [Los Alamos National Laboratory; Romero, Jerry J [Los Alamos National Laboratory


    The study of solid-solid phase transformations is hindered by the difficulty of finding a volumetric probe to use as a progress variable. Solids are typically optically opaque and heterogeneous. Over the past several years, second harmonic generation (SHG) has been used as a kinetic probe for a solid-solid phase transition in which the initial and final phases have different symmetries. Bulk generation of SHG is allowed by symmetry only in noncentrosymmetric crystallographic space groups. For the organic energetic nitramine octahydro-1,3 ,5,7 -tetranitro-1,3 ,5,7 -tatrazocine (HMX), the beta phase is centro symmetric (space group P2{sub 1}/c) and the delta phase iS noncentrosymmetric (space group P6{sub 1}22) making SHG an extremely sensitive, essentially zero background probe of the phase change progress. We have used SHG as a tool to follow the progress of the transformation from beta to delta phase during the solid-solid transformation. However, kinetic models of the transformation derived using different observables from several other groups have differed, showing later onset for the phase change and faster progression to completion. In this work, we have intercompared several techniques to understand these differences. The three techniques discussed are second harmonic generation, Raman spectroscopy, and differential scanning calorimetry (DSC). The progress of the beta to delta phase transition in HMX observed with each of these different probes will be discussed and advantages and disadvantages of each technique described. This paper compares several different observables for use in measuring the kinetics of solid-solid phase transitions. Relative advantages and disadvantages for each technique are described and a direct comparison of results is made for the beta to delta polymorphic phase transition of the energetic nitramine, octahydro-1,3,5,7-tetranitro-1,3,5,7-tatrazocine.

  7. Distribution of Dechlorinating Bacteria between the Aqueous and Solid Phases

    Cápiro, N. L.; Hatt, J. K.; Wang, Y.; Loeffler, F. E.; Pennell, K. D.


    Microbial monitoring of aquifers relies on nucleic acid biomarker analysis, which is typically performed with biomass recovered from groundwater samples; however, it is unclear what fraction of the target population(s) is associated with groundwater (i.e., planktonic cells) or is attached to solid phases (i.e., biofilms). Understanding how the titer of target organism(s) in groundwater correlates with the true cell titers of the target organism in the aquifer (i.e., planktonic plus attached cells) is critical for a meaningful interpretation of the data, the prediction of bioremediation performance, and the implementation of site management strategies. To evaluate the distribution of active cells between resident solid phase and the aqueous phase, one-dimensional columns were packed under water-saturated conditions with Bio-Dechlor INOCULUM, a PCE-to ethene-dechlorinating bacterial consortium containing both multiple Dehalococcoides (Dhc) strains and Geobacter lovleyi strain SZ (GeoSZ). The columns were packed with two distinct solid matrices: a low organic content sandy Federal Fine Ottawa soil or Appling soil with higher organic matter content. Influent reduced mineral salts medium supplied at a groundwater pore-water velocity of 0.3 m/day contained both 10 mM lactate as electron donor and 0.33 mM PCE as electron acceptor. Routine collection of biomass from column side ports and effluent samples measured the titers of target cells in the aqueous phase and determined when steady state conditions had been reached. A second set of column experiments evaluated delivery and filtration effects by the solid matrix (i.e., Federal Fine Ottawa sand versus Appling soil) under the same conditions except that electron donor or acceptor were omitted (no growth conditions). Quantitative real-time PCR (qPCR) analysis using Dhc and GeoSZ 16S rRNA gene-targeted primer and probe sets determined the planktonic cell counts, and destructive sampling of the columns allowed measurement

  8. Solid Phase Peptide Synthesis of Fusukang for AIDS

    甘一如; 戴琦; 张雪竹; 高晨昊


    A 36-residue peptide is designed to cure acquired immunodeficiency syndrome(AIDS), and is synthesized by the manual solid phase peptide synthesis technique. Different reaction conditions of the synthesis process were discussed. Stirring efficiency of mechanics and nitrogen was compared. The mechanical method displays a predominant performance. Although the coupling efficiencies of diisopropylcarbodiimide(DIC) and dicyclohexylcarbodiimide(DCC) are virtually identical, DIC offers several advantages over DCC in practice due to different physical characters. Wash conditions after deprotection and coupling were investigated to monitor washing efficiency. 0.369 2 g crude peptide was obtained.

  9. Solid-phase colorimetric method for the quantification of fucoidan.

    Lee, Jung Min; Shin, Z-U; Mavlonov, Gafurjon T; Abdurakhmonov, Ibrokhim Y; Yi, Tae-Hoo


    We described the simple, selective, and rapid method for determination of fucoidans using methylene blue staining of sulfated polysaccharides, immobilized into filter paper and consequent optic density (at A (663) nm) measurement of the eluted dye from filter paper. This solid-phase method allows selective determination of 1-20 μg fucoidan in presence of potentially interfering compounds (alginic acid, DNA, salts, proteins, and detergents). Further, we demonstrated the alternative way of using image processing software for fucoidan quantification without extraction of methylene blue dye from stained spots of fucoidan-dye complex.

  10. Immunoassays in Biotechnology

    Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patient’s serum to develop a...

  11. Immunoassays in Biotechnology

    Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patient’s serum to develop a...

  12. Solid-Phase Preparation and Characterization of Chitosan

    GaoLe-ping; DuYu-min; ZhangDao-bin; ShiXiao-wen; ZhanHuai-yu; SongWen-hua


    Chitosan was prepared with stressing method by blending chitin and solid alkali in a single-screw extruder at given temperature and characterized by potentiometric titration, gel permeation chromatography (GPC), infrared spectrum (IR) and carborr13 magnetic resonance sperctroscopy (13C NMR). Chitosan with a deacetylation degree (DD) of 76. 1% was obtained at a mass ratio 0.2 : 1 : 1 for H20/chitin/NaOH at 160℃ for 12 mirL Compared to conventional solution method(usually 1 : 10 for chitin/NaOH), the alkali assumption greatly decreased. Molecular weight of chitosan obtained by solid-phase method(S3,M. 1.54 X 10s ) was lower than that obtained by suspension method(Y2,Mw3. 34×105). During deacetylation, molecular weight decreased with high reaction temperature and long reaction time but remained same at different initial ratios of NaOH/chitirL It might be concluded that degradation of chitosan was caused by breakout of the main chain of the oxidized chitosan catalyzed by alkali during the deactylation. IR and 13C NMR showed that structures of chitosans prepared by solid-phase method were not changed.

  13. Solid-Phase Purification of Synthetic DNA Sequences.

    Grajkowski, Andrzej; Cieslak, Jacek; Beaucage, Serge L


    Although high-throughput methods for solid-phase synthesis of DNA sequences are currently available for synthetic biology applications and technologies for large-scale production of nucleic acid-based drugs have been exploited for various therapeutic indications, little has been done to develop high-throughput procedures for the purification of synthetic nucleic acid sequences. An efficient process for purification of phosphorothioate and native DNA sequences is described herein. This process consists of functionalizing commercial aminopropylated silica gel with aminooxyalkyl functions to enable capture of DNA sequences carrying a 5'-siloxyl ether linker with a "keto" function through an oximation reaction. Deoxyribonucleoside phosphoramidites functionalized with the 5'-siloxyl ether linker were prepared in yields of 75-83% and incorporated last into the solid-phase assembly of DNA sequences. Capture of nucleobase- and phosphate-deprotected DNA sequences released from the synthesis support is demonstrated to proceed near quantitatively. After shorter than full-length DNA sequences were washed from the capture support, the purified DNA sequences were released from this support upon treatment with tetra-n-butylammonium fluoride in dry DMSO. The purity of released DNA sequences exceeds 98%. The scalability and high-throughput features of the purification process are demonstrated without sacrificing purity of the DNA sequences.

  14. Detection of bacterial protein toxins by solid phase magnetic immunocapture and mass spectrometry.

    Pocsfalvi, Gabriella; Schlosser, Gitta


    Bacterial protein toxins are involved in a number of infectious and foodborne diseases and are considered as potential biological warfare agents as well. Their sensitive multiplex detection in complex environmental, food, and biological samples are an important although challenging task. Solid-phase immunoaffinity capture provides an efficient way to enrich and purify a wide range of proteins from complex mixtures. We have shown that staphylococcal enterotoxins, for example, can be efficiently enriched by means of magnetic immunocapture using antibody functionalized paramagnetic beads. The method was successfully interfaced by the on-beads and off-beads detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry at the protein level and by the off-beads nano-electrospray ionization-MS/MS detection at the enzyme digests level, enabling thus the unambiguous identification of the toxin. The method is applicable to any bacterial toxin to which an antibody is available.

  15. Kinase Activity Studied in Living Cells Using an Immunoassay

    Bavec, Aljos?a


    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  16. A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

    Tsai Chueh-Jen


    Full Text Available Abstract There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α and nuclear factor-kappa B (NF-κB were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.

  17. Cost-effectiveness of a modified two-step algorithm using a combined glutamate dehydrogenase/toxin enzyme immunoassay and real-time PCR for the diagnosis of Clostridium difficile infection.

    Vasoo, Shawn; Stevens, Jane; Portillo, Lena; Barza, Ruby; Schejbal, Debra; Wu, May May; Chancey, Christina; Singh, Kamaljit


    The analytical performance and cost-effectiveness of the Wampole Toxin A/B EIA, the C. Diff. Quik Chek Complete (CdQCC) (a combined glutamate dehydrogenase antigen/toxin enzyme immunoassay), two RT-PCR assays (Progastro Cd and BD GeneOhm) and a modified two-step algorithm using the CdQCC reflexed to RT-PCR for indeterminate results were compared. The sensitivity of the Wampole Toxin A/B EIA, CdQCC (GDH antigen), BD GeneOhm and Progastro Cd RT-PCR were 85.4%, 95.8%, 100% and 93.8%, respectively. The algorithm provided rapid results for 86% of specimens and the remaining indeterminate results were resolved by RT-PCR, offering the best balance of sensitivity and cost savings per test (algorithm ∼US$13.50/test versus upfront RT-PCR ∼US$26.00/test). Copyright © 2012. Published by Elsevier B.V.

  18. Clostridium difficile testing algorithms using glutamate dehydrogenase antigen and C. difficile toxin enzyme immunoassays with C. difficile nucleic acid amplification testing increase diagnostic yield in a tertiary pediatric population.

    Ota, Kaede V; McGowan, Karin L


    We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens.

  19. Solid-solid phase transitions via melting in metals

    Pogatscher, S.; Leutenegger, D.; Schawe, J. E. K.; Uggowitzer, P. J.; Löffler, J. F.


    Observing solid-solid phase transitions in-situ with sufficient temporal and spatial resolution is a great challenge, and is often only possible via computer simulations or in model systems. Recently, a study of polymeric colloidal particles, where the particles mimic atoms, revealed an intermediate liquid state in the transition from one solid to another. While not yet observed there, this finding suggests that such phenomena may also occur in metals and alloys. Here we present experimental evidence for a solid-solid transition via the formation of a metastable liquid in a `real' atomic system. We observe this transition in a bulk glass-forming metallic system in-situ using fast differential scanning calorimetry. We investigate the corresponding transformation kinetics and discuss the underlying thermodynamics. The mechanism is likely to be a feature of many metallic glasses and metals in general, and may provide further insight into phase transition theory.

  20. Solid phase epitaxial regrowth of (001) anatase titanium dioxide

    Barlaz, David Eitan; Seebauer, Edmund G., E-mail: [Department of Chemical and Biomolecular Engineering, University of Illinois, 600 S Mathews Ave., Urbana, Illinois 61801 (United States)


    The growing interest in metal oxide based semiconductor technologies has driven the need to produce high quality epitaxial films of one metal oxide upon another. Largely unrecognized in synthetic efforts is that some metal oxides offer strongly polar surfaces and interfaces that require electrostatic stabilization to avoid a physically implausible divergence in the potential. The present work examines these issues for epitaxial growth of anatase TiO{sub 2} on strontium titanate (001). Solid phase epitaxial regrowth yields only the (001) facet, while direct crystalline growth by atomic layer deposition yields both the (112) and (001). The presence of amorphous TiO{sub 2} during regrowth may provide preferential stabilization for formation of the (001) facet.

  1. Nanoscale doping of compound semiconductors by solid phase dopant diffusion

    Ahn, Jaehyun, E-mail:; Koh, Donghyi; Roy, Anupam; Banerjee, Sanjay K., E-mail: [Department of Electrical and Computer Engineering, The University of Texas at Austin, Austin, Texas 78712 (United States); Chou, Harry [Materials Science and Engineering Program, University of Texas at Austin, Austin, Texas 78712 (United States); Kim, Taegon [Department of Electrical and Computer Engineering, The University of Texas at Austin, Austin, Texas 78712 (United States); Semiconductor R& D Center, Samsung Electronics Corporation, 1 Samsungjeonja-ro, Hwasung, Kyounggi 445-330 (Korea, Republic of); Song, Jonghan [Advanced Analysis Center, Korea Institute of Science and Technology, Cheongryang, P.O. Box 131, Seoul 130-650 (Korea, Republic of)


    Achieving damage-free, uniform, abrupt, ultra-shallow junctions while simultaneously controlling the doping concentration on the nanoscale is an ongoing challenge to the scaling down of electronic device dimensions. Here, we demonstrate a simple method of effectively doping ΙΙΙ-V compound semiconductors, specifically InGaAs, by a solid phase doping source. This method is based on the in-diffusion of oxygen and/or silicon from a deposited non-stoichiometric silicon dioxide (SiO{sub x}) film on InGaAs, which then acts as donors upon activation by annealing. The dopant profile and concentration can be controlled by the deposited film thickness and thermal annealing parameters, giving active carrier concentration of 1.4 × 10{sup 18 }cm{sup −3}. Our results also indicate that conventional silicon based processes must be carefully reviewed for compound semiconductor device fabrication to prevent unintended doping.

  2. A rapid easy—to—perform solid phase digoxin radioimmunoassay

    LiBin; ZhouMei-Ying; 等


    A solid-phase-radioimmunoassay(SPRIA) for the monitoring of blood digoxin level has been developed,in which a secondary antibody-coated polystyrene tubes are used.This noval method seems to be simple to use and only takes about an half hour.The standard curve is linear from 0.25to 4μg/L.The sensitivity of the detection is 0.1μg/L.Reproducibility studies with 3 control sera of 0.5-2.5μg/L give intraassay CV<5% and interassay CV<10%.The specimens are measured and compared with those of the conventional radioimmunoassay and the values are well correlated(r=0.96,Y=1.022X+0.04μg/L)。

  3. Solid-phase synthesis of siRNA oligonucleotides.

    Beaucage, Serge L


    Since the discovery of RNA interference (RNAi) as a means to silence the expression of specific genes, small interfering RNA (siRNA) oligonucleotides have been recognized as powerful tools for targeting therapeutically important mRNAs and eliciting their destruction. This discovery has created a high demand for synthetic oligoribonucleotides as potential therapeutics and has spurred a renaissance in the development of rapid, efficient methods for solid-phase RNA synthesis. The design and implementation of 2'-hydroxyl protecting groups that provide ribonucleoside phosphoramidites with coupling kinetics and coupling efficiencies comparable to those of deoxyribonucleoside phosphoramidites are key to the production of RNA oligonucleotides in sufficient quantity and purity for pharmaceutical applications. In this context, various siRNAs were chemically modified to identify the biophysical and biochemical parameters necessary for effective and stable RNAi-mediated gene-silencing activities.

  4. Altering the interfacial activation mechanism of a lipase by solid-phase selective chemical modification.

    López-Gallego, Fernando; Abian, Olga; Guisán, Jose Manuel


    This study presents a combined protein immobilization, directed mutagenesis, and site-selective chemical modification approach, which was used to create a hyperactivated semisynthetic variant of BTL2. Various alkane chains were tethered at three different positions in order to mimic the lipase interfacial activation exogenously triggered by detergents. Optimum results were obtained when a dodecane chain was introduced at position 320 by solid-phase site-selective chemical modification. The resulting semisynthetic variant showed a 2.5-fold higher activity than the wild-type nonmodified variant in aqueous conditions. Remarkably, this is the maximum hyperactivation ever observed for BTL2 in the presence of detergents such as Triton X-100. We present evidence to suggest that the endogenous dodecane chain hyperactivates the enzyme in a similar fashion as an exogenous detergent molecule. In this way, we also observe a faster irreversible enzyme inhibition and an altered detergent sensitivity profile promoted by the site-selective chemical modification. These findings are also supported by fluorescence studies, which reveal that the structural conformation changes of the semisynthetic variant are different to those of the wild type, an effect that is more pronounced in the presence of detergent. Finally, the optimal immobilized semisynthetic variant was successfully applied to the selective synthesis of oxiran-2-yl butyrate. Significantly, this biocatalyst is 12-fold more efficient than the immobilized wild-type enzyme, producing the S-enantiomer with higher enantiospecificity (ee = 92%).

  5. Magnetic Affinity Immunoassay Based Enzyme-Labeled Phage Displayed Antibody%基于酶标噬菌体抗体的磁分离免疫分析方法

    穆晞惠; 童朝阳; 黄启斌; 刘冰; 刘志伟; 郝兰群; 张金平


    A new magnetic affinity immunoassay (MAIA) strategy based on enzyme-labeled phage displayed antibody was developed. The assay consisted of a sandwich format in which immobilized polyclonal antibody (pcAb) on magnetic microparticle was used for capture probe, and enzyme-labeled phage displayed antibody for specific detection probe to increase enzyme amount and enhance detection signal. By the proposed method,β-bungarotoxin (β-BGT) was successfully detected. A linear relationship between absorbance value and the concentration of β-BGT in the range of 0. 016-62. 5 μg / L was obtained. The linear regression equation was Y=0. 641X+1. 355 (R =0. 9925, n = 13, p<0. 0001) with a detection limit of 0. 016 μg / L. In comparison with the traditional ELISA, this method gave a 10-fold better sensitivity in β-BGT detection. This strategy also gave a 4-fold better sensitivity comparing with the MAIA based on enzyme labeled monoclonal antibody (mcAb). Due to low detection limit, acceptable reproducibility and high specificity, this method holds great promise in toxin trace detection.%以磁微粒偶联多抗为磁性捕获探针,酶标噬菌体抗体为特异信号检测探针,采用“磁性捕获探针-待测物-酶标噬菌体抗体探针”的检测模式,成功建立了一种基于酶标噬菌体抗体的磁分离免疫分析方法。本方法检测β-银环蛇毒素线性范围为0.016~62.5μg/ L,回归方程为 Y =0.641X+1.355( R =0.9925,n =13, p<0.0001),检出限为0.016μg/ L。本方法比传统 ELISA 法检测灵敏度提高了10倍,与采用酶标单抗复合物探针的双抗体夹心磁分离免疫分析法相比,检测灵敏度提高4倍。本方法灵敏度高,具有较好重现性与特异性,在毒素的痕量检测方面具有广阔的应用前景。

  6. Influence of matrix effect upon cyclosporine A test by fluorescence polarization immunoassay and enzyme-multiplied immunoassay technique methods%基质效应对荧光偏振免疫测定技术和酶放大免疫测定方法检测环孢素A结果的影响

    顾志冬; 陈皓; 周佩军; 冯晓静; 林孝怡; 徐达; 樊绮诗


    Objective To explore the matrix effect on cyclosporine A (CsA) test by fluorescence polarization immunoassay (FPIA) and enzyme-multiplied immunoassay technique (EMIT), explain the discrepancy of external quality control results between these two methods and find the corrective action.Methods One hundred whole blood samples with various concentrations were adopted and CsA levels were detected by FPIA and EMIT.The results were compared with each other.Moreover, the influence of residual metal ions upon immunoreactions was assessed by adding Cu2+ and Zn2+.The effect of non-whole blood matrix on extraction efficiency for quality control materials and CsA calibrator was evaluated by adding identical volume of Hb-rich reagents followed with re-extraction.Results There is good correlation between results measured with FPIA(X) and EMIT(Y) methods ( Y=0.926 8X -8.115,R2 =0.996 9).Neither FPIA nor EMIT was affected by residual metal ions ( P > 0.05 ). Non-whole blood matrix decreased the extraction efficiency of two methods, but it could be corrected by supplementation of the Hb-rich reagents (≥30 g/L).Conclusions Non-whole blood matrix may be the main reason for the inconsistent results measured by FPIA and EMIT methods.It could be corrected by using Hb-rich reagents.In addition,we should consider the influence of low lib on CsA test,espocially for organ transplant patients with lower Hb ( <30 g/L).%目的 通过荧光偏振免疫测定(fluorescence polarization immunoassay,FPIA)和酶放大免疫测定技术(enzyme-multiplied immunoassay technique,EMIT)测定环孢素A(cyciosporine,CsA),了解基质效应对检测结果的影响,解释在CsA室问质评结果中两方法的检测结果的差别,并找到纠正方法.方法 选择不同浓度的临床全血标本100份,用FPIA和EMIT技术进行检测,对比检测结果;通过添加Cu2+,zn2+评估离子残留对免疫反应的影响;用添加等体积血红蛋白富集液后再次抽提的方法,

  7. Detection of neuron specific enolase concentrations in cerebrospinal fluid from patients with neurological disorders by means of a sensitive enzyme immunoassay.

    Vermuyten, K; Lowenthal, A; Karcher, D


    An enzyme linked immunosorbent assay (ELISA) for the detection of neuron specific enolase (NSE) in cerebrospinal fluid (CSF) was developed. The sensitivity of the ELISA was less than 1 microgram/ml. This sensitivity is comparable with radioimmunoassays which have the disadvantage that radiolabelled products are used. The developed assay was used to measure cerebrospinal fluid neuron specific enolase (CSF-NSE) levels in 1178 patients with neurological disorders to establish its potential usefulness and clinical application. CSF-NSE levels in this group of patients were independent of sex and no correlation with age was found. CSF-NSE was significantly increased in Creutzfeldt-Jacob disease, meningeal hemorrhage, thrombosis, Guillain-Barré syndrome and in schizophrenia.

  8. Updates in immunoassays: parasitology.

    Josko, Deborah


    Although most clinical laboratories use microscopy and routine O&P procedures when identifying parasitic infections, there are several parasites that are better detected through serological means. Toxoplasma, Giardia, and Cryptosporidium were discussed along with immunoassays used for their detection. Immunoassays provide quick results and are less labor intensive than specimen concentration and slide preparation for microscopic examination. These assays are easy to use and provide sensitive and specific results. Some clinical laboratories no longer perform O&Ps in house and refer specimens to reference laboratories for evaluation. By using immunoassays, some of the more common parasites can be identified in a timely manner reducing turn-around times. Some controversy exists over the use of IIF and EIA tests used for ANA testing along with measuring CRPs and PCT as predictors of bacterial sepsis and septic shock. Regardless of the methodology discussed in this series of articles, there are pros and cons to the various immunoassays available. Determining the most appropriate assay based on patient population and volume is governed by the institution and its patients' needs. In conclusion, immunoassays, whether manual or automated, are easy to use, cost effective and allow the medical laboratory professional to provide quick and accurate results to the clinician so the most appropriate treatment can be administered to the patient. The ultimate goal of healthcare professionals is to provide the highest quality of medical care in a timely manner. The use of immunoassays in the clinical laboratory allows the healthcare team to successfully achieve this goal.

  9. Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma.

    Lim, Chun Shen; Goh, Siang Ling; Kariapper, Leena; Krishnan, Gopala; Lim, Yat-Yuen; Ng, Ching Ching


    Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC). Thioredoxin fusion VCA p18 (VCA-Trx) and IBs of VCA p18 without fusion tags (VCA-IBs) were purified from E. coli. The diagnostic performances of IgG/VCA-IBs, IgG/VCA-Denat-IBs (using VCA-IBs coated in 8mol/l urea), IgG/VCA-Trx, and IgG/VCA-Peptide assays were compared by screening 100 NPC case-control pairs. The IgG/VCA-Denat-IBs assay showed the best area under the receiver operating characteristic curve (AUC: 0.802; p<0.05), while the AUCs for the IgG/VCA-IBs, IgG/VCA-Trx, and IgG/VCA-Peptide assays were comparable (AUC: 0.740, 0.727, and 0.741, respectively). We improved the diagnostic performance of the ELISA significantly using IBs of recombinant VCA p18. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. A Linker for the Solid-Phase Synthesis of Hydroxamic Acids and Identification of HDAC6 Inhibitors.

    Bang, Claus G; Jensen, Jakob F; O'Hanlon Cohrt, Emil; Olsen, Lasse B; Siyum, Saba G; Mortensen, Kim T; Skovgaard, Tine; Berthelsen, Jens; Yang, Liang; Givskov, Michael; Qvortrup, Katrine; Nielsen, Thomas E


    We herein present broadly useful, readily available and nonintegral hydroxylamine linkers for the routine solid-phase synthesis of hydroxamic acids. The developed protocols enable the efficient synthesis and release of a wide range of hydroxamic acids from various resins, relying on high control and flexibility with respect to reagents and synthetic processes. A trityl-based hydroxylamine linker was used to synthesize a library of peptide hydroxamic acids. The inhibitory effects of the compounds were examined for seven HDAC enzyme subtypes using a chemiluminescence-based assay.

  11. Immunochemical cross-reactivity between albumin and solid-phase adsorbed histamine

    Poulsen, L K; Nolte, H; Søndergaard, I


    For production of an antibody against histamine, this was coupled to human serum albumin (HSA) and used for immunization of rabbits. To test the antiserum, an immunoradiometric assay was developed comprising solid-phase bound histamine, antisera and radiolabelled protein A. Titration and inhibition...... experiments revealed that histamine adsorbed onto a solid-phase could bind the antiserum. However, neither free histamine nor histamine coupled to unrelated carriers could inhibit the binding of antiserum to the solid-phase histamine. Cross-reactivity was demonstrated between HSA and solid-phase bound...

  12. Enzyme immunoassay and proteomic characterization of troponin I as a marker of mammalian muscle compounds in raw meat and some meat products.

    Zvereva, Elena A; Kovalev, Leonid I; Ivanov, Alexei V; Kovaleva, Marina A; Zherdev, Anatoly V; Shishkin, Sergey S; Lisitsyn, Andrey B; Chernukha, Irina M; Dzantiev, Boris B


    The skeletal muscle protein troponin I (TnI) has been characterized as a potential thermally stable and species-specific biomarker of mammalian muscle tissues in raw meat and meat products. This study proposed a technique for the quantification of TnI comprising protein extraction and sandwich enzyme-linked immunosorbent assay (ELISA). The technique is characterized by a TnI detection limit of 4.8 ng/ml with quantifiable concentrations ranging from 8.7 to 52 ng/ml. The method was shown to be suitable for detection of TnI in mammalian (beef, pork, lamb, and horse) meat but not in poultry (chicken, turkey, and duck) meat. In particular, the TnI content in beef was 0.40 3 ± 0.058 mg/g of wet tissue. The TnI estimations obtained for the pork and beef samples using ELISA were comparable to the proteomic analysis results. Thus, the quantitative study of TnI can be a convenient way to assess the mammalian muscle tissue content of various meat products. Copyright © 2015. Published by Elsevier Ltd.

  13. Evaluation of an enzyme immunoassay for the detection of the mycotoxin tenuazonic acid in sorghum grains and sorghum-based infant food.

    Gross, Madeleine; Asam, Stefan; Rychlik, Michael


    An enzyme-linked immunosorbent assay (ELISA) for the Alternaria mycotoxin tenuazonic acid (TeA) was evaluated by comparative analysis of naturally contaminated sorghum grains and sorghum-based infant food, using a stable isotope dilution LC-MS assay (SIDA; limit of detection (LOD) 1.0 μg/kg) as the reference method. LODs of the ELISA were 30 μg/kg in sorghum grains and 220 μg/kg in sorghum-based infant cereals. With SIDA, 100% of the samples (n = 28) had been positive for TeA in a concentration range of 6-584 μg/kg (mean 113 μg/kg). The ELISA consistently detected TeA in all naturally contaminated samples at cut-off levels of 30-60 μg/kg (sorghum) and 200-300 μg/kg (infant cereals), as based on corresponding to SIDA values. Although the ELISA was much less sensitive than the SIDA method, it may be useful as a screening method for sorghum and sorghum-based infant foods and can be employed to identify samples containing elevated concentrations of TeA in food, well below the proposed level of concern (500 μg/kg).

  14. Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immunoassay in horses from São Paulo State, Brazil.

    Baldani, Cristiane Divan; Hilario, Eduardo; Nakaghi, Andréa Cristina Higa; Bertolini, Maria Célia; Machado, Rosangela Zacarias


    The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding the entire EMA-1 of Theileria equi Jaboticabal strain was cloned and expressed in Escherichia coli as a histidine-tagged protein (His6-EMA1). The expressed EMA-1 reacted with specific antibodies in Western blot and had an apparent molecular mass of 34 kDa which was largely consistent with its theoretical value. The nucleotide sequence of the EMA-1 gene of Jaboticabal strain was comparatively analyzed with other published sequences. The results indicated a high degree of homology with EMA-1 genes of all other strains isolated from various countries. The recombinant purified His6-EMA1 protein was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies anti-T. equi in horses. The ELISA clearly differentiated T. equi-infected from Babesia caballi-infected horse sera or normal horse sera. Field serum samples collected from horses in the State of São Paulo, Southeastern Brazil, were examined for the diagnosis of T. equi infection by ELISA. Of 170 samples analyzed, 95.88% (163/170) were positive for T. equi infection. These results suggest that the His6-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for ELISA test and that T. equi infection is a serious concern in the State of São Paulo, Brazil.

  15. Comparison of enzyme immunoassays detecting Helicobacter pylori specific IgG in serum and saliva with endoscopic and biopsy findings in patients with dyspepsia

    A El-Mekki


    Full Text Available Purpose: To compare the performance of two indirect enzyme-linked immunosorbent assays (ELISA detecting Helicobacter pylori (HP-specific IgG antibodies in serum and saliva with endoscopic observations and histologic findings of biopsies from dyspeptic patients, in an area of high HP prevalence. Materials and Methods : Sera, saliva and antral biopsies were obtained from 55 dyspeptic patients. IgG antibodies against HP were assayed in sera and saliva utilizing two indirect ELISAs. Biopsies were processed according to standard procedures in order to detect histological changes and the presence or absence of Helicobacter pylori. Laboratory data thus obtained were compared and statistically analyzed. Results: Forty-two (76.36% biopsies were positive for HP. The organisms were detected in 4 of 16 (25% cases with normal endoscopic findings, in all 16 cases of gastritis and in 22 of the 23 (95.6% cases of duodenal ulcers (DU. Serum and saliva HP-specific IgG antibodies were detected in 4 normal cases with positive biopsies, in 12 and 14 cases of gastritis, respectively, and in all 22 (100% biopsy positive cases of DU. The sensitivities of the serum and saliva tests were 90.5% and 95%, respectively, while the specificities were 84.5% and 70%, respectively. Conclusion: Due to their high sensitivity and specificity in diagnosing HP-associated DU and gastritis, serum and saliva antibody testing seems to offer a valuable alternative to invasive procedures especially in areas of high HP prevalence such as ours; saliva antibody testing is simple and practical especially in children and in difficult patients who resent venipuncture.

  16. Validity and reliability of enzyme immunoassays using Leishmania major or L. infantum antigens for the diagnosis of canine visceral leishmaniasis in Brazil.

    Mauro Maciel de Arruda

    Full Text Available BACKGROUND: American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. METHODS: A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. RESULTS: The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. INTERPRETATION: ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil.

  17. Molecularly imprinted solid phase extraction of fluconazole from pharmaceutical formulations.

    Manzoor, S; Buffon, R; Rossi, A V


    This work encompasses a direct and coherent strategy to synthesise a molecularly imprinted polymer (MIP) capable of extracting fluconazole from its sample. The MIP was successfully prepared from methacrylic acid (functional monomer), ethyleneglycoldimethacrylate (crosslinker) and acetonitrile (porogenic solvent) in the presence of fluconazole as the template molecule through a non-covalent approach. The non-imprinted polymer (NIP) was prepared following the same synthetic scheme, but in the absence of the template. The data obtained from scanning electronic microscopy, infrared spectroscopy, thermogravimetric and nitrogen Brunauer-Emmett-Teller plot helped to elucidate the structural as well as the morphological characteristics of the MIP and NIP. The application of MIP as a sorbent was demonstrated by packing it in solid phase extraction cartridges to extract fluconazole from commercial capsule samples through an offline analytical procedure. The quantification of fluconazole was accomplished through UPLC-MS, which resulted in LOD≤1.63×10(-10) mM. Furthermore, a high percentage recovery of 91±10% (n=9) was obtained. The ability of the MIP for selective recognition of fluconazole was evaluated by comparison with the structural analogues, miconazole, tioconazole and secnidazole, resulting in percentage recoveries of 51, 35 and 32%, respectively.

  18. Automated Solid-Phase Radiofluorination Using Polymer-Supported Phosphazenes

    Bente Mathiessen


    Full Text Available The polymer supported phosphazene bases PS-P2tBu and the novel PS-P2PEG allowed for efficient extraction of [18F]F− from proton irradiated [18O]H2O and subsequent radiofluorination of a broad range of substrates directly on the resin. The highest radiochemical yields were obtained with aliphatic sulfonates (69% and bromides (42%; the total radiosynthesis time was 35–45 min. The multivariate analysis showed that the radiochemical yields and purities were controlled by the resin load, reaction temperature, and column packing effects. The resins could be reused several times with the same or different substrates. The fully automated on-column radiofluorination methodology was applied to the radiosynthesis of the important PET radiotracers [18F]FLT and [18F]FDG. The latter was produced with 40% yield on a 120 GBq scale and passed GMP-regulated quality control required for commercial production of [18F]FDG. The combination of compact form factor, simplicity of [18F]F− recovery and processing, and column reusability can make solid phase radiofluorination an attractive radiochemistry platform for the emerging dose-on-demand instruments for bedside production of PET radiotracers.

  19. Microwave heating in solid-phase peptide synthesis.

    Pedersen, Søren L; Tofteng, A Pernille; Malik, Leila; Jensen, Knud J


    The highly refined organic chemistry in solid-phase synthesis has made it the method of choice not only to assemble peptides but also small proteins - mainly on a laboratory scale but increasingly also on an industrial scale. While conductive heating occasionally has been applied to peptide synthesis, precise microwave irradiation to heat the reaction mixture during coupling and N(α)-deprotection has become increasingly popular. It has often provided dramatic reductions in synthesis times, accompanied by an increase in the crude peptide purity. Microwave heating has been proven especially relevant for sequences which might form β-sheet type structures and for sterically difficult couplings. The beneficial effect of microwave heating appears so far to be due to the precise nature of this type of heating, rather than a peptide-specific microwave effect. However, microwave heating as such is not a panacea for all difficulties in peptide syntheses and the conditions may need to be adjusted for the incorporation of Cys, His and Asp in peptides, and for the synthesis of, for example, phosphopeptides, glycopeptides, and N-methylated peptides. Here we provide a comprehensive overview of the advances in microwave heating for peptide synthesis, with a focus on systematic studies and general protocols, as well as important applications. The assembly of β-peptides, peptoids and pseudopeptides are also evaluated in this critical review (254 references).

  20. Ionic liquids in solid-phase microextraction: a review.

    Ho, Tien D; Canestraro, Anthony J; Anderson, Jared L


    Solid-phase microextraction (SPME) has undergone a surge in popularity within the field of analytical chemistry in the past two decades since its introduction. Owing to its nature of extraction, SPME has become widely known as a quick and cost-effective sample preparation technique. Although SPME has demonstrated extraordinary versatility in sampling capabilities, the technique continues to experience a tremendous growth in innovation. Presently, increasing efforts have been directed towards the engineering of novel sorbent material in order to expand the applicability of SPME for a wider range of analytes and matrices. This review highlights the application of ionic liquids (ILs) and polymeric ionic liquids (PILs) as innovative sorbent materials for SPME. Characterized by their unique physico-chemical properties, these compounds can be structurally-designed to selectively extract target analytes based on unique molecular interactions. To examine the advantages of IL and PIL-based sorbent coatings in SPME, the field is reviewed by gathering available experimental data and exploring the sensitivity, linear calibration range, as well as detection limits for a variety of target analytes in the methods that have been developed.

  1. Solid-phase microextraction and the human fecal VOC metabolome.

    Emma Dixon

    Full Text Available The diagnostic potential and health implications of volatile organic compounds (VOCs present in human feces has begun to receive considerable attention. Headspace solid-phase microextraction (SPME has greatly facilitated the isolation and analysis of VOCs from human feces. Pioneering human fecal VOC metabolomic investigations have utilized a single SPME fiber type for analyte extraction and analysis. However, we hypothesized that the multifarious nature of metabolites present in human feces dictates the use of several diverse SPME fiber coatings for more comprehensive metabolomic coverage. We report here an evaluation of eight different commercially available SPME fibers, in combination with both GC-MS and GC-FID, and identify the 50/30 µm CAR-DVB-PDMS, 85 µm CAR-PDMS, 65 µm DVB-PDMS, 7 µm PDMS, and 60 µm PEG SPME fibers as a minimal set of fibers appropriate for human fecal VOC metabolomics, collectively isolating approximately 90% of the total metabolites obtained when using all eight fibers. We also evaluate the effect of extraction duration on metabolite isolation and illustrate that ex vivo enteric microbial fermentation has no effect on metabolite composition during prolonged extractions if the SPME is performed as described herein.

  2. Automated solid-phase peptide synthesis to obtain therapeutic peptides

    Veronika Mäde


    Full Text Available The great versatility and the inherent high affinities of peptides for their respective targets have led to tremendous progress for therapeutic applications in the last years. In order to increase the drugability of these frequently unstable and rapidly cleared molecules, chemical modifications are of great interest. Automated solid-phase peptide synthesis (SPPS offers a suitable technology to produce chemically engineered peptides. This review concentrates on the application of SPPS by Fmoc/t-Bu protecting-group strategy, which is most commonly used. Critical issues and suggestions for the synthesis are covered. The development of automated methods from conventional to essentially improved microwave-assisted instruments is discussed. In order to improve pharmacokinetic properties of peptides, lipidation and PEGylation are described as covalent conjugation methods, which can be applied by a combination of automated and manual synthesis approaches. The synthesis and application of SPPS is described for neuropeptide Y receptor analogs as an example for bioactive hormones. The applied strategies represent innovative and potent methods for the development of novel peptide drug candidates that can be manufactured with optimized automated synthesis technologies.

  3. Headspace solid-phase microextraction for wine volatile analysis.

    Azzi-Achkouty, Samar; Estephan, Nathalie; Ouaini, Naïm; Rutledge, Douglas N


    The most commonly used technique to prepare samples for the analysis of wine volatile is the headspace solid-phase microextraction (HS-SPME). This method has gained popularity in last few years, as it is a unique solventless preparation technique. In this paper, a summary of recently published studies using HS-SPME for the analysis of wine aromas, with special emphasis on the method developed, has been compiled. Several papers are discussed in detail, mainly with respect to the SPME conditions used. A brief summary of the reviews related to HS-SPME analysis is given and discussed. Several parameters affecting the HS-SPME, such as the salt concentration and the agitation conditions, are used in the same way as used in several papers. The HS-SPME extraction proved to be sufficiently sensitive to satisfy legislative requirements related to low detection and quantification limits as well as method accuracy and precision requirements. However, in order to achieve the best performance and precision, the protocol needs to be optimized for each case. The effect of different parameters must be well characterized to ensure correct extraction and desorption to ensure the transfer of extracted compounds into the analytical system. The operating parameters, such as time, temperature, and agitation, must then be kept constant for all the samples.


    E.Q. Xie; W.W. Wang; N. Jiang; D.Y. He


    Manganese silicide MnSi2-x thin films have been prepared on n-type silicon substratesthrough solid phase reaction. The heterostructures were analyzed by X-ray diffraction,Rutherford backscattering spectroscopy, Fourier transform infrared transmittance spec-troscopy and the four-point probe technique. The results show that two manganese sili-cides have been formed sequentially via the reaction of thin layer Mn with Si substrateat different irradiation annealing stages, i.e., MnSi at 450℃ and MnSi1.73 at 550℃.MnSi1.73 phase exhibits preferred growth after irradiation with infrared. In situ four-point probe measurements of sheet resistance during infrared irradiation annealingshow that nucleation of MnSi and phase transformation of MnSi to MnSi1. 73 occur at410℃ and 530℃, respectively; the MnSi phase shows metallic behavior, while MnSi1.73exhibits semiconducting behavior. Characteristic phonon bands of MnSi2-x silicides,which can be used for phase identification along with conventional XRD techniques,have been observed by FTIR spectroscopy.

  5. Solid Phase Formylation of N-Terminus Peptides

    Anna Lucia Tornesello


    Full Text Available Formylation of amino groups is a critical reaction involved in several biological processes including post-translational modification of histones. The addition of a formyl group (CHO to the N-terminal end of a peptide chain generates biologically active molecules. N-formyl-peptides can be produced by different methods. We performed the N-formylation of two chemotactic hexapetides, Met1-Leu2-Lys3-Leu4-Ile5-Val6 and Met1-Met2-Tyr3-Ala4-Leu5-Phe6, carrying out the reaction directly on peptidyl-resin following pre-activation of formic acid with N,N-dicyclohexylcarbodiimmide (DCC in liquid phase. The overnight incubation at 4 °C resulted in a significant increase in production yields of formylated peptides compared to the reaction performed at room temperature. The method is consistently effective, rapid, and inexpensive. Moreover, the synthetic strategy can be applied for the formylation of all primary amines at N-terminus of peptide chains or amino groups of lysine side-chains in solid phase.

  6. Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

    Choudhary, Nandlal; Roy, Avijit; Govindarajulu, A; Nakhla, M K; Levy, L; Brlansky, R H


    Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing. Published by Elsevier B.V.

  7. Electrochemical immunoassay of benzo[a]pyrene based on dual amplification strategy of electron-accelerated Fe{sub 3}O{sub 4}/polyaniline platform and multi-enzyme-functionalized carbon sphere label

    Lin Mouhong [Institute of Biomaterials, College of Sciences, South China Agricultural University, Guangzhou 510642, Guangdong Province (China); Liu Yingju, E-mail: [Institute of Biomaterials, College of Sciences, South China Agricultural University, Guangzhou 510642, Guangdong Province (China); Sun Zihong; Zhang Shenglai; Yang Zhuohong [Institute of Biomaterials, College of Sciences, South China Agricultural University, Guangzhou 510642, Guangdong Province (China); Ni Chunlin, E-mail: [Institute of Biomaterials, College of Sciences, South China Agricultural University, Guangzhou 510642, Guangdong Province (China)


    Graphical abstract: Schematic representation of Fe{sub 3}O{sub 4}/PANI/Nafion-based immunosensor using multi-HRP-HCS-Ab{sub 2} bioconjugates as labels. Highlights: Black-Right-Pointing-Pointer An electrochemical immunosensor for high sensitive detection of BaP. Black-Right-Pointing-Pointer A dual amplification strategy by Fe{sub 3}O{sub 4}/PANI/Nafion film and multi-HRP-HCS-Ab{sub 2} label. Black-Right-Pointing-Pointer An accelerated electron transfer pathway by the Fe{sub 3}O{sub 4}/PANI/Nafion film. - Abstract: An electrochemical immunosensor, basing on a dual amplification strategy by employing a biocompatible Fe{sub 3}O{sub 4}/polyaniline/Nafion (Fe{sub 3}O{sub 4}/PANI/Nafion) layer as sensor platform and multi-enzyme-antibody functionalized highly-carbonized spheres (multi-HRP-HCS-Ab{sub 2}) as label, was constructed for sensitive detection of benzo[a]pyrene (BaP). The stable film, Fe{sub 3}O{sub 4}/PANI/Nafion, can not only immobilize biomolecules, but also catalyze the reduction of hydrogen peroxide, indicating an accelerated electron transfer pathway of the platform. The experimental conditions, including the concentration of Nafion, concentration of Fe{sub 3}O{sub 4}/polyaniline (Fe{sub 3}O{sub 4}/PANI), pH of the detection solution and concentrations of biomolecules, were studied in detail. Basing on a competitive immunoassay, the current change was proportional to the logarithm of BaP concentration in the range of 8 pM and 2 nM with the detection limit of 4 pM. The proposed immunosensor exhibited acceptable reproducibility and stability. This new type of dual amplification strategy may provide potential applications for the detection of environmental pollutants.

  8. 化学发光酶免疫法测牛奶中3种喹诺酮类药物%Determination of QNs Residues in Milk by Enhanced Chemiluminescent Enzyme Immunoassay

    李源珍; 生威; 刘恩梅; 韩静; 秦沛; 王硕


    A direct competitive enhanced chemiluminescent enzyme immunoassay(dc-CLEIA) for determination of ofloxacin (OFL), marbofloxacin (MAR), flerofloxacin (FLE) residues in milk was developed. The detection limit of assay were 0.01, 0.03, 0.04 ng/mL respectively. The spiked milk samples were extracted with trichloroacetic acid and analyzed by dc-CLEIA. The average recoveries at five spiked levels of 200, 100, 50, 20, 10 ng/mL were between 75.4%and 94.1%. There was a good correlation between data obtained using the dc-CLEIA and HPLC, indicating the good performance of this dc-CLEIA. Therefore, the proposed method was simple, accurateand and suitable for the rapid determination of ofloxacin, marbofloxacin and flerofloxacin in milk samples.%建立可同时检测牛奶中氧氟沙星(OFL)、麻保沙星(MAR)、氟罗沙星(FLE)残留的直接竞争化学发光酶免疫法(dc-CLEIA),方法的检出限分别为0.01、0.03、0.04 ng/mL。牛奶中的氧氟沙星、麻保沙星、氟罗沙星用7.5%的三氯乙酸提取,测定200、100、50、20、10 ng/mL 5个添加水平的回收率,平均回收率在75.4%~94.1%之间。HPLC与dc-CLEIA测定的结果有很好的相关性,说明所建立的dc-CLEIA可用于实际样品的检测,结果准确可靠。

  9. Evaluation of an IgM/IgG sensitive enzyme immunoassay and the utility of index values for the screening of syphilis infection in a high-risk population.

    Wong, Ernest H; Klausner, Jeffrey D; Caguin-Grygiel, Gloria; Madayag, Carmela; Barber, Kim O; Qiu, Julia S; Liska, Sally; Pandori, Mark W


    Increasing interest in the use of enzyme immunoassays (EIA) for syphilis screening has generated a considerable need for data on the performance of such tests. We compared the performance of 1 EIA, the TREP-SURE EIA to that of the Venereal Disease Research Laboratory (VDRL) and Treponema pallidum particle agglutination assay (TPPA) in the detection of infection with Treponema pallidum. In total, 674 specimens were tested by VDRL and EIA (356 VDRL-nonreactive and 318 VDRL-reactive). All specimens that were found to be reactive by either the VDRL or EIA were subsequently analyzed by TPPA. We found that the TREP-SURE EIA was marginally less sensitive than the VDRL test for screening, but was significantly more specific. All EIA-TPPA discordant specimens were analyzed by multiple tests, including Immunoglobulin M- and G-specific Western blots and an IgM-specific EIA. Signal-to-cutoff ratios (index values) generated by the TREP-SURE EIA were also investigated. It was found that these values may be instructive regarding the interpretation of test results, as they were found to correlate strongly with the probability of positivity on a TPPA assay. Specimens that reacted positively on the EIA with very high index values were found overwhelmingly to be reactive by TPPA, perhaps obviating the need for the testing of most EIA positive specimens with a secondary treponemal test. An IgM/IgG sensitive EIA would be an effective alternative to VDRL for syphilis screening. Using the EIA index values may provide additional, helpful information to the diagnostic process.

  10. Polystyrene Based SPR Biosensor Chip for Use in Immunoassay


    Biosensors are widely used in immunoassay.The biosensor chip carries a receptor which is used in immunoassay and the chip properties have an important influence on the detecting sensitivity of the biosensor.This paper describes a polystyrene-based biosensor chip developed and used as part of a surface plasmon resonance (SPR) biosensor.The SPR biosensor has a much higher detecting sensitivity than enzyme-linked immunoserbent assay (ELISA).

  11. An acid-stable tert-butyldiarylsilyl (TBDAS) linker for solid-phase organic synthesis.

    Diblasi, Christine M; Macks, Daniel E; Tan, Derek S


    [reaction: see text] A new, robust tert-butyldiarylsilyl (TBDAS) linker has been developed for solid-phase organic synthesis. This linker is stable to both protic and Lewis acidic reaction conditions, overcoming a significant limitation of previously reported silyl linkers. Solid-phase acetal deprotection, olefination, asymmetric allylation, and silyl protecting group deblocking reactions have been demonstrated with TBDAS-linked substrates.

  12. 40 CFR 227.32 - Liquid, suspended particulate, and solid phases of a material.


    ... MATERIALS Definitions § 227.32 Liquid, suspended particulate, and solid phases of a material. (a) For the... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Liquid, suspended particulate, and solid phases of a material. 227.32 Section 227.32 Protection of Environment ENVIRONMENTAL...


    A. R. Koohpaei ، S. J. Shahtaheri ، M. R. Ganjali ، A. Rahimi Forushani


    Full Text Available Solid phase extraction is one of the major applications of molecularly imprinted polymers fields for clean-up of environmental and biological samples namely molecularly imprinted solid-phase extraction. In this study, solid phase extraction using the imprinted polymer has been optimized with the experimental design approach for a triazine herbicide, named atrazine with regard to the critical factors which influence the molecular imprinted solid phase extraction efficiency such as sample pH, concentration, flow-rate, volume, elution solvent, washing solvent and sorbent mass. Optimization methods that involve changing one factor at a time can be laborious. A novel approach for the optimization of imprinted solid-phase extraction using chemometrics is described. The factors were evaluated statistically and also validated with spiked water samples and showed a good reproducibility over six consecutive days as well as six within-day experiments. Also, in order to the evaluate efficiency of the optimized molecularly imprinted solid-phase extraction protocols, enrichment capacity, reusability and cross-reactivity of cartridges have been also evaluated. Finally, selective molecularly imprinted solid-phase extraction of atrazine was successfully demonstrated with a recovery above 90% for spiked drinking water samples. It was concluded that the chemometrics is frequently employed for analytical method optimization and based on the obtained results, it is believed that the central composite design could prove beneficial for aiding the molecularly imprinted polymer and molecularly imprinted solid-phase extraction development.

  14. New methods and materials for solid phase extraction and high performance liquid chromatography

    Dumont, Philip John [Iowa State Univ., Ames, IA (United States)


    This paper describes methods for solid phase extraction and high performance liquid chromatography (HPLC). The following are described: Effects of Resin Sulfonation on the Retention of Polar Organic Compounds in Solid Phase Extraction; Ion-Chromatographic Separation of Alkali Metals In Non-Aqueous Solvents; Cation-Exchange Chromatography in Non-Aqueous Solvents; and Silicalite As a Stationary Phase For HPLC.

  15. Facile synthesis of aliphatic isothiocyanates and thioureas on solid phase using peptide coupling reagents

    Boas, Ulrik; Andersen, Heidi Gertz; Christensen, Jørn B.;


    Peptide coupling reagents can be used as versatile reagents for the formation of aliphatic isothiocyanates and thioureas on solid phase from the corresponding solid-phase anchored aliphatic primary amines. The formation of the thioureas is fast and highly chemoselective, and proceeds via formation...

  16. Complement fixation by solid phase immune complexes. Reduced capacity in SLE sera

    Baatrup, G; Jonsson, H; Sjöholm, A


    We describe an ELISA for assessment of complement function based on the capacity of serum to support fixation of complement components to solid phase immune complexes (IC). Microplates were coated with aggregated bovine serum albumin (BSA) followed by rabbit anti-BSA IgG. The solid phase IC were ...

  17. Comparative solution and solid-phase glycosylations toward a disaccharide library

    Agoston, K.; Kröger, Lars; Agoston, Agnes


    A comparative study on solution-phase and solid-phase oligosaccharide synthesis was performed. A 16-member library containing all regioisomers of Glc-Glc, Glc-Gal, Gal-Glc, and Gal-Gal disaccharides was synthesized both in solution and on solid phase. The various reaction conditions for different...

  18. Steady-state diffusion regime in solid-phase micro extraction kinetics

    Benhabib, K.; Laak, ter T.L.; Leeuwen, van H.P.


    The temporal evolution of diffusion-controlled analyte accumulation in solid-phase microextraction (SPME) is critically discussed in terms of the various aspects of steady-state diffusion in the two phases under conditions of fast exchange of the analyte at the solid phase film/water interface. For

  19. Hydrogel nanoparticle based immunoassay

    Liotta, Lance A; Luchini, Alessandra; Petricoin, Emanuel F; Espina, Virginia


    An immunoassay device incorporating porous polymeric capture nanoparticles within either the sample collection vessel or pre-impregnated into a porous substratum within fluid flow path of the analytical device is presented. This incorporation of capture particles within the immunoassay device improves sensitivity while removing the requirement for pre-processing of samples prior to loading the immunoassay device. A preferred embodiment is coreshell bait containing capture nanoparticles which perform three functions in one step, in solution: a) molecular size sieving, b) target analyte sequestration and concentration, and c) protection from degradation. The polymeric matrix of the capture particles may be made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. This device is useful for point of care diagnostic assays for biomedical applications and as field deployable assays for environmental, pathogen and chemical or biological threat identification.

  20. Novel materials and methods for solid-phase extraction and liquid chromatography

    Ambrose, Diana [Iowa State Univ., Ames, IA (United States)


    This report contains a general introduction which discusses solid-phase extraction and solid-phase micro-extraction as sample preparation techniques for high-performance liquid chromatography, which is also evaluated in the study. This report also contains the Conclusions section. Four sections have been removed and processed separately: silicalite as a sorbent for solid-phase extraction; a new, high-capacity carboxylic acid functionalized resin for solid-phase extraction; semi-micro solid-phase extraction of organic compounds from aqueous and biological samples; and the high-performance liquid chromatographic determination of drugs and metabolites in human serum and urine using direct injection and a unique molecular sieve.

  1. Development of Chemiluminescence Enzyme Immunoassay for the Determination of Ochratoxin A in Cereal%谷物中赭曲霉毒素A化学发光酶免疫分析法的建立

    刘星; 许杨; 何庆华


    建立了间接竞争化学发光酶免疫法检测赭曲霉毒素A(ochratoxin A,OTA),该方法IC50为112 pg/mL,检出限是2.47 pg/mL,平均批内和批间变异系数分别为7.03%和14.7%.在大米和小麦样本中添加浓度1.5~6 μg/kg的OTA标品,平均回收率在66.97%~97.96%之间,与其他常见真菌毒素未见交叉反应.将该方法应用于30份谷物样本(包括20份大米样本和10份小麦样本)中OTA的检测,检测结果与商品化ELISA试剂盒的相关系数R2= 0.942 4.该方法简单、灵敏、快速、准确适用于谷物中OTA的检测.%A sensitive indirect competitive chemiluminescence enzyme immunoassay (CLEIA)for OTA was developed. The concentration of OTA causing 50% inhibition of binding enzyme marker (IC50) was 112 pg/mL and the detection limit was 2.47 pg/mL, the mean coefficients of variation of within groups and between groups were 7.03,14.7% respectively. The recoveries from rice and wheat samples spiked with 1.5 ~ 6 Ig/kg of OTA varied between 66.97% and 97.96% ,and the OTA antibody showed no cross -reactivity to the else common mycotoxins. OTA in 30 cereal samples were screened by the CLEIA, including 20 rice samples and 10 wheat samples. The results show good coefficient with commercial ELISA kit (R2 = 0. 9424). The assay was sensitive, fast and accurate, which proved to be suitable for the screening of cereal samples for the presence of OTA.

  2. Development of novel solid-phase protein formulations

    Montalvo Ortiz, Brenda Liz

    Proteins are the next-generation drugs for the treatment of several diseases. However, the number of protein drugs is still limited due to the physical or chemical instability of proteins during processing, formulation, storage, and delivery. The formulation of proteins at the solid state has advantages over liquid state, such as improved stability during long-term storage and delivery and decreases transportation costs. In this dissertation, we developed new solid-phase protein formulations in which the integrity of the protein was not compromised. The long term goal of this research was to use these protein formulations to improve protein stability in drug delivery devices, such as poly(lactic-co-glycolic) acid (PLGA). The first solid-phase protein formulation developed in this investigation was named "glassification". We proposed glassification as an alternative protein dehydration technique to the common used one, lyophilization, because this last method involves a series of steps which are detrimental to protein structure and stability. The glassification method consisted on protein dehydration by the use of organic solvents. As a result of the glassification process a small (micrometer size range) protein solid bead was obtained. The proteins used to study the glassification process were lysozyme (LYS), alpha-chymotrypsin (CHYMO) and horseradish peroxidase (HRP). These studies revealed that the glassification process itself did not alter protein structure and the activity was preserved. Ethyl acetate was the most effective organic solvent for protein glassification because it led to the highest protein residual activity, no insoluble aggregate formation and is a relatively non-toxic solvent, which allow the incorporation of these protein microparticles in PLGA microspheres. The incorporation of spherical HRP microparticles into PLGA microspheres resulted in superior properties when compared with encapsulated lyophilized HRP powder, such as improved release

  3. Solid phase epitaxial regrowth of (100)GaAs

    Almonte, Marlene Isabel [California Univ., Berkeley, CA (United States). Dept. of Materials Science and Mineral Engineering


    This thesis showed that low temperature (250°C) SPE of stoichiometrically balanced ion implanted GaAs layers can yield good epitaxial recovery for doses near the amorphization threshold. For 250°C anneals, most of the regrowth occurred in the first 10 min. HRTEM revealed much lower stacking fault density in the co-implanted sample than in the As-only and Ga-only samples with comparable doses. After low temp annealing, the nonstoichiometric samples had a large number of residual defects. For higher dose implants, very high temperatures (700°C) were needed to remove residual defects for all samples. The stoichiometrically balanced layer did not regrow better than the Ga-only and As-only samples. The co-implanted sample exhibited a thinner amorphous layer and a room temperature (RT) annealing effect. The amorphous layer regrew about 5 nm, suggesting that stoichiometrically balanced amorphous layers can regrow even at RT. Mechanisms for solid phase crystallization in (100)GasAs is discussed: nucleation and growth of randomly oriented crystallites and SPE. These two mechanisms compete in compound semiconductors at much lower temperatures than in Si. For the low dose As-only and Ga-only samples with low-temp anneals, both mechanisms are active. For this amorphization threshold dose, crystallites remain in the amorphous layer for all as-implants. 250°C annealing showed recrystallization from the surface and bulk for these samples; for the co-implant, the mechanism is not evident.

  4. Application of solid phase microextraction on dental composite resin analysis.

    Wang, Ven-Shing; Chang, Ta-Yuan; Lai, Chien-Chen; Chen, San-Yue; Huang, Long-Chen; Chao, Keh-Ping


    A direct immersion solid phase microextraction (DI-SPME) method was developed for the analysis of dentin monomers in saliva. Dentine monomers, such as triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA) and 2,2-bis-[4-(2-hydroxy-3-methacryloyloxypropoxy) phenyl]-propane (Bis-GMA), have a high molecular weight and a low vapor pressure. The polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber with a medium polarity was employed for DI-SPME, and 215 nm of detection wavelength was found to be optimum in the chromatogram of HPLC measurement. The calibration range for DI-SPME was 0.30-300 μg/mL with correlation coefficients (r) greater than 0.998 for each analyte. The DI-SPME method achieved good accuracy (recovery 96.1-101.2%) and precision (2.30-8.15% CV) for both intra- and inter-day assays of quality control samples for three target compounds. Method validation was performed on standards dissolved in blank saliva, and there was no significant difference (p>0.2) between the DI-SPME method and the liquid injection method. However, the detection limit of DI-SPME was as low as 0.03, 0.27 and 0.06 μg/mL for TEGDMA, UDMA and Bis-GMA, respectively. Real sample analyses were performed on commercial dentin products after curing for the leaching measurement. In summary, DI-SPME is a more sensitive method that requires less sample pretreatment procedures to measure the resin materials leached in saliva.

  5. Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage

    Rowley, G.; Saad, S.; Giannelli, F.; Green, P.M. [Guy`s & St. Thomas`s Hospitals, London (United Kingdom)


    The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segments ({approximately}1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5{prime} or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microliter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5{prime} labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost twofold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day. 26 refs., 7 figs., 1 tab.

  6. Mass spectrometric immunoassay

    Nelson, Randall W; Williams, Peter; Krone, Jennifer Reeve


    Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

  7. Tetrodoxtoxin Immunoassays. Phase 2


    TTX CIEIA warrants comment. Development of such a system was discussed with Dr. John Hewetson during a visit by James Raybould to Fort Detrick in...T.J.G. Raybould , Ph.D. - directed immunoassay development using T20G10 MAb and T20G10-AP conjugates. Gary S. Bignami, M.S. - responsible for hybridoma



    Velocities of solid phase and liquid phase in debris flow are one key problem to research on impact and abrasion mechanism of banks and control structures under action of debris flow. Debris flow was simplified as two-phase liquid composed of solid phase with the same diameter particles and liquid phase with the same mechanical features. Assume debris flow was one-dimension two-phase liquid moving to one direction,then general equations of velocities of solid phase and liquid phase were founded in twophase theory. Methods to calculate average pressures, volume forces and surface forces of debris flow control volume were established. Specially, surface forces were ascertained using Bingham's rheology equation of liquid phase and Bagnold's testing results about interaction between particles of solid phase. Proportional coefficient of velocities between liquid phase and solid phase was put forward, meanwhile, divergent coefficient between theoretical velocity and real velocity of solid phase was provided too. To state succinctly before, method to calculate velocities of solid phase and liquid phase was obtained through solution to general equations. The method is suitable for both viscous debris flow and thin debris flow. Additionally, velocities every phase can be identified through analyzing deposits in-situ after occurring of debris flow. It is obvious from engineering case the result in the method is consistent to that in real-time field observation.

  9. Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.

    Lundqvist, Magnus; Edfors, Fredrik; Sivertsson, Åsa; Hallström, Björn M; Hudson, Elton P; Tegel, Hanna; Holmberg, Anders; Uhlén, Mathias; Rockberg, Johan


    We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.

  10. Determining the solid phases hosting arsenic in Mekong Delta sediments

    Wucher, M.; Stuckey, J. W.; McCurdy, S.; Fendorf, S.


    The major river systems originating from the Himalaya deposit arsenic bearing sediment into the deltas of South and Southeast Asia. High rates of sediment and organic carbon deposition combined with frequent flooding leads to anaerobic processes that release arsenic into the pore-water. Arsenic concentrations in the groundwater of these sedimentary basins are often above the World Health Organization drinking water standard of 10 μg As L-1. As a result, 150 million people are at risk of chronic arsenic poisoning through water and rice consumption. The composition of the iron bearing phases hosting the arsenic in these deltaic sediments is poorly understood. Here we implemented a suite of selective chemical extractions to help constrain the types of arsenic bearing solid phases, which were complimented with synchrotron-based X-ray absorption spectroscopy and X-ray diffraction analyses to define the arsenic and iron mineralogy of the system. Sediment cores were collected in triplicate from a seasonally-inundated wetland in Cambodia at depths of 10, 50, 100, and 150 centimeters. We hypothesize that (i) arsenic will be predominantly associated with iron oxides, and (ii) the ratio of crystalline to amorphous iron oxides will increase with sediment depth (and age). We performed four selective extractions in parallel to quantify the various pools of arsenic. First, 1 M MgCl2 was used to extract electrostatically-bound arsenic (labile forms) from the sediment. Second, 1 M NaH2PO4 targeted strongly adsorbed arsenic. Third, 1 M HCl was used to liberated arsenic coprecipitated with amorphous Fe/Mn oxides, carbonates, and acid-volatile sulfides. Finally, a dithionite extraction was used to account for arsenic associated with reducible Fe/Mn oxides. Through this work, we identified the composition of the phases hosting arsenic at various depths through the soil profile, improving our understanding of how arsenic persists in the aquifer. In addition, defining the arsenic and

  11. Porous, High Capacity Coatings for Solid Phase Microextraction by Sputtering.

    Diwan, Anubhav; Singh, Bhupinder; Roychowdhury, Tuhin; Yan, DanDan; Tedone, Laura; Nesterenko, Pavel N; Paull, Brett; Sevy, Eric T; Shellie, Robert A; Kaykhaii, Massoud; Linford, Matthew R


    We describe a new process for preparing porous solid phase microextraction (SPME) coatings by the sputtering of silicon onto silica fibers. The microstructure of these coatings is a function of the substrate geometry and mean free path of the silicon atoms, and the coating thickness is controlled by the sputtering time. Sputtered silicon structures on silica fibers were treated with piranha solution (a mixture of concd H2SO4 and 30% H2O2) to increase the concentration of silanol groups on their surfaces, and the nanostructures were silanized with octadecyldimethylmethoxysilane in the gas phase. The attachment of this hydrophobic ligand was confirmed by X-ray photoelectron spectroscopy and contact angle goniometry on model, planar silicon substrates. Sputtered silicon coatings adhered strongly to their surfaces, as they were able to pass the Scotch tape adhesion test. The extraction time and temperature for headspace extraction of mixtures of alkanes and alcohols on the sputtered fibers were optimized (5 min and 40 °C), and the extraction performances of SPME fibers with 1.0 or 2.0 μm of sputtered silicon were compared to those from a commercial 7 μm poly(dimethylsiloxane) (PDMS) fiber. For mixtures of alcohols, aldehydes, amines, and esters, the 2.0 μm sputtered silicon fiber yielded signals that were 3-9, 3-5, 2.5-4.5, and 1.5-2 times higher, respectively, than those of the commercial fiber. For the heavier alkanes (undecane-hexadecane), the 2.0 μm sputtered fiber yielded signals that were approximately 1.0-1.5 times higher than the commercial fiber. The sputtered fibers extracted low molecular weight analytes that were not detectable with the commercial fiber. The selectivity of the sputtered fibers appears to favor analytes that have both a hydrophobic component and hydrogen-bonding capabilities. No detectable carryover between runs was noted for the sputtered fibers. The repeatability (RSD%) for a fiber (n = 3) was less than 10% for all analytes tested

  12. Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs.

    Oliver-Calixte, Nyoté J; Uba, Franklin I; Battle, Katrina N; Weerakoon-Ratnayake, Kumuditha M; Soper, Steven A


    The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. We report the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA. Enzyme immobilization was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our results suggest that the efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. We obtained a clipping rate of 1.0 × 10(3) nucleotides s(-1) for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. The kinetic behavior of the solid-phase reactor could be described by a fractal Michaelis-Menten model with a catalytic efficiency nearly 17% better than the homogeneous solution-phase reaction. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.

  13. Solid phase precipitates in (Zr,Th)-OH-(oxalate, malonate) ternary aqueous system

    Kobayashi, T.; Sasaki, T.; Takagi, I.; Moriyama, H. [Kyoto Univ. (Japan). Dept. of Nuclear Engineering


    The solubility-limiting solid phases in the ternary aqueous systems of Zr(IV)/OH/oxalate, Zr(IV)/OH/malonate, Th(IV)/OH/oxalate and Th(IV)/OH/malonate were characterized by elemental analysis, X-ray diffraction, thermogravimetric analysis and differential thermal analysis. The ternary solid phase of M(IV)/OH/carboxylate was observed to form, even under acidic conditions, depending on the pH and the concentration of carboxylate ligand. In the presence of a large excess of carboxylic acid, however; the binary M(IV)-carboxylate solid phase formed. (orig.)

  14. Design and Synthesis of a Dual Linker for Solid Phase Synthesis of Oleanolic Acid Derivatives

    Shaorong Wang


    Full Text Available A hydrophilic amino-terminated poly(ethylene glycol-type dual linker for solid phase synthesis of oleanolic acid derivatives using trityl chloride resin was designed and synthesized for the first time. Model reactions in both liquid and solid phase were performed to show the feasibility of its selective cleavage at two different sites. The biological assay results indicated that the long and flexible alkyl ether functionality in the linker is less likely to be critical for the binding event. Following the successful solid-phase synthesis of model compounds, the potential of this dual linker in reaction monitoring and target identification is deemed worthy of further study.

  15. MEIA检测低水平血清HBsAg及结果分析%Determination of Low Level HBsAg in Serum by Microparticle Enzyme Immunoassay and Analysis of Results

    谭德安; 龙智钢; 罗胜权; 马双双


    目的 了解低水平血清乙肝病毒表面抗原(HBsAg)的人群分布及其相关乙肝病毒血清标志物模式特征.方法 微粒子酶免疫分析技术(microparticle enzyme immunoassay,MEIA)测定8 089例非肝炎病区住院患者血清HBsAg及其表面抗体(抗-HBs)、乙肝病毒e抗原及其e抗体(HBeAg、抗-Hbe)和乙肝病毒核心抗体(抗-HBc);根据定值参比血清和样本的HBsAg荧光速率值/阴性对照荧光速率值之比值(S/N值),并结合中和试验结果,确定浓度在5μg/L以下的HBsAg阳性例数,并分析相关乙肝病毒(HBV)血清标志物模式.结果 共检出HBsAg阳性816例,HBsAg浓度在5 μg/L以下的有189例,占总数的2.34%,占HBsAg阳性人群的23.16%,其中,HBsAg浓度在1 μg/L以下的有84例(44.04%);1~2 μg/L的有33例(17.5%);2~5 μg/L的有72例(38.10%).对上述低水平HBsAg人群的5项HBV血清标志物检测获得8种模式,以"HBsAg、抗-HBc、抗-Hbe阳性","HBsAg和抗-HBs阴性"模式为主(74.60%);累计HB-sAg和抗-HBc同时阳性者占94.17%;HBsAg与HBs同时阳性只出现在HBsAg 1 μg/L以下人群中(6.45%). 结论 低水平血清HBsAg人群不容忽视,提高HBsAg检测灵敏度有重要意义;同时检测相关HBV血清标志物,对于确定上述人群有帮助.

  16. Role of signal-to-cut-off ratios of anti-hepatitis C virus antibody by enzyme immunoassays along with ID-NAT for screening of whole blood donors in India

    Satyam Arora


    Full Text Available Background: The use of elevated signal-to-cut off ratios (S/CO as an alternate to further supplemental testing (i.e., RIBA has been included in the guidelines provided by the Centres for Disease Control and Prevention for HCV diagnostic purposes since 2003. With availability of screening by NAT and non availability of RIBA, further confirmation of HCV infection has been possible at the molecular level (RNA. Aims: To study the role of S/CO ratios of anti hepatitis C virus antibody detection by enzyme immunoassays (EIA along with ID-NAT for screening of whole blood donors. Methods: In this study we reviewed the donor screening status for anti HCV from January 2013 to May 2014. All the donations were screened for anti HCV with fourth generation ELISA (BioRad Monolisa Ag-Ab Ultra as well as with ID NAT (Procleix Ultrio. The S/CO ratio of all the anti-HCV reactive samples were analysed for their presence of HCV RNA. Results: On screening 21,115 donors for HCV, 83 donors (0.39% were found reactive on pilot tube and repeat plasma bag testing (S/Co ratio ≥1 by ELISA. 41 donors were HCV RNA reactive with ID-NAT. 4 samples out of 41 were NAT yields and 37 were concordant reactive with ELISA. The S/Co ratio of anti-HCV reactive samples ranged from 0.9-11.1 [mean = 5.1; SD ΁ 2.9] whereas S/Co ratio of anti HCV and NAT reactive samples (concordant positives ranged from 4.1-11.1 [mean 7.3]. In our analysis we found that S/CO ratio of 4 showed positive predictive value (PPV and sensitivity of 100%. Summary/Conclusions: Our study showed that S/CO of 4 for anti HCV on ELISA would have maximum positive predictive value of having donor with HCV RNA. S/CO ratio of 4 is very close to 3.8 which was the CDC guideline. The presence of anti-HCV does not distinguish between current or past infections but a confirmed anti-HCV-positive result indicates the need for counseling and medical evaluation for HCV infection.

  17. Enzyme-linked Immunoassay (ELISA) for Testing and Evaluation of Low Concentration of HBsAg%酶联免疫法(ELISA)对低浓度HBsAg的检测与评价



    目的:探讨酶联免疫法(ELISA)在低浓度HBsAg检测中的效果。方法以本院2014年1~12月门诊与住院患者中53例采用科华ELISA法检测HBsAg呈阳性且S/CO≤10的标本为研究对象,对所有标本采用HBsAg确认试剂盒以及配套使用的ELISA法进行检测,比较检测结果。结果53例科华ELISA检测HBsAg阳性患者,经确认试剂盒及其配套ELISA法检测,阳性的45例(84.9%),8例(15.1%)确认试验为阴性,并经科华试剂复测结果亦为阴性。结论对低浓度阳性标本进行HBsAg ELISA法复检,并用中和试验去除假阳性结果,能够有效避免报告错误率,减少手术,输血及器官移植等安全隐患。%ObjectiveTo investigate the enzyme-linked immunoassay (ELISA) in low concentration effect of HBsAg detection.Methods In January 2014~December 2014 at our hospital outpatient and hospitalized patients in 53 patients with progress ELISA method to detect HBsAg positive and S/CO specimens of 10 or less as the research object, for all specimens using HBsAg conifrm kit as wel as supporting the use of ELISA method for testing, comparing test results.Results Kehua ELISA detected 53 cases of HBsAg positive patients, conifrmed kit and supporting by ELISA. Positive results of 45 cases (84.9%), 8 cases (15.1%) of the validation test were negative, and the progress reagent of retest results are negative. Conclusion HBsAg ELISA method for low concentration samples recheck, and neutralization test to remove false positive results, can effectively avoid the report error rate, reduce the surgery, blood transfusion and organ transplantation and other security hidden danger.

  18. 对硫磷化学发光酶联免疫吸附分析方法的建立和评价%Development of an Indirect Competitive Chemiluminescence Enzyme-linked Immunoassay for Parathion

    邓浩; 孔德彬; 杨金易; 徐振林; 沈玉栋; 杨星星; 孙远明


    Polyclonal antibody (PcAb) against parathion was raised and used to develop an indirect competi?tive chemiluminescence enzyme-linked immunoassay (icCLEIA). The hapten was prepared from thiophosphor-yl chloride after a three-step substitution reaction. The hapten was coupled to bovine serum albumin (BSA) and ovalbumin(OVA) as immounogen and coating antigen respectively by active ester method. New Zealand rabbits were immunized by the immunogen to obtain anti-parathion polyclonal antibody. Several parameters that might affect icCLEIA performance were carefully optimized. Under the optimum conditions, the linear range of the developed icCLEIA was 0.24-15.83 μg/L, the IC50 was 1. 14 μg/L and the limit of detection was 0. 09 μg/L. The average recovery of parathion from spiked vegetables and water samples ranged from 74.6% to 121.0%. In conclusion, the icCLEIA is a practical method for trace detection of parathion in real samples.%建立了基于多克隆抗体的对硫磷间接竞争化学发光酶联免疫吸附分析方法(icCLEIA).以三氯硫磷为原料,经三步取代反应合成对硫磷半抗原,通过活泼酯法将半抗原分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,制备免疫抗原和包被抗原.经免疫新西兰大白兔,获得对硫磷抗血清.通过优化条件参数,建立了对硫磷的icCLEIA分析方法.本方法的检测线性范围为0.24~ 15.83 μg/L;半抑制浓度IC50为1.14 μg/L;检出限为0.09 μg/L;对蔬菜样品和水样品的平均添加回收率为74.6%~121.0%.本方法可用于实际样品中痕量对硫磷残留检测.

  19. Bio-solid-phase extraction/tandem mass spectrometry for identification of bioactive compounds in mixtures.

    Forsberg, Erica M; Brennan, John D


    We describe a two-step column-based bioassay method with tandem mass spectrometric detection for rapid identification of bioactive species in mixtures. The first step uses an immobilized enzyme reactor (IMER) column interfaced to an electrospray ionization mass spectrometer (ESI-MS) to identify mixtures containing bioactive compounds (i.e., enzyme inhibitors), while the second step uses bioselective solid-phase extraction (bioSPE) columns to isolate compounds from "hit" mixtures, which are then identified online by data-dependent ESI-MS. IMER columns were prepared by entrapment of adenosine deaminase (ADA) into sol-gel derived monolithic silica columns, and used to perform a primary IMER screen of mixtures prepared from a bioactive library, which resulted in four apparent hit compounds. Such columns did not provide sufficient binding site density to allow bioSPE, and thus a new column format was developed using ADA that was covalently immobilized to monolithic silica capillary columns, providing ∼500-fold more protein binding sites than were present in columns containing entrapped proteins. Using the covalently linked ADA columns, bioactive mixtures identified by IMER were infused until a maximum total ion current was achieved, followed by washing with a buffer to remove unbound compounds. A harsh wash with 3% acetic acid eluted the strongly bound ligands and the resulting peak triggered data dependent MS/MS to identify the ligand, showing that two of the apparent hits were true ADA inhibitors and demonstrating the ability of this method to rapidly identify bioactive compounds in mixtures.

  20. Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics

    Mysling, Simon; Palmisano, Giuseppe; Højrup, Peter;


    Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE...

  1. Study on New Sensitive Method of Determination of Phosphorus by Solid Phase Spectrophotometry


    The use of solid phase spectrophotometry for the determination of trace phosphorus in the system of phosphomolybdate-fructose is described. The adsorption of the system on anion-exchange resin is reported.

  2. Recent Application of Solid Phase Based Techniques for Extraction and Preconcentration of Cyanotoxins in Environmental Matrices.

    Mashile, Geaneth Pertunia; Nomngongo, Philiswa N


    Cyanotoxins are toxic and are found in eutrophic, municipal, and residential water supplies. For this reason, their occurrence in drinking water systems has become a global concern. Therefore, monitoring, control, risk assessment, and prevention of these contaminants in the environmental bodies are important subjects associated with public health. Thus, rapid, sensitive, selective, simple, and accurate analytical methods for the identification and determination of cyanotoxins are required. In this paper, the sampling methodologies and applications of solid phase-based sample preparation methods for the determination of cyanotoxins in environmental matrices are reviewed. The sample preparation techniques mainly include solid phase micro-extraction (SPME), solid phase extraction (SPE), and solid phase adsorption toxin tracking technology (SPATT). In addition, advantages and disadvantages and future prospects of these methods have been discussed.

  3. Design and Solid-Phase Synthesis of Multiple Muramyl Dipeptide (MMD)


    As a non-specific modulator of macrophage, multiplied muramyl dipeptide (MMD) is solid-phase synthesized by application of standard Fmoc chemistry strategy. Tam's multiple antigen system (MAS) is used as our four branched-linker on Lysine.

  4. Solid Phase Equilibria in the Pi-Ga-As and Pt-Ga-Sb Systems


    OFFICE OF NAVAL RESEARCH Research Contract N00014-87-K-0014 R&T Code 413E026---01 AD-A 198 654 TECHNICAL REPORT No. 9 SOLID PHASE EQUILIBRIA IN THE...Classtcation) UNCLASSLFIED: Tech.Rept.#9 SOLID PHASE EQUILIBRIA IN T11: Pt-Ga-As AND Pt-Ga-Sb SYST’IS 12 PERSONAL AuTiOR(S) C.T. Tsai and R.S. Williats 13a TYPE

  5. Solid-Phase Organic Synthesis and Catalysis: Some Recent Strategies Using Alumina, Silica, and Polyionic Resins

    Basudeb Basu; Susmita Paul


    Solid-phase organic synthesis (SPOS) and catalysis have gained impetus after the seminal discovery of Merrifield’s solid-phase peptide synthesis and also because of wide applicability in combinatorial and high throughput chemistry. A large number of organic, inorganic, or organic-inorganic hybrid materials have been employed as polymeric solid supports to promote or catalyze various organic reactions. This review article provides a concise account on our approaches involving the use of (i) al...

  6. Molecularly imprinted polymers: New molecular recognition materials for selective solid-phase extraction of organic compounds

    Martín Esteban, A.


    During the last few years molecularly imprinted polymers have appeared as new selective sorbents for solid-phase extraction of organic compounds in different samples. Molecular imprinting technology involves the preparation of a polymer with specific recognition sites for certain molecules. Once the polymer has been obtained, it can be used in solid-phase extraction protocols, where a careful selection of the most appropriate solvents to be used in the different steps (sample loading, washing...

  7. Expedient protocol for solid-phase synthesis of secondary and tertiary amines

    Olsen, Christian A; Witt, Matthias; Jaroszewski, Jerzy W


    [reaction: see text] An expedient solid-phase synthetic approach to secondary and tertiary amines was developed. The protocol employs conversion of resin-bound amino alcohols to the corresponding iodides, followed by iodide displacement with primary or secondary amines or with unprotected amino...... alcohols. This two-step procedure, affording products in good to excellent yields, is suitable for solid-phase synthesis of polyamines....

  8. Preparation of Pt/C Catalyst with Solid Phase Reaction Method


    The Pt/C catalyst was prepared with solid phase reaction method (Pt/C(S)) for the first time. Its performances were compared with that prepared by the traditional liquid phase reaction method. The results demonstrate that the electrocatalytic activity of Pt/C catalyst with solid phase reaction method for methanol oxidation is higher than that with liquid phase reaction method. XRD and TEM measurements indicate that the Pt/C(S) possesses low crystalline extent and small particle size.

  9. Updates in immunoassays: virology.

    Josko, Deborah


    Virus identification is a challenge to the clinical microbiologist since growing viruses in traditional cell culture is labor intensive, time consuming, and subject to contamination. The advent of rapid and automated immunoassays has eliminated this problem by generating positive results in minutes to hours. For example, testing for infectious mononucleosis can yield a positive result in 3-8 minutes as seen with the Beckman Coulter, Inc. ICON Mono test or in 5-15 minutes with the MONO Mononucleosis Rapid Test Device marketed by ACON Laboratories, Inc. Fully automated immunoassay analyzers provide fast, accurate, sensitive results that aid in a prompt and accurate diagnosis for the patient. Turnaround times are shortened, allowing for timely medical intervention and treatment. The priority in any hospital or medical facility is to treat the patient as quickly and appropriately as possible. By using immunoassays, clinical laboratory professionals are able to report out correct results in a timely manner, ensuring overall positive patient outcomes and improved quality of healthcare.

  10. Solid phase fermentation of Helianthus tuberosus for ethanol

    Baerwald, G.; Hamad, S.H.


    The direct fermentation of pure inulin and hammer mill crushed Helianthus tuberosus tubers (topinambur, Jerusalem artichoke) was studied using two heat-tolerant yeasts, namely Kluyveromyces marxianus and Candida kefyr. A Saccharomyces cerevisiae was included in the study so as to compare the yields of these two yeasts with that of a commercial distiller's yeast. The inulin fermentation was carried out in an 18-L bioreactor using the fed-batch and the batch-fermentation methods. The final ethanol concentration was 6.1% (L/L) which represents 82% of the theoretical yield. Commercial scale experiments with hammer mill crushed tubers gave yields lower than those found in the laboratory: 69% of the theoretical yield for direct fermentation without enzyme addition, and about 91% when cellolytic enzymes were added.

  11. Screening for Anabolic Steroids in Urine of Forensic Cases Using Fully Automated Solid Phase Extraction and LC–MS-MS

    Andersen, David Wederkinck; Linnet, Kristian


    A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated. The method involves a fully automated sample preparation including enzyme treatment, addition of internal standards...... and solid phase extraction followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with adduct formation for two compounds. Urine samples from 580 forensic cases were analyzed to determine the T/E ratio and occurrence of exogenous anabolic steroids....... Extraction recoveries ranged from 77 to 95%, matrix effects from 48 to 78%, overall process efficiencies from 40 to 54% and the lower limit of identification ranged from 2 to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9%) were found positive for one or more anabolic...

  12. Study of two different enzyme immunoassays for the detection of Mayaro virus antibodies Estudo com dois diferentes ensaios imuno-enzimáticos para a detecção de anticorpos contra o vírus Mayaro

    Luiz Tadeu Moraes Figueiredo


    Full Text Available This paper presents the evaluation of an enzyme immunoassay in which Mayaro virus-infected cultured cells ara used as antigen (EIA-ICC and an IgM antibody capture ELISA (MAC-ELISA for Mayaro serologic diagnosis using 114 human sera obtained during a Mayaro outbreak occurred in Bolivia, in 1987. Results were compared with those obtained by haemagglutination-inhibition test (HAI. MAC-ELISA was the most sensitive technique for anti-Mayaro IgM detection. MAC-ELISA was twice sensitive as IgM EIA-ICC. The data shows that MAC-ELISA is a practical and valid technique for diagnosis of recent mayaro infection. IgG-ICC showed hight sensitivity and high specificity compared to HAI. The combination of anti-Mayaro IgG and IgM EIA-ICC results presented the highest sensitivity of the study. Anti-Mayaro IgG and IgM simultaneous detection by ELISA-ICC can be used for recent infection diagnosis (in spite of a less sensitive IgM detection than by MAC-ELISA, for surveillance and sero-epidemiologic studies, and for studies of IgG and IgM responses to Mayaro infection.Apresentamos a avaliação de um teste imuno-enzimático no qual células infectadas com o vírus Mayaro são usadas como antígeno (EIA-ICC e a de um teste de captura de IgM (MAC-ELISA, no diagnóstico sorológico de infecções por Mayaro. Soros humanos em número de 114, obtidos durante uma epidemia ocorrida em 1987 na Bolívia, foram utilizados neste estudo. Os resultados foram comparados com aqueles obtidos pelo teste de inibição da hemaglutinação (HAI. MAC-ELISA mostrou-se duas vezes mais sensível que EIA-ICC na detecção de anticorpos do tipo IgM para Mayaro. MAC-ELISA mostrou-se uma técnica válida e prática para o diagnóstico de infecções recentes por Mayaro. EIA-ICC para detecção de IgG mostrou-se mais sensível e também mais específico que HAI. Os resultados da combinação de EIA-ICC para detecção de IgM e IgG de Mayaro apresentaram a maior sensibilidade dentre os testes

  13. Determination of melamine in aquaculture feed samples based on molecularly imprinted solid-phase extraction.

    Lian, Ziru; Liang, Zhenlin; Wang, Jiangtao


    This research highlights the application of highly efficient molecularly imprinted solid-phase extraction for the preconcentration and analysis of melamine in aquaculture feed samples. Melamine-imprinted polymers were synthesized employing methacrylic acid and ethylene glycol dimethacrylate as functional monomer and cross-linker, respectively. The characteristics of obtained polymers were evaluated by scanning electron microscopy, Fourier transform infrared spectroscopy and binding experiments. The imprinted polymers showed an excellent adsorption ability for melamine and were applied as special solid-phase extraction sorbents for the selective cleanup of melamine. An off-line molecularly imprinted solid-phase extraction procedure was developed for the separation and enrichment of melamine from aquaculture feed samples prior to high-performance liquid chromatography analysis. Optimum molecularly imprinted solid-phase extraction conditions led to recoveries of the target in spiked feed samples in the range 84.6-96.6% and the relative standard deviation less than 3.38% (n = 3). The aquaculture feed sample was determined, and there was no melamine found. The results showed that the molecularly imprinted solid-phase extraction protocols permitted the sensitive, uncomplicated and inexpensive separation and pre-treatment of melamine in aquaculture feed samples.

  14. Two-dimensional solid-phase extraction strategy for the selective enrichment of aminoglycosides in milk.

    Shen, Aijin; Wei, Jie; Yan, Jingyu; Jin, Gaowa; Ding, Junjie; Yang, Bingcheng; Guo, Zhimou; Zhang, Feifang; Liang, Xinmiao


    An orthogonal two-dimensional solid-phase extraction strategy was established for the selective enrichment of three aminoglycosides including spectinomycin, streptomycin, and dihydrostreptomycin in milk. A reversed-phase liquid chromatography material (C18 ) and a weak cation-exchange material (TGA) were integrated in a single solid-phase extraction cartridge. The feasibility of two-dimensional clean-up procedure that experienced two-step adsorption, two-step rinsing, and two-step elution was systematically investigated. Based on the orthogonality of reversed-phase and weak cation-exchange procedures, the two-dimensional solid-phase extraction strategy could minimize the interference from the hydrophobic matrix existing in traditional reversed-phase solid-phase extraction. In addition, high ionic strength in the extracts could be effectively removed before the second dimension of weak cation-exchange solid-phase extraction. Combined with liquid chromatography and tandem mass spectrometry, the optimized procedure was validated according to the European Union Commission directive 2002/657/EC. A good performance was achieved in terms of linearity, recovery, precision, decision limit, and detection capability in milk. Finally, the optimized two-dimensional clean-up procedure incorporated with liquid chromatography and tandem mass spectrometry was successfully applied to the rapid monitoring of aminoglycoside residues in milk. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences.

    Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L


    An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5'-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5'-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  16. Silica supported Fe(3)O(4) magnetic nanoparticles for magnetic solid-phase extraction and magnetic in-tube solid-phase microextraction: application to organophosphorous compounds.

    Moliner-Martinez, Y; Vitta, Yosmery; Prima-Garcia, Helena; González-Fuenzalida, R A; Ribera, Antonio; Campíns-Falcó, P; Coronado, Eugenio


    This work demonstrates the application of silica supported Fe3O4 nanoparticles as sorbent phase for magnetic solid-phase extraction (MSPE) and magnetic on-line in-tube solid-phase microextraction (Magnetic-IT-SPME) combined with capillary liquid chromatography-diode array detection (CapLC-DAD) to determine organophosphorous compounds (OPs) at trace level. In MSPE, magnetism is used as separation tool while in Magnetic-IT-SPME, the application of an external magnetic field gave rise to a significant improvement of the adsorption of OPs on the sorbent phase. Extraction efficiency, analysis time, reproducibility and sensitivity have been compared. This work showed that Magnetic-IT-SPME can be extended to OPs with successful results in terms of simplicity, speed, extraction efficiency and limit of detection. Finally, wastewater samples were analysed to determine OPs at nanograms per litre.

  17. Simultaneous analysis of cortisol and cortisone in saliva using XLC-MS/MS for fully automated online solid phase extraction.

    Jones, Rachel L; Owen, Laura J; Adaway, Joanne E; Keevil, Brian G


    Salivary cortisol measurements are increasingly being used in the investigation of disorders of the hypothalamic-pituitary-adrenal axis. In the salivary gland, cortisol is metabolised to cortisone by the action of 11β-hydroxysteroid dehydrogenase type 2, and cortisone is partly responsible for the variable interference observed in current salivary cortisol immunoassays. The aim of this study was to validate an assay for the simultaneous analysis of salivary cortisol and cortisone using the Spark Holland Symbiosis™ in eXtraction liquid chromatography-tandem mass spectrometry (XLC-MS/MS) mode for fully automated online solid phase extraction (SPE). Saliva samples were diluted in water with the addition of internal standard (d4-cortisol and d7-cortisone). Online SPE was performed using the Spark Holland Symbiosis™ with HySphere™ C18 SPE cartridges and compounds were eluted onto a Phenomenex® C18 guard column attached to a Phenomenex® Onyx monolithic C18 column for chromatography. Mass spectrometry used the Waters® Xevo™ TQ MS in electrospray positive mode. Cortisol and cortisone eluted with their internal standards at 1.95 and 2.17 min, respectively, with a total run time of four minutes. No evidence of ion-suppression was observed. The assay was linear up to 3393 nmol/L for cortisol and 3676 nmol/L for cortisone, with lower limits of quantitation of 0.75 nmol/L and 0.50 nmol/L, respectively. Intra- and inter-assay imprecision was cortisone across three levels of internal quality control, with accuracy and recovery within accepted limits. High specificity was demonstrated following interference studies which assessed 29 structurally-related steroids at supra-physiological concentrations. We have successfully validated an assay for the simultaneous analysis of salivary cortisol and cortisone using XLC-MS/MS and fully automated online SPE. The assay benefits from increased specificity compared to immunoassay and minimal sample preparation which allows high

  18. Solid-Phase Synthesis of Amine/Carboxyl Substituted Prolines and Proline Homologues: Scope and Limitations.

    Zhou, Ziniu; Scott, William L; O'Donnell, Martin J


    A solid-phase procedure is used to synthesize racemic peptidomimetics based on the fundamental peptide unit. The peptidomimetics are constructed around proline or proline homologues variably substituted at the amine and carbonyl sites. The procedure expands the diversity of substituted peptidomimetic molecules available to the Distributed Drug Discovery (D3) project. Using a BAL-based solid-phase synthetic sequence the proline or proline homologue subunit is both constructed and incorporated into the peptidomimetic by an α-alkylation, hydrolysis and intramolecular cyclization sequence. Further transformations on solid-phase provide access to a variety of piperazine derivatives representing a class of molecules known to exhibit central nervous system activity. The procedure works well with proline cores, but with larger six- and seven-membered ring homologues the nature of the carboxylic acid acylating the cyclic amine can lead to side reactions and result in poor overall yields.

  19. Solid-phase microextraction for bioconcentration studies according to OECD TG 305

    Duering, Rolf-Alexander; Boehm, Leonard [Land Use and Nutrition (IFZ) Justus Liebig University Giessen, Institute of Soil Science and Soil Conservation, Research Centre for BioSystems, Giessen (Germany); Schlechtriem, Christian [Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Schmallenberg (Germany)


    An important aim of the European Community Regulation on chemicals and their safe use is the identification of (very) persistent, (very) bioaccumulative, and toxic substances. In other regulatory chemical safety assessments (pharmaceuticals, biocides, pesticides), the identification of such (very) persistent, (very) bioaccumulative, and toxic substances is of increasing importance. Solid-phase microextraction is especially capable of extracting total water concentrations as well as the freely dissolved fraction of analytes in the water phase, which is available for bioconcentration in fish. However, although already well established in environmental analyses to determine and quantify analytes mainly in aqueous matrices, solid-phase microextraction is still a rather unusual method in regulatory ecotoxicological research. Here, the potential benefits and drawbacks of solid-phase microextraction are discussed as an analytical routine approach for aquatic bioconcentration studies according to OECD TG 305, with a special focus on the testing of hydrophobic organic compounds characterized by log K{sub OW}> 5. (orig.)

  20. Proposed method for controlling turbid particles in solid-phase bioluminescent toxicity measurement.

    Yeo, Seul-Ki; Park, Jun-Boum; Ahn, Joo-Sung; Han, Young-Soo


    In the recent half century, numerous methods have been developed to assess ecological toxicity. However, the presence of solid-particle turbidity sometimes causes such tests to end with questionable results. Many researchers focused on controlling this arbitrary turbidity effect when using the Microtox® solid-phase toxicity system, but there is not yet a standard method. In this study, we examined four solid-phase sample test methods recommended in the Microtox® manual, or proposed from the literature, and compared the existing methods with our proposed method (centrifuged basic solid-phase test, c-BSPT). Four existing methods use the following strategies to control turbid particles: complete separation of liquid and solid using 0.45-μm filtration before contacting solid samples and bacteria, natural settlement, moderate separation of large particles using coarser pore size filtration, and exclusion of light loss in the toxicity calculation caused by turbidity after full disturbance of samples. Our proposed method uses moderate centrifugation to separate out the heavier soil particles from the lighter bacteria after direct contact between them. Among the solid-phase methods tested, in which the bacteria and solid particles were in direct contact (i.e., the three existing methods and the newly proposed one, c-BSPT), no single method could be recommended as optimal for samples over a range of turbidity. Instead, a simple screening strategy for selecting a sample-dependent solid-phase test method was suggested, depending on the turbidity of the solid suspension. The results of this study highlight the importance of considering solid particles, and the necessity for optimal selection of test method to reduce errors in the measurement of solid-phase toxicity.

  1. Extraction of Pb2+ using Silica from Rice Husks Ash (RHA – Chitosan as Solid Phase

    Hanandayu Widwiastuti


    Full Text Available The existence of lead (Pb compounds in waters can be caused of waste pollution from industrial activities such as dye and battery industries. Lead has toxic characteristic and is able to causing deseases. The levels of Cr(VI can be decreased by methods such as electroplating, oxidation, reduction, and membrane separation. But this methods require high cost and produce a lot of waste. Furthermore, those methods cannot determine the small concentration of Pb2+. Therefore, solid phase extraction is used because it’s a simple method and can be used to preconcentrate Pb2+ ion. The aim of this study is to create solid phase from nature material as an alternative method to determine Pb2+ in water samples. The solid phase is silica from rice husks ash (RHA that was modified using chitosan. To achieve that aim, the optimization of silica : chitosan composition was done. The influence of Pb2+ concentration and citric acid concentration was studied to obtain optimum recovery of Pb2+. Interaction between Pb2+ ion and solid phase silica – chitosan could be estimated based on the result. The result showed the optimum composition of silica : chitosan is 65% silica : 35% chitosan with Cation Exchange Capacity (CEC 0.00455 mek/g. Mass Adsorbed Pb2+for 1 g silica : chitosan 65% is 9.715 mg/g. Optimum recovery of Pb2+ on solid phase extraction is reached at concentration of Pb2+ 10 ppm and citric acid concentration 0.05 M (88.25 % and 81.18 %. This result showed that solid phase extraction using silica – chitosan can be used as an alternative method to determine Pb2+ in water.

  2. Novel functionalized polymeric fabric and fiber material as solid support for solid-phase synthesis and biomedical applications

    Xiang, Bei

    The aim of the research is to develop novel polymer solid support by modifying or fabricating polymeric fibrous materials for peptide synthesis and biomedical applications. Originally chemical inert isotactic polypropylene (iPP) fabric was utilized and modified to serve as a functional flexible planar solid support for solid phase peptide synthesis. The modification was achieved through thermal initiated radical grafting polymerization using acrylic acid, poly (ethylene glycol) diacrylate as monomers, and benzoyl peroxide as radical initiator. The iPP fabric was successfully functionalized and possessing as high as 0.7mmol/g carboxylic acid groups. Peptide ligand LHPQF was successfully synthesized on the new functional planar support. Specific enzyme immobilization was fulfilled on the functional iPP fabric support. A commercially available ethylene-acrylic acid copolymer was made into ultrafine copolymer fiber bundles which are composed of nanofibers with diameters ranging from 200nm to 800nm. Various mixing ratios of copolymer/matrix materials were utilized to explore the effect on the final nanofiber physical properties including morphology and stability in solvents. The surface carboxylic acid groups were further converted to amino groups before the functional nanofibers can be applied in solid phase peptide synthesis. Two peptide ligands, LHPQF and HWRGWV, were also successfully synthesized on the nanofiber bundles. Streptavidin and human immunoglobulin G specific binding with the corresponding ligand which was anchored on the nanofibers was conducted successfully to illustrate the potential applications of the nanofiber materials in biomedical field. Further study on the dispersion of the ethylene-acrylic acid nanofiber bundles was pursued to take advantage of the super high active surface area of functional nanofibers. To manipulate the polymer nanofibers during synthesis and bio-assays, a technique was developed to controllably assemble and disperse the

  3. A photolabile linker for the solid-phase synthesis of peptide hydrazides and heterocycles.

    Qvortrup, Katrine; Komnatnyy, Vitaly V; Nielsen, Thomas E


    A photolabile hydrazine linker for the solid-phase synthesis of peptide hydrazides and hydrazine-derived heterocycles is presented. The developed protocols enable the efficient synthesis of structurally diverse peptide hydrazides derived from the standard amino acids, including those with side-chain protected residues at the C-terminal of the resulting peptide hydrazide, and are useful for the synthesis of dihydropyrano[2,3-c]pyrazoles. The linker is compatible with most commonly used coupling reagents and protecting groups for solid-phase peptide synthesis.

  4. Development of orthogonally protected hypusine for solid-phase peptide synthesis.

    Song, Aimin; Tom, Jeffrey; Yu, Zhiyong; Pham, Victoria; Tan, Dajin; Zhang, Dengxiong; Fang, Guoyong; Yu, Tao; Deshayes, Kurt


    An orthogonally protected hypusine reagent was developed for solid-phase synthesis of hypusinated peptides using the Fmoc/t-Bu protection strategy. The reagent was synthesized in an overall yield of 27% after seven steps from Cbz-Lys-OBzl and (R)-3-hydroxypyrrolidin-2-one. The side-chain protecting groups (Boc and t-Bu) are fully compatible with standard Fmoc chemistry and can be readily removed during the peptide cleavage step. The utility of the reagent was demonstrated by solid-phase synthesis of hypusinated peptides.

  5. Synthesis of indium tin oxide powder by solid-phase reaction with microwave heating

    Fukui, Kunihiro; Kanayama, Keiji; Katoh, Manabu; Yamamoto, Tetsuya; Yoshida, Hideto


    Indium tin oxide (ITO) powder was synthesized from indium oxide and tin oxide powders by a solid-phase method using microwave heating and conventional heating methods. Microwave heating could reduce the treatment time necessary for the completion of the solid-phase reaction by 1/30. This decrease was attributed to an increase in the diffusion rate of Sn at the local heat spot in the indium oxide formed by microwave irradiation. However, microwave heating also decreased the amount of ITO produ...

  6. Detection of ibuprofen and ciprofloxacin by solid-phase extraction and UV/Vis spectroscopy

    Zhou, Zhengwei; Jiang, Jia Qian


    A simple and economic solid-phase extraction coupled with UV/Vis spectrophotometric method is described for the analysis of ibuprofen and ciprofloxacin. Following solid-phase extraction from model wastewater samples containing standard ibuprofen or ciprofloxacin, elutes were analyzed by a UV/Vis spectrophotometer at 225 nm for ibuprofen and 280 nm for ciprofloxacin. The assay was linear for both compounds with good coefficients of correlation. This method shows good recoveries for both compounds with 101.0 ± 9.8% for ibuprofen and 99.4 ± 11.8% ciprofloxacin.

  7. A Photolabile Linker for the Solid-Phase Synthesis of Peptide Hydrazides and Heterocycles

    Qvortrup, Katrine; Komnatnyy, Vitaly V.; Nielsen, Thomas Eiland


    A photolabile hydrazine linker for the solid-phase synthesis of peptide hydrazides and hydrazine-derived heterocycles is presented. The developed protocols enable the efficient synthesis of structurally diverse peptide hydrazides derived from the standard amino adds, including those with side-cha......-chain protected residues at the C-terminal of the resulting peptide hydrazide, and are useful for the synthesis of dihydropyrano[2,3-c]pyrazoles. The linker is compatible with most commonly used coupling reagents and protecting groups for solid-phase peptide synthesis....

  8. CuAAC: An Efficient Click Chemistry Reaction on Solid Phase.

    Castro, Vida; Rodríguez, Hortensia; Albericio, Fernando


    Click chemistry is an approach that uses efficient and reliable reactions, such as Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), to bind two molecular building blocks. CuAAC has broad applications in medicinal chemistry and other fields of chemistry. This review describes the general features and applications of CuAAC in solid-phase synthesis (CuAAC-SP), highlighting the suitability of this kind of reaction for peptides, nucleotides, small molecules, supramolecular structures, and polymers, among others. This versatile reaction is expected to become pivotal for meeting future challenges in solid-phase chemistry.

  9. Matrix solid phase dispersion assisted enzymatic hydrolysis as a novel approach for cocaine and opiates isolation from human hair.

    Míguez-Framil, Martha; Cabarcos, Pamela; Tabernero, María Jesús; Bermejo, Ana María; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio


    The possibility of assisting enzymatic hydrolysis (EH) procedures by sample disruption mechanisms inherent to matrix solid phase dispersion (MSPD) has been explored in the current study. EH of hair specimens from poly-drug abusers was assisted by dispersing/blending the sample (0.05 g) with alumina (2.25 g) before loading the dissolved enzyme (6 mL of 1 mg mL(-1) Pronase E in 1.4 M/1.4 M Tris/HCl, pH 7.3) through the hair-alumina solid phase packaged inside a disposable MSPD syringe. The MSPD-EH method was developed, and it proved to offer quantitative results when isolating cocaine, benzoylecgonine (BZE), codeine, morphine and 6-monoacethylmorphine (6-MAM) from human hair samples. The procedure allows an on column clean-up/pre-concentration procedure of the isolated targets by attaching a previously conditioned Oasis HLB cartridge to the end of the MSPD syringe. The EH procedure of human hair with Pronase E can therefore be shortened to approximately 30 min. Within this time, sample blending/dispersion, MSPD syringe package, elution (EH when dissolved Pronase E is passing through the sample-dispersant bed), and extract clean-up and target pre-concentration stages are achieved. Gas chromatography-mass spectrometry (GC-MS) was used for determining each target after elution from the Oasis HLB cartridges with 2 mL of 2% (v/v) acetic acid in methanol, concentration by N2 stream evaporation, and dried extract derivatization with N-methyl-tert-butylsilyltrifluoroacetamide (BSTFA) and chlorotrimethylsilane (TMCS). The method was validated according to the guidance for bioanalytical method validation of the US Department of Health and Human Services, Food and Drug Administration. The simplicity of the proposed approach makes it a useful procedure for screening/quantifying drugs of abuse in hair specimens from poly-drug abusers.

  10. Solid-phase synthesis of an apoptosis-inducing tetrapeptide mimicking the Smac protein

    Le Quement, Sebastian Thordal; Ishøy, Mette; Petersen, Mette Terp;


    An approach for the solid-phase synthesis of apoptosis-inducing Smac peptidomimetics is presented. Using a Rink linker strategy, tetrapeptides mimicking the N-4-terminal residue of the Smac protein [(N-Me)AVPF sequence] were synthesized on PEGA resin in excellent purities and yields. Following tw...

  11. Detection and specifity of class specific antibodies to whole bacteria cells using a solid phase radioimmunoassay

    Czerkinsky, C.; Rees, A.S.; Bergimeier, L.A.; Challacombe, S.J. (Guy' s Hospital Medical and Dental Schools, London (UK))


    A solid phase radioimmunoassay has been developed which can be used for the detection of isotype specific antibodies to whole bacteria and other particulate antigens, and is applicable to a variety of species. Bacteria are bound to the solid phase by the use either of antibodies, or of methyl glyoxal. Both methods result in a sensitive and reproducible assay, and bacteria do not appear to desorb from the solid phase. The specificity of antibodies to whole bacteria was examined by absorption of antisera with various species of bacteria and retesting, or by determining the binding of antisera to various bacteria bound to the solid phase. Both methods revealed specificity for the bacteria examined. Inhibition studies showed that antibodies to Streptococcus mutans whole cells could be inhibited by purified cell surface antigens glucosyltransferase and antigen I/II, but only minimally by lipoteichoic acid, c polysaccharide or dextran. In murine antisera antibodies of the IgG, IgM, and IgA classes could be detected at amounts of less than 1 ng/ml.

  12. New method for the preparation of solid-phase bound isocyanocarboxylic acids and Ugi reactions therewith

    Henkel, Bernd; Sax, Michael; Dömling, Alexander


    A novel method of synthesizing solid-phase bound isocyanocarboxylic acids is reported. The potassium salts of four different isocyanocarboxylic acids are coupled onto a brominated resin in DMF in good yields. 32 Ugi reactions were performed using these resins and 24 products were obtained in good to

  13. Solid phase microextraction speciation analysis of triclosan in aqueous mediacontaining sorbing nanoparticles

    Zielinska, K.


    Solid phase microextraction (SPME) is applied in the speciation analysis of the hydrophobic compound triclosan in an aqueous medium containing sorbing SiO2 nanoparticles (NPs). It is found that these NPs, as well as their complexes with triclosan, partition between the bulk medium and the solid

  14. A Discovery-Oriented Approach to Solid-Phase Peptide Synthesis

    Bockman, Matthew R.; Miedema, Christopher J.; Brennan, Brian B.


    In this discovery-oriented laboratory experiment, students use solid-phase synthesis techniques to construct a dipeptide containing an unknown amino acid. Following synthesis and cleavage from the polymeric support, electrospray ionization-mass spectrometry is employed to identify the unknown amino acid that was used in the peptide coupling. This…

  15. Solid Phase Extraction: Applications to the Chromatographic Analysis of Vegetable Oils and Fats

    Panagiotopoulout, P. M.; Tsimidou, M.


    Applications of solid-phase extraction for the isolation of certain lipid classes prior to chromatographic analysis are given. More information was found for sterols and related compounds, polar phenols and contaminants such as polycyclic aromatic hydrocarbons. Detailed analytical protocols are presented and discussed in many cases. (Author) 120 refs.

  16. Solid-phase synthesis of polyfunctional polylysine dendrons using aldehyde linkers

    Svenssen, Daniel K.; Mirsharghi, Sahar; Boas, Ulrik


    A straightforward method for the solid-phase synthesis of C-terminally modified polylysine dendrons has been developed by applying bisalkoxybenzaldehyde and trisalkoxybenzaldehyde linkers. The method has been used for the synthesis of polylysine dendrons with a variety of C-terminal ‘tail groups’...

  17. Solid phase microextraction speciation analysis of triclosan in aqueous mediacontaining sorbing nanoparticles

    Zielinska, K.


    Solid phase microextraction (SPME) is applied in the speciation analysis of the hydrophobic compound triclosan in an aqueous medium containing sorbing SiO2 nanoparticles (NPs). It is found that these NPs, as well as their complexes with triclosan, partition between the bulk medium and the solid phas

  18. Micro versus macro solid phase extraction for monitoring water contaminants: a preliminary study using trihalomethanes.

    Alexandrou, Lydon D; Spencer, Michelle J S; Morrison, Paul D; Meehan, Barry J; Jones, Oliver A H


    Solid phase extraction is one of the most commonly used pre-concentration and cleanup steps in environmental science. However, traditional methods need electrically powered pumps, can use large volumes of solvent (if multiple samples are run), and require several hours to filter a sample. Additionally, if the cartridge is open to the air volatile compounds may be lost and sample integrity compromised. In contrast, micro cartridge based solid phase extraction can be completed in less than 2 min by hand, uses only microlitres of solvent and provides comparable concentration factors to established methods. It is also an enclosed system so volatile components are not lost. The sample can also be eluted directly into a detector (e.g. a mass spectrometer) if required. However, the technology is new and has not been much used for environmental analysis. In this study we compare traditional (macro) and the new micro solid phase extraction for the analysis of four common volatile trihalomethanes (trichloromethane, bromodichloromethane, dibromochloromethane and tribromomethane). The results demonstrate that micro solid phase extraction is faster and cheaper than traditional methods with similar recovery rates for the target compounds. This method shows potential for further development in a range of applications.

  19. Solid-phase oligosaccharide synthesis with tris(alkoxy)benzyl amine (BAL) safety-catch anchoring

    Tolborg, Jakob Fjord; Jensen, Knud Jørgen


    A tris(alkoxy)benzylamine (BAL) handle strategy was developed for safety-catch anchoring of D-glucosamine derivatives in solid-phase synthesis of oligosaccharides; the linkage between the BAL handle and the amine proved stable to conc. TFA and Lewis acids, but after N-acylation the amide could...

  20. A Long Chain Alcohol as Support in Solid Phase Organic Synthesis

    Nurlela, Yeni; Minnaard, Adrian J.; Achmad, Sadijah; Wahyuningrum, Deana


    The solid phase synthesis is a method by which organic compound synthesis are performed on a support. With this method, the purification can be carried out easily by simple filtration and washing procedures. Long-chain alcohol (C-100 alcohol) can be used as a support because of its insolubility in o

  1. Determination of Roxarsone in feeds using solid phase extraction and liquid chromatography with ultraviolet detection.

    Sapp, R E; Davidson, S


    A method is presented for detection and quantitation of Roxarsone in poultry feed by liquid chromatography. The drug is extracted by phosphate buffer and determined by solid phase extraction and reversed-phase liquid chromatography. Recoveries of the sample spikes and fortified field samples agree closely with those obtained by the standard spectrophotometric method.

  2. A Solid Phase Synthesis of Chalcones by Claisen-Schmidt Condensations


    In order to accelerate the development of relatively inexpensive antimalarials that are effective against chloroquine-resistant strains of Plasmodium falclparum, a methodology for the solid phase synthesis of chalcone (l, 3-diphenyl-2-propen-l-one) analogues in reasonably high yields has been developed.

  3. Development of a Solid Phase Extraction Method for Agricultural Pesticides in Large-Volume Water Samples

    An analytical method using solid phase extraction (SPE) and analysis by gas chromatography/mass spectrometry (GC/MS) was developed for the trace determination of a variety of agricultural pesticides and selected transformation products in large-volume high-elevation lake water sa...

  4. Microwave-assisted solid-phase Ugi four-component condensations

    Nielsen, John


    An 18-member library was constructed from 2 isocyanides, 3 aldehydes and 3 carboxylic acids via microwave-assisted solid-phase Ugi reactions on TentaGel S RAM. Products of high purity were obtained in moderate to excellent yields after reaction times of 5 minutes or less (irradiation at 60W). (C...

  5. Determination of lidocaine in plasma by direct solid-phase microextraction combined with gas chromatography

    Koster, EHM; Wemes, C; Morsink, JB; de Jong, GJ


    Direct-immersion solid-phase microextraction (SPME) has been used to extract the local anesthetic lidocaine from human plasma. A simplified model shows the relationship between the total amount of drug in plasma and the amount of drug extracted. The model takes into account that the drug participate

  6. Solid-phase micro-extraction in bioanalysis, exemplified by lidocaine determination

    de Jong, GJ; Koster, EHM


    Solid-phase micro-extraction (SPME) is a never sample preparation technique that can be used for gaseous, liquid or solid samples in conjunction with GC, HPLC or CE (e.g. [1]). The use of SPME for the analysis of drugs in biofluids is also becoming popular (e.g. [2]). The principle is that a fused s

  7. Side-chain-anchored N(alpha)-Fmoc-Tyr-OPfp for bidirectional solid-phase synthesis

    Olsen, Christian A; Jørgensen, Malene; Hansen, Steen H;


    [reaction: see text] A mild resin-immobilization strategy employing a readily prepared trityl bromide resin for anchoring building blocks via a phenol group has been developed. With N(alpha)-Fmoc-Tyr-OPfp as a starter building block, it was possible to prepare asymmetrically substituted hybrids o...... of spider- and wasp-type polyamine toxins using solid-phase peptide synthesis conditions....

  8. Speciation analysis of aqueous nanoparticulate diclofenac complexes by solid-phase microextraction

    Zielinska, K.; Leeuwen, van H.P.; Thibault, S.; Town, R.M.


    The dynamic sorption of an organic compound by nanoparticles (NPs) is analyzed by solid-phase microextraction (SPME) for the example case of the pharmaceutical diclofenac in dispersions of impermeable (silica, SiO(2)) and permeable (bovine serum albumin, BSA) NPs. It is shown that only the protonate

  9. Recent developments and future trends in solid phase microextraction techniques towards green analytical chemistry.

    Spietelun, Agata; Marcinkowski, Łukasz; de la Guardia, Miguel; Namieśnik, Jacek


    Solid phase microextraction find increasing applications in the sample preparation step before chromatographic determination of analytes in samples with a complex composition. These techniques allow for integrating several operations, such as sample collection, extraction, analyte enrichment above the detection limit of a given measuring instrument and the isolation of analytes from sample matrix. In this work the information about novel methodological and instrumental solutions in relation to different variants of solid phase extraction techniques, solid-phase microextraction (SPME), stir bar sorptive extraction (SBSE) and magnetic solid phase extraction (MSPE) is presented, including practical applications of these techniques and a critical discussion about their advantages and disadvantages. The proposed solutions fulfill the requirements resulting from the concept of sustainable development, and specifically from the implementation of green chemistry principles in analytical laboratories. Therefore, particular attention was paid to the description of possible uses of novel, selective stationary phases in extraction techniques, inter alia, polymeric ionic liquids, carbon nanotubes, and silica- and carbon-based sorbents. The methodological solutions, together with properly matched sampling devices for collecting analytes from samples with varying matrix composition, enable us to reduce the number of errors during the sample preparation prior to chromatographic analysis as well as to limit the negative impact of this analytical step on the natural environment and the health of laboratory employees.

  10. Solid-phase synthesis of succinylhydroxamate peptides : Functionalized matrix metalloproteinase inhibitors

    Leeuwenburgh, MA; Geurink, PP; Klein, T; Kauffman, HF; van der Marel, GA; Bischoff, R; Overkleeft, HS


    A novel solid-phase synthesis strategy toward succinylhydroxamate peptides, using an appropriately protected hydroxamate building block, is described. Rapid and efficient access is gained to amine-functionalized peptides, which can be decorated with, for instance, a fluorescent label. In addition, w

  11. A review on solid phase extraction of actinides and lanthanides with amide based extractants.

    Ansari, Seraj A; Mohapatra, Prasanta K


    Solid phase extraction is gaining attention from separation scientists due to its high chromatographic utility. Though both grafted and impregnated forms of solid phase extraction resins are popular, the later is easy to make by impregnating a given organic extractant on to an inert solid support. Solid phase extraction on an impregnated support, also known as extraction chromatography, combines the advantages of liquid-liquid extraction and the ion exchange chromatography methods. On the flip side, the impregnated extraction chromatographic resins are less stable against leaching out of the organic extractant from the pores of the support material. Grafted resins, on the other hand, have a higher stability, which allows their prolong use. The goal of this article is a brief literature review on reported actinide and lanthanide separation methods based on solid phase extractants of both the types, i.e., (i) ligand impregnation on the solid support or (ii) ligand functionalized polymers (chemically bonded resins). Though the literature survey reveals an enormous volume of studies on the extraction chromatographic separation of actinides and lanthanides using several extractants, the focus of the present article is limited to the work carried out with amide based ligands, viz. monoamides, diamides and diglycolamides. The emphasis will be on reported applied experimental results rather than on data pertaining fundamental metal complexation. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. A convenient procedure for the solid-phase synthesis of hydroxamic acids on PEGA resins

    Nandurkar, Nitin Subhash; Petersen, Rico; Qvortrup, Katrine


    An efficient method for the solid-phase synthesis of hydroxamic acids is described. The method comprises the nucleophilic displacement of esters immobilized on PEGA resins with hydroxylamine/sodium hydroxide in isopropanol. The hydroxyaminolysis protocol is compatible with a broad range of PEGA-s...

  13. A Rapid Solid-phase Synthesis to Soluble Oligothiophene Molecular Wires


    A novel method for the preparation of oligothiophene molecular wires is described via a bi-directional solid-phase synthesis. Using an alternating sequence of bromination and Stille coupling reactions, oligomers were obtained up to the heptamer in excellent yield and purity.

  14. Linkers, resins, and general procedures for solid-phase peptide synthesis

    Shelton, Anne Pernille Tofteng; Jensen, Knud Jørgen


    and linkers for solid-phase synthesis is a key parameter for successful peptide synthesis. This chapter provides an overview of the most common and useful resins and linkers for the synthesis of peptides with C-terminal amides, carboxylic acids, and more. The chapter finishes with robust protocols for general...

  15. Total synthesis of human urotension-Ⅱ by microwave-assisted solid phase method


    Human urotension-Ⅱ was synthesized efficiently on Wang resin under microwave irradiation using Fmoc/tBu orthogonal protection strategy. Disulphide bridge was formed on solid phase with the irradiation of microwave, then the whole peptide was cleaved from the resin. The purity of crude peptide cyclized under microwave irradiation was higher than that under room temperature.

  16. Solid-phase Synthesis of a Novel Kind of Hydroxylated Heterocyclic Ketene Aminals

    Tao PENG; Chu Yi YU; Zhi Tang HUANG


    An efficient solid-phase synthesis method for novel heterocyclic ketene aminals containing a hydroxyl group has been developed. The loading of the substrate on the resin through the hydroxyl group and the protection of the amine by the Schiff base were the key steps in the synthesis.

  17. Fibers coated with molecularly imprinted polymers for solid-phase microextraction

    Koster, E.H M; Crescenzi, C; den Hoedt, W; Ensing, K; de Jong, G.J.


    The simplicity and flexibility of solid-phase microextraction have been combined with the selectivity of molecularly imprinted polymers (MIPs), Silica fibers were coated reproducible with a 75-mum layer of methacrylate polymer either nonimprinted or imprinted with clenbuterol to compare their extrac

  18. Dynamic speciation analysis of atrazine in aqueous latex nanoparticle dispersions using solid phase microextraction (SPME)

    Benhabib, K.; Town, R.M.; Leeuwen, van H.P.


    Solid phase microextraction (SPME) is applied in the dynamic speciation analysis of the pesticide atrazine in an aqueous medium containing sorbing latex nanoparticles. It is found that the overall rate of extraction of the analyte is faster than in the absence of nanoparticles and governed by the

  19. Characterization of rhamnolipids by liquid chromatography/mass spectrometry after solid-phase extraction.

    Behrens, Beate; Engelen, Jeannine; Tiso, Till; Blank, Lars Mathias; Hayen, Heiko


    Rhamnolipids are surface-active agents with a broad application potential that are produced in complex mixtures by bacteria of the genus Pseudomonas. Analysis from fermentation broth is often characterized by laborious sample preparation and requires hyphenated analytical techniques like liquid chromatography coupled to mass spectrometry (LC-MS) to obtain detailed information about sample composition. In this study, an analytical procedure based on chromatographic method development and characterization of rhamnolipid sample material by LC-MS as well as a comparison of two sample preparation methods, i.e., liquid-liquid extraction and solid-phase extraction, is presented. Efficient separation was achieved under reversed-phase conditions using a mixed propylphenyl and octadecylsilyl-modified silica gel stationary phase. LC-MS/MS analysis of a supernatant from Pseudomonas putida strain KT2440 pVLT33_rhlABC grown on glucose as sole carbon source and purified by solid-phase extraction revealed a total of 20 congeners of di-rhamnolipids, mono-rhamnolipids, and their biosynthetic precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) with different carbon chain lengths from C8 to C14, including three rhamnolipids with uncommon C9 and C11 fatty acid residues. LC-MS and the orcinol assay were used to evaluate the developed solid-phase extraction method in comparison with the established liquid-liquid extraction. Solid-phase extraction exhibited higher yields and reproducibility as well as lower experimental effort.

  20. Solid-Phase Synthesis of Smac Peptidomimetics Incorporating Triazoloprolines and Biarylalanines

    Le Quement, Sebastian T.; Ishoey, Mette; Petersen, Mette T.;


    -Me)AVPF sequence, peptides incorporating triazoloprolines and biarylalanines were synthesized by means of Cu(I)-catalyzed azide–alkyne cycloaddition and Pd-catalyzed Suzuki cross-coupling reactions. Solid-phase procedures were optimized to high efficiency, thus accessing all products in excellent crude purities...

  1. Rapid and convenient semi-automated microwave-assisted solid-phase synthesis of arylopeptoids

    Rasmussen, Jakob Ewald; Boccia, Marcello Massimo; Nielsen, John


    A facile and expedient route to the synthesis of arylopeptoid oligomers (N-alkylated aminomethyl benz-amides) using semi-automated microwave-assisted solid-phase synthesis is presented. The synthesis was optimized for the incorporation of side chains derived from sterically hindered or unreactive...

  2. Use of aptamers in immunoassays.

    Nezlin, Roald


    Aptamers, short single-chain DNA or RNA oligonucleotides, react specifically with small molecules, as well as with proteins. Unlike antibodies, they may be obtained relatively easily. Aptamers are now widely employed in immunological studies and could replace antibodies in immunoassays. In this short review, methods for immobilizing aptamers on various insoluble materials (so-called apta-sorbents) are described. Recent findings on their use in the detection and isolation of immunoglobulins and their application in various immunoassays are also discussed.

  3. A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR

    Yi, Sun; Dhumpa, Raghuram; Bang, Dang Duong


    This paper describes a lab-on-a-chip device for fast AIV screening by integrating DNA microarray-based solid-phase PCR on a microfluidic chip.......This paper describes a lab-on-a-chip device for fast AIV screening by integrating DNA microarray-based solid-phase PCR on a microfluidic chip....

  4. A photolabile linker for the solid-phase synthesis of 4-substituted NH-1,2,3-triazoles

    Qvortrup, Katrine; Nielsen, Thomas Eiland


    A novel photolabile linker for solid-phase synthesis is presented. The linker displays an azido handle for copper-catalyzed azide–alkyne cycloaddition reactions with a variety of alkynes, remains intact under typical solid-phase reaction conditions, and enables a mild photolytic release of 4-subs...

  5. Detection of drug residues in fish and fishery products:comparison between chemiluminescence enzyme-linked immunoassay and enzyme linked immunosorbent assay%化学发光免疫分析与酶联免疫分析法检测水产品药物残留的比较研究

    萨仁托雅; 张峰; 郑有虎; 卢亚楠


    Detection of furazolidone metabolite ( AOZ ) and chloramphenicol ( CAP ) residues in fish and fishery products was comparatively studied by chemiluminescence enzyme-linked immunoassay ( CLEIA ) and traditional colorimetric enzyme-linked immunosorbent assay ( ELISA) . The results showed that CLEIA had better linear range and detection limit than ELISA, and a close correlation of recovery rate was shown in the two methods. In CLISA, the detection limit was about 0. 01 μg/kg for AOZ, and about 0. 016 μg/kg for CAP and the linear range was from 0. 01 to 2. 56μg/kg for AOZ, and from 0. 025μg/kg to 6. 400μg/kg for CAP. In ELISA, however, the detection limit was 0 . 1 μg/kg for AOZ and about 0 . 05 μg/kg for CAP and the linear range was from 0 . 1 to 1 . 62 μg/kg for AOZ and from 0. 05 to 4. 05 μg/kg for CAP. The satisfactory precisions of the assay were found in CLEIA, with variation coefficient of 5. 5%-11. 3% in the intra-assay and 12. 3%-20. 9% in the inter-assay for CAP, and with variation coefficient of 6. 6-11. 1% in the intra-assay and 15. 6%-18. 3% in the inter-assay for AOZ, within the record reference evaluation standard of ELISA residues of veterinary drugs ( kit) .%对检测水产品中呋喃唑酮代谢物( AOZ)和氯霉素( CAP)药物残留的两种方法---化学发光免疫分析( CLEIA)和酶联免疫分析( ELISA)法进行了比较研究。结果表明:两种药物残留检测中, CLEIA分析方法的线性范围和检出限均优于ELISA法,且两种免疫分析法的添加回收率均具有很好的相关性;用CLEIA法检测AOZ的检出限为0.01μg/kg,线性范围为0.01~2.56μg/kg,检测CAP的检出限为0.016μg/kg,线性范围为0.025~6.400μg/kg;用ELISA法检测AOZ的检出限为0.1μg/kg,线性范围为0.10~1.62μg/kg,检测CAP的检出限为0.05μg/kg,线性范围为0.05~4.05μg/kg;用CLEIA法检测CAP的批内变异系数( RSD)为5.5%~11.3%,批间RSD为12.3%~20.9%,检测AOZ的批内RSD为6.6%~11.1%,批间RSD为15.6%~18.3%

  6. Oriented antibody immobilization to polystyrene macrocarriers for immunoassay modified with hydrazide derivatives of poly(methacrylic acid

    Vinokurova Ludmila G


    Full Text Available Abstract Background Hydrophobic polystyrene is the most common material for solid phase immunoassay. Proteins are immobilized on polystyrene by passive adsorption, which often causes considerable denaturation. Biological macromolecules were found to better retain their functional activity when immobilized on hydrophilic materials. Polyacrylamide is a common material for solid-phase carriers of biological macromolecules, including immunoreagents used in affinity chromatography. New macroformats for immunoassay modified with activated polyacrylamide derivatives seem to be promising. Results New polymeric matrices for immunoassay in the form of 0.63-cm balls which contain hydrazide functional groups on hydrophilic polymer spacer arms at their surface shell are synthesized by modification of aldehyde-containing polystyrene balls with hydrazide derivatives of poly(methacrylic acid. The beads contain up to 0.31 μmol/cm2 active hydrazide groups accessible for covalent reaction with periodate-oxidized antibodies. The matrices obtained allow carrying out the oriented antibody immobilization, which increases the functional activity of immunosorbents. Conclusions An efficient site-directed antibody immobilization on a macrosupport is realized. The polymer hydrophilic spacer arms are the most convenient and effective tools for oriented antibody coupling with molded materials. The suggested scheme can be used for the modification of any other solid supports containing electrophilic groups reacting with hydrazides.

  7. The Effects of Solid Phase Additives on Sintering Properties of Alumina Bioceramic

    WANG Xin-yu; LI Shi-pu; HE Jian-hua; JIANG Xin; LI Jian-hua


    In order to reduce the sintering temperature and improve the preparing conditions of alumina bioceramics,the Mg-Zr-Y composite solid phase additives were added into high purity Al2O3 micro-powder by chemical coprecipitation method.The powder was shaped under 200MPa cold isostatic pressure,and then the biscuits were sintered at 1600℃ under normal pressure.The sintered alumina materials were tested and the sintering mechanism was discussed.The results show that physical properties of the material were improved comparatively.The Mg-Zr-Y composite solid additives could promote the sintering of alumina bioceramics and the mechanism is solid phase sintering.

  8. Solid-Phase Organic Synthesis and Catalysis: Some Recent Strategies Using Alumina, Silica, and Polyionic Resins

    Basudeb Basu


    Full Text Available Solid-phase organic synthesis (SPOS and catalysis have gained impetus after the seminal discovery of Merrifield’s solid-phase peptide synthesis and also because of wide applicability in combinatorial and high throughput chemistry. A large number of organic, inorganic, or organic-inorganic hybrid materials have been employed as polymeric solid supports to promote or catalyze various organic reactions. This review article provides a concise account on our approaches involving the use of (i alumina or silica, either having doped with metal salts or directly, and (ii polyionic resins to either promote various organic reactions or to immobilize reagents/metal catalysts for subsequent use in hydrogenation and cross-coupling reactions. The reaction parameters, scopes, and limitations, particularly in the context of green chemistry, have been highlighted with pertinent approaches by other groups.

  9. Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

    Houen, G.; Olsen, D.T.; Hansen, P.R.;


    A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated......, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates...... of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings...

  10. Advances in automatic, manual and microwave-assisted solid-phase peptide synthesis.

    Sabatino, Giuseppina; Papini, Anna M


    Solid-phase strategies speed up the production of both short- and long-sequence peptides compared with solution methodologies. Therefore, solid-phase peptide synthesis (SPPS), proposed by Merrifield in the early 1960s, contributed to the 'Peptide Revolution' in the fields of diagnostics, and drug and vaccine development. Since then, peptide chemistry research has aimed to optimize these synthetic procedures, focusing on areas such as amide bond formation (the coupling step), solid supports and automation. Particular attention was devoted to the environmental impact of SPPS: the requirement for large amounts of organic solvents meant high costs for industrial peptide manufacturing that needed to be reduced. SPPS, alone or in hybrid technologies, has become strategic for the production of peptides as active pharmaceutical ingredients on a commercial scale.

  11. A Facile, Choline Chloride/Urea Catalyzed Solid Phase Synthesis of Coumarins via Knoevenagel Condensation

    Hosanagara N. Harishkumar


    Full Text Available The influence of choline chloride/urea ionic liquid in solid phase on the Knoevenagel condensation is demonstrated. The active methylene compounds such as meldrum’s acid, diethylmalonate, ethyl cyanoacetate, dimethylmalonate, were efficiently condensed with various salicylaldehydes in presence of choline chloride/urea ionic liquid without using any solvents or additional catalyst. The reaction is remarkably facile because of the air and water stability of the catalyst, and needs no special precautions. The reactions were completed within 1hr with excellent yields (95%. The products formed were sufficiently pure, and can be easily recovered. The use of ionic liquid choline chloride/urea in solid phase offered several significant advantages such as low cost, greater selectivity and easy isolation of products.

  12. Silica-Based Solid Phase Extraction of DNA on a Microchip

    陈晓芳; 沈科跃; 刘鹏; 郭旻; 程京; 周玉祥


    Micro total analysis systems for chemical and biological analysis have attracted much attention.However,microchips for sample preparation and especially DNA purification are still underdeveloped.This work describes a solid phase extraction chip for purifying DNA from biological samples based on the adsorption of DNA on bare silica beads prepacked in a microchannel.The chip was fabricated with poly-dimethylsiloxane.The silica beads were packed in the channel on the chip with a tapered microchannel to form the packed bed.Fluorescence detection was used to evaluate the DNA adsorbing efficiency of the solid phase.The polymerase chain reaction was used to evaluate the quality of the purified DNA for further use.The extraction efficiency for the DNA extraction chip is approximately 50% with a 150-nL extraction volume.Successful amplification of DNA extracted from human whole blood indicates that this method is compatible with the polymerase chain reaction.

  13. Solid phase epitaxy amorphous silicon re-growth: some insight from empirical molecular dynamics simulation

    Krzeminski, Christophe; 10.1140/epjb/e2011-10958-7


    The modelling of interface migration and the associated diffusion mechanisms at the nanoscale level is a challenging issue. For many technological applications ranging from nanoelectronic devices to solar cells, more knowledge of the mechanisms governing the migration of the silicon amorphous/crystalline interface and dopant diffusion during solid phase epitaxy is needed. In this work, silicon recrystallisation in the framework of solid phase epitaxy and the influence on orientation effects have been investigated at the atomic level using empirical molecular dynamics simulations. The morphology and the migration process of the interface has been observed to be highly dependent on the original inter-facial atomic structure. The [100] interface migration is a quasi-planar ideal process whereas the cases [110] and [111] are much more complex with a more diffuse interface. For [110], the interface migration corresponds to the formation and dissolution of nanofacets whereas for [111] a defective based bilayer reor...

  14. Experimental setup for investigating silicon solid phase crystallization at high temperatures.

    Schmidt, Thomas; Gawlik, Annett; Schneidewind, Henrik; Ihring, Andreas; Andrä, Gudrun; Falk, Fritz


    An experimental setup is presented to measure and interpret the solid phase crystallization of amorphous silicon thin films on glass at very high temperatures of about 800 °C. Molybdenum-SiO(2)-silicon film stacks were irradiated by a diode laser with a well-shaped top hat profile. From the relevant thermal and optical parameters of the system the temperature evolution can be calculated accurately. A time evolution of the laser power was applied which leads to a temperature constant in time in the center of the sample. Such a process will allow the observation and interpretation of solid phase crystallization in terms of nucleation and growth in further work.

  15. Microwave spectroscopic observation of distinct electron solid phases in wide quantum wells.

    Hatke, A T; Liu, Yang; Magill, B A; Moon, B H; Engel, L W; Shayegan, M; Pfeiffer, L N; West, K W; Baldwin, K W


    In high magnetic fields, two-dimensional electron systems can form a number of phases in which interelectron repulsion plays the central role, since the kinetic energy is frozen out by Landau quantization. These phases include the well-known liquids of the fractional quantum Hall effect, as well as solid phases with broken spatial symmetry and crystalline order. Solids can occur at the low Landau-filling termination of the fractional quantum Hall effect series but also within integer quantum Hall effects. Here we present microwave spectroscopy studies of wide quantum wells that clearly reveal two distinct solid phases, hidden within what in d.c. transport would be the zero diagonal conductivity of an integer quantum-Hall-effect state. Explanation of these solids is not possible with the simple picture of a Wigner solid of ordinary (quasi) electrons or holes.

  16. Anisotropic kinetics of solid phase transition from first principles: alpha-omega phase transformation of Zr.

    Guan, Shu-Hui; Liu, Zhi-Pan


    Structural inhomogeneity is ubiquitous in solid crystals and plays critical roles in phase nucleation and propagation. Here, we develop a heterogeneous solid-solid phase transition theory for predicting the prevailing heterophase junctions, the metastable states governing microstructure evolution in solids. Using this theory and first-principles pathway sampling simulation, we determine two types of heterophase junctions pertaining to metal α-ω phase transition at different pressures and predict the reversibility of transformation only at low pressures, i.e. below 7 GPa. The low-pressure transformation is dominated by displacive Martensitic mechanism, while the high-pressure one is controlled by the reconstructive mechanism. The mechanism of α-ω phase transition is thus highly pressure-sensitive, for which the traditional homogeneous model fails to explain the experimental observations. The results provide the first atomic-level evidence on the coexistence of two different solid phase transition mechanisms in one system.

  17. The Use of Aryl Hydrazide Linkers for the Solid Phase Synthesis of Chemically Modified Peptides

    Woo, Y; Mitchell, A R; Camarero, J A


    Since Merrifield introduced the concept of solid phase synthesis in 1963 for the rapid preparation of peptides, a large variety of different supports and resin-linkers have been developed that improve the efficiency of peptide assembly and expand the myriad of synthetically feasible peptides. The aryl hydrazide is one of the most useful resin-linkers for the synthesis of chemically modified peptides. This linker is completely stable during Boc- and Fmoc-based solid phase synthesis and yet it can be cleaved under very mild oxidative conditions. The present article reviews the use of this valuable linker for the rapid and efficient synthesis of C-terminal modified peptides, head-to-tail cyclic peptides and lipidated peptides.

  18. 西仑吉肽的固相合成%Solid phase synthesis of cilengitide

    张波; 王卫国; 康武; 智小霞; 姚忠; 徐红岩


    Fully-protected linear peptide was synthesized by Fmoc solid phase peptide synthesis methods. The solid phase carrier was 2-chlorotrityl chloride resin. HATU/HOBt and HBTU/HOBt were used as the coupling reagents. The synthesis of fully-protected cyclic peptide used THF/DCM ( at a ratio of 1: 1 by volume) as the solvent and HATU/HOBt as the coupling reagents. Finally cilengitide could be obtained by deprotection reaction.%采用Fmoc固相合成法,选用2-氯三苯甲基氯树脂作为固相载,HBTU/HOBt和HATU/HOBt为缩合剂,合成全保护线性肽.以V(DCM)∶V(THF)= 1∶1为溶剂,HATU/HOBt为缩合剂,合成全保护环肽.最后脱除保护基得终产物西仑吉肽.

  19. The Solid Phase Curing Time Effect of Asbuton with Texapon Emulsifier at the Optimum Bitumen Content

    Sarwono, D.; Surya D, R.; Setyawan, A.; Djumari


    Buton asphalt (asbuton) could not be utilized optimally in Indonesia. Asbuton utilization rate was still low because the processed product of asbuton still have impracticable form in the term of use and also requiring high processing costs. This research aimed to obtain asphalt products from asbuton practical for be used through the extraction process and not requiring expensive processing cost. This research was done with experimental method in laboratory. The composition of emulsify asbuton were 5/20 grain, premium, texapon, HCl, and aquades. Solid phase was the mixture asbuton 5/20 grain and premium with 3 minutes mixing time. Liquid phase consisted texapon, HCl and aquades. The aging process was done after solid phase mixing process in order to reaction and tie of solid phase mixed become more optimal for high solubility level of asphalt production. Aging variable time were 30, 60, 90, 120, and 150 minutes. Solid and liquid phase was mixed for emulsify asbuton production, then extracted for 25 minutes. Solubility level of asphalt, water level, and asphalt characteristic was tested at extraction result of emulsify asbuton with most optimum ashphal level. The result of analysis tested data asphalt solubility level at extract asbuton resulted 94.77% on 120 minutes aging variable time. Water level test resulted water content reduction on emulsify asbuton more long time on occurring of aging solid phase. Examination of asphalt characteristic at extraction result of emulsify asbuton with optimum asphalt solubility level, obtain specimen that have rigid and strong texture in order that examination result have not sufficient ductility and penetration value.

  20. Determination of zinc in environmental samples by solid phase spectrophotometry: optimization and validation study

    Molina, Mar??a Francisca; Nechar, Mounir; Bosque-Sendra, Juan M.


    A simple and specific solid-phase spectrophotometric (SPS) determination of zinc in ??g dm-3 level has been developed based on the reaction of Zn(II) with 4-(2-pyridylazo)resorcinol (PAR) in the presence of potassium iodide; the product was then fixed on an anionic exchanger. The absorbance of the gel, packed in a 1 mm cell, is measured directly. PAR and KI concentrations were optimized simultaneously using response surface methodology (RSM) from sequential experimental Doehlert designs. The ...

  1. Zirconyl chloride promoted highly efficient solid phase synthesis of amide derivatives


    An efficient solid phase route for the synthesis of amide derivatives by the reaction of carboxylic acids with urea in the presence of catalytic amount of zirconyl chloride under microwave irradiation conditions was described. In this way, a range of interesting amide derivatives was obtained in good to excellent yields. The catalyst was recycled with fresh reactants and it gave almost similar results without significant loss of activity up to the third run.

  2. R. Bruce Merrifield and Solid-Phase Peptide Synthesis: A Historical Assessment

    Mitchell, A R


    Bruce Merrifield, trained as a biochemist, had to address three major challenges related to the development and acceptance of solid-phase peptide synthesis (SPPS). The challenges were (1) to reduce the concept of peptide synthesis on a insoluble support to practice, (2) overcome the resistance of synthetic chemists to this novel approach, and (3) establish that a biochemist had the scientific credentials to effect the proposed revolutionary change in chemical synthesis. How these challenges were met is discussed in this article.

  3. A New Molecularly Imprinted Polymer for Solid-phase Extraction of Cotinine from Human Urine

    Jun YANG; Xiao Lan ZHU; Ji Bao CAI; Qing De SU; Yun GAO; Liang ZHANG


    A molecularly imprinted polymer (MIP), prepared around a cotinine template, has been synthesized. The feasibility of using the polymer for solid-phase extraction (SPE) of cotinine from biological samples has been investigated. The results show that cotinine can be quantitatively retained and eluted from the polymer. Experiments with human urine samples indicate that clean target analyte is obtained for HPLC with UV detection using the protocol.

  4. Selective fiber used for headspace solid-phase microextraction of abused drugs in human urine

    Sunanta Wangkarn


    A sensitive and selective fiber for simultaneous analysis of three drugs of abuse (amphetamine, methamphetamine and ephedrine) in urine samples was explored using headspace solid phase microextraction and gas chromatography with flame ionization detection. Several parameters affecting extraction such as extraction time, extraction temperature, pH of solution and salt concentrations were investigated. Among five commercially available fibers, divinylbenzene/carboxen/ polydimethylsiloxane is th...

  5. Dynamics of Vibrio cholerae abundance in Austrian saline lakes, assessed with quantitative solid-phase cytometry


    International audience; In order to elucidate the main predictors of Vibrio cholerae dynamics and to estimate the risk of Vibrio cholera-related diseases, a recently developed direct detection approach based on fluorescence in situ hybridization and solid-phase cytometry (CARD-FISH/ SPC) was applied in comparison to cultivation for water samples from the lake Neusiedler See, Austria and three shallow alkaline lakes over a period of 20 months. Vibrio cholerae attached to crustacean zoo-plankto...

  6. Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

    Lundqvist, Magnus; Edfors, Fredrik; Sivertsson, Åsa


    We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We...... at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations....

  7. Fmoc solid-phase synthesis of peptide thioesters by masking as trithioortho esters

    Brask, Jesper; Albericio, F.; Jensen, Knud Jørgen


    Total chemical synthesis of proteins by chemoselective ligation relies on C-terminal peptide thioesters as building blocks. Their preparation by standard Fmoc solid-phase peptide synthesis is made difficult by the lability of thioesters to aminolysis by the secondary amines used for removal of th...... of the Fmoc group. Here we present a novel backbone amide linker (BAL) strategy for their synthesis in which the thioester functionality is masked as a trithioortho ester throughout the synthesis....

  8. Solid-phase microextraction for flavor analysis in Harari Khat (Catha edulis) stimulant

    AL-FLAHI Abdulsalam; ZOU Jian-kai (邹建凯); YIN Xue-feng (殷学锋)


    This research examined the typical flavor compounds in the commonest type of Khat called Harari Khat grown in the region of Ethiopia. Twenty-eight compounds, which includes 1,2-Propanedione, 1-Phenyl, Hexanol, Hexanal compounds, Limonene, Benzaldehyde with other flavors, were extracted by polydimethylsiloxane at room temperature for 30 min from Khat samples, and identified by solid-phase microextraction-gas chromatography-mass spectrometry (SPME- GC-MS). This method needs no organic solvents and required minimal sample.

  9. Solid-Phase Organic Synthesis of Aryl Vinyl Ethers Using Sulfone-Linking Strategy

    余腊妹; 汤妮; 盛寿日; 陈茹冰; 刘晓玲; 蔡明中


    A novel facile solid-phase organic synthesis of aryl vinyl ethers by reaction of polystyrene-supported 2-phenylsulfonylethanol with phenols under Mitsunobu conditions and subsequent elimination reaction with DBU has been developed. The advantages of this method include straightforward operation, good yield and high purity of the products. Alternatively, a typical example of Suzuki coupling reaction on-resin was further applied to prepare 4-phenylphenyl vinyl ether for extending this method.

  10. Solid-phase reduction of Cr2O3 under chemical catalytic conditions

    Simonov, V. K.; Grishin, A. M.


    The kinetics of the solid-phase reduction of Cr2O3 with carbon under chemical catalytic action on the reacting system is studied. A significant intensification of the process in the presence of small amounts of potassium and sodium salts is established. The concepts of the catalyst action mechanism are considered and experimentally substantiated. Manufacture of iron-chromium master alloys with a restricted content of carbon can be organized at low temperatures, and they can be used in steelmaking.

  11. Preparation of high-quality poly-Si films by a solid phase crystallizing method

    Yao Ruo He


    A solid phase crystallizing method has been developed to grow a Si crystal at temperatures as low as 550 degree C. Using this method, a high-quality thin-film polycrystalline silicon (Poly-Si) was obtained. The largest grain size, examined with X-ray diffraction spectroscopy and scanning electron microscopy images of recrystallized samples, is approximately 1 mu m for substrate temperature at 300 degree C and annealed at 550 degree C for 3 hours

  12. Determination of Trace Amount of Yttrium with Bromopyrogallol Red by Solid-phase Spectrophotometry


    A simple and sensitive method for the determination of trace amount of yttrium by solid-phase spectrophotometry has been studied. Yttrium can form a 1∶1 complex with bromopyrogallol red (BPR) on resin, which was determined directly at 605 nm, pH=6.5. It has a highly sensitivity ( = 6.3€?06) which is 300-fold higher than the corresponding spectrophotometry in solution. The method was applied to the determination of yttrium in churchite.

  13. Studies on solid phase synthesis,characterization and fluorescent property of the new rare earth complexes

    Shi, Jianwei; Xiaoxu TENG; Wang, Linling; Long, Rong


    Rare earth-β-diketone ligand complex luminescent material has stable chemical properties and excellent luminous property. Using europium oxide and (γ-NTA) as raw materials, novel rare earth-β-dione complexes are synthesized by solid state coordination chemistry. The synthesis temperature and milling time are discussed for optimization. Experimental results show that the suitable reaction situation is at 50 ℃ and 20 h for solid-phase synthesis. The compositions and structures of the complexes...

  14. The Synthesis of QADMAA and its Application to the Solid Phase ...

    and the solid phase extraction of the Co(II)-QADMAA chelate with C18 membrane disks was .... For up to 2.4 µg of Co(II), the use of about 5~10 mL 5 × 10–4 ..... 22 benzoic acid. 2-(3,5-Dibromo-2-pyridylazo)-diaminotoluene. pH 4–8. 590. 13.4.

  15. Determination of formaldehyde in Brazilian alcohol fuels by flow-injection solid phase spectrophotometry

    Teixeira, Leonardo Sena Gomes; Leão, Elsimar S.; Dantas,Alailson Falcão; Pinheiro, Heloísa Lúcia C.; Costa, Antonio Celso Spinola; Andrade,Jailson Bittencourt de


    p. 711–715 In thiswork, a solid phase spectrophotometric method in association with flowinjection analysis for formaldehyde determination has been developed with direct measurement of light-absorption in C18 material. The 3,5-diacetyl-1,4-dihydrolutidine produced from the reaction between formaldehyde and fluoral P was quantitatively retained on C18 support and the spectrophotometric detection was performed simultaneously at 412 nm. The retained complex was quickly eluted from C18 mater...


    S. Manna


    Full Text Available High performance liquid chromatographic determination of organophosphorous compound has been done by reverse phase chromatography in goats. The goats were dying showing the symptoms of organophosphorous poisoning. The viscera and stomach contents sample were received from Project Co-Ordinator, Animal Disease Research Institute, Phulnakhara, Cuttack, Orissa. The analysis of samples by HPLC with UV detector after cleaning up in Solid Phase Extraction (SPE revealed presence of malathion that was later quantified.

  17. Different Biotinylation Strategies for Competitive Immunoassay of Estradiol

    ZHAO,Jin-Fu(赵金富); WANG,Yong-Cheng(王永成); LI,Yuan-Zong(李元宗); CHANG,Wen-Bao(常文保)


    Study on biotinylation strategies for competitive immunoassay of estradiol (E2) was carried out. Two types of competitive enzyme immunoassay (EIA) with Biotin-Avidin amplification system were established and optimized.The E2-Biotin conjugate was used as a tracer in one assay, and biotinylated antibody was used as a tracer in the other. In both of EIAs, horseradish peroxidase-labelled Avidin (Avidin-HRP) was used with a spectrometric determination of enzyme activity. The precision, sensitivity and specificity were measured and compared. The results showed that although both were satisfactory in specificity, the EIA with hapten-Biotin showed to be superior to the EIA with biotinylated antibody in sensitivity and precision. The limit of detection of serum E2 was 8 and 50 pg/mL with E2-Biotin and biotinylated antibody as tracer, respectively.

  18. Direct molecular dynamics simulation of liquid-solid phase equilibria for a three-component plasma.

    Hughto, J; Horowitz, C J; Schneider, A S; Medin, Zach; Cumming, Andrew; Berry, D K


    The neutron-rich isotope ²²Ne may be a significant impurity in carbon and oxygen white dwarfs and could impact how the stars freeze. We perform molecular dynamics simulations to determine the influence of ²²Ne in carbon-oxygen-neon systems on liquid-solid phase equilibria. Both liquid and solid phases are present simultaneously in our simulation volumes. We identify liquid, solid, and interface regions in our simulations using a bond angle metric. In general we find good agreement for the composition of liquid and solid phases between our MD simulations and the semianalytic model of Medin and Cumming. The trace presence of a third component, neon, does not appear to strongly impact the chemical separation found previously for two-component carbon and oxygen systems. This suggests that small amounts of ²²Ne may not qualitatively change how the material in white dwarf stars freezes. However, we do find systematically lower melting temperatures (higher Γ) in our MD simulations compared to the semianalytic model. This difference seems to grow with impurity parameter Q_{imp} and suggests a problem with simple corrections to the linear mixing rule for the free energy of multicomponent solid mixtures that is used in the semianalytic model.

  19. Direct MD simulation of liquid-solid phase equilibria for three-component plasma

    Hughto, J; Schneider, A S; Medin, Zach; Cumming, Andrew; Berry, D K


    The neutron rich isotope 22Ne may be a significant impurity in carbon and oxygen white dwarfs and could impact how the stars freeze. We perform molecular dynamics simulations to determine the influence of 22Ne in carbon-oxygen-neon systems on liquid-solid phase equilibria. Both liquid and solid phases are present simultaneously in our simulation volumes. We identify liquid, solid, and interface regions in our simulations using a bond angle metric. In general we find good agreement for the composition of liquid and solid phases between our MD simulations and the semi analytic model of Medin and Cumming. The trace presence of a third component, neon, does not appear to strongly impact the chemical separation found previously for two component carbon and oxygen systems. This suggests that small amounts of 22Ne may not qualitatively change how the material in white dwarf stars freezes. However, we do find systematically lower melting temperatures (higher Gamma) in our MD simulations compared to the semi analytic ...


    S. J. Shahtaheri, H. R. Heidari, F. Golbabaei, M. Alimohammadi, A. Rahimi Froshani


    Full Text Available Conventional analytical method for organic pollutants in water requires extraction of the pollutants, using hazardous solvent. Solid phase microextraction is a solvent free equilibrium extraction method, in which, proper calibration can allow quantitative determinations of organic pollutants at a very good sensitivity without the use of any organic solvent. Because individual volatile organic carbons are generally exposed environmentally and present in urine only at trace levels, a sensitive and accurate determination technique is essential. So, this study describes the optimization of headspace solid phase microextraction (HS-SPME followed by GC-FID for benzene in spiked urine. Through this investigations, the parameters affecting the extraction and gas chromatographic determination of analytes, including extraction time, temperature, desorption temperature, desorption time, salt addition, sample pH, sample volume and sample agitation were studied. An optimized headspace extraction was carried out at 30°C for 6 min in the presence of 0.2 g/mL of NaCl in the sample solution. Desorption of the analytes was carried out for 60 sec. at 250°C. The optimized procedure was also validated with three different pools of spiked urine samples and showed a good reproducibility over six consecutive days as well as six within-day experiments. The accuracy, linearity, detection limits were also determined. The headspace solid phase microextraction, GC-FID technique provides a relatively simple, convenient, practical procedure, which was here successfully applied to determine benzene in spiked urine.

  1. Solid phase speciation of Zn and Cd in zinc smelter effluent-irrigated soils

    Prasenjit Ray


    Full Text Available Solubility of metal in contaminated soils is a key factor which controls the phytoavailability and toxic effects of metals on soil environment. The chemical equilibria of metal ions between soil solution and solid phases govern the solubility of metals in soil. Hence, an attempt was made to identify the probable solid phases (minerals, which govern the solubility of Zn2+ and Cd2+ in zinc smelter effluent-irrigated soils. Estimation of free ion activities of Zn2+ (pZn2+ and Cd2+ (pCd2+ by Baker soil test indicated that metal ion activities were higher in smelter effluent-irrigated soils as compared to that in tubewell water-irrigated soils. Identification of solid phases further reveals that free ion activity of Zn2+ and Cd2+ in soil highly contaminated with Zn and Cd due to long-term irrigation with zinc smelter effluent is limited by the solubility of willemite (Zn2SiO4 in equilibrium with quartz and octavite (CdCO3, respectively. However, in case of tubewell water-irrigated soil, franklinite (ZnFe2O4 in equilibrium with soil-Fe and exchangeable Cd are likely to govern the activity of Zn2+ and Cd2+ in soil solution, respectively. Formation of highly soluble minerals namely, willemite and octavite indicates the potential ecological risk of Zn and Cd, respectively in smelter effluent irrigated soil.

  2. The isolation of soyasaponins by fractional precipitation, solid phase extraction, and low pressure liquid chromatography.

    Gurfinkel, D M; Reynolds, W F; Rao, A V


    Bioactive soyasaponins are present in soybean (Glycine max). In this study, the isolation of soyasaponins in relatively pure form (>80%) using precipitation, solid phase extraction and reverse phase low pressure liquid chromatography (RP-LPLC) is described. Soy flour soyasaponins were separated from non-saponins by methanol extraction and precipitation with ammonium sulphate. Acetylated group A soyasaponins were isolated first by solid phase extraction followed by RP-LPLC (solvent: ethanol-water). Soyasaponins, from a commercial preparation, were saponified and fractionated into deacetylated group A and group B soyasaponins by solid phase extraction (methanol-water). Partial hydrolysis of group B soyasaponins produced a mixture of soyasaponin III and soyasapogenol B monoglucuronide. RP-LPLC of deacetylated group A soyasaponins separated soyasaponin A1 and A2 (38% methanol); of group B soyasaponins isolated soyasaponin I (50% ethanol); and of the partial hydrolysate separated soyasaponin III from soyasapogenol B monoglucuronide (50% ethanol). This methodology provides soyasaponin fractions that are suitable for biological evaluation.

  3. Comparative evaluation of four trityl-type amidomethyl polystyrene resins in Fmoc solid phase peptide synthesis.

    Zikos, Christos; Livaniou, Evangelia; Leondiadis, Leondios; Ferderigos, Nikolas; Ithakissios, Dionyssis S; Evangelatos, Gregory P


    Four trityl-type (i.e. non-substituted trityl-, o-Cl-trityl-, o-F-trityl- and p-CN-trityl-) amidomethyl polystyrene resins were evaluated comparatively, in terms of the stability of the trityl-ester bond in slightly acidic dichloromethane solutions, and the p-CN-trityl-amidomethyl polystyrene resin was found to be the most stable of them. The above resins were applied, in parallel with Wang benzyl-type resin, well known for its stability in mild acidic conditions, to the Fmoc solid phase synthesis of the 43-amino acid residue long bioactive peptide thymosin beta-4. Independent of their differences in acid sensitivity, the resins seemed to function equally well under the conditions used, since pure thymosin beta-4 was obtained with a final yield of approximately 30% from each resin. The trityl-type amidomethyl polystyrene resins were also applied, in parallel with the Wang resin, to the Fmoc solid phase synthesis of a bioactive peptide containing proline at its C-terminus, i.e. the N-terminal tetrapeptide of thymosin beta-4, AcSDKP. In this case, the best yield (87%) was obtained with the o-Cl-trityl-amidomethyl polystyrene resin, which may be the resin of choice, of those studied, for the Fmoc solid phase peptide synthesis.

  4. Preparation of fluorescent DNA probe by solid-phase organic synthesis


    Full Text Available Fluorescent DNA probe based on fluorescence resonance energy transfer (FRET was prepared by solid-phase organic synthesis when CdTe quantum dots (QDs were as energy donors and Au nanoparticles (AuNPs were as energy accepters. The poly(divinylbenzene core/poly(4-vinylpyridine shell microspheres, as solid-phase carriers, were prepared by seeds distillation-precipitation polymerization with 2,2′-azobisisobutyronitrile (AIBN as initiator in neat acetonitrile. The CdTe QDs and AuNPs were self-assembled on the surface of core/shell microspheres, and then the linkage of CdTe QDs with oligonucleotides (CdTe-DNA and AuNPs with complementary single-stranded DNA (Au-DNA was on the solid-phase carriers instead of in aqueous solution. The hybridization of complementary double stranded DNA (dsDNA bonded to the QDs and AuNPs (CdTe-dsDNA-Au determined the FRET distance of CdTe QDs and AuNPs. Compared with the fluorescence of CdTe-DNA, the fluorescence of CdTe-dsDNA-Au conjugates (DNA probes decreased extremely, which indicated that the FRET occurred between CdTe QDs and AuNPs. The probe system would have a certain degree recovery of fluorescence when the complementary single stranded DNA was introduced into this system, which showed that the distance between CdTe QDs and AuNPs was increased.

  5. Screening for anabolic steroids in urine of forensic cases using fully automated solid phase extraction and LC-MS-MS.

    Andersen, David W; Linnet, Kristian


    A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated. The method involves a fully automated sample preparation including enzyme treatment, addition of internal standards and solid phase extraction followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with adduct formation for two compounds. Urine samples from 580 forensic cases were analyzed to determine the T/E ratio and occurrence of exogenous anabolic steroids. Extraction recoveries ranged from 77 to 95%, matrix effects from 48 to 78%, overall process efficiencies from 40 to 54% and the lower limit of identification ranged from 2 to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9%) were found positive for one or more anabolic steroids. Only seven different steroids including testosterone were found in the material, suggesting that only a small number of common steroids are likely to occur in a forensic context. The steroids were often in high concentrations (>100 ng/mL), and a combination of steroids and/or other drugs of abuse were seen in the majority of cases. The method presented serves as a fast and automated screening procedure, proving the suitability of LC-MS-MS for analyzing anabolic steroids.

  6. The Role of 4-Hydroxyphenylpyruvate Dioxygenase in Enhancement of Solid-Phase Electron Transfer by Shewanella oneidensis MR-1

    Turick, Charles E. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Beliaev, Alex S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Zakrajsek, Brian A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Reardon, Catherine L. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lowy, Daniel A. [Nova Research Inc., Alexandria, VA (United States); Poppy, Tara E. [Univ. of South Carolina, Aiken, SC (United States); Maloney, Andrea [Winthrop Univ., Rock Hill, SC (United States); Ekechukwu, Amy A. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)


    ABSTRACT - While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane associated c-type cytochromes and electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of the tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione ([2-(2- chloro- 4- methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA, which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates at which MR-1 reduces hydrous ferric oxide were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E°') of S. oneidensis MR-1. Based on our findings, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in S. oneidensis MR-1.


    Turick, C; Amy Ekechukwu, A


    While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane-associated c-type cytochromes and redox active electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. In this study, we determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione (2-(2-chloro-4-methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates, with which MR-1 reduces hydrous ferric oxide, were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E{sup o}{prime}) of S. oneidensis MR-1. Based on this work, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in Shewanella oneidensis.

  8. Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

    Hunt, J S; McGiven, A R; Groufsky, A; Lynn, K L; Taylor, M C


    Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.

  9. Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

    Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa


    In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening.

  10. Solid phase total synthesis of the 3-amino-6-hydroxy-2-piperidone (Ahp) cyclodepsipeptide and protease inhibitor Symplocamide A.

    Stolze, Sara C; Meltzer, Michael; Ehrmann, Michael; Kaiser, Markus


    The solid phase total synthesis of the marine cyanobacterial Ahp-cyclodepsipeptide Symplocamide A is reported as a model for a general route for the synthesis of tailor-made non-covalent serine protease inhibitors.

  11. Comparison of solid-phase and eluate assays to gauge the ecotoxicological risk of organic wastes on soil organisms.

    Domene, Xavier; Alcañiz, Josep M; Andrés, Pilar


    Development of methodologies to assess the safety of reusing polluted organic wastes in soil is a priority in Europe. In this study, and coupled with chemical analysis, seven organic wastes were subjected to different aquatic and soil bioassays. Tests were carried out with solid-phase waste and three different waste eluates (water, methanol, and dichloromethane). Solid-phase assays were indicated as the most suitable for waste testing not only in terms of relevance for real situations, but also because toxicity in eluates was generally not representative of the chronic effects in solid-phase. No general correlations were found between toxicity and waste pollutant burden, neither in solid-phase nor in eluate assays, showing the inability of chemical methods to predict the ecotoxicological risks of wastes. On the contrary, several physicochemical parameters reflecting the degree of low organic matter stability in wastes were the main contributors to the acute toxicity seen in collembolans and daphnids.

  12. Structural control of Fe-based alloys through diffusional solid/solid phase transformations in a high magnetic field.

    Ohtsuka, Hideyuki


    A magnetic field has a remarkable influence on solid/solid phase transformations and it can be used to control the structure and function of materials during phase transformations. The effects of magnetic fields on diffusional solid/solid phase transformations, mainly from austenite to ferrite, in Fe-based alloys are reviewed. The effects of magnetic fields on the transformation temperature and phase diagram are explained thermodynamically, and the transformation behavior and transformed structures in magnetic fields are discussed.

  13. Solid Phase Red Cell Adherence Assay: a tubeless method for pretransfusion testing and other applications in transfusion science.

    Ching, Eric


    Solid Phase Red Cell Adherence Assay (SPRCA) is one of the two tubeless methods developed to improve sensitivity and specificity in blood group serology. The SPRCA (solid phase) and the column agglutination (gel) technology have gained wide acceptance following successful adaptation to fully automated platforms, The purpose of this paper is to discuss the development, principle, procedures as well as laboratory and clinical applications of the SPRCA in transfusion medicine.

  14. Matrix solid-phase dispersion and solid-phase microextraction applied to study the distribution of fenbutatin oxide in grapes and white wine.

    Montes, R; Canosa, P; Lamas, J Pablo; Piñeiro, A; Orriols, I; Cela, R; Rodríguez, I


    The fate of the acaricide fenbutatin oxide (FBTO) during the elaboration of white wine is evaluated. Matrix solid-phase dispersion (MSPD) and solid-phase microextraction (SPME) were used as sample preparation techniques applied to the semi-solid and the liquid matrices involved in this research, respectively. Selective determination of FBTO was achieved by gas chromatography with atomic emission detection (GC-AED). GC coupled to mass spectrometry was also used to establish the identity of FBTO by-products detected in must and wine samples. MSPD extractions were accomplished using C18 as dispersant and co-sorbent. Sugars and other polar interferences were first removed with water and water/acetone mixtures, then FBTO was recovered with 8 mL of acetone. When used in combination with GC-AED, the MSPD method provided limits of quantification (LOQs) in the low nanogram per gram range, recoveries around 90% and relative standard deviations below 13% for extractions performed in different days. Performance of SPME for must and wine was mainly controlled by the extraction temperature, time and fibre coating. Under final conditions, FBTO was extracted in the headspace mode for 45 min at 100 degrees C, using a 100 microm poly(dimethylsiloxane)-coated fibre. The achieved LOQs remained around or below 0.1 ng mL(-1), depending on the type of sample, and the inter-day precision ranged from 10% to 13%. FBTO residues in grapes stayed mostly on the skin of the fruit. Although FBTO was not removed during must and white wine elaboration, it remained associated with suspended particles existing in must and lees, settled after must fermentation, with a negligible risk of being transferred to commercialised wine. On the other hand, two by-products of FBTO (bis and mono (2-methyl-2-phenylpropyl) tin) were identified, for first time, in must and final white wines obtained from FBTO treated grapes. Found values for the first species ranged from 0.03 to 0.9 ng mL(-1).


    Yuliya Modna


    Full Text Available The acute destructive pneumonias (ADP occupy up to 80% of the total number of pneumonias. They require constant improvement of treatment strategy. Nowadays the use of surfactants is a part of most treatment protocols. The aim was to study the features of the solid phase bronchoalveolar lavage in children with the ADPs in the dynamics of complex treatment with exogenous surfactant.Material and methods: We examined 39 patients of contaminated surgery. We identified 2 groups of patients. The patients of first group (n=27 had pulmonary pleural form of ADP, the second group (n=12 had pulmonary form of ADP. All patients got classical treatment and the earlier draining of pleural cavity. We used as an antiseptic reamberin 1.5% by 10 ml/kg and endobronchially injected exogenous surfactant Bl in dose12 mg/kg body weight a day, 6 mg/kg every 12 hours. All the children were made a bronchoscopy to obtain BAL to study the crystallization properties. The solid phase of BAL was studied by method of cuneal dehydration.Results: All facies before treatment were divided into two groups according to classification of facies of biological fluids. Only the facies of the second and the third types were detected there. It was revealed that the sizes of the zones of the facies were different in the comparison groups before treatment and after. And the level of crystalline structures and amorphous aggregates were different in the groups with different degrees of inflammation.Conclusion: So, we can assume that the change in surfactant system is characterized by changes in the morphological structure of solids phases of BAL. And the morphological structure of BAL depends on the chemical composition of BAL.

  16. Formation of organic solid phases in hydrocarbon reservoir fluids. Final report

    Andersen, S.I.; Lindeloff, N.; Stenby, E.H.


    The occurrence of solid phases during oil recovery is a potential problem. The present work has mainly been concerned with wax formation due to cooling of oils with a large paraffin content. 8 oils have been included in this project, although only a few of these have till now been subject to all the experimental techniques applied. The oils and wax fractions from these have been characterized using techniques such as GC-MS and Ftir. The goal has in part been to get a detailed description of the oil composition for use in model evaluation and development and in part to get a fundamental understanding of waxy oil properties and behaviour. A high pressure (200 bar) equipment has been developed for automatic detection of wax appearance using a filtration technique and laser light turbidimetry. The latter was found to be far superior to the filtration. The filtration was used to sample the incipient solid phase for characterization. However entrapment of liquid in the filters currently used have hampered this part. A number of model systems and one gas condensate have been investigated. The GC-MS procedure was found only to been able to detect molecules up to n-C45 and the group type analysis was not accurate enough for modelling purposes. Using Ftir it was obvious that incipient phases may contain very complex molecules (asphaltenes) which are not captured by GC-MS especially when fractionation is done using the acetone precipitation at elevated temperature. The latter fractionation procedure has been investigated thoroughly as a tool for understanding wax distribution etc. Within thermodynamic modelling a delta lattice parameter model has been developed which incorporates the non-ideality of the solid phases into the calculation of SLE. The non-ideality is estimated from pure component properties. A new algorithm for phase equilibria involving gas-liquid-solid has been developed. Currently both the model work and the experimental works are continued. (au)

  17. Solid-phase extraction and HPLC assay of nicotine and cotinine in plasma and brain.

    Dawson, Ralph; Messina, S M; Stokes, C; Salyani, S; Alcalay, N; De Fiebre, N C; De Fiebre, C M


    The aim of this study was to develop a simple and reliable assay for nicotine (NIC) and its major metabolite, cotinine (COT), in plasma and brain. A method was developed that uses an extraction method compatible with reverse-phase high-performance liquid chromatography (HPLC) separation and ultraviolet (UV) detection. Sequential solid-phase extraction on silica columns followed by extraction using octadecyl (C18) columns resulted in mean percent recovery (n = 5) of 51 +/- 5, 64 +/- 10, and 52 +/- 10% for NIC, COT, and phenylimidazole (PI), respectively, in spiked 1-mL serum samples. Recovery (mean +/- SEM) of the internal standard (PI) from spiked samples of nicotine-injected rats averaged 64.1 +/- 1.5% (n = 138) from plasma, and 20.7+/-0.8% (n = 128) from brain. The limits of detection of NIC in plasma samples were approximately 8 ng per mL, and of COT, 13.6 ng per mL. Further optimization of our extraction method, using slower flow rates and solid-phase extraction on silica columns, followed by C18 column extraction, yielded somewhat better recoveries (38 +/-3%) for 1-mL brain homogenates. Interassay precision (coefficient of variation) was determined on the basis of daily calibrations for 2 months and was found to be 7%, 9%, and 9% for NIC, COT, and PI, respectively, whereas intra-assay variability was 3.9% for both NIC and COT. Limited studies were performed on analytical columns for comparison of retention, resolution, asymmetry, and column capacity. We concluded that a simple two-step solid-phase extraction method, coupled with HPLC separation and UV detection, can be used routinely to measure NIC and COT in biological fluids and tissues.

  18. Preconcentration of indapamide from human urine using molecularly imprinted solid-phase extraction.

    Yılmaz, Hüma; Basan, Hasan


    A simple, sensitive, and selective molecularly imprinted solid-phase extraction and spectrophotometric method has been developed for the clean-up and preconcentration of indapamide from human urine. Molecularly imprinted polymers were prepared by a non-covalent imprinting approach using indapamide as a template molecule, 2-(trifluoromethyl) acrylic acid as a functional monomer, ethylene glycol dimethacrylate as a crosslinker, N,N-azobisisobutyronitrile as a thermal initiator and acetonitrile as a porogenic solvent. A non-imprinted polymer was also prepared in the same way, but in the absence of template. Molecularly imprinted polymer and non-imprinted polymer sorbents were dry-packed into solid-phase extraction cartridges. Eluates from cartridges were analyzed using a spectrophotometer for the determination of indapamide by referring to the calibration curve in the range 0.14-1.50 μg/mL. Preconcentration factor, limit of detection, and limit of quantification were 16.30, 0.025 μg/mL, and 0.075 μg/mL, respectively. A relatively high imprinting factor (9.3) was also achieved and recovery values for the indapamide spiked into human urine were in the range of 80.1-81.2%. In addition, relatively low within-day (0.17-0.42%) and between-day (1.1-1.4%) precision values were obtained as well. The proposed molecularly imprinted solid-phase extraction and spectrophotometric method was successfully applied to selective extraction, preconcentration, and determination of indapamide from human urine samples.

  19. Solid-Phase Iminium Cyclization Reactions for the Synthesis of Natural Product-Like Diketopiperazines

    Petersen, Rico

    The development of methodology for the solid-phase synthesis of fused 2,5-diketopiperazines with an emphasis on structural and stereochemical control, has been accomplished through two different approaches. The first approach was based on a highly trans-stereoselective (82% d.e.) intramolecular N......]tetrahydroisoquinoline product in a high purity (71%). Unfortunately an issue with incomplete conversion in the final step (20%) rendered separation of the diastereoisomers, by preparative RP-HPLC, problematic. Employing the developed methods, a diastereoisomeric matrix of aminomethylthiophene hydroxamic acid biased HDAC...

  20. Solid-phase synthesis and biological evaluation of a combinatorial library of philanthotoxin analogues

    Strømgaard, K; Brier, T J; Andersen, K


    The modular structure of philanthotoxins was exploited for construction of the first combinatorial library of these compounds using solid-phase parallel synthesis. (S)-Tyrosine and (S)-3-hydroxyphenylalanine were used as amino acid components, spermine, 1,12-dodecanediamine, and 4,9-dioxa-1...... former compounds may bind to nAChR in a similar fashion but differently from that of PhTX-12. The combinatorial library approach described in this work represents a prototype methodology for future exploration of structure-activity relationships of philanthotoxins....

  1. Preparation of A New Fiber by Sol-gel Technology in Solid-phase Microextraction (SPME)

    Li Ming WEI; Qing Yu OU; Ju Bai LI


    The sol-gel technology is applied for the preparation of solid-phase microextraction (SPME) fiber. The fiber demonstrates high thermal stability, efficient extraction rate and the selectivity for non-polar or low-polar analytes. Efficient SPME-GC-FID analyses of benzene- toluene-ethylbenzene-xylenes (BTEXs) and low-polar halocarbon were achieved by the sol-gel coated DSDA-DDBT-TiO2 fiber. Some parameters of the SPME fiber for the determination of halocarbon in aqueous sample were investigated.

  2. Challenges of infrared reflective spectroscopy of solid-phase explosives and chemicals on surfaces

    Phillips, Mark C.; Suter, Jonathan D.; Bernacki, Bruce E.; Johnson, Timothy J.


    Reliable active and passive hyperspectral imaging and detection of explosives and solid-phase chemical residue on surfaces remains a challenge and an active area of research and development. Both methods rely on reference libraries for material identification, but in many cases the reference spectra do not sufficiently resemble those instrumental signals scattered from real-world objects. We describe a physics-based model using the dispersive complex dielectric constant to explain what is often thought of as anomalous behavior of scattered or non-specular signatures encountered in active and passive sensing of explosives or chemicals on surfaces and show modeling and experimental results for RDX.

  3. Solid Phase Characterization of Tank 241-AY-102 Annulus Space Particulate

    Cooke, G. A.


    The Special Analytical Studies Group at the 222-S Laboratory (222-S) examined the particulate recovered from a series of samples from the annular space of tank 241-AY-102 (AY-102) using solid phase characterization (SPC) methods. These include scanning electron microscopy (SEM) using the ASPEX®1 scanning electron microscope, X-ray diffraction (XRD) using the Rigaku®2 MiniFlex X-ray diffractometer, and polarized light microscopy (PLM) using the Nikon®3 Eclipse Pol optical microscope. The SEM is equipped with an energy dispersive X-ray spectrometer (EDS) to provide chemical information.

  4. Selective Solid-phase Extraction of Aloe Emodin from Aloe by Molecularly Imprinted Polymers

    TIAN Ming-lei; LEE Yu-ri; PARK Dong-wha; ROW Kyung-ho


    The extraction and separation of aloe emodin were optimized via selective molecularly imprinted solid-phase extraction.Molecularly imprinted polymer was prepared from the functional monomer,methacrylic acid and a mixture of ethanol/dodecanol(90/10,volume ratio) as porogen.It overcomes the common problems of imprinting biological polar compounds and shows high selectivity compared favorably with those of non-imprinted polymer and commercially available C18 and silica cartridges in similar aloe emodin tests.Good linearity was obtained between 0.002 and 2.5 mg/mL(r2=0.998) with relative standard deviations below 3.3%.

  5. Reducing the sulfur-dioxide binding power of sweet white wines by solid-phase extraction.

    Saidane, Dorra; Barbe, Jean-Christophe; Birot, Marc; Deleuze, Hervé


    The high sulfur-dioxide binding power of sweet white wines may be reduced by extracting the naturally present carbonyl compounds from wine that are responsible for carbonyl bisulphites formation. The carbonyl compounds mainly responsible for trapping SO2 are acetaldehyde, pyruvic acid, and 2-oxoglutaric acid. The method employed was selective solid phase extraction, using phenylsulfonylhydrazine as a scavenging agent. The scavenging function was grafted onto a support prepared from raw materials derived from lignin. This approach is more acceptable to winemakers than the polymer media previously reported, as it reduces the possible contamination of wine to molecules already present in the wine making process.

  6. Solid-phase enolate chemistry investigated using HR-MAS NMR spectroscopy.

    Fruchart, Jean-Sébastien; Lippens, Guy; Kuhn, Cyrille; Gras-Masse, Hélène; Melnyk, Oleg


    Supported P4-t-Bu enolate chemistry of phenylacetyloxymethyl polystyrene (PS) resin was investigated using high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Direct analysis of the crude reaction suspensions through the use of a diffusion filter (DF) allowed a rapid selection of the optimal experimental conditions, but also the characterization of the enolate on the solid phase. Comparison with solution experiments and literature data allowed us to address partially the structure of the enolate. HR-MAS NMR spectra of the enolate revealed also a tight interaction of P4-t-Bu base with the polymer matrix.

  7. Structurally Diverse Polyamines: Solid-Phase Synthesis and Interaction with DNA.

    Umezawa, Naoki; Horai, Yuhei; Imamura, Yuki; Kawakubo, Makoto; Nakahira, Mariko; Kato, Nobuki; Muramatsu, Akira; Yoshikawa, Yuko; Yoshikawa, Kenichi; Higuchi, Tsunehiko


    A versatile solid-phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher-order structures of DNA.

  8. Solid-phase microextraction for flavor analysis in Harari Khat (Catha edulis) stimulant

    AL-FLAHIAbdulsalam; 邹建凯; 殷学锋


    This research examined the typical flavor compounds in the commonest type of Khat called Harari Khat grown in the region of Ethiopia.Twenty-eight compounds, which includes 1,2-Propanedione,1-Phenyl,Hexanol,Hexanal compounds,Limonene, Benzaldehyde with other flavors, were extracted by polydimethylsiloxane at room temperature for 30min from Khat samples,and identified by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS).This method needs no organic solvents and required minimal sample.

  9. Different methods to select the best extraction system for solid-phase extraction.

    Bielicka-Daszkiewicz, Katarzyna


    The optimization methods for planning a solid-phase extraction experiment are presented. These methods are based on a study of interactions between different parts of an extraction system. Determination of the type and strength of interaction depends on the physicochemical properties of the individual components of the system. The main parameters that determine the extraction properties are described in this work. The influence of sorbents' and solvents' polarity on extraction efficiency, Hansen solubility parameters and breakthrough volume determination on sorption and desorption extraction step are discussed.

  10. Screening of Brazilian fruit aromas using solid-phase microextraction-gas chromatography-mass spectrometry.

    Augusto, F; Valente, A L; dos Santos Tada, E; Rivellino, S R


    Manual headspace solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) was used for the qualitative analysis of the aromas of four native Brazilian fruits: cupuassu (Theobroma grandiflorum, Spreng.), cajá (Spondias lutea. L.), siriguela (Spondias purpurea, L.) and graviola (Anona reticulata, L). Industrialized pulps of these fruits were used as samples, and extractions with SPME fibers coated with polydimethylsiloxane, polyacrylate, Carbowax and Carboxen were carried out. The analytes identified included several alcohols, esters, carbonyl compounds and terpernoids. The highest amounts extracted, evaluated from the sum of peak areas, were achieved using the Carboxen fiber.

  11. Synthesis of silicon carbide nanowires by solid phase source chemical vapor deposition

    NI Jie; LI Zhengcao; ZHANG Zhengjun


    In this paper,we report a simple approach to synthesize silicon carbide(SiC)nanowires by solid phase source chemical vapor deposition(CVD) at relatively low temperatures.3C-SiC nanowires covered by an amorphous shell were obtained on a thin film which was first deposited on silicon substrates,and the nanowires are 20-80 am in diameter and several μm in length,with a growth direction of[200].The growth of the nanowires agrees well on vapor-liquid-solid (VLS)process and the film deposited on the substrates plays an important role in the formation of nanowires.

  12. An efficient protocol for the solid-phase synthesis of glycopeptides under microwave irradiation.

    Garcia-Martin, Fayna; Hinou, Hiroshi; Matsushita, Takahiko; Hayakawa, Shun; Nishimura, Shin-Ichiro


    A standardized and smooth protocol for solid-phase glycopeptides synthesis under microwave irradiation was developed. Double activation system was proved to allow for highly efficient coupling of Tn-Ser/Thr and bulky core 2-Ser/Thr derivatives. Versatility and robustness of the present strategy was demonstrated by constructing a Mucine-1 (MUC1) fragment and glycosylated fragments of tau protein. The success of this approach relies on the combination of microwave energy, a resin consisting totally of polyethylene glycol, a low excess of sugar amino acid and the "double activation" method.



    We have developed a simple method for the extraction of gamma-hydroxybutyrate (GHB) in human whole blood using headspace solid-phase microextraction (SPME). The procedure involves the conversion of GHB to gamma-butyrolactone (GBL) with acid catalysis; gamma-valerolactone (GVL) was used as internal standard (IS). After heating a vial containing a whole blood sample with GHB and IS at 80℃ for 5 min in the presence of H3PO4 solution, a Carboxen/polydimethylsiloxane -coated fiber was exposed to t...

  14. Chromatographic Separations Using Solid-Phase Extraction Cartridges: Separation of Wine Phenolics

    Brenneman, Charles A.; Ebeler, Susan E.


    We describe a simple laboratory experiment that demonstrates the principles of chromatographic separation using solid-phase extraction columns and red wine. By adjusting pH and mobile phase composition, the wine is separated into three fractions of differing polarity. The content of each fraction can be monitored by UV-vis spectroscopy. When the experiment is combined with experiments involving HPLC or GC separations, students gain a greater appreciation for and understanding of the highly automated instrumental systems currently available. In addition, they learn about the chemistry of polyphenolic compounds, which are present in many foods and beverages and which are receiving much attention for their potentially beneficial health effects.

  15. Exploring solid-phase approaches for the preparation of new beta-lactams from amino acids.

    Gerona-Navarro, Guillermo; Royo, Miriam; García-López, Ma Teresa; Albericio, Fernando; González-Muñiz, Rosario


    Two solid-phase approaches, involving the base-assisted intramolecular alkylation of N-chloroacetyl-Phe derivatives anchored to appropriate solid supports, were investigated for the preparation of novel beta-lactams. When a BAL-type strategy was used, the resin-bound azetidinones were easily formed, as established by MAS-NMR, but final compounds could not be removed from the resin, unless a suitable two linkers system was used. In the second approach, in which the Phe residue is anchored to a Wang-type resin through the carboxylate group, the corresponding 1,4,4-trisubstituted 2-azetidinone was obtained in moderate to good yield and high purity.

  16. Assessment of 4-nitrogenated benzyloxymethyl groups for 2'-hydroxyl protection in solid-phase RNA synthesis.

    Cieślak, Jacek; Kauffman, Jon S; Kolodziejski, Michelle J; Lloyd, John R; Beaucage, Serge L


    The search for a 2'-OH protecting group that would impart ribonucleoside phosphoramidites with coupling kinetics and coupling efficiencies comparable to those of deoxyribonucleoside phosphoramidites led to an assessment of 2'-O-(4-nitrogenated benzyloxy)methyl groups through solid-phase RNA synthesis using phosphoramidites 2a-d, 12a, and 14a. These phosphoramidites exhibited rapid and efficient coupling properties. Particularly noteworthy is the cleavage of the 2'-O-[4-(N-methylamino)benzyloxy]methyl groups in 0.1 M AcOH, which led to U19dT within 15 min at 90 degrees C. [reaction: see text

  17. Novel nanoporous sorbent for solid-phase extraction in petroleum fingerprinting

    Alayande, S. Oluwagbemiga; Hlengilizwe, Nyoni; Dare, E. Olugbenga; Msagati, Titus A. M.; Akinlabi, A. Kehinde; Aiyedun, P. O.


    Sample preparation is crucial in the analysis of petroleum and its derivatives. In this study, developing affordable sorbent for petroleum fingerprinting analysis using polymer waste such expanded polystyrene was explored. The potential of electrospun expanded polystyrene (EPS) as a sorbent for the solid-phase extraction (SPE) technique was investigated, and its efficiency was compared with commercial cartridges such as alumina, silica and alumina/silica hybrid commercial for petroleum fingerprinting analysis. The chromatograms showed that the packed electrospun EPS fibre demonstrated excellent properties for SPE applications relative to the hybrid cartridges.

  18. A Convergent Solid-Phase Synthesis of Actinomycin Analogues - Towards Implementation of Double-Combinatorial Chemistry

    Tong, Glenn; Nielsen, John


    The actinomycin antibiotics bind to nucleic acids via both intercalation and hydrogen bonding. We found this 'double-action attack' mechanism very attractive in our search for a novel class of nucleic acid binders. A highly convergent, solid-phase synthetic strategy has been developed for a class...... with the requirements for combinatorial synthesis and furthermore, the final segment condensation allows, for the first time, double-combinatorial chemistry to be performed where two combinatorial libraries can be reacted with each other. Copyright (C) 1996 Elsevier Science Ltd....

  19. The model of solid phase crystallization of amorphous silicon under elastic stress


    Solid phase crystallization of an amorphous silicon (a-Si) film stressed by a Si3N4 cap was studied by laser Raman spectroscopy. The a-Si films were deposited on Si3N4 (50 nm)/Si(100) substrate by rf sputtering. The stress in an a-Si film was controlled by thickness of a Si3N4 cap layer. The Si3N4 films were also deposited by rf sputtering. It was observed that the crystallization was affected by the stress in a-Si films introduced by the Si3N4 cap layer. The study suggests that the elastic s...

  20. [Anaerobic solid-phase fermentation of plant substrates by Bacillus subtilis].

    Ushakova, N A; Brodskiĭ, E S; Kozlova, A A; Nifatov, A V


    Solid-phase growth of Bacillus subtilis 8130 on cellulose-rich plant substrates (presscakes or pulp) under hypoxic conditions was accompanied by cellulose depolymerization, protein hydrolysis, and degradation of other plant components, including some processes of mixed-type carbohydrate fermentation. The bacterial fermentation yielded propionic, butyric, and hexanoic acids and butyric acid derivatives. The bacterial metabolism and fermentation degree can be characterized by the proportions of fatty acids in the reaction mixture. The product of sea buckthorn cake fermentation has a good sorption quality.

  1. Solid-phase synthesis and biological evaluation of Joro spider toxin-4 from Nephila clavata

    Barslund, Anne Fuglsang; Poulsen, Mette Homann; Bach, Tinna Brøbech


    Polyamine toxins from orb weaver spiders are attractive pharmacological tools particularly for studies of ionotropic glutamate (iGlu) receptors in the brain. These polyamine toxins are biosynthesized in a combinatorial manner, providing a plethora of related, but structurally complex toxins...... to be exploited in biological studies. Here, we have used solid-phase synthetic methodology for the efficient synthesis of Joro spider toxin-4 (JSTX-4) (1) from Nephila clavata, providing sufficient amounts of the toxin for biological evaluation at iGlu receptor subtypes using electrophysiology. Biological...

  2. Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.

    Hanson, Cynthia; Israelsen, Nathan D; Sieverts, Michael; Vargis, Elizabeth


    Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available. Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags(1). Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

  3. Studies on solid phase synthesis,characterization and fluorescent property of the new rare earth complexes

    Jianwei SHI


    Full Text Available Rare earth-β-diketone ligand complex luminescent material has stable chemical properties and excellent luminous property. Using europium oxide and (γ-NTA as raw materials, novel rare earth-β-dione complexes are synthesized by solid state coordination chemistry. The synthesis temperature and milling time are discussed for optimization. Experimental results show that the suitable reaction situation is at 50 ℃ and 20 h for solid-phase synthesis. The compositions and structures of the complexes are characterized by means of elemental analysis, UV-Vis and FTIR methods, and the phase stability of the complex is determined by using TG-DTA technique. It is proved that preparation of waterless binary rare earth complexes by the solid phase reaction method results in a higher product yield. The fluorescence spectra show that between Eu (Ⅲ and γ-NTA, there exists efficient energy transfer, and the rare earth complexes synthesis is an excellent red bright light-emitting material with excellent UV excited luminescence properties.

  4. Dense Pellicular Agarose-Glass Beads for Expanded Bed Application: Flow Hydrodynamics and Solid Phase Classifications

    周鑫; 史清洪; 白姝; 孙彦


    Two dense pellicular agarose-glass matrices of different sizes and densities, i.e., AG-S and AG-L, have been characterized for their bed expansion behavior, flow hydrodynamics and particle classifications in an expanded bed system. A 26 mm ID column with side ports was used for sampling the liquid-solid suspension during expanded bed operations. Measurements of the collected solid phase at different column positions yielded the particle size and density distribution data. It was found that the composite matrices showed particle size as well as density classifications along the column axis, i.e., both the size and density of each matrix decreased with increasing the axial bed height. Their axial classifications were expressed by a correlation related to both the particle size and density as a function of the dimensionless axial bed height. The correlation was found to fairly describe the solid phase classifications in the expanded bed system. Moreover, it can also be applied to other two commercial solid matrices designed for expanded bed applications.

  5. Solid phase synthesis and antiprotozoal evaluation of di- and trisubstituted 5'-carboxamidoadenosine analogues.

    Rodenko, Boris; Detz, Remko J; Pinas, Victorine A; Lambertucci, Catia; Brun, Reto; Wanner, Martin J; Koomen, Gerrit-Jan


    The rapid increase of resistance to drugs commonly used in the treatment of tropical diseases such as malaria and African sleeping sickness calls for the prompt development of new safe and efficacious drugs. The pathogenic protozoan parasites lack the capability of synthesising purines de novo and they take up preformed purines from their host through various transmembrane transporters. Adenosine derivatives constitute a class of potential therapeutics due to their selective internalisation by these transporters. Automated solid-phase synthesis can speed up the process of lead finding and we pursued the solid-phase synthesis of di- and trisubstituted 5'-carboxamidoadenosine derivatives by using a safety-catch approach. While efforts with Kenner's sulfonamide linker remained fruitless, successful application of the hydrazide safety-catch linker allowed the construction of two representative combinatorial libraries. Their antiprotozoal evaluation identified two compounds with promising activity: N(6)-benzyl-5'-N-phenylcarboxamidoadenosine with an IC(50) value of 0.91 microM against Trypanosoma brucei rhodesiense and N(6)-diphenylethyl-5'-phenylcarboxamidoadenosine with an IC(50) value of 1.8 microM against chloroquine resistant Plasmodium falciparum.

  6. A comparison of methods to predict solid phase heats of formation of molecular energetic salts.

    Byrd, Edward F C; Rice, Betsy M


    In this study a variety of methods were used to compute the energies for lattice enthalpies and gas phase heats of formation of the ionic constituents used in Born-Fajans-Haber cycles to produce solid phase heats of formation of molecular ionic energetic crystals. Several quantum mechanically based or empirical approaches to calculate either the heat of formation of the ionic constituents in the gas phase (deltaH(o)f(g)) or the lattice enthalpy (deltaH(o)Lattice) were evaluated. Solid phase heats of formation calculated from combinations of deltaH(o)f(g) and deltaH(o)Lattice determined through various approaches are compared with experimental values for a series of molecular energetic salts with 1:1, 2:1 and 2:2 charge ratios. Recommendations for combinations of deltaH(o)f(g) and deltaH(o)Lattice to produce best agreement with experiment are given, along with suggestions for improvements of the methods.

  7. Matrix compatible solid phase microextraction coating, a greener approach to sample preparation in vegetable matrices.

    Naccarato, Attilio; Pawliszyn, Janusz


    This work proposes the novel PDMS/DVB/PDMS fiber as a greener strategy for analysis by direct immersion solid phase microextraction (SPME) in vegetables. SPME is an established sample preparation approach that has not yet been adequately explored for food analysis in direct immersion mode due to the limitations of the available commercial coatings. The robustness and endurance of this new coating were investigated by direct immersion extractions in raw blended vegetables without any further sample preparation steps. The PDMS/DVB/PDMS coating exhibited superior features related to the capability of the external PDMS layer to protect the commercial coating, and showed improvements in terms of extraction capability and in the cleanability of the coating surface. In addition to having contributed to the recognition of the superior features of this new fiber concept before commercialization, the outcomes of this work serve to confirm advancements in the matrix compatibility of the PDMS-modified fiber, and open new prospects for the development of greener high-throughput analytical methods in food analysis using solid phase microextraction in the near future.

  8. Solid Phase Extraction Disk Procedure to Determine 239Pu in Soils

    ZHANG Ji-qiao;ZHAO Ya-ping;DING You-qian;ZHANG Sheng-dong;YANG Jin-ling


    Full Text Available 239Pu in many soil samples should be analyzed to survey radioactive pollution level in nuclear facilities and its affinity environment efficiently. In order to input the opt conditions for column experiment, the experiments of the static adsorption coefficient of 239Pu to solid phase extraction disk with different contact time, concentration of HNO3 and different temperature were carried out. The chemical procedure for the rapid separation and determination of 239Pu in soils had been formulated, which using solid phase extraction disk (EmporeTM Anion Exchange-SR as extraction material and liquid scintillation spectrometry counting as measurement. In the procedure, soil sample usage was 10 g, and were leached by 8 mol/L HNO3, the chemical recovery of the procedure was about 78.9%, and the minimum detectable concentration was 3.7 Bq/kg. It took less than 3 hours once and the presence of 137Cs, 90Sr-90Y and natural uranium, 241Am, 99Tc did not interfere with the procedure, owning high DF of them. The procedure can be used extensively in determination of 239Pu in soils.

  9. Determination of amphetamines in hair by integrating sample disruption, clean-up and solid phase derivatization.

    Argente-García, A; Moliner-Martínez, Y; Campíns-Falcó, P; Verdú-Andrés, J; Herráez-Hernández, R


    The utility of matrix solid phase dispersion (MSPD) for the direct analysis of amphetamines in hair samples has been evaluated, using liquid chromatography (LC) with fluorescence detection and precolumn derivatization. The proposed approach is based on the employment of MSPD for matrix disruption and clean-up, followed by the derivatization of the analytes onto the dispersant-sample blend. The fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) has been used for derivatization. Different conditions for MSPD, analyte purification and solid phase derivatization have been tested, using amphetamine (AMP), methamphetamine (MET), ephedrine (EPE) and 3,4-methylenedioxymethamphetamine (MDMA) as model compounds. The results have been compared with those achieved by using ultrasound-assisted alkaline digestion and by MSPD combined with conventional solution derivatization. On the basis of the results obtained, a methodology is proposed for the analysis of amphetamines in hair which integrates sample disruption, clean-up and derivatization using a C18 phase. Improved sensitivity is achieved with respect to that obtained by the alkaline digestion or by the MSPD followed by solution derivatization methods. The method can be used for the quantification of the tested amphetamines within the 2.0-20.0ng/mg concentration interval, with limits of detection (LODs) of 0.25-0.75ng/mg. The methodology is very simple and rapid (the preparation of the sample takes less than 15min).

  10. Determination of amphetamines in human urine by headspace solid-phase microextraction and gas chromatography.

    Raikos, Nikolaos; Christopoulou, Klio; Theodoridis, Georgios; Tsoukali, Heleni; Psaroulis, Dimitrios


    Solid-phase microextraction (SPME) is under investigation for its usefulness in the determination of a widening variety of volatile and semivolatile analytes in biological fluids and materials. Semivolatiles are increasingly under study as analytical targets, and difficulties with small partition coefficients and long equilibration times have been identified. Amphetamines were selected as semivolatiles exhibiting these limitations and methods to optimize their determination were investigated. A 100- micro m polydimethylsiloxane (PDMS)-coated SPME fiber was used for the extraction of the amphetamines from human urine. Amphetamine determination was made using gas chromatography (GC) with flame-ionization detection (FID). Temperature, time and salt saturation were optimized to obtain consistent extraction. A simple procedure for the analysis of amphetamine (AMP) and methamphetamine (MA) in urine was developed and another for 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methamphetamine (MDMA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) using headspace solid-phase microextraction (HS-SPME) and GC-FID. Higher recoveries were obtained for amphetamine (19.5-47%) and methamphetamine (20-38.1%) than MDA (5.1-6.6%), MDMA (7-9.6%) and MDEA (5.4-9.6%).

  11. Rapid solid-phase extraction and analysis of resveratrol and other polyphenols in red wine.

    Hashim, Shima N N S; Schwarz, Lachlan J; Boysen, Reinhard I; Yang, Yuanzhong; Danylec, Basil; Hearn, Milton T W


    Red wine has long been credited as a good source of health-beneficial antioxidants, including the bioactive polyphenols catechin, quercetin, and (E)-resveratrol. In this paper, we report the application of reusable molecularly imprinted polymers (MIPs) for the selective and robust solid-phase extraction (SPE) and rapid analysis of (E)-resveratrol (LOD=8.87×10(-3) mg/L, LOQ=2.94×10(-2) mg/L), along with a range of other polyphenols from an Australian Pinot noir red wine. Optimization of the molecularly imprinted solid-phase extraction (MISPE) protocol resulted in the significant enrichment of (E)-resveratrol and several structurally related polyphenols. These secondary metabolites were subsequently identified by RP-HPLC and μLC-ESI ion trap MS/MS methods. The developed MISPE protocol employed low volumes of environmentally benign solvents selected according to the Green Chemistry principles, and resulted in the recovery of 99% of the total (E)-resveratrol present. These results further demonstrate the potential of generic protocols for the analysis of target compound with health beneficial properties within the food and nutraceutical industries using tailor-made MIPs.

  12. Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands.

    Dirin, Mehrdad; Urban, Ernst; Noe, Christian R; Winkler, Johannes


    Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2'-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Selective fiber used for headspace solid-phase microextraction of abused drugs in human urine

    Sunanta Wangkarn


    Full Text Available A sensitive and selective fiber for simultaneous analysis of three drugs of abuse (amphetamine, methamphetamine and ephedrine in urine samples was explored using headspace solid phase microextraction and gas chromatography with flame ionization detection. Several parameters affecting extraction such as extraction time, extraction temperature, pH of solution and salt concentrations were investigated. Among five commercially available fibers, divinylbenzene/carboxen/ polydimethylsiloxane is the most sensitive and selective fiber at pH 10.0, extraction temperature at 80 C for 20 min and desorption temperature at 220 C for 2 min. Under the optimal conditions, the proposed solid phase microextraction method provided good linearity in the ranges 0.1-10 µg/ml for amphetamine and methamphetamine and 0.5-20 µg/ml for ephedrine. The detection limits for amphetamine, methamphetamine and ephedrine were 9, 3 and 30 ng/ml, respectively. The recoveries of three drugs in urine samples were exceeding 85%.

  14. Improvement of multilayer graphene crystallinity by solid-phase precipitation with current stress application during annealing

    Sahab Uddin, Md.; Ichikawa, Hiroyasu; Sano, Shota; Ueno, Kazuyoshi


    To improve the crystallinity of multilayer graphene (MLG) films by solid-phase precipitation, a new method by which current stress is introduced during annealing of a carbon-doped cobalt (Co-C) layer using cobalt (Co) as the catalyst has been investigated. The effects of current stress on the formation and crystallinity of MLG films were investigated by comparing the characteristics of the films annealed at the same temperature with and without current by taking into account the temperature rise due to Joule heating. The characteristics obtained by Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD) measurements revealed that the MLG films produced were crystalline in nature and their crystallinity increased with applied current stress at the same temperature. From SEM observations, beside Joule heating, enhancement of Co grain size by agglomeration induced by current stress may be the potential reason for the improvement of the crystallinity of MLG films. We have also improved the uniformity of MLG films by depositing an additional copper (Cu) capping layer over the Co-C layer. Current stress application can lead to low-temperature fabrication of MLG with higher crystallinity by solid-phase precipitation.

  15. Solid-phase microextraction for the analysis of short-chain chlorinated paraffins in water samples.

    Castells, P; Santos, F J; Galceran, M T


    A novel solid-phase microextraction (SPME) method coupled to gas chromatography with electron capture detection (GC-ECD) was developed as an alternative to liquid-liquid and solid-phase extraction for the analysis of short-chain chlorinated paraffins (SCCPs) in water samples. The extraction efficiency of five different commercially available fibres was evaluated and the 100-microm polydimethylsiloxane coating was the most suitable for the absorption of the SCCPs. Optimisation of several SPME parameters, such as extraction time and temperature, ionic strength and desorption time, was performed. Quality parameters were established using Milli-Q, tap water and river water. Linearity ranged between 0.06 and 6 microg l(-1) for spiked Milli-Q water and between 0.6 and 6 microg l(-1) for natural waters. The precision of the SPME-GC-ECD method for the three aqueous matrices was similar and gave relative standard deviations (RSD) between 12 and 14%. The limit of detection (LOD) was 0.02 microg l(-1) for Milli-Q water and 0.3 microg l(-1) for both tap water and river water. The optimised SPME-GC-ECD method was successfully applied to the determination of SCCPs in river water samples.

  16. Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support

    Saiyed, Z. M.; Ramchand, C. N.; Telang, S. D.


    In recent years, techniques employing magnetizable solid-phase supports (MSPS) have found application in numerous biological fields. This magnetic separation procedure offers several advantages in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap, and often highly scalable. The current paper details a genomic DNA isolation method optimized in our laboratory using magnetic nanoparticles as a solid-phase support. The quality and yields of the isolated DNA from all the samples using magnetic nanoparticles were higher or equivalent to the traditional DNA extraction procedures. Additionally, the magnetic method takes less than 15 min to extract polymerase chain reaction (PCR) ready genomic DNA as against several hours taken by traditional phenol-chloroform extraction protocols. Moreover, the isolated DNA was found to be compatible in PCR amplification and restriction endonuclease digestion. The developed procedure is quick, inexpensive, robust, and it does not require the use of organic solvents or sophisticated instruments, which makes it more amenable to automation and miniaturization.

  17. Solid phase extraction and spectrophotometric determination of palladium with 2-(2-quinolylazo-5-diethylaminobenzoic acid



    Full Text Available Asensitive, selective and rapid method for the determination of palladium based on the rapid reaction of palladium(II with 2-(2-quinolylazo-5-diethylaminobenzoic acid (QADEAB and the solid phase extraction of the Pd(II –QADEAB chelate with a reversed phase polymer-based C18 cartridge was developed. In the presence of 0.05 – 0. 5 mol/L of hydrochloric acid solution and cetyl trimethylammonium bromide (CTAB medium, QADEAB reacts with palladium(II to form a violet complex with a mole ratio 1:2 (palladium to QADEAB. The chelate was enriched by solid phase extraction with a reversed phase polymer-based C18 cartridge. An enrichment factor of 200 was obtained by elution of the chelate form the cartridge with the minimal amount of isopentyl alcohol. The molar absorptivity of the chelate in the isopentyl alcohol medium was 1.43 × 105 L mol-1 cm-1 at 628 nm. Beer’s law was obeyed in the range of 0.01 – 1.2 mg/mL. The relative standard deviation for eleven replicate samples at the 0.2 mg/L level was 2.18 %. The attained detection limit amounted to 0.02 mg/L in the original samples. This method was applied to the determination of palladium in environmental samples with good results.

  18. DNA display III. Solid-phase organic synthesis on unprotected DNA.

    David R Halpin


    Full Text Available DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl-based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide-DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation.

  19. Carbon nanocones/disks as new coating for solid-phase microextraction.

    Jiménez-Soto, Juan Manuel; Cárdenas, Soledad; Valcárcel, Miguel


    In this article, the potential of carbon nanocones/disks as coating for solid-phase microextraction has been evaluated for the first time. The nanostructures were immobilized on a stainless steel needle by means of an organic binder. The fiber coating obtained was ca. 50 microm of thickness and 35 mm in length. The evaluation of the sorbent capacity was carried out through the determination of toluene, ethylbenzene, xylene isomers and styrene in water samples following the headspace sampling modality (15 min, 30 degrees C). The fiber was then transferred to a 10 mL vial which was sealed and heated at 110 degrees C for 15 min in the headspace module of the instrument to achieve the thermal desorption of the analytes. Then 2.5 mL of the headspace generated were injected in the gas chromatograph-mass spectrometer for analytes separation and quantitation. The detection and quantitation limits obtained for 10 mL of sample were 0.15 and 0.5 ng mL(-1) (0.6 and 2 ng mL(-1) for toluene). The optimized procedure was applied to the determination of the selected volatile compounds in waters collected from different locations. The recovery values obtained (average recovery ca. 92%) demonstrated the usefulness of the carbon nanocones/disks as sorbent material in solid-phase microextraction.

  20. Solid-phase microextraction-gas chromatographic determination of volatile monoaromatic hydrocarbons in soil.

    Zygmunt, B; Namiesnik, J


    Benzene, toluene, ethylbenzene, three isomers of xylene, and cumene have been isolated and enriched from soil samples by a combination of water extraction at room and elevated temperature and headspace-solid-phase microextraction before their gas chromatographic-mass spectrometric (GC-MS) determination. The conditions used for all stages of sample preparation and chromatographic analysis were optimized. Analytes sampled on a polydimethylsiloxane-coated solid-phase microextraction fiber were thermally desorbed in the split/splitless injector of a gas chromatograph (GC) coupled with a mass spectrometer (MS). The desorption temperature was optimized. The GC separation was performed in a capillary column. Detection limits were found to be of the order of ca. 1 ng g(-1). Relative recoveries of the analytes from soils were found to be highly dependent on soil organic-matter content and on compound identity; they ranged from ca 92 to 96% for sandy soil (extraction at room temperature) and from ca 27 to 55% for peaty soil (extraction at elevated temperature). A few real-world soil samples were analyzed; the individual monoaromatic hydrocarbon content ranged from below detection limits to 6.4 ng g(-1) for benzene and 8.1 for the total of p- + m-xylene.

  1. Improved detection limits for phthalates by selective solid-phase micro-extraction

    Zia, Asif I.


    Presented research reports on an improved method and enhanced limits of detection for phthalates; a hazardous additive used in the production of plastics by solid-phase micro-extraction (SPME) polymer in comparison to molecularly imprinted solid-phase extraction (MISPE) polymer. The polymers were functionalized on an interdigital capacitive sensor for selective binding of phthalate molecules from a complex mixture of chemicals. Both polymers owned predetermined selectivity by formation of valuable molecular recognition sites for Bis (2-ethylhexyl) phthalate (DEHP). Polymers were immobilized on planar electrochemical sensor fabricated on a single crystal silicon substrate with 500 nm sputtered gold electrodes fabricated using MEMS fabrication techniques. Impedance spectra were obtained using electrochemical impedance spectroscopy (EIS) to determine sample conductance for evaluation of phthalate concentration in the spiked sample solutions with various phthalate concentrations. Experimental results revealed that the ability of SPME polymer to adsorb target molecules on the sensing surface is better than that of MISPE polymer for phthalates in the sensing system. Testing the extracted samples using high performance liquid chromatography with photodiode array detectors validated the results.

  2. The effect of surfactant and solid phase concentration on drug aggregates in model aerosol propellent suspensions.

    Bower, C; Washington, C; Purewal, T S


    The effect of increasing solid phase concentration on the morphology and flocculation rate of model aerosol suspensions has been investigated. Suspensions of micronized salbutamol sulphate and lactose in trichlorotrifluoroethane (P113) were studied under conditions of increasing shear stress. By use of image analysis techniques, measurement of aggregate size, fractal dimension and rate of aggregation was performed. The effect of the surfactant sorbitan monooleate on morphology and flocculation rate was also studied. Increased solid phase concentration caused an increase in the rate of aggregation and average aggregate size at a given value of shear stress. Surfactant addition retarded the aggregation rate, and caused a shift from a diffusion-limited cluster aggregation to a reaction-limited cluster aggregation mechanism. The aggregate profiles showed a corresponding change from rugged and crenellated without surfactant, to increasingly smooth and Euclidian with increasing surfactant concentration. The morphological changes were characterized by a decrease in the average boundary fractal dimension which also correlated well with the corresponding reduction in aggregation rate.

  3. Kinetic study of solid phase crystallisation of expanding thermal plasma deposited a-Si:H

    Law, F., E-mail: [Solar Energy Research Institute of Singapore (SERIS), National University of Singapore (Singapore); Department of Materials Science and Engineering, Faculty of Engineering, National University of Singapore (Singapore); Hoex, B. [Solar Energy Research Institute of Singapore (SERIS), National University of Singapore (Singapore); Wang, J. [Department of Materials Science and Engineering, Faculty of Engineering, National University of Singapore (Singapore); Luther, J. [Solar Energy Research Institute of Singapore (SERIS), National University of Singapore (Singapore); Department of Materials Science and Engineering, Faculty of Engineering, National University of Singapore (Singapore); Sharma, K.; Creatore, M.; Van de Sanden, M.C.M. [Department of Applied Physics, Eindhoven University of Technology, Eindhoven (Netherlands)


    In-situ X-ray diffraction was used to study the dynamics of the solid phase crystallisation (SPC) of hydrogenated amorphous silicon (a-Si:H) films deposited by expanding thermal plasma technique. The Johnson-Mehl-Avrami-Kolmogorov model was used for the analysis of the dynamic data and the activation energy associated with the SPC process was 2.9 eV, which was lower than a-Si:H films deposited by other techniques. Relationships between the Avrami exponent n, the SPC process stability and the subsequent grain structure were demonstrated. Under certain conditions, the films exhibited columnar grain structure with indications of good grain quality, suggesting that these films are suitable to be further developed into solar cell devices. Structure of the grains and the SPC dynamics in this work lend support to prior work that vacancies decorated by hydrogen clusters are related to nucleation sites. - Highlights: Black-Right-Pointing-Pointer The crystallisation of expanding thermal plasma (ETP) deposited a-Si:H was studied. Black-Right-Pointing-Pointer Johnson-Mehl-Avrami-Kolmogorov model was used to model the crystallisation process. Black-Right-Pointing-Pointer Activation energy of the solid phase crystallisation process was 2.9 eV. Black-Right-Pointing-Pointer Vacancies decorated by hydrogen clusters are suggested nucleation sites. Black-Right-Pointing-Pointer ETP is promising in the fabrication process of pc-Si thin film solar cells.

  4. Novel polymeric resin for solid phase extraction and determination of lead in waters

    Karaaslan, Nagihan M.; Cengiz, Emine; Yaman, Mehmet [Science Faculty, Department of Chemistry, Firat University, Elazig (Turkey); Senkal, B. Filiz [Science and Arts Faculty, Department of Chemistry, Istanbul Technical University, Istanbul (Turkey)


    Interest in preconcentration techniques for the determination of metals at ultratrace levels still continues increasingly because of some disadvantages of flameless atomic absorption spectrometry and the high costs of other sensitive methods in compared to flame atomic absorption spectrometry (FAAS). Among preconcentration techniques, solid-phase extraction is the most popular because of a number of advantages. In this work, thiol-containing sulfonamide resin was synthesized, characterized, and applied as a new sorption material for solid phase extraction and determination of lead in natural water samples. The optimization of experimental conditions was performed using the parameters including pH, contact time, and volumes of initial and elution solutions. After preconcentration procedure, FAAS was used for determinations. The synthesized resin exhibits the superiority in compared to the other adsorption reagents because of the fact that there is no necessity of any complexing reagent as well as high sorption capacity. Consequently, 280-fold improvement in the sensitivity of analytical scheme was achieved by combining the slotted tube atom trap-atomic absorption spectrometry (STAT-FAAS) and the developed preconcentration method. The limit of detection was found to be 0.15 ng mL{sup -1}. The Pb{sup 2+} concentrations in the studied water samples were found to be in the range of 0.9-6.7 ng mL{sup -1}. (Copyright copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  5. Molecularly imprinted polymer microspheres for solid-phase extraction of protocatechuic acid in Rhizoma homalomenae.

    Chen, Fang-Fang; Wang, Guo-Ying; Shi, Yan-Ping


    Molecularly imprinted polymers (MIPs) had been prepared by precipitation polymerization method using acrylamide as the functional monomer, ethylene glycol dimethacrylate as the cross-linker, acetonitrile as the porogen solvent and protocatechuic acid (PA), one of phenolic acids, as the template molecule. The MIPs were characterized by scanning electron microscopy and Fourier transform infrared, and their performance relative to non-imprinted polymers was assessed by equilibrium binding experiments. Six structurally similar phenolic acids, including p-hydroxybenzoic acid, gallic acid, salicylic acid, syringic acid, vanillic acid, ferulic acid were selected to assess the selectivity and recognition capability of the MIPs. The MIPs were applied to extract PA from the traditional Chinese medicines as a solid-phase extraction sorbent. The resultant cartridge showed that the MIPs have a good extraction performance and were able to selectively extract almost 82% of PA from the extract of Rhizoma homalomenae. Thus, the proposed molecularly imprinted-solid phase extraction-high performance liquid chromatography method can be successfully used to extract and analyse PA in traditional Chinese medicines.

  6. Titanium dioxide solid phase for inorganic species adsorption and determination: the case of arsenic.

    Vera, R; Fontàs, C; Anticó, E


    We have evaluated a new titanium dioxide (Adsorbsia As600) for the adsorption of both inorganic As (V) and As (III) species. In order to characterize the sorbent, batch experiments were undertaken to determine the capacities of As (III) and As (V) at pH 7.3, which were found to be 0.21 and 0.14 mmol g(-1), respectively. Elution of adsorbed species was only possible using basic solutions, and arsenic desorbed under batch conditions was 50 % when 60 mg of loaded titanium dioxide was treated with 0.5 M NaOH solution. Moreover, its use as a sorbent for solid-phase extraction and preconcentration of arsenic species from well waters has been investigated, without any previous pretreatment of the sample. Solid-phase extraction was implemented by packing several minicolumns with Adsorbsia As600. The method has been validated showing good accuracy and precision. Acceptable recoveries were obtained when spiked waters at 100-200 μg L(-1) were measured. The presence of major anions commonly found in waters did not affect arsenic adsoption, and only silicate at 100 mg L(-1) level severely competed with arsenic species to bind to the material. Finally, the measured concentrations in water samples containing arsenic from the Pyrinees (Catalonia, Spain) showed good agreement with the ICP-MS results.

  7. Fixed bed reactor for solid-phase surface derivatization of superparamagnetic nanoparticles.

    Steitz, Benedikt; Salaklang, Jatuporn; Finka, Andrija; O'Neil, Conlin; Hofmann, Heinrich; Petri-Fink, Alke


    The functionalization of nanoparticles is conditio sine qua non in studies of specific interaction with a biological target. Often, their biological functionality is achieved by covalent binding of bioactive molecules on a preexisting single surface coating. The yield and quality of the resulting coated and functionalized superparamagnetic iron oxide nanoparticles (SPIONs) can be significantly improved and reaction times reduced by using solid-phase synthesis strategies. In this study, a fixed bed reactor with a quadrupole repulsive arrangement of permanent magnets was assayed for SPION surface derivatization. The magnet array around the fixed bed reactor creates very high magnetic field gradients that enables the immobilization of SPIONs with a diameter as low as 9 nm. The functionalization on the surface of immobilized 25 nm 3-(aminopropyl)trimethoxysilane-coated SPIONs (APS-SPIONs) was performed using fluorescein-isothiocyanate directly, and by the SV40 large T-antigen nuclear localization signal peptide (PKKKRKVGC) conjugated to acryloylpoly(ethylene glycol)-N-hydroxysuccinimide, where the PEG reagent is conjugated first to create a functionalized nanoparticle and the peptide is added to the acryloyl group. We show that the yield of reactant grafted on the surface of the APS-coated SPIONs was higher in solid-phase within the fixed bed reactor compared to conventional liquid-phase chemistry. In summary, the functionalization of SPIONs using a magnetically fixed bed reactor was superior to the liquid-phase reaction in terms of the yield, reaction times required for derivatization, size distribution, and scalability.

  8. Determination of aflatoxins in rice samples by ultrasound-assisted matrix solid-phase dispersion.

    Manoochehri, Mahboobeh; Asgharinezhad, Ali Akbar; Safaei, Mahdi


    This work describes the application of ultrasound-assisted matrix solid-phase dispersion as an extraction and sample preparation approach for aflatoxins (B1, B2, G1 and G2) and subsequent determination of them by high-performance liquid chromatography-fluorescence detection. A Box-Behnken design in combination with response surface methodology was used to determine the affecting parameters on the extraction procedure. The influence of different variables including type of dispersing phase, sample-to-dispersing phase ratio, type and quantity of clean-up phase, ultrasonication time, ultrasonication temperature, nature and volume of the elution solvent was investigated in the optimization study. C18, primary-secondary amine (PSA) and acetonitrile were selected as dispersing phase, clean-up phase and elution solvent, respectively. The obtained optimized values were sample-to-dispersing phase ratio of 1 : 1, 60 mg of PSA, 11 min ultrasonication time, 30°C ultrasonication temperature and 4 mL acetonitrile. Under the optimal conditions, the limits of detection were ranged from 0.09 to 0.14 ng g(-1) and the precisions [relative standard deviation (RSD%)] were <8.6%. The recoveries of the matrix solid-phase dispersion process ranged from 78 to 83% with RSD <10% in all cases. Finally, this method was successfully applied to the extraction of trace amounts of aflatoxins in rice samples. © The Author [2014]. Published by Oxford University Press. All rights reserved. For Permissions, please email:


    Márcia Marques Jericó


    Full Text Available Non-radiometric immunoassays offer many advantages over radiometric assays, such as higher stability of kit compounds and absence of potential hazardous effects for users and environment. The comparison of cortisol measurements by fluoroimmunoassay (FIA and fluorometric enzyme immunoassay (FEIA with radioimmunoassay (RIA in adrenal function evaluation of normal (n=50 and hypercortisolemic dogs (n=12 was proposed. Serum concentrations of cortisol were measured in basal conditions and 8 hours after dexamethasone (DEX suppression (0.01mg/kg/IV. All our reference values were based on the 5th and 95th percentile. The values for basal cortisol of healthy dogs were 0.20 to 2.35mug/d for FIA, 0.30 to 5.39mug/d for FEIA, and 0.65 to 4.64mug/d for RIA. After DEX suppression the values were , and for FIA, FEIA and RIA, respectively. In hypercortisolemic dogs, the values of cortisol (mean ± SD in basal and post-DEX conditions were 2.71 + 0.41mug/d and 1.73 + 1.15mug/d for FIA, 7.05 + 2.85mug/d and 4.93 + 2.26mug/d for FEIA, and 4.80 + 1.43mug/d and 3.52 + 1.08mug/d for RIA. Statistically significant differences (pOs métodos de dosagem hormonal por técnicas não-radioativas apresentam inúmeras vantagens sobre os que utilizam radioisótopos como marcadores de hormônios ou anticorpos. Dentre tais vantagens, incluem-se maior meia-vida útil dos reagentes por maior estabilidade dos compenentes, inexistência de riscos de contaminação radioativa, tanto pessoal quanto ambiental. Para avaliar a aplicação destes novos métodos na prática da endocrinologia clínica de pequenos animais, comparamos o método de radioimunoensaio (RIE aos métodos alternativos fluoroimunoensaio (FIE e enzimaimunoensaio (EIE na avaliação da função adrenal canina. Para tanto, padronizaram-se os níveis de cortisol em cães normais (n=50 e em cães com hiperadrenocorticismo (n=12, sob condições basais e oito horas após supressão com dexametasona (DEX (0,01mg/kg IV. Os

  10. Poly(dimethylsiloxane) microchip-based immunoassay with multiple reaction zones: Toward on-chip multiplex detection platform

    Shao, Guocheng; Wang, Jun; Li, Zhaohui; Saraf, Laxmikant V.; Wang, Wanjun; Lin, Yuehe


    In this work, a poly(dimethylsiloxane) (PDMS) microchip-based immuno-sensing platform with integrated pneumatic micro valves is described. The microchip was fabricated with multiple layer soft lithography technology. By controlling the activation status of corresponding valves, reagent flows in the microchannel network can be well manipulated so that immuno-reactions only take place at designated reaction zones (DRZs). Four DRZs are included in the prototype microchip. Since these DRZs are all isolated from each other by micro valves, cross contamination is prevented. Using the inner surface of the all-PDMS microchannel as immunoassay substrate, on-chip sandwich format solid phase immunoassay was performed to demonstrate the feasibility of this immuno-sensing platform. Mouse IgG and fluorescein isothiocyanate (FITC) were used as the model analyte and the signal reporter respectively. Only 10 ul sample is needed for the assay and low detection limit of 5 ng/ml (≈33 pM) was achieved though low-cost polyclonal antibodies were used in our experiment for feasibility study only. The encouraging results from mouse IgG immunoassay proved the feasibility of our microchip design. With slight modification of the assay protocol, the same chip design can be used for multi-target detection and can provide a simple, cost-effective and integrated microchip solution for multiplex immunoassay applications.

  11. Determination of designer drug cross-reactivity on five commercial immunoassay screening kits.

    Regester, Laura E; Chmiel, Jeffrey D; Holler, Justin M; Vorce, Shawn P; Levine, Barry; Bosy, Thomas Z


    The detection of new designer drugs is often a difficult issue in forensic urine drug testing as immunoassays are the primary screening methodology for drugs of abuse in many of these laboratories. Cross-reactivity of compounds with immunoassay kits can either aid or complicate the detection of a variety of drug and drug metabolites. For instance, emerging designer drugs that share structural similarities to amphetamines and phencyclidine (PCP) have the potential to cross-react with assays designed to detect these compounds. This study evaluates the cross-reactivity of five commercially available immunoassay reagent kits for 94 designer drugs on a Roche/Hitachi Modular P automated screening instrument. The compounds used in this study are grouped by structural class as follows: 2,5-dimethoxyamphetamines, 2C (2,5-dimethoxyphenethylamines), β-keto amphetamines, substituted amphetamines, piperazines, α-pyrrolidinopropiophenones, tryptamines and PCP analogs. A drug concentration of 100 µg/mL was used to determine cross-reactivity for each assay and resulted in the following positive rates: Microgenics DRI(®) Ecstasy enzyme assay (19%), Microgenics DRI(®) Phencyclidine enzyme assay (20%), Lin-Zhi Methamphetamine enzyme immunoassay (39%), Siemens/Syva(®) EMIT(®)II Plus Amphetamines assay (43%) and CEDIA(®) DAU Amphetamine/Ecstasy assay (57%). Of the 94 designer drugs tested, 14% produced a negative response for all five kits. No designer drug used in this study generated a positive result for all five immunoassay kits.

  12. Improved buprenorphine immunoassay performance after urine treatment with β-glucuronidase.

    Snyder, Marion L; Darragh, Alicia; Flood, James G; Jones, Jenny; Ropar, Kaitlin; Jarolim, Petr; Melanson, Stacy E F


    Buprenorphine (BUP), a semi-synthetic opioid analgesic, is increasingly prescribed for the treatment of chronic pain and opioid dependence. Urine immunoassay screening methods are available for monitoring BUP compliance and misuse; however, these screens may have poor sensitivity or specificity. We evaluated whether the pretreatment of urine with β-glucuronidase (BG) improves the sensitivity and overall accuracy of three BUP enzyme immunoassays when compared with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Urine samples sent to our laboratories for BUP testing (n = 114) were analyzed before and after BG pretreatment by cloned enzyme donor immunoassay (CEDIA), enzyme immunoassay (EIA) and homogenous EIA (HEIA) immunoassays using a common 5 ng/mL cutoff. Total BUP and norbuprenorphine (NBUP) concentrations were measured by LC-MS-MS as the reference method. Urine BG pretreatment improved EIA, HEIA and CEDIA sensitivities from 70, 82 and 94%, respectively, to 97% for each of the three methods, when compared with LC-MS-MS. While the specificity of the EIA and HEIA remained 100% after BG pretreatment, the specificity of the CEDIA decreased from 74 to 67%. Urine pretreatment with BG is recommended to improve sensitivity of the EIA and HEIA BUP screening methods. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email:

  13. Development of a monoclonal antibody to urinary degradation products from the C-terminal telopeptide alpha 1 chain of type I collagen. Application in an enzyme Immunoassay and comparison to CrossLaps(TM) ELISA

    C, Fledelius; I, Kolding; P, Quist


    A monoclonal antibody MAbA7 was raised against a synthetic peptide having a sequence (EKAHDGGR) specific for a part of the C-telopeptide alpha 1 chain of type I collagen. MAbA7 was labelled with horseradish peroxide and used in a competitive one-step enzyme-linked immunosorbent assay (ELISA...... averaged 96.6%+/-5.3 (mean+/-1SD). Values obtained in the ELISA were highly correlated (r=0.93) to values obtained by a commercially available assay (CrossLaps(TM) ELISA) known to measure urinary degradation products derived from the C-telopeptide of type I collagen reflecting the rate of bone resorption....... Investigation of the urinary fragments responsible for the immunological response in the two assays revealed, however, that they are not identical. Values obtained in urine samples from postmenopausal women (n=108) and patients with Paget's disease (n=6) increased 43% (p

  14. Development and validation of an enzyme-linked immunosorbent assay for detection of cortisol in human saliva.

    Ozgocer, Tuba; Yildiz, Sedat; Uçar, Cihat


    Non-invasive measurement of cortisol in saliva is of prime importance as it represents a bioavailable neuroendocrine marker for stress. Therefore, in this study, we developed an enzyme immune assay that was suitable for salivary cortisol measurements. For that purpose, rabbit polyclonal antibody was raised against cortisol-3-CMO:BSA conjugate. The test was based on competition of liquid phase cortisol with conjugated cortisol on the solid phase. Primary antibody was used to bind available sites on the conjugate, which was proportional to numbers of cortisol in liquid phase. Biotinylated secondary anti-rabbit antibody was used to detect primary antibodies by addition of streptavidin peroxidase and substrate, respectively. Color formation was stopped and yellow color was read by a plate-reader spectrophotometer. Additionally, validated test was used to met all validation criteria including. Test developed was used to establish cortisol awakening response (CAR) in saliva samples collected in the morning after awakening (0, 15, 30, and 60(th) min) from women (n = 4) and men (n = 4) at 8 or 4 different days, respectively. Diurnal cortisol levels were assessed (n = 8) at after awaking 60 min at morning, 12:00, 19:00, and 22:00 hr. In conclusion, an enzyme immunoassay test was successfully produced, validated and used for cortisol measurement in saliva samples.

  15. Halogen bonding: A new retention mechanism for the solid phase extraction of perfluorinated iodoalkanes

    Yan Xiaoqing; Shen Qianjin; Zhao Xiaoran; Gao Haiyue; Pang Xue [College of Chemistry, Beijing Normal University, Beijing 100875 (China); Jin Weijun, E-mail: [College of Chemistry, Beijing Normal University, Beijing 100875 (China)


    Highlights: Black-Right-Pointing-Pointer Halogen bonding (XB) is firstly utilised in solid phase extraction. Black-Right-Pointing-Pointer The perfluorinated iodine alkanes can be extracted by C-I Midline-Horizontal-Ellipsis Cl{sup -} halogen bonding. Black-Right-Pointing-Pointer The C-I Midline-Horizontal-Ellipsis Cl{sup -} halogen bond is well characterised by spectroscopy methods. Black-Right-Pointing-Pointer The analytes with strong halogen-bonding abilities can be selectively extracted. - Abstract: For the first time, halogen-bonding interaction is utilised in the solid phase extraction of perfluorinated iodoalkane (PFI). Nine PFIs, as model analytes, were tested, and analyses by UV, {sup 19}F NMR and Raman spectroscopies demonstrate that the PFIs are extracted by a strong anion exchange (SAX) sorbent from n-hexane due to the C-I Midline-Horizontal-Ellipsis Cl{sup -} halogen-bonding interactions. The results also show that the adsorptivities of SAX for the diiodoperfluoro-alkanes (diiodo-PFIs) were much stronger than those for the perfluoroalkyl iodides (monoiodo-PFIs). Specifically, the recoveries for 1,6-diiodoperfluorohexane and 1,8-diiodoperfluorooctane were higher than 80% when 100 mL of sample spiked with a 5 ng mL{sup -1} analyte mixture was extracted. Interestingly, SAX had no adsorption for hexafluorobenzene at all, which is known to be unable to form a halogen bond with Cl{sup -}. The analytical performance of the halogen bond-based SPE-GC-MS method for the diiodo-PFIs was also examined in soil samples. The sorbent SAX enabled the selective extraction of four diiodo-PFIs successfully from soil samples. The recoveries of the diiodo-PFIs extracted from 5 g soil sample at the 100 ng g{sup -1} spike level were in the range of 73.2-93.8% except 26.8% for 1,2-diiodoperfluoroethane. The limit of detection varied from 0.02 to 0.04 ng g{sup -1} in soil samples. Overall, this work reveals the great application potential of halogen bonding in the field of solid

  16. Comparison of simultaneous distillation extraction and solid-phase micro-extraction for determination of volatile constituents in tobacco flavor

    ZHONG Ke-jun; WEI Wan-zhi; GUO Fang-qiu; HUANG Lan-fang


    The volatile and semi-volatile components in tobacco flavor additives were extracted by both simultaneous distillation extraction and solid-phase micro-extraction. Extraction conditions for solid-phase micro-extraction were optimized with information theory. Then, detection were accomplished by gas chromatography-mass spectrometry. Characteristic of each method was compared. Qualitative analysis and quantitative analysis of 6# tobacco flavor sample were accomplished through both simultaneous distillation extraction and solid-phase micro-extraction. The experimental results show that solid-phase micro-extraction method is the first choice for qualitative analysis and simultaneous distillation extraction is another good selection for quantitative analysis. By means of simultaneous distillation extraction, 20 components are identified, accounting for 92.77% of the total peak areas. Through solid-phase micro-extraction, there are 17 components identified accounting for 91.49% of the total peak areas. The main aromatic components in 6# tobacco flavor sample are propanoic acid, 2-hydroxy-, ethyl ester, menthol and menthyl acetate. The presented method has been successfully used for quality control of tobacco flavor.

  17. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences.

    Coin, Irene; Beyermann, Michael; Bienert, Michael


    This protocol for solid-phase peptide synthesis (SPPS) is based on the widely used Fmoc/tBu strategy, activation of the carboxyl groups by aminium-derived coupling reagents and use of PEG-modified polystyrene resins. A standard protocol is described, which was successfully applied in our lab for the synthesis of the corticotropin-releasing factor (CRF), >400 CRF analogs and a countless number of other peptides. The 41-mer peptide CRF is obtained within approximately 80 working hours. To achieve the so-called difficult sequences, special techniques have to be applied in order to reduce aggregation of the growing peptide chain, which is the main cause of failure for peptide chemosynthesis. Exemplary application of depsipeptide and pseudoproline units is shown for synthesizing an extremely difficult sequence, the Asn(15) analog of the WW domain FBP28, which is impossible to obtain using the standard protocol.

  18. Direct MD simulation of liquid-solid phase equilibria for two-component plasmas

    Schneider, A S; Horowitz, C J; Berry, D K


    We determine the liquid-solid phase diagram for carbon-oxygen plasma mixtures using two-phase MD simulations. We identified liquid, solid, and interface regions using a bond angle metric. To study finite size effects, we perform 55296 ion simulations and compare to earlier 27648 ion results. To help monitor non-equilibrium effects, we calculate diffusion constants $D_i$. We find that $D_O$ for oxygen ions in the solid is much smaller than $D_C$ for carbon ions and that both diffusion constants are 80 or more times smaller than diffusion constants in the liquid phase. There is excellent agreement between our phase diagram and that predicted by Medin and Cumming. This suggests that errors from finite size and non-equilibrium effects are small and that the carbon-oxygen phase diagram is now accurately known.

  19. Critical Regimes of Two-Phase Flows with a Polydisperse Solid Phase

    Barsky, Eugene


    This book brings to light peculiarities of the formation of critical regimes of two-phase flows with a polydisperse solid phase. A definition of entropy is formulated on the basis of statistical analysis of these peculiarities. The physical meaning of entropy and its correlation with other parameters determining two-phase flows are clearly defined. The interrelations and main differences between this entropy and the thermodynamic one are revealed. The main regularities of two-phase flows both in critical and in other regimes are established using the notion of entropy. This parameter serves as a basis for a deeper insight into the physics of the process and for the development of exhaustive techniques of mass exchange estimation in such flows. The book is intended for graduate and postgraduate students of engineering studying two-phase flows, and to scientists and engineers engaged in specific problems of such fields as chemical technology, mineral dressing, modern ceramics, microelectronics, pharmacology, po...

  20. Defect structure of erbium-doped <1 1 1> silicon layers formed by solid phase epitaxy

    Kyutt, R.N.; Sobolev, Nikolai A. E-mail:; Nikolaev, Yu. A.; Vdovin, V.I


    Erbium-doped layers have been produced on <1 1 1>-oriented silicon wafers using high-energy amorphizing Er implants and solid phase epitaxy (SPE). Transmission electron microscopy (TEM) and X-ray diffraction (XRD) techniques, used to study the microstructure of these layers, revealed the presence of microtwins and dislocations. The twins were found to be platelets with lateral dimensions of 15-30 nm and a thickness of about 2-9 nm, and their density throughout the regrown layer was nonuniform. The dislocation densities observed in the regrown layers were very high with densities exceeding 10{sup 10} cm{sup -2}. Within the implant fluence range studied, between 1x10{sup 14} and 9x10{sup 14} Er cm{sup -2}, the twin and dislocation densities were observed to increase with fluence, while the twin dimensions were found to decrease.