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Sample records for fluorometric enzyme immunoassay

  1. Enzyme immunoassay for methamphetamine.

    PubMed

    Aoki, K; Kuroiwa, Y

    1983-01-01

    A competitive enzyme immunoassay for methamphetamine with alkaline phosphatase labeled methamphetamine, Sepharose-antibody and p-nitrophenylphosphate as substrate was developed. The anti-methamphetamine antisera produced in rabbits by immunization with N-(4-aminobutyl) methamphetamine-BSA conjugate were specific for methamphetamine and showed low cross-reactivities with p-OH methamphetamine and amphetamine (metabolites of methamphetamine). The range of methamphetamine measurable by the enzyme immunoassay was 1 to 300 ng/tube. According to the assay, methamphetamine could be detected from urine and extract of hair.

  2. A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology.

    PubMed

    Swartzman, E E; Miraglia, S J; Mellentin-Michelotti, J; Evangelista, L; Yuan, P M

    1999-07-01

    We have developed a simple, homogeneous bead-based immunoassay for use with fluorometric microvolume assay technology (FMAT). The FLISA (fluorescence-linked immunosorbent assay) can be easily adapted from existing immunoassays, is comparable to traditional ELISAs with respect to linear dynamic range and sensitivity, and can be readily performed in 96- and 384-well plates. Additionally, the FLISA utilizes 100-fold less primary antibody than the conventional immunoassay. The scanner uses a helium/neon laser to image and measure bead-bound fluorescence while the background fluorescence is ignored. Consequently, no wash steps are required to remove unbound antibody, ligand, and fluorophore. Furthermore, the instrument is capable of detecting two different fluorescent dyes, allowing for multiplexed assays based on color. Fluorescent bead-based immunoassays were developed for the cytokines IL-6 and IL-8, and their use in both one-color and two-color FLISAs is demonstrated. Although no wash steps were employed, the FLISA was able to accurately measure the concentrations of IL-6 and IL-8 in the growth media of cytokine-stimulated HUVEC cells. In addition, a simulated high-throughput two-color FLISA positively identified those wells in a 384-well plate that contained different amounts of IL-6 and/or IL-8 peptide. The homogeneous, multiplex and multiplate format of the FLISA reduces hands-on time and reagent usage, and is therefore ideally suited for high-throughput screening.

  3. Electrochemical Enzyme Immunoassay for Detection of Toxins.

    DTIC Science & Technology

    developed a new biosensor design that combines advantages of immunoassay with electrochemical response for this purpose. The technology permits developing... amperometric enzyme immunoelectrode for immunoassays of small chemical molecules, based on the principle of coupling the immunochemical reaction to the electrode...response by using a soluble electrochemically active mediator. Toxin detection; Electrochemical; Enzyme immunoassay; Biosensor ; Biological sample.

  4. An enzyme immunoassay for plasma betamethasone

    SciTech Connect

    Kominami, G.; Yamauchi, A.; Ishihara, S.; Kono, M.

    1981-03-01

    A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-beta-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-beta-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using /sup 3/H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol as 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.

  5. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  6. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1997-01-01

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups.

  7. A fluorometric microarray with ZnO substrate-enhanced fluorescence and suppressed ``coffee-ring'' effects for fluorescence immunoassays

    NASA Astrophysics Data System (ADS)

    Li, Shuying; Dong, Minmin; Li, Rui; Zhang, Liyan; Qiao, Yuchun; Jiang, Yao; Qi, Wei; Wang, Hua

    2015-11-01

    A glass slide was first patterned with hydrophobic hexadecyltrimethoxysilane (HDS) and then microspotted with hydrophilic ZnO nanoparticles in an aminopropyltriethoxysilane (APS) matrix. The resulting HDS-ZnO-APS microarray could present the capability of suppressing the undesirable ``coffee-ring'' effects through its hydrophobic pattern so as to allow the fabrication of ZnO-APS testing microspots with a highly dense and uniform distribution. The lotus-like ``self-cleaning'' function could also be expected to effectively curb the cross contamination of multiple sample droplets. More importantly, the introduction of ZnO nanoparticles could endow the testing microspots with substrate-enhanced fluorescence leading to signal-amplification microarray fluorometry. The practical application of the developed HDS-ZnO-APS microarray was investigated by the sandwiched fluorometric immunoassays of human IgG, showing a linear detection range from 0.010 to 10.0 ng mL-1. Such a throughput-improved fluorometric microarray could be tailored for probing multiple biomarkers in complicated media like serum or blood.A glass slide was first patterned with hydrophobic hexadecyltrimethoxysilane (HDS) and then microspotted with hydrophilic ZnO nanoparticles in an aminopropyltriethoxysilane (APS) matrix. The resulting HDS-ZnO-APS microarray could present the capability of suppressing the undesirable ``coffee-ring'' effects through its hydrophobic pattern so as to allow the fabrication of ZnO-APS testing microspots with a highly dense and uniform distribution. The lotus-like ``self-cleaning'' function could also be expected to effectively curb the cross contamination of multiple sample droplets. More importantly, the introduction of ZnO nanoparticles could endow the testing microspots with substrate-enhanced fluorescence leading to signal-amplification microarray fluorometry. The practical application of the developed HDS-ZnO-APS microarray was investigated by the sandwiched fluorometric

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

    PubMed

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

    2013-01-01

    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. Rapid dioxin screening by enzyme immunoassay

    SciTech Connect

    Harrison, R.O.; Carlson, R.E.; Shirkhan, H.; Keimig, T.

    1995-12-31

    A system has been developed for rapid screening of 2,3,7,8-Tetra-ChloroDibenzo-p-Dioxin (TCDD). The system uses a competitive inhibition Enzyme ImmunoAssay (EIA) based on a mouse monoclonal antibody which is specific for TCDD and related congeners. Sample preparation can be performed with a programmable automated extraction and cleanup system which uses disposable Teflon clad columns. The extraction and cleanup system has been extensively validated by GC-MS for a variety of sample types. The sample preparation system allows immunoassay analysis of soil, serum, water, and other matrices by taking each sample type to the same sample preparation endpoint. Concentration factors and endpoint conditions are completely flexible and programmable. Immunoassay analysis is performed by the addition of a prepared sample extract in organic solvent to an antibody coated microwell containing an aqueous sample diluent. This is mixed and incubated for 30 minutes to allow the immobilized antibody to capture analyte from the sample. The liquid is then removed and the well is washed to remove unbound materials. The well is then incubated with a competitor-HRP conjugate capable of binding specifically to the antibody sites not occupied by TCDD. After 30 minutes, the unbound conjugate is washed away and enzyme substrate is added for color development. The color generated is directly related to the amount of competitor-HRP bound in the second step, which is inversely related to the amount of analyte bound in the first step. After 30 minutes, a stop solution is added and the developed color is read on a microplate reader. The total time required for the EIA analysis of a prepared extract is less than 2 hours.

  10. Enzyme immunoassays with special reference to ELISA techniques.

    PubMed Central

    Voller, A; Bartlett, A; Bidwell, D E

    1978-01-01

    In this review outlines are given on various types of enzyme immunoassay. The applications to such enzyme immunoassays, especially ELISA, are dealth with in detail. It is concluded that these techniques have high sensitivity and will be suitable in due course as routine laboratory tests. PMID:78929

  11. Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay.

    PubMed

    Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

    2017-06-01

    The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Screening for antinuclear antibodies by enzyme immunoassay.

    PubMed

    Jaskowski, T D; Schroder, C; Martins, T B; Mouritsen, C L; Litwin, C M; Hill, H R

    1996-04-01

    Indirect fluorescent antibody (IFA) ia most widely used method in clin clinical laboratories to screen for autoantibodies against a wide variety of nuclear antigens. Recently, a number of antinuclear antibody (ANA) enzyme immunoassay (EIA) screens have become commercially available and claim to be an alternative method to screen for ANAs. Given the subjectivity of technical interpretation of IFA and the high number of ANA negative samples, a suitable EIA method for ANA screening would be beneficial to clinical laboratories with large samples volumes. Five ANA EIA screens were compared (Elias, Helix, Sanofi, TheraTest and Zeus) to IFA using a human epithelial cell line (HEp-2). Sera from 601 patients submitted to our reference laboratory for autoimmune testing, and from 202 normal healthy blood donors, were included in this study. Samples with discordant results between IFA and EIA were further analyzed using single antigen EIAs for SSA, SSB, Sm, RNP, Scl-70, histones, dsDNA, and ssDNA. Analyses were based on clinically significant IFA titers of > or equal to 1:160 as positive and <1:40 as negative. When compared to IFA, agreement, sensitivity and specificity for each ANA EIA screen were as follows: Elias: 87.0%, 69.5% and 97.9%; Helix: 94.6%, 90.2%, and 97.3%; Sanofi: 95.0%, 93.7%, and 95.9%; TheraTest: 95.3%, 97.7%, and 93.5%; Zeus: 87.1%, 96.2%, and 81.4%, respectively. In conclusion, screening for ANAs by EIA using several commercial assays was both sensitive and specific when compared to IFA. Moreover, the EIA is objective and much less labor intensive when screening a large number of clinical specimens. None of the EIAs were 100% sensitive and, thus, may fail to detect a few of the nonspecific ANAs that demonstrate atypical as well as classical IFA patterns. The advantages of employing these nonsubjective assays to screen out the vast majority of ANA negative sera is clear. The authors still recommend confirming titers and patterns of sera with positive EIA

  13. Application Of Laser Fluorimetry To Enzyme-Linked Immunoassay

    NASA Astrophysics Data System (ADS)

    Hinsberg, William D.; Milby, Kristin H.; Lidofsky, Steven D.; Zare, Richard N.

    1981-09-01

    An enzyme-linked sandwich immunoassay for insulin is described. Horseradish peroxidase is employed as an enzyme label for antibody, and enzyme activity is measured via the fluorogenic substrate, p-hydroxyphenylacetic acid. The product is detected by excitation of fluorescence with the 325 nm line of a cw helium-cadmium ion laser on-line with reverse phase high performance liquid chromatography. The method requires a total incubation time of 45 minutes, and the limit of insulin detection is 1.1 μU/ml (6.6 pM). This assay is applicable to the analysis of human serum samples.

  14. Monensin concentrations measured in feeder cattle using enzyme immunoassay.

    PubMed

    Mount, M E; Cullor, J S; Kass, P H; Garret, W

    1996-06-01

    Thirty heifers were fed a ration containing 30 g monensin/ton. Fecal, urinary and seral samples were collected at varying intervals prior to and after initiating administration of the monensin-containing feed, and monensin concentrations were determined using a modified indirect enzyme immunoassay. Fecal samples contained measurable (micrograms/g; ppm) concentrations of monensin in most samples. The majority of sera and urine samples contained monensin at ng/ml (ppb) concentrations, which were above background levels prior to monensin feeding. Twelve head were fed monensin at 60 g/ton and 90 g/ton for 5 d with collection of similar samples. Higher concentrations of monensin were detected with increasing ration amounts in all 3 sample types. Enzyme immunoassay for monensin in these biological samples identified presence of the feed additive.

  15. Multicentre evaluation of dengue IgM dot enzyme immunoassay.

    PubMed

    Lam, S K; Fong, M Y; Chungue, E; Doraisingham, S; Igarashi, A; Khin, M A; Kyaw, Z T; Nisalak, A; Roche, C; Vaughn, D W; Vorndam, V

    1996-11-01

    The traditional methods used in the diagnosis of dengue infection do not lend themselves to field application. As such, clinical specimens have to be sent to a central laboratory for processing which invariably leads to delay. This affects patient management and disease control. The development of the dengue IgM dot enzyme immunoassay has opened up the possibility of carrying out the test in peripheral health settings. This multicentre study was conducted to evaluate a new, commercial nitrocellulose membrane based IgM capture enzyme immunoassay. The sensitivity and specificity of the test were compared with in-house dengue IgM enzyme-linked immunoassays routinely performed by each of the selected centres. Known positive and negative dengue specimens, as well as specimens from non-dengue cases, were included in the evaluation. Based on 402 specimens tested by the six centres, the sensitivity was 92.1% and specificity 88.1%, with an overall agreement of 92.8% when compared with IgM EIA assays performed on microplates. The results suggest that this commercial kit has a role to play in the diagnosis of dengue infection, especially in peripheral health settings.

  16. Enzyme immunoassay for the detection of group A streptococcal antigen.

    PubMed Central

    Knigge, K M; Babb, J L; Firca, J R; Ancell, K; Bloomster, T G; Marchlewicz, B A

    1984-01-01

    A competitive inhibition enzyme immunoassay for the detection of Streptococcus pyogenes directly from throat specimens or from solid bacteriological medium is described. Group A-specific polysaccharide adsorbed onto treated polystyrene beads, in conjunction with rabbit antibody to S. pyogenes, was used to determine the presence of the polysaccharide antigen. Inhibition values in excess of 65% were observed with 10(4) or more CFU of S. pyogenes per test. An inhibition of 25% was demonstrated with as few as 10(3) CFU per test. Heterologous microorganisms tested at 10(6) CFU per test reacted at levels of inhibition less than 25%. Two types of bacterial transport medium and swabs of different fiber compositions did not alter the assay performance. Accurate identification of S. pyogenes was achieved by testing single colonies picked directly from blood agar plates which had been incubated for 18 to 24 h. In addition, the assay was performed on throat specimens from children and adults having pharyngitis. A single-swab, blind study was conducted in which enzyme immunoassay reactivity was compared with results of blood agar culture and bacitracin sensitivity. When there were discordant results, serological identification was used as the confirmatory test. At an optimal cutoff value of 40% inhibition, sensitivity and specificity by enzyme immunoassay were 97.0% and 97.9%, respectively, as compared with confirmed culture results. The assay has an incubation time of 3 h and is a sensitive and specific method for the detection of S. pyogenes antigen. PMID:6386878

  17. Enzyme immunoassays and related procedures in diagnostic medical virology

    PubMed Central

    Kurstak, Edouard; Tijssen, Peter; Kurstak, Christine; Morisset, Richard

    1986-01-01

    This review article describes several applications of the widely used enzyme immunoassay (EIA) procedure. EIA methods have been adapted to solve problems in diagnostic virology where sensitivity, specificity, or practicability is required. Concurrent developments in hybridoma and conjugation methods have increased significantly the use of these assays. A general overview of EIA methods is given together with typical examples of their use in diagnostic medical virology; attention is drawn to possible pitfalls. Recent advances in recombinant DNA technology have made it possible to produce highly specific nucleic acid probes that have a sensitivity approximately 100 times greater than that of EIA. Some applications of these probes are described. Although the non-labelled nucleic acid probes for use in the field are not as refined as non-labelled immunoassays, their range of applications is expected to expand rapidly in the near future. ImagesFig. 4 PMID:3533302

  18. Fluorometric immunoassay for human serum albumin based on its inhibitory effect on the immunoaggregation of quantum dots with silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Marukhyan, Seda S.; Gasparyan, Vardan K.

    2017-02-01

    Quantitative determination of HSA was conducted by competitive immunoassay. Inhibition of aggregation of antibody conjugated quantum dots (QD) with albumin conjugated silver nanoparticles (AgNPs) in the presence of HSA was conducted. If antibody-loaded CdSe QDs aggregate with HSA-coated silver nanoparticles the distance between the two kinds of nanoparticles will be reduced enough to cause fluorescence resonance energy transfer (FRET). In this case the yellow fluorescence of the Ab-QDs is quenched. However if HSA (antigen) is added to the Ab-QDs their surface will be blocked and they cannot aggregate any longer with the HSA-AgNPs. Hence, fluorescence will not be quenched. The drop of the intensity of fluorescence (peaking at 570 nm) is inversely correlated with the concentration of HSA in the sample. The method allows to determine HSA in the 30-600 ng·mL-1 concentration range.

  19. Detection of Trichomonas vaginalis antigen in women by enzyme immunoassay.

    PubMed Central

    Yule, A; Gellan, M C; Oriel, J D; Ackers, J P

    1987-01-01

    An enzyme immunoassay (EIA) was developed for the detection of Trichomonas vaginalis antigen in vaginal swabs. Four hundred and eighty two women attending a sexually transmitted disease (STD) clinic were tested; 44 (9.1%) were positive by culture, 32 (6.6%) were positive by wet film examination, and 54 (11.2%) were considered to be positive for trichomonal antigen by EIA. Taking culture as the reference method, the EIA had a sensitivity of 93.2% and a specificity of 97.5%. The predictive value of a positive test was 82% and that of a negative test was 99.3%. PMID:3495554

  20. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    NASA Astrophysics Data System (ADS)

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

    1999-04-01

    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.

  1. Particle counting immunoassay for urinary cotinine. Comparison with chromatography, enzyme-linked immunoassay and fluorescence polarization immunoassay.

    PubMed

    Galanti, L M; Dell'Omo, J; Vanbeckbergen, D; Dubois, P; Masson, P L; Cambiaso, C L

    1999-07-01

    Urinary cotinine was measured according to its inhibitory activity on the agglutination of cotinine-coated latex particles by anti-cotinine antibodies, the agglutination being measured by optical counting of the remaining non-agglutinated particles (particle counting, PaC). The detection limit was 0.03 microgram/ml and the practical range extended from 0.03 to 3.9 micrograms/ml. The correlation results of 320 urine samples with those of high pressure liquid chromatography, enzyme-linked (Coti-Tracq EIA, Serex Inc., Maywood, NJ, USA), and fluorescence polarization immunoassay (TDX instrument, Abbott, Abbott Park, IL, USA) were r = 0.90, r = 0.69, r = 0.87, respectively, whereas the correlation coefficients between the assays other than particle counting ranged from 0.62 to 0.88. PaC does not require any separation step and can thus be easily automated.

  2. Quality assurance in immunoassay performance--comparison of different enzyme immunoassays for the determination of caffeine in consumer products.

    PubMed

    Grandke, Julia; Oberleitner, Lidia; Resch-Genger, Ute; Garbe, Leif-Alexander; Schneider, Rudolf J

    2013-02-01

    Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3',5,5'-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format.

  3. Immunoassays

    NASA Astrophysics Data System (ADS)

    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.

  4. DEVELOPMENT OF A CLASS-SELECTIVE ENZYME IMMUNOASSAY FOR URINARY PHENOLIC GLUCURONIDES. (R825433)

    EPA Science Inventory

    Class-selective immunoassays for the measurement of glucuronides in human urine can aid evaluation of human exposure to complex mixtures of xenobiotics. Therefore, an enzyme immunoassay (EIA) for the group-selective detection of phenolic DEVELOPMENT OF A CLASS-SELECTIVE ENZYME IMMUNOASSAY FOR URINARY PHENOLIC GLUCURONIDES. (R825433)

    EPA Science Inventory

    Class-selective immunoassays for the measurement of glucuronides in human urine can aid evaluation of human exposure to complex mixtures of xenobiotics. Therefore, an enzyme immunoassay (EIA) for the group-selective detection of phenolic High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

    PubMed Central

    Bell, Colin W.; Fricks, Barbara E.; Rocca, Jennifer D.; Steinweg, Jessica M.; McMahon, Shawna K.; Wallenstein, Matthew D.

    2013-01-01

    Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample). Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil

  5. High-throughput fluorometric measurement of potential soil extracellular enzyme activities.

    PubMed

    Bell, Colin W; Fricks, Barbara E; Rocca, Jennifer D; Steinweg, Jessica M; McMahon, Shawna K; Wallenstein, Matthew D

    2013-11-15

    Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample). Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil

  6. Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

    PubMed

    Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

    2015-01-01

    The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs.

  7. Treponemal antibody-absorbent enzyme immunoassay for syphilis.

    PubMed Central

    Chen, J; Lin, T M; Schubert, C M; Halbert, S P

    1986-01-01

    An enzyme immunoassay for the diagnosis of syphilis (ELISA-SY) was developed with solid-phase extracts of Treponema pallidum, specimen diluent containing Reiter treponeme absorbent, and three 30-min incubations. The ELISA-SY results were determined in comparison with a standardized positive control and reported as a percentage of strong positive control. In tests with 1,005 serum samples from a venereal disease clinic and other sources, 98.2% agreement was found with fluorescent treponemal antibody-absorption (FTA-ABS) results, and 98.3% agreement was found with T. pallidum passive hemagglutination (PHA) findings. Only 1 of 29 sera originally considered to be biologically false-positive by ELISA-SY; the latter specimen was also positive by PHA and FTA-ABS tests performed in our laboratories. Serum samples from clinically diagnosed syphilitics (16 primary-stage isolates, 7 secondary-stage isolates, and 3-latent-stage isolates) were all positive by ELISA-SY, FTA-ABS, and PHA. Serum samples from 51 newborns suspected of having syphilis on the basis of positive cardiolipin flocculation tests showed 98% agreement of ELISA-SY results with FTA-ABS and PHA findings. Sera from all 61 patients with a variety of autoimmune and other diseases known to be associated with biologically false-positive reactions for syphilis were negative by this ELISA-SY. The specificity of the ELISA procedure for T. pallidum antibody was also confirmed immunologically by blocking experiments. PMID:3519659

  8. Rapid dioxin screening of milk and water by enzyme immunoassay

    SciTech Connect

    Harrison, R.O.; Carlson, R.E.; Shirkhan, H.

    1995-12-01

    A simple and easy to use enzyme immunoassay (EIA) system has been developed for rapid screening of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378D). This EIA has been adapted to analysis of water and milk using an automated system for extraction of liquid samples. Water analysis can be performed directly following extraction and solvent exchange with no extract clean-up. The same automated system is used for milk extraction and the extract is then cleaned chromatographically using the automated FMS Dioxin-Prep{trademark} System. Sensitivity for 2378D in the EIA is approximately 100 pg per analysis. Thus sensitivity to 10 ppt 2378D (whole weight basis) in milk is possible using only 50 ml or less of sample and sensitivity to 0.1 ppt 2378D in water is possible using 1-2 liters of sample. Total time for sample preparation and analysis is about 3 hours for water and 4.5 hours for milk.

  9. Blastomyces dermatitidis Antigen Detection by Quantitative Enzyme Immunoassay

    PubMed Central

    Hage, Chadi A.; Bariola, J. Ryan; Bensadoun, Eric; Rodgers, Mark; Bradsher, Robert W.; Wheat, L. Joseph

    2012-01-01

    The second-generation MVista Blastomyces antigen enzyme immunoassay was not quantitative; therefore, specimens obtained previously were tested in the same assay as new specimens to assess the change in antigen levels. Furthermore, the sensitivity in serum had not been fully evaluated. The purpose of this study was to evaluate a quantitative Blastomyces antigen assay and detection of antigen in serum. Calibrators containing known concentrations of Blastomyces galactomannan were used to quantify antigen in urine and serum from patients with proven blastomycosis and from controls. Paired current and previously obtained urine specimens were tested to determine if quantification eliminated the need for concurrent testing to assess change in antigen. Pretreatment of serum with EDTA at 104°C was evaluated to determine if dissociation of immune complexes improved detection of antigenemia. Antigenuria was detected in 89.9% of patients with culture- or histopathology-proven blastomycosis. Specificity was 99.0% in patients with nonfungal infections and healthy subjects, but cross-reactions occurred in 95.6% of patients with histoplasmosis. Change in antigen level categorized as increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in ng/ml determined from different assays. Pretreatment increased the sensitivity of detection of antigenemia from 35.7% to 57.1%. Quantification eliminated the need for concurrent testing of current and previously obtained specimens for assessment of changes in antigen concentration. Pretreatment increased the sensitivity for detection of antigenemia. Differentiation of histoplasmosis and blastomycosis is not possible by antigen detection. PMID:22116687

  10. Optimization of condition for conjugation of enrofloxacin to enzymes in chemiluminescence enzyme immunoassay

    NASA Astrophysics Data System (ADS)

    Yu, Songcheng; Yu, Fei; Zhang, Hongquan; Qu, Lingbo; Wu, Yongjun

    2014-06-01

    In this study, in order to find out a proper method for conjugation of enrofloxacin to label enzymes, two methods were compared and carbodiimide condensation was proved to be better. The results showed that the binding ratio of enrofloxacin and alkaline phosphatase (ALP) was 8:1 and that of enrofloxacin and horseradish peroxidase (HRP) was 5:1. This indicated that conjugate synthesized by carbodiimide condensation was fit for chemiluminescence enzyme immunoassay (CLEIA). Furthermore, data revealed that dialysis time was an important parameter for conjugation and 6 days was best. Buffer to dilute conjugate had little effect on CLEIA. The storage condition for conjugates was also studied and it was shown that the conjugate was stable at 4 °C with no additive up to 30 days. These data were valuable for establishing CLEIA to quantify enrofloxacin.

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

    PubMed

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

    2002-01-01

    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.

  12. Competitive Enzyme Immunoassay for Diagnosis of Human Brucellosis

    PubMed Central

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

    1999-01-01

    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

  13. Fluorometric flow-immunoassay for alkylphenol polyethoxylates on a microchip containing a fluorescence detector comprised of an organic light emitting diode and an organic photodiode.

    PubMed

    Liu, Rong; Ishimatsu, Ryoichi; Yahiro, Masayuki; Adachi, Chihaya; Nakano, Koji; Imato, Toshihiko

    2015-03-01

    A compact fluorescence detector was constructed on a microchip from an organic light emitting diode (OLED) as the light source and an organic photodiode (OPD) as the photo-detector and was used in an immunoassay for alkylphenol polyethoxylates (APE). The OLED based on a terbium complex emitted a sharp light at the main wavelength of 546 nm with a full width at half maximum of 9 nm. The incident photo-to-current conversion efficiency (IPCE) of the OPD fabricated with Fullerene 70 (C70) and tris[4-(5-phenylthiopen-2-yl)phenyl]-amine (TPTPA) was approximately 44% for light at a wavelength of 586 nm. The performance of the fluorescence detector was evaluated for the determination of resorufin (λ(em)=586 nm) and the photocurrent of the OPD due to the fluorescence of resorufin was proportional to the concentration of resorufin in the range from 0 to 18 µM with a detection limit (S/N=3) of 0.6 µM. The fluorescence detector was successfully utilized in a competitive enzyme-linked immunosorbent assay for APE, where an anti-APE antibody was immobilized on the surface of the channel of the Polydimethylsiloxane (PDMS) microchip or on the surface of magnetic microbeads. After an immunoreaction with a sample solution of APE containing a horse radish peroxidase (HRP)-labeled APE, the fluorescence of resorufin generated just after introduction of a mixed solution of Amplex Red and H2O2 was measured using the fluorescence detector. The calibration curve for the photocurrent signals of the OPD due to the fluorescence of resorufin against the logarithmic concentration of APE was sigmoidal in shape. The detection limits defined as IC80 were ca. 1 ppb and ca. 2 ppb, respectively, for the methods using the anti-APE antibody immobilized on the surface of the microchannel and in the case where the antibody was immobilized on the surface of magnetic microbeads. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. False-Positive Results of Enzyme Immunoassays for Human Immunodeficiency Virus in Patients with Uncomplicated Malaria

    PubMed Central

    Gasasira, Anne F.; Dorsey, Grant; Kamya, Moses R.; Havlir, Diane; Kiggundu, Moses; Rosenthal, Philip J.; Charlebois, Edwin D.

    2006-01-01

    Malaria may impact upon human immunodeficiency virus (HIV) test results. We evaluated two HIV enzyme immunoassays (EIAs) by testing 1,965 Ugandans with malaria. We found poor positive predictive values (53% and 76%), particularly with younger age. Combining EIAs eliminated false positives but missed 21% of true positives. Performance of HIV EIAs in malaria may be unsatisfactory. PMID:16891532

  15. Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes.

    PubMed

    Parolo, Claudio; de la Escosura-Muñiz, Alfredo; Merkoçi, Arben

    2013-02-15

    The use of gold nanoparticles (AuNPs) as labeling carriers in combination with the enzymatic activity of the horseradish peroxidase (HRP) in order to achieve an improved optical lateral flow immunoassay (LFIA) performance is presented here. Briefly in a LFIA with an immune-sandwich format AuNPs are functionalized with a detection antibody already modified with HRP, obtaining an 'enhanced' label. Two different detection strategies have been tested: the first one following just the red color of the AuNPs and the second one using a substrate for the HRP (3 different substrates are evaluated), which produces a darker color that enhances the intensity of the previous red color of the unmodified AuNPs. In such very simple way it is gaining sensitivity (up to 1 order of magnitude) without losing the simplicity of the LFIA format, opening the way to other LFIA applications including their on-demand performance tuning according to the analytical scenario.

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

    PubMed

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

    2017-01-01

    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.

  17. Enzyme immunoassays for detection of gypsy moth nuclear polyhedrosis virus

    Treesearch

    Michael Ma

    1985-01-01

    Enzyme-linked immunosorbent assays (ELISA) were developed for detecting gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (NPV). They were used to detect the presence of NPV in hemoplymph samples collected from infected larvae. The incorporation of hybridoma antibodies with these procedures would make them even more specific for gypsy moth...

  18. Improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Gevkan, Ivan I.; Overchuk, Marta O.

    2014-02-01

    Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations - 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

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

    PubMed

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

    2006-04-30

    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.

  2. Comparison of galactomannan enzyme immunoassay performance levels when testing serum and plasma samples.

    PubMed

    White, P Lewis; Jones, Tim; Whittle, Katie; Watkins, Joanne; Barnes, Rosemary A

    2013-04-01

    Diagnostic galactomannan (GM) enzyme immunoassay (EIA) testing is formally validated only for serum, though in practice, plasma is occasionally tested. It is assumed, but not confirmed, that results will be comparable to those for serum. GM EIA when testing plasma was evaluated, providing sensitivity (85.7%) and specificity (85.4%) comparable to those for serum. Plasma index values were higher than those for serum; if plasma GM EIA were used to define probable cases, four additional cases would have been diagnosed.

  3. Evaluation of ractopamine cross-reactivity in several commercially available beta-agonist enzyme immunoassay kits.

    PubMed

    Wicker, A L; Turberg, M P; Coleman, M R

    1995-12-01

    Several enzyme immunoassay test kits are commercially available for screening of matrices including cattle feed, tissues, faeces and urine for the presence of beta-agonists. The kits utilized for this evaluation offer sensitivity as low as 0.1 ng ml-1 (assay solution concentration, not test sample). Evaluation of ractopamine hydrochloride (LY31537) solutions at concentrations as high as 1000 ng ml-1, using six different kits from four different manufacturers, showed cross-reactivity of less than 0.5%.

  4. Surveillance of giardiasis in Northwest England 1996-2006: impact of an enzyme immunoassay test.

    PubMed

    Ellam, H; Verlander, N Q; Lamden, K; Cheesbrough, J S; Durband, C A; James, S

    2008-09-11

    The incidence of giardiasis in Central Lancashire increased following the introduction of a sensitive enzyme immunoassay diagnostic test in November 2002. We compared the epidemiological trends for 1996-2006 in Central Lancashire with a control area which used a standard wet preparation diagnostic method throughout. Poisson regression modelling was used to investigate trends in giardiasis before and after the introduction of the test. In the control area, incidence of giardiasis was four per 100,000 in 2005. In contrast, in Central Lancashire, the rates increased in temporal association with the introduction of the enzyme immunoassay test from 10.1 per 100,000 population in 2002 to 33.6 per 100,000 in 2006. The increase in giardiasis was unexplained by local factors including travel, outbreaks or sampling trends. The increase in giardiasis occurred in all age groups except for males aged 0-14 years and was most marked in males aged 25-44 years. The relative risk for trend post-test introduction in Central Lancashire was 1.11 (95% CI, 1.01-1.23). This suggests that the increase in giardiasis following the introduction of the sensitive enzyme immunoassay test was at least in part due to improved detection. There appears to be considerable under-diagnosis of giardiasis, particularly in adults. Additional research is required to evaluate the enzyme immunoassay test more widely. The test may assist in standardisation of diagnostic methods for giardiasis and enable more accurate estimation of disease burden and transmission routes.

  5. Competitive solid phase enzyme-linked immunoassay for the quantification of limonin in citrus.

    PubMed

    Jourdan, P S; Mansell, R L; Oliver, D G; Weiler, E W

    1984-04-01

    A solid-phase enzyme immunoassay for the quantitative determination of the bitter triterpene-lactone, limonin, in citrus juice samples is described. As little as 0.1 ppm of limonin can be detected. Quantitative results are available within 1 h of total assay time. The assay makes use of a limonin-alkaline phosphatase tracer of high immunoreactivity and has been semiautomated using antibody-coated polystyrene microcuvettes, a vertical light path photometer, and a forced-air microplate incubator.

  6. Cytomegalovirus Antibody in Cerebrospinal Fluid of Schizophrenic Patients Detected by Enzyme Immunoassay

    NASA Astrophysics Data System (ADS)

    Fuller Torrey, E.; Yolken, Robert H.; Winfrey, C. Jack

    1982-05-01

    By means of enzyme immunoassay techniques to detect the presence of antibody to cytomegalovirus, the cerebrospinal fluid of 178 patients with schizophrenia, 17 patients with bipolar disorders, and 11 other psychiatric patients was compared with that of 79 neurological patients and 41 normal control subjects. The cerebrospinal fluid of 20 of the schizophrenic patients and 3 of the patients with bipolar disorders showed significant increases in immunoglobulin M antibody to cytomegalovirus; no difference was found in patients on or off psychotropic medications.

  7. Progress in enzyme immunoassays: production of reagents, experimental design, and interpretation*

    PubMed Central

    Kurstak, Edouard

    1985-01-01

    Enzyme immunoassays represent in many cases the preferred procedure for the detection of antigens or corresponding antibodies. However, many of the current procedures are performed suboptimally. This article reviews the available designs, auxiliary recognition systems, production and purification of antibodies, conjugation procedures, solid-phase materials, recording and interpretation of results, and quality control and standardization of procedures to improve the reproducibility of tests. PMID:3910300

  8. Multicenter evaluation of a fluorometric enzyme immunocapture assay to detect toxoplasma-specific immunoglobulin M in dried blood filter paper specimens from newborns.

    PubMed Central

    Eaton, R B; Petersen, E; Seppänen, H; Tuuminen, T

    1996-01-01

    An easy-to-perform fluorometric enzyme immunocapture assay (FEIA) was developed by Labsystems, Helsinki, Finland, to detect toxoplasma-specific immunoglobulin M (IgM) in dried blood spots. Assay materials were distributed to two sites that have programs in place designed to identify infants born with congenital toxoplasma infection: the Statens Serum Institut, Copenhagen, Denmark, and the New England Regional Newborn Screening Program, Boston, Mass. Each site tested over 700 dried blood samples from healthy newborns to define a cutoff at the 99.5 percentile (5 enzyme immunounits for Copenhagen and 4 enzyme immunounits for Boston). Each site then applied its own cutoff of interpret results for dried blood spots prepared from either adults with serology suggestive of acute infection (Copenhagen) or infants determined to be congenitally infected on the basis of serological criteria (Boston). In Copenhagen, 35 of 38 adult samples were either positive to a small degree or borderline positive for IgA. These samples thus may not represent acute infection. In Boston, of 26 congenitally infected infants, 22 were positive by FEIA. The four infant specimens not positive by FEIA were either negative or borderline positive by the standard Boston assay. These results demonstrate that the IgM FEIA is a potential alternative to other filter paper assay for toxoplasma-specific IgM currently in use for newborns. PMID:8940462

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

    PubMed

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

    2016-04-09

    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.

  10. The detection of cocaine in hair specimens using micro-plate enzyme immunoassay.

    PubMed

    Moore, C; Deitermann, D; Lewis, D; Feeley, B; Niedbala, R S

    1999-05-01

    The analysis of hair for drugs of abuse is becoming increasingly popular and is under consideration by the Division of Health and Human Services as a possible alternative or adjunct to urinalysis in workplace programs. The detection of cocaine in human hair using a commercially available micro-plate enzyme immunoassay is described for the first time. Sample size and incubation time were the major variables in the optimization of the method. In order to validate the procedure, the method was applied to 105 consecutive hair samples routinely received into our laboratory. The samples were simultaneously analyzed by the Micro-Plate immunoassay (EIA), as well as our current fluorescence polarization immunoassay (FPIA) procedure and gas chromatography-mass spectrometry (GC/MS). The sensitivity of the EIA and FPIA assays were 75% and 67.8% respectively; specificity 97.4% and 80.5% respectively; and efficiency 91.4 and 77.1% respectively. The Micro-Plate EIA was shown to be a valid alternative to other immunoassay screening methods for the detection of cocaine in hair by demonstrating increased sensitivity, specificity and efficiency over our current technique.

  11. Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal.

    PubMed

    Liu, Xing; Xu, Yang; Wan, De-bin; Xiong, Yong-hua; He, Zhen-yun; Wang, Xian-xian; Gee, Shirley J; Ryu, Dojin; Hammock, Bruce D

    2015-01-20

    A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

  12. Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal

    PubMed Central

    Liu, Xing; Xu, Yang; Wan, De-bin; Xiong, Yong-hua; He, Zhen-yun; Wang, Xian-xian; Gee, Shirley J.; Ryu, Dojin; Hammock, Bruce D.

    2015-01-01

    A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double mutant gene. The Nb28-AP construct was transformed into E. coli BL21(DE3)plysS and soluble expression in bacteria was confirmed by SDS-PAGE and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 ng/mL and 0.04 ng/mL, respectively, with a linear range of 0.06–0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal. PMID:25531426

  13. Rabies virus binding to cellular membranes measured by enzyme immunoassay.

    PubMed

    Lentz, T L; Chester, J; Benson, R J; Hawrot, E; Tignor, G H; Smith, A L

    1985-05-01

    The binding of rabies virus to cellular membranes was measured using an enzyme-linked immunosorbent assay (ELISA). Virus binding to membranes adsorbed to the wells of microtiter plates was detected with rabies virus antibody and alkaline phosphatase-linked second antibody. The greatest degree of binding was to myotube, neuroblastoma, and salivary gland membranes; intermediate levels occurred in striated muscle and nerve membranes; and low levels of binding were found in other membranes, including those of most parenchymal organs. Binding of rabies virus to myotube membranes was saturable, dependent on pH (with an optimum of pH 6.0), facilitated by the divalent cations Ca++, Mn++, and Mg++, and was temperature dependent. Binding was greatly reduced by inactivation of virus with beta-propiolactone or treatment of virus with trypsin. In embryonic chick myotubes, total acetylcholine receptor content and acetylcholinesterase activity undergo marked changes during development, first increasing and then decreasing at the time of hatching. Binding of rabies virus followed a similar pattern, indicating that the virus may interact with the acetylcholine receptor or other surface molecules undergoing similar developmental changes.

  14. [Enzyme immunoassay for the determination of hexestrol in meat].

    PubMed

    Vdovenko, M M; Peng, C F; Xu, C L; Vylegzhanina, E S; Komarov, A A; Sakharov, I Iu

    2011-01-01

    An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an antibiotic forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC10) equaled 0.01 ng/ml, IC50 equaled 0.17 ng/ml, and the working range (IC20-IC80) equaled 0.03-0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05 ng/ml, 2.9 ng/ml, and 0.26-32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.

  15. Validation of a monoclonal enzyme immunoassay for the determination of carbofuran in fruits and vegetables.

    PubMed

    Moreno, M J; Abad, A; Pelegrí, R; Marínez, M J; Sáez, A; Gamón, M; Montoya, A

    2001-04-01

    The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput.

  16. Flow-through membrane-based enzyme immunoassay for rapid detection of ochratoxin A in wheat.

    PubMed

    De Saeger, S; Van Peteghem, C

    1999-01-01

    A flow-through membrane-based enzyme immunoassay for the rapid detection of ochratoxin A in wheat was developed (patent pending). An Immunodyne ABC membrane was coated with rabbit anti-mouse immunoglobulins and free protein binding sites were blocked. After these antibody-coated membranes were placed on an absorbent layer in a plastic test device, a sequential competitive enzyme immunoassay was performed. The following reactants were successively dropped onto the membrane: wash solution, a dilution of monoclonal anti-ochratoxin A immunoglobulins, wash solution, ochratoxin A standard solution or sample extract solution, a dilution of ochratoxin A-horseradish peroxidase conjugate, and wash solution. Finally, substrate solution (H2O2-3,3',5,5'-tetramethylbenzidine) was added for color reaction. The dot color intensity on the membrane was visually compared with that of the negative control, which showed the most intense blue color because of the inverse relationship between toxin concentration and color development. A portable colorimeter was used to confirm and quantify the visual observations. An ochratoxin A concentration of 0.4 ng/ml in buffer solution suppressed the color development completely. With the use of a simple sample preparation procedure it was possible to eliminate matrix interference. A wheat sample spiked with 4 microg/kg resulted in a complete color suppression. With coated membranes, the immunoassay could be performed in less than 15 min.

  17. Determination of atrazine in rainfall and surface water by enzyme immunoassay

    USGS Publications Warehouse

    Dankwardt, Andrea; Wüst, Susanne; Elling, Wolfram; Thurman, E. Michael; Hock, Bertold

    1994-01-01

    Rainwater and surface water from four sites in Germany (Bavaria and Lower Saxony) were analyzed for atrazine by enzyme immunoassay from June 1990 until October 1992. The limit of quantification of the immunoassay was 0.02 μg/L with a middle of the test at 0.2 μg/L. About 60 % of the samples contained measurable amounts of atrazine. Seasonal trends were observed, with the highest concentration in the summer months of up to 4 μg/L for rainwater and up to 15 μg/L for surface waters. The highest concentrations were found in agricultural areas, while in the investigated national parks up to 0.56 μg/L could be detected in rain water. This points to long-range atmospheric transport from agricultural areas to pristine national parks. Samples from forest stands usually showed higher atrazine concentrations than samples from open fields. Deposition rates of 10 – 50 μg/m2 · yr were observed in the national parks and 10–180 μg/m2 · yr at the agricultural sites. Comparison of results obtained by enzyme immunoassay and GC/MS showed a good correlation of r = 0.95.

  18. A competitive chemiluminescence enzyme immunoassay for rapid and sensitive determination of enrofloxacin

    NASA Astrophysics Data System (ADS)

    Yu, Fei; Wu, Yongjun; Yu, Songcheng; Zhang, Huili; Zhang, Hongquan; Qu, Lingbo; Harrington, Peter de B.

    With alkaline phosphatase (ALP)-adamantane (AMPPD) system as the chemiluminescence (CL) detection system, a highly sensitive, specific and simple competitive chemiluminescence enzyme immunoassay (CLEIA) was developed for the measurement of enrofloxacin (ENR). The physicochemical parameters, such as the chemiluminescent assay mediums, the dilution buffer of ENR-McAb, the volume of dilution buffer, the monoclonal antibody concentration, the incubation time, and other relevant variables of the immunoassay have been optimized. Under the optimal conditions, the detection linear range of 350-1000 pg/mL and the detection limit of 0.24 ng/mL were provided by the proposed method. The relative standard deviations were less than 15% for both intra and inter-assay precision. This method has been successfully applied to determine ENR in spiked samples with the recovery of 103%-96%. It showed that CLEIA was a good potential method in the analysis of residues of veterinary drugs after treatment of related diseases.

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

    PubMed

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

    2014-06-17

    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.

  20. Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies.

    PubMed

    Yoshimoto, T; Magata, K; Ehara, H; Mizuno, K; Yamamoto, S

    1986-06-11

    Prostaglandin endoperoxide synthase transforms arachidonic acid to prostaglandin H2 via prostaglandin G2. The enzyme purified from bovine vesicular gland was given to mice as antigen, and monoclonal antibodies were raised by the hybridoma technique. Two species of the monoclonal antibody recognizing different sites of the enzyme were utilized to establish a peroxidase-linked immunoassay of prostaglandin endoperoxide synthase. Fab' fragment of one of the antibodies was prepared and conjugated to horseradish peroxidase. The conjugate was then bound to prostaglandin endoperoxide synthase, and the labeled enzyme was precipitated by the addition of the other antibody. The peroxidase activity of the immunoprecipitate correlated linearly with the amount of prostaglandin endoperoxide synthase. This sensitive and convenient method to determine the enzyme amount rather than the enzyme activity was utilized to extensively screen the amount of prostaglandin endoperoxide synthase in various bovine tissues. In addition to vesicular gland, platelets and kidney medulla previously known as rich enzyme sources, the immunoenzymometric assay demonstrated a high content of the enzyme in various parts of alimentary tract and a low but significant amount of enzyme in some parts of brain.

  1. Open-sandwich enzyme immunoassay for one-step noncompetitive detection of corticosteroid 11-deoxycortisol.

    PubMed

    Ihara, Masaki; Suzuki, Tatsuya; Kobayashi, Norihiro; Goto, Junichi; Ueda, Hiroshi

    2009-10-15

    A noncompetitive immunoassay has the potential for improved sensitivity and working range compared with corresponding competitive assays. However, monovalent antigens with less than 1000 in molecular weight are not susceptible to sandwich assays due to their small size. As a noncompetitive immunoassay that can be performed with a clone of an antibody, an open-sandwich immunoassay (OS-IA) based on the antigen-dependent stabilization of the antibody variable region (V(H) + V(L)) was applied to the quantification of 11-deoxycortisol (11-DC; M(r) 346.5), a corticosteroid serving as a diagnostic index for pituitary-adrenal function, as a model target hapten. By one step OS-IA detection of enzyme-labeled V(H) fragment bound to immobilized V(L) in the presence of sample in microplate wells, 11-DC was measured with a femtomolar detection limit and the working range was wider than that with corresponding competitive assay. In addition, the selectivity against analogues was found almost identical to that of conventional assays. The effect of the mutagenesis of a V(H) residue at the V(H)/V(L) interface to reduce background signal was also shown, implying the wider application of OS-IA in small molecule analyses.

  2. [Detection of IgE specific for egg yolk by enzyme immunoassay. Description of a case].

    PubMed

    Carrillo Díaz, T; Cuevas Agustín, M; Moneo Goiri, I; Ibáñez Sandín, M D; Ureña Vilardell, V

    1986-01-01

    Food allergy is a common disease in our country, especially affecting atopic children. Egg-white hypersensitivity is frequently found in these patients. However, egg-yolk hypersensitivity is not usually reported in patients with egg allergy. This article describes a young patient with egg-yolk hypersensitivity, a 12 year old female patient with a medical history of contact urticaria, angioedema and severe acute bronchospasm shortly after the intake of small amounts of egg-yolk. All these episodes required treatment in emergency care units because of the severity of the symptoms. The patient did not describe any other food hypersensitivity and remained symptom-free after the intake of boiled or fried egg-white. She had clinical symptoms of grass pollen hypersensitivity and was therefore on specific immunotherapy at the time of the study. The skin prick-tests were positive to grass pollen and egg-yolk and were negative to mites, moulds, animal dander and to the common food tested (milk, fish, peanut, almond and hazel-nut). Total serum IgE was 1.160 UL/ml. The patient had a positive RAST to egg-white (0.0 PRU/ml) as well as to egg-yolk (8.6 PRU/ml). Furthermore, an indirect enzyme immunoassay as well as a reverse enzyme immunoassay also revealed the presence of specific IgE antibodies. The reverse enzyme immunoassay uses microtiter plates as a solid surface. These plates are coated with a monospecific antihuman IgE antibody. Thereafter, the serum samples are incubated overnight in the wells. After several washings, the presence of specific antibodies is revealed by means of a peroxidase conjugated allergen.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Immunocapture-based fluorometric assay for the measurement of insulin-degrading enzyme activity in brain tissue homogenates.

    PubMed

    Miners, James Scott; Kehoe, Patrick Gavin; Love, Seth

    2008-03-30

    Internally quenched fluorogenic substrates are commonly used for measuring enzyme activity in biological samples and allow high sensitivity and continuous real-time measurement that is well suited for high throughput analysis. We describe the development and optimisation of an immunocapture-based assay that uses the fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)) and allows the specific measurement of insulin-degrading enzyme (IDE) activity in brain tissue homogenates. This fluorogenic substrate can be cleaved by a number of enzymes including neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1) and angiotensin-converting enzyme (ACE), as well as IDE, and we have previously shown that discrimination between these individual enzymes is not readily achieved in tissue homogenates, even in the presence of selective inhibitors and pH conditions. We tested a panel of IDE antibodies to isolate and capture IDE from brain tissue homogenates and found that immunocapture with antibody to the inactive domain of IDE prior to the addition of fluorogenic substrate allows sensitive (linear at 156-2500ng/ml) and specific measurement of IDE activity and negligible cross-reactivity with NEP, ACE or ECE-1. This assay should allow the measurement of IDE enzyme levels in a variety of biological tissues and may be useful in study of diseases such as Alzheimer's disease and insulin-dependent diabetes.

  4. Application of magnetic nanoparticles in full-automated chemiluminescent enzyme immunoassay

    NASA Astrophysics Data System (ADS)

    Xie, Xiaomao; Ohnishi, Noriyuki; Takahashi, Yuki; Kondo, Akihiko

    2009-05-01

    The magnetic nanoparticles (MNPs) Therma-Max™ were used as a carrier to develop an automated sandwich chemiluminescent enzyme immunoassay (CLEIA) to detect thyroid-stimulating hormone (TSH) in a sensitive and specific way. The Therma-Max™ particles allow for automation because, unlike magnetic microspheres, they are completely dispersed in aqueous solution and allow for accurate automatic handling. Signal intensities detected with MNPs were 8-fold higher than those found with conventional micron-sized magnetic particles. A reproducibility study suggests that these particles allow for a stable detection method, as the coefficient of variation (CV) is less than 6% ( n=10).

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

    NASA Technical Reports Server (NTRS)

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

    1987-01-01

    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.

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

    PubMed

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

    2008-10-01

    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.

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

    NASA Technical Reports Server (NTRS)

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

    1987-01-01

    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.

  8. Comparison of Enzyme Immunoassays for Detection of Antibodies to Hepatitis D Virus in Serum

    PubMed Central

    Chow, Siu-Kei; Atienza, Ederlyn E.; Cook, Linda; Prince, Harry; Slev, Patricia; Lapé-Nixon, Mary

    2016-01-01

    Serology remains critical for diagnosing hepatitis D virus (HDV) infection, which affects 15 to 20 million people worldwide, but the literature on characterizing commercial enzyme immunoassays (EIAs) dates back to 15 years ago. We evaluated 2 commercial EIAs currently available for detecting anti-HDV antibodies. The DiaSorin assay demonstrated 100% sensitivity and specificity. Using a modified cutoff value, the Cusabio assay demonstrated a sensitivity of 81.3% and specificity of 90.9%. Our data show that recently developed EIAs are reliable for anti-HDV antibody detection. PMID:27280621

  9. Determination of thiabendazole in fruit juices by a new monoclonal enzyme immunoassay.

    PubMed

    Abad, A; Manclús, J J; Moreno, M J; Montoya, A

    2001-01-01

    A competitive, indirect enzyme-linked immunosorbent assay (ELISA) for thiabendazole has been developed and applied to the analysis of fruit juices spiked with this fungicide. The immunoassay is based on a new monoclonal antibody derived from a hapten functionalized at the nitrogen atom in the 1-position of the thiabendazole structure. To our knowledge, such a structure has not been previously used to obtain antibodies to thiabendazole. The I50 value and the detection limit of the ELISA for standards were 0.2 and 0.05 ng/mL, respectively. Fruit juices were analyzed by diluting samples in assay buffer, without extraction or cleanup. Samples were not even centrifuged or filtered to remove fruit pulp. Under these conditions, the immunoassay was able to accurately determine thiabendazole down to 1 ng/mL in orange and grapefruit juices, down to 5 ng/mL in banana juice, and down to 20 ng/mL in apple and pear juices. Sensitivity differences of the ELISA were caused by the minimum dilution required by each juice to minimize matrix effects: 1/10 for orange and grapefruit juices, 1/50 for banana juice, and 1/100 for apple and pear juices. In an attempt to further increase the sensitivity of the immunoassay for matrixes showing the strongest interferences, apple and pear juices spiked with thiabendazole at low levels (1-20 ng/mL) were extracted with ethyl acetate before analysis. This simple procedure entailed a significant reduction of matrix effects, which in fact allowed us to determine accurately as low as 5 ng/mL thiabendazole in apple and pear juices. Irrespective of whether samples were analyzed by the direct dilution method or after extraction, the simplicity, sensitivity, and sample throughput of this monoclonal immunoassay makes it a very convenient method for the routine monitoring of thiabendazole residues in fruit juices.

  10. Development of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spots.

    PubMed

    Ullal, Anirudh J; Millington, David S; Bali, Deeksha S

    2014-01-01

    Mucopolysaccharidosis type IVA or Morquio type-A disease is a hereditary lysosomal storage disorder caused by deficient activity of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The disease is caused by lysosomal accumulation of unprocessed glycosaminoglycans (GAGs) that manifests with severe to mild skeletal and cardiopulmonary abnormalities. We have developed a modified microtiter plate-based enzyme activity assay using dried blood spots and a fluorescent substrate for measuring specific GALNS activity to identify patients with MPS IVA.

  11. A rapid fluorometric assay for the S-malonyltransacylase FabD and other sulfhydryl utilizing enzymes.

    PubMed

    Marcella, Aaron M; Barb, Adam W

    The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist. Here we present two variations of a fluorescence assay that is readily extendable to high-throughput screening and is appropriate for thiol consuming and generating enzymes including the Escherichia coli enzymes malonyl-coenzyme A transacylase (FabD) and keto-acylsynthase III (FabH). We measured KM values of 60 ± 20 µM (acetyl-CoA) and 20 ± 10 µM (malonyl-ACP) and a kcat of 7.4-9.0 s(-1) with FabH. Assays of FabD included a precipitation step to remove the thiol-containing substrate holo-ACP from the reaction product coenzyme-A to estimate reaction rates. Analysis of initial velocity measurements revealed KM values of 60 ± 20 µM (malonyl-CoA) and 40 ± 10 µM (holo-ACP) and a kcat of 2100-2600 s(-1)for the FabD enzyme. Our data show similar results when compared to existing radioactive and continuous coupled assays in terms of sensitivity while providing the benefit of simplicity, scalability and repeatability. Fluorescence detection also eliminates the need for radioactive substrates traditionally used to assay these enzymes.

  12. The management of isolated positive syphilis enzyme immunoassay results in HIV-negative patients attending a sexual health clinic.

    PubMed

    Thorley, Nicola; Adebayo, Michael; Smit, Erasmus; Radcliffe, Keith

    2016-08-01

    An unconfirmed positive treponemal enzyme immunoassay (enzyme immunoassay positive, Treponema pallidum particle agglutination negative and rapid plasma reagin negative) presents a clinical challenge to distinguish early syphilis infection from false-positive results. These cases are referred for syphilis line assay (INNO-LIA) and recalled for repeat syphilis serology. We performed a retrospective audit to establish the proportion of HIV-negative cases with unconfirmed positive enzyme immunoassay results, the proportion of these cases that received an INNO-LIA test and repeat syphilis serology testing and reviewed the clinical outcomes; 0.35% (80/22687) cases had an unconfirmed positive treponemal enzyme immunoassay result. Repeat syphilis serology was performed in 80% (64/80) cases, but no additional cases of syphilis were identified. Eighty-eight per cent (70/80) received an INNO-LIA test; 14% (5/37) unconfirmed enzyme immunoassay-positive cases with no prior history of syphilis were confirmed on INNO-LIA assay, supporting a diagnosis of latent syphilis. As a confirmatory treponemal test, the INNO-LIA assay may be more useful than repeat syphilis serological testing. © The Author(s) 2016.

  13. Stool diagnosis of giardiasis using a commercially available enzyme immunoassay to detect Giardia-specific antigen 65 (GSA 65).

    PubMed

    Rosoff, J D; Sanders, C A; Sonnad, S S; De Lay, P R; Hadley, W K; Vincenzi, F F; Yajko, D M; O'Hanley, P D

    1989-09-01

    A commercially available enzyme immunoassay for the diagnosis of giardiasis was evaluated in a clinical trial. The ProSpecT/Giardia diagnostic test (Alexon, Inc., Mountain View, Calif.) was compared with the standard ova and parasite (O&P) microscopic examination. Additionally, several widely used stool fixatives and a commonly used transport medium were assessed for compatibility with the immunoassay. A total of 325 stool specimens were collected and used to evaluate assay performance. Of those, 93 specimens were collected from symptomatic Giardia O&P-positive patients and 232 specimens were randomly collected from patients as part of a routine health screening procedure. All 93 Giardia O&P-positive stool specimens were strongly positive by visual and spectrophotometric examination using the immunoassay. Of the 232 randomly collected specimens, 16 were positive by O&P examination and immunoassay, 6 were negative by O&P examination but positive by immunoassay, and 1 was positive by O&P examination and negative by immunoassay. There was substantial supportive evidence that indicated that the six immunoassay-positive, O&P-negative specimens were true-positives. When these six specimens were accepted as true-positives, the immunoassay detected almost 30% more cases of Giardia infection than did O&P examination. Its sensitivity and specificity were 96 and 100%, respectively, while the sensitivity and specificity of O&P examination were 74 and 100%, respectively. The immunoassay also performed well on specimens treated with 10% neutral Formalin, sodium acetate-Formalin fixative, and Cary-Blair transport medium. However, the test was not compatible with polyvinyl alcohol-treated specimens. Overall, the ProSpecT/Giardia test was a sensitive, specific immunoassay which was easy to run and interpret. It offers a simple solution to traditional difficulties encountered in diagnosing Giardia infection.

  14. Rapid diagnosis of rabies in humans and animals by a dot blot enzyme immunoassay.

    PubMed

    Madhusudana, Shampur Narayan; Paul, Joel Prem Vasanth; Abhilash, Venugopal Karavattu; Suja, Mooriyath Sukumaran

    2004-11-01

    The presently advocated tests for rapid diagnosis of rabies, such as the fluorescent antibody test (FAT) are expensive and require expertise to carry out and interpret the results. In this study, a simple direct dot blot enzyme immunoassay (DIA) has been developed and evaluated to detect the rabies antigen in brain specimens of animals and humans. The utility of this test in the ante-mortem diagnosis of human rabies has also been evaluated. Brain homogenates of suspected rabid animals (n = 250), humans (n = 16) and clinical samples like saliva (n = 12) and cerebrospinal fluid (CSF) (n = 12) were directly spotted on polyvinylidene difluoride membrane (PVDF) and the absorbed rabies nucleoprotein antigen was detected using biotinylated antinucleoprotein antibody followed by treatment with streptavidin peroxidase conjugate and color development with diamino benzedine (DAB). Rabies-infected and normal mouse brain homogenates were used as positive and negative controls, respectively. The results of this test were evaluated with fluorescent antibody technique (for brain samples) and mouse inoculation test (for saliva and CSF samples). A distinct dark brown color was seen in the positive control and all positive samples, while there was no color development with either the negative control or the negative samples. The concordance between the fluorescent antibody test (FAT) and dot immunoassay was 98.4% for brain samples, 83.3% for saliva and 91.6% for CSF samples. The specificity of the test was found to be 100%. The dot blot enzyme immunoassay (DIA) test described here is a sensitive, specific and rapid test for the post-mortem diagnosis of rabies in animals and humans. The utility of this test for the ante-mortem diagnosis of rabies needs to be further evaluated.

  15. Development of a fluorometric microtiter plate-based enzyme assay for arylsulfatase B (MPS VI) using dried blood spots.

    PubMed

    Ullal, Anirudh J; Millington, David S; Bali, Deeksha S

    2014-01-01

    Mucopolysaccharidosis type VI or Maroteaux-Lamy syndrome is an autosomal recessive lysosomal storage disorder caused by deficiency of arylsulfatase B (ARS-B) enzyme activity. It results in mild to severe multi-organ system failure from accumulation of undigested glycosaminoglycans (GAGs); dermatan sulfate and chondroitin-4-sulfate. We have developed a single-step enzyme assay using a fluorescent substrate and dried blood spots to measure ARS-B activity to identify disease patients. This assay is robust, reproducible, specific and convenient to perform.

  16. Monoclonal antibody capture enzyme immunoassay for detection of Paracoccidioides brasiliensis antibodies in paracoccidioidomycosis.

    PubMed Central

    Camargo, Z P; Gesztesi, J L; Saraiva, E C; Taborda, C P; Vicentini, A P; Lopes, J D

    1994-01-01

    Four murine monoclonal antibodies (MAbs 17C, 21A, 21F, and 32B) raised against the 43-kDa glycoprotein of Paracoccidioides brasiliensis were tested in a capture enzyme immunoassay (EIA) for the detection of specific human anti-gp43 immunoglobulin G in patients with paracoccidioidomycosis (PCM). All MAbs reacted similarly in the assay. These MAbs, which detected anti-gp43 at levels of as low as 500 pg/ml, were demonstrated to specifically recognize at least two different epitopes in gp43 binding assays. Specific antibodies in the sera of patients with active PCM were detected at dilutions of as high as 1:819,200, and the reactivities of patient sera, as measured by optical densities, were found to be significantly higher than those of control sera. The comparison between classical ELISA and our capture enzyme immunoassay showed that both sensitivity and specificity were greatly improved by the latter. These MAbs represent the first specific reagents to P. brasiliensis described for use in serological tests for PCM. Images PMID:7814469

  17. Activity monitoring and an enzyme immunoassay for milk progesterone to aid in the detection of estrus.

    PubMed

    Moore, A S; Spahr, S L

    1991-11-01

    The accuracy and efficiency of estrus detection using an electronic activity monitor tag in conjunction with an enzyme immunoassay for milk progesterone were studied during 55 observed estrous periods in 37 cows. At approximately 30 d postpartum, cows were equipped with an activity tag and visually observed for estrus, an activity tag with a flashing light-emitting diode, or both twice daily. Milk progesterone concentrations were determined from cows observed in estrus or with an activated tag. Mean daily activity was greater on the day of estrus than during any of the 3 d preceding or 3 d following estrus. Functioning activity tags correctly identified 55% of all visually observed estrous periods that coincided with low milk progesterone levels. The overall accuracy of a flag by the tag for identifying true estrus was 21%. The enzyme immunoassay for milk progesterone was in agreement with 98% of all visually observed estrous periods and false flags committed by the tags. Although the activity tags detected some cows in estrus, a more durable and reliable tag must be developed before it is of practical value to dairy producers.

  18. Dipstick enzyme immunoassay to detect Fusarium T-2 toxin in wheat.

    PubMed Central

    De Saeger, S; Van Peteghem, C

    1996-01-01

    A dipstick enzyme immunoassay for the rapid detection of Fusarium T-2 toxin in wheat was developed. An Immunodyne ABC membrane was precoated with rabbit anti-mouse immunoglobulins. After the strips were immersed in a solution of monoclonal anti-T-2 toxin antibodies, a direct competitive enzyme immunoassay was performed. This assay included the incubation of the antibody-coated dipsticks in a mixture of sample and T-2 toxin-horseradish peroxidase conjugate. Afterwards, the strips were placed in a chromogen-containing substrate solution (H202-3,3',5,5'-tetramethylbenzidine) for color reaction. The dot color intensity of toxin-positive dipsticks was visually distinguishable from that of the negative control. A portable colorimeter was used to confirm and quantify the visual observations. With coated strips, the tests could be performed in 45 min. The visual detection limit for T-2 toxin in buffer solution was 0.25 ng/ml. Artificially infected wheat samples were extracted with 80% methanol-water. A dilution of the raw extract of 1:8 was sufficient to avoid matrix effects. It was possible to make visually a clear distinction between the negative control and a wheat extract spiked with 12 ng/g. PMID:8787386

  19. Monoclonal enzyme immunoassay for the analysis of carbaryl in fruits and vegetables without sample cleanup.

    PubMed

    Abad, A; Moreno, M J; Pelegrí, R; Martínez, M I; Sáez, A; Gamón, M; Montoya, A

    2001-04-01

    The N-methylcarbamate pesticide carbaryl is one of the most important insecticides used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) for the determination of this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 101.0 +/- 26.9 ng/L and with a dynamic range between 31.6 and 364.0 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbaryl at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on both extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop and the fortification level, recoveries in the 59.0--120.0% range were obtained for purified samples and in the 70.0--137.7% range for crude extracts. The carbaryl immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbaryl at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was excellent (y = 1.04x + 0.71, r(2) = 0.992, n = 33), with HPLC being more precise than ELISA (mean coefficients of variation of 5.2 and 12.0%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.28x - 0.59, r(2) = 0.987, n = 33) while maintaining similar precision. Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbaryl in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases sample throughput.

  20. Comparative studies with penicillinase, horseradish peroxidase, and alkaline phosphatase as enzyme labels in developing enzyme immunoassay of cortisol.

    PubMed

    Kumari, G Lakshmi; Dhir, Ravindra N

    2003-01-01

    Relative merit of different enzyme labels for measuring cortisol directly in serum by competitive enzyme immunoassay (ELISA) was examined. Cortisol-21-hemisuccinate was labeled separately with penicillinase, horseradish peroxidase (HRP), and alkaline phosphatase (ALP) under identical reaction conditions. Antibody developed in rabbits against cortisol-3-0-(carboxymethyl)-oxime-bovine serum albumin was used to coat polystyrene tubes that were precoated with anti-rabbit gamma globulin (ARGG). Cortisol standards were prepared in steroid-free human serum in buffer (1:4) contaning 8-anilino-1-naphthalene sulfonic acid (8-ANS). Assay buffer also consisted 8-ANS. The assay involved adding standard cortisol or serum sample to antibody-coated tubes, followed by addition of enzyme label and buffer, and incubation for 2 h at 37 degrees C. The whole procedure took 3 h for completion. All three labels proved to be sensitive, with a slope around -2.0. Although penicillinase as an enzyme label was highly sensitive and stable compared with others, the assays were not always accurate and precise, especially at low concentrations of cortisol. This was mainly due to the color reagent used for measuring penicillinase activity. Serum samples that underwent 2-3 freeze-thaw cycles gave high values with HRP label compared with ALP. Therefore, utilizing ALP as an enzyme label, an ELISA was developed and its performance was comparable with some of the commercial kits already in the market.

  1. Mesoporous carbon-enriched palladium nanostructures with redox activity for enzyme-free electrochemical immunoassay of brevetoxin B.

    PubMed

    Lin, Youxiu; Zhou, Qian; Lin, Yuping; Lu, Minghua; Tang, Dianping

    2015-08-05

    A new signal amplification strategy based on mesoporous carbon-enriched palladium nanostructure (MSC-PdNS) was designed for enzyme-free electrochemical immunoassay of brevetoxin B (BTB) in marine toxins. The assay was carried out on a BTB-bovine serum albumin-functionalized electrode by using monoclonal mouse anti-BTB-labeling MSC-PdNS as the signal-transduction tag. A competitive-type assay protocol was successfully introduced to develop a high-efficiency enzyme-free immunoassay accompanying the doped palladium nanostructure into MSC-PdNS toward reduction of H2O2. Under the optimal conditions, the catalytic current decreased with the increment of BTB concentration in the range from 0.01 to 10 ng mL(-1) with a detection limit (LOD) of 5.0 pg mL(-1) BTB at the 3s(blank) criterion. The selectivity and precision were acceptable. In addition, the methodology was further validated for assaying spiked seafood samples, and consistent results between the electrochemical immunoassay and the referenced enzyme immunoassay were obtained. Importantly, the enzyme-free electrochemical immunoassay provides a promising approach for rapid screening of marine toxin because of its simplicity, low cost, sensitivity, specificity and without the need of sample pretreatment.

  2. A simple competitive enzyme immunoassay for the detection of the trypanocidal drug isometamidium.

    PubMed

    Eisler, M C; Elliott, C T; Holmes, P H

    1996-02-01

    A new competitive enzyme immunoassay technique has been developed for the determination of concentrations of the trypanocidal drug isometamidium chloride (Samorin) in bovine serum. The method has been shown to be highly repeatable and reproducible, and it has several advantages over previous immunoassay techniques for the drug. There are fewer incubation steps overall; microtitre plates may be of coated in batches and stored frozen for future use; and the competition incubation is overnight and is followed only by a brief colour development stage of 10 min. Coefficients of variation (CVs) of duplicate samples were approximately 5%, and mean response variances of untreated cattle (n = 57) were small (CV, 10%). Partitioning of variance showed 77% of this variability to be intrinsic to the samples, and the remaining 23% was due to the procedure. The limit of detection was approximately 0.5 ng ml-1, which was considered to be satisfactory for the intended use of the method. The drug could be detected in serum of treated cattle for up to 10 weeks following treatment, and determinations showed a high level of reproducibility.

  3. A competitive enzyme immunoassay for the quantitative detection of cocaine from banknotes and latent fingermarks.

    PubMed

    van der Heide, Susan; Garcia Calavia, Paula; Hardwick, Sheila; Hudson, Simon; Wolff, Kim; Russell, David A

    2015-05-01

    A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162ngmL(-1) achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrate the effectiveness and robustness of the assay. The immunoassay presents a simple and cost

  4. A model system for a fluorometric biosensor using permeabilized Zymomonas mobilis or enzymes with protein confined dinucleotides

    SciTech Connect

    Thordsen, O.; Lee, S.J.; Degelau, A. ); Scheper, T. ); Loos, H.; Rehr, B.; Sahm, H. )

    1993-07-01

    Using permeabilized Zymomonas mobilis or glucose-fructose oxidoreductase isolated from this microorganism a model system for biosensors with a protein confined NADP(H) cofactor for the determination of glucose, fructose, gluconolactone, and sorbitol was developed. Either permeabilized microorganisms containing the oxidoreductase or the pure enzyme were confined via membrane separation in a small measuring chamber, that was integrated into a flow injection analysis system (FIA). The measuring principle was the monitoring of the NAD(P)H fluorescence, excited at 360 nm and measured at 450 nm. NADP(H), which is confined in the protein complex, was oxidized or reduced during the enzymatic reactions and the changes in the fluorescence intensity were related to the substrate concentration. The sensitivity of the system covered a range from 0.001 to 100 g/L of the analyte depending on substrate and operating conditions. The applicability of this model system for bioprocess monitoring was proved using samples from a Pseudomonas pseudoflava cultivation.

  5. Detection of Pesticides and Pesticide Metabolites Using the Cross Reactivity of Enzyme Immunoassays

    USGS Publications Warehouse

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

    2001-01-01

    Enzyme immunoassay is an important environmental analysis method that may be used to identify many pesticide analytes in water samples. Because of similarities in chemical structure between various members of a pesticide class, there often may be an unwanted response that is characterized by a percentage of cross reactivity. Also, there may be cross reactivity caused by degradation products of the target analyte that may be present in the sample. In this paper, the concept of cross reactivity caused by degradation products or by nontarget analytes is explored as a tool for identification of metabolites or structurally similar compounds not previously known to be present in water samples. Two examples are examined in this paper from various water quality studies. They are alachlor and its metabolite, alachlor ethane sulfonic acid, and atrazine and its class members, prometryn and propazine. A method for using cross reactivity for the detection of these compounds is explained in this paper.

  6. The return of postpartum fertility monitored by enzyme-immunoassay for salivary progesterone.

    PubMed

    Bolaji, I I; Tallon, D F; Meehan, F P; O'Dwyer, E M; Fottrell, P F

    1992-03-01

    A longitudinal study in which daily salivary progesterone and estrone were measured by solid-phase enzyme-immunoassays was performed in 30 postpartum women to monitor the return of ovarian activity. Ovulation was inferred from a sustained rise in salivary progesterone over 251 pmol/l, but salivary estrone measurements were not as informative as progesterone in this regard. Recovery of ovarian activity was slower in lactating women compared with non-lactators; the mean delivery-menstruation interval were 123 (+/- 10) and 57 (+/- 7) days, respectively. An abnormal luteal phase was noted in 35% of the first ovulatory cycles, 20% had short luteal phases and 15% were less than the 5th percentile of a normal control corridor. The pregnancy rate in this study of 3.3% was lower than the anticipated rate of 8.8%. We conclude that salivary progesterone measurements are useful for monitoring the return of ovarian activity postnatally.

  7. Dot enzyme immunoassay: an alternative diagnostic aid for dengue fever and dengue haemorrhagic fever.

    PubMed Central

    Cardosa, M. J.; Tio, P. H.

    1991-01-01

    A dot enzyme immunoassay (DEIA) for the detection of antibodies to dengue virus was tested for use as a tool in the presumptive diagnosis of dengue fever and dengue haemorrhagic fever. Paired sera from the following groups of patients were tested using the DEIA and the haemagglutination inhibition (HI) test: those with primary dengue fever; those experiencing a second dengue infection; and febrile patients who did not have dengue. The data obtained show that the DEIA can be effectively used at a serum dilution of 1:1000 to confirm presumptive recent dengue in patients with a second dengue infection. However, demonstration of seroconversion proved necessary for patients with primary dengue. At a serum dilution of 1:1000 the DEIA has a specificity of 97.3%. The role of this simple and rapid test in improving the effectivity of programmes for the control of dengue virus infection is discussed. PMID:1786623

  8. Detection of pesticides and pesticide metabolites using the cross reactivity of enzyme immunoassays.

    PubMed

    Thurman, E M; Aga, D S

    2001-01-01

    Enzyme immunoassay is an important environmental analysis method that may be used to identify many pesticide analytes in water samples. Because of similarities in chemical structure between various members of a pesticide class, there often may be an unwanted response that is characterized by a percentage of cross reactivity. Also, there may be cross reactivity caused by degradation products of the target analyte that may be present in the sample. In this paper, the concept of cross reactivity caused by degradation products or by nontarget analytes is explored as a tool for identification of metabolites or structurally similar compounds not previously known to be present in water samples. Two examples are examined in this paper from various water quality studies. They are alachlor and its metabolite, alachlor ethane sulfonic acid, and atrazine and its class members, prometryn and propazine. A method for using cross reactivity for the detection of these compounds is explained in this paper.

  9. Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate.

    PubMed

    Tao, Xiaoqi; Wang, Wenjun; Wang, Zhanhui; Cao, Xingyuan; Zhu, Jinghui; Niu, Lanlan; Wu, Xiaoping; Jiang, Haiyang; Shen, Jianzhong

    2014-06-01

    In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N-azoles as secondary enhancer was also presented.

  10. Enzyme immunoassays as a method for quantifying hair reproductive hormones in two felid species

    PubMed Central

    Terwissen, C. V.; Mastromonaco, G. F.; Murray, D. L.

    2014-01-01

    Non-invasive monitoring of wild felid reproductive states is important, given that many species reproduce poorly in captivity. Despite extensive work in faecal hormone analysis in felids, continued development of techniques is necessary, particularly with wild populations. In this study, our aims were as follows: (i) biochemical validation of enzyme immunoassays for estrogen, testosterone and progesterone in Canada lynx and domestic cat hair extracts; (ii) assessment of the use of hair reproductive hormones to differentiate between reproductive states (intact, estrus, pregnant and spayed/neutered), using domestic cats as a model; and (iii) assessment of the use of hair reproductive hormones to differentiate between age and sex, accounting for potential regional variability in wild lynx populations. Analysis of hair hormone levels showed prospective value in detecting pregnancy states, with pregnant domestic cats having higher levels of progesterone than spayed females. However, intact and pregnant cats did not differ in progesterone levels. Yet, two female domestic cats had higher levels of hair progesterone following a 38-day oral progestin treatment, perhaps providing a preliminary pharmacological validation of the method. Estrogen and testosterone did not differ statistically according to reproductive states of domestic cats, although intact males had higher levels of hair testosterone than neutered males. When we applied these techniques to lynx fur, we determined that hormone levels were not sufficiently precise to differentiate age classes. Hair reproductive hormone ratios differed between sexes, with the estrogen-to-progesterone ratio demonstrating the highest accuracy in differentiating males from females. Hair hormone levels differed regionally for wild lynx, indicating that spatial variability should be a consideration in wildlife hormone studies spanning large spatial scales. We conclude that use of hair hormone analysis by enzyme immunoassay may

  11. A class capture enzyme immunoassay for immunoglobulin level determinations in bovine sera.

    PubMed Central

    Nielsen, K; Rosenbaum, B; Stiller, J

    1985-01-01

    An enzyme immunoassay for determination of individual isotype concentrations in bovine serum was developed. Polystyrene tubes were coated with affinity purified goat antibovine IgA, IgG1, IgG2 or IgM, washed and then incubated with purified isotypes to ascertain crossreactivity and sensitivity limits. Bound isotype was detected using the homologous affinity purified antibody conjugated to horseradish peroxidase and hydrogen peroxide-2,2-azino-di-(3-ethylbenzthiazoline sulfonic acid), as the substrate/chromogen. A standardized serum was diluted and used as a control for comparison. Several dilutions were used initially, however, determinations may be made with a single dilution, 1:200, for all isotypes. Results for 100 sera were compared to data obtained with the same samples using a radial immunodiffusion technique. A low correlation coefficient was noted between results from the two assays. Day to day variation and within test repeatability were determined for both assays using ten samples. For the enzyme immunoassay method, day to day variation for IgA, IgM, IgG1 and IgG2 determinations was 17.5, 19.3, 7.6 and 7.3% while variation in repeatability (within a test) was 6.2, 5.9, 3.3 and 4.5%, respectively. Day to day variation for the single radial immunodiffusion test for IgA, IgM, IgG1 and IgG2 was 15.4, 26.0, 11.5 and 18.3% and variation repeatability (within a test) was 11.6, 13.9, 5.9 and 8.3%, respectively. The procedures consistently detected 0.1 micrograms of immunoglobulin whereas the radial diffusion sensitivity limit was approximately 500 micrograms. PMID:3935300

  12. Cross-reactivity of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Cryptococcus species in the commercial Platelia Aspergillus enzyme immunoassay.

    PubMed

    Xavier, Melissa O; Pasqualotto, Alessandro C; Cardoso, Isabel Cristina E; Severo, Luiz Carlos

    2009-01-01

    Cross-reactivity in the Platelia Aspergillus enzyme immunoassay was evaluated using 120 sera from patients with paracoccidioidomycosis, histoplasmosis, and cryptococcosis. At a cutoff value of 0.5, positivity rates were 50%, 67%, and 50%, respectively. The implications for these findings are discussed.

  13. Pretreatment-free lateral flow enzyme immunoassay for progesterone detection in whole cows' milk.

    PubMed

    Samsonova, J V; Safronova, V A; Osipov, A P

    2015-01-01

    New rapid method of lateral flow enzyme immunoassay (LFEIA) for progesterone detection in whole cows' milk was developed. The test system utilized horseradish peroxidase as a label along with the substrate solution containing 3,3',5,5'-tetramethylbenzidine and dextran sulfate to obtain an insoluble blue colored product of the enzyme reaction on a surface of analytical membrane (test and control lines). Several aspects of LFEIA were optimized: time of the signal detection, membrane materials and assay conditions. Resulting competitive LFEIA can be performed within 15 minutes with the limit of progesterone detection of 0.8 ng/ml. Progesterone concentration in whole milk samples was determined by LFEIA and enzyme-linked immunosorbent assay (ELISA). The results obtained were in good correlation (R=0.97, n=46). Thus new sensitive LFEIA can be successfully used for on-site monitoring of oestrus status of cows' reproductive system and for early none-pregnancy detection. The method is fast, easy to perform and needs no preliminary sample preparation.

  14. Reverse enzyme immunoassay for detection of specific anti-Toxoplasma immunoglobulin M antibodies.

    PubMed Central

    Franco, E L; Walls, K W; Sulzer, A J

    1981-01-01

    A reverse enzyme immunoassay (R-EIA) is described, in which polystyrene muplates are sensitized with anti-immunoglobulin M (IgM) (mu chain) antibodies and then sequentially allowed to react with patient's serum, peroxidase-labeled Toxoplasma gondii soluble antigen, and substrate. Measurement of activity of the solid-phase bound enzyme conjugate was done by colorimetric reading of the final developed color and kinetically by the initial rate of color development. This R-EIA allowed full resolution between absorbance values of a group of 36 sera which presented positive results in the Toxoplasma IgM immunofluorescence test and the remaining groups, which consisted of 39 normal individuals, 22 rheumatoid factor-positive sera, 8 Waldenstrom's macroglobulinemic sera, 3 infectious mononucleosis samples, and 6 high-titered IgG anti-T. gondii sera. No interference of rheumatoid factor IgM or inhibition by high-titered specific IgG was detected, even in the false IgM immunofluorescence-positive rheumatoid factor samples. Likewise, false-negative IgM immunofluorescence samples gave positive R-EIA even without adsorption with Staphylococcus aureus protein A. The possibility of direct tagging of the antigen with the enzyme eliminates the need for using antigen and anti-antigen conjugates as separate layers, therefore eliminating one step in the assay. PMID:7016911

  15. Self-Assembly of Ferritin Nanoparticles into an Enzyme Nanocomposite with Tunable Size for Ultrasensitive Immunoassay.

    PubMed

    Men, Dong; Zhang, Ting-Ting; Hou, Li-Wei; Zhou, Juan; Zhang, Zhi-Ping; Shi, Yuan-Yuan; Zhang, Jin-Li; Cui, Zong-Qiang; Deng, Jiao-Yu; Wang, Dian-Bing; Zhang, Xian-En

    2015-11-24

    The self-assembly of nanoparticles into larger superstructures is a powerful strategy to develop novel functional nanomaterials, as these superstructures display collective properties that are different to those displayed by individual nanoparticles or bulk samples. However, there are increasing bottlenecks in terms of size control and multifunctionalization of nanoparticle assemblies. In this study, we developed a self-assembly strategy for construction of multifunctional nanoparticle assemblies of tunable size, through rational regulation of the number of self-assembling interaction sites on each nanoparticle. As proof-of-principle, a size-controlled enzyme nanocomposite (ENC) was constructed by self-assembly of streptavidin-labeled horseradish peroxidase (SA-HRP) and autobiotinylated ferritin nanoparticles (bFNP). Our ENC integrates a large number of enzyme molecules, together with a streptavidin-coated surface, allowing for a drastic increase in enzymatic signal when the SA is bound to a biotinylated target molecule. As result, a 10 000-fold increase in sensitivity over conventional enzyme-linked immunosorbent assays (ELISA) methods was achieved in a cardiac troponin immunoassay. Our method presented here should provide a feasible approach for constructing elaborate multifunctional superstructures of tunable size useful for a broad range of biomedical applications.

  16. Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads.

    PubMed

    Tang, Dianping; Su, Biling; Tang, Juan; Ren, Jingjing; Chen, Guonan

    2010-02-15

    A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method.

  17. Monoclonal antibody-based enzyme immunoassays for the sensitive detection of s-triazines in water

    NASA Astrophysics Data System (ADS)

    Hock, Bertold; Giersch, Thomas; Kramer, Karl-Josef

    1993-03-01

    Immunoassays in pesticide residue analysis significantly profit from the monoclonal antibody (mAb) technology because a sufficient supply of standardized antibodies can be provided. For the production of atrazine-specific mAbs hybridoma cells were produced by fusion of mouse myeloma cells and spleen cells from mice which were immunized with 4-chloro-6-ethylamino- 1,3,5-triazine-2-(6-aminohexanoic acid) coupled to keyhole limped hemocyanin. After screening with a competitive enzyme immunoassay (EIA) a mAb with high binding affinity towards atrazine was selected. A sensitive EIA was developed detecting atrazine with a range from 0.05 to 1 (mu) g/l with a test midpoint of 0.1 (mu) g/l. The mAb cross-reacts predominantly with propazine (136%). Since this herbicide is not used in most European countries, the test allows a rapid and inexpensive screening for atrazine in the ppt range. Another EIA has been constructed for the detection of terbuthylazine. The limiting factor in EIA development is the screening for cell lines secreting mAbs with high affinity and selectivity towards the analyte. Super paramagnetic beads being coated with suitable immonoconjugates are shown to bind to hybridomas presenting hapten-specific receptors on their surface. Hybridomas secreting hapten-specific mAbs can be removed by a magnet and be cloned subsequently by standard procedures. A considerable demand of mAbs is expected in the future due to new emerging techniques such as immunosensors.

  18. Cross-reactivity of tapentadol specimens with DRI methadone enzyme immunoassay.

    PubMed

    Collins, Ayodele A; Merritt, A Paola; Bourland, James A

    2012-10-01

    A substantial incidence of positive methadone screens for pain management urine specimens using a commercial enzyme immunoassay (EIA) was observed in the absence of a methadone prescription, with negative methadone confirmation by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS). Tapentadol was the only common prescription among the investigated specimens. Tapentadol or one of its three major metabolites was tested at various concentrations (100-200,000 ng/mL) against the DRI EIAs for methadone and methadone metabolite, to evaluate cross-reactivity. Ninety-seven authentic tapentadol urine specimens that produced false-positive methadone EIA results (cutoff = 130 ng/mL) were analyzed for methadone and tapentadol in compound-specific UPLC-MS-MS confirmation tests. Tapentadol, tapentadol glucuronide, tapentadol sulfate and N-desmethyltapentadol exhibited cross-reactivity with the methadone EIA at 6,500 (2.2%), 25,000 (0.6%), 3,000 (4.4%) and 20,000 ng/mL (0.9%), respectively. No cross-reactivity was observed with the methadone metabolite 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine EIA. All authentic urine specimens were confirmed to be negative for methadone, but positive for tapentadol and all monitored metabolites. Individual concentrations indicated that separate or combined urinary concentrations of tapentadol and its conjugates may produce false-positive methadone screens through cross-reactivity with the methadone immunoassay. The potential for false-positive results for methadone EIA screening of urine specimens associated with tapentadol prescriptions should be considered when interpreting results.

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

    PubMed

    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

    2013-11-15

    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. Evaluation of an Enzyme Immunoassay for Detection of Dengue Virus NS1 Antigen in Human Serum▿

    PubMed Central

    Dussart, Philippe; Labeau, Bhety; Lagathu, Gisèle; Louis, Philippe; Nunes, Marcio R. T.; Rodrigues, Sueli G.; Storck-Herrmann, Cécile; Cesaire, Raymond; Morvan, Jacques; Flamand, Marie; Baril, Laurence

    2006-01-01

    We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories. PMID:16988003

  1. Long-wavelength homogeneous enzyme immunoassay for the determination of amikacin in water samples.

    PubMed

    Sánchez-Martínez, M L; Aguilar-Caballos, M P; Gómez-Hens, A

    2009-04-15

    A simple and rapid homogeneous enzyme immunoassay involving the use of the malic dehydrogenase enzyme and a long-wavelength fluorophor, the oxazine Cresyl Violet, is proposed for the determination of the antibiotic amikacin in water samples. An enzymatic tracer has been synthesized by covalent binding of amikacin to malic dehydrogenase via a carbodiimide derivative. Free tracer catalyses the reaction between Cresyl Violet and malic acid giving rise to a decrease in the fluorescence of the fluorophor. Kinetic curves for this reaction have been monitored at lambda(ex) 585 and lambda(em) 624 nm using the stopped-flow mixing technique, being the initial rate measured in only 2-3s. The dynamic range of the method is 1-15 ng mL(-1) and the detection limit is 0.3 ng mL(-1), using aqueous standard solutions or water samples. The precision, obtained at 1 and 5 ng mL(-1) and expressed as relative standard deviation, was 6.0 and 9.6%, respectively. The method has been applied to the analysis of drinking, river and wastewater samples. The sample pre-treatment involved a solid-phase extraction step for the clean-up of the samples. A recovery study was carried out to validate the method, being the values obtained in the range 80-114%, with a mean value of 96.7%.

  2. Enzyme immunoassay for rat growth hormone: applications to the study of growth hormone variants

    SciTech Connect

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

    1987-06-29

    A sensitive and specific competitive enzyme immunoassay (EIA) for rat growth hormone was developed. In this assay soluble growth hormone and growth hormone adsorbed to a solid-phase support compete for monkey anti-growth hormone antibody binding sites. The immobilized antibody-growth hormone complex is detected and quantified using goat anti-monkey immunoglobin G covalently conjugated to horse radish peroxidase. Therefore, a high concentration of soluble growth hormone in the sample will result in low absorbance detection from the colored products of the enzyme reaction. Assay parameters were optimized by investigating the concentration of reagents and the reaction kinetics in each of the assay steps. The assay can be performed in 27 hours. A sensitivity range of 0.19 ng to 25 ng in the region of 10 to 90% binding was obtained. Near 50% binding (3 ng) the intraassay coefficient of variation (CV) was 5.54% and the interassay CV was 5.33%. The correlation coefficient (r/sup 2/) between radioimmunoassay and EIA was 0.956 and followed the curve Y = 0.78X + 1.0. 9 references, 6 figures.

  3. Accurate detection of Campylobacter spp. antigens by immunochromatography and enzyme immunoassay in routine microbiological laboratory.

    PubMed

    Regnath, Thomas; Ignatius, Ralf

    2014-09-01

    Campylobacter spp. are fastidious microorganisms, and their detection by culture depends on the freshness of the stool sample and the skills of the laboratory staff. To improve laboratory diagnosis, assays for the detection of specific antigens have been developed. Here, we evaluated two assays for the detection of Campylobacter spp.-specific antigens, i.e., one immunochromatographic test and one enzyme-linked immunosorbent assay (EIA), in 38 frozen Campylobacter spp.-positive specimens and prospectively in 533 fresh stool samples with a conventional enzyme immunoassay (EIA) and culture. Both assays were positive for 36 samples with Campylobacter jejuni and one with Campylobacter coli among 38 Campylobacter spp.-positive frozen samples. One Campylobacter lari-positive sample was identified by the immunochromatographic assay (ICA) only. In a prospective study performed within the course of routine microbiology, both assays were positive for 24/25 C. jejuni culture-positive samples (positive percent agreement, 96.0% [95% CI: 78.9-100%]). ICA and EIA also were positive for 14 and 10 culture-negative samples, respectively (negative percent agreement: ICA, 97.2% [95% CI: 95.4-98.4%]; EIA, 98.0% [95% CI: 96.4-99.0%]). In conclusion, the high agreement between both antigen-detection assays and culture indicates that both assays may be initially performed followed by culture only upon a positive test result.

  4. Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies.

    PubMed Central

    Märtlbauer, E; Gareis, M; Terplan, G

    1988-01-01

    Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay). PMID:3278686

  5. Enzyme-controlled dissolution of MnO2 nanoflakes with enzyme cascade amplification for colorimetric immunoassay.

    PubMed

    Lai, Wenqiang; Wei, Qiaohua; Xu, Mingdi; Zhuang, Junyang; Tang, Dianping

    2017-03-15

    A new colorimetric immunosensing platform accompanying enzyme cascade amplification strategy was fabricated for quantitative screening of small-molecular mycotoxins (aflatoxin B1, AFB1 used in this case) coupling with enzyme-controlled dissolution of MnO2 nanoflakes. The visual colored assay was executed by high-efficient MnO2-3,3',5,5'-tetramethylbenzidine (TMB) system (blue). In the presence of ascorbic acid, MnO2 nanoflakes were dissolved into Mn(2+) ions, thus resulting in a perceptible color change from blue to colorless. The reaction could be weakened through ascorbate oxidase to catalyze ascorbic acid into dehydroascorbic acid, which indirectly depended on the concentration of ascorbate oxidase. By using ascorbate oxidase/ anti-AFB1 antibody-labeled gold nanoparticles, a novel competitive-type colorimetric enzyme immunoassay was developed for detection of AFB1 on AFB1-bovine serum albumin (BSA)-conjugated magnetic beads. Upon addition of target AFB1, the analyte competed with the conjugated AFB1-BSA on the magnetic beads for the labeled anti-AFB1 antibody on the gold nanoparticles. Under optimal conditions, the absorbance decreased with increasing target AFB1 within the dynamic range of 0.05-150ngmL(-1) with a detection limit of 6.5pgmL(-1) at the 3Sblank level. The precision and specificity of the MnO2-TMB-based immunosensing system were acceptable. In addition, method accuracy was further validated for monitoring spiked peanut samples, giving results matched well with those obtained from commercialized AFB1 ELISA kit.

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

    PubMed

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

    2014-12-16

    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.

  7. Design of a single-step immunoassay principle based on the combination of an enzyme-labeled antibody release coating and a hydrogel copolymerized with a fluorescent enzyme substrate in a microfluidic capillary device.

    PubMed

    Wakayama, Hideki; Henares, Terence G; Jigawa, Kaede; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-11-21

    A combination of an enzyme-labeled antibody release coating and a novel fluorescent enzyme substrate-copolymerized hydrogel in a microchannel for a single-step, no-wash microfluidic immunoassay is demonstrated. This hydrogel discriminates the free enzyme-conjugated antibody from an antigen-enzyme-conjugated antibody immunocomplex based on the difference in molecular size. A selective and sensitive immunoassay, with 10-1000 ng mL(-1) linear range, is reported.

  8. 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.

    PubMed Central

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

    1985-01-01

    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. PMID:2985650

  9. 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.

    PubMed

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

    1985-04-01

    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.

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

    SciTech Connect

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

    1986-07-18

    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.

  11. Assessment of an enzyme immunoassay for the detection of salmonellas in foods and animal feeding stuffs.

    PubMed

    Todd, L S; Roberts, D; Bartholomew, B A; Gilbert, R J

    1987-06-01

    The Salmonella Bio-EnzaBead Screening Kit, in its modified form with both the MOPC 467 and the 6H4 antibodies, was used for the detection of salmonellas in naturally contaminated foods and animal feeding stuffs in parallel with a traditional cultural procedure. Initial results showed an 82% agreement between the enzyme immunoassay (EIA) and cultural methods when using the criterion recommended by the manufacturer as a cut-off for all types of foods. By adjusting the cut-off for each type of food, the number of EIA positive, culture negative samples was reduced although the number of EIA negative, culture positive samples increased. The EIA may be more sensitive than the cultural methods as in many cases the EIA positive, culture negative results could be real positives which were not detected by the cultural methods. The screening kit provides a simple and convenient method for the detection of salmonella in foods and feeds and a presumptive positive result can be reported within 48 h. The advantages and disadvantages of the method are discussed.

  12. Performance of plasma fractionated free metanephrines by enzyme immunoassay in the diagnosis of pheochromocytoma and paraganglioma.

    PubMed

    Sarathi, Vijaya; Pandit, Reshma; Jagtap, Varsha; Lila, Anurag R; Bandgar, Tushar R; Menon, Padma S; Varthakavi, Prema; Raghavan, Vijaya P; Shah, Nalini S

    2011-01-01

    To study the performance of measuring plasma fractionated free metanephrines by enzyme immunoassay (EIA) in the diagnosis of pheochromocytoma and catecholamine-secreting paraganglioma. Consecutive patients attending the endocrine clinic at King Edward Memorial Hospital, Mumbai, India, for suspicion of catecholamine-secreting tumors were included. Plasma fractionated free metanephrines were measured by EIA, and computed tomography of the neck, chest, abdomen, and pelvis was performed. Those with tumor identified by imaging underwent 131I m-iodobenzylguanidine scintigraphy. All patients with adrenal masses larger than 3 cm and patients with secretory tumors, irrespective of their size, underwent tumor excision. The rest were followed up for 6 to 12 months. One hundred patients with a clinical suspicion of pheochromocytoma or paraganglioma were included. Plasma free normetanephrine alone had a sensitivity of 94.1% (cutoff: 180 ng/mL), while plasma free metanephrine had a sensitivity of 14.7% (cutoff: 90 pg/mL). Both had 96.9% specificity. When combined (either test positive), the sensitivity was 94.1% with a specificity of 93.75%. Thirty-four patients had a histopathologically proven pheochromocytoma or paraganglioma. It was concluded that 66 patients did not harbor a pheochromocytoma or catecholamine-secreting paraganglioma. Plasma fractionated free metanephrines measured by EIA have good sensitivity and specificity in the diagnosis of pheochromocytoma and catecholamine-secreting paraganglioma.

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

    PubMed

    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

    2013-09-24

    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.

  14. The Rapid Screening of Triazophos Residues in Agricultural Products by Chemiluminescent Enzyme Immunoassay

    PubMed Central

    Chen, Ge; Yang, Lihua; Jin, Maojun; Du, Pengfei; Zhang, Chan; Wang, Jian; Shao, Hua; Jin, Fen; Zheng, Lufei; Wang, Shanshan; She, Yongxin; Wang, Jing

    2015-01-01

    A highly sensitive chemiluminescent enzyme immunoassay (CLEIA) method was developed in this study for efficient screening of triazophos residues in a large number of samples. Based on the maximum residue limits (MRLs) set by China and CAC for triazophos in different agro-products, the representative apple, orange, cabbage, zucchini, and rice samples were selected as spiked samples, and the triazophos at the concentrations of the MRL values were spiked to blank samples. Subsequently, the five samples with the spiked triazophos standard were measured by CLEIA 100 times, and the detection results indicated that the correction factors of the apple, orange, cabbage, zucchini, and rice were determined as 0.79, 0.66, 0.85, 0.76, and 0.91, respectively. In this experiment, 1500 real samples were detected by both the CLEIA and the GC-MS methods. With the GC-MS method, 1462 samples were identified as negative samples and 38 samples as positive samples. Based on the correction factors, the false positive rate of the CLEIA method was 0.13%, and false negative rate was 0. The results showed that the established CLEIA method could be used to screen a large number of real samples. PMID:26218576

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

    PubMed

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

    1996-09-01

    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.

  16. Enzyme immunoassay for urogenital trichomoniasis as a marker of unsafe sexual behaviour.

    PubMed

    Mason, P R; Gregson, S; Gwanzura, L; Cappuccinelli, P; Rapelli, P; Fiori, P L

    2001-02-01

    Enzyme immunoassay (EIA) was used to detect antibodies to Trichomonas vaginalis in sera from Zimbabwe. The EIA showed a sensitivity of 95 and 94% when compared with vaginal swab culture among women attending a family planning clinic (FPC) and female commercial sex workers (CSW) respectively. The specificity was 85 and 77% in the two groups. Culture-negative FPC women were sub-divided into high risk or low risk of exposure to trichomoniasis. The seroprevalence was 10% (6/61) among low risk women, 21% (10/48) among high risk women and 23% (9/39) among culture negative CSW. The EIA was positive in 46% (18/39) men with genital discharge but only 5% (2/37) healthy blood donors. None of 31 sera from prepubescent children was positive. The EIA may be useful for community surveys of trichomoniasis. Because T. vaginalis is a common sexually transmitted disease, the test may indicate behaviour that increases the risk of STD transmission.

  17. Determination of capsaicinoids in salsa by liquid chromatography and enzyme immunoassay.

    PubMed

    Perkins, Brian; Bushway, Rodney; Guthrie, Kelly; Fan, Titan; Stewart, Bonnie; Prince, Alison; Williams, Mark

    2002-01-01

    Two simple and rapid methods were developed to monitor pungency of salsa in production. Capsaicin (C) and dihydrocapsaicin (DHC) were quantitated in 17 commercially available tomato-based salsas by enzyme immunoassay (EIA) and liquid chromatography (LC) with fluorescent detection. Samples were extracted with methanol and the extracts were subjected to solid-phase extraction (SPE) using polystyrene-divinylbenzene columns. Analysis of SPE eluates showed good correlation (r2 = 0.953) between LC and EIA, with a slightly high bias for EIA. Salsa fortified with C and DHC from 0.118 to 103.2 microg/g resulted in recoveries of 90-112% (C) and 76-97% (DHC). Limits of detection by LC were 0.1 microg/g for each capsaicinoid and 0.1 microg/g by EIA for total capsaicinoids. The LC on-column response was linear from 0.2 to 100 ng for both C and DHC, whereas the working range for EIA was 0.1-2.0 ppm. Pungency varied between different salsa brands labeled mild, medium, and hot.

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

    PubMed

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

    1986-08-01

    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.

  19. Diagnosis of Oropouche virus infection using a recombinant nucleocapsid protein-based enzyme immunoassay.

    PubMed

    Saeed, M F; Nunes, M; Vasconcelos, P F; Travassos Da Rosa, A P; Watts, D M; Russell, K; Shope, R E; Tesh, R B; Barrett, A D

    2001-07-01

    Oropouche (ORO) virus is an emerging infectious agent that has caused numerous outbreaks of an acute febrile (dengue-like) illness among humans in Brazil, Peru, and Panama. Diagnosis of ORO virus infection is based mainly on serology. Two different antigens, hamster serum antigen (HSA) and Vero cell lysate antigen (VCLA), are currently used in enzyme immunoassays (EIAs) in Brazil and Peru, respectively, to investigate the epidemiology of ORO virus infection. Both antigens involve use of infectious virus, and for this reason their use is restricted. Consequently, the frequency and distribution of ORO virus infection are largely unexplored in other countries of South America. This report describes the use of a bacterially expressed recombinant nucleocapsid (rN) protein of ORO virus in EIAs for the diagnosis of ORO virus infection. The data revealed that the purified rN protein is comparable to the authentic viral N protein in its antigenic characteristics and is highly sensitive and specific in EIAs. Among 183 serum samples tested, a high degree of concordance was found between rN protein-based EIA and HSA- and VCLA-based EIAs for the detection of both ORO virus-specific immunoglobulin M (IgM) and IgG antibodies. The high sensitivity, specificity, and safety of the rN protein-based EIA make it a useful diagnostic technique that can be widely used to detect ORO virus infection in South America.

  20. Diagnosis of Oropouche Virus Infection Using a Recombinant Nucleocapsid Protein-Based Enzyme Immunoassay

    PubMed Central

    Saeed, Mohammad F.; Nunes, Marcio; Vasconcelos, Pedro F.; Travassos Da Rosa, Amelia P. A.; Watts, Douglas M.; Russell, Kevin; Shope, Robert E.; Tesh, Robert B.; Barrett, Alan D. T.

    2001-01-01

    Oropouche (ORO) virus is an emerging infectious agent that has caused numerous outbreaks of an acute febrile (dengue-like) illness among humans in Brazil, Peru, and Panama. Diagnosis of ORO virus infection is based mainly on serology. Two different antigens, hamster serum antigen (HSA) and Vero cell lysate antigen (VCLA), are currently used in enzyme immunoassays (EIAs) in Brazil and Peru, respectively, to investigate the epidemiology of ORO virus infection. Both antigens involve use of infectious virus, and for this reason their use is restricted. Consequently, the frequency and distribution of ORO virus infection are largely unexplored in other countries of South America. This report describes the use of a bacterially expressed recombinant nucleocapsid (rN) protein of ORO virus in EIAs for the diagnosis of ORO virus infection. The data revealed that the purified rN protein is comparable to the authentic viral N protein in its antigenic characteristics and is highly sensitive and specific in EIAs. Among 183 serum samples tested, a high degree of concordance was found between rN protein-based EIA and HSA- and VCLA-based EIAs for the detection of both ORO virus-specific immunoglobulin M (IgM) and IgG antibodies. The high sensitivity, specificity, and safety of the rN protein-based EIA make it a useful diagnostic technique that can be widely used to detect ORO virus infection in South America. PMID:11427552

  1. Evaluation of enzyme immunoassay for Candida cytoplasmic antigens in neutropenic cancer patients.

    PubMed Central

    Morhart, M; Rennie, R; Ziola, B; Bow, E; Louie, T J

    1994-01-01

    A Candida albicans cytoplasmic antigen enzyme immunoassay (CACP antigen EIA) was developed with antibodies raised against antigens prepared from yeast cells grown under standardized growth conditions. The C. albicans components reactive in the EIA were shown to be predominantly proteins with associated carbohydrates. Denaturing gel electrophoresis revealed the presence of five major CACP proteins with molecular weights between 36,000 and 44,000. The clinical usefulness of the CACP EIA was evaluated by retrospective blinded measurement of 89 serum samples from 31 granulocytopenic patient episodes. Twice-weekly surveillance cultures, sequential serum samples (approximately once per week or with change of the clinical course), and standard diagnostic criteria of fungal infection were used to categorize patients. The sensitivity and specificity of the CACP assay on the basis of serum samples were 82 and 100%, respectively (67 and 100% on the basis of patient episodes). The positive and negative predictive values were 100 and 97% for serum (100 and 93% for patient episodes). By comparison, the CANDTEC assay had low sensitivity (33%) and poor positive predictive values (50%). The CACP EIA may be a useful test suitable for further evaluations as a method for the diagnosis of invasive Candida infection in neutropenic cancer patients. Images PMID:8195392

  2. Potentiometric enzyme immunoassay using miniaturized anion-selective electrodes for detection

    PubMed Central

    Szűcs, Júlia; Pretsch, Ernö; Gyurcsányi, Róbert E.

    2010-01-01

    An enzyme-linked immunosorbent assay (ELISA) for prostate specific antigen (PSA) detection in human serum was developed based on the potentiometric detection of 6,8-difluoro-4-methylumbelliferone (DiFMU). The assays were carried out in anti-human PSA capture-antibody modified microtiter plates (150 µl volume). After incubation in the PSA containing serum samples, β-galactosidase-labeled PSA tracer antibody was added. The β-galactosidase label catalyzed the hydrolysis of 6,8-difluoro-4-methylumbelliferyl-β-D-galactopyranoside (DiFMUG) and the resulting DiFMU− anion was detected by potentiometric microelectrodes with anion-exchanger membrane. The selectivity of the anion-exchanger electrode is governed by the lipophilicity of the anions in the sample. Since DiFMU− is much more lipophilic (log P = 1.83) than any of the inorganic anions normally present in the working buffers and occurs in its anionic form at the physiological pH (pKa = 4.19), it was chosen as the species to be detected. The potentiometric ELISA-based method detects PSA in serum with a linear concentration range of 0.1–50 ng/mL. These results confirm the applicability of potentiometric detection in diagnostic PSA assays. Owing to simple methodology and low cost, potentiometric immunoassays seem to offer a feasible alternative to the development of in vitro diagnostic platforms. PMID:20448926

  3. Evaluation of an automated, homogeneous enzyme immunoassay for serum thyroxine measurement in dog and cat serum.

    PubMed

    Horney, Barbara S.; MacKenzie, Allan L.; Burton, Shelley A.; Olexson, Dennis W.; Mitton, Katherine L.; Coty, William A.; Rinne, Sherrie G.

    1999-01-01

    A homogenous enzyme immunoassay (EIA) for measurement of serum thyroxine (T4) concentration was evaluated for use with canine and feline serum. The EIA method was linear from 0 to 150 nmol T4/L for human serum, 0 to 94 nmol T4/L for feline serum and 10 to 60 nmol T4/L for canine serum. Intra- and interassay precision studies yielded coefficients of variation

  4. Detection of Snake Venom in Post-Antivenom Samples by Dissociation Treatment Followed by Enzyme Immunoassay

    PubMed Central

    Maduwage, Kalana P.; O’Leary, Margaret A.; Silva, Anjana; Isbister, Geoffrey K.

    2016-01-01

    Venom detection is crucial for confirmation of envenomation and snake type in snake-bite patients. Enzyme immunoassay (EIA) is used to detect venom, but antivenom in samples prevents venom detection. We aimed to detect snake venom in post-antivenom samples after dissociating venom-antivenom complexes with glycine-HCl (pH 2.2) and heating for 30 min at 950 °C. Serum samples underwent dissociation treatment and then Russell’s viper venom or Australian elapid venom measured by EIA. In confirmed Russell’s viper bites with venom detected pre-antivenom (positive controls), no venom was detected in untreated post-antivenom samples, but was after dissociation treatment. In 104 non-envenomed patients (negative controls), no venom was detected after dissociation treatment. In suspected Russell’s viper bites, ten patients with no pre-antivenom samples had venom detected in post-antivenom samples after dissociation treatment. In 20 patients with no venom detected pre-antivenom, 13 had venom detected post-antivenom after dissociation treatment. In another 85 suspected Russell’s viper bites with no venom detected pre-antivenom, 50 had venom detected after dissociation treatment. Dissociation treatment was also successful for Australian snake envenomation including taipan, mulga, tiger snake and brown snake. Snake venom can be detected by EIA in post-antivenom samples after dissociation treatment allowing confirmation of diagnosis of envenomation post-antivenom. PMID:27136587

  5. Detection of eastern equine encephalomyelitis viral antigen in avian blood by enzyme immunoassay: a laboratory study.

    PubMed

    Scott, T W; Olson, J G

    1986-05-01

    An enzyme immunoassay (EIA) was evaluated for its efficacy at detecting eastern equine encephalomyelitis (EEE) virus in avian blood and brain specimens. Preliminary analysis of blood from experimentally infected house sparrows and naturally infected whooping cranes showed that EEE antigen could be detected with the EIA. Polyclonal mouse antibodies were selected for antigen capture, and rabbit antibodies were selected for antigen detection. Overnight antigen incubation increased sensitivity. The lower limit of EEE antigen detection was 10(3.5) TCID50/ml for a stock of virus. Sensitivity was 10% (2/20) for antigen detection in the blood of chicks inoculated with EEE virus less than 24 hr earlier. At 24 and 48 hr after infection, sensitivity was 100% (10/10). Sensitivity and specificity of antigen detection were excellent (100% for both) in house sparrows experimentally inoculated with EEE, Highlands J (HJ), western equine encephalomyelitis (WEE), or St. Louis encephalitis (SLE) virus and bled at 24 hr intervals. Cross-reactivity was observed, however, with high concentrations (10(5.5) TCID50/ml) of HJ virus. EEE antigen was detected in avian blood by the EIA after infectious virus had declined to undetectable levels. The EIA is a useful alternative to virus isolation in cell culture for diagnosis or detection of EEE virus infections in birds. The test has the advantages of being simple, rapid, and capable of detecting antigen in the absence of infectious virus.

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

    PubMed

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

    1989-01-01

    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 and radioimmunoassay methods for the measurement of plasma oxytocin

    PubMed Central

    Szeto, Angela; McCabe, Philip M.; Nation, Daniel A.; Tabak, Benjamin A.; Rossetti, Maria A.; McCullough, Michael E.; Schneiderman, Neil; Mendez, Armando J.

    2011-01-01

    Objective There is increased interest in measuring peripheral oxytocin levels to better understand the role of this peptide in mammalian behavior, physiology, and disease. The purpose of this study was to compare methods for plasma oxytocin measurement using a commercially available enzyme immunoassay (EIA) and radioimmunoassay (RIA), to evaluate the need for sample extraction, and to assess the immunospecificity of the assays. Methods Oxytocin was measured in extracted and unextracted human plasma samples (n = 39). Oxytocin and its degradation products were separated by high performance liquid chromatography and gel filtration chromatography and then assayed by EIA or RIA to identify oxytocin immunoreactive peaks. Results Without extraction, plasma measured by EIA was more than 100-fold higher than in extracted plasma, and the correlation between oxytocin levels in extracted and unextracted plasma was minimal (Spearman’s rho = −0.10, p = 0.54). Using the RIA, most samples (> 90%) were below the level of detection with or without extraction. Following chromatographic fractionation of sample extracts, multiple immunoreactive products were found to be present in addition to oxytocin, which casts doubts on the specificity of the assays. Conclusions Changes in oxytocin levels have been reported in social and behavioral challenge studies. This study indicates that sample extraction is necessary to obtain valid assay results. Changes in oxytocin degradation products are likely to contribute to the previously observed responses in circulating oxytocin levels to behavioral and social challenge. There is a critical need for validated and reliable methods to measure oxytocin in biological samples. PMID:21636661

  8. An enzyme immunoassay for advanced glycosylation end-products in serum.

    PubMed

    Nazaimoon, W; Bak, K

    1998-12-01

    We successfully developed an in-house, competitive enzyme immunoassay to measure advanced glycosylation end-products (AGE) in serum. The assay involved coating microtitre wells with AGE-BSA at 8 micrograms/ml for 4 hours, followed by overnight incubation of 20 microliters sample (prediluted at 1:6) with 80 microliters antiserum (1:8000). HRP-labelled goat anti-rabbit was used as the second antibody and 3,5',5,5'-tetramethylbenzidine dihydrochloride as the substrate. Incubation was carried out at 4 degrees C. As suggested in an earlier study, we standardised the AGE units against normal human serum (NHS). Thus, one AGE unit was defined as the inhibition that resulted when the 1:6 diluted NHS was assayed. Mean (+/- SD) AGE level in normal subjects (n = 37) was significantly lower than in diabetes subjects with microalbuminuria (n = 57) (6.0 +/- 0.7 versus 10.2 +/- 4.7 units/ml, p = 0.0001). With the availability of in-house assay and by standardising the AGE unit with the other laboratories, more studies could be undertaken and results compared, and possibly, further elucidate the roles of AGE in the pathogenesis of diabetic complications.

  9. Detection of the organophosphorus nerve agent sarin by a competitive inhibition enzyme immunoassay.

    PubMed

    Zhou, Y X; Yan, Q J; Ci, Y X; Guo, Z Q; Rong, K T; Chang, W B; Zhao, Y F

    1995-01-01

    Two artificial antigens, NalphaNepsilon-di(O,O-diisopropyl) phosphoryl L-lysine (DIP)- bovine serum albumin (BSA) conjugate (DIP-BSA) and DIP-KLH (keyhole limpet hemocyanin), were synthesized. Antibodies against sarin (O-isopropyl methylphosphonofluoridate) were obtained after immunization of rabbits with DIP-KLH conjugate. A competitive inhibition enzyme immunoassay (CIEIA) was developed to detect the organophosphorus nerve agent sarin. The antibody solutions could be inhibited by sarin as low as 10(-6) mol/l, and the standard curve was linear over 3 orders of magnitude. The coefficients of intraassay and interassay variation of this method were 5.4-6.2% (n = 11) and 8.0-9.5% (n = 6) at a sarin concentration range of 10(-3)-10(-6) mol/l, respectively. The recovery of sarin in water samples at the concentration of 5 x 10(-5) mol/l was in the range of 96.8-102.5%. The specificity of the antiserum was assessed by comparing the inhibition induced by sarin with soman, Vx, isopropyl alcohol and isopropyl methyl phosphonic acid. The results showed that less than 5 mmol/l soman, 2 mmol/l Vx, 16 mmol/l isopropyl alcohol and 8 mmol/l isopropyl methyl phosphonic acid did not influence the determination of sarin in water samples.

  10. A competitive chemiluminescence enzyme immunoassay method for β-defensin-2 detection in transgenic mice.

    PubMed

    Yang, Xi; Zhou, Tao; Yu, Lei; Tan, Wenwen; Zhou, Rui; Hu, Yonggang

    2015-03-01

    A competitive chemiluminescence enzyme immunoassay (CLEIA) method for porcine β-defensin-2 (pBD-2) detection in transgenic mice was established. Several factors that affect detection, including luminol, p-iodophenol and hydrogen peroxide concentrations, as well as pH, were studied and optimized. The linear range of the proposed method for pBD-2 detection under optimal conditions was 0.05-80 ng/mL with a correlation coefficient of 0.9960. Eleven detections of a 30 ng/mL pBD-2 standard sample were performed. Reproducible results were obtained with a relative standard deviation of 3.94%. The limit of detection of the method for pBD-2 was 3.5 pg/mL (3σ). The proposed method was applied to determine pBD-2 expression levels in the tissues of pBD-2 transgenic mice, and compared with LC-MS/MS and quantitative real-time reverse-transcriptase polymerase chain reaction. This suggests that the CLEIA can be used as a valuable method to detect and quantify pBD-2.

  11. The Rapid Screening of Triazophos Residues in Agricultural Products by Chemiluminescent Enzyme Immunoassay.

    PubMed

    Chen, Ge; Yang, Lihua; Jin, Maojun; Du, Pengfei; Zhang, Chan; Wang, Jian; Shao, Hua; Jin, Fen; Zheng, Lufei; Wang, Shanshan; She, Yongxin; Wang, Jing

    2015-01-01

    A highly sensitive chemiluminescent enzyme immunoassay (CLEIA) method was developed in this study for efficient screening of triazophos residues in a large number of samples. Based on the maximum residue limits (MRLs) set by China and CAC for triazophos in different agro-products, the representative apple, orange, cabbage, zucchini, and rice samples were selected as spiked samples, and the triazophos at the concentrations of the MRL values were spiked to blank samples. Subsequently, the five samples with the spiked triazophos standard were measured by CLEIA 100 times, and the detection results indicated that the correction factors of the apple, orange, cabbage, zucchini, and rice were determined as 0.79, 0.66, 0.85, 0.76, and 0.91, respectively. In this experiment, 1500 real samples were detected by both the CLEIA and the GC-MS methods. With the GC-MS method, 1462 samples were identified as negative samples and 38 samples as positive samples. Based on the correction factors, the false positive rate of the CLEIA method was 0.13%, and false negative rate was 0. The results showed that the established CLEIA method could be used to screen a large number of real samples.

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

    PubMed

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

    2014-06-15

    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; p<0.0001) showing IMMULITE's robustness against the complex milk matrix and its ability to measure low milk B12 concentrations.

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

    PubMed

    Gaafar, Maha R

    2011-08-01

    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.

  14. Establishment and clinical application of enzyme immunoassays for determination of luteinizing hormone releasing hormone and metastin.

    PubMed

    Katagiri, Fumihiko; Tomita, Kenji; Oishi, Shinya; Takeyama, Masaharu; Fujii, Nobutaka

    2007-06-01

    Metastin, a 54-residue peptide, was identified as the cognate ligand of human G-protein-coupled receptor GPR54. Since metastin is a gene product of the human metastasis suppressor gene 'KiSS-1', early studies on metastin were focused on its activity as a tumor metastasis suppressor. Recently, there have been some reports that metastin is found in human plasma and is particularly abundant in the plasma of pregnant women. Dysfunction of the GPR54 receptor causes diseases that are characterized by an insufficient release of gonadotropin and lack or delay of pubertal maturation. This information strongly suggests that metastin is involved in the regulation of reproductive endocrine functions. In order to determine the plasma levels of metastin and luteinizing hormone releasing hormone (LHRH) in an isolated hypogonadotropic hypogonadism (IHH) patient, who received intermittent administrations of LHRH, we tried to establish a sensitive and specific enzyme immunoassay. The plasma LHRH levels of the patient were very high, while plasma metastin levels were at almost the same levels as circadian rhythms of healthy male humans. In the central nervous system, metastin stimulates the neuroendocrine reproductive axis. However, the effects of peripheral metastin are not known. Our result suggested that peripheral metastin had a genesis and activity different from central metastin.

  15. Sensitive enzyme-amplified electrical immunoassay for protein A-bearing Staphylococcus aureus in foods.

    PubMed Central

    Brooks, J L; Mirhabibollahi, B; Kroll, R G

    1990-01-01

    An amperometric electrochemical immunoassay specific for protein A-bearing Staphylococcus aureus was developed. The method was based on a sandwich immunosorbent assay and incorporated an enzyme amplification step, using a NAD-specific redox cycle generating NADH (C. H. Stanley, A. Johannsson, and C. H. Self, J. Immunol. Methods 83:89-95, 1985). Reduction of the mediator, ferricyanide, was dependent on the initial concentration of antigen. The final potential was measured by using a Pt disk electrode polarized at +0.8 V to the Ag/AgCl reference electrode. The assay was rapid (4 h) and generated protein A- and cell (S. aureus)-dependent signals. The system was highly sensitive and could detect 10 pg of protein A ml-1 and less than 100 CFU of S. aureus ml-1. Similar sensitivities were observed with S. aureus cultures inoculated into beef and milk, but the sensitivity was reduced slightly (ca. 10(3) g-1) with samples of Cheddar cheese. PMID:2268148

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

    PubMed

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

    2000-12-01

    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. Fecal steroid evaluation to monitor reproductive status in wild ungulate females using enzyme immunoassay commercial kits.

    PubMed

    Borque, Conception; Pérez-Garnelo, Sonia S; Delclaux, Maria; Martínez, Eva; De la Fuente, Julio

    2011-12-01

    Analysis of reproductive hormones in fecal samples is necessary for the noninvasive monitoring of reproductive status in free-ranging species. The aim of the present study was to establish an easy noninvasive method to monitor reproductive status in wild ungulate females. Feces were collected daily, weekly, or three or four times a week directly from the soil for a period ranging from 1 to 9.8 mo. Fecal estradiol and progestagens were monitored in nine wild ungulate females (Barbary sheep, Ammotragus lervia [n = 3]; European bison, Bison bonasus [n = 1]; auroch, Bos taurus primigenius [n = 2]; sitatunga, Tragelaphus spekii gratus [n = 2]; and Indian rhinoceros, Rhinoceros unicornis [n = 1]) by using commercially available enzyme immunoassay kits prepared for human serum or plasma. In the species evaluated in this study, luteal phase, abortion, and gestation patterns corresponded closely with changes in fecal progestagens. Luteal phase and gestation values differed significantly (P < 0.001) from basal values, whereas progestagens values after abortion were not significantly different (P > 0.05) from basal values. For estradiol excretory patterns, follicular phase and pregnancy values differed significantly (P < 0.001) from basal values, but differences between values after abortion and basal values were not significant (P > 0.05); length of estrous cycles were clearly defined through estradiol data. This study demonstrates that ovarian function in the wild ungulate females studied can be monitored by enzyme-linked immunosorbent assay (ELISA). Therefore, ELISA methodologies used here could be a practical alternative to other ELISAs that require more complex procedures or whose commercial availability is difficult.

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

    PubMed

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

    2015-04-15

    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.

  19. Spin-amplified culture followed by enzyme immunoassay for detection of herpes simplex virus in patient specimens: a comparative study.

    PubMed

    Verano, L; Michalski, F J

    1993-03-01

    In a comparative study it was found that a combination technique of spin-amplified culture and detection of herpes simplex virus (HSV) antigen in 48-h incubated cell culture lysate by a HSV antigen detection enzyme immunoassay kit (Dupont Herpchek) detected the largest number (227) of confirmed HSV positives when compared to standard cell culture (191) and direct Herpchek (146) on the same 415 clinical specimens.

  20. Multicentric evaluation of new commercial enzyme immunoassays for the detection of immunoglobulin M and total antibodies against hepatitis A virus.

    PubMed

    Arcangeletti, M C; Dussaix, E; Ferraglia, F; Roque-Afonso, A M; Graube, A; Chezzi, C

    2011-08-01

    A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively).

  1. Multicentric Evaluation of New Commercial Enzyme Immunoassays for the Detection of Immunoglobulin M and Total Antibodies against Hepatitis A Virus▿

    PubMed Central

    Arcangeletti, M. C.; Dussaix, E.; Ferraglia, F.; Roque-Afonso, A. M.; Graube, A.; Chezzi, C.

    2011-01-01

    A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively). PMID:21653739

  2. Comparison of a new, rapid enzyme immunoassay with a latex agglutination test for qualitative detection of rubella antibodies.

    PubMed Central

    Ferraro, M J; Kallas, W M; Welch, K P; Lau, A Y

    1987-01-01

    A total of 450 sera were tested for rubella virus antibodies by using a new, rapid enzyme immunoassay, SUDS Rubella. The results were compared with those obtained by using the Rubascan test, a well-established latex agglutination method. The sensitivity of the SUDS Rubella was 99.5%, and the specificity was 100%, when compared with Rubascan. The SUDS Rubella test can be performed in 10 min and provides an accurate screening test for the detection of rubella antibodies. Images PMID:3308952

  3. Comparison of a new, rapid enzyme immunoassay with a latex agglutination test for qualitative detection of rubella antibodies.

    PubMed

    Ferraro, M J; Kallas, W M; Welch, K P; Lau, A Y

    1987-09-01

    A total of 450 sera were tested for rubella virus antibodies by using a new, rapid enzyme immunoassay, SUDS Rubella. The results were compared with those obtained by using the Rubascan test, a well-established latex agglutination method. The sensitivity of the SUDS Rubella was 99.5%, and the specificity was 100%, when compared with Rubascan. The SUDS Rubella test can be performed in 10 min and provides an accurate screening test for the detection of rubella antibodies.

  4. Cross-reaction of propyl and butyl alcohol glucuronides with an ethyl glucuronide enzyme immunoassay.

    PubMed

    Arndt, Torsten; Beyreiß, Reinhild; Schröfel, Stefanie; Stemmerich, Karsten

    2014-08-01

    Ethyl glucuronide (EtG) in urine is considered a marker of recent alcohol consumption. Using immunoassays for EtG screening without confirmatory analysis bears a risk of getting false-positives as shown for trichloroethyl glucuronide from chloral hydrate medication and 1-propyl glucuronide from propanol-based hand disinfection. The aim of the study was to check whether glucuronides of frequently used aliphatic short chain alcohols aside from EtG and 1-propyl glucuronide can cross-react with the DRI(®) Ethyl Glucuronide Assay. Aliquots of EtG-free urine were individually spiked with methyl β-D-glucuronide, 1-propyl β-D-glucuronide, 2-propyl β-D-glucuronide, 1-butyl β-D-glucuronide, 2-butyl β-D-glucuronide, and tert-butyl β-D-glucuronide. To check the response rate of the DRI(®) Ethyl Glucuronide Assay to its target analyte, EtG was also added to a native EtG-free urine sample. The spiked alcohol glucuronide concentrations (seven levels up to 10mg/L) and the DRI(®) Ethyl Glucuronide Assay results were evaluated by Passing-Bablok regression analysis. The 95% confidence interval ranges for the slope of the regression function were considered a measure of cross-reaction of the individual alcohol glucuronides with the enzyme immunoassay. 2-Propyl glucuronide showed a cross-reactivity of 69-84% at the 95% probability level, methyl glucuronide, 1-propyl glucuronide, and 1- and 2-butyl glucuronide of 4-9%, and tert-butyl glucuronide almost no cross-reactivity. The response rate for EtG was 87-94% at the 95% probability level. The DRI(®) Ethyl Glucuronide Assay shows cross-reaction rates with aliphatic short chain alcohol glucuronides aside from EtG which bear a risk of getting false-positives regarding ethanol consumption. Mass spectrometric detection of EtG is mandatory for confirmation of positive immunological EtG screenings. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Development of a Highly Sensitive and Specific Blastomycosis Antibody Enzyme Immunoassay Using Blastomyces dermatitidis Surface Protein BAD-1

    PubMed Central

    Smedema, Melinda L.; Durkin, Michelle M.; Brandhorst, T. Tristan; Hage, Chadi A.; Connolly, Patricia A.; Leland, Diane S.; Davis, Thomas E.; Klein, Bruce S.; Wheat, L. Joseph

    2014-01-01

    Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis. PMID:24285817

  6. Development of a highly sensitive and specific blastomycosis antibody enzyme immunoassay using Blastomyces dermatitidis surface protein BAD-1.

    PubMed

    Richer, Sarah M; Smedema, Melinda L; Durkin, Michelle M; Brandhorst, T Tristan; Hage, Chadi A; Connolly, Patricia A; Leland, Diane S; Davis, Thomas E; Klein, Bruce S; Wheat, L Joseph

    2014-02-01

    Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis.

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

    PubMed

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

    2016-05-01

    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. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Development of enzyme linked immunoassay for the simultaneous detection of carbaryl and metolcarb in different agricultural products.

    PubMed

    Sun, Jingwei; Dong, Tingting; Zhang, Yan; Wang, Shuo

    2010-05-07

    A direct competitive enzyme linked immunosorbent assay in multi-enzyme tracers format for the simultaneous analysis of carbaryl and metolcarb in agricultural products is described in this study. The concentrations of coating antibodies and enzyme tracer were studied. Under the optimum conditions, the limits of detection of carbaryl and metolcarb were 0.15 microg L(-1) and 1.2 microg L(-1), respectively. Determination of carbaryl and metolcarb in fruit juices and vegetables was accomplished by simple, rapid and efficient extraction methods. Recoveries of spiked samples were great than 70%. Validation of the immunosorbent assay was conducted by comparison of results from high performance liquid chromatography (HPLC). The correlations between the data obtained using multi-enzyme tracers enzyme linked immunosorbent assay and high performance liquid chromatography were good. Results indicated that the new strategy for developing immunoassay for simultaneous quantitative determination of carbaryl and metolcarb residues was suitable in this study. 2010. Published by Elsevier B.V.

  9. Sensitivity of HIV rapid tests compared with fourth-generation enzyme immunoassays or HIV RNA tests.

    PubMed

    Tan, Wei Sheng; Chow, Eric P F; Fairley, Christopher K; Chen, Marcus Y; Bradshaw, Catriona S; Read, Tim R H

    2016-07-31

    Determine the sensitivity of HIV rapid tests compared with fourth-generation enzyme immunoassays (EIA) or nucleic acid amplification tests (NAAT) in clinical settings. Systematic review and meta-analysis. Medline, PubMed, Embase, Cochrane Controlled Trials Register, Cochrane reviews and Cumulative Index to Nursing and Allied Health Literature were searched until 14 July 2015 for studies of adults comparing point-of-care HIV rapid tests to fourth-generation HIV EIA antibody/p24 antigen or HIV NAAT. From 953 titles, 18 studies were included, involving 110 122 HIV rapid test results. Compared with EIA, the estimated sensitivity (random effects) of HIV rapid tests was 94.5% [95% confidence interval (CI): 87.4-97.7]. Compared with NAAT, the sensitivity of HIV rapid tests was 93.7% (95% CI: 88.7-96.5). The sensitivity of HIV rapid tests in high-income countries was 85.7% (95% CI: 81.9-88.9) and in low-income countries was 97.7% (95% CI: 95.2-98.9) compared with either EIA or NAAT (P < 0.01 for difference between settings). Proportions of antibody negative acute infections were 13.6 (95% CI: 10.1-18.0) and 4.7% (95% CI: 2.8-7.7) in studies from high-income and low-income countries, respectively (P < 0.01). In clinical settings, HIV rapid tests were less sensitive in high-income countries compared with low-income countries, missing about one in seven infections, possibly because of the larger proportion of acute infections in targeted populations. This suggests that in high-income countries, HIV rapid tests should be used in combination with fourth-generation EIA or NAAT tests, except in special circumstances. Prospective Registration of Systematic Reviews registration number CRD42015020154.Supplementary video link: http://links.lww.com/QAD/A924.

  10. Enzyme immunoassays for invasive Candida infections: reactivity of somatic antigens of Candida albicans.

    PubMed Central

    Zöller, L; Krämer, I; Kappe, R; Sonntag, H G

    1991-01-01

    The main problem encountered with serodiagnostic tests for Candida infections is their failure to differentiate between invasive and superficial candidosis. Recent immunoblotting studies suggested that the use of selective somatic proteins of Candida albicans as antigens might be a promising approach toward developing a new generation of serodiagnostic assays. In this study major cytoplasmic protein antigens with molecular weights of 47,000 (47K), 46,000 (46K), 45,000 (45K), and 29,000 (29K) were identified as potential marker antigens for antibody detection in invasive candidosis. Continuous-flow isoelectric focusing was employed to enrich the proteins in two fractions, one of them containing the 47K and 29K proteins and the other one containing predominantly the 47K and 45K major proteins. These antigens and a whole somatic antigen extract were used to establish enzyme immunoassays (EIAs) for antibody detection. Whereas all tests were able to discriminate between patients with invasive candidosis (n = 27) and normal healthy volunteers (n = 167), as proved by graphic marker analysis, the selective antigen EIAs were highly superior to the whole somatic antigen EIA and two serological standard assays (indirect immunofluorescence assay and indirect hemagglutination assay) when a panel of sera from patients with superficial candidosis (n = 34) was used as a negative control group. The use of the 47K-29K antigen fraction allowed the best differentiation between invasive and noninvasive candidosis. The corresponding immunoglobulin G class-specific EIA had a sensitivity of 81.5% and a specificity of 97% for both negative control groups as well. Images PMID:1774309

  11. Determination of Babesia microti seroprevalence in blood donor populations using an investigational enzyme immunoassay

    PubMed Central

    Levin, Andrew E; Williamson, Phillip C; Erwin, James L; Cyrus, Sherri; Bloch, Evan M; Shaz, Beth H; Kessler, Debra; Telford, Sam R; Krause, Peter J; Wormser, Gary P; Ni, Xiaoyan; Wang, Haihong; Krueger, Neil X; Caglioti, Sally; Busch, Michael P

    2014-01-01

    Background Transfusion-transmitted babesiosis caused by Babesia microti has emerged as a significant risk to the US blood supply. This study estimated the prevalence of B. microti antibodies in blood donors using an investigational enzyme immunoassay (EIA). Study Design and Methods A peptide-based EIA that detects both immunoglobulin (Ig)G and IgM antibodies to B. microti was developed and validated. Donor samples randomly selected from areas defined as high-risk endemic, lower-risk endemic, and nonendemic for B. microti were deidentified and tested using the investigational EIA. Samples that were EIA repeat reactive were further tested by B. microti immunofluorescent assay (IFA), polymerase chain reaction (PCR) on red blood cell lysates, and peripheral blood smear examination. A random subset of 1272 samples from high-risk endemic areas was tested by IFA, PCR, and peripheral blood smear in parallel with EIA. Results Among 15,000 donations tested with the investigational B. microti EIA, EIA repeat-reactive rates were 1.08% (54/5000) in a high-risk endemic area, 0.74% (37/5000) in a lower-risk area, and 0.40% (20/5000) in a nonendemic area. After application of a revised cutoff, these values were reduced to 0.92%, (46/5000), 0.54% (27/5000), and 0.16% (8/5000). Overall concordance between EIA and IFA among donor samples was 99.34%. One seropositive sample was positive by PCR. Conclusion The seroprevalence of B. microti in blood donors in a high-risk area measured by an investigational EIA was approximately 1%. The EIA shows promise as an efficient high-throughput blood donor screening assay for B. microti. PMID:24995863

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

    PubMed

    Adel Ahmed, Heba; Azzazy, Hassan M E

    2013-11-15

    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.

  13. Development and application of an enzyme immunoassay for coronavirus OC43 antibody in acute respiratory illness.

    PubMed Central

    Gill, E P; Dominguez, E A; Greenberg, S B; Atmar, R L; Hogue, B G; Baxter, B D; Couch, R B

    1994-01-01

    Study of coronavirus OC43 infections has been limited because of the lack of sensitive cell culture systems and serologic assays. To improve this circumstance, we developed an indirect enzyme immunoassay (EIA) to detect serum antibody to OC43. Antigen (100 ng) prepared by polyethylene glycol precipitation provided optimal results without a postcoat procedure. Evaluation of intraplate variation indicated that a > or = 2.5-fold increase in serum titer was significant. Sixteen of 18 (89%) paired serum samples with previously identified, reproducible increases in the level of hemagglutination inhibition (HAI) antibody to OC43 also showed significant increases as detected by EIA. Specificity for the EIA was established with paired sera obtained from persons given influenza immunizations or experiencing a respiratory infection. No rise in antibody titers occurred among 33 persons with documented coronavirus 229E infection. EIA was then performed on each of 419 paired serum samples from ambulatory chronic obstructive pulmonary disease patients and healthy older adults, from asthmatic adults presenting for emergency room treatment, and from persons hospitalized with acute respiratory symptoms. Twenty-three antibody rises to OC43 were detected; only nine of these were detected by the HAI test, and the HAI test did not detect any increases in antibody titers that were not detected by EIA. Nineteen of 25 coronavirus OC43 infections for which a month of infection could be assigned occurred between November and February. Overall, 4.4% of acute respiratory illnesses in the studied populations were associated with a coronavirus OC43 infection. PMID:7814468

  14. Detection of circulating toxocaral antigens in dogs by sandwich enzyme-immunoassay.

    PubMed Central

    Matsumura, K; Kazuta, Y; Endo, R; Tanaka, K

    1984-01-01

    This study describes the presence of circulating toxocaral antigens (CTA) in the sera of dogs infected with Toxocara canis (T. canis) by using a sandwich enzyme-immunoassay (SEIA). A specificity of this assay with different antigens was observed, i.e. the EIA values, which express the antigen concentration, of excretory-secretory antigen from T. canis larvae were higher than those of other antigens (Ascaris lumbricoides, Dirofilaria immitis and Fasciola hepatica). The variability in intra-assay was below 10%. In age distribution of CTA levels, the highest level was observed at 1 month of age. Thereafter, the levels decreased gradually until 6 months of age and then the same levels were maintained until adult age. Also, slightly elevated levels were found in the sera of foetuses. A significant correlation was obtained between age and CTA levels. The positive correlation between the number of worms and CTA levels was significant. As for the IgG, IgM and IgA antibodies, a significant correlation was observed between the IgM antibody activities and CTA levels, but this was not observed with IgG and IgA antibodies. From these results, it was indicated that the immunological response to T. canis infection in dogs may not be reached until 1 or 2 months after birth, although detectable CTA levels were observed in foetal and early life. It was also suggested that the immunological stimulation for canine toxocariasis may be maintained by the excretory-secretory materials from the larvae through life and as a result, IgM antibody production may be observed even in chronically infected adult dogs. The SEIA technique reported in this study may be useful as a diagnostic tool of human toxocariasis, since the CTA can be directly demonstrated by the technique. PMID:6365746

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

    PubMed

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

    1995-12-01

    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.

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

    PubMed

    Martin, Christin A; Keren, David F

    2002-01-01

    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.

  17. Comparison of Murex Single-Use Diagnostic System with Traditional Enzyme Immunoassay for Detection of Exposure to Human Immunodeficiency Virus

    PubMed Central

    Martin, Christin A.; Keren, David F.

    2002-01-01

    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. PMID:11777853

  18. Caffeine clearance by enzyme multiplied immunoassay technique: a simple, inexpensive, and useful indicator of liver function.

    PubMed Central

    McDonagh, J E; Nathan, V V; Bonavia, I C; Moyle, G R; Tanner, A R

    1991-01-01

    The clinical value and sensitivity of serum caffeine clearance measurement has been evaluated as an indicator of hepatic disease. After a 17 hour caffeine exclusion period, 300 mg of caffeine citrate was administered orally to the study subjects. Serum samples were taken four and 16 hours later. Serum caffeine concentrations were measured using an enzyme multiplied immunoassay technique (EMIT) and a clearance value derived. Conventional liver function tests were measured at the same time. A total of 103 subjects attending the medical unit in a district general hospital were studied. Twenty one had alcoholic liver disease, 11 non-alcoholic cirrhosis, nine non-cirrhotic liver disease, 21 suspected liver disease, six hepatic tumours, and 35 were hospital and normal control subjects. Caffeine clearance values were lowest in subjects with alcoholic liver disease (median 0.19 ml/min/kg, range 0.04-0.61 ml/min/kg) and significantly reduced in all subjects with liver disease (median 0.32 ml/min/kg, range 0.04-2.68 ml/min/kg) compared with control subjects (median 1.27 ml/min/kg, p less than 0.001). In subjects with suspected liver disease subsequently shown to have another explanation for abnormal liver function test results, caffeine clearance values were normal (median 1.31 ml/min/kg, range 0.23-2.64 ml/min/kg) and significantly different, p less than 0.001, from those of subjects with liver disease. Serum albumen values were not different for these latter two groups. Using a cut off value of 0.86 ml/min/kg, caffeine clearance measurement was 100% sensitive for alcoholic liver disease and 89% sensitive for all liver disease. The respective sensitivities for conventional liver function test measurement were 76% and 83%. In the suspected liver disease group, caffeine clearance was abnormal in only 24%, conventional liver function tests were abnormal in 95%. The respective specificities for caffeine clearance and liver function test measurement in control subjects were 93

  19. Evaluation of a novel chemiluminescent microplate enzyme immunoassay for hepatitis B surface antigen detection.

    PubMed

    Yang, Lin; Song, Liu-Wei; Fang, Lin-Lin; Wu, Yong; Ge, Sheng-Xiang; Li, Hui; Yuan, Quan; Zhang, Jun; Xia, Ning-Shao

    2016-02-01

    Hepatitis B virus surface antigen (HBsAg) is an important biomarker used in the diagnosis of hepatitis B virus (HBV) infection, but false-negative results are still reported in the detection of HBsAg using commercial assays. In this study, we evaluated the qualitative properties of a novel HBsAg chemiluminescence enzyme immunoassay (CLEIA) assay--WTultra. WHO standard sample dilution series and samples from low-level HBsAg carriers (<1 ng/mL) were used to evaluate the sensitivity of the WTultra assay. Boston Biomedica, Inc. (BBI) hepatitis B seroconversion panels were used to assess the ability of the WTultra assay to detect the window period. In addition, dilution series of 22 serum samples with different genotypes, serotypes and HBsAg mutations were used to assess the WTultra assay, and these were compared with other commercial assays. The lower detection limit of the WTultra assay was 0.012 IU/mL, and it showed a high sensitivity (97.52%, 95% CI, 94.95-99.00) in the detection of 282 low-level HBsAg carriers (<1 ng/mL). In samples with various HBV genotypes, serotypes and HBsAg mutations, the WTultra assay yielded 117 positive results in 132 samples, which was significantly higher than the results with the other four commercial assays (89, 83, 65 and 45, respectively, p<0.01). In the assays of mutant strains, the WTultra assay detected 82 positive results in 90 samples, which was significantly better than the results for the Hepanostika HBsAg Ultra (58 positive) and Architect (55 positive) (p<0.01) assays, which in turn were significantly better than the Murex V.3 (41 positive, p=0.026) and AxSYM V2 (29 positive, p<0.01) assays. However, in the detection of 42 samples of wild-type strains with various genotypes and serotypes, no significant differences were observed among the WTultra (35 positive), Architect (28 positive) and Hepanostika HBsAg Ultra (31 positive) assays. However, the WTultra assay detected significantly more samples than the Murex V.3 (24

  20. Development of a sensitive enzyme immunoassay for measuring taipan venom in serum.

    PubMed

    Kulawickrama, S; O'Leary, M A; Hodgson, W C; Brown, S G A; Jacoby, T; Davern, K; Isbister, G K

    2010-07-01

    The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3-3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1-3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough

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

    PubMed Central

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

    2014-01-01

    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

  2. Development of dual-enzyme-based simultaneous immunoassay for measurement of progesterone and human chorionic gonadotropin.

    PubMed

    Basu, Anupam; Maitra, Saumen Kumar; Shrivastav, Tulsidas G

    2007-07-15

    The development of a simultaneous multianalyte immunoassay for the detection of progesterone and human chorionic gonadotropin (hCG) in serum is described. In this simultaneous multianalyte assay, two different enzymes, viz. horse radish peroxidase (HRP) and alkaline phosphatase (ALP), were used as markers. To the simultaneous immobilized progesterone and hCG antibody microwells, 50 microL of different concentrations of combined standards or serum samples was added in duplicate and then 100 microL of combined conjugate reagent, composed of 17-alpha-OH-P-ALP and hCG-biotin was added to all the wells and incubated for 1h at 37 degrees C. After incubation, the contents of the wells were decanted and washed thoroughly with running tap water. After washing, 100 microL alkaline phosphatase substrate along with streptavidin-horseradish peroxidase was added to all the wells and incubated for 0.5 h at 37 degrees C. After incubation, the developed color was measured at 405 nm. The absorbency at this stage provides the result for the progesterone assay. The contents of the wells were decanted and washed. In the next step, 100 microL of tetramethylbenzidene/H2O2 reagent was added to all the wells. After 15 min of incubation, 100 microL of 0.5 M H2SO4 was added to all the wells and the color was read at 450 nm. The absorbency at this stage provides the result for the hCG assay. Sensitivity of the progesterone and hCG assays were 0.118 ng/ml and 0.124 IU/ml respectively. Intra- and inter assay percentage coefficients of variation ranged from 1.8 to 7.1 and 9.1 to 11.5 for progesterone and from 2.1 to 10.4 and 7.2 to 11.3 for hCG. There was good correlation between the discrete and the simultaneous assays. For progesterone assay, R2 was 0.99 and for hCG R2 was also 0.99. The developed dual assay for progesterone and hCG may be useful for the diagnosis of abnormal pregnancies such as miscarriages and ectopic pregnancies.

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

    PubMed

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

    2014-08-01

    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. 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. Semi-quantitative 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. 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). The Immunalysis HEIA K2 Spice kit required no

  4. Dried venous blood samples for the detection and quantification of measles IgG using a commercial enzyme immunoassay.

    PubMed Central

    Riddell, Michaela A.; Byrnes, Graham B.; Leydon, Jennie A.; Kelly, Heath A.

    2003-01-01

    OBJECTIVES: To determine whether samples of dried venous blood (DVB) were an acceptable alternative to serum for detecting measles-specific IgG in a commercial enzyme immunoassay. METHODS: Paired samples of serum and DVB were collected from 98 suspected cases of measles and 1153 schoolchildren in Victoria, Australia. All samples were tested using the Dade Behring Enzygnost Anti-Measles-Virus/IgG immunoassay. DVB samples were eluted using either the sample buffer provided with the kit or 5% dry milk powder in phosphate-buffered saline-Tween 20. FINDINGS: DVB samples eluted by sample buffer showed significantly better linear correlation to the serum samples than did DVB samples eluted in 5% dry milk in phosphate-buffered saline-Tween 20. To improve the comparability of serum and DVB samples an adjustment factor of 1.28 was applied to the optical density (OD) values of DVB. This adjustment also enabled quantification of the titre of measles IgG in mIU/ml directly from the OD value using the alpha calculation as specified by the kit protocol. For DVB samples stored for less than six months at 4 degrees C, the assay showed an overall sensitivity of 98.4% and a specificity of 97.2% compared with the results of serum testing. CONCLUSION: These results illustrate the potential for DVB samples to be widely used with the Dade Behring enzyme immunoassay system for determining the immunity of the individual and the population to the measles virus. PMID:14758429

  5. A Semester-long Student-directed Research Project Involving Enzyme Immunoassay: Appropriate for Immunology, Endocrinology, or Neuroscience Courses

    PubMed Central

    DeLuca, Jane

    2007-01-01

    The following project aimed at promoting integrated and long-lasting learning is described for an Immunology course, but it may be adapted to other disciplines. Students were asked to develop and carry out a research project to examine the relationship between immune function and stress. The experiments were required to include the assessment of salivary cortisol and salivary IgA (sIgA) with enzyme immunoassays. All other aspects of the experiments were developed by student groups with appropriate guidance from the instructor. Data are presented for one group project that assessed the effect of music on cortisol and sIgA. Overall levels of sIgA and cortisol were consistent with reported values. Students found a significant decrease in cortisol over time. Additionally, there was a trend that supported the overall student hypothesis regarding the effect of stress and immune function. Compared with the same Immunology course that included an instructor-designed experiment using enzyme immunoassays for cortisol and sIgA, several assessments (e.g., final grades and comments on student evaluations) show that overall learning seemed to be much better in the course with the student-directed research project. PMID:18056304

  6. Naked-eye detection as a universal approach to lower the limit of detection of enzyme-linked immunoassays.

    PubMed

    O'Connor, Erin F; Paterson, Sureyya; de la Rica, Roberto

    2016-05-01

    Colorimetric biosensors for the detection of analytes with the naked eye are required in environmental monitoring, point-of-care diagnostics, and analyses in resources constrained settings, where detection instruments may not be available. However, instrument-based detection methods are usually more adequate for detecting small variations in the signal compared to naked-eye detection schemes, and consequently the limit of detection of the latter is usually higher than the former. Here, we demonstrate that the limit of detection of colorimetric enzyme-linked immunoassays can be decreased several orders of magnitude when using naked-eye detection instead of a spectrophotometer for detecting the signal. The key step to lower the limit of detection is adding a small volume of chromogenic substrate during the signal generation step. This generates highly colored solutions that can be easily visualized with the naked eye and recorded with the camera of a mobile phone. The proposed method does not require expensive equipment or complex protocols to enhance the signal, and therefore it is a universal approach to lower the limit of detection of colorimetric enzyme-linked immunoassays.

  7. A semester-long student-directed research project involving enzyme immunoassay: appropriate for immunology, endocrinology, or neuroscience courses.

    PubMed

    Ramos Goyette, Sharon; DeLuca, Jane

    2007-01-01

    The following project aimed at promoting integrated and long-lasting learning is described for an Immunology course, but it may be adapted to other disciplines. Students were asked to develop and carry out a research project to examine the relationship between immune function and stress. The experiments were required to include the assessment of salivary cortisol and salivary IgA (sIgA) with enzyme immunoassays. All other aspects of the experiments were developed by student groups with appropriate guidance from the instructor. Data are presented for one group project that assessed the effect of music on cortisol and sIgA. Overall levels of sIgA and cortisol were consistent with reported values. Students found a significant decrease in cortisol over time. Additionally, there was a trend that supported the overall student hypothesis regarding the effect of stress and immune function. Compared with the same Immunology course that included an instructor-designed experiment using enzyme immunoassays for cortisol and sIgA, several assessments (e.g., final grades and comments on student evaluations) show that overall learning seemed to be much better in the course with the student-directed research project.

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

    SciTech Connect

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

    2011-09-09

    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.

  9. Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

    PubMed Central

    Gao, Zhuangqiang; Hou, Li; Xu, Mingdi; Tang, Dianping

    2014-01-01

    Methods based on enzyme labels have been developed for colorimetric immunoassays, but most involve poor sensitivity and are unsuitable for routine use. Herein, we design an enhanced colorimetric immunoassay for prostate-specific antigen (PSA) coupling with an enzyme-cascade-amplification strategy (ECAS-CIA). In the presence of target PSA, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification. Results indicated that the ECAS-CIA presents good responses toward PSA, and allows detection of PSA at a concentration as low as 0.05 ng mL−1. Intra- and inter-assay coefficients of variation are below 9.5% and 10.7%, respectively. Additionally, the methodology is validated for analysis of clinical serum specimens with consistent results obtained by PSA ELISA kit. Importantly, the ECAS-CIA opens a new horizon for protein diagnostics and biosecurity. PMID:24509941

  10. Magnetic affinity enzyme-linked immunoassay for diagnosis of Schistosomiasis japonicum in persons with low-intensity infection.

    PubMed

    Yu, Qin; Yang, Hai; Feng, Youmei; Zhu, Yanhong; Yang, Xiangliang

    2012-10-01

    Most schistosome-endemic areas in China are characterized by low-intensity infections that are independent of prevalence. To establish an effective diagnostic method, we developed a magnetic affinity enzyme-linked immunoassay based on soluble egg antigens (SEA-MEIA) for diagnosing schistosomiasis in persons with low-intensity infection with Schistosoma japonicum by comparing it with a conventional enzyme-linked immunosorbent assay (ELISA). Our results showed that the SEA-MEIA had a higher sensitivity and greater precision in the diagnosis of low-intensity S. japonicum infections than the ELISA. In addition, when we used Pearson's correlation in associating SEA-MEIA with ELISA, a significant correlation existed between the two assays (r = 0.845, P < 0.001). Our data indicated that SEA-MEIA, with a higher sensitivity and greater ease of performance, would be valuable for diagnosis of schistosomiasis japonicum in persons with low-intensity infections.

  11. Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

    PubMed Central

    Takehara, Shizuka; Takahashi, Masaharu

    2013-01-01

    Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals. PMID:23761660

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

    PubMed

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

    2014-02-01

    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.

  13. Paraquat enzyme-immunoassays in biological samples: assessment of the effects of hapten-protein bridge structures on assay sensitivity.

    PubMed

    Abuknesha, Ramadan A; Luk, Connie

    2005-06-01

    Previously unreported paraquat derivatives were prepared and used to develop enzyme-immunoassay methods for paraquat in serum and urine matrices. The study involved comparison of the effects of novel paraquat derivatives made of methyl and ethyl-4,4'-bipyridinium and cyanuric chloride (heterologous bridges) or valeric acid (homologous bridges) on the ability of paraquat standards to inhibit binding of the antibody to adsorbed hapten-protein plate coating antigens prepared by coupling the derivatives to gelatine. The comparison showed striking differences in assay sensitivity due to the hapten bridge binding phenomenon where the heterologous bridge conjugates enabled achievement of sensitivity levels several orders of magnitude greater than the homologous structures. The constructed ELISA showed minimal detection limit in the range 4 pg mL(-1) in the buffer systems and less then 100 pg mL(-1) in charcoal-stripped human and horse sera and human urine. The study presents details of synthesis of novel paraquat derivatives and a highly sensitive ELISA. In addition the investigation demonstrates the critical importance of judicious selection of hapten-bridge structures to achieve improved levels of detection limits of paraquat immunoassays. The reported assay is suitable for use in monitoring of paraquat levels in exposed persons or animals and for emergency diagnostic tests.

  14. Screening for cocaine on Euro banknotes by a highly sensitive enzyme immunoassay.

    PubMed

    Abdelshafi, Nahla A; Panne, Ulrich; Schneider, Rudolf J

    2017-04-01

    This study focused on quantitative detection of cocaine on Euro banknotes in Germany. A sensitive direct competitive immunoassay was developed and optimized with a limit of detection (LOD) of 5.6ng/L. Exhaustive cocaine extraction by solvent was tested using different methanol concentrations and buffered solutions. Cross-reactivity studies were performed to determine the degree of interference of cocaine metabolites with the immunoassay. Sixty-five Euro banknotes obtained from different districts in Berlin were evaluated. A 100% contamination frequency with cocaine was detected. A comparison between the amount of cocaine extracted by cotton swabbing of one square centimeter of the banknote showed a good correlation for lower contamination levels. This assay showed high sensitivity of detecting pg of cocaine per 1cm(2) of one banknote by swabbing 1cm(2): 0, 14, and 21pg/cm(2). Moreover, three notes of different denominations revealed high cocaine concentration; 1.1mg/note, and twice 55µg/note.

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

    PubMed

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

    2004-01-01

    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.

  16. Europium nanoparticle-based high performing immunoassay for the screening of treponemal antibodies.

    PubMed

    Talha, Sheikh M; Hytönen, Jukka; Westhorpe, Adam; Kumar, Sushil; Khanna, Navin; Pettersson, Kim

    2013-01-01

    Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p=0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening

  17. Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

    PubMed Central

    Talha, Sheikh M.; Hytönen, Jukka; Westhorpe, Adam; Kumar, Sushil; Khanna, Navin; Pettersson, Kim

    2013-01-01

    Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a

  18. A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay.

    PubMed

    Baykov, A A; Evtushenko, O A; Avaeva, S M

    1988-06-01

    An improved procedure for phosphate determination based on a highly colored complex of phosphomolybdate and malachite green is described. All necessary reagents are combined in one concentrated solution, making the assay sensitive and convenient. The procedure is based on the finding that the dye is easily soluble and stable in the presence of 6 N acid. The addition of Tween 20 is required to stabilize the dye-phosphomolybdate complex at phosphate concentrations above 10 microM. The time of color development at 25 degrees C is about 3 min. The procedure was adopted to measure alkaline phosphate activity in heterogeneous enzyme immunoassay with rho-nitrophenyl phosphate and pyrophosphate as substrates. In both cases, a 4-fold increase in sensitivity in terms of absorbance readings was obtained compared to the standard method based on rho-nitrophenol measurement. In visual analysis, the gain in sensitivity was as high as 20-fold, due to contrast color change (yellow to greenish blue).

  19. A prospective field evaluation of an enzyme immunoassay: Detection of eastern equine encephalomyelitis virus antigen in pools of Culiseta melanura

    USGS Publications Warehouse

    Scott, T.W.; Olson, J.G.; Lewis, T.E.; Carpenter, J.W.; Lorenz, L.H.; Lembeck, L.A.; Joseph, S.R.; Pagac, B.B.

    1987-01-01

    A prospective field study was conducted to determine the sensitivity and specificity of an enzyme immunoassay (EIA) compared to virus isolation in cell culture for the detection of eastern equine encephalomyelitis (EEE) virus in naturally infected mosquitoes. A total of 10,811 adult female Culiseta melanura were collected in light traps during 1985 from four locations in Maryland. Eastern equine encephalomyelitis virus was isolated from 5 of 495 mosquito pools in African green monkey kidney and baby hamster kidney cell cultures. All five virus-infected pools were detected by the EIA, and all 490 uninfected pools were correctly scored as not containing virus. The EIA did not produce false positive or false negative results. Results support the assertion of previous researchers that the antigen detection EIA is a rapid, sensitive, specific, and simple alternative to traditional bioassays for the detection of EEE virus in mosquitoes.

  20. 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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. Enzyme-triggered tyramine-enzyme repeats on prussian blue-gold hybrid nanostructures for highly sensitive electrochemical immunoassay of tissue polypeptide antigen.

    PubMed

    Xu, Tisen; Zhang, Haiying; Li, Xuegui; Xie, Zhaohui; Li, Xiangyong

    2015-11-15

    A novel sandwich-type electrochemical immunoassay with sensitivity enhancement was developed for quantitative detection of tissue polypeptide antigen (TPA) by coupling with target-induced tyramine signal amplification on prussian blue-gold hybrid nanostructures. The immunosensor was prepared through immobilizing anti-TPA capture antibody on a cleaned screen-printed carbon electrode (SPCE). Prussian blue-gold hybrid nanostructures (PBGNS) labeled with horseradish peroxidase (HRP) and detection antibody were utilized as the signal-transduction tags. Upon target TPA introduction, the sandwiched immunocomplex was formed between capture antibody and detection antibody on the electrode. The carried HRP could trigger the formation of tyramine-HRP repeats on the PBGNS in the presence of H2O2. Using the doped prussian blue as the electron mediator, the conjugated HRP could catalyze the reduction of H2O2. Under the optimal conditions, the catalytic currents increased with the increasing target TPA in the dynamic range from 1.0 pg mL(-1) to 100 ng mL(-1) with a detection limit of 0.3 pg mL(-1). The reproducibility and specificity of the electrochemical immunoassay were acceptable. In addition, the contents of target TPA in nine human serum specimens were evaluated by using the developed electrochemical immunosensor, and the obtained results correlated well with those from commercially enzyme-linked immunosorbent assay (ELISA) method with a correlation coefficient of 0.9975. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Fluorometric procedures for dye tracing

    USGS Publications Warehouse

    Wilson, James F.

    1968-01-01

    This manual describes the current fluorometric procedures used by the U.S. Geological Survey in dye tracer studies such as time of travel, dispersion, reaeration, and dilution-type discharge measurements. The advantages of dye tracing are (1) low detection and measurement limits and (2) simplicity and accuracy in measuring dye tracer concentrations using fluorometric techniques. The manual contains necessary background information about fluorescence, dyes, and fluorometers and a description of fluorometric operation and calibration procedures as a guide for laboratory and field use. The background information should be useful to anyone wishing to experiment with dyes, fluorometer components, or procedures different from those described. In addition, a brief section on aerial photography is included because of its possible use to supplement ground-level fluorometry.

  3. Fluorometric procedures for dye tracing

    USGS Publications Warehouse

    Wilson, James E.; Cobb, E.D.; Kilpatrick, F.A.

    1984-01-01

    This manual describes the current fluorometric procedures used by the U.S. Geological Survey in dye tracer studies such as time of travel, dispersion, reaeration, and dilution-type discharge measurements. The outstanding characteristics of dye tracing are: (1) the low detection and measurement limits, and (2) the simplicity and accuracy of measuring dye tracer concentrations using fluorometric techniques. The manual contains necessary background information about fluorescence, dyes, and fluorometers and a description of fluorometric operation and calibration procedures as a general guide for laboratory and field use. The background information should be useful to anyone wishing to experiment with dyes, fluorometer components, or procedures different from those described. In addition, a brief section is included on aerial photography because of its possible use to supplement ground-level fluorometry. (USGS)

  4. Fluorometric procedures for dye tracing

    USGS Publications Warehouse

    Wilson, James F.; Cobb, Ernest D.; Kilpatrick, F.A.

    1986-01-01

    This manual describes the current fluorometric procedures used by the U.S. Geological Survey in dye tracer studies such as time of travel, dispersion, reaeration, and dilution-type discharge measurements. The advantages of dye tracing are (1) low detection and measurement limits and (2) simplicity and accuracy in measuring dye tracer concentrations using fluorometric techniques. The manual contains necessary background information about fluorescence, dyes, and fluorometers and a description of fluorometric operation and calibration procedures as a guide for laboratory and field use. The background information should be useful to anyone wishing to experiment with dyes, fluorometer components, or procedures different from those described. In addition, a brief section on aerial photography is included because of its possible use to supplement ground-level fluorometry.

  5. Fluorometric procedures for dye tracing

    USGS Publications Warehouse

    Wilson, James E.; Cobb, Ernest D.; Kilpatrick, Frederick A.

    1984-01-01

    This manual describes the current fluorometric procedures used by the U.S. Geological Survey in dye tracer studies such as time of travel, dispersion, reaeration, and dilution-type discharge measurements. The outstanding characteristics of dye tracing are: (1) the low detection and measurement limits, and (2) the simplicity and accuracy of measuring dye tracer concentrations using fluorometric techniques. The manual contains necessary background information about fluorescence, dyes, and fluorometers and a description of fluorometric operation and calibration procedures as a general guide for laboratory and field use. The background information should be useful to anyone wishing to experiment with dyes, fluorometer components, or procedures different from those described. In addition, a brief section is included on aerial photography because of its possible use to supplement ground-level fluorometry.

  6. Enzyme-catalysed deposition of ultrathin silver shells on gold nanorods: a universal and highly efficient signal amplification strategy for translating immunoassay into a litmus-type test.

    PubMed

    Yang, Xinjian; Gao, Zhiqiang

    2015-04-25

    On the basis of enzyme-catalysed reduction of silver ions and consequent deposition of ultrathin silver shells on gold nanorods, a highly efficient signal amplification method for immunoassay is developed. For a model analyte prostate-specific antigen, a 10(4)-fold improvement over conventional enzyme-linked immunosorbent assay is accomplished by leveraging on the cumulative nature of the enzymatic reaction and the sensitive response of plasnomic gold nanorods to the deposition the silver shells.

  7. Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.

    PubMed

    Miners, James Scott; Verbeek, Marcel M; Rikkert, Marcel Olde; Kehoe, Patrick Gavin; Love, Seth

    2008-01-30

    Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimer's disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and cerebrospinal fluid (CSF). The assay uses a neprilysin-specific antibody, previously used in a commercially available ELISA kit, to isolate and immobilise NEP from brain homogenates and CSF, prior to the addition of a fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)). This fluorogenic substrate is ordinarily cleaved by multiple enzymes. We have shown that without the immunocapture phase, even under reaction conditions reported to be specific for neprilysin - i.e. in the presence of thiorphan, at pH above 7 - the fluorogenic peptide substrate does not allow neprilysin activity in brain homogenates and CSF to be discriminated from that of other closely related enzymes. The specificity of the immunocapture enzyme activity assay was confirmed by >80% inhibition of substrate cleavage in brain homogenates and CSF in the presence of thiorphan. The assay allows high-throughput analysis and, critically, also ensures a high level of enzyme specificity even when assaying crude tissue homogenates or CSF.

  8. A competitive enzyme immunoassay Subclass for the determination of total IgG-subclass levels in human serum. Comparison with single radial immunodiffusion.

    PubMed

    Marañón, F; Casanovas, M; Berrens, L; Ollés, J M; Dieguez, M A

    1994-05-01

    A quantitative immunoassay has been developed for the determination of the total IgG subclass levels in human serum. The method is based on an enzyme immunoassay in which IgG subclass proteins IgGx in an unknown serum sample compete with a known quantity of peroxidase (PO)-labelled IgGx in fluid phase for subclass-specific monoclonal antibodies coated to the solid phase of microtiter wells. Using a series of human blood samples an excellent correlation was observed with the IgG-subclass levels determined by single radial immunodiffusion.

  9. 3,3',5,5' - Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay.

    PubMed

    Bos, E S; van der Doelen, A A; van Rooy, N; Schuurs, A H

    1981-01-01

    The use of 3,3',5,5' - tetramethylbenzidine as non-mutagenic chromogen for the end point determination in enzyme-immunoassay (EIA) is described. In sandwich EIAs for HCG and HBsAg and in a competitive EIA for testosterone, the colour yield with TMB was superior to that obtained with o-phenylene diamine (OPD), which was by far the best chromogen for horse-radish peroxidase until now. This led to an improvement of sensitivity and precision of the assays and makes EIA even more competitive with other types of immunoassays.

  10. Multiplexed electrochemical immunoassay of phosphorylated proteins based on enzyme-functionalized gold nanorod labels and electric field-driven acceleration.

    PubMed

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

    2011-09-01

    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 a multienzyme amplification strategy using gold nanorods (AuNRs) as nanocarrier for coimmobilization of horseradish peroxidase (HRP) and detection antibody (Ab(2)) at a high ratio of HRP/Ab(2), 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. Under optimal conditions, this method could simultaneously detect phospho-p53(392), phospho-p53(15), phospho-p53(46), and total p53 ranging from 0.01 to 20 nM, 0.05 to 20 nM, 0.1 to 50 nM, and 0.05 to 20 nM with detection limits of 5 pM, 20 pM, 30 pM, and 10 pM, respectively. Accurate determinations of these proteins in human plasma samples were demonstrated by comparison to the standard ELISA method. The disposable immunosensor array shows excellent promise for clinical screening of phosphorylated proteins and convenient point-of-care diagnostics.

  11. Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species

    PubMed Central

    Sircar, Subhankar; Saurabh, Sharad; Gulati, Baldev R.; Singh, Neeraj; Singh, Arvind Kumar; Joshi, Vinay G.; Banyai, Krisztian; Dhama, Kuldeep

    2016-01-01

    We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831–0.956 at 95% CI = 0.711–1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings. PMID:27391106

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

    PubMed

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

    2016-10-01

    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.

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

    PubMed

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

    2001-01-01

    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.

  14. Rapid determination of recent cocaine use with magnetic particles-based enzyme immunoassays in serum, saliva, and urine fluids.

    PubMed

    Vidal, Juan C; Bertolín, Juan R; Bonel, Laura; Asturias, Laura; Arcos-Martínez, M Julia; Castillo, Juan R

    2016-06-05

    Cocaine is one of the most worldwide used illicit drugs. We report a magnetic particles-based enzyme-linked immunoassay (mpEIA) method for the rapid and sensitive determination of cocaine (COC) in saliva, urine and serum samples. Under optimized conditions, the limits of detections were 0.09ngmL(-1) (urine), 0.15ngmL(-1) (saliva), and 0.06ngmL(-1) COC (human serum). Sensitivities were in the range EC50=0.6-2.5ngmL(-1) COC. The cross-reactivity with the principal metabolite benzoylecgonine (BZE) was only 1.6%. Recovering percentages of doped samples (0, 10, 50, and 100ngmL(-1) of COC) ranged from about 86-111%. Some advantages of the developed mpEIA over conventional ELISA kits are faster incubations, improved reproducibility, and consumption of lower amounts of antibody and enzyme conjugates due to the use of magnetic beads. The reported method was validated following the guidelines on bioanalytical methods of the European Medicines Agency (2011). Unmetabolized COC detection has a great interest in pharmacological, pharmacokinetics, and toxicokinetics studies, and can be used to detect a very recent COC use (1-6h). Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Fluorometric enzymatic assay of L-arginine

    NASA Astrophysics Data System (ADS)

    Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

    2017-01-01

    The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

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

    PubMed

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

    2011-03-15

    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. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Comparison of sensor structures for the signal amplification of surface plasmon resonance immunoassay using enzyme precipitation

    NASA Astrophysics Data System (ADS)

    Yang, Chih-Tsung; Thierry, Benjamin

    2015-12-01

    Surface plasmon resonance (SPR) biosensing has been successfully applied for the label-free detection of a broad range of bioanalytes ranging from bacteria, cell, exosome, protein and nucleic acids. When it comes to the detection of small molecules or analytes found at low concentration, amplification schemes are desirable to enhance binding signals and in turn increase sensitivity. A number of SPR signal amplification schemes have been developed and validated; however, little effort has been devoted to understanding the effect of the SPR sensor structures on the amplification of binding signals and therefore on the overall sensing performance. The physical phenomenon of long-range SPR (LRSPR) relies on the propagation of coupled surface plasmonic waves on the opposite sides of a nanoscale-thick noble metal film suspended between two dielectrics with similar refractive indices. Importantly, as compared with commonly used conventional SPR (cSPR), LRSPR is not only characterized by a longer penetration depth of the plasmonic waves in the analyzed medium but also by a greater sensitivity to bulk refractive index changes. In this work, an immunoassay signal amplification platform based on horseradish peroxidase (HRP) catalyzed precipitation was utilized to investigate the sensing performance with regards to cSPR and LRSPR. The enzymatic precipitation of 3, 3'-diaminobenzidine tetrahydrochloride (DAB)/H2O2 was used to amplify SPR signals. The structure-function relationship of cSPR and LRSPR sensors is presented for both standard refractometric measurements and the enzymatic precipitation scheme. Experimental data shows that despite its inherent higher sensitivity to bulk refractive index changes and higher figure of merit, LRSPR was characterized by a lower angular sensitivity in the model enzymatic amplification scheme used here.

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

    USDA-ARS?s Scientific Manuscript database

    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. Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal.

    PubMed

    Shu, Mei; Xu, Yang; Liu, Xing; Li, Yanping; He, Qinghua; Tu, Zhui; Fu, Jinheng; Gee, Shirley J; Hammock, Bruce D

    2016-06-14

    A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB1 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 FB1 were 2.69 and 0.35 ng mL(-1), respectively, with a linear range of 0.93-7.73 ng mL(-1). The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL(-1), and the IC50 was 0.89 ± 0.09 ng mL(-1) with a linear range of 0.29-2.68 ng mL(-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 FB1 contamination in cereals. The Ab2β-Nb-AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Diagnosis of acute toxoplasmosis by an enzyme immunoassay for specific immunoglobulin m antibodies.

    PubMed Central

    Wielaard, F; van Gruijthuijsen, H; Duermeyer, W; Joss, A W; Skinner, L; Williams, H; van Elven, E H

    1983-01-01

    A recently developed enzyme-linked immunosorbent assay for detection of immunoglobulin M (IgM) class antibodies to Toxoplasma gondii was evaluated with respect to specificity and sensitivity. By using an antibody capture principle and F(ab')2 conjugates, interference of rheumatoid factors was absent. No cross-reactions with anti-toxoplasma IgG occurred, and no interference with antinuclear antibodies was found. A large-scale study with about 1,500 clinical specimens revealed a 100% specificity. By testing 79 sera from patients with acute-phase acquired toxoplasmosis, sensitivity was found to be 97%. In routine clinical practice, the IgM-enzyme-linked immunosorbent assay proved to be a more sensitive tool for diagnosis than the immunofluorescent-antibody test. The course of IgM-enzyme-linked immunosorbent assay antibodies in acute patients was studied; IgM reached peak levels within 1 month after onset of illness, and could be demonstrated up to an average of 8 months after onset. PMID:6874915

  1. Comparison of a lateral flow milk progesterone test with enzyme immunoassay as an aid for reproductive status determination in cows.

    PubMed

    Waldmann, A; Raud, A

    2016-03-12

    The lateral flow test (LFT) is an immunochromatographic method that utilises an immunostrip for non-laboratory diagnostic purposes. The present study evaluated a milk progesterone LFT against the enzyme immunoassay (EIA) to confirm oestrus and a non-pregnancy diagnosis. In total, 277 milk samples from 70 cows were analysed, collected on the day of artificial insemination and at 19 days, 21 days and 24 days post insemination. The level of accuracy of the LFT compared with the EIA was 95.0 per cent for milk samples containing <2 ng/ml progesterone and 97.0 per cent for milk samples containing >10 ng/ml progesterone. The validation of oestrus by the LFT was 98.6 per cent accurate using 2 ng/ml progesterone as the EIA estimate for oestrus. The test performance for a non-pregnancy diagnosis was subject to the day of milk sampling, showing the highest accuracy on day 24 post insemination for both tests. When optimised for maximum specificity, and compared with rectal palpation, the LFT had a sensitivity and specificity for non-pregnancy diagnosis on day 24 post insemination of 75.0 per cent and 100.0 per cent, respectively, with an overall accuracy of 84.4 per cent. The corresponding characteristics for the quantitative EIA were 85.0 per cent, 100.0 per cent and 90.6 per cent, respectively. The LFT results compared favourably with the quantitative milk progesterone EIA.

  2. Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay

    PubMed Central

    de Aguirre, Liliana; Hurst, Steven F.; Choi, Jong Soo; Shin, Jong Hee; Hinrikson, Hans Peter; Morrison, Christine J.

    2004-01-01

    We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor, differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium, Exophiala, Candida, Fusarium, Mucor, Paecilomyces, Penicillium, Rhizopus, Scedosporium, Sporothrix, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species (A. flavus, A. fumigatus, A. nidulans, A. niger, A. terreus, A. ustus, and A. versicolor) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi. PMID:15297489

  3. Comparison of free plasma metanephrines enzyme immunoassay with (131)I-MIBG scan in diagnosis of pheochromocytoma.

    PubMed

    Gao, Yun-Chao; Lu, Han-Kui; Luo, Quan-Yong; Chen, Li-Bo; Ding, Ying; Zhu, Rui-Sen

    2008-06-01

    Measurement of free plasma metanephrines (metanephrine and normetanephrine), usually performed by high-performance liquid chromatography with electrochemical detection (HPLC-ECD), has been recommended as the single biochemical test of choice for the diagnosis of pheochromocytoma. Alternatively, a widely available, simple means to measure these biomarkers with enzyme immunoassay (EIA) needs to be studied. The aim of this study was to investigate the diagnostic efficacy of such a method in comparison with (131)I-metaiodobenzylguanidine (MIBG) whole body scan (WBS) in patients with pheochromocytoma. We enrolled patients undergoing (131)I-MIBG WBS due to clinical findings suggestive of pheochromocytoma (n = 45), and patients with primary hypertension (n = 36). All subjects had blood tests for free plasma metanephrine (MN) and normetanephrine (NM) with a commercially available EIA kit. WBS was positive in 30 pheochromocytoma patients and negative in 15 refuted ones, with 100% accuracy. The sensitivity, specificity and accuracy of MN and NM in combination (either or both positive) were 96.7%, 86.3% and 90.1%, showing comparable diagnostic performance both to (131)I-MIBG WBS (all p > 0.1), and also to the same markers measured with HPLC-ECD reported in the literature. These results showed that the EIA method may be eligible as an alternative to HPLC-ECD for plasma metanephrine determination in the identification of pheochromocytoma.

  4. Evaluation of an enzyme immunoassay for plasma-free metanephrines in the diagnosis of catecholamine-secreting tumors.

    PubMed

    Procopiou, Michel; Finney, Hazel; Akker, Scott A; Chew, Shern L; Drake, William M; Burrin, Jacky; Grossman, Ashley B

    2009-07-01

    To define the test characteristics of an enzyme immunoassay (EIA) for plasma-free metanephrines (metanephrine and normetanephrine) in the diagnosis of pheochromocytoma and paraganglioma. Prospective observational design from a single University Hospital. Twenty-four hour urine for catecholamines and plasma for free metanephrines were collected from patients with a clinical suspicion of pheochromocytoma or paraganglioma. Patient records were reviewed for clinical data, follow-up, imaging and laboratory results to establish or exclude the diagnosis of pheochromocytoma. Out of 178 consecutive patients, 10 had a paraganglioma and 12 had a pheochromocytoma: 156 were finally judged not to harbour active tumors and were therefore considered as controls. The main outcome measure was the diagnosis or exclusion of paraganglioma or pheochromocytoma and test characteristics of plasma-free metanephrines measured by EIA. Urinary epinephrine had a sensitivity of 45.5% and norepinephrine a sensitivity of 75% (98.8% specificity) for the diagnosis of pheochromocytoma. Plasma-free metanephrine and normetanephrine both had a sensitivity of 66.7% and a specificity of 100%, but when combined (either positive) they demonstrated a 91.7% sensitivity with a preserved specificity of 100%. For the diagnosis of paraganglioma, urinary norepinephrine gave slightly better results than plasma-free metanephrines, but combined testing was of no additional value. Plasma-free metanephrines measured by EIA have better diagnostic test characteristics than urinary catecholamines in the diagnosis of pheochromocytoma. The EIA offers a simple and effective measurement of plasma-free metanephrines.

  5. Fluoroimmunoassay for detection of rubella-specific immunoglobulin M: comparison with indirect enzyme immunoassay and mu-chain capture.

    PubMed Central

    Echevarria, J M; de Ory, F; Najera, R

    1985-01-01

    The performance of a commercially-available method of fluoroimmunoassay (Rubella M FIAX, International Diagnostic Technology, Santa Clara, Calif.), designed for the detection of rubella-specific immunoglobulin M, was tested with 137 selected sera, including 52 from cases of primary rubella, 29 from healthy pregnant women, 21 containing rheumatoid factor, and 35 from cases of infectious mononucleosis (IM) caused by Epstein-Barr virus. The results were compared with those obtained by commercial indirect enzyme immunoassay (EIA) and EIA anti-mu chain capture tests. The fluoroimmunoassay technique showed a satisfactory level of sensitivity, but low values had to be interpreted with caution as false-positive results were detected with sera with rheumatoid factor and from IM cases, even after preliminary treatment of sera with the anti-human immunoglobulin G antisera provided in the kit. On the other hand, no false-positive results in the analysis of IM sera were seen in the EIA anti-mu chain capture method. Because of its sensitivity and specificity, we recommend the use of the latter technique for the diagnosis of primary rubella. PMID:2995439

  6. Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples.

    PubMed

    Xu, Zhen-Lin; Sun, Wen-Jia; Yang, Jin-Yi; Jiang, Yue-Ming; Campbell, Katrina; Shen, Yu-Dong; Lei, Hong-Tao; Zeng, Dao-Ping; Wang, Hong; Sun, Yuan-Ming

    2012-03-07

    Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.

  7. Comparison of PCR, enzyme immunoassay and conventional culture for adenovirus detection in bone marrow transplant patients with hemorrhagic cystitis.

    PubMed

    Raboni, S M; Siqueira, M M; Portes, S R; Pasquini, R

    2003-08-01

    Adenovirus-associated hemorrhagic cystitis (HC) has become a recognized sequel of immunosuppression. The diagnosis of viral infection is usually determined by viral cultures. Analysis of different diagnostic methods for adenovirus (AdV) detection in bone marrow transplant patients with hemorrhagic cystitis. We describe a prospective study for AdV detection in the urine of patients with hematuria in the first 100 days after bone marrow transplant (BMT), comparing different laboratory techniques, PCR, enzyme immunoassay (EIA) and conventional culture. A total of 143 urine samples were analyzed, 75 collected in the pre-transplant period with and without hematuria and 68 post-transplant, only with microscopic or macroscopic hematuria. After BMT, hematuria occurred in 38.9% of patients, being more frequent in unrelated donor transplants. AdV was isolated in one pre-transplant patient without symptoms and in three post-transplant patients with HC grades 3 and 4 (severe), who were in month 2 or 3 post-transplant. Compared to culture as the gold standard, the accuracy, specificity and sensitivity of EIA were 95, 30 and 100% and for PCR were 63, 100 and 60%, respectively. We concluded that despite technical difficulties and the long time that elapsed before results were obtained, cell culture still remains the best method for adenovirus detection in the urine of patients with hemorrhagic cystitis.

  8. Evaluation of an enzyme immunoassay for the detection of central nervous system tissue contamination at the slaughterhouse.

    PubMed

    Bozzetta, E; Nappi, R; Ru, G; Negro, M; Maurella, C; Caramelli, M

    2006-09-01

    To protect public health from bovine spongiform encephalopathy, European Commission Regulation EC 1139/2003 on monitoring programs and specified risk material requires that as of 1 October 2003, each member state has in place a sampling plan with an appropriate laboratory test to detect central nervous system (CNS) tissue in bovine head meat harvested at slaughterhouses or cutting plants. With this study, we wanted to evaluate the accuracy and reliability of an enzyme immunoassay, the RIDASCREEN Risk Material 10/5, in targeting a CNS-specific marker, the glial fibrillary acidic protein. A receiver operating characteristics curve was plotted to identify the best cutoff of CNS concentration. Reliability was calculated by Cohen's kappa on data from two diagnostic sessions. Test performance showed high sensitivity and specificity (97.9 and 97.4%, respectively) for a cutoff value between positive and negative at a CNS concentration of 0.049%; reliability of test precision was also very good. When these criteria are applied, the RIDASCREEN Risk Material 10/5 test appears to be a reliable tool for monitoring CNS tissue contamination in meat. This diagnostic procedure should therefore be recommended for national application in monitoring programs.

  9. Validation of a commercial enzyme immunoassay for detection of Clostridium difficile toxins in feces of horses with acute diarrhea.

    PubMed

    Medina-Torres, C E; Weese, J S; Staempfli, H R

    2010-01-01

    Clostridium difficile infection (CDI) is a recognized cause of colitis in the horse. Identification of its toxins is important for management of individual cases and for prevention of transmission and zoonosis. In humans, CDI diagnosis is performed with enzyme immunoassays, none of which have been validated for horses. (1) Establish which test for CDI diagnosis was more frequently used by diagnostic laboratories, (2) determine the identified test's performance, sensitivity, and specificity, and (3) validate its use in diarrheic horses. Samples were obtained from 72 horses presented with acute diarrhea and hospitalized at the Ontario Veterinary College, University of Guelph. A survey was conducted to establish which of the tests for CDI diagnosis in horses is most commonly used throughout North America. A questionnaire was sent to all laboratories registered in the Veterinary Infection Control Society and the American Association of Veterinary Laboratory Diagnosticians. The performance of the test was evaluated by comparison to a cell cytotoxicity assay (CTA), the accepted Gold Standard for C. difficile toxin detection. The Techlab C. difficile Tox A/B II ELISA was the most frequently used test. Compared with the CTA, no significant difference was observed, and a good level of agreement (93%) was obtained. The diagnostic performance of the ELISA test was adequate (84% sensitivity and 96% specificity). Results demonstrate that the Techlab C. difficile Tox A/B II ELISA is a reliable, adequate, and practical tool for identification of C. difficile toxins in horse feces.

  10. Laboratory diagnosis of Mycoplasma pneumoniae infection. 1. Direct detection of antigen in respiratory exudates by enzyme immunoassay.

    PubMed Central

    Kok, T. W.; Varkanis, G.; Marmion, B. P.; Martin, J.; Esterman, A.

    1988-01-01

    Direct and indirect antigen capture enzyme immunoassays (Ag-EIA) have been developed for the detection of Mycoplasma pneumoniae in nasopharyngeal aspirates or sputum from respiratory infection. The sensitivity of the two Ag-EIA were similar, but the indirect method using polyclonal rabbit and guinea-pig antisera was more convenient. The Ag-EIA had a detection limit of 10(4-4.5) colony-forming units/ml of sample. It was specific for M. pneumoniae and gave a low level response with M. genitalium. There were no cross-reactions with 10 other species of mycoplasmas. Tests with a wide range of bacteria and chlamydia group antigen, representing agents sometimes found in the respiratory tract, were also negative. At the current level of development, the Ag-EIA detected about 90% of specimens that were also positive for culture; 43% of specimens from culture-negative--seropositive patients gave a positive result. The overall pattern of results indicated that while antigen detection is a quick and effective substitute for the slow culture method, serological examination for specific IgM antibody is also necessary to give a complete diagnostic coverage. PMID:3145891

  11. Quantification of amebae specific antibodies as "Multiple of normal activity (MONA)" with a standardized enzyme immunoassay (EIA).

    PubMed

    Funke, M; Felgner, P; Geister, R

    1981-12-01

    Sera of 853 returnees from tropical countries, of 24 cases with amebic liver abscess and of 172 nonexposed German individuals were tested for antibodies to E. histolytica in the enzyme immunoassay (EIA). Antibody results were expressed as "multiple of normal activity (MONA)". The qualification of the EIA as a routine screening procedure for amebae specific antibodies was investigated and compared to that of the complement fixation test. Based on the symmetric frequency distribution of results from the 172 non-exposed controls the upper one sided limits for 95% (less than 2.8 MONA) and 99% (less than 4.2 MONA) specificity were determined. Routine results below the 95% specificity limit were considered "inconspicuous", such between both limits "borderline" and all MONA values exceeding the 99% specificity limit "conspicuous". The intention was to thereby secure a high degree of sensitivity for amebae antibody in the test. Cases with clinically confirmed liver abscess revealed a one sided lower 95% sensitivity limit (greater than or equal to 1.2 MONA), far above the onset of the defined sensitivity thresholds for conspicuous MONA values.

  12. Detection of bovine viral diarrhea virus by amplification on polycation-treated cells followed by enzyme immunoassay.

    PubMed

    Gogorza, L M; Morán, P E; Larghi, J L; Braun, M; Esteban, E N

    2006-01-01

    A bovine viral diarrhea virus (BVDV) amplification method combined with an enzyme immunoassay was developed to detect BVDV antigens in seropositive cattle. Reconstitution assays conducted by adding decreasing amounts of BVDV (Singer strain) to Madin-Darby bovine kidney (MDBK) cells showed that the sensitivity threshold of the combined assay was 10(-7) TCID50. BVDV amplification was carried out in polycation (DEAE-Dextran and polybrene)-treated MDBK cells. Treated cells were able to replicate both ether-treated virus and neutralizing antibody-coated virus. Ammonium chloride decreased virus replication in polycation-treated cells, suggesting viral penetration by endocytosis. BVDV detection was tested in leukocytes from 104 seropositive cattle from 2 unvaccinated commercial closed dairy herds with high seroprevalence. Lysates and co-cultures of peripheral blood leukocytes (PBL) were tested, directly or after up to 6 blind passages in normal or polycation-treated cells. BVDV was detected in 10/104 cattle after only one co-culture of PBL in treated cells. No virus was detected in whole blood or plasma samples. BVDV positive and negative cattle were retested three times, achieving consistent results. The finding of immune carriers supports the possibility that these animals may constitute an epidemiological risk.

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

    PubMed

    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

    2015-09-01

    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.

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

    PubMed

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

    2002-04-01

    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.

  15. Development and validation of a simple, sensitive enzyme immunoassay (EIA) for quantification of prolactin in buffalo plasma.

    PubMed

    Roy, K S; Prakash, B S

    2007-02-01

    A simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for prolactin quantification in buffalo plasma (on a microtitreplate) using the biotin-streptavidin-peroxidase amplification and immobilized antiserum in a competitive assay. Prolactin standards (range: 5-5000 pg/(well 50 microL)) were prepared in hormone-free plasma collected from minimal stress non-lactating buffalo heifers in temperate weather. The sensitivity of the EIA procedure was 5 pg/(well 50 microL) (corresponds to 0.1 ng/mL plasma); the 50% relative binding sensitivity occurred at 160 ng/(well 50 microL). Plasma volumes for the EIA, viz. 12.5, 25, and 50 microL, did not influence the shape of standard curve. A parallelism test was carried out to compare the endogenous buffalo plasma prolactin with bovine prolactin standard. To validate the assay biologically, 11 Murrah buffaloes were given a third-generation antiprolactin (Norprolac; 10 mg/animal, i.m.). Blood samples were collected 1 d prior to the start of Norprolac administration and continued up to seventh day in an Ovsynch treatment program. In all animals, there were abrupt declines in prolactin concentrations following Norprolac treatments, which confirmed the biological validation of the EIA. After development and validation of EIA procedure, the concentration of plasma prolactin was determined efficiently in samples collected during both summer and winter samples.

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

    PubMed Central

    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

    2015-01-01

    The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma 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. PMID:26517651

  17. Antigenic diversity of the serotype antigen complex of Neisseria gonorrhoeae: analysis by an indirect enzyme-linked immunoassay.

    PubMed Central

    Johnston, K H

    1980-01-01

    An indirect enzyme-linked immunoassay (ELISA) has been developed to analyze the antigenic profile of the outer membrane serotype complex of Neisseria gonorrhoeae. Antisera raised in rabbits to serotype-specific vesicles (SSV) reacted primarily with homologous SSV; however, there was significant cross-reactivity (less than 50%) with heterologous SSV. N. meningitidis SSV cross-reacted with all antigonococcal SSV but at a lower degree (less than 20%). Preimmune sera did not cross-react significantly with all antigonoccoccal SSV. The sensitivity of the ELISA was enhanced when the integral SSV proteins 1a and 2 were used as adsorbed antigen. Heterologous anti-SSV cross-reacted slightly, having ELISA values less than 15% of the homologous reaction. Antisera prepared by immunoabsorbent affinity columns were highly specific. Homologous affinity anti-SSV reacted only with proteins 1a and 2. The reaction of immune sera was inhibited by homologous proteins 1a and 2; lipopolysaccharide and proteins 1a and 2 isolated from heterologous serotypes did not inhibit the reaction. The reaction of affinity-purified antisera could be inhibited only by homologous protein 1a. By the use of affinity-purified antisera, a specific and highly sensitive ELISA was developed to analyze the antigenic profile of strains of N. gonorrhoeae. PMID:6769815

  18. A synthetic parvovirus B19 capsid protein can replace viral antigen in antibody-capture enzyme immunoassays.

    PubMed

    Kock, W C

    1995-09-01

    To establish a renewable source of parvovirus B19 antigens for diagnostic tests, gene sequences for the viral capsid proteins, VP1 and VP2, were cloned into baculovirus expression vectors and the recombinant viruses used to infect Sf9 insect cells. Cell lysates examined by immunoblotting demonstrated reactive proteins corresponding to the expected sizes of native VP1 (83 kDa) and VP2 (58 kDa). The VP2 protein was produced efficiently in quantity and self-assembled into empty capsids as shown by density equilibration in a CsCl step gradient. The VP2 protein was purified and used as an antigen in antibody-capture enzyme immunoassays for the detection of B19 IgG and IgM antibodies. Compared to a standard antibody-capture EIA based on whole viral antigen, the VP2-EIA gave a sensitivity of 100% and specificity of 97% in detection of B19 IgM in 138 patients suspected of B19 infection. No IgM-positive specimens were missed. IgG detection yielded a sensitivity of 100% and specificity of 96% in the same population. Recombinant VP2 capsid proteins expressed in baculovirus-infected insect cells can substitute for serum-derived B19 virus in standard antibody-capture EIA for the detection of B19 IgG and IgM with comparable results.

  19. Evaluation of an enzyme immunoassay for detection of Chlamydia psittaci in vaginal secretions, placentas, and fetal tissues from aborting ewes.

    PubMed

    Sanderson, T P; Andersen, A A

    1989-10-01

    A commercially available enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in human urogenital and conjunctival specimens was compared with isolation in cell culture for the detection of Chlamydia psittaci in vaginal and placental swabs from aborting ewes and swabs of aborted fetal tissues. The EIA on vaginal swabs collected from 10 ewes experimentally infected with C. psittaci had a sensitivity of 85.7% and a specificity of 85.7%. Vaginal swabs collected at the time of abortion or within 3 days were the best samples for detection of chlamydial infection. The 29 vaginal swabs collected during this period from experimentally infected ewes were all strongly EIA-positive, and chlamydia were isolated from 28. The EIA on vaginal swabs from 78 field cases of abortion had a sensitivity of 78.0% and a specificity of 76.8%. The EIA on swabs of cotyledons from 65 placentas had a sensitivity of 100% and a specificity of 75.0% compared with isolation in cell culture. The EIA on 57 swabs of fetal tissues or body fluids from 10 aborted fetuses or weak lambs from experimentally infected ewes had a sensitivity of 26.6% and a specificity of 88.1% compared with isolation in cell culture. Limitations of the EIA are discussed.

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

    PubMed

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

    2015-11-01

    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%, P<.001, specificity 98% vs. 84%, P = .014). Fourteen specimens from patients with clinically characterized histoplasmosis (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. Validation of a shed skin corticosterone enzyme immunoassay in the African House Snake (Lamprophis fuliginosus) and its evaluation in the Eastern Massasauga Rattlesnake (Sistrurus catenatus catenatus).

    PubMed

    Berkvens, Charlene N; Hyatt, Crystal; Gilman, Christine; Pearl, David L; Barker, Ian K; Mastromonaco, Gabriela F

    2013-12-01

    This study investigates the use of an enzyme immunoassay to measure keratin glucocorticoid concentrations in reptilian shed skins. Keratin glucocorticoid concentrations were compared to fecal glucocorticoid concentrations during the period of keratin growth in the African House Snake (Lamprophis fuliginosus) and the Eastern Massasauga Rattlesnake (Sistrurus catenatus catenatus). Biochemical validation was performed for the shed skin and fecal corticosterone enzyme immunoassays in the African House Snake. Biological and physiological validations were attempted in the African House Snake. A statistically significant positive association was detected between shed skin corticosterone and the mean fecal corticosterone metabolites from 3 weeks before to 1 week after the previous ecdysis in the African House Snake. A statistically significant difference was not detected between the shed skin corticosterone concentrations of the minimally handled control and the weekly handled (or experimentally stressed) African House Snakes. Adrenocorticotropic hormone stimulation did not result in the physiological validation anticipated for shed skin corticosterone concentrations in the African House Snake.

  2. New enzyme immunoassay for detecting total, type I, and type II intrinsic factor antibodies.

    PubMed

    Waters, H M; Smith, C; Howarth, J E; Dawson, D W; Delamore, I W

    1989-03-01

    A method for the detection of total, type I, and type II intrinsic factor antibodies was devised. The technique comprises a two-site solid phase enzyme linked immunosorbent assay (ELISA), with human intrinsic factor conjugated with horseradish peroxidase as label and attached to polystyrene tubes as solid phase. One conjugation provides sufficient material to assay more than 10,000 patient samples. The label proved stable during the course of this evaluation and was still in use more than 12 months after preparation. When applied to 45 serum samples from cases of pernicious anaemia, intrinsic factor antibodies were shown in 30 (67%). Simplicity, high capacity, low cost and label stability, combined with relatively high clinical sensitivity make the method suitable for cost effective screening of large numbers of samples. Simple modifications to the basic assay reagents permitted type I and type II intrinsic factor antibodies to be differentiated.

  3. Development of an indirect enzyme linked immunoassay for abscisic acid. [Pisum sativum

    SciTech Connect

    Ross, G.S.; Elder, P.A.; McWha, J.A.; Pearce, D.; Pharis, R.P.

    1987-09-01

    AN INDIRECT METHOD OF ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) IS REPORTED FOR ABSCISIC ACID (ABA), UTILIZING A THYROGLOBULIN-ABA CONJUGATE FOR COATING WELLS. THE ASSAY CAN USE COMMERCIALLY AVAILABLE MONOCLONAL ANTIBODIES, IS SENSITIVE TO AS LITTLE AS 20 PICOGRAMS ABA PER WELL, AND IS MUCH MORE CONSERVATIVE OF ANTIBODY THAN DIRECT METHODS. THE MOST DILUTE ABA STANDARDS DID NOT RETAIN THEIR ANTIGENICITY DURING STORAGE, SO ABA STANDARD SETS WERE DILUTED IMMEDIATELY PRIOR TO USE. THE INDIRECT ELISA WAS USED SUCCESSFULLY TO ESTIMATE ABA CONCENTRATIONS IN DEVELOPING COTYLEDONS OF PISUM SATIVUM L., AFTER ONLY LITTLE PRELIMINARY PURIFICATION. IT WAS VALIDATED FOR THIS TISSUE THROUGH THE USE OF GAS CHROMATOGRAPHY-ELECTRON CAPTURE DETECTION (GC-EC), AND CAPILLARY GC-SELECTED ION MONITORING (GC-MS-SIM) USING LABELLED ABA AS AN INTERNAL STANDARD. FULL SPECTRUM GC-MASS SPECTROMETRY WAS ALSO USED TO VERIFY THAT ABA WAS PRESENT IN A SAMPLE ASSAYED QUANTITATIVELY BY BOTH ELISA AND GC-MS-SIM.

  4. An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor.

    PubMed

    Joung, Hyou-Arm; Oh, Young Kyoung; Kim, Min-Gon

    2014-03-15

    Microfluidic integrated enzyme immunosorbent assay (EIA) sensors are efficient systems for point-of-care testing (POCT). However, such systems are not only relatively expensive but also require a complicated manufacturing process. Therefore, additional fluidic control systems are required for the implementation of EIAs in a lateral flow immunosensor (LFI) strip sensor. In this study, we describe a novel LFI for EIA, the use of which does not require additional steps such as mechanical fluidic control, washing, or injecting. The key concept relies on a delayed-release effect of chemiluminescence substrates (luminol enhancer and hydrogen peroxide generator) by an asymmetric polysulfone membrane (ASPM). When the ASPM was placed between the nitrocellulose (NC) membrane and the substrate pad, substrates encapsulated in the substrate pad were released after 5.3 ± 0.3 min. Using this delayed-release effect, we designed and implemented the chemiluminescent LFI-based automatic EIA system, which sequentially performed the immunoreaction, pH change, substrate release, hydrogen peroxide generation, and chemiluminescent reaction with only 1 sample injection. In a model study, implementation of the sensor was validated by measuring the high sensitivity C-reactive protein (hs-CRP) level in human serum.

  5. Epidemiologic survey of sylvatic plague by serotesting coyote sentinels with enzyme immunoassay.

    PubMed

    Willeberg, P W; Ruppanner, R; Behymer, D E; Higa, H H; Franti, C E; Thompson, R A; Bohannan, B

    1979-09-01

    The geographic distribution and areas of high sylvatic plague activity in California were verified by using coyotes (Canis latrans) as sentinel animals. Antibody levels against Yersinia pestis were tested using the enzyme-labelled antibody (ELA) test and the microtiter passive hemagglutination and hemagglutination inhibition. A survey using the ELA test indicated that the overall antibody prevalence among 143 coyotes was 21%. By geographic regions, the highest antibody prevalence was 27% among coyotes from mountain areas on the northern and eastern borders of the state. This was followed by 19% in the central coastal area and 12% in the central valley. Areas with a high prevalence of seropositive coyotes or high antibody levels in individual coyotes matched the four areas of human plague exposures reported in 1977 and 1978. These areas included the central Sierra mountains adjacent to Lake Tahoe, southeastern Kern County, the central coastal area and Scott Valley near the Oregon border. The ELA test appears to be a promising tool for future epidemiologic studies of plague.

  6. New screening system for simultaneous determination of two marker proteins by homogeneous enzyme immunoassay.

    PubMed

    Hoshino, N; Miyai, K

    1992-03-01

    To save time and labour in mass screening, by detecting two marker proteins on one specimen using only one test. alpha Fetoprotein and ferritin were chosen to demonstrate the principal of this system. The assay reagents were horseradish peroxidase (HRP)-labelled anti-alpha fetoprotein and HRP-labelled anti-ferritin antibodies. After the serum sample had been incubated with these reagents the substrate for HRP was added and the absorbance measured. An absorbance value below the cutoff point indicated that both parameters were within normal limits; a value above the cutoff point indicated that at least one of the two parameters was abnormally high. Fifty sera from healthy Japanese subjects were assayed by the simultaneous assay method. All samples gave absorbancy values below the cutoff point. Fifty serum samples from patients with high alpha fetoprotein concentrations (over 20 ng/ml) and 50 samples with high ferritin concentrations (over 200 ng/ml) were also assayed. The absorbancy values of all samples with high alpha fetoprotein concentrations, and all but one sample with high ferritin concentrations gave values above the cutoff point. Although this homogeneous enzyme assay method was applied to the combination of alpha fetoprotein and ferritin, it could be used in mass screening for any other combination of two markers.

  7. New screening system for simultaneous determination of two marker proteins by homogeneous enzyme immunoassay.

    PubMed Central

    Hoshino, N.; Miyai, K.

    1992-01-01

    AIMS: To save time and labour in mass screening, by detecting two marker proteins on one specimen using only one test. METHODS: alpha Fetoprotein and ferritin were chosen to demonstrate the principal of this system. The assay reagents were horseradish peroxidase (HRP)-labelled anti-alpha fetoprotein and HRP-labelled anti-ferritin antibodies. After the serum sample had been incubated with these reagents the substrate for HRP was added and the absorbance measured. An absorbance value below the cutoff point indicated that both parameters were within normal limits; a value above the cutoff point indicated that at least one of the two parameters was abnormally high. RESULTS: Fifty sera from healthy Japanese subjects were assayed by the simultaneous assay method. All samples gave absorbancy values below the cutoff point. Fifty serum samples from patients with high alpha fetoprotein concentrations (over 20 ng/ml) and 50 samples with high ferritin concentrations (over 200 ng/ml) were also assayed. The absorbancy values of all samples with high alpha fetoprotein concentrations, and all but one sample with high ferritin concentrations gave values above the cutoff point. CONCLUSIONS: Although this homogeneous enzyme assay method was applied to the combination of alpha fetoprotein and ferritin, it could be used in mass screening for any other combination of two markers. PMID:1372918

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

    PubMed

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

    2013-09-27

    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.

  9. Use of enzyme immunoassay for large water-quality surveys of major herbicides

    SciTech Connect

    Thurman, E.M.; Aga, D.S.; Zimmerman, L.R.; Goolsby, D.A.

    1996-10-01

    Commercially available enzyme-linked immunosorbent assay (ELISA) was used for the determination of major herbicides in several large water-quality surveys of surface water, rainwater, and ground water throughout the United States. The ELISA results were compared with gas chromatography/mass spectrometry (GC/MS) for accuracy and cross reactivity. In total, five compounds were analyzed: alachlor, atrazine, cyanazine, metolachlor, and (2,4-dichlorophenoxy) acetic acid (2,4-D). Results indicated that the ELISA and GC/MS results were comparable for cyanazine and metolachlor. The atrazine ELISA correlated well with GC/MS for surface- and ground-water samples from the central United States but did not correlate with samples from Texas where the cotton triazine, prometryn, is used. Results using the alachlor ELISA were poor because of cross reactivity with the metabolite, alachlor ethane-sulfonic acid. The ELISA for (2,4-dichlorophenoxy) acetic acid was insensitive at concentrations that occur in most surface water.

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

    PubMed Central

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

    2016-01-01

    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

  11. Factors affecting the detection of cytomegalovirus in urine by sandwich enzyme immunoassays.

    PubMed

    el-Mekki, A; Al-Nakib, W; Bibi, R

    1987-01-01

    Some factors influencing the detection of human cytomegalovirus (HCMV) in urine were investigated employing 2 enzyme-linked immunosorbent assays (ELISAs); one utilised anti-CMV DNA polymerase while the other anti-CMV mouse monoclonals as the detecting antibodies. The use of anti-CMV DNA polymerase was found to be superior in detecting HCMV in both urine and tissue culture fluids than anti-CMV monoclonals. Furthermore, alkaline phosphatase conjugates produced much lower background than did peroxidase conjugates. In reconstruction experiments, the extremes of pH in the urine clearly had an adverse effect on the detection rate of extracellular virus. pH correction of urines to neutrality improved the detection rate considerably. On the other hand, pH correction had little effect on the detection rate of intracellular HCMV in urine, although it was improved when specimens were subjected to repeated cycles of freeze-thawing, ultrasonication, and storage at 4 degrees C. It was concluded that, in addition to the factors investigated which all appear to affect virus detection rate, there may well be additional factors that interfere with CMV detection in the urine by ELISA particularly with intracellular virus.

  12. Rapid screening of flonicamid residues in environmental and agricultural samples by a sensitive enzyme immunoassay.

    PubMed

    Liu, Zhenjiang; Zhang, Zhen; Zhu, Gangbing; Sun, Jianfan; Zou, Bin; Li, Ming; Wang, Jiagao

    2016-05-01

    A fast and sensitive polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the analysis of flonicamid in environmental and agricultural samples. Two haptens of flonicamid differing in spacer arm length were synthesized and conjugated to proteins to be used as immunogens for the production of polyclonal antibodies. To obtain most sensitive combination of antibody/coating antigen, two antibodies were separately screened by homologous and heterologous assays. After optimization, the flonicamid ELISA showed that the 50% inhibitory concentration (IC50 value) was 3.86mgL(-1), and the limit of detection (IC20 value) was 0.032mgL(-1). There was no cross-reactivity to similar tested compounds. The recoveries obtained after the addition of standard flonicamid to the samples, including water, soil, carrot, apple and tomato, ranged from 79.3% to 116.4%. Moreover, the results of the ELISA for the spiked samples were largely consistent with the gas chromatography (R(2)=0.9891). The data showed that the proposed ELISA is an alternative tool for rapid, sensitive and accurate monitoring of flonicamid in environmental and agricultural samples.

  13. Comparative evaluation of the WHO and DAKOPATTS enzyme-linked immunoassay kits for rotavirus detection.

    PubMed Central

    Flewett, T. H.; Arias, C. F.; Avendano, L. F.; Ghafoor, A.; Mathan, M. M.; Mendis, L.; Moe, K.; Bishop, R. F.

    1989-01-01

    Faeces obtained from 1,163 children (including 66 newborn babies) were analysed in parallel for the presence of rotavirus particles using two enzyme-linked immunosorbent assay kits. The kits had been formulated by the WHO Collaborating Centre for Reference and Research on Rotavirus (WHO-ELISA kit) and by DAKOPATTS (DAKO-ELISA kit) to be suitable for use in laboratories in developing countries. The kits were evaluated in laboratories in Burma, Chile, India, Mexico, Pakistan, Sri Lanka and the United Kingdom. Comparison of the results obtained with the two kits indicated that the DAKO-ELISA had an overall sensitivity of 97% and a specificity of 97% relative to the WHO-ELISA. In individual laboratories the DAKO-ELISA (K349) kit had a sensitivity in the range 90-100%, and a specificity of 85-100%. The kit showed a sensitivity of 100% and a specificity of 98% in assays on faeces obtained from newborn babies. We conclude that the DAKO-ELISA is as sensitive and specific as the WHO-ELISA for the detection of rotavirus antigen in faeces. PMID:2680139

  14. Development of an enzyme immunoassay for urinary pregnanediol-3-glucuronide in a female giant panda (Ailuropoda melanoleuca).

    PubMed

    Hama, Natsuki; Kanemitsu, Hideyasu; Tanikawa, Michiyo; Shibaya, Masami; Sakamoto, Kensuke; Oyama, Yujiro; Acosta, Tomas J; Ishikawa, Osamu; Pengyan, Wang; Okuda, Kiyoshi

    2009-07-01

    In order to enable monitoring of the reproductive status of the female giant panda after observation of estrus behavior, we developed an enzyme immunoassay (EIA) system for urinary pregnanediol-3-glucuronide (PdG), a progesterone metabolite, using commercial reagents and examined the changes in the urinary concentration of PdG in a female giant panda that showed pseudopregnancy and suspicious pseudopregnancy in 6 consecutive years. The developed EIA system had good reproducibility (intra- and interassay CVs 6.1% and 16.3%, respectively), good parallelism between the standard curve and the dose response curve of serial diluted samples and positive correlation (r=0.836) with the data for PdG in the same samples measured by gas chromatography. Urinary PdG in the female panda showed two phases of increase. The first elevation was observed immediately after estrus with the levels of PdG below 100 ng/Crmg, while the second phase was characterized by a drastic elevation above 100 ng/Crmg until the level began to decrease at the end of pseudopregnancy or suspicious pseudopregnancy. The length of the second phase had wider range than that of the first phase. In the present study, a new EIA assay system for urinary PdG in the female giant panda was developed, and we found that the length of the second phase is unstable in the pseudopregnant and suspicious pseudopregnant giant panda, in contrast with the unstable length of the first phase caused by delayed implantation in the pregnant giant panda.

  15. Performance of plasma fractionated free metanephrines by enzyme immunoassay in the diagnosis of pheochromocytoma and paraganglioma in children.

    PubMed

    Sarathi, Vijaya; Pandit, Reshma; Patil, Varsha K; Lia, Anurag R; Bandgar, Tushar R; Shah, Nalini S

    2012-01-01

    To establish pediatric reference ranges for plasma fractionated free metanephrines by enzyme immunoassay (EIA) and to evaluate its performance in the diagnosis of catecholamine-secreting tumors in the pediatric population. Normotensive children and children with suspected catecholamine-secreting tumors underwent measurement of plasma fractionated metanephrines by EIA to establish pediatric reference ranges. Children with suspected pheochromocytoma or paraganglioma also underwent magnetic resonance imaging or computed tomography from the neck to the pelvis and were followed up for a minimum of 1 year. Diagnosis of pheochromocytoma/paraganglioma was confirmed by histologic examination. Pheochromocytoma/paraganglioma was excluded in children who had a histologic diagnosis other than pheochromocytoma/paraganglioma and in those who had no imaging evidence of tumor and no progression on follow-up. Plasma fractionated metanephrines were measured in 78 normotensive children (age range, 1.5-17 years) and in 38 children with suspected catecholamine-secreting tumors. Of the 38 children (age range, 6-17 years) with suspected pheochromocytoma/paraganglioma, 17 had a histopathologically proven catecholamine-secreting tumor. The newly derived pediatric upper reference limit for metanephrine (128 pg/mL) was higher than in adults (90 pg/mL), whereas the pediatric upper reference limit for normetanephrine (149 pg/mL) was lower than in adults (180 pg/mL). The manufacturer's reference range for plasma fractionated metanephrines yielded a sensitivity of 100% and a specificity of 85.7%. Use of newly established pediatric reference ranges increased the specificity to 95.2% without altering the sensitivity (100%). Plasma fractionated metanephrines by EIA provide an accurate test with good sensitivity and specificity for the diagnosis of pheochromocytoma and paraganglioma in children. Use of pediatric reference ranges improves accuracy of the test.

  16. Evaluation of monoclonal antibody-based capture enzyme immunoassays for detection of specific antibodies to measles virus.

    PubMed Central

    Erdman, D D; Anderson, L J; Adams, D R; Stewart, J A; Markowitz, L E; Bellini, W J

    1991-01-01

    Monoclonal antibodies to the hemagglutinin protein, fusion protein, phosphoprotein, matrix protein, and nucleoprotein of measles virus were evaluated as detector antibodies in capture enzyme immunoassays (EIAs) for the detection of specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to measles virus. A pool of monoclonal antibodies to hemagglutinin protein and nucleoprotein proved optimal and was further evaluated. Specific IgM was detected in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of clinical measles cases and vaccinees, in 26% of healthy persons, and in 36% of infants 8 months postvaccination; consequently, IgA antibodies were not a useful indicator of recent measles infection. A significant increase in IgG antibodies between paired specimens was detected in 92% of clinical cases and all vaccinees. Only 59% of infant specimens had persistent IgG antibodies as detected by capture EIA at 8 months postvaccination, whereas all specimens had antibodies as detected by hemagglutination inhibition and plaque neutralization. An alternative indirect EIA, in which antigen was directly absorbed to the solid phase, was more sensitive than the capture design, detecting IgG antibodies in all infants postvaccination. When standardized with a microneutralization assay for the detection of persistent antibodies, the indirect IgG EIA gave predictive values for positive and negative tests exceeding 90%. Our capture IgM and indirect IgG EIAs provide a practical combination of serologic tests for the determination of acute measles virus infection and past exposure to measles virus or vaccine, respectively. PMID:1885743

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

    PubMed

    Islam, Kamrun Nahar; Ihara, Masaki; Dong, Jinhua; Kasagi, Noriyuki; Mori, Toshihiro; Ueda, Hiroshi

    2011-02-01

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

  18. Establishment of hybridomas secreting monoclonal antibodies to placental alkaline phosphatase and development of an enzyme immunoassay for its determination.

    PubMed

    Kinoshita, Y; Okamoto, T; Mano, H; Furuhashi, Y; Goto, S; Tomoda, Y

    1990-06-01

    We established seven hybridomas secreting murine IgG monoclonal antibodies (MoAbs) to placental alkaline phosphatase (PLAP). The seven hybridomas were designated (1) 7C6, (2) 6G10, (3) 5B9, (4) 6D5, (5) 6B5, (6) 11G6 and (7) 3E10, respectively. The characteristics of these hybridomas were evaluated by radioimmunoassay (RIA) with 125I-PLAP. Their reactivity with the intestinal alkaline phosphatase, one of the alkaline phosphatase isozymes, was (1) 0.04, (2) 0.2, (3) 1.4, (4) 1.8, (5) 0, (6) 4.0 and (7) 6.2(%), respectively. None of them showed signs of cross-reactivity with the liver-type alkaline phosphatase, also one of the alkaline phosphatase isozymes, within a PLAP concentration of 2,000 IU/l. The subtype of 5B9 was IgG1, and that of the others was IgG2a. We then used 7C6, to develop a sensitive, specific and convenient enzyme immunoassay (EIA) for the determination of PLAP, and assayed sera from patients with various gynecologic diseases. The incidence of increased PLAP was 6.4% in patients with benign diseases, 21.5% in cervical cancer, 36.4% in endometrial carcinoma, and 39.5% in malignant ovarian tumors. The specificity for malignant diseases seemed to be higher than that of CA125. Among endometrial carcinomas, well-differentiated adenocarcinoma had the highest incidence of an increased concentration. Among malignant ovarian tumors, serous cystadenocarcinoma, endometrioid carcinoma, dysgerminoma and Krukenberg's tumor showed a higher incidence than the other types.

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

    PubMed

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

    2016-10-01

    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 < .002). Addition of a supplemental immunoblot improved the specificity of the C6 EIA to 98.6%. For children and adolescents undergoing evaluation for 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 journals.permissions@oup.com.

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

    PubMed

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

    2017-03-11

    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: journals.permissions@oup.com.

  1. [Diagnosis of leptospirosis: evaluation of a solid-phase enzyme immunoassay in different stages of the disease].

    PubMed

    Vanasco, Norma B; Lottersberger, Javier; Schmeling, María F; Gardner, Ian A; Tarabla, Héctor D

    2007-06-01

    To develop a solid-phase enzyme immunoassay (ELISA) for genus-specific immunoglobulin G (IgG) determination with leptospirosis and to evaluate the ELISA in different stages of the disease. A total of 1,077 serum samples from 812 patients with suspected leptospirosis were analyzed. The samples had come from diagnoses done in the laboratory of the National Institute of Respiratory Diseases (Instituto Nacional de Enfermedades Respiratorias), in the city of Santa Fe, Argentina, between 1999 and 2005. Included in the study were 182 confirmed cases (267 samples), 167 negative cases (293 samples), and 40 probable cases (60 samples) (based on case definitions based on the results from the microscopic agglutination test (MAT), leukocyte counts, and neutrophilia values). Each sample was classified, according to the days of the natural history of disease, into one of three stages: first (< 10 days), second (10-25 days), or third (> 25 days). The antigen used in the ELISA was an extract of a mixture of pyrogenes and tarassovi serovars cultivated in a liquid medium, treated with ultrasound, and immobilized by adsorption on polystyrene plates. As a secondary antibody, a peroxidase-conjugated goat anti-human IgG monoclonal antibody was used. The cutoff value, sensitivity, and specificity of the ELISA were determined using the definitions of confirmed cases and of negatives cases as the standard. In order to determine the optimal cutoff value, the area under the receiver operating characteristic curve was calculated. The sensitivity of the evaluated test was much higher in the second stage (93.2%) than in either the first stage (68.1%) or the third stage (78.8%). The specificity increased gradually from 96.3% in the first stage to 100% in the third stage. Our results indicate that this ELISA test can be a very useful complement to the MAT for the diagnosis of leptospirosis in all the stages and, in particular, in order to diagnose acute disease sooner.

  2. Direct detection of influenza virus antigen in nasopharyngeal specimens by direct enzyme immunoassay in comparison with quantitating virus shedding.

    PubMed Central

    Döller, G; Schuy, W; Tjhen, K Y; Stekeler, B; Gerth, H J

    1992-01-01

    We developed a direct enzyme immunoassay [EIA; Enzygnost Influenza A(Ag) and Enzygnost Influenza B(Ag)] for the direct detection of influenza A and B virus antigens in nasopharyngeal secretion specimens (NPS). The test is performed without sonification of specimens, and results are obtained within 4 h. A direct comparison between direct EIA and quantitation of virus shedding for influenza A and B virus antigen detection was carried out. A total of 210 NPS and 98 nasopharyngeal wash specimens (NPW) were investigated. We isolated influenza A viruses from 79 (37.6%) of 210 NPS; of these 79 cell-culture-positive NPS, 70 (88.6%) were also positive by direct EIA. Of 29 (13.8%) NPS from which influenza B virus was isolated, 24 (82.8%) NPS were positive by direct EIA. Virus shedding was determined quantitatively in 48 NPS from patients with influenza A and in 24 NPS from patients with influenza B. Only a crude correlation between optical density values and virus concentrations was observed. Detection of influenza virus antigens in NPS by direct EIA showed sensitivities of 89.7% for influenza A virus and 87.9% for influenza B virus and specificities of 99.3% for influenza A virus and 100% for influenza B virus. With direct EIA, all NPW were negative for influenza A virus, although virus was isolated from 21 (21.4%) NPW. Of 15 NPW from which influenza B virus was isolated, 7 showed positive results in direct EIA. In addition, direct EIA is suitable for detecting influenza A and B viruses in cell cultures before the appearance of any cytopathic effects and can be used as a cell culture confirmation test. PMID:1572972

  3. Development of improved enzyme-based and lateral flow immunoassays for rapid and accurate serodiagnosis of canine brucellosis.

    PubMed

    Cortina, María E; Novak, Analía; Melli, Luciano J; Elena, Sebastián; Corbera, Natalia; Romero, Juan E; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

    2017-09-01

    Brucellosis is a widespread zoonotic disease caused by Brucella spp. Brucella canis is the etiological agent of canine brucellosis, a disease that can lead to sterility in bitches and dogs causing important economic losses in breeding kennels. Early and accurate diagnosis of canine brucellosis is central to control the disease and lower the risk of transmission to humans. Here, we develop and validate enzyme and lateral flow immunoassays for improved serodiagnosis of canine brucellosis using as antigen the B. canis rough lipopolysaccharide (rLPS). The method used to obtain the rLPS allowed us to produce more homogeneous batches of the antigen that facilitated the standardization of the assays. To validate the assays, 284 serum samples obtained from naturally infected dogs and healthy animals were analyzed. For the B. canis-iELISA and B. canis-LFIA the diagnostic sensitivity was of 98.6%, and the specificity 99.5% and 100%, respectively. We propose the implementation of the B. canis-LFIA as a screening test in combination with the highly accurate laboratory g-iELISA. The B. canis-LFIA is a rapid, accurate and easy to use test, characteristics that make it ideal for the serological surveillance of canine brucellosis in the field or veterinary laboratories. Finally, a blind study including 1040 serum samples obtained from urban dogs showed a prevalence higher than 5% highlighting the need of new diagnostic tools for a more effective control of the disease in dogs and therefore to reduce the risk of transmission of this zoonotic pathogen to humans. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Development of magnetic particle-based chemiluminescence enzyme immunoassay for the detection of 17beta-estradiol in environmental water.

    PubMed

    Xin, Tian-Bing; Wang, Xu; Jin, Hui; Liang, Shu-Xuan; Lin, Jin-Ming; Li, Zhen-Jia

    2009-09-01

    In the present work, a simple, fast, and highly sensitive chemiluminescence enzyme immunoassay for 17beta-estradiol (E2) in environmental water samples was developed, using magnetic particles (MPs) labeled with secondary antibody as both the immobilization matrix and the separation tools. The specific anti-E2 polyclonal antibody (PcAb) was produced against a conjugate of estradiol-bovine serum albumin. The specificity of the anti-E2 antibody was studied. The results showed that the antibody did not cross-react with the structurally related endocrine-disrupting compounds, including estrone, ethinyl E2, estriol, E2-17-glucuronide, E2-3-sulfate-17-glucuronide, androstenedione, and dihydrotestosterone. The water samples were pretreated with solid-phase extraction using C18 cartridges for the removal of matrix effects. Several physicochemical parameters including the dilution ratios of E2-6-horseradish peroxidase conjugate and anti-E2 PcAb, immunoreaction time, volume of chemiluminescent substrate and MPs, chemiluminescence reaction time, and pH of assay solution were studied and optimized. At optimal experimental conditions, it was found that the proposed method exhibited high performance with detection limit of 2.0 pg/mL, linear range of 20-1,200 pg/mL, and total assay time of 45 min. Both inter- and intra-assay coefficient of variation were less than 10%. The average recoveries of three different spiked concentration samples ranged from 86.3% to 108%. The method was successfully applied to the determination of E2 in river, waste, and tap water, and showed a good correlation with the commercially available radioimmunoassay kit.

  5. Development of an enzyme immunoassay for detection of antibodies against Coccidioides in dogs and other mammalian species.

    PubMed

    Chow, Nancy A; Lindsley, Mark D; McCotter, Orion Z; Kangiser, Dave; Wohrle, Ron D; Clifford, Wayne R; Yaglom, Hayley D; Adams, Laura E; Komatsu, Kenneth; Durkin, Michelle M; Baker, Rocky J; Shubitz, Lisa F; Derado, Gordana; Chiller, Tom M; Litvintseva, Anastasia P

    2017-01-01

    Coccidioides is a soil-dwelling fungus that causes coccidioidomycosis, a disease also known as Valley fever, which affects humans and a variety of animal species. Recent findings of Coccidioides in new, unexpected areas of the United States have demonstrated the need for a better understanding of its geographic distribution. Large serological studies on animals could provide important information on the geographic distribution of this pathogen. To facilitate such studies, we used protein A/G, a recombinant protein that binds IgG antibodies from a variety of mammalian species, to develop an enzyme immunoassay (EIA) that detects IgG antibodies against Coccidioides in a highly sensitive and high-throughput manner. We showed the potential of this assay to be adapted to multiple animal species by testing a collection of serum and/or plasma samples from dogs, mice, and humans with or without confirmed coccidioidomycosis. We then evaluated the performance of the assay in dogs, using sera from dogs residing in a highly endemic area, and found seropositivity rates significantly higher than those in dogs of non-endemic areas. We further evaluated the specificity of the assay in dogs infected with other fungal pathogens known to cross-react with Coccidioides. Finally, we used the assay to perform a cross-sectional serosurvey investigating dogs from Washington, a state in which infection with Coccidioides has recently been documented. In summary, we have developed a Coccidioides EIA for the detection of antibodies in canines that is more sensitive and has higher throughput than currently available methods, and by testing this assay in mice and humans, we have shown a proof of principle of its adaptability for other animal species.

  6. Detection of Campylobacter in stool and determination of significance by culture, enzyme immunoassay, and PCR in developing countries.

    PubMed

    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; Houpt, Eric R

    2014-04-01

    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 < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, 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.

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

    PubMed Central

    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

    2014-01-01

    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 < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, 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

  8. Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in spotted hyenas (Crocuta crocuta).

    PubMed

    Benhaiem, Sarah; Dehnhard, Martin; Bonanni, Roberto; Hofer, Heribert; Goymann, Wolfgang; Eulenberger, Klaus; East, Marion L

    2012-09-01

    The use of enzyme immunoassays (EIAs) to measure faecal glucocorticoid metabolites (fGCM) is a useful non-invasive technique to monitor adrenocortical activity in vertebrates. The first objective of this study was to validate an 'in-house' EIA (cortisol-3-CMO) for the measurement of fGCM concentrations in spotted hyenas. High-performance liquid chromatography (HPLC) was used to characterise fGCM in samples from a captive hyena that received an i.v. injection of [(3)H] cortisol. All HPLC fractions were analysed with the EIA for the presence and quantities of radiolabelled fGCM. Radiolabelled fGCM consisted of substances with a higher polarity than cortisol and substances of lower polarity that eluted between cortisol and corticosterone. Authentic radiolabelled cortisol was not detected. The EIA measured substantial amounts of immunoreactivity corresponding to the radioactive peaks. It also detected a significant increase in fGCMs after an adrenocorticotropic hormone (ACTH) challenge in two other captive animals and a significant increase in fGCMs in a fourth captive animal after anaesthesia. The second objective was to investigate an age effect on fGCM: we conducted pairwise comparisons of fGCM concentrations in individual free-ranging juvenile spotted hyenas when less than 6 months of age and when between 6 and 24 months of age. We expected juveniles to experience a more unpredictable and therefore more stressful environment when younger than when older. When younger, juveniles had significantly higher fGCM concentrations than when they were older. Our results demonstrate that our assay can be used to assess adrenocortical activity in spotted hyenas.

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

    PubMed

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

    2012-01-01

    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.

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

    PubMed

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

    2016-01-21

    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.

  11. A novel enzyme immunoassay based on potentiometric measurement of molecular adsorption events by an extended-gate field-effect transistor sensor.

    PubMed

    Kamahori, Masao; Ishige, Yu; Shimoda, Maki

    2007-06-15

    We developed a novel enzyme immunoassay based on a potentiometric measurement of molecular adsorption events by using an extended-gate field-effect transistor (FET) sensor. The adsorbing rate of a thiol compound on a gold surface was found to depend on the concentration of the compound. To construct an electrochemical enzyme immunoassay system by using the sensor, the enzyme chemistry of acetylcholinesterase (AChE) to generate a thiol compound was used and combined with the enzyme-linked immunosorbent assays (ELISA). After the AChE-catalyzed reaction, the amount of the antigen was obtained by detecting the adsorbing rate of the generated thiol compound on the gold electrode using the FET sensor. The measurement stability was also found to improve when a high frequency voltage of 10 kHz or more was superimposed to the reference electrode. The signal corresponding to a range between 1 and 250 pg/mL of Interleukin 1 beta was obtained by the FET sensor when a voltage of 1 MHz was superimposed onto the reference electrode. The FET sensor based ELISA used in this measurement technique can successfully detect Interleukin 1 beta at concentrations as low as 1 pg/mL.

  12. Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System.

    PubMed

    Gorton, R L; White, P L; Bagkeris, E; Cotterall, D; Desai, R; McHugh, T; Kibbler, C C

    2015-07-01

    The galactomannan enzyme immunoassay (GM-EIA) is widely utilized for the diagnosis of invasive aspergillosis (IA). There is inconsistent reproducibility of results between centers when the assay is processed manually. Automation of EIAs can reduce variation. This study investigated the semiautomation of the GM-EIA on the DS2 (Dynex) platform in the following three stages: (i) DS2 GM-EIA method validation with experimental samples, (ii) DS2 retesting of case-defined clinical samples, and (iii) a 12-month audit of DS2 GM-EIA performance. In stage i, Bland-Altman analysis demonstrated a reduced variance between optical density index (ODI) values for samples processed on two DS2 platforms (mean difference, -0.02; limits of agreement [LOA], -0.19 to 0.14) compared with the variance between samples processed manually and on a DS2 platform (mean difference, 0.02; LOA, -0.25 to 0.3). In stage ii, 100% (14/14 samples) qualitative agreement was observed for serum samples from patients with IA, with no significant change in the ODI values when samples were processed on the DS2 platform. A significant decrease in ODI values was observed for control serum samples on the DS2 platform (difference, 0.01; P = 0.042). In stage iii, a significant reduction in the frequency of equivocal results, from 5.56% (136/2,443 samples) to 1.56% (15/961 samples), was observed after DS2 automation (difference, 4.0%; 95% confidence interval [CI], 2.7 to 5.2%; P < 0.01), with an equivalent increase in negative results. This study demonstrates that GM-EIA automation may reduce intersite variability. Automation does not have an impact on the repeatability of truly positive results but contributes to a reduction in false-positive (equivocal) GM-EIA results, reducing the need to retest a significant proportion of samples.

  13. Development of a fluorescent microbead-based immunoassay for the detection of hepatitis E virus IgG antibodies in pigs and comparison to an enzyme-linked immunoassay.

    PubMed

    Owolodun, Olajide A; Giménez-Lirola, Luis G; Gerber, Priscilla F; Sanford, Brenton J; Feagins, Alicia R; Meng, Xiang-Jin; Halbur, Patrick G; Opriessnig, Tanja

    2013-11-01

    Swine hepatitis E virus (HEV) is a zoonotic virus and pigs are considered as an important reservoir. Swine HEV infection is widespread and most pig herds are infected. Humans can be infected with swine HEV via consumption of undercooked pork or through direct contact with infected pigs. To minimize the risk of zoonotic transmission, sensitive tools to assess the HEV infection status of pigs and pork products are needed. The objective of this study was to develop a fluorescent microbead-based immunoassay (FMIA) for the detection of IgG antibodies against swine HEV and compare it to an in-house enzyme-linked immunoassay (ELISA). Three sets of samples were utilized: (A) samples from pigs infected experimentally with different strains of HEV (positive controls, n=72), (B) samples from known HEV-negative pigs (negative controls, n=62) and (C) samples from pigs of unknown HEV infection status (n=182). All samples were tested by both ELISA and FMIA. The results on the experimental samples with known HEV exposure indicate that both assays have a specificity of 100% while the sensitivity ranges from 84.6% (ELISA) to 92.3% (FMIA). The overall prevalence of HEV IgG antibodies in field samples from pigs with unknown HEV exposure was 21.9% (40/182) for the ELISA and 21.4% (39/182) for the FMIA. The two assays had an almost perfect overall agreement (Kappa=0.92).

  14. [Evaluation of an immunochromatography test using enzyme immunoassay for rapid detection of influenza A and B viruses].

    PubMed

    Mitamura, Keiko; Yamazaki, Masahiko; Ichikawa, Masataka; Kimura, Kazuhiro; Kawakami, Chiharu; Shimizu, Hideaki; Watanabe, Sumi; Imai, Mitsunobu; Shinjo, Masayoshi; Takeuchi, Yoshinao; Sugaya, Norio

    2004-07-01

    We evaluated the performance of an improved version of Espline Influenza A & B-N (Fujirebio Inc., Japan), an immunochromatography test using enzyme immunoassay for rapid diagnosis of influenza A and B. The test produced positive results for four strains of influenza viruses and thirty-one influenza viral antigens and negative results for all of thirty strains of other respiratory viruses that were tested. The detection limit of this test was 5.8 x 10(2) to 5.8 x 10(3) pfu/assay, which is more sensitive than the old version of Espline. Furthermore, 715 respiratory specimens collected from the patients (children, 79.4%; adults, 18.5%; unknown, 2.1%) with influenza-like illnesses during the 2002/2003 influenza season in Japan were tested as part of a clinical evaluation of this test. The relative performance of this test compared to cell culture and nested RT-PCR results were examined. In the cell cultures, influenza viruses were detected in 488 of the 715 specimens (overall, 68.3%; AH3, 41.7%; B, 26.4%; AH3 and B, 0.1%). For influenza A, the sensitivity of this test was 95.4% (125/131) for nasal aspirates, 96.8% (92/95) for nasal swabs, and 85.1% (63/74) for throat swabs. For influenza B, the sensitivity of this test was 91.2% (52/57) for nasal aspirates, 88.1% (59/67) for nasal swabs, and 71.6% (48/67) for throat swabs. The new test exhibited a remarkably higher sensitivity to influenza A in throat swabs than the old version of Espline. Only two false positive results were obtained out of a total of 223 virus negative specimens; the specificity of the test was 100% (88/88) for nasal aspirates, 97.6% (81/83) for nasal swabs, and 100% (52/52) for throat swabs. We conclude that the new Espline Influenza A&B-N rapid diagnostic test is easy to use and has a high sensitivity and specificity, especially for influenza A.

  15. An enzyme immunoassay and immunoblot analysis for curculin, a new type of taste-modifying protein: cross-reactivity of curculin and miraculin to both antibodies.

    PubMed

    Nakajo, S; Akabane, T; Nakaya, K; Nakamura, Y; Kurihara, Y

    1992-02-01

    We have developed an enzyme immunoassay method for curculin, a new type of taste-modifying protein. This method can accurately quantify 0.05-20 ng of curculin, a sensitivity about 3000-times that of the psychometric method. The content of curculin in the fruit of Curculigo latifolia increased gradually until 3 weeks after artificial pollination and dramatically at 4 weeks, to finally reach 1.3 mg per fruit. Immunoblot analysis indicated that antiserum to curculin was faintly reactive with miraculin, but not with thaumatin or monellin.

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

    PubMed Central

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

    1992-01-01

    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

  17. Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time, age and genetic background.

    PubMed

    Lin, L; Wisor, J; Shiba, T; Taheri, S; Yanai, K; Wurts, S; Lin, X; Vitaterna, M; Takahashi, J; Lovenberg, T W; Koehl, M; Uhl, G; Nishino, S; Mignot, E

    2002-12-01

    The hypocretins (1 and 2) have emerged as key regulators of sleep and wakefulness. We developed a high-throughput enzyme immunoassay (EIA) to measure total brain hypocretin levels from large numbers of mice. Hypocretin levels were not altered by circadian time or age. However, significant differences in one or both hypocretin peptides were observed between different mouse strains. We studied hypocretin levels in knockout and transgenic mouse models with obesity, circadian gene mutations or monoaminergic defects. Compared to controls, only histamine receptor knockouts had lower hypocretin levels. This was most pronounced in H1 receptor knockouts suggesting the existence of a positive feedback loop between hypocretin and histaminergic neurons.

  18. [DIAGNOSTIC VALUE OF COMBINED USE OF COMBINED METHOD OF ENZYME IMMUNOASSAY AND POLYMERASE CHAIN REACTION TO DETECT OF INTRAUTERINE FETAL INFECTION BY PARVOVIRUS B19].

    PubMed

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

    2015-01-01

    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.

  19. 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.

    PubMed

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

    1995-06-01

    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.

  20. Rapid detection and counting of viable beer-spoilage lactic acid bacteria using a monoclonal chemiluminescence enzyme immunoassay and a CCD camera.

    PubMed

    March, Carmen; Manclús, Juan J; Abad, Antonio; Navarro, Alfonso; Montoya, Angel

    2005-08-01

    A chemiluminescence enzyme immunoassay carried out with a monoclonal antibody (MAb) and a charge-coupled device (CCD) camera was developed for rapid enumeration of viable beer-spoilage lactic acid bacteria. LA-4 MAb, which recognizes a broad spectrum of lactic acid bacteria isolated from several breweries across Spain, was produced and characterized. Test samples were filtered through polycarbonate membranes, and the membranes with retained bacteria were incubated at 31 degrees C for 2 days. They were then subjected to a two-step chemiluminescence enzyme immunoassay with MAb LA-4, and light-emitting points were detected and counted with a CCD camera. Eighteen out of 19 beer-spoilage lactic acid bacteria analysed produced luminous spots that could be enumerated. Results provided by the immunochemiluminescence assay correlated very well with those obtained by visual plate counting within a range of 3-100 CFU/100 ml. Correlation coefficients were 0.994 for four strains in sterile saline solution and 0.984 for 14 strains in artificially contaminated beer. The excellent agreement suggests that luminous spots detected within 2 days of culture are produced only by viable cells.

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

    PubMed

    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

    2017-03-01

    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.

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

    PubMed

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

    2014-06-01

    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.

  3. Optimetric system facilitates colorimetric and fluorometric measurements

    NASA Technical Reports Server (NTRS)

    Haley, F. C.

    1968-01-01

    Compact, unitary optimetric systems uses a single device for colorimetric, fluorometric and spectral absorption measurements. The basic element of the unitary systems is a test cell containing filter elements with uniquely fabricated lenses.

  4. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  5. 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).

    PubMed

    Ogata, Norio

    2006-04-01

    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 < 0.0001). Of 26 subjects with CLIA measurements below 10 mIU/mL, 15 samples (57.7%) showed CLEIA measurements more than 10 mIU/mL. Thus, in the subtype adr-vaccinees CLEIA demonstrated considerably high serum 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.

  6. Development of a novel fluorescent microbead-based immunoassay and comparison with three enzyme-linked immunoassays for detection of anti-Erysipelothrix spp. IgG antibodies in pigs with known and unknown exposure.

    PubMed

    Giménez-Lirola, L G; Xiao, C T; Halbur, P G; Opriessnig, T

    2012-10-01

    A novel fluorescent microbead immunoassay (FMIA) using the recombinant polypeptide SpaA415 was developed for detection of anti-Erysipelothrix spp. IgG in pig sera. The diagnostic performance of the FMIA was evaluated on samples from pigs with known and unknown Erysipelothrix spp. exposure and compared to an in-house enzyme-linked immunosorbent assay (ELISA-1) based on the same capture antigen, and two commercially available ELISAs (ELISA-2 and ELISA-3). Sera from pigs experimentally infected with Erysipelothrix rhusiopathiae serotype 1a (n=60) or 19 (n=12), sera from pigs vaccinated with a commercial attenuated-live vaccine based on serotype 1a (n=12) or a commercial bacterin based on serotype 2 (n=12), and 90 field samples were utilized. The sensitivity on 22 true positive samples collected in the later stages of infection/post-vaccination was 100% for the FMIA and ELISA-1, 63.6% for ELISA-2 and 81.8% for ELISA-3. The earliest antibody response was detected 7days post inoculation with the FMIA (77.8%) and ELISA-1 (11.1%), and at 14days post-vaccination (dpv) with FMIA (50%) and ELISA-1 (50%). On field samples, a higher seroprevalence was found in pigs older than 21days with all four assays. Kappa analysis indicated that the FMIA and ELISA-1 had almost complete agreement whereas the agreement was slight with ELISA-2 and fair with ELISA-3. The sensitivity of both immunoassays based on the rSpaA415 antigen was higher compared to that of the two commercial ELISAs. The rSpaA415 FMIA has great potential as an inexpensive ELISA alternative for detection of antibodies against E. rhusiopathiae in the future.

  7. IMMUNOASSAY HUMAN EXPOSURE STUDIES

    EPA Science Inventory

    The Human Exposure Research Branch has developed several enzyme-linked immunosorbent assay (ELISA) methods to support human exposure assessment studies. Immunoassays to detect low levels (<10 ng/mL) of chlorpyrifos in food, track-in dirt and house dust have been applied to sam...

  8. IMMUNOASSAY HUMAN EXPOSURE STUDIES

    EPA Science Inventory

    The Human Exposure Research Branch has developed several enzyme-linked immunosorbent assay (ELISA) methods to support human exposure assessment studies. Immunoassays to detect low levels (<10 ng/mL) of chlorpyrifos in food, track-in dirt and house dust have been applied to sam...

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

    PubMed

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

    2013-01-01

    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.

  10. Identification of human skin from a tissue fragment by detection of squamous cell carcinoma-related antigen using an enzyme immunoassay.

    PubMed

    Kitao, T; Miyaishi, S; Yamamoto, Y; Ishizu, H

    1996-12-02

    A new method of identifying human skin from a tissue fragment by detection of squamous cell carcinoma-related (SCC) antigen, using an enzyme immunoassay, was developed. When an extract was prepared from 0.1 g human skin homogenized with 1 ml of phosphate buffered saline, this method was able to detect SCC antigen in extracts diluted 10(2)-fold. There was no difference in the detection limit between individuals. Species specificity was good, and there was no cross reaction observed with skins from animals. Our method could also discriminate between skin and other organs or tissues, except for esophagus and lung. A practical case to which this method was applied is presented.

  11. Rapid Detection of Campylobacter jejuni in Stool Specimens by an Enzyme Immunoassay and Surveillance for Campylobacter upsaliensis in the Greater Salt Lake City Area

    PubMed Central

    Hindiyeh, Musa; Jense, Sandra; Hohmann, Sheri; Benett, Hilary; Edwards, Cheryl; Aldeen, William; Croft, Ann; Daly, Judy; Mottice, Susan; Carroll, Karen C.

    2000-01-01

    The Alexon-Trend, Inc. (Ramsey, Minn.), ProSpecT Campylobacter microplate assay was compared with culture on a Campy-CVA plate (Remel, Lenexa, Kans.) and blood-free campylobacter agar with cefoperazone (20 μg/ml), amphotericin B (10 μg/ml), and teicoplanin (4 μg/ml) (CAT medium; Oxoid Limited, Hampshire, England) with 631 patient stool samples. The CAT medium was used to isolate Campylobacter upsaliensis. The enzyme immunoassay (EIA) had a sensitivity and a specificity of 89 and 99%, respectively, and the positive and negative predictive values were 80 and 99%, respectively. Even though we extensively looked for C. upsaliensis in stool samples from patients from the greater Salt Lake City area, we did not isolate this species during the study period. The overall excellent specificity of the EIA allows rapid detection and treatment of positive patients; however, a negative result should be confirmed by culture when clinical suspicion is high. PMID:10921981

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

    PubMed

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

    2014-01-01

    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.

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

    PubMed Central

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

    2013-01-01

    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

  14. A novel rapid enzyme immunoassay (Fluorophos BetaScreen) for detection of beta-lactam residues in ex-farm raw milk.

    PubMed

    Sternesjö, A; Johnsson, G

    1998-07-01

    A novel, qualitative enzyme immunoassay based on fluorescence detection for determination of beta-lactam antibiotics in raw, commingled milk (Fluorophos BetaScreen E. U. test) was evaluated. A dose-response profile for penicillin G was constructed by analysis of spiked milk samples. The limit of detection, defined as the concentration of penicillin G that resulted in 95% of the samples being evaluated as positive, was 1.8 micrograms/kg. The repeatability of the assay was very high both within and between the three participating milk quality testing laboratories. In total 5,061 randomly selected tanker milk samples were analyzed with the BetaScreen test and compared with the Delvotest SP. The agreement between the two tests was 99.7%. Probably due to a higher sensitivity to penicillin G, the BetaScreen test detected almost twice as many suspect positive tanker milk samples (0.45%) as the Delvotest SP (0.26%).

  15. Preferential labeling of alpha-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays.

    PubMed

    Sélo, I; Négroni, L; Créminon, C; Grassi, J; Wal, J M

    1996-12-15

    Experimental conditions (pH 6.5, 24 h reaction, peptide:biotin ratio 1:5) were defined for preferential incorporation of the biotin molecule in the N-terminal alpha-amino group of peptides. This strategy could be helpful in numerous applications when an entire peptide chain must remain accessible for antibody or receptor binding. We illustrate this advantage in a solid-phase enzyme immunoassay designed to detect antibodies specific for bovine beta-lactoglobulin present in rabbit or human sera. This test involves synthetic peptides biotinylated in different positions and immobilized on a solid phase. The use of biotin/streptavidin interactions permitted more efficient detection of specific anti-peptide antibodies than solid phases prepared using conventional passive-adsorption techniques. The highest levels of antibody binding were measured when biotinylation occurred at the N-terminal extremity of immobilized peptides.

  16. 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.

    PubMed

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

    2016-11-01

    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.

  17. Comparison of a whole-virus enzyme immunoassay (EIA) with a peptide-based EIA for detecting rubella virus immunoglobulin G antibodies following rubella vaccination.

    PubMed Central

    Zrein, M; Joncas, J H; Pedneault, L; Robillard, L; Dwyer, R J; Lacroix, M

    1993-01-01

    A total of 250 human serum samples were tested for rubella virus immunoglobulin G antibodies by two enzyme immunoassays (EIAs), one using whole rubella virus antigen and the other based on the use of synthetic peptide antigen. The samples were taken from 125 volunteers before and after their immunization with the RA 27/3 rubella vaccine. This study indicates that a synthetic peptide-based EIA can favorably replace current viral lysate-based EIAs to detect rubella virus antibodies following immunization. Because the synthetic peptide used in this newly developed EIA represents a putative neutralization epitope of the rubella virus, it could also be instrumental in determining rubella immune status and in assessing vaccine program efficiency. PMID:8314994

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

    SciTech Connect

    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.

    2006-06-16

    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.

  19. Rapid detection of Campylobacter jejuni in stool specimens by an enzyme immunoassay and surveillance for Campylobacter upsaliensis in the greater Salt Lake City area.

    PubMed

    Hindiyeh, M; Jense, S; Hohmann, S; Benett, H; Edwards, C; Aldeen, W; Croft, A; Daly, J; Mottice, S; Carroll, K C

    2000-08-01

    The Alexon-Trend, Inc. (Ramsey, Minn.), ProSpecT Campylobacter microplate assay was compared with culture on a Campy-CVA plate (Remel, Lenexa, Kans.) and blood-free campylobacter agar with cefoperazone (20 microg/ml), amphotericin B (10 microg/ml), and teicoplanin (4 microg/ml) (CAT medium; Oxoid Limited, Hampshire, England) with 631 patient stool samples. The CAT medium was used to isolate Campylobacter upsaliensis. The enzyme immunoassay (EIA) had a sensitivity and a specificity of 89 and 99%, respectively, and the positive and negative predictive values were 80 and 99%, respectively. Even though we extensively looked for C. upsaliensis in stool samples from patients from the greater Salt Lake City area, we did not isolate this species during the study period. The overall excellent specificity of the EIA allows rapid detection and treatment of positive patients; however, a negative result should be confirmed by culture when clinical suspicion is high.

  20. Plasma and urinary metanephrines determined by an enzyme immunoassay, but not serum chromogranin A for the diagnosis of pheochromocytoma in patients with adrenal mass.

    PubMed

    Unger, N; Hinrichs, J; Deutschbein, T; Schmidt, H; Walz, M K; Mann, K; Petersenn, S

    2012-09-01

    Adrenal pheochromocytomas are neoplasms characterized by catecholamine excess. Determination of metanephrines by high-pressure liquid chromatography has been well established for the diagnosis of pheochromocytomas, demonstrating high sensitivity and specificity. This study evaluates the diagnostic value of newly available enzyme immunoassays for metanephrines in plasma and urine. Chromogranin A was studied as a potential additional diagnostic tool. Spontaneous blood samples and 24-h urine samples were collected in 149 subjects, including 24 histologically proven pheochromocytomas, 17 aldosterone-secreting and 21 cortisol-secreting adrenal adenomas, 30 nonfunctioning adrenal masses, 15 patients with essential hypertension and 42 healthy normotensive volunteers. Plasma and urinary metanephrine and normetanephrine as well as chromogranin A were determined and putative thresholds were calculated by ROC analysis. Plasma free normetanephrine was found to be the best single parameter with the highest sensitivity (89.5%) and specificity (98.3%) using a threshold of 167 pg/ml. Analysis of the combination of plasma free metanephrines revealed a similar sensitivity with lower specificity of 90.0%. Considering both urinary parameters demonstrated a slightly higher sensitivity (92.9%) with lower specificity (77.6%). ROC analysis revealed a threshold of 215 µg/l for chromogranin A with rather low sensitivity (73.9%) and specificity (74.2%). A weak positive correlation was found between the tumor size of pheochromocytomas and plasma metanephrine (r = 0.53, p ≤ 0.05) as well as chromogranin A (r = 0.60, p ≤ 0.01). In conclusion, plasma free and urinary metanephrines measured by enzyme immunoassays are convenient and reliable parameters for the diagnosis of pheochromocytoma. In contrast, CgA demonstrated poor sensitivity and specificity. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.

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

    PubMed

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

    2005-03-01

    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 < 0.001) between baseline and peak concentrations of both hormone measures. Female ZGG-12301, which conceived, but this pregnancy resulted in an unobserved spontaneous abortion, showed no significant difference (P > 0.05) between baseline and gestation 17beta-estradiol values; fecal 17beta-estradiol excretion during pregnancy was statistically different (P < 0.001) from excretion during the nonpregnancy period. Baseline progestogen concentrations were different from pregnancy (P < 0.001) and postovulatory (P < 0.01) concentrations, and progestogen concentrations during pregnancy period were different (P < 0.001) from postovulatory concentrations. In the nontreated cheetah (ZGT-3301), basal and increased progestogen concentrations were statistically different (P < 0.01). On the basis of 17beta-estradiol excretory patterns, duration of the estrous cycle (x +/- SEM) was 13.2 +/- 2.2 days. These results suggest that the enzyme-linked immunosorbent methods reported in this study were capable of quantifying reproductive hormones in fecal extracts of cheetahs and could be a practical alternative to other enzyme-linked immunosorbent assays which require more complex procedures.

  2. Immunoassay techniques.

    PubMed

    Wheeler, Michael J

    2013-01-01

    No other development has had such a major impact on the measurement of hormones as immunoassay. Reagents and assay kits can now be bought commercially but not for the more esoteric or new hormones. This chapter explains the basics of the immunoassay reaction and gives simple methods for immunoassays and immunometric assays and for the production of reagents for both antigenic and hapten hormones. Alternative methods are given for the preparation of labeled hormones as well as several possible separation procedures. The methods described here have been previously used in a wide range of assays and have stood the test of time. They will allow the production of usable immunoassays in a relatively short period of time.

  3. Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay.

    PubMed

    Li, Xiangmei; Wang, Wenjun; Wang, Limiao; Wang, Qi; Pei, Xingyao; Jiang, Haiyang

    2015-10-01

    Phenylethanolamine A (PA) is a β-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other β-agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.

  4. Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810).

    PubMed

    Paul, Vijay; Steinke, Kerstin; Meyer, Heinrich H D

    2008-01-21

    The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development. The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL(-1) with a decision limit (CCalpha) of 1.5 ng mL(-1) and detection capability (CCbeta) of 2.3 ng mL(-1). Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma. In total, 20 plasma samples from cows (n=7) fed non-transgenic maize and 24 samples from cows (n=8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL(-1); CCalpha). No plasma sample was positive for the presence of the Cry1Ab protein at CCalpha and CCbeta of the assay.

  5. Nanobody-based enzyme immunoassay for aflatoxin in agro-products with high tolerance to cosolvent methanol.

    PubMed

    He, Ting; Wang, Yanru; Li, Peiwu; Zhang, Qi; Lei, Jiawen; Zhang, Zhaowei; Ding, Xiaoxia; Zhou, Haiyan; Zhang, Wen

    2014-09-02

    A phage-displayed library of variable domain of heavy chain of the heavy chain antibody (VHH) or nanobody (Nb) was constructed after immunizing an alpaca with aflatoxin B1 (AFB1) conjugated with bovine serum albumin (AFB1-BSA). Two AFB1-specific nanobodies were selected. The obtained nanobodies were compared to an aflatoxin-specific monoclonal antibody B5 with respect to stability under organic solvents and high temperature. The two nanobodies could bind antigen specifically after exposure to temperatures as high as 95 °C. Besides, the nanobodies showed better or similar tolerance to organic solvents. A competitive ELISA with nanobody Nb26 was developed for the analysis of AFB1, exhibiting an IC50 value of 0.754 ng/mL (2.4 μM), linear range from 0.117 to 5.676 ng/mL. Due to the high tolerance to methanol, sample extracts were analyzed by nanobody-based ELISA without dilution. The recovery from spiked peanut, rice, corn and feedstuff ranged from 80 to 115%. In conclusion, the isolated nanobodies are excellent candidates for immunoassay application in aflatoxin determination.

  6. Novel colorimetric immunoassay for ultrasensitive monitoring of brevetoxin B based on enzyme-controlled chemical conversion of sulfite to sulfate.

    PubMed

    Lai, Wenqiang; Zhuang, Junyang; Tang, Dianping

    2015-02-25

    A simple colorimetric immunoassay for quantitative monitoring of brevetoxin B on a functionalized magnetic bead by using glucose oxidase (GOx)/antibrevetoxin antibody-labeled gold nanoparticle as the signal transduction tag was developed. The assay was carried out on the basis of GOx-controlled sulfite-to-sulfate chemical conversion with a silver(I)-3,3',5,5'-tetramethylbenzidine [Ag(I)-TMB] system. Initially, the sulfite was used as an inhibitor of Ag(I) to hinder the color development of TMB due to the formation of insoluble silver sulfite. Accompanying H2O2 generation with GOx-catalyzed glucose, the sulfite was converted into the sulfate, thus resulting in the colorless-to-blue change. Under the optimal conditions, the absorbance decreased with increasing brevetoxin B from 0.5 to 200 ng/kg with a detection limit of 0.1 ng/kg (ppt). The precision and specificity were acceptable. Furthermore, the methodology gave results matching well with the referenced brevetoxin ELISA kit for monitoring of spiked Musculista senhousia samples.

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

    SciTech Connect

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

    2009-03-01

    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.

  8. Evaluation of the performance of 5 commercialized enzyme immunoassays for the detection of Taenia solium antibodies and for the diagnosis of neurocysticercosis.

    PubMed

    Carod, Jean-François; Randrianarison, Mickaël; Razafimahefa, Julien; Ramahefarisoa, Rondro Mamitiana; Rakotondrazaka, Mahenintsoa; Debruyne, Monique; Dautigny, Mélanie; Cazal, Pierrette; Andriantseheno, Marcellin Lalaoarisoa; Charles, Emile Ramarokoto

    2012-01-01

    This study aimed to evaluate 5 enzyme immunoassays for detecting human antibodies against Taenia solium in human serum and for the diagnosis of neurocysticercosis (NCC): DRG™, RIDASCREEN™, NOVATECH™, CYPRESS™, and IVD™. A collection of 114 reference serum samples were used. All sera were tested both by ELISA and by an immunoblot method (enzyme-linked immunoelectrotransfer blot [EITB]). When compared with EITB, the Ridascreen™ test had the best positive concordance rate (85.1-91.2%) and the NovaLisa test™ showed the optimal negative concordance rate (93.7-95.6%). All tests had a sensitivity under 72% and a specificity above 60%. The best sensitivity was obtained using Ridascreen™ test (71.4%). An optimal specificity was achieved by the NovaLisa test™. T. solium-positive sera all cross-reacted with E. granulosus positive samples. In the commercial assays evaluated here, the most appropriate ELISA test for screening may be the Ridascreen™ assay. Antibody detection seems to be not appropriate for NCC diagnosis because of its overall lack of sensitivity.

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

    PubMed

    Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

    2015-02-15

    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 for the ZZ protein.

  10. Fluorometric Beam Profiling of UV MALDI Lasers

    NASA Astrophysics Data System (ADS)

    Steven, Rory T.; Palmer, Andrew D.; Bunch, Josephine

    2013-07-01

    The photon distribution (beam profile) of the laser as projected onto the sample is an important variable in matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Measurement of the beam profile is, therefore, an important factor within MALDI-MS. In this study a simple, low-cost fluorometric laser beam profiling technique is presented and applied in conjunction with MALDI-MS experiments. A comparison of the beam profile information afforded by a commercial system and the fluorometric method is carried out to determine the variation of beam profile for an Nd:YVO4 laser operated between 1 and 25 kHz. The beam profile information can be used, in conjunction with corresponding ion yields, to inform MALDI-MS experiments. The fluorometric beam profiling technique is used to obtain information about the beam dimensions as incident upon the MALDI-MS sample plate in-source. These values are compared with equivalent information obtained from ablation of thin film α-cyano-4-hydroxycinnamic acid (CHCA). In this study, area estimation by ablation provided a value 1.6 times smaller than that obtained by the fluorometric method, demonstrating the need for caution when measuring beam profile and, therefore, fluence, in MALDI-MS. [Figure not available: see fulltext.

  11. Immunoassays for diagnosis of coagulation disorders.

    PubMed

    Kappel, A; Ehm, M

    2010-11-01

    Immunoassays play a pivotal role in the clinical laboratory. In the coagulation section of the laboratory, they are used as an aid for diagnosis of deep vein thrombosis or pulmonary embolism, thrombophilia screening, or detection of coagulation factor deficiencies, respectively. Enzyme-linked immunosorbent assay (ELISA) and latex agglutination immunoassay technologies are currently most widely used, while Luminescent Oxygen Channeling Immunoassay (LOCI®) and other chemiluminescence-based immunoassays are emerging technologies for the coagulation laboratory. However, not all immunoassay technologies employed are compatible with the workflow requirements of the coagulation laboratory, and, not all technologies are suitable for detection or quantification of every marker. This review focuses on technical and performance aspects of those immunoassay technologies that are most widely used in the coagulation laboratory, and provides a description of markers that are typically tested by immunoassays.

  12. Antibodies for immunoassays.

    PubMed

    Newman, D J

    2000-01-01

    What is an immunoassay without an antibody? Clearly the name provides the answer to this question; without antibodies there would be no immunoassays. An immunoassay is an analytical technique, quantitative or qualitative, that relies absolutely on the specificity and affinity of the interaction between epitope and paratope for generation of a detectable response. The actual detection of this binding interaction can be via one of literally hundreds of different signal transduction mechanisms, e.g., fluorimetry, chemiluminescence, agglutination (turbidimetry or nephelometry) enzyme reactions, and so forth (1 -4), but these are simply transducing systems for the primary binding interaction. Antibodies thus provide us with an exquisitely sensitive and specific analytical technology for detecting and quantifying epitopic structures. These structures include amino-acid derivatives, e.g., thyroid hormones, peptides, e.g., vasopressin, proteins, e.g., cytokines, as well as carbohydrate structures, e.g., CA-125. Immunoassay technology has developed to such an extent that it is probably the most versatile analytical tool available able to identify and quantify epitopic structures across the milli- to zeptomolar concentration ranges (2).

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

    PubMed

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

    2016-03-01

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. [Relevance of anti-nucleosome antibodies detected by enzyme-based immunoassays in lupus diagnosis. Comparative analysis of four commercial kits].

    PubMed

    Lepers, S; Hachulla, E; Leleux, E; Hatron, P Y; Prin, L; Dubucquoi, S

    2002-12-01

    Among the biological assays used for the diagnosis of systemic lupus erythematosus (SLE), the detection of anti-double strand DNA antibodies (dsDNA Ab) is regarded as highly specific. However this biological parameter is negative among 20 to 40% of patients. Recent studies have revealed potential interest of the anti-nucleosome antibodies in the diagnosis of the lupus, in particular when any anti-dsDNA antibody activity could be detected. We selected 80 sera in order to evaluate four commercial anti-nucleosome enzyme-based immunoassays (EIA) kits. Their sensitivity and specificity values were compared with those obtained by the detection of anti-dsDNA Ab, carried out with both a Farr assay and two EIA kits. No anti-nucleosome EIA kits reached performances of the Farr assay for the diagnosis of lupus. On the other hand, our results show an higher diagnostic value for some anti-nucleosome EIA kits compared with 2 anti-dsDNA EIA kits. Apart from SLE, anti-nucleosome antibodies can be observed in others auto-immune diseases, in particular Sjögren's syndromes, the primary antiphospholipid syndrome, the systemic sclerosis and the mixed connective tissue disease. Compared results of the four anti-nucleosome EIA kits highlight many discordances. These variations, testifying to the absence of standardization for this new parameter, must encourage with a careful interpretation of results, according to the clinical context.

  15. Comparative evaluation of enzyme-linked immunoassay and reference methods for the detection of shellfish hydrophilic toxins in several presentations of seafood.

    PubMed

    Garet, Elina; González-Fernández, Africa; Lago, Jorge; Vieites, Juan M; Cabado, Ana G

    2010-02-10

    A comparative study was conducted to determine the feasibility of enzyme-linked immunosorbent assays (ELISAs) for the detection of amnesic shellfish poisoning (ASP) and paralytic shellfish poisoning (PSP) toxins in nine naturally contaminated species in fresh, frozen, boiled and canned fish and shellfish. PSP and ASP were analyzed in 138 shellfish samples (mussels, clams, barnacles, razor shells, scallops and cockles) and anchovies by mouse bioassay (MBA) and high performance liquid chromatography with ultraviolet detection (HPLC-UV), respectively. Results were compared with toxin concentrations obtained using two commercial competitive ELISAs, saxitoxin and ASP kits. Immunoassays were able to quantify toxins in different matrices showing excellent Pearson's correlation coefficients (r = 0.974 for saxitoxin ELISA and r = 0.973 for ASP ELISA) and to detect PSP and ASP with a lower limit of detection (LOD), namely, 50 microg saxitoxin equivalent/kg shellfish meat for PSP and 60 microg/kg domoic acid in shellfish flesh for ASP, than the reference methods (350 microg saxitoxin equivalent/kg shellfish meat and 1.6 mg/kg domoic acid in shellfish flesh, respectively). These results suggest that the ELISA method could be used as screening systems in a variety of species without matrix interference.

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

    PubMed

    Verebey, K; DePace, A

    1989-01-01

    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.

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

    PubMed

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

    1988-06-01

    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.

  18. Characterization of the third generation enzyme immunoassay IEA-HIV1/2-III for the detection of anti-HIV specific antibodies in human sera.

    PubMed

    Rayevskaya, G; Pilipenko, V G; Tkáciková, L; Spivak, N Y; Mikula, I; Chumak, R M

    2000-01-01

    The sensitivity and specificity of the developed anti-HIV1/2 third generation enzyme immunoassay, the IEA-HIV1/2-III, was examined. The test system for the detection of anti-HIV antibodies included peroxidase-conjugated HIV-specific recombinant Gag protein fragments (epitopes of p24 and p17 proteins), Env-1 (epitopes of p41 and p120 proteins), and Env-2 (p36 epitopes). Sensitivity was evaluated with 346 sera from HIV1-seropositive subjects, Anti-HIV1 Low Titer panels no. 10 and PRB-106 and seropositive panel PRB-931 in comparison with other third- and second-generation assays. The IEA-HIV1/2-III assays are characterized with high sensitivity comparable to the other third generation assays and the better sensitivity with respect to the second generation test-kit to determine HIV-specific antibodies in human sera. The specificity was determined using three hundred sixty-seven potentially cross-reactive samples (but negative for anti-HIV1/2). Only one specimen among them was reactive by IEA-HIV1/2-III.

  19. Vidas UP-enzyme-linked fluorescent immunoassay based on recombinant phage protein and fluorescence in situ hybridization as alternative methods for detection of Salmonella enterica serovars in meat.

    PubMed

    Zadernowska, Anna; Chajęcka-Wierzchowska, Wioleta; Kłębukowska, Lucyna

    2014-09-01

    Several methods for the rapid and specific detection of Salmonella spp. in meat have been described. This study was conducted to evaluate the capability of the VIDAS(®) UP (SPT [Salmonella Phage Technology]), an enzyme-linked fluorescent immunoassay method, and fluorescence in situ hybridization (FISH) to complement the International Organization for Standardization Method 6579 (ISO) in detecting Salmonella spp. from beef, pork, and poultry meat samples. The meat was inoculated with a mixture of Salmonella spp. on three levels of contamination. It was also checked that the tests did not produce cross-reactions with other Enterobacteriaceae rods. On the basis of the results, the relative specificity, relative accordance, and relative sensitivity of the method were determined. In meat samples, Vidas UP and FISH detection results were in substantial agreement with ISO, with relative specificity, accordance, and sensitivity rates of 90%, 96.3%, and 100%, respectively, for Vidas UP and 100%, 100%, and 99.4%, respectively, for FISH. This is the first report on the evaluation of both Vidas UP and FISH compared to ISO for the rapid detection of Salmonella enterica serovars in meat.

  20. Equivalence of assurance Gold Enzyme Immunoassay for visual or instrumental detection of motile and nonmotile Salmonella in all foods to AOAC culture method: collaborative study.

    PubMed

    Feldsine, P T; Mui, L A; Forgey, R L; Kerr, D E

    2000-01-01

    Six foods representative of a wide variety of processed, dried powder processed, and raw food types were analyzed by the Assurance Gold Salmonella Enzyme Immunoassay (EIA) and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the Assurance method and one by the AOAC culture method. The results for Assurance method were read visually and instrumentally with a microplate reader. A total of 24 laboratories representing federal government agencies and private industry, in the United States and Canada, participated in this collaborative study. Food types were inoculated with species of Salmonella with the exception of raw ground chicken, which was naturally contaminated. No statistical differences (p < 0.05) were observed between Assurance Gold Salmonella EIA with either visual or instrumental interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The Assurance visual and instrumental options of reading sample reactions produced the same results for 1277 of the 1296 sample and controls analyzed.

  1. Development and evaluation of a rapid enzyme-immunoassay system for measurement of the urinary concentration of estrone-3-glucuronide in a female giant panda (Ailuropoda melanoleuca).

    PubMed

    Hama, Natsuki; Kanemitsu, Hideyasu; Sakamoto, Kensuke; Oyama, Yujiro; Acosta, Tomas J; Ishikawa, Osamu; Pengyan, Wang; Okuda, Kiyoshi

    2008-08-01

    To detect estrus for reproductive management, and to determine the relationship between urinary estrogen and estrous behavior, in a female giant panda, we developed and evaluated a rapid enzyme immunoassay (EIA) system for urinary Estrone-3-glucuronide (E1G) using commercial reagents. The developed EIA system took only around 3 hours, including all procedures to obtain a result. It indicated good reproducibility (intra-assay CV of 5.16%, interassay CV of 15.4%) and sensitivity (lowest standard concentration was 0.0156 ng/ml) for measurement of the urinary concentrations of E1G in the giant panda. There was a positive correlation (r=0.934) with the data for estrone (E1) in the same samples, as measured by radioimmunoassay (RIA) performed in a commercial laboratory. The changes in the E1G concentrations were almost synchronous with the changes in E1 assayed by RIA in urine collected during 4 consecutive estrous seasons. The dynamics of urinary E1G measured by this system highly correlated with the occurrence of the presenting estrous behavior in the giant panda. The above results indicate that this assay system may be normally, rapidly and practically used for measurement of the urinary concentration of E1G in the giant panda.

  2. 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.

    PubMed

    Johansson, Karin; Karlsson, Hanna; Norén, Torbjörn

    2016-11-01

    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.

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

    PubMed

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

    2015-12-15

    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

  4. [Detection of AlpA and AlpB lytic endopeptidase propeptides of Lysobacter sp. XL1 by sandwich-enzyme immunoassay based on monoclonal antibodies].

    PubMed

    Rudenko, N V; Tsfasman, I M; Latypov, O R; Ledova, L A; Krasovskaia, L A; Karatovskaia, A P; Brovko, F A; Vasil'eva, N V; Stepnaia, O A

    2014-01-01

    The extracellular lytic endopeptidases AlpA and AlpB of the bacterium Lysobacter sp. XL1 are highly homologous and synthesized as precursors consisting of signal peptide, propeptide and mature form. In this work, two monoclonal antibodies against propeptide endopeptidase AlpA (ProA) and eleven against propeptide endopeptidase AlpB (ProB) were obtained to study the AlpA and AlpB endopeptidases secretion. The affinity constants of the antibodies against ProA were 2.9 x 10(9) and 3.5 x 10(9) M(-1), and the affinity constants of the antibodies against ProB were from 1.5 x 10(8) to 2.2 x 10(9) M(-1). The obtained antibodies did not have cross-reactivity between themselves, as well as mature forms of the enzymes. The monoclonal antibodies based sandwich-type enzyme immunoassay has been developed for measuring the propeptide in a native form. The linear range of determination ProA was 1.5-100 ng/mL with 6% error of measurement, and for determining ProB 0.2-6.25 ng/mL with 6% error. Using the developed assay, ProA and ProB propeptides have been detected in cell lysates of Lysobacter sp. XL1 in an amount 1.18 ± 0.03 ng and 0.096 ± 0.002 ng per 1 OD540 of the bacterial culture, respectively. The immunochemical assay for detection various forms of AlpA and AlpB lytic endopeptidases can be useful when dealing with issues related to their secretion into the environment.

  5. 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.

    PubMed

    Dong, Jie-Xian; Li, Zhen-Feng; Lei, Hong-Tao; Sun, Yuan-Ming; Ducancel, Frédéric; Xu, Zhen-Lin; Boulain, Jean-Claude; Yang, Jin-Yi; Shen, Yu-Dong; Wang, Hong

    2012-07-29

    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(H) and V(L)) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V(H) and V(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(-1), respectively, and the linear response range extended from 0.05 to 1.45 ng mL(-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 the data of dc-CLEIA and HPLC-MS (R(2)>0.99), indicating that the assay was an efficient analytical method for monitoring food safety. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Diagnosing Human Anisakiasis: Recombinant Ani s 1 and Ani s 7 Allergens versus the UniCAP 100 Fluorescence Enzyme Immunoassay

    PubMed Central

    Anadón, A. M.; Rodríguez, E.; Gárate, M. T.; Cuéllar, C.; Romarís, F.; Chivato, T.; Rodero, M.; González-Díaz, H.; Ubeira, F. M.

    2010-01-01

    Commercially available serological methods for serodiagnosis of human anisakiasis either are poorly specific or do not include some of the most relevant Anisakis allergens. The use of selected recombinant allergens may improve serodiagnosis. To compare the diagnostic and clinical values of enzyme-linked immunosorbent assay (ELISA) methods based on Ani s 1 and Ani s 7 recombinant allergens and of the UniCAP 100 fluorescence enzyme immunoassay (CAP FEIA) system, we tested sera from 495 allergic and 25 non-food-related allergic patients. The decay in specific IgE antibodies in serum was also investigated in 15 positive patients over a period of 6 to 38 months. Considering sera that tested positive by either Ani s 1 or Ani s 7 ELISA, the CAP FEIA classified 25% of sera as falsely positive, mainly in the group of patients with the lowest levels of anti-Anisakis IgE antibodies, and 1.28% of positive sera as falsely negative. Considering allergens individually, the overall sensitivities of Ani s 7 ELISA and Ani s 1 ELISA were 94% and 61%, respectively. The results also showed that anti-Anisakis IgE antibodies can be detected in serum for longer with Ani s 1 ELISA than with Ani s 7 ELISA and CAP FEIA (P < 0.01). Our findings suggest that ELISA methods with Ani s 7 and Ani s 1 allergens as targets of IgE antibodies are currently the best option for serodiagnosis of human anisakiasis, combining specificity and sensitivity. The different persistence of anti-Ani s 1 and anti-Ani s 7 antibodies in serum may help clinicians to distinguish between recent and old Anisakis infections. PMID:20107002

  7. Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay.

    PubMed Central

    Ng, S P; Tsui, C O; Roberts, D; Chau, P Y; Ng, M H

    1996-01-01

    We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples. PMID:8779567

  8. Bisphenol A determination in baby bottles by chemiluminescence enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry.

    PubMed

    Maiolini, Elisabetta; Ferri, Elida; Pitasi, Agata Laura; Montoya, Angel; Di Giovanni, Manuela; Errani, Ermanno; Girotti, Stefano

    2014-01-07

    Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4'-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL(-1), respectively. The LOD of LFIA was 0.1 μg mL(-1). A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL(-1), with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL(-1) and 17.4 ng mL(-1), respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 μg mL(-1)) and by the Directive 2004/19/EC (0.6 μg mL(-1)), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one

  9. IgA anticardiolipin and IgA anti-β2 glycoprotein I antibody positivity determined by fluorescence enzyme immunoassay in primary antiphospholipid syndrome.

    PubMed

    Mattia, Elena; Ruffatti, Amelia; Tonello, Marta; Meneghel, Lauro; Robecchi, Bianca; Pittoni, Marina; Gallo, Nicoletta; Salvan, Elisa; Teghil, Vera; Punzi, Leonardo; Plebani, Mario

    2014-09-01

    Primary antiphospholipid syndrome (PAPS) is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity as well as blood antiphospholipid (aPL) antibodies such as anticardiolipin (aCL), anti-β2 glycoprotein I (anti-β2GPI) antibodies of the IgG/IgM isotype and lupus anticoagulant (LA). The clinical significance of aCL and anti-β2GPI antibodies of the IgA isotype in PAPS is still a controversial issue. Sera and plasma were collected from 84 PAPS patients (54 with thrombosis and/or pregnancy morbidity and 30 with pregnancy morbidity alone), 66 seronegative patients (subjects with clinical manifestations of PAPS although with negative results on conventional antiphospholipid antibody testing), and 78 healthy blood donors. IgA aCL and IgA anti-β2GPI were determined using fluorescence enzyme immunoassay (FEIA), (EliATM, Phadia AB, Uppsala, Sweden). For comparison purposes, the sera were also tested for IgG/IgM aCL/anti-β2GPI antibodies using the same immunoassay method. LA was assayed following internationally accepted guidelines. Present respectively in 19% and 50% of the PAPS patients studied, IgA aCL and IgA anti-β2GPI antibody frequencies were both statistically significant (p=0.001 and p<0.001, respectively). The mean titers of both IgA aCL and IgA anti-β2GPI antibodies were higher in the thrombotic patients, but only the latter were significantly associated with thrombosis. Isolated IgA anti-β2GPI antibody positivity was significantly prevalent (p=0.04) in seven (10.6%) of the seronegative patients. Positivity to IgA anti-β2GPI antibody detected using FEIA was found to be clinically relevant in PAPS patients. Moreover the prevalence of isolated IgA anti-β2GPI antibody positivity was significant in the seronegative patients.

  10. Effect of bentonites on fluorometric selenium determination

    SciTech Connect

    Olson, O.E.; Herr, A.A.; Palmer, I.S.

    1981-01-01

    Bentonites in feeds cause error in the analysis for Se by the AOAC (3.097-3.101) fluorometric method for Se in plants. The error apparently results from the binding of the piazselenol by insoluble residue from the bentonite. This effect is avoided by diluting digests to volume after reduction with HCl, centrifuging or allowing to stand, and analyzing a portion of the clear supernatant liquid. Insoluble residues present after digestion of plant materials do not appear to cause a similar error.

  11. Rapid enumeration of Staphylococcus aureus in foods by direct demonstration of enterotoxigenic colonies on membrane filters by enzyme immunoassay.

    PubMed Central

    Peterkin, P I; Sharpe, A N

    1984-01-01

    Based on enzyme-linked immunosorbent assay, a convenient method has been devised for the direct demonstration of enterotoxin B production by Staphylococcus aureus colonies grown for 24 h on membrane filters. The problem of false-positive reactions due to binding of immunoglobulin G to protein A was turned to advantage by conjugating horseradish peroxidase directly to protein A, which then mediated the labeling of the antitoxin. The test requires 3 h to complete and yields a purple stain at the site of enterotoxin B-producing colonies, thus allowing direct enumeration of confirmed S. aureus in foods within 27 h. The method should be applicable to other enterotoxins of S. aureus. Images PMID:6742823

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

    PubMed

    Phillips, K Scott; Han, Jong Ho; Cheng, Quan

    2007-02-01

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

  13. 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.

    PubMed

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

    2015-08-05

    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(-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.

  14. Comparative activity of peroxidase-antibody conjugates with periodate and glutaraldehyde coupling according to an enzyme immunoassay.

    PubMed

    Tresca, J P; Ricoux, R; Pontet, M; Engler, R

    1995-01-01

    Horseradish peroxidase is often used as an antibody-coupled enzyme and several procedures have been developed to obtain IgG-peroxidase conjugates. The most widely used are coupling with periodate or glutaraldehyde. To compare the efficiency of these methods, the authors conducted periodate coupling or glutaraldehyde coupling in one or two steps, using the same batches of peroxidase, C-reactive protein (CRP) and anti-CRP monoclonal antibodies to develop a specially sensitive Elisa for CRP. Comparison of immunoenzymatic activities showed that periodate-mediated conjugation was much more efficient, because the activity of the coupling products was about 100 times greater than that of the products obtained after one or two-step conjugation with glutaraldehyde. The lower coupling efficiency observed with glutaraldehyde was not due to inactivation of the coupling agent or to a possible decrease in the affinity of the conjugates for CRP due to the coupling procedure. The differences in efficiency can be ascribed to the fact that periodate induced more coupling sites than glutaraldehyde. Periodate is therefore a better coupling agent for preparing conjugates to be used in Elisa or related techniques, in which conjugate size does not hinder accessibility to the antigen.

  15. Immunoassay standardization.

    PubMed

    Ekins, R

    1991-01-01

    Assays employed in the biological sciences fall into two categories, which may be respectively termed "comparative" (or "functionally-specific") and "analytical" (or "structurally-specific"). The former are intended to compare the relative effects of substances, or mixtures of substances--not necessarily of identical chemical structure--on a biological system (e.g. whole animal, tissue, cell, etc). Results are represented by units of effect (i.e. they are not units of "amount" of the substance(s) measured), and differ depending on the biological system used. Such assays cannot be "standardised" by the use of a calibrant. In contrast, analytical assays are intended to measure the number of molecules (or mass) of a single substance of unique chemical structure in a test sample, and cannot legitimately be employed to measure mixtures of substances of different structure. Results are represented by units of molecular number or mass, and should be identical for any test sample irrespective of the assay system used. Immunoassays generally fall into this category. Insofar as the antigenic substances present in standards or test samples are dissimilar and/or molecularly heterogeneous, an immunoassay is invalid, and the results it yields have no universal significance. Attempts to standardize "analytically-invalid" immunoassays inevitably fail. Many substances of biological interest (e.g. TSH)--initially defined in terms of their biological function--have subsequently been shown to be molecularly heterogenous. Problems thus arise in the standardization of immunoassays used for their measurement, reflecting the fact that the measurement of a mixture of substances of differing molecular structure (and function) is a meaningless concept. It is thus impossible to "measure TSH"; it is only possible to measure the effect TSH exerts in a particularly assay system. The only long-term solution to this problem is the development of assay systems measuring individual components of

  16. Evaluation of an enzyme immunoassay for the detection of the insect growth regulator fenoxycarb in environmental and biological samples.

    PubMed

    Le, Hong T M; Szurdoki, Ferenc; Székács, András

    2003-04-01

    A competitive enzyme-linked immunosorbent assay (ELISA) for fenoxycarb was adapted for quantitative detection of this insect growth regulator in various environmental, agricultural, food and biological matrices. Environmental samples were taken from soil and surface waters in Hungary. The ELISA enabled fenoxycarb detection in surface waters in the 1.1-125 ng ml(-1) concentration range without sample cleanup. In contrast, soil produced a strong matrix effect due to humic acids and other soil components. Several fruit homogenates and commercial fruit juices (eg apple, pear, grape) were analyzed by the ELISA. The assay was found to be suitable for analysis of fenoxycarb in fruit juices diluted 1:40. Biological samples included insect, fish and bovine tissues. The ELISA was applied to detect fenoxycarb in various biological matrices from larvae of the silkworm, Bombyx mori L. The assay proved useful for the analysis of haemolymph diluted 1:10 or at higher dilutions. Fat body and whole body homogenates, however, caused severe matrix effects. Fenoxycarb was detected in liver homogenates (diluted 1:40) from fish treated with various doses of fenoxycarb, and the concentrations determined correlated with the applied doses. The method was used to analyze spiked bovine urine samples diluted 1:10 or at greater dilutions. Fenoxycarb content determined by the ELISA in water and fruit juice samples was validated using GC-MS with solid-phase microextraction (SPME) sample preparation. The results of these studies demonstrated both the value and limitations of the assay when used for monitoring fenoxycarb in environmental, food and biological samples.

  17. Evaluation of five enzyme immunoassays compared with the cytotoxicity assay for diagnosis of Clostridium difficile-associated diarrhea in dogs.

    PubMed

    Chouicha, Nadira; Marks, Stanley L

    2006-03-01

    Clostridium difficile-associated-diarrhea (CDAD) is a nosocomial infection in dogs. Diagnosis of this infection is dependent on clinical signs of disease supported by laboratory detection of C. difficile toxins A or B, or both, in fecal specimens via enzyme-linked immunosorbent assay (ELISA). Unfortunately, to the authors' knowledge, commercially available ELISAs have not been validated in dogs to date. We evaluated 5 ELISAs done on 143 canine fecal specimens (100 diarrheic and 43 nondiarrheic dogs) and on 29 C. difficile isolates. The results of each ELISA were compared with the cytotoxin B tissue culture assay (CTA). Clostridium difficile was isolated from 23% of the fecal specimens. Eighteen of the 143 fecal specimens were toxin positive (15 diarrheic and 3 nondiarrheic dogs). On the basis of multiplex polymerase chain reaction (PCR) analysis for toxin-A and -B genes, 72% of the isolates were toxigenic. The carriage rate of toxigenic isolates in diarrheic dogs was higher than that in the nondiarrheic dogs; however, these differences were not statistically significant. A good correlation was found between CTA, PCR, and culture results. The ELISAs done on fecal specimens collected from diarrheic dogs had low sensitivity (7-33%). In contrast, ELISA for toxin A or B, or both, performed on toxigenic isolates had high sensitivity (93%). These results suggest that commercially available human ELISAs are inadequate for the diagnosis of canine C. difficile-associated diarrhea when tested on fecal specimens. In contrast, the Premier ToxinA/B and Techlab ToxinA/B ELISAs may be useful for the diagnosis of canine CDAD when used on toxigenic isolates.

  18. New Commercially Available IgG Kits and Time-Resolved Fluorometric IgE Assay for Diagnosis of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis

    PubMed Central

    Barrera, Coralie; Richaud-Thiriez, Bénédicte; Rocchi, Steffi; Rognon, Bénédicte; Roussel, Sandrine; Grenouillet, Frédéric; Laboissière, Audrey; Dalphin, Jean-Charles; Reboux, Gabriel

    2015-01-01

    Allergic bronchopulmonary aspergillosis (ABPA) is difficult to diagnose; diagnosis relies on clinical, radiological, pathological, and serological criteria. Our aim was to assess the performance of two new commercially available kits and a new in-house assay: an Aspergillus fumigatus enzyme-linked immunosorbent assay (ELISA) IgG kit (Bordier Affinity Products), an Aspergillus Western blotting IgG kit (LDBio Diagnostics), and a new in-house time-resolved fluorometric IgE assay (dissociation-enhanced lanthanide fluorescent immunoassay, or DELFIA) using recombinant proteins from an Aspergillus sp. recently developed by our laboratory for ABPA diagnosis in a retrospective study that included 26 cystic fibrosis patients. Aspergillus fumigatus-specific IgG levels measured by a commercial ELISA kit were in accordance with the level of precipitins currently used in our lab. The ELISA kit could accelerate and help standardize ABPA diagnosis. Aspergillus fumigatus-specific IgE levels measured by ImmunoCAP (Phadia) with A. fumigatus M3 antigen and by DELFIA with a purified protein extract of A. fumigatus were significantly correlated (P < 10−6). The results with recombinant antigens glucose-6-phosphate isomerase and mannitol-1-phosphate dehydrogenase were encouraging but must be confirmed with sera from more patients. The DELFIA is an effective tool that can detect specific IgE against more fungal allergens than can be detected with other commercially available tests. PMID:26698651

  19. Performance of Three Enzyme Immunoassays and Two Direct Fluorescence Assays for Detection of Giardia lamblia in Stool Specimens Preserved in ECOFIX

    PubMed Central

    Fedorko, Daniel P.; Williams, Esther C.; Nelson, Nancy A.; Calhoun, Leslie B.; Yan, Sizhuang S.

    2000-01-01

    ECOFIX is a single-vial stool preservative that is both formalin- and mercury-free. We evaluated the abilities of three commercial Giardia lamblia-specific enzyme immunoassays (EIAs) (ProSpecT Giardia Microplate Assay [Alexon-Trend Inc.], Giardia Test [Techlab], and Premier Giardia lamblia [Meridian Diagnostics, Inc.]) and two commercial direct fluorescent-antibody (FA) assays for G. lamblia (Crypto/Giardia IF Test [Techlab] and Merifluor Cryptosporidium/Giardia [Meridian Diagnostics, Inc.]) to detect G. lamblia in 34 G. lamblia-positive and 44 G. lamblia-negative stool specimens (determined by traditional examination for ova and parasites) preserved in ECOFIX compared to their abilities to detect G. lamblia in the same specimens preserved in formalin as the “gold standard” for each assay. Of the 34 formalin-fixed positive specimens, the number detected by each assay was as follows:, Alexon EIA, 34; Meridian EIA, 27; Techlab EIA, 29; Meridian FA assay, 31; and Techlab FA assay, 28. Both FA tests and the Alexon EIA performed well with ECOFIX, but the other two EIAs detected fewer positive specimens (the difference was statistically significant with the Techlab EIA) when ECOFIX was the preservative. Use of G. lamblia cyst antigen from cultured organisms preserved in formalin and ECOFIX demonstrated that the Alexon EIA could detect smaller amounts of antigen in ECOFIX than the other two EIAs could and suggested that cyst antigen is more stable in formalin. We recommend that laboratories use an FA assay or the Alexon EIA if they plan to use ECOFIX as their stool preservative. PMID:10878088

  20. Performance of three enzyme immunoassays and two direct fluorescence assays for detection of Giardia lamblia in stool specimens preserved in ECOFIX.

    PubMed

    Fedorko, D P; Williams, E C; Nelson, N A; Calhoun, L B; Yan, S S

    2000-07-01

    ECOFIX is a single-vial stool preservative that is both formalin- and mercury-free. We evaluated the abilities of three commercial Giardia lamblia-specific enzyme immunoassays (EIAs) (ProSpecT Giardia Microplate Assay [Alexon-Trend Inc.], Giardia Test [Techlab], and Premier Giardia lamblia [Meridian Diagnostics, Inc.]) and two commercial direct fluorescent-antibody (FA) assays for G. lamblia (Crypto/Giardia IF Test [Techlab] and Merifluor Cryptosporidium/Giardia [Meridian Diagnostics, Inc.]) to detect G. lamblia in 34 G. lamblia-positive and 44 G. lamblia-negative stool specimens (determined by traditional examination for ova and parasites) preserved in ECOFIX compared to their abilities to detect G. lamblia in the same specimens preserved in formalin as the "gold standard" for each assay. Of the 34 formalin-fixed positive specimens, the number detected by each assay was as follows:, Alexon EIA, 34; Meridian EIA, 27; Techlab EIA, 29; Meridian FA assay, 31; and Techlab FA assay, 28. Both FA tests and the Alexon EIA performed well with ECOFIX, but the other two EIAs detected fewer positive specimens (the difference was statistically significant with the Techlab EIA) when ECOFIX was the preservative. Use of G. lamblia cyst antigen from cultured organisms preserved in formalin and ECOFIX demonstrated that the Alexon EIA could detect smaller amounts of antigen in ECOFIX than the other two EIAs could and suggested that cyst antigen is more stable in formalin. We recommend that laboratories use an FA assay or the Alexon EIA if they plan to use ECOFIX as their stool preservative.

  1. Development and validation of an Australian in-house anti-pertussis toxin IgG and IgA enzyme immunoassay.

    PubMed

    May, Meryta L; Evans, Jenny; Riley, Jennifer; Lambkin, Geoffrey; Robson, Jennifer M

    2013-02-01

    Although anti-pertussis toxin (PT) immunoglobulin G (IgG) is considered one of the most specific serological markers for Bordetella pertussis infection, there are few commercial kits available in Australia. We aimed to present the process of development, quality control and on-going clinical validation of an anti-PT IgG and IgA enzyme immunoassay (EIA) in use since 1999, and discuss the application of such tests in the diagnosis of B. pertussis infections. A total of 1311 serum samples were used during multiple clinical validations from 1998 to 2010. The samples were drawn from healthy adults, children, patients with other respiratory infections, and patients with confirmed pertussis. Assay reproducibility, accuracy and precision criteria conformed to National Pathology Accreditation Advisory Council (NPAAC) guidelines. Using the World Health Organization clinical and/or laboratory definition of a definite case as the comparative standard, sensitivity was 84% [95% confidence interval (CI) 75-93] and specificity was 98% (95%CI: 90-100) for anti-PT IgG. Sensitivity was 72% (95%CI 64-80) and specificity was 98% (95%CI 90-100) for anti-PT IgA. There was minimal background positivity in either healthy adults or children using the established cut-offs. There was no appreciable effect of immunisation or cross reactions with other respiratory pathogens. Serological evaluation of various populations enabled the development of an anti-PT IgG and IgA EIA assay which was suitable for the diagnosis of acute infection in convalescent samples from clinically confirmed cases. Repeated evaluations of population-based cut-offs are required for in-house assays to ensure they remain clinically relevant. The subsequent validation of the cut-offs with WHO international standards has been published in a recent prospective study.

  2. Application of the BED capture enzyme immunoassay for HIV incidence estimation among female sex workers in Kaiyuan City, China, 2006–2007

    PubMed Central

    Xu, Junjie; Wang, Haibo; Jiang, Yan; Ding, Guowei; Jia, Manhong; Wang, Guixiang; Chu, Jennifer; Smith, Kumi; Sharp, Gerald B.; Chen, Ray Y.; Jin, Xia; Dong, Ruiling; Han, Xiaoxu; Wang, Ning

    2009-01-01

    Objective To estimate HIV incidence among female sex workers (FSWs) by serial cross-sectional surveys and IgG-capture BED-enzyme immunoassay (BED-CEIA). Methods We conducted three cross-sectional surveys six months apart among all consenting FSWs in Kaiyuan City, China. HIV-antibody-positive samples were also tested by BED-CEIA. Results Among 1412 unique participants, 475 tested HIV-negative and attended >1 survey (longitudinal cohort). Compared to 786 HIV-negative FSWs who only participated once, the longitudinal cohort reported more illicit drug use (10.9% vs 7.4%, p=0.03), injected drugs more often in previous three months (8.8% vs. 5.3%, p=0.02), and had more positive urine opiate tests (13.7% vs. 8.9%, p=0.008). Four participants in the longitudinal cohort seroconverted over the year, with an overall incidence of 1.1 (95% confidence interval [CI] 0.3–2.8)/100 person-years. Crude BED-CEIA incidence was 3.4 (95% CI 2.3–4.4)/100 person-years with adjusted rates (McDougal, 1.5/100 person-years [95% CI 1.0–2.0]; Hargrove, 1.6/100 person-years [95% CI 1.1–2.1]) similar to the cohort incidence. BED-CEIA false positive rate was 4.4% (10/229) among samples from FSWs known to be infected ≥365 days. Conclusions Although limited by power, this study provides additional data towards validating BED-CEIA in China. If confirmed by other studies, BED-CEIA will be a useful tool to estimate HIV incidence rates and trends. PMID:20102792

  3. Large-scale evaluation of the immuno-mycologics lateral flow and enzyme-linked immunoassays for detection of cryptococcal antigen in serum and cerebrospinal fluid.

    PubMed

    Hansen, Jessica; Slechta, E Susan; Gates-Hollingsworth, Marcellene A; Neary, Brandon; Barker, Adam P; Bauman, Sean; Kozel, Thomas R; Hanson, Kimberly E

    2013-01-01

    Cryptococcosis is a systemic infection caused by the pathogenic yeasts Cryptococcus neoformans and C. gattii. Detection of cryptococcal capsular antigen (CrAg) in serum and cerebrospinal fluid (CSF) plays an important diagnostic role. We prospectively compared the new Immuno-Mycologics Inc. (IMMY) lateral flow assay (LFA) and enzyme immunoassay (EIA) to our current CrAg test (Premier EIA; Meridian Bioscience Inc.). Discordant samples were retested with the latex-Cryptococcus antigen test (IMMY) and using serotype-specific monoclonal antibodies (MAbs). A total of 589 serum and 411 CSF specimens were tested in parallel. Qualitative agreement across assays was 97.7%. In all, 56 (41 serum and 15 CSF) samples were positive and 921 (527 serum and 394 CSF) samples were negative by all three assays. The 23 discrepant specimens were all Meridian EIA negative. Of 23 discordant specimens, 20 (87.0%) were positive by both the IMMY LFA and EIA, 2 were LFA positive only, and 1 was EIA positive only. Eleven discrepant specimens had adequate volume for latex agglutination (LA) testing; 8 were LA positive, and 3 were LA negative. LA-negative samples (2 CSF samples and 1 serum) had low IMMY LFA/EIA titers (≤1:10). Serotype-specific MAb analysis of the LA-positive samples suggested that these specimens contained CrAg epitopes similar to those of serotype C strains. In conclusion, the IMMY assays showed excellent overall concordance with the Meridian EIA. Assay performance differences were related to issues of analytic sensitivity and possible serotype bias. Incomplete access to patient-level data combined with low specimen volumes limited our ability to fully resolve discrepant results.

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

    PubMed

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

    2006-01-01

    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%).

  5. Detection of Helicobacter pylori Antibodies in a Pediatric Population: Comparison of Three Commercially Available Serological Tests and One In-House Enzyme Immunoassay

    PubMed Central

    Sunnerstam, Bengt; Kjerstadius, Torbjörn; Jansson, Lillemor; Giesecke, Johan; Bergström, Mats; Ejderhamn, Jan

    1999-01-01

    A serum immunoglobulin G enzyme immunoassay (EIA) for Helicobacter pylori antibodies already in use in adults was evaluated with 99 pediatric serum samples to determine its usefulness for the study of H. pylori disease in children. The reference method used was either the 13C-urea breath test or a biopsy culture of gastric mucosa. In children, an EIA cutoff of 0.35 absorbancy unit yielded sensitivity, specificity, and positive and negative predictive values of 93, 97, 93, and 97%, respectively. The cutoff recommended when this EIA was published for use in adults was 0.70 absorbancy unit (H. Gnarpe, P. Unge, C. Blomqvist, and S. Mäkitalo, APMIS 96:128–132, 1988). Another subset of 169 serum samples taken from children was analyzed by four serological tests in order to compare the performance of the in-house EIA with the Pyloriset, HM-CAP, and Helico-G kits. For the 169 samples, 10 (5.9%) false-positives and no false-negatives occurred with the Helico-G, 3 (1.8%) false-positives and no false-negatives occurred with the Pyloriset, and 3 (1.8%) false-positives and 1 (0.6%) false-negative occurred with the HM-CAP. For the 169 samples, 1 (0.6%) false-positive and no false-negatives occurred with the in-house EIA. Serological detection of H. pylori antibodies with our EIA seems to be valuable in diagnosing H. pylori infection in children, but only if a lowered, specific pediatric cutoff is established. The commercial kits, particularly the Helico-G, seem to overdiagnose pediatric H. pylori infection. A positive serological test for H. pylori infection, particularly for children, needs to be confirmed by a reference method because of the possibility of spontaneous eradication of infection, with a lingering serological response. PMID:10488200

  6. Detection of Helicobacter pylori antibodies in a pediatric population: comparison of three commercially available serological tests and one in-house enzyme immunoassay.

    PubMed

    Sunnerstam, B; Kjerstadius, T; Jansson, L; Giesecke, J; Bergström, M; Ejderhamn, J

    1999-10-01

    A serum immunoglobulin G enzyme immunoassay (EIA) for Helicobacter pylori antibodies already in use in adults was evaluated with 99 pediatric serum samples to determine its usefulness for the study of H. pylori disease in children. The reference method used was either the (13)C-urea breath test or a biopsy culture of gastric mucosa. In children, an EIA cutoff of 0.35 absorbancy unit yielded sensitivity, specificity, and positive and negative predictive values of 93, 97, 93, and 97%, respectively. The cutoff recommended when this EIA was published for use in adults was 0.70 absorbancy unit (H. Gnarpe, P. Unge, C. Blomqvist, and S. Mäkitalo, APMIS 96:128-132, 1988). Another subset of 169 serum samples taken from children was analyzed by four serological tests in order to compare the performance of the in-house EIA with the Pyloriset, HM-CAP, and Helico-G kits. For the 169 samples, 10 (5.9%) false-positives and no false-negatives occurred with the Helico-G, 3 (1.8%) false-positives and no false-negatives occurred with the Pyloriset, and 3 (1.8%) false-positives and 1 (0.6%) false-negative occurred with the HM-CAP. For the 169 samples, 1 (0.6%) false-positive and no false-negatives occurred with the in-house EIA. Serological detection of H. pylori antibodies with our EIA seems to be valuable in diagnosing H. pylori infection in children, but only if a lowered, specific pediatric cutoff is established. The commercial kits, particularly the Helico-G, seem to overdiagnose pediatric H. pylori infection. A positive serological test for H. pylori infection, particularly for children, needs to be confirmed by a reference method because of the possibility of spontaneous eradication of infection, with a lingering serological response.

  7. Evaluation of an enzyme immunoassay for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen as an aid in the diagnosis of blastomycosis in dogs.

    PubMed

    Mourning, Alyssa C; Patterson, Edward E; Kirsch, Emily J; Renschler, Janelle S; Wolf, Linda A; Paris, Jasmin K; Durkin, Michelle M; Wheat, Lawrence J

    2015-11-15

    To evaluate the sensitivity and specificity of an enzyme immunoassay (EIA) for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen (rBAD-1) to aid in the diagnosis of blastomycosis in dogs and compare the findings with results from other tests used for this purpose. Prospective analytic study. Serum and urine from 70 dogs with and without blastomycosis. Serum and urine samples were collected from dogs with blastomycosis (n = 21), histoplasmosis (8), or nonfungal pulmonary disease (21) and from healthy control dogs living in a blastomycosis-endemic area (20). Serum was tested for antibodies against Blastomyces dermatitidis with the rBAD-1 antibody EIA and an A-antigen antibody agar gel immunodiffusion (AGID) assay. Serum and urine were tested for B dermatitidis antigen with a quantitative EIA. Sensitivity of the quantitative antigen EIA was 100% in serum and urine samples from dogs with blastomycosis, with specificity of 95% in urine samples from dogs with nonfungal pulmonary disease and 100% in urine samples from healthy dogs. Sensitivity of the rBAD-1 antibody EIA (95%) was significantly greater than that of the A-antigen antibody AGID assay (65%). Specificity of the antibody EIA was 88% in dogs with histoplasmosis, 95% in healthy dogs, and 100% in dogs with nonfungal pulmonary disease. The rBAD-1 antibody EIA had greater sensitivity than the A-antigen antibody AGID assay in dogs with blastomycosis. This antibody EIA may assist in distinguishing histoplasmosis from blastomycosis. Further evaluation in a larger prospective study is needed to verify these results.

  8. Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in Eurasian (Lynx lynx) and Iberian lynx (Lynx pardinus).

    PubMed

    Pribbenow, Susanne; Jewgenow, Katarina; Vargas, Astrid; Serra, Rodrigo; Naidenko, Sergey; Dehnhard, Martin

    2014-09-15

    Stress hormone levels are important indicator of an animal's well-being, as stress has harmful effects on reproduction, growth and immune function. The development of enzyme immunoassays (EIA) to monitor faecal glucocorticoid metabolites (fGM) contributes a powerful tool to assess an animal's adrenal status non-invasively. We aimed to identify a suitable EIA for monitoring fGM by assessing the suitability of six different EIAs for detecting quantitative changes in fGM concentrations in response to an ACTH challenge test in Eurasian lynx. FGM were characterised in a male Eurasian lynx that received an injection of (3)H-cortisol. Using HPLC analyses radiolabeled metabolites were compared with immunoreactive metabolites. The second aim was to biologically validate the established EIA for monitoring adrenocortical activity of captive Iberian lynxes after a translocation to new enclosures in relation to behaviour. Additionally faecal samples of ten pregnant Iberian lynxes from the peripartal period were analysed. The ACTH challenge revealed an 11β-hydroxyetiocholanolone EIA as the most sensitive assay to reflect acute fGM elevations in the Eurasian lynx. HPLC immunograms demonstrated that the 11β-hydroxyetiocholanolone EIA measured significant amounts of immunoreactivities corresponding to radiolabeled metabolites with strong similarities across both lynx species. Additionally, HPLC and GC-MS analyses confirmed the presence of 11β-hydroxyetiocholanolone in faeces of both, the Eurasian and the Iberian lynx. Longitudinal fGM profiles of Iberian lynx revealed increases in concentrations associated with management events. During the peripartal period, however, fGM concentrations were not significantly elevated. Our results show that the 11β-hydroxyetiocholanolone EIA is a reliable tool to assess fGM in both lynx species.

  9. Human chorionic gonadotropin detection in cerebrospinal fluid of patients with a germinoma and its prognostic significance: assessment by using a highly sensitive enzyme immunoassay.

    PubMed

    Fukuoka, Kohei; Yanagisawa, Takaaki; Suzuki, Tomonari; Shirahata, Mitsuaki; Adachi, Jun-Ichi; Mishima, Kazuhiko; Fujimaki, Takamitsu; Katakami, Hideki; Matsutani, Masao; Nishikawa, Ryo

    2016-11-01

    OBJECTIVE Human chorionic gonadotropin (HCG) can be detected in a certain population of patients with a germinoma, but the frequency of germinoma HCG secretion and the prognostic value of HCG in the CSF are unknown. METHODS The authors measured HCG levels in sera and CSF in patients with a histologically confirmed germinoma by using a highly sensitive assay known as an immune complex transfer enzyme immunoassay (EIA), which is more than 100 times as sensitive as the conventional method, and they analyzed the correlation between HCG levels and the prognoses of patients with a germinoma. RESULTS HCG levels in sera and CSF of 35 patients with a germinoma were examined with the immune complex transfer EIA. The median CSF HCG levels in patients with a germinoma during the pretreatment and posttreatment evaluations were 192.5 pg/ml (range 1.2-13,116.5 pg/ml) and 18.7 pg/ml (1.2-283.9 pg/ml), respectively. Before treatment, the CSF HCG level was greater than the cutoff value in 85.7% of the patients with a germinoma. The authors compared survival rates among the patients by using a CSF HCG cutoff level of 1000 pg/ml, and the difference was statistically significant between the groups (p = 0.029, log-rank test). CONCLUSIONS Results of this study demonstrate that most germinomas secrete HCG. Patients with a germinoma that secretes higher amounts of HCG in their CSF experienced recurrence more frequently than those with lower CSF HCG levels.

  10. Diagnosing Clostridium difficile-associated diarrhea using enzyme immunoassay: the clinical significance of toxin negativity in glutamate dehydrogenase-positive patients

    PubMed Central

    Yuhashi, Kazuhito; Yagihara, Yuka; Misawa, Yoshiki; Sato, Tomoaki; Saito, Ryoichi; Okugawa, Shu; Moriya, Kyoji

    2016-01-01

    Purpose The enzyme immunoassay (EIA) has lower sensitivity for Clostridium difficile toxins A and B than the polymerase chain reaction in the diagnosis of C. difficile-associated diarrhea (CDAD). Furthermore, toxin positivity with EIA performed on C. difficile isolates from stool cultures may be observed even in patients with EIA glutamate dehydrogenase (GDH)-positive and toxin-negative stool specimens. It is unclear whether such patients should be treated as having CDAD. Methods The present study retrospectively compared patient characteristics, treatment, and diarrhea duration among three groups of patients who underwent stool EIA testing for CDAD diagnosis: a toxin-positive stool group (positive stool group; n=39); a toxin-negative stool/toxin-positive isolate group (discrepant negative/positive group, n=14); and a dual toxin-negative stool and isolate group (dual negative group, n=15). All cases included were confirmed to be GDH positive on EIA test. Results Patients’ backgrounds and comorbidities were not significantly different among three groups. No difference was observed among the three groups with regard to antimicrobial drug use before diarrhea onset. Treatment was received by 82.1% of the positive stool group compared to 7.1% of the discrepant positive/negative group and 0% of the dual negative group, while mean diarrhea duration was 10.6 days compared to 7.9 days (P=0.6006) and 3.4 days (P=0.0312), respectively. Conclusion Even without treatment, patients with toxin-negative stool specimens had shorter diarrhea duration than those with toxin-positive stool specimens even with toxin-positive isolates. These findings may suggest a limited need for CDAD treatment for GDH-positive patients and toxin-negative stool specimens. PMID:27313472

  11. Imaging of Lactobacillus brevis single cells and microcolonies without a microscope by an ultrasensitive chemiluminescent enzyme immunoassay with a photon-counting television camera.

    PubMed Central

    Yasui, T; Yoda, K

    1997-01-01

    An ultrasensitive chemiluminescent enzyme immunoassay (CLEIA) was developed for the rapid detection and quantification of Lactobacillus brevis contaminants in beer and pitching yeast (Saccharomyces cerevisiae slurry collected for reinoculation). L. brevis cells trapped on a 47-mm nucleopore membrane (0.4-micron pore size) were reacted with a peroxidase-labelled Lactobacillus group E antibody and then subjected to an enhanced CLEIA analysis with 4-iodophenol as the enhancer. The combination of a nucleopore membrane with low background characteristics that enables the antigen-antibody reaction to proceed through the pores of the membrane and a labelled antibody prepared by the maleimide hinge method with minimal nonspecific binding characteristics was essential to minimize background in the detection of single cells. An ultrahigh sensitive charge-coupled device (CCD) camera equipped with a fiber optics image intensifier permitted the imaging of single cells. A clear correlation existed between the number of luminescent spots observed and the plate count [y (CLEIA) = 0.990x (plate count) + 15.9, where n = 7, r = 0.993, and P < 0.001]. Microscopic observation confirmed that the luminescent spots were produced by single cells. This assay could be used to detect approximately 20 L. brevis cells in 633 ml of beer within 4 h. Our ultrasensitive CLEIA could also be used to detect microcolonies approximately 20 microns in diameter which had formed on a membrane after 15 to 18 h of incubation. This method, which we called the microcolony immunoluminescence (MIL) method, increased the signal-to-noise ratio dramatically. The MIL method could be used to detect a 10(0) level of L. brevis contamination in 633 ml of beer and a 1/10(8) level of L. brevis contamination in pitching yeast within 1 day (15 to 18 h to form microcolonies and 2 h for CLEIA). PMID:9361439

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

    PubMed

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

    2012-01-01

    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.

  13. An Efficient Sample Preparation Method for High-Throughput Analysis of 15(S)-8-iso-PGF2α in Plasma and Urine by Enzyme Immunoassay

    PubMed Central

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

    2012-01-01

    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. PMID:22989424

  14. Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection

    PubMed Central

    Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

    2016-01-01

    Background: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Objective: Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. Materials and Methods: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. Results: In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). Conclusion: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed. PMID:27525325

  15. 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)

    PubMed Central

    KAJAYSRI, Jatuporn; NOKKAEW, Weerapun

    2013-01-01

    ABSTRACT 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. PMID:24257195

  16. 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.

    PubMed

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

    2013-11-01

    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 < 0.001). Group 2 samples had lower fecal loads than those from groups 3 and 4 (P < 0.001). There was a significant correlation between PCR CT and fecal loads (ρ = -0.697; P < 0.001). NAP1 strains were associated with the detection of toxins by EIA or CCA (P = 0.041). This study demonstrates an association between C. difficile fecal load and the results of routinely used diagnostic tests.

  17. ImmunoCard STAT! cartridge antigen detection assay compared to microplate enzyme immunoassay and modified Kinyoun's acid-fast staining technique for detection of Cryptosporidium in fecal specimens.

    PubMed

    El-Moamly, Amal Abdul-Rasheed; El-Sweify, Mohamed Aly

    2012-02-01

    Cryptosporidium species infect humans and a wide range of animals worldwide; outbreaks of cryptosporidiosis have been reported in several countries. Routine diagnostic methods may be insufficient to demonstrate the presence of these organisms. The study assessed the diagnostic accuracy of the antigen detection immuno-cartridge test, ImmunoCard STAT! (Meridian Bioscience Inc., Cincinnati, OH, USA), compared to the combined gold standard: modified Kinyoun's acid-fast technique confirmed with the microplate enzyme immunoassay (EIA) for the detection of Cryptosporidium in fecal specimens. Three hundred fifteen formalin-fixed stool specimens were submitted for testing. The Kinyoun's acid-fast-stained smear revealed 24 positive samples for Cryptosporidium (of which 23 specimens were confirmed by the EIA) and 291 negative samples (of which 289 were negative by EIA). Agreement between the three used tests was shown in 22 positive and 288 negative samples for Cryptosporidium. Kappa score of agreement between the immuno-cartridge test and EIA was 0.957, p = 0.000. The sensitivity of the immuno-cartridge test was 96% (95% confidence interval (CI), 87% to 104%) and the total accuracy of the test was 97% (95% CI, 93-103). The ImmunoCard STAT! Cryptosporidium cartridge assay is easy to use and does not require specialized training or equipment and is useful in routine diagnosis and screening for Cryptosporidium especially where rapid, point of care testing is needed or where other reliable tests are unfeasible with a performance comparable to the EIA and acid-fast technique.

  18. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader.

    PubMed

    Pick, E; Mizel, D

    1981-01-01

    Two simple semiautomated microassays for the measurement of superoxide (O-2) and hydrogen peroxide (H2O2) production by cultured macrophages (MPs) are described. The measurement of O-2 is based on the reduction of ferricytochrome c as assayed by the increase in its absorbance at 550 nm. Quantitation of H2O2 is based on the horseradish peroxidase (HRPO)-dependent oxidation of phenol red which is assayed by its increased absorbance at 600 nm. MPs are cultured in monolayers in 96-well flat-bottom tissue culture plates and covered with 100 mul amounts per well of either a ferricytochrome c solution containing phenol red and HRPO. Following the addition of an agent eliciting an oxidative burst (OB) and incubation of the plates at 37 degrees C for various time intervals, the changes in the absorbance of ferricytochrome c and phenol red, respectively, are measured directly in the wells of the tissue culture plates with the cells in situ, by using an automatic 8-channel photometer which reads absorbances vertically through individual wells. This instrument, which was originally designed for reading enzyme immunoassays in microtitration plates, can be easily adapted for use in the above test, when fitted with interference filters with wave lengths of 550 nm (for the assay of O-2) and 600 nm (for the assay of H2O2). The principal advantages of this techniques are: the ability to perform the assays directly in the culture plates with cells in situ; the small amounts of cells and reagents needed; its sensitivity and reproducibility; the ease with which kinetic experiments can be done; the large number of samples which can be tested in parallel, and especially the speed and convenience offered by the automated reading and printout of absorbance values.

  19. 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).

    PubMed

    Kajaysri, Jatuporn; Nokkaew, Weerapun

    2014-03-01

    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.

  20. Performance of the fourth-generation Bio-Rad GS HIV Combo Ag/Ab enzyme immunoassay for diagnosis of HIV infection in Southern Africa.

    PubMed

    Piwowar-Manning, Estelle; Fogel, Jessica M; Richardson, Paul; Wolf, Shauna; Clarke, William; Marzinke, Mark A; Fiamma, Agnès; Donnell, Deborah; Kulich, Michal; Mbwambo, Jessie K K; Richter, Linda; Gray, Glenda; Sweat, Michael; Coates, Thomas J; Eshleman, Susan H

    2015-01-01

    Fourth-generation HIV assays detect both antigen and antibody, facilitating detection of acute/early HIV infection. The Bio-Rad GS HIV Combo Ag/Ab assay (Bio-Rad Combo) is an enzyme immunoassay that simultaneously detects HIV p24 antigen and antibodies to HIV-1 and HIV-2 in serum or plasma. To evaluate the performance of the Bio-Rad Combo assay for detection of HIV infection in adults from Southern Africa. Samples were obtained from adults in Soweto and Vulindlela, South Africa and Dar es Salaam, Tanzania (300 HIV-positive samples; 300 HIV-negative samples; 12 samples from individuals previously classified as having acute/early HIV infection). The samples were tested with the Bio-Rad Combo assay. Additional testing was performed to characterize the 12 acute/early samples. All 300 HIV-positive samples were reactive using the Bio-Rad Combo assay; false positive test results were obtained for 10 (3.3%) of the HIV-negative samples (sensitivity: 100%, 95% confidence interval [CI]: 98.8-100%); specificity: 96.7%, 95% CI: 94.0-98.4%). The assay detected 10 of the 12 infections classified as acute/early. The two infections that were not detected had viral loads<400 copies/mL; one of those samples contained antiretroviral drugs consistent with antiretroviral therapy. The Bio-Rad Combo assay correctly classified the majority of study specimens. The specificity reported here may be higher than that seen in other settings, since HIV-negative samples were pre-screened using a different fourth-generation test. The assay also had high sensitivity for detection of acute/early infection. False-negative test results may be obtained in individuals who are virally suppressed. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Imaging of Lactobacillus brevis single cells and microcolonies without a microscope by an ultrasensitive chemiluminescent enzyme immunoassay with a photon-counting television camera.

    PubMed

    Yasui, T; Yoda, K

    1997-11-01

    An ultrasensitive chemiluminescent enzyme immunoassay (CLEIA) was developed for the rapid detection and quantification of Lactobacillus brevis contaminants in beer and pitching yeast (Saccharomyces cerevisiae slurry collected for reinoculation). L. brevis cells trapped on a 47-mm nucleopore membrane (0.4-micron pore size) were reacted with a peroxidase-labelled Lactobacillus group E antibody and then subjected to an enhanced CLEIA analysis with 4-iodophenol as the enhancer. The combination of a nucleopore membrane with low background characteristics that enables the antigen-antibody reaction to proceed through the pores of the membrane and a labelled antibody prepared by the maleimide hinge method with minimal nonspecific binding characteristics was essential to minimize background in the detection of single cells. An ultrahigh sensitive charge-coupled device (CCD) camera equipped with a fiber optics image intensifier permitted the imaging of single cells. A clear correlation existed between the number of luminescent spots observed and the plate count [y (CLEIA) = 0.990x (plate count) + 15.9, where n = 7, r = 0.993, and P < 0.001]. Microscopic observation confirmed that the luminescent spots were produced by single cells. This assay could be used to detect approximately 20 L. brevis cells in 633 ml of beer within 4 h. Our ultrasensitive CLEIA could also be used to detect microcolonies approximately 20 microns in diameter which had formed on a membrane after 15 to 18 h of incubation. This method, which we called the microcolony immunoluminescence (MIL) method, increased the signal-to-noise ratio dramatically. The MIL method could be used to detect a 10(0) level of L. brevis contamination in 633 ml of beer and a 1/10(8) level of L. brevis contamination in pitching yeast within 1 day (15 to 18 h to form microcolonies and 2 h for CLEIA).

  2. Development of highly sensitive chemiluminescence enzyme immunoassay based on the anti-recombinant H(C) subunit of botulinum neurotoxin type A monoclonal antibodies.

    PubMed

    Liu, Zhijia; Song, Chaojun; Li, Yongming; Liu, Fei; Zhang, Kui; Sun, Yuanjie; Li, Haitao; Wei, Yuying; Xu, Zhuwei; Zhang, Chunmei; Yang, Angang; Xu, Zhikai; Yang, Kun; Jin, Boquan

    2012-07-20

    Botulinum neurotoxins (BoNTs) are the most poisonous substances ever known. The early detection of these toxins could bear more time for appropriate medical intervention. The standard method for detecting BoNTs is the mouse bioassay, which is time consuming (up to 4 days) and requires a large number of laboratory animals. The immunologic detection methods could detect the toxins within a day, but most of these methods are less sensitive compared with the mouse bioassay due to the lack of high-affinity antibodies. Recently, the recombinant H(C) subunit of botulinum neurotoxin type A (rAH(C)) was expressed as an effective vaccine against botulism, indicating that the rAH(C) could be an effective immunogen that raises the monoclonal antibody (mAb) for detecting BoNT/A. After immunized BALB/c mice with rAH(C), 56 mAbs were generated. Two of these mAbs were selected to establish a highly sensitive sandwich chemiluminescence enzyme immunoassay (CLEIA), in which FMMU-BTA-49 and FMMU-BTA-22 were used as capture antibody and detection antibody, respectively. The calculated limit of detection (LOD) based on molecular weight of rAH(C) and BoNT/A reached 0.45 pg mL(-1). This CLEIA can be used in the detection of BoNT/A in matrices such as milk and beef extract. This method has 20-40 fold lower LOD than that of the mouse bioassay and takes only 3 h to complete the detection, indicating that it can be used as a valuable method to detect and quantify BoNT/A.

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

    PubMed

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

    2016-08-01

    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.

  4. The laboratory impact of changing syphilis screening from the rapid-plasma reagin to a treponemal enzyme immunoassay: a case-study from the Greater Toronto Area.

    PubMed

    Mishra, Sharmistha; Boily, Marie-Claude; Ng, Victoria; Gold, Wayne L; Okura, Tom; Shaw, Marsha; Mazzulli, Tony; Fisman, David N

    2011-03-01

    In 2005, syphilis screening in the Greater Toronto Area of Canada moved from the rapid plasma reagin (RPR) to a treponemal enzyme immunoassay (EIA). We sought to understand the consequences of this change on laboratory results and testing patterns with a population-based retrospective study of laboratory-based diagnoses of syphilis. Samples positive under RPR (1998-2005) and EIA (2005-2008) screening were confirmed with an alternate treponemal test, and during the latter period underwent RPR testing. We compared monthly rates and the forecasting relationship between positives and future submissions with time-series methods, and assessed risk factors for EIA(+)/RPR(-) results using Poisson regression. A total of 3,092,938 submissions were included. Following EIA implementation, confirmed positive rates increased by 10.3 per 100,000 population (P<0.001). 0.59% of EIA(+)/RPR(-) individuals converted to RPR(+) within 2 months. EIA(+)/RPR(-) patients were more likely to be male (incidence rate ratio [IRR]: 2.3, 95% confidence interval [CI]: 1.6-2.5), asymptomatic (IRR: 1.8, 95% CI: 1.3-2.8), and aged>50 years (IRR: 2.4, 95% CI: 1.6-3.5) than those with EIA(+)/RPR(+) results. We detected a significant positive feedback loop between positive tests and subsequent submissions. This relationship was only transiently evident for EIA(+)/RPR(-) results up to 1 year following the changeover. EIA screening facilitates identification of probable latent syphilis and earlier serological detection of infectious syphilis, but may transiently cause increases in testing and indirectly suggests that physicians' interpretation of RPR(-) serology may lead to partner testing. In the absence of a true gold standard, implementation of EIA screening warrants careful communication regarding serological interpretation.

  5. Quantifying estrogen metabolism: an evaluation of the reproducibility and validity of enzyme immunoassays for 2-hydroxyestrone and 16alpha-hydroxyestrone in urine.

    PubMed Central

    Ziegler, R G; Rossi, S C; Fears, T R; Bradlow, H L; Adlercreutz, H; Sepkovic, D; Kiuru, P; Wahala, K; Vaught, J B; Donaldson, J L; Falk, R T; Fillmore, C M; Siiteri, P K; Hoover, R N; Gail, M H

    1997-01-01

    Rapid and simple enzyme immunoassays (EIAs) were recently developed to measure 2-hydroxyestrone and 16alpha-hydroxyestrone in unextracted urine. The balance between these competing estrogen metabolism pathways may serve as a biomarker of breast cancer risk. Before testing these assays in epidemiologic studies, we evaluated their reproducibility, and validity relative to gas chromatography-mass spectroscopy (GC-MS). Overnight 12-hr urine collections from five midfollicular premenopausal women, five midluteal premenopausal women, and five postmenopausal women were aliquoted and stored at -70 degrees C. Two aliquots from each woman were assayed with the EIAs in a random, blinded order, monthly over 4 months and 1 year later. Reproducibility over 4 months was good for both metabolites in premenopausal women (coefficient of variation = 8-14%) and satisfactory in postmenopausal women (approximately 19%). Reproducibility over 12 months remained good in premenopausal women, but was poor in postmenopausal women, with mean readings increasing 50 to 100%. Wide variation in estrogen metabolite levels enabled a single EIA measurement to characterize individual differences among premenopausal women in midfollicular (intraclass correlation coefficient = 98-99%) and midluteal phase (85-91%). A narrower range in metabolite levels among postmenopausal women reduced discrimination (78-82%). The correlation between EIA and GC-MS measurement was excellent for both metabolites (r>0.9), except for 2-hydroxyestrone in postmenopausal women (r=0.6). Analysis of absolute agreement suggested that both EIAs were less sensitive than GC-MS, and each detected nonspecific background. The low concentration of estrogen metabolites in urine from postmenopausal women may explain the problems with reproducibility and validity in this menstrual group. Accordingly, more sensitive EIAs have been developed and are now being evaluated. PMID:9168003

  6. Sensitive Enzyme Immunoassay with β-d-Galactosidase—Fab Conjugate for Detection of Type A Influenza Virus Antigen in Clinical Specimens

    PubMed Central

    Harmon, Maurice W.; Russo, Lorrie L.; Wilson, Samuel Z.

    1983-01-01

    The most sensitive method for diagnosis of type A influenza virus infection is isolation of the agent in cell culture. However, detection and identification may require several days to complete. This delay in diagnosis prevents effective use of the antiviral agents available for treatment of type A influenza infection. As a rapid diagnostic method, enzyme immunoassay (EIA) is attaining increased usage for direct detection of viral antigen in clinical specimens. Standard EIA techniques, however, are usually not sensitive enough for reliable detection of viral antigen in respiratory secretions. We developed a conjugate consisting of the antigen-binding fragment of goat antirabbit immunoglobulin G coupled to β-d-galactosidase, using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Other immunoreagents in our EIA consisted of guinea pig and rabbit antisera to influenza A/Brazil/11/78 (H1N1) for microtiter plate coating and primary antiserum, respectively. The sensitivity of this EIA was tested with 60 clinical specimens containing influenza A/England/333/80 (H1N1) which closely resembles A/Brazil. Of 31 initial specimens, collected within 24 h of the onset of symptoms, 27 (87%) were positive, using a fluorgenic substrate, and 18 of 29 (62%) specimens obtained 12 to 60 h after the initial specimens were positive, for a total of 75% (45 of 60). All positive reactions were specific, as shown in a confirmatory test with preimmune and hyperimmune guinea pig globulins. Clinical specimens negative for virus (n = 33) or containing heterologous respiratory viruses (n = 26) were negative in this system. These results indicate that EIA systems can be developed with a sensitivity approaching that required for clinical usefulness. PMID:6403573

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

    PubMed

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

    2013-01-01

    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.

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

    PubMed

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

    2015-07-09

    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.

  9. Europium nanoparticle-based simple to perform dry-reagent immunoassay for the detection of hepatitis B surface antigen.

    PubMed

    Talha, Sheikh M; Salminen, Teppo; Juntunen, Etvi; Spangar, Anni; Gurramkonda, Chandrasekhar; Vuorinen, Tytti; Khanna, Navin; Pettersson, Kim

    2016-03-01

    Hepatitis B infection, caused by hepatitis B virus (HBV), presents a huge global health burden. Serological diagnosis of HBV mainly relies on the detection of hepatitis B surface antigen (HBsAg). Although there are high sensitivity commercial HBsAg enzyme immunoassays (EIAs) available, many low-resource laboratories lacking trained technicians continue to use rapid point-of-care assays with low sensitivities for HBsAg detection, due to their simplicity to operate. We developed a time-resolved fluorometric dry-reagent HBsAg immunoassay which meets the detection limit of high sensitivity EIAs but is simple to operate. To develop the assay, anti-HBsAg monoclonal antibody coated on europium nanoparticles was dried atop of biotinylated anti-HBsAg polyclonal antibody immobilized on streptavidin-coated microtiter wells. To test a sample in dry-reagent assay, serum sample and assay buffer were added to the wells, incubated, washed and europium signals were measured. The assay showed a detection limit of 0.25 ng/ml using HBsAg spiked in serum sample. When evaluated with 24 HBV positive and 37 negative serum samples, assay showed 100% sensitivity and specificity. Assay wells are stable for at least 26 weeks when stored at 4°C, and can tolerate elevated temperatures of up to 35°C for two weeks. The developed assay has high potential to be used in low-resource laboratories.

  10. Determination of lithium in rocks: Fluorometric method

    USGS Publications Warehouse

    White, C.E.; Fletcher, M.H.; Parks, J.

    1951-01-01

    The gravimetric method in general use for the determination of lithium is tedious, and the final weighed product often contains other alkali metals. A fluorometric method was developed to shorten the time required for the analysis and to assure that the final determination is for lithium alone. This procedure is based on the complex formed between lithium and 8-hydroxyquinoline. The fluorescence is developed in a slightly alkaline solution of 95% alcohol and measurement is made on a photoelectric fluorometer. Separation from the ore is carried out by the wet method or by the distillation procedure. Sodium and potassium are removed by alcohol and ether, but complete separation is not necessary. Comparison of analyzed samples shows excellent agreement with spectrographic and gravimetric methods. The fluorometric method is more rapid than the gravimetric and produces more conclusive results. Another useful application is in the preparation of standard lithium solutions from reagent quality salts when a known standard is available. In this case no separations are necessary.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23946517

  12. Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

    PubMed

    Halling, V W; Jones, M F; Bestrom, J E; Wold, A D; Rosenblatt, J E; Smith, T F; Cockerill, F R

    1999-10-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a

  13. Concordance of four commercial enzyme immunoassay and three immunoblot formats for the detection of Lyme borreliosis antibodies in human serum: the two-tier approach remains.

    PubMed

    Dickeson, David J; Chen, Sharon C-A; Sintchenko, Vitali G

    2016-04-01

    Serological tests show considerable variation in their ability to correctly diagnose Lyme borreliosis (LB). This study compared four commercially available screening enzyme immunoassays (EIA) for the detection of LB IgG using either whole cell lysate (WCL) antigens, purified proteins or recombinant antigens with the second-tier whole cell sonicate (WCS) western immunoblots or recombinant antigen line blots. A consensus between three EIA results from 222 patient sera was designated as a point of comparison for each method which gave 66 positive and 156 negative results. The positive predictive values (PPV) of WCL EIA were 40% for the MarDx Diagnostics Borrelia burgdorferi EIA 'combined' IgG and IgM (Trinity Biotech) and 55% for the EUROIMMUN plus VlsE IgG. These were significantly lower PPVs than that produced by the recombinant antigen-based EIA NovaLisa Borrelia burgdorferi IgG-ELISA (NovaTec Immunodiagnostica) and the EUROIMMUN Anti-Borrelia Select ELISA IgG (90% and 100%, respectively; p = 0.02). The WCS western immunoblot using B. burgdorferi and B. afzelii separately showed a high PPV of 91% but its positive agreement with consensus EIA result was only 65%. Another WCL western immunoblot with purified extracts of Osp C and VlsE, the Trinity Biotech EU Lyme + VlsE IgG Western Blot had a PPV of 92% while the recombinant line blot from EUROIMMUN, the Anti-Borrelia (IgG) EUROLINE-RN-AT, demonstrated a significantly reduced PPV of 70% with some non-specific reactions in sera containing antibodies to Leptospira species, Helicobacter pylori and Treponema pallidum. The use of recombinant antigens in EIA for LB IgG screening significantly improves the predictive values of serological results above those of WCL antigen EIA. Second tier WCS western immunoblots offer high PPVs, especially with added specific purified proteins, more so than in one recombinant line blot.

  14. 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).

    PubMed

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

    2006-07-01

    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 < 0.001). Two peaks of 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

  15. 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.

    PubMed

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

    2008-03-01

    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)).

  16. Immunoassays of soy proteins.

    PubMed

    Brandon, David L; Friedman, Mendel

    2002-10-23

    Proteins of soybeans (Glycine max) are widely used in animal and human nutrition. In addition to the bulk of the seed storage proteins, which are classified as albumins and globulins, approximately 6% of soybean proteins are classified as inhibitors of trypsin and chymotrypsin and approximately 0.5% are sugar-binding lectins. The two major classes of inhibitors are the Kunitz trypsin inhibitor, which inhibits trypsin, and the Bowman-Birk inhibitor (BBI), which inhibits both trypsin and chymotrypsin. Unless removed or inactivated, these inhibitors and lectins can impair the nutritional quality and safety of soy-based diets. On the other hand, several studies suggest that BBI can also function as an anticarcinogen, possibly through interaction with a cellular serine protease. Good-quality soybean proteins contribute to the nutritional value of many specialty foods including infant soy formulas and milk replacers for calves, and provide texture to many processed foods. However, they may also induce occasional allergic responses in humans. This paper outlines immunoassays developed to analyze for soy proteins in different soybean lines, in processed foods, and in nonsoy foods fortified with soy proteins. An assessment of the current status of immunoassays, especially of enzyme-linked immunosorbent assays for soybean inhibitors of digestive enzymes, soy globulins, and soy lectins, demonstrates the usefulness of these methods in plant and food sciences and in medicine.

  17. A competitive indirect enzyme-linked immunoassay for lead ion measurement using mAbs against the lead-DTPA complex.

    PubMed

    Xiang, Jun-jian; Zhai, Yi-fan; Tang, Yong; Wang, Hong; Liu, Bin; Guo, Chang-wei

    2010-05-01

    Immunoassays for quantitative measurement of environmental heavy metals offer several advantages over other traditional methods. To develop an immunoassay for lead, Balb/c mice were immunized with a lead-chelate-protein conjugate to allow maximum exposure of the metal to the immune system. Three stable hybridoma cell lines were obtained through spleen cells fusion with Sp2/0 cells. One cell line, 2A11D11, produced mAbs with preferential selectivity and sensitivity for Pb-DTPA than DTPA, exhibiting an affinity constant of 3.34 + or - 0.24 x 10(9) M(-1). Cross reactivity (CR) with other metals were below 1%, except for Fe(III) with a CR less than 5%. This quantitative indirect ELISA for the lead ion was used to detect environmental lead content in local water sources; importantly, the results from the immunoassay were in excellent agreement with those from ICP-MS. Development of immunoassays for metal ions may thus facilitate the detection and regulation of environmental pollution.

  18. Target-induced nano-enzyme reactor mediated hole-trapping for high-throughput immunoassay based on a split-type photoelectrochemical detection strategy.

    PubMed

    Zhuang, Junyang; Tang, Dianyong; Lai, Wenqiang; Xu, Mingdi; Tang, Dianping

    2015-09-15

    Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on a microplate using a secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthesized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nanoenzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of the photocurrent signal. To elaborate, the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system), was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL(-1), with a low detection limit (LOD) of 0.32 pg mL(-1). Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format.

  19. New sensitive fluorometric method for measurement of vascular permeability

    SciTech Connect

    Watanabe, K.; Nakagawa, H.; Tsurufuji, S.

    1984-06-01

    A sensitive fluorometric method has been developed for the measurement of vascular permeability in carrageenin air-pouch inflammation in rats. Fluorescein isothiocyanate-labeled bovine serum albumin (F-BSA) was used as a tracer. This fluorometric method is as simple and reliable as the method using radioiodine-labeled human serum albumin and has the advantages of low cost, no health hazard, and the fact that F-BSA can be stored over a long period. This fluorometric method is probably applicable to other inflammation models such as pleurisy and peritonitis in which inflammatory exudate can be collected.

  20. 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

    PubMed Central

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

    2004-01-01

    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. PMID:15472364

  1. 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.

    PubMed

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

    2004-10-01

    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.

  2. Bioelectrochemical Immunoassay of Polychlorinated Biphenyl

    SciTech Connect

    Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.; Lin, Yuehe

    2008-04-01

    A simple, rapid, and highly sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) and disposable screen-printed electrodes (SPE) has been developed to detect polychlorinated biphenyls (PCBs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PCB-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PCB (HRP-PCB). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreactions among PCB-antibody-coated MBs, PCB analyte, and HRP-PCB. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing o-aminophenol and hydrogen peroxide for electrochemical detection. The different parameters, including the amount of HRP-PCB conjugates, immunoreaction time, and the concentration of substrate that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 5 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical immunoassay was successfully evaluated with untreated river water spiked with PCBs, and the results were validated by commercial PCB enzyme-linked immunosorbent assay kit, indicating that this convenient and sensitive technique offers great promise for decentralized environmental application and trace PCBs monitoring.

  3. Quantitative fluorometric assay for the measurement of endo-1,4-β-glucanase.

    PubMed

    Mangan, D; McCleary, B V; Liadova, A; Ivory, R; McCormack, N

    2014-08-18

    There is a growing demand for research tools to aid the scientific community in the search for improved cellulase enzymes for the biofuel industry. In this work, we describe a novel fluorometric assay for cellulase (endo-1,4-β-glucanase) which is based on the use of 4,6-O-benzylidene-4-methylumbelliferyl-β-cellotrioside (BzMUG3) in the presence of an ancillary β-glucosidase. This assay can be used quantitatively over a reasonable linear range, or qualitatively as a solution screening tool which may find extensive use in the area of metagenomics.

  4. 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

    NASA Astrophysics Data System (ADS)

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

    2008-10-01

    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).

  5. Fluorometric determination of hydrogen peroxide in groundwater

    SciTech Connect

    Holm, T.R.; George, G.K.; Barcelona, M.J.

    1987-02-15

    The fluorometric scopoletin-horseradish peroxidase method has been modified for field determinations of hydrogen peroxide concentrations in groundwaters. Standard additions calibration compensates for background fluorescence and inconsistent stoichiometry of the fluorescence quenching reaction due to interferences by the matrix. The detection limit, defined as the blank plus three standard deviations, ranged from 3.6 to 44.6 nM. However, this limit was more an indication of the difficulty of preparing peroxide-free water than the actual limit imposed by the sensitivity of the method for the peroxide contamination introduced with the reagents. For 111 field determinations the weighted average (uncorrected) hydrogen peroxide concentration was 20.2 nM and the pooled standard deviation was 7.7 nM. The average of 45 field blanks was 7.8 nM with a pooled standard deviation of 5.2 nM. At nanomolar concentration levels, it is essential that samples are analyzed for H/sub 2/O/sub 2/ in the field. Storage periods exceeding 1 h caused serious errors and irreproducible results.

  6. Fluorometric determination of zirconium in minerals

    USGS Publications Warehouse

    Alford, W.C.; Shapiro, L.; White, C.E.

    1951-01-01

    The increasing use of zirconium in alloys and in the ceramics industry has created renewed interest in methods for its determination. It is a common constituent of many minerals, but is usually present in very small amounts. Published methods tend to be tedious, time-consuming, and uncertain as to accuracy. A new fluorometric procedure, which overcomes these objections to a large extent, is based on the blue fluorescence given by zirconium and flavonol in sulfuric acid solution. Hafnium is the only element that interferes. The sample is fused with borax glass and sodium carbonate and extracted with water. The residue is dissolved in sulfuric acid, made alkaline with sodium hydroxide to separate aluminum, and filtered. The precipitate is dissolved in sulfuric acid and electrolysed in a Melaven cell to remove iron. Flavonol is then added and the fluorescence intensity is measured with a photo-fluorometer. Analysis of seven standard mineral samples shows excellent results. The method is especially useful for minerals containing less than 0.25% zirconium oxide.

  7. Fluorometric determination of histamine in cheese.

    PubMed

    Chambers, T L; Staruszkiewicz, W F

    1978-09-01

    Thirty-one samples of cheese obtained from retail outlets were analyzed for histamine, using an official AOAC fluorometric method. The types of cheese analyzed and the ranges of histamine found were: colby, 0.3--2.8; camembert, 0.4--4.2; cheddar, 1.2--5.8; gouda, 1.3--2.4; provolone, 2.0--23.5; roquefort, 1.0--16.8; mozzarella 1.6--5.0; and swiss, 0.4--250 mg histamine/100 g. Ten of the 12 samples of swiss cheese contained less than 16 mg histamine/100 g. The remaining 2 samples which contained 116 and 250 mg histamine/100 g were judged organoleptically to be of poor quality. An investigation of one processing facility showed that the production of histamine in swiss cheese may have been a result of a hydrogen peroxide/low temperature treatment of the milk supply. Recovery of histamine added to methanol extracts of cheese ranged from 93 to 105%. Histamine content was confirmed by high pressure liquid chromatographic analysis of the methanol extracts.

  8. A new dual immunoassay for tumor markers based on chemiluminescence signal amplification by magnetic mesoporous silica and enzyme modified gold nanoparticles.

    PubMed

    Lin, Jiehua; Chu, Pengfei; Wei, Zhijing

    2012-01-01

    A sensitive dual immunoassay was proposed for the determination of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) based on signal amplification. Monoclonal antibodies immobilized on magnetic mesoporous silica particles (Fe(3)O(4)/SiO(2)) were prepared as the primary probe. Horseradish peroxidase (HRP) labeled antibodies co-coated with HRP on gold nanoparticles (AuNPs) were used as the secondary probe to achieve signal amplification. HRP tags were retained in the flow cells after a sandwich immunoassay. By controlling two switches on the two channels, chemiluminescent substrates were injected orderly man way, and then signals for CEA and AFP were sequentially detected by HRP-luminol-H(2)O(2). Due to the increased amount of HRP on AuNPs and the increased amount of monoclonal antibodies on Fe(3)O(4)/SiO(2), the signals were largely amplified. Under the optimal conditions, CEA and AFP could be detected in the linear ranges of 1.0 - 80 and 1.0 - 75 ng mL(-1) with detection limits of 0.25 and 0.5 ng mL(-1), respectively.

  9. [Possibility of the species identification using blood stains located on the material evidences and bone fragments with the method of solid phase enzyme immunoassay with "IgG general-EIA-BEST" kit and human immunoglobulin G].

    PubMed

    Sidorov, V L; Shvetsova, I V; Isakova, I V

    2007-01-01

    The authors give the comparative analysis of Russian and foreign forensic medical methods of species character identification of the blood from the stains on the material evidences and bone fragments. It is shown that for this purpose it is feasible to apply human immunoglobulin G (IgG) and solid phase enzyme immunoassay (EIA) with the kit "IgG general-EIA-BEST". In comparison with the methods used in Russia this method is more sensitive, convenient for objective registration and computer processing. The results of experiments shown that it is possible to use the kit "IgG general-EIA-BEST" in forensic medicine for the species character identification of the blood from the stains on the material evidences and bone fragments.

  10. Enzyme

    MedlinePlus

    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 ...

  11. The differences in short- and long-term varicella-zoster virus (VZV) immunoglobulin G levels following varicella vaccination of healthcare workers measured by VZV fluorescent-antibody-to-membrane-antigen assay (FAMA), VZV time-resolved fluorescence immunoassay and a VZV purified glycoprotein enzyme immunoassay.

    PubMed

    Maple, P A C; Haedicke, J; Quinlivan, M; Steinberg, S P; Gershon, A A; Brown, K E; Breuer, J

    2016-08-01

    Healthcare workers (HCWs) reporting no history of varicella frequently receive varicella vaccination (vOka) if they test varicella-zoster virus (VZV) immunoglobulin G (IgG) negative. In this study, the utilities of VZV-IgG time-resolved fluorescence immunoassay (VZV-TRFIA) and a commercial VZV-IgG purified glycoprotein enzyme immunoassay (gpEIA) currently used in England for confirming VZV immunity have been compared to the fluorescent-antibody-to-membrane-antigen assay (FAMA). A total of 110 HCWs received two doses of vOka vaccine spaced 6 weeks apart and sera collected pre-vaccination (n = 100), at 6 weeks post-completion of vaccination (n = 86) and at 12-18 months follow-up (n = 73) were analysed. Pre-vaccination, by FAMA, 61·0% sera were VZV IgG negative, and compared to FAMA the sensitivities of VZV-TRFIA and gpEIA were 74·4% [95% confidence interval (CI) 57·9-87·0] and 46·2% (95% CI 30·1-62·8), respectively. Post-completion of vaccination the seroconversion rate by FAMA was 93·7% compared to rates of 95·8% and 70·8% determined by VZV-TRFIA and gpEIA, respectively. At 12-18 months follow-up seropositivity rates by FAMA, VZV-TRFIA and gpEIA were 78·1%, 74·0% and 47·9%, respectively. Compared to FAMA the sensitivities of VZV-TRFIA and gpEIA for measuring VZV IgG following vaccination were 96·4% (95% CI 91·7-98·8) and 74·6% (95% CI 66·5-81·6), respectively. Using both FAMA and VZV-TRFIA to identify healthy adult VZV susceptibles and measure seroconversion showed that vOka vaccination of HCWs is highly immunogenic.

  12. Immunoassay as a screening tool for industrial toxicants

    SciTech Connect

    Pierce, T.

    1986-08-01

    Immunoassay techniques may represent useful screening tools to assist analysts interested in the presence and amounts of organic toxicants in biological fluids. The widespread application of immunoassay methods in medicinal and forensic (drugs of abuse) chemistry has resulted in such screening methodologies. Four methodologies of potential benefit are considered: the free radical assay technique, the enzyme-mediated immunoassay technique, radioimmunoassay, and hemagglutination. Each of these immunoassays is based on the competitive displacement of the labeled drug (or toxicant) from the antibody complex by the unlabeled drug-toxicant in the sample.

  13. Immunoassays in Biotechnology

    EPA Science Inventory

    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...

  14. Immunoassays in Biotechnology

    EPA Science Inventory

    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...

  15. Direct detection of Helicobacter pylori resistance to macrolides by a polymerase chain reaction/DNA enzyme immunoassay in gastric biopsy specimens

    PubMed Central

    Marais, A; Monteiro, L; Occhialini, A; Pina, M; Lamouliatte, H; Megraud, F

    1999-01-01

    reaction (PCR); immunoassay PMID:10075951

  16. Colloidal nanomaterial-based immunoassay.

    PubMed

    Teste, Bruno; Descroix, Stephanie

    2012-06-01

    Nanomaterials have been widely developed for their use in nanomedicine, especially for immunoassay-based diagnosis. In this review we focus on the use of nanomaterials as a nanoplatform for colloidal immunoassays. While conventional heterogeneous immunoassays suffer from mass transfer limitations and consequently long assay time, colloidal immunosupports allow target capture in the entire volume, thus speeding up reaction kinetics and shortening assay time. Owing to their wide range of chemical and physical properties, nanomaterials are an interesting candidate for immunoassay development. The most popular colloidal nanomaterials for colloidal immunoassays will be discussed, as well as their influence on immune reactions. Recent advances in nanomaterial applications for different formats of immunoassays will be reported, such as nanomaterial-based indirect immunoassays, optical-based agglutination immunoassays, resonance energy transfer-based immunoassays and magnetic relaxation-based immunoassays. Finally, the future of using nanomaterials for homogeneous immunoassays dedicated to clinical diagnosis will be discussed.

  17. Microfluidic paper-based analytical device for photoelectrochemical immunoassay with multiplex signal amplification using multibranched hybridization chain reaction and PdAu enzyme mimetics.

    PubMed

    Lan, Feifei; Sun, Guoqiang; Liang, Linlin; Ge, Shenguang; Yan, Mei; Yu, Jinghua

    2016-05-15

    promising platform of clinical immunoassay for other biomolecules. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Kinase Activity Studied in Living Cells Using an Immunoassay

    ERIC Educational Resources Information Center

    Bavec, Aljos?a

    2014-01-01

    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…

  19. Kinase Activity Studied in Living Cells Using an Immunoassay

    ERIC Educational Resources Information Center

    Bavec, Aljos?a

    2014-01-01

    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…

  20. Discrimination of methicillin-resistant Staphylococcus aureus from methicillin-susceptible Staphylococcus aureus or coagulase-negative staphylococci by detection of penicillin-binding protein 2 and penicillin-binding protein 2' using a bioluminescent enzyme immunoassay.

    PubMed

    Shiga, Kazuki; Gomi, Keiko; Nishimura, Motoi; Watanabe, Masaharu; Nomura, Fumio; Kajiyama, Naoki

    2013-02-28

    For the discrimination of methicillin-resistant S. aureus (MRSA) from methicillin-susceptible S. aureus (MSSA) or coagulase-negative staphylococci (CNS), we developed a bioluminescent enzyme immunoassay (BLEIA) for detecting penicillin-binding protein 2 (PBP2) and penicillin-binding protein 2' (PBP2') using biotinylated firefly luciferase. The BLEIA was able to detect recombinant PBP2 at 50 pg/ml and recombinant PBP2' at 500 pg/ml. PBP2 and PBP2' present in the membranes of S. aureus were extracted by acid and detergent treatment. The method was able to detect PBP2 or PBP2' extracted from 10(6) colony forming units of S. aureus because of efficient extraction and the high sensitivity of luciferase. In a study of clinical isolates previously characterized as either MRSA or MSSA by antibiotic susceptibility testing, all 34 specimens identified as MRSA were both PBP2 and PBP2' positive. The 34 MSSA specimens were PBP2 positive and PBP2' negative. Moreover, the BLEIA could detect PBP2' extracted from four species of methicillin-resistant CNS, but not PBP2 extracted from four species of methicillin-resistant and methicillin-susceptible CNS. This result suggested that PBP2 could be a unique marker for discrimination of S. aureus from CNS. A BLEIA that is able to detect PBP2 and PBP2' may be useful in clinical diagnostics.

  1. Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children

    PubMed Central

    Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

    2015-01-01

    Background: The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Methods: Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9–11 years were determined by two methods of CPBA and HPLC. Results: Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Conclusions: Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes. PMID:26330983

  2. Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9-12 Years Children.

    PubMed

    Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

    2015-01-01

    The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9-11 years were determined by two methods of CPBA and HPLC. Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes.

  3. 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.

    PubMed

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

    2014-02-01

    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.

  4. 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.

    PubMed

    Ota, Kaede V; McGowan, Karin L

    2012-04-01

    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.

  5. Sequential injection immunoassay for environmental measurements.

    PubMed

    Soh, Nobuaki; Tanaka, Mayumi; Hirakawa, Koji; Zhang, RuiQi; Nakajima, Hizuru; Nakano, Koji; Imato, Toshihiko

    2011-01-01

    Sequential injection immunoassay systems for environmental measurements based on the selective immunoreaction between antigen and antibody were described. A sequential injection analysis (SIA) technique is suitable to be applied for the procedure of enzyme-linked immunosorbent assay (ELISA), because the washing and the addition of reagent solutions can be automated by using a computer-controlled syringe pump and switching valve. We selected vitellogenin (Vg), which is a biomarker for evaluating environmental risk caused by endocrine-disrupting chemicals in the hydrosphere, and linear alkylbenzene sulfonates (LAS) and alkylphenol polyethoxylates (APEO), which are versatile surfactants, as target analytes in the flow immunoassay systems. For Vg monitoring, SIA systems based on spectrophotometric, chemiluminescence, and electrochemical determinations were constructed. On the other hand, chemiluminescence determination was applied to the detection of LAS and APEO. For APEO, an SIA system combined with surface plasmon resonance (SPR) sensor was also developed. These new sequential injection immunoassay systems are expected to be useful systems for environmental analysis.

  6. BrucELISA: an enzyme-antibody immunoassay for detection of Brucella abortus antibodies in milk: correlation with the Brucella ring test and with shedding of viable organisms.

    PubMed Central

    Boraker, D K; Stinebring, W R; Kunkel, J R

    1981-01-01

    An indirect enzyme-antibody immunosorbent assay (BrucELISA) is described for the detection of antibody to Brucella abortus in cow's milk. Three series of milk samples were obtained from an adult-vaccinated dairy herd infected with B. abortus. The BrucELISA system was used as a screening test for individual milks diluted 1:200 (BE 200 test), for undiluted bulk milks, and to determine antibody titer (BrucELISA titration assay). The BrucELISA results correlated highly with positive Brucella ring test reactions and culture positivity, eliminated false-positive Brucella ring test reactions, detected antibody in some samples which were Brucella ring test negative, and distinguished between vaccinated and infected animals. BrucELISA titration assay titers of greater than 1:800 were correlated with shedding, or were prognostic for animals which eventually became shedders. Binding of the enzyme-antibody conjugate to bovine immunoglobulin in the absence of rabbit anti-bovine immunoglobulin occurred with culture-positive or -negative milks showing titers of greater than 1:1,600 (the beta effect); the effect was also of predictive value in identifying eventual shedders. The BrucELISA system is a sensitive, specific, and inexpensive method for screening large numbers of individual or bulk milk samples for the presence of antibody to B. abortus. PMID:6793622

  7. Fluorometric Method for Determination of Uric Acid in Flour

    DTIC Science & Technology

    1980-07-01

    of the excreta of adult Tribolium used in this study for natural infestation, accumulates in infested products providing a quantitative measure of past...by Adult Tribolium 8 4 FLUOROMETRIC METHOD FOR DETERMINATION OF URIC ACID IN FLOUR Introduction Chemical and microanalytical techniques have been...determining uric acid content.’ Approximately 18% of the total excreta from Tribolium confusum is uric acid.2 Various investigators have established

  8. Updates in immunoassays: parasitology.

    PubMed

    Josko, Deborah

    2012-01-01

    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.

    PubMed

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

    2015-08-25

    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 Fluorometric Method of Measuring Carboxypeptidase Activities for Angiotensin II and Apelin-13

    PubMed Central

    Liu, Pan; Wysocki, Jan; Serfozo, Peter; Ye, Minghao; Souma, Tomokazu; Batlle, Daniel; Jin, Jing

    2017-01-01

    Degradation of the biologically potent octapeptide angiotensin Ang II-(1-8) is mediated by the activities of several peptidases. The conversion of Ang II to the septapeptide Ang-(1-7) is of particular interest as the latter also confers organ protection. The conversion is catalyzed by angiotensin-converting enzyme 2 and other enzymes that selectively cleave the peptide bond between the proline and the phenylalanine at the carboxyl terminus of Ang II. The contribution of various enzyme activities that collectively lead to the formation of Ang-(1-7) from Ang II, in both normal conditions and in disease states, remains only partially understood. This is largely due to the lack of a reliable and sensitive method to detect these converting activities in complex samples, such as blood and tissues. Here, we report a fluorometric method to measure carboxypeptidase activities that cleave the proline-phenylalanine dipeptide bond in Ang II. This method is also suitable for measuring the conversion of apelin-13. The assay detects the release of phenylalanine amino acid in a reaction with the yeast enzyme of phenylalanine ammonia lyase (PAL). When used in cell and mouse organs, the assay can robustly measure endogenous Ang II and apelin-13-converting activities involved in the renin-angiotensin and the apelinergic systems, respectively. PMID:28378780

  11. Novel approach to activity evaluation for release-active forms of anti-interferon-gamma antibodies based on enzyme-linked immunoassay.

    PubMed

    Gavrilova, Elena S; Bobrovnik, Sergey A; Sherriff, Gordon; Myslivets, Andrey A; Tarasov, Sergey A; Epstein, Oleg I

    2014-01-01

    Selection of a suitable assay to measure the activity of drug agents based on release-active forms of anti-interferon-gamma antibodies (RA forms of Abs) is an important step forward in the investigation of such agents. In this study, the enzyme-linked immunosorbent assay was utilized to examine the effect of RA forms of Abs specific for human interferon gamma on the interaction between monoclonal anti-interferon gamma antibodies and recombinant human interferon gamma. The experimental data and the results obtained by using relevant mathematical analysis showed that such RA forms of Abs are able to modulate the monoclonal antibody interaction with both soluble and immobilized (to the assay plate well) interferon gamma. These data demonstrated the importance of using relatively low concentrations of both soluble and plate-immobilized interferon gamma to detect the effects of RA forms of Abs to interferon gamma on the binding of monoclonal antibodies to interferon gamma. It has been suggested that the observed influence of RA forms of Abs on 'antibody-antigen' interaction could be used to detect and analyze the activity of drugs containing RA forms of Abs.

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

    PubMed

    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

    2015-07-01

    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.

  13. Use of enzyme-linked immunoassays for antibody to types C and D botulinum toxins for investigations of botulism in cattle.

    PubMed

    Gregory, A R; Ellis, T M; Jubb, T F; Nickels, R J; Cousins, D V

    1996-02-01

    The development of specific enzyme-linked immunosorbent assays (ELISA) for antibody to types C and D Clostridium botulinum toxins for investigation of botulism in cattle is described. Partially purified type C and D toxins were used as antigens to develop these ELISAs. Specificity of the ELISAs was evaluated on sera from 333 adult beef and dairy cattle from areas with no history or evidence of botulism in animals or water birds. The test was also evaluated on sera from 41 herds that included herds vaccinated against botulism, confirmed botulism cases and herds from areas where the disease is considered endemic. The ELISAs detected the presence of antibody to botulinum toxins in samples from vaccinated cattle and both convalescent and clinically normal animals from unvaccinated herds with outbreaks of botulism. Antibody was also found in unvaccinated animals from herds in which there had been no diagnosed botulism cases in areas where botulism was considered endemic. Sera from some unvaccinated cattle with high ELISA reactivity was shown to be protective for mice in botulinum toxin neutralisation tests. The use of these tests in investigations of botulism in cattle is discussed.

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

    PubMed

    Gross, Madeleine; Asam, Stefan; Rychlik, Michael

    2017-02-01

    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).

  15. Enzymatic diagnosis of Fabry disease using a fluorometric assay on dried blood spots: An alternative methodology.

    PubMed

    Caudron, Eric; Prognon, Patrice; Germain, Dominique P

    2015-12-01

    Fabry disease (FD, OMIM#301500) is an X-linked lysosomal storage disorder caused by the functional deficiency of α-galactosidase A, a lysosomal enzyme. A method to screen for FD in large populations has been developed using a fluorometric assay of α-galactosidase A activity in dried blood spots (DBS) on filter paper. However, results can be influenced by quenching of fluorescence by haemoglobin which, together with small sample size, may result in a low light emission signal. An alternative, simple and sensitive fluorometric assay was developed for the determination of α-galactosidase A activity in DBS. The assay uses 4-methylumbelliferyl-α-d-galactose as an artificial substrate. To minimize the risk of false-positives, zinc sulfate was used for protein precipitation to stop the enzymatic reaction and eliminate interfering species (hemoglobin). Samples from 209 individuals (60 hemizygotes, 68 heterozygotes, and 81 controls) were tested to establish reference values for the assay. The mean α-galactosidase A activity of the 81 controls was 9.1 ± 3.3 μmol h(-1) L(-1) (mean ± SD). All 60 hemizygotes affected with FD had AGAL activities below 1.7 μmol h(-1) L(-1) (0.2 ± 0.3 μmol h(-1) L(-1)). For the 68 heterozygous females, AGAL activity ranged from 0 to 12.6 μmol h(-1) L(-1) (3.5 ± 2.7 μmol h(-1) L(-1)). Two-thirds of the female patients could be identified using the enzymatic assay and a cut-off level of 40% of the median control value (<3.4 μmol h(-1) L(-1)). Our fluorometric assay using zinc sulfate protein precipitation was shown to have similar sensitivity and robustness while reducing the risk of false positive results due to quenching of 4-MU fluorescence by haemoglobin. Copyright © 2015. Published by Elsevier Masson SAS.

  16. Indirect enzyme-linked immunosorbent assay for immunoglobulin G and four immunoassays for immunoglobulin M to Toxoplasma gondii in a series of heart transplant recipients.

    PubMed Central

    Sluiters, J F; Balk, A H; Essed, C E; Mochtar, B; Weimar, W; Simoons, M L; Ijzerman, E P

    1989-01-01

    Toxoplasma gondii infections in heart transplant recipients were monitored by indirect enzyme-linked immunosorbent assay for immunoglobulin G (ELISA-IgG), indirect ELISA-IgM in serum IgM fractions, antibody capture ELISA-IgM, IgM-immunosorbent agglutination assay (ISAGA), and IgM immunoblotting. Basic immunosuppression consisted of cyclosporine and low-dose steroids. Before transplantation, 26 of 43 recipients showed serological evidence of infection. In serum samples from 15 (35%) recipients, specific antibodies were not detected. Approximately 50% of the heart donors, were toxoplasma seropositive. Eight of the fifteen seronegative recipients received hearts from toxoplasma-seropositive donors. In four of the eight recipients, seroconversion could be demonstrated with all tests used. In three of these four patients, clinical disease developed. One patient with strong serological evidence of toxoplasmosis died, but toxoplasma parasites and antigens were not detected at autopsy. In two patients, toxoplasma cysts were found in cardiac biopsies. Seroconversion was not prevented by the use of spiramycin prophylaxis in two recipients. Reactivations of latent infections or reinfections were detected by indirect ELISA in six (23%) seropositive recipients, but symptoms and signs of active T. gondii infection were not seen. Seroconversion and reactivation of infection were readily found by a combined use of indirect ELISA-IgG and ELISA-IgM and antibody capture ELISA-IgM. Discrepancies in results could be examined by immunoblotting. IgM-ISAGA retained stable positive values longer than IgM-ELISAs did. Cyclosporine treatment did not hamper detection of seroconversion but could cause antibody levels to remain relatively low in primary infections. Seronegative recipients should receive antitoxoplasma treatment on seroconversion. PMID:2654182

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

    PubMed

    de Arruda, Mauro Maciel; Figueiredo, Fabiano Borges; Cardoso, Fernanda Alvarenga; Hiamamoto, Roberto Mitsuyoshi; Brazuna, Júlia Cristina Macksoud; de Oliveira, Maria Regina Fernandes; Noronha, Elza Ferreira; Romero, Gustavo Adolfo Sierra

    2013-01-01

    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. 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. 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. 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.

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

    PubMed

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

    2014-09-01

    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.

  19. Diagnostic Accuracy of the InBios Scrub Typhus Detect Enzyme-Linked Immunoassay for the Detection of IgM Antibodies in Northern Thailand

    PubMed Central

    Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Nawtaisong, Pruksa; Kantipong, Pacharee; Laongnualpanich, Achara; Day, Nicholas P. J.

    2015-01-01

    In this study, we examined the diagnostic accuracy of the InBios Scrub Typhus Detect IgM enzyme-linked immunosorbent assay (ELISA) and determined the optimal diagnostic optical density (OD) cutoffs for screening and diagnostic applications based on prospectively collected, characterized samples from undifferentiated febrile illness patients in northern Thailand. Direct comparisons with the serological gold standard, indirect immunofluorescence assay (IFA), revealed strong statistical correlation of ELISA OD values and IFA IgM titers. Determination of the optimal ELISA cutoff for seroepidemiology or screening purposes compared to the corresponding IFA reciprocal titer of 400 as previously described for Thailand was 0.60 OD, which corresponded to a sensitivity (Sn) of 84% and a specificity (Sp) of 98%. The diagnostic performance against the improved and more-stringent scrub typhus infection criteria (STIC), correcting for low false-positive IFA titers, resulted in an Sn of 93% and an Sp of 91% at an ELISA cutoff of 0.5 OD. This diagnostic ELISA cutoff corresponds to IFA reciprocal titers of 1,600 to 3,200, which greatly reduces the false-positive rates associated with low-positive IFA titers. These data are in congruence with the recently improved serodiagnostic positivity criteria using the Bayesian latent class modeling approach. In summary, the InBios Scrub Typhus Detect IgM ELISA is affordable and easy-to-use, with adequate diagnostic accuracy for screening and diagnostic purposes, and should be considered an improved alternative to the gold standard IFA for acute diagnosis. For broader application, regional cutoff validation and antigenic composition for consistent diagnostic accuracy should be considered. PMID:26656118

  20. 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.

    PubMed

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

    2011-01-01

    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.

  1. Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment.

    PubMed

    Tao, Xiaoqi; Chen, Min; Jiang, Haiyang; Shen, Jianzhong; Wang, Zhanhui; Wang, Xia; Wu, Xiaoping; Wen, Kai

    2013-09-01

    A chemiluminescent competitive indirect enzyme-linked immunosorbent assay, based on a mutant single-chain variable fragment (scFv), was developed to detect a broad range of fluoroquinolones (FQs) in fish and shrimp matrices. In this study, the best scFvC4A9H1_mut2 was adopted, which showed 10-fold improved affinity to sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO), while the affinity to other FQs was fully inherited from wild-type scFvC4A9H1. In the optimized generic test, scFvC4A9H1_mut2 in combination with norfloxacin-ovalbumin conjugate and horseradish peroxidase-labeled anti-c-myc 9E10 antibody showed 50 % binding inhibition (IC50) at 0.12 μg kg(-1) for norfloxacin in buffer. Screening for the class of FQ antibiotics is accomplished using a simple, rapid extraction carried out with ethanol/acetic acid (99:1, v/v). This common extraction was able to detect 20 FQ residues such as s ciprofloxacin (CIP), danofloxacin, DIF, enoxacin, enrofloxacin (ENR), fleroxacin, amifloxacin, flumequine, levofloxacin, lomefloxacin hydrochloride, marbofloxacin, norfloxacin (NOR), ofloxacin, orbifloxacin, pazufloxacin, pefloxacin-d5 (PEF), prulifloxacin, SAR, sparfloxacin, and TRO in fish and shrimp. The limit of detection (LOD) for NOR was 0.2 μg kg(-1) and the LODs for CIP and ENR were all <0.2 μg kg(-1). Values of LODs inferred from the cross-reactivity data will range from approximately 0.23 μg kg(-1) for PEF to 2.1 μg kg(-1) for TRO. Field fish and shrimp samples were analyzed and compared to the results obtained from liquid chromatography tandem mass spectrometric method. All five instances (from 0.25 to 15.6 μg kg(-1)) in which FQs were present at concentrations near or above the assay LOD were identified as positive by the newly developed assay, demonstrating the usefulness of this assay as a screening tool.

  2. Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins

    PubMed Central

    Biagini, Raymond E.; Sammons, Deborah L.; Smith, Jerome P.; MacKenzie, Barbara A.; Striley, Cynthia A. F.; Semenova, Vera; Steward-Clark, Evelen; Stamey, Karen; Freeman, Alison E.; Quinn, Conrad P.; Snawder, John E.

    2004-01-01

    Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 μg/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 μg of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 μg/ml, while the dynamic range was 0.06 to 1.7 μg/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 μg of anti-PA IgG per ml, the RDL was 0.016 μg/ml, and the whole-serum equivalent MDC was 1.5 μg/ml. The dynamic range was 0.006 to 6.8 μg/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r2 = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection. PMID:14715544

  3. Comparison of Premier CAMPY Enzyme Immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY Tests with Culture for Laboratory Diagnosis of Campylobacter Enteric Infections ▿ †

    PubMed Central

    Granato, Paul A.; Chen, Li; Holiday, Iris; Rawling, Russell A.; Novak-Weekley, Susan M.; Quinlan, Tammy; Musser, Kimberlee A.

    2010-01-01

    Campylobacter enteritis is a food-borne or waterborne illness caused almost exclusively by Campylobacter jejuni and, to a lesser extent, by Campylobacter coli. These organisms produce indistinguishable clinical diseases and together represent the second most common cause of bacterial diarrhea in the United States and the leading cause of enteric infection throughout the world. The conventional approach to the laboratory diagnosis of Campylobacter enteritis is based on the recovery of the organism from a stool specimen, which requires the use of a specialized medium incubated at 42°C for several days in an artificially created microaerophilic environment. Recently, several commercially available enzyme immunoassays (EIAs) have been developed for the direct detection of C. jejuni and C. coli in stool specimens. This study compared conventional culture with three EIA methods, the Premier CAMPY EIA (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter EIA (Remel, Lenexa, KS), and the ImmunoCard STAT! CAMPY test (Meridian Bioscience, Cincinnati, OH), for the detection of C. jejuni and C. coli in 485 patient stool samples. Discordant results were arbitrated by using an in-house, real-time PCR assay that was developed and validated by a public health reference laboratory. Following analyses of the discrepant specimens by PCR, the sensitivity and specificity of both the Premier CAMPY and ProSpecT Campylobacter EIAs were 99.3% and 98%, respectively, while the ImmunoCard STAT! CAMPY test had a sensitivity of 98.5% and a specificity of 98.2%. By use of the PCR test as the reference standard, culture detected 127 of 135 Campylobacter-positive stool specimens, yielding a sensitivity of 94.1%. These results showed that the three EIAs evaluated in this study provide a rapid and reliable alternative for the laboratory diagnosis of enteric infections with C. jejuni and C. coli and that conventional culture may no longer be recognized as the “gold standard” for

  4. Comparison of Performance and Cost-Effectiveness of Direct Fluorescent-Antibody, Ligase Chain Reaction, and PCR Assays for Verification of Chlamydial Enzyme Immunoassay Results for Populations with a Low to Moderate Prevalence of Chlamydia trachomatis Infection

    PubMed Central

    Dean, Deborah; Ferrero, Dennis; McCarthy, Michael

    1998-01-01

    Many laboratories use a commercial enzyme immunoassay (EIA) with verification testing to diagnose Chlamydia trachomatis infections in an effort to contain costs. This study was designed to compare the performance and cost-effectiveness of direct fluorescent-antibody assay (DFA), commercial PCR, and ligase chain reaction (LCR) for the verification of EIA results. Cervical specimens were screened by EIA. DFA, PCR, and LCR were compared as verification tests for EIA-reactive specimens and negative greyzone (NGZ) specimens at 50% below the cutoff value. These samples were also tested by in-house PCR, which was used in the analysis of verification results. A total of 477 (7%) of 6,571 samples were reactive or within the NGZ. EIA results with verification by DFA testing (EIA/DFA results) agreed with 93% of the true results compared with 97% for EIA/PCR results for one set of 242 samples; there was 97% agreement with true results for EIA/DFA results versus 95% for EIA/LCR results for another set of 235 samples. Ten samples were false positive by LCR. Time and costs were equivalent for EIA with the DFA, PCR, or LCR as the verification test but were two- to threefold greater for PCR or LCR alone than for EIA with verification. Since it is important to balance cost containment with public health objectives, DFA, PCR, and LCR as EIA verification tests for cervical samples offer acceptable sensitivities and specificities at reasonable cost for low- to moderate-risk populations and therefore can be extended to a broader spectrum of at-risk populations. PMID:9431928

  5. Comparison of the Binax Legionella Urinary Antigen Enzyme Immunoassay (EIA) with the Biotest Legionella Urin Antigen EIA for Detection of Legionella Antigen in both Concentrated and Nonconcentrated Urine Samples

    PubMed Central

    Domínguez, J. A.; Galí, N.; Pedroso, P.; Fargas, A.; Padilla, E.; Manterola, J. M.; Matas, L.

    1998-01-01

    We evaluated a newly commercial enzyme immunoassay (EIA) (Biotest Legionella Urin Antigen EIA; Biotest AG, Dreieich, Germany) for detection of antigens of all Legionella pneumophila serogroups with a relatively wide spectrum of cross-reactivity as well as antigens of other Legionella spp. by comparing its sensitivity and specificity with those of an EIA for detection of L. pneumophila serogroup 1 antigen (Legionella urinary antigen EIA; Binax, Portland, Maine). Both tests were performed with both concentrated and nonconcentrated urine samples. We also evaluated the capabilities of both EIAs to detect extracted soluble antigens of American Type Culture Collection (ATCC) Legionella strains (L. pneumophila serogroups 1 to 14, L. bozemanii, and L. longbeachae). The sensitivity of the Biotest EIA was 66.66% in nonconcentrated urine and 86.66% in concentrated urine. The sensitivity of the Binax EIA was 63.76% and 88.88% in nonconcentrated and concentrated urine, respectively. The specificity was 100% in nonconcentrated and concentrated urine for both assays. The Binax EIA and Biotest EIA detected extracted soluble antigens of L. pneumophila serogroups 1 to 14 and L. bozemanii ATCC strains. The cross-reactions observed with the Binax EIA were probably due to common epitopes directly related to lipopolysaccharide. Further studies are required to determine the usefulness of the Binax EIA for detection of urinary antigens from Legionella species and serogroups other than L. pneumophila serogroup 1. The Biotest EIA proved to be as rapid, sensitive, and specific as the Binax EIA for the diagnosis of legionellosis. Concentration of antigen present in urine increased the sensitivities of both techniques with no reduction in specificity. PMID:9705420

  6. 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.

    PubMed

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

    2011-06-01

    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.

  7. Rough lipopolysaccharide of Brucella abortus RB51 as a common antigen for serological detection of B. ovis, B. canis, and B. abortus RB51 exposure using indirect enzyme immunoassay and fluorescence polarization assay.

    PubMed

    Nielsen, K; Smith, P; Conde, S; Draghi de Benitez, G; Gall, D; Halbert, G; Kenny, K; Massengill, C; Muenks, Q; Rojas, X; Perez, B; Samartino, L; Silva, P; Tollersrud, T; Jolley, M

    2004-01-01

    Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA.

  8. Hydrogel nanoparticle based immunoassay

    DOEpatents

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

    2015-04-21

    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.

  9. Interferences in Immunoassay

    PubMed Central

    Tate, Jill; Ward, Greg

    2004-01-01

    Substances that alter the measurable concentration of the analyte or alter antibody binding can potentially result in immunoassay interference. Interfering, endogenous substances that are natural, polyreactive antibodies or autoantibodies (heterophiles), or human anti-animal antibodies together with other unsuspected binding proteins that are unique to the individual, can interfere with the reaction between analyte and reagent antibodies in immunoassay. Lipaemia, cross-reactivity, and exogenous interferences due to pre-analytical variation, matrix and equipment reaction also affect immunoassay. Interfering substances may lead to falsely elevated or falsely low analyte concentration in one or more assay systems depending on the site of the interference in the reaction and possibly result in discordant results for other analytes. The prevalence of interference is generally low in assays containing blocking agents that neutralise or inhibit the interference but is often higher in new, untested immunoassays. A wide range of analytes measured by immunoassay including hormones, tumour markers, drugs, cardiac troponin and microbial serology may be affected. Interference in immunoassay may lead to the misinterpretation of a patient's results by the laboratory and the wrong course of treatment being given by the physician. Laboratories should put processes in place to detect, test and report suspected interferences. It is equally important that physicians communicate any clinical suspicion of discordance between the clinical and the laboratory data to the laboratory. The detection of interference may require the use of an alternate assay or additional measurements, before and after treatment with additional blocking reagent, or following dilution of the sample in non-immune serum. It is imperative that laboratories inform physicians of the follow-up procedure and report on the presence of any interference. The establishment of on-going laboratory-physician contact is

  10. Fluorometric study of the beryllium-morin system

    USGS Publications Warehouse

    Fletcher, M.H.

    1965-01-01

    Three principal beryllium-morin complexes, a (1 + 1) monomer, a (1 + 1) dimer, and a (1 + 2) complex are found and conditional equilibrium constants for their formation are evaluated. Approximate ionization constants, absorption spectra, and the relative fluorescence intensities for five ionic species of morin are also determined in a spectrophotometric and fluorometric study of morin. The following interrelationships are discussed: pH, ionization of morin, absorption spectra of the various ionic species of morin and of the berylliummorin complexes, equilibria for the reactions between beryllium and morin, the period of time between preparation of the solution and measurement of the fluorescence, and fluorescence intensity.

  11. Mass spectrometric immunoassay

    DOEpatents

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

    2013-07-16

    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.

  12. Mass spectrometric immunoassay

    DOEpatents

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

    2005-12-13

    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.

  13. Mass spectrometric immunoassay

    DOEpatents

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

    2007-12-04

    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.

  14. Laser fluorometric analysis of plants for uranium exploration

    USGS Publications Warehouse

    Harms, T.F.; Ward, F.N.; Erdman, J.A.

    1981-01-01

    A preliminary test of biogeochemical exploration for locating uranium occurrences in the Marfa Basin, Texas, was conducted in 1978. Only 6 of 74 plant samples (mostly catclaw mimosa, Mimosa biuncifera) contained uranium in amounts above the detection limit (0.4 ppm in the ash) of the conventional fluorometric method. The samples were then analyzed using a Scintrex UA-3 uranium analyzer* * Use of trade names in this paper is for descriptive purposes only and does not constitute endorsement by the U.S. Geological Survey. - an instrument designed for direct analysis of uranium in water, and which can be conveniently used in a mobile field laboratory. The detection limit for uranium in plant ash (0.05 ppm) by this method is almost an order of magnitude lower than with the fluorometric conventional method. Only 1 of the 74 samples contained uranium below the detection limit of the new method. Accuracy and precision were determined to be satisfactory. Samples of plants growing on mineralized soils and nonmineralized soils show a 15-fold difference in uranium content; whereas the soils themselves (analyzed by delayed neutron activation analysis) show only a 4-fold difference. The method involves acid digestion of ashed tissue, extraction of uranium into ethyl acetate, destruction of the ethyl acetate, dissolution of the residue in 0.005% nitric acid, and measurement. ?? 1981.

  15. Modern Aspects Of Fluorometric Detection In Liquid-Phase Chromatography

    NASA Astrophysics Data System (ADS)

    Bousquet, Bernard; Garnier, Jean P.; Dreux, Claude

    1983-10-01

    Recent advances are described in the combined use of fluorometric derivatization and high performance liquid chromatography (HPLC) for clinical chemistry determinations. Derivatives (especially dansyl derivatives) can be formed prior to chromatography in the case of estrogens, amino acids, and catecholamines. In post-column reactions, we preferred to use air-segmented reactions as they conform better to all the optimized chromatographic and spectrofluorometric parameters. Fluorescent derivatives produced from cate-cholamines, tryptophan and its metabolites, hydroxyindoles, tryptamine, amino acids, sugars, polyamines, and other substances are often sufficiently sensitive to be detected in picogram quantities by HPLC. Their reaction principle and some of their applications to samples are described. Recently, chemical excitation of fluorophore-like dansyl amino acid was proposed as a detection system for HPLC. By a post-column reaction, a fluorophore can be made to emit light by its reaction with trichlorophenyl oxalate (TCPO) and hydrogen peroxide. The detection limit of this system is about 10 fmol for each dansyl amino acid. Application of this new reaction to catecholamines opens up new prospects for fluorometric detection.

  16. [Comparison study of a real-time reverse transcription polymerase chain reaction assay with an enzyme immunoassay and shell vial culture for influenza A and B virus detection in adult patients].

    PubMed

    Reina, Jordi; Plasencia, Virginia; Leyes, Maria; Nicolau, Antonio; Galmés, Antonia; Arbona, Gabriel

    2010-02-01

    The age of the patients and the type of sample are major problems in the diagnosis of influenza. Most available diagnostic techniques are highly effective in pediatric patients and in nasopharyngeal aspirates. However, in the adult population and using throat swabs, these techniques are much less reliable. We performed a prospective study comparing the efficacy of a commercial real-time reverse transcription PCR assay (RT-PCR) with that of an enzyme immunoassay (EIA) or shell vial culture (SV) in the detection of influenza A and B viruses in 125 throat swabs from adults with clinically suspected influenza during the 2007-2008 flu season. Throat swabs were subjected to rapid antigen detection for influenza viruses by means of a commercial dot-blot EIA. For the RT-PCR technique, RNA was extracted from 200 microL of each sample by the automated extraction system, EZ1 virus minikit (version 2.0). Genomic amplification of the extracted viral RNA was carried out using the OneStep RT-PCR FluA+FluB automated system with the SmartCycler amplification system. Each sample was inoculated into 2 SV of the MDCK cell line. Turnaround times were calculated from the time specimens were received in the laboratory to the time the result was reported to clinicians. The EIA system detected 27 (21.6%) positive samples, RT-PCR 62 (49.6%) positive samples, and SV 56 (44.8%) positive samples. Among the 62 positive samples, EIA detected 27 (43.5%), RT-PCR 62 (100%) and SV 56 (90.3%). With the use of RT-PCR, 38.4% of the adults studied were diagnosed on the same day samples were received. Among the total, 67.2% of diagnostic results were obtained within the first 24 hours; turnaround time was 1.1 days. The real-time RT-PCR method studied displayed high sensitivity and specificity in the detection of influenza virus in adult patients, when compared with the conventional techniques. With real-time RT-PCR, large numbers of samples can be rapidly tested and results provided the same day samples

  17. Validation of an enzyme immunoassay and comparison of fecal cortisol metabolite levels in black and gold howler monkeys (Alouatta caraya) inhabiting fragmented and continuous areas of the humid Chaco region, Argentina.

    PubMed

    Cantarelli, Verónica Inés; Perez-Rueda, Maria Amparo; Kowalewski, Martin M; Mastromonaco, Gabriela F; Ponzio, Marina Flavia

    2017-03-01

    In the last years, the study of how environmental stimuli influence the physiology and specifically the endocrinology of an organism became increasingly important, relying mainly on the quantification of glucocorticoids to monitor animal welfare. Most studies investigating cortisol levels in primates were focused on the impact of social stressors; however, a major concern for the conservation of howler monkeys is the increased habitat fragmentation led by the advancement of the agricultural frontier. We compared fecal cortisol metabolite levels (FGCM) in howler monkeys (Alouatta caraya) living in fragmented and continuous forests of the Argentine humid Chaco region, throughout the warm season (spring-summer). Fecal samples (n = 114) were collected from adult individuals, and steroid extracts analyzed with an enzyme immunoassay also validated in this work. Parallel displacement curves were obtained between dilutions of pooled fecal extracts and the cortisol standard curve (r(2)  = 0.99; P = 0.23). Efficiency of the fecal extraction procedure was 79.4% ± 38%; recovery of exogenous hormone added to fecal extracts indicated a low interference of components in the feces with antibody binding. The exogenous administration of ACTH in captive-bred animals demonstrated a "cause-and-effect" relationship between the adrenal gland activation and increased FGCM levels. Contrary to our initial prediction, we were not able to demonstrate a significant difference in FGCM levels of caraya monkeys inhabiting the continuous versus fragmented habitats in our study site (83.2 ± 4.9 ng/g [n = 10 individuals] vs. 71.5 ± 4.9 ng/g [n = 7 individuals]; P = 0.29); this could be the result of low levels of disturbance imposed by a moderate and selective logging, which has proved to be beneficial for this species with high resilience by adjusting their diet to cope with feeding in degraded habitats but with new leaves and buds. Regardless of the

  18. Defining the smallest analyte concentration an immunoassay can measure.

    PubMed

    Brown, E N; McDermott, T J; Bloch, K J; McCollom, A D

    1996-06-01

    An immunoassay's minimal detectable concentration (MDC), the smallest analyte concentration the assay can reliably measure, is one of its most important properties. Bayes' theorem is used to unify the five current mathematical MDC definitions. The unified definition has significant implications for defining positive results for screening and diagnostic tests, setting criteria for immunoassay quality control and optimal design, reliably measuring biological substances at low concentrations, and, in general, measuring small analyte concentrations with calibrated analytic methods. As an illustration, we apply the unified definition to the microparticle capture enzyme immunoassay for prostate-specific antigen (PSA) developed for the Abbott IMx automated immunoassay system. The MDC of this assay as estimated by our unifying approach is shown to be 4.1-7.1 times greater than currently reported. As a consequence, the ability of the assay to measure reliably small concentrations of PSA to detect early recurrences of prostate cancer is probably overstated.

  19. Homogeneous substrate-labeled fluorescent immunoassay for human serum albumin.

    PubMed

    Patinkin, J; Inbar, D; Ben-Gigi, C; Derfler, S; Klausner, Y; Fridlender, B

    1983-01-01

    A homogeneous substrate-labeled fluorescent immunoassay for human serum albumin (HSA) has been developed, similar to previously described immunoassays for Immunoglobulin G and Immunoglobulin M. HSA was covalently linked to 6-(7-beta-galactosylcoumarin-3-carboxamide) hexylamine. The resulting conjugate had minimal fluorescence at 450 nm (with excitation at 400 nm). However, when the acetal linkage of the galactosyl moiety was hydrolyzed by beta-galactosidase, a substantial increase in the fluorescence was obtained. This increase was specifically inhibited by antibody to HSA. A competitive binding immunoassay was established by letting the conjugate compete with HSA in the serum for the limited number of antibody-binding sites. The level of fluorescence resulting from the addition of enzyme was proportional to the amount of HSA in the serum. Precision, analytical recovery and serum dilution studies were carried out on the assay. The immunoassay was compared to an albumin assay using the dye-binding method.

  20. Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis.

    PubMed

    Oyama, Takehiro; Yodohsi, Masahiro; Yamane, Ayako; Kakehi, Kazuaki; Hayakawa, Takao; Suzuki, Shigeo

    2011-10-01

    Asparagine-type oligosaccharides are released from core proteins as N-glycosylamines in the initial step of the action of the peptide N(4)-(N-acetyl-β-D-glucosaminyl)asparagine amidase F (PNGase F). The released N-glycosylamine-type oligosaccharides (which are exclusively present at least during the course of the enzyme reaction) could therefore be derivatized with amine-labeling reagents. Here we report a method using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a labeling reagent for glycosylamine-type oligosaccharides. We applied the method for the sensitive analysis of some oligosaccharide mixtures derived from well-characterized glycoproteins including human transferrin, α(1)-acid glycoprotein, bovine fetuin, and ribonuclease B. NBD-labeled oligosaccharides were successfully separated on an amide-bonded column or a diol-silica column. This labeling method included the release of oligosaccharides from glycoproteins and derivatization of oligosaccharides in a one-pot reaction and was completed within 3h. The method showed approximately fivefold higher sensitivity than that involving labeling with ethyl p-aminobenzoate (ABEE) in HPLC using fluorometric detection and a one order of magnitude higher response in ESI-LC/MS. We also applied this method for the sensitive analysis of glycoprotein-derived oligosaccharides by capillary electrophoresis with laser-induced fluorometric detection (LIF-CE). The limit of detection in HPLC and LIF-CE were 100fmol and 4fmol, respectively.

  1. Fluorometric Biosniffer Camera "Sniff-Cam" for Direct Imaging of Gaseous Ethanol in Breath and Transdermal Vapor.

    PubMed

    Arakawa, Takahiro; Sato, Toshiyuki; Iitani, Kenta; Toma, Koji; Mitsubayashi, Kohji

    2017-03-31

    Various volatile organic compounds can be found in human transpiration, breath and body odor. In this paper, a novel two-dimensional fluorometric imaging system, known as a "sniffer-cam" for ethanol vapor released from human breath and palm skin was constructed and validated. This imaging system measures ethanol vapor concentrations as intensities of fluorescence through an enzymatic reaction induced by alcohol dehydrogenase (ADH). The imaging system consisted of multiple ultraviolet light emitting diode (UV-LED) excitation sheet, an ADH enzyme immobilized mesh substrate and a high-sensitive CCD camera. This imaging system uses ADH for recognition of ethanol vapor. It measures ethanol vapor by measuring fluorescence of nicotinamide adenine dinucleotide (NADH), which is produced by an enzymatic reaction on the mesh. This NADH fluorometric imaging system achieved the two-dimensional real-time imaging of ethanol vapor distribution (0.5-200 ppm). The system showed rapid and accurate responses and a visible measurement, which could lead to an analysis of metabolism function at real time in the near future.

  2. Automated chemiluminescence immunoassay measurements

    NASA Astrophysics Data System (ADS)

    Khalil, Omar S.; Mattingly, G. P.; Genger, K.; Mackowiak, J.; Butler, J.; Pepe, C.; Zurek, T. F.; Abunimeh, N.

    1993-06-01

    Chemiluminescence (CL) detection offers potential for high sensitivity immunoassays (CLIAs). Several approaches were attempted to automate CL measurements. Those include the use of photographic film, clear microtitration plates, and magnetic separation. We describe a photon counting detection apparatus that performs (CLIA) measurements. The CL detector moves toward a disposable reaction vessel to create a light-tight seal and then triggers and integrates a CL signal. The capture uses antibody coated polystyrene microparticles. A porous matrix, which is a part of a disposable reaction tray, entraps the microparticle-captured reaction product. The CL signal emanated off the immune complex immobilized by the porous matrix is detected. The detection system is a part of a fully automated immunoassay analyzer. Methods of achieving high sensitivities are discussed.

  3. Mass Spectrometric Immunoassay Revisited

    PubMed Central

    Nelson, Randall W.; Borges, Chad R.

    2013-01-01

    The progressive understanding and improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), realized over the years through the considerable efforts of Dr. Marvin Vestal, have made possible numerous comparable efforts involving its application in the biological sciences. Here we revisit the concepts behind one such analytical approach, Mass Spectrometric Immunoassay, which is designed to selectively detect and quantify proteins present in biological milieu. PMID:21953037

  4. Updates in immunoassays: virology.

    PubMed

    Josko, Deborah

    2012-01-01

    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.

  5. Development of an automated handheld immunoaffinity fluorometric biosensor

    NASA Astrophysics Data System (ADS)

    Phillips, Terry E.; Bargeron, C. Brent; Benson, Richard C.; Carlson, Micah A.; Fraser, Allan B.; Groopman, John D.; Ko, Harvey W.; Strickland, Paul T.; Velky, James

    2000-04-01

    A new immunoaffinity fluorometric biosensor has been developed for detecting and quantifying aflatoxins, a family of potent fungi-produced carcinogens that are commonly found in a variety of agriculture products. They have also been cited as a biological agent under weapons development. The handheld, self-contained biosensor is fully automatic, highly sensitive, quick, quantitative, and requires no special storage. Concentration from 0.1 parts per billion to 50 ppb can be determined in less than 2 minutes with a 1 ml sample volume. Higher concentrations can be determined by simply reducing the sample volume. The device operates on the principles of immunoaffinity for specificity and fluorescence for a quantitative assay. The analytic procedure is flexible so that other chemical and biological analytes could be detected with minor modifications to the current device.

  6. Detection of bound residues in soils by sandwich-immunoassay

    NASA Astrophysics Data System (ADS)

    Dosch, M.; Weller, Michael G.; Niessner, Reinhard

    1995-10-01

    Immunoassays are useful analytical instruments for the detection of many environmental compounds. This method is now introduced for the detection of non-extractable compounds in soil. So-called 'bound residues' consist of a soil component, e.g. humic acids, and an irreversibly bound pollutant. Because of the complexity of those macromolecules conventional analytical methods in general do not work. Enzyme immunoassays, in contrast, seem to have a large potential for applications and further developments in this field. The use of antibodies with high affinity to the analytes makes a selective detection of environmental pollutants possible. With the development of an enzyme-labeled sandwich-immunoassay polycyclic aromatic hydrocarbons (PAHs), irreversibly bound to humic acids, were determined for the first time.

  7. A Fluorometric Activity Assay for Light-Regulated Cyclic-Nucleotide-Monophosphate Actuators.

    PubMed

    Schumacher, Charlotte Helene; Körschen, Heinz G; Nicol, Christopher; Gasser, Carlos; Seifert, Reinhard; Schwärzel, Martin; Möglich, Andreas

    2016-01-01

    As a transformative approach in neuroscience and cell biology, optogenetics grants control over manifold cellular events with unprecedented spatiotemporal definition, reversibility, and noninvasiveness. Sensory photoreceptors serve as genetically encoded, light-regulated actuators and hence embody the cornerstone of optogenetics. To expand the scope of optogenetics, ever more naturally occurring photoreceptors are being characterized, and synthetic photoreceptors with customized, light-regulated function are being engineered. Perturbational control over intracellular cyclic-nucleotide-monophosphate (cNMP) levels is achieved via sensory photoreceptors that catalyze the making and breaking of these second messengers in response to light. To facilitate discovery, engineering and quantitative characterization of such light-regulated cNMP actuators, we have developed an efficient fluorometric assay. Both the formation and the hydrolysis of cNMPs are accompanied by proton release which can be quantified with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). This assay equally applies to nucleotide cyclases, e.g., blue-light-activated bPAC, and to cNMP phosphodiesterases, e.g., red-light-activated LAPD. Key benefits include potential for parallelization and automation, as well as suitability for both purified enzymes and crude cell lysates. The BCECF assay hence stands to accelerate discovery and characterization of light-regulated actuators of cNMP metabolism.

  8. Development of highly fluorescent detection reagents for the construction of ultrasensitive immunoassays.

    PubMed

    Qin, Q P; Lövgren, T; Pettersson, K

    2001-04-01

    We developed two kinds of highly fluorescent streptavidin-based conjugates for use as universal detection reagents in ultrasensitive immunoassays. The direct conjugate was produced by covalently linking streptavidin to poly(Glu: Lys) which was labeled heavily with Eu chelates; the indirect conjugate was made by first conjugating bovine serum albumin (BSA) to poly(Glu:Lys) labeled heavily with Eu chelates and then further linking streptavidin to the conjugate of BSA-poly(Glu:Lys)-Eu chelate. Both direct and indirect conjugates were used to construct a highly sensitive time-resolved fluorometric assay for prostate-specific antigen (PSA). Of two monoclonal antibodies used in the assay, one was coated on the well surface of the microtitration strips, and the other was biotinylated. When 10 microL of sample volume was used, we found that the assay using the indirect conjugate had a detection limit of 0.006 microg/L, which was approximately 5.6-fold more sensitive than the one using Eu chelate directly labeled detection antibody and 6.8-fold more sensitive than the one using Eu chelate-labeled streptavidin. However, the assay that used the direct conjugate was 1.5-fold more sensitive than the one that utilized the indirect conjugate. When 45 microL of sample volume was used, a detection limit of 0.001 microg/L was achieved by using the direct conjugate. This improvement in sensitivity should be equally obtainable for the analytes other than PSA. We further demonstrated that the final immunoassay performance was affected not only by the quality of the streptavidin-based conjugate used but also by the quality of the biotinylated antibody reagent. The universal detection reagents described here are believed to be particularly useful for the construction of ultrasensitive time-resolved fluorometric immunoassays and are potentially applicable in other fields such as immunohistochemistry and nucleic acid detection.

  9. [A noninstrumental Immunoassay based on colloidal dyes].

    PubMed

    Liubavina, I A; Salomatina, I S; Zinchenko, A A; Zherdeev, A V; Dzantiev, B B

    2000-03-01

    Detecting labels based on water dispersions of colloidal textile dyes were developed that are useful in various analytical and diagnostic test systems for a simple visual assessment of the assay. Colored water-insoluble particles of dyes were used for the sorptional immobilization of streptavidin on their surface. The resulting streptavidin-dye (STR-DYE) complexes possessed a high visualizing capacity and were used for the combined detection of pesticides (simazine and 2,4-dichlorophenoxyacetic acid) by noninstrumental immunoassay (DYE-comb-assay, competitive dot-immunoassay in the comb format). The detection limits and the duration of our DYE-comb-assay (4 ng/ml, 20-25 min), HRP-comb-assay (competitive dot-immunoassay in the comb format using the enzymic conjugate of STR with horseradish peroxidase) (16 ng/ml), and the traditional competitive ELISA (12-16 ng/ml, 1.5 h) were compared. This DYE-comb-assay is simple enough and can be used under field conditions.

  10. Homogeneous Immunoassays: Historical Perspective and Future Promise

    NASA Astrophysics Data System (ADS)

    Ullman, Edwin F.

    1999-06-01

    The founding and growth of Syva Company is examined in the context of its leadership role in the development of homogeneous immunoassays. The simple mix and read protocols of these methods offer advantages in routine analytical and clinical applications. Early homogeneous methods were based on insensitive detection of immunoprecipitation during antigen/antibody binding. The advent of reporter groups in biology provided a means of quantitating immunochemical binding by labeling antibody or antigen and physically separating label incorporated into immune complexes from free label. Although high sensitivity was achieved, quantitative separations were experimentally demanding. Only when it became apparent that reporter groups could provide information, not only about the location of a molecule but also about its microscopic environment, was it possible to design practical non-separation methods. The evolution of early homogenous immunoassays was driven largely by the development of improved detection strategies. The first commercial spin immunoassays, developed by Syva for drug abuse testing during the Vietnam war, were followed by increasingly powerful methods such as immunochemical modulation of enzyme activity, fluorescence, and photo-induced chemiluminescence. Homogeneous methods that quantify analytes at femtomolar concentrations within a few minutes now offer important new opportunities in clinical diagnostics, nucleic acid detection and drug discovery.

  11. Evaluation of a fluorometric-enzymatic method based on 3alpha-hydroxysteroid dehydrogenase for the mycotoxin zearalenone determination in corn.

    PubMed

    Andreu, Yolanda; Castillo, Juan R; de Marcos, Susana; Galbán, Javier

    2004-09-08

    A new method for the fluorometric determination of zearalenone (ZEN) based on its reaction with betaNADH in the presence of the enzyme 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) is described. The procedure is based on the change in fluorescence intensity that takes place during the enzymatic reaction (excitation at 340nm and emission at 454nm). The optimum reaction conditions and the analytical characteristics were studied; linear response range (1-10mgl(-1)) and reproducibility (8mgl(-1), 2.7%, n=7). Moreover, a mathematical model explaining the analytical signal is proposed. The method has been applied to zearalenone determination in a spiked corn sample.

  12. Fluorometric determination of urea in alcoholic beverages by using an acid urease column-FIA system.

    PubMed

    Iida, Y; Ikeda, M; Aoto, M; Satoh, I

    2004-12-15

    An acid urease column was applied to a fluorometric flow-injection analysis (FIA) system as a recognition element for determination of urea in rice wines. The acid urease has specific properties of showing its catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans. The enzymes were covalently immobilized onto porous glass beads with controlled pore size and then, packed into a small polymer column. The flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, and a flow-through quartz cell attached to a fluorescent spectrophotometer. Citrate buffer (50mM, pH 5.0) as the carrier solution was continuously pumped through the system. Sample solutions were introduced into the system via a rotary injection valve. A standard urea solution was measured through monitoring variations in fluorescent intensity attributable to fluorescent isoindole derivatives formed by coupling with ammonia molecules released in the enzymatic hydrolysis of urea and orthophthalaldehyde reagents. The fluorescent intensity was measured under the conditions of lambda(ex) = 415nm and lambda(em) = 485nm. A wide, linear relationship was obtained between the concentration of urea (1.0-100muM) and the variation in fluorescent intensity. The monitoring did not suffer from ethanol and various amino acids contained in rice wines. Real samples pretreated with ion exchange resins for removal of endogenous ammonia were introduced into the FIA system and urea in the samples was determined. These results were compared with those obtained with use of an F-kit method. The proposed FIA system should present sensitive, selective and convenient analysis of urea in alcoholic beverages.

  13. Comparison of the performance of the LIAISON VZV-IgG and VIDAS automated enzyme linked fluorescent immunoassays with reference to a VZV-IgG time-resolved fluorescence immunoassay and implications of choice of cut-off for LIAISON assay.

    PubMed

    Maple, P A C; Rathod, P; Smit, E; Gray, J; Brown, D; Boxall, E H

    2009-01-01

    Determination of Varicella Zoster virus (VZV) immune status in pregnant women without history of chickenpox is important in identifying those who genuinely need VZV immune globulin prophylaxis following significant exposure to chickenpox or shingles. Immune status testing requires highly sensitive and specific immunoassays for timely and accurate results. To compare the performance of DiaSorin LIAISON and Biomerieux VIDAS VZV-IgG assays with reference to a VZV-IgG time-resolved fluorescence immunoassay (TRFIA). A panel of sera collected from 65 pregnant contacts of VZV and 62 individuals tested for VZV immunity was tested in all three assays. Dose-response curves were generated using International Standards W1044 and 90/690. Sensitivity and specificity of VIDAS compared to VZV-TRFIA was 54.5% and 97.9% respectively and for LIAISON compared to VZV-TRFIA was 67% and 100% respectively. Both assays correlated well with TRFIA with R2 correlation coefficients of 0.79 and 0.76 respectively. Dose-response curves showed both Standards behaved in a similar manner in each assay. For VIDAS, the test cut-off value of 0.9 correlated with 275-280mIU/ml and for LIAISON a cut-off value of 150mIU/ml correlated with 208-219mIU/ml. By dose-response data and in comparison with TRFIA, LIAISON is more sensitive and specific than VIDAS.

  14. Protein microchips : use for immunoassay and enzymatic reactions.

    SciTech Connect

    Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloschuk, S.; Chupeeva, V.; Mirzabekov, A.; Biochip Technology Center; Russian Academy of Sciences

    2000-02-15

    Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-{mu}m gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.

  15. Fluorometric Index for Sensing Oil in the Sea Environment.

    PubMed

    Baszanowska, Emilia; Otremba, Zbigniew

    2017-06-02

    Excitation-emission matrix spectroscopy (EEMS) was applied to determine the fluorometric index (FI) as a parameter indicating the presence of a source of oil pollution in a specific area of the sea. Seawater from the Polish coast (the Baltic Sea) and the same water combined with various amounts of crude oil extracted from the Baltic Sea shelf (Petrobaltic-type oil) were used in this study. The FI values were calculated for excitation and emission wavelengths found at the maximal peak, taking into account the natural seawater and the seawater artificially contaminated (for an oil-to-water ratio range of 0.5 × 10(-6) - 500 × 10(-6)). The wavelength configurations (Ex/Em) (225/355 and 225/340) for the FI index were applied. It was found that, independent of the amount of oil, the FI achieves a higher value for natural seawater than for seawater that has had contact with oil. These results provide the basis to design a sensor signaling the appearance of oil in a defined sea area.

  16. Fluorometric enzymatic determination of total cholesterol in serum.

    PubMed

    Huang, H; Kauan, J W; Guilbault, G G

    1975-10-01

    We describe a fluorometric enzymatic method for determining total serum cholesterol, based on hydrolysis of cholesterol esters to free cholesterol by cholesterol ester hydrolase (EC 3.1.1.13). The free cholesterol formed, as well as that initially present, is then oxidized by cholesterol oxidase (EC 1.1.3.6) to cholest-4-en-3-one with simultaneous production of hydrogen peroxide. The latter catalytically oxidizes homovanillic acid in the presence of peroxidase (EC 1.11.1.7) to form the highly fluorescent 2,2'-dihydroxy-3,3'-dimethoxy-biphenyl-5,5'-diacetic acid. A calibration curve is constructed from data on a series of standard cholesterol solutions vs. the corresponding fluorescence change (deltaf/5 min). This curve is linear up to 4.0 g of total serum cholesterol per liter of serum. The method is specific, precise, accurate, rapid, and simple, and results correlate well with those obtained by both the Liebermann-Burchard procedure and the colorimetric enzymatic method (correlation coefficients, 0.984 and 0.981, respectively).

  17. Mass spectrometric immunoassay

    SciTech Connect

    Nelson, R.W.; Krone, J.R.; Bieber, A.L.; Williams, P.

    1995-04-01

    A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin from the venoms of the prairie rattlesnake, Crotalus virdis virdis, and the Mojave rattlesnake, Crotalus scutulatus scutulatus. 18 refs., 8 figs.

  18. Microbead-based electrochemical immunoassay with interdigitated array electrodes.

    PubMed

    Thomas, Jennifer H; Kim, Sang Kyung; Hesketh, Peter J; Halsall, H Brian; Heineman, William R

    2004-05-15

    The objective of this study was to develop a sensitive and miniaturized immunoassay by coupling a microbead-based immunoassay with an interdigitated array (IDA) electrode. An IDA electrode amplifies the signal by recycling an electrochemically redox-reversible molecule. The microfabricated platinum electrodes had 25 pairs of electrodes with 1.6-microm gaps and 2.4-microm widths. An enzyme-labeled sandwich immunoassay on paramagnetic microbeads with mouse IgG as the analyte and beta-galactosidase as the enzyme label was used as the model system. beta-Galactosidase converted p-aminophenyl beta-D-galactopyranoside to p-aminophenol (PAP). This enzyme reaction was measured continuously by positioning the microbeads near the electrode surface with a magnet. Electrochemical recycling occurred with PAP oxidation to p-quinone imine (PQI) at +290 mV followed by PQI reduction to PAP at -300 mV vs Ag/AgCl. Dual-electrode detection amplified the signal fourfold compared to single-electrode detection, and the recycling efficiency reached 87%. A calibration curve of PAP concentration vs anodic current was linear between 10(-4) and 10(-6)M. A signal from 1000 beads in a 20-microL drop was detectable and the immunoassay was complete within 10 min with a detection limit of 3.5x10(-15)mol mouse IgG.

  19. A Wash-Free Homogeneous Colorimetric Immunoassay Method

    PubMed Central

    Liu, Huiqiao; Rong, Pengfei; Jia, Hongwei; Yang, Jie; Dong, Bo; Dong, Qiong; Yang, Cejun; Hu, Pengzhi; Wang, Wei; Liu, Haitao; Liu, Dingbin

    2016-01-01

    Rapid and convenient biosensing platforms could be beneficial to timely diagnosis and treatment of diseases in virtually any care settings. Sandwich immunoassays, the most commonly used methods for protein detection, often rely on expensive tags such as enzyme and tedious wash and incubation procedures operated by skilled labor. In this report, we revolutionized traditional sandwich immunoassays by providing a wash-free homogeneous colorimetric immunoassay method without requirement of any separation steps. The proposed strategy was realized by controlling the growth of gold nanoparticles (AuNPs) to mediate the interparticle spacing in the protein-AuNP oligomers. We have demonstrated the successful in vitro detection of cancer biomarker in serum samples from patients with high clinical sensitivity and specificity. PMID:26722373

  20. Immunoassay as an analytical tool in agricultural biotechnology.

    PubMed

    Grothaus, G David; Bandla, Murali; Currier, Thomas; Giroux, Randal; Jenkins, G Ronald; Lipp, Markus; Shan, Guomin; Stave, James W; Pantella, Virginia

    2006-01-01

    Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.

  1. Morphological resonances for multicomponent immunoassays

    NASA Astrophysics Data System (ADS)

    Whitten, W. B.; Shapiro, M. J.; Ramsey, J. M.; Bronk, B. V.

    1995-06-01

    An immunoassay technique capable of detecting and identifying a number of species of microorganisms in a single analysis is described. The method uses optical-resonance size discrimination of microspheres to identify antibodies to which stained microorganisms are bound.

  2. Optimizing the fluorometric β-glucuronidase assay in ruminant milk for a more precise determination of mastitis.

    PubMed

    Larsen, Torben; Aulrich, Karen

    2012-02-01

    Activity of the enzyme β-glucuronidase (EC 3.2.1.31) is found in milk from ruminants with mastitis. However, the use of this enzymic activity as an indicator of mastitis has gained little attention possibly because of its low activity when compared with other mastitis indicators. The determination may therefore be less precise and the analytical procedure very time consuming and labour intensive. The present study optimized the fluorometric determination of the β-glucuronidase activity with respect to substrate concentration, pH, incubation time etc., validated the assay, and developed it into large scale analyses. The assay performance is satisfactory regarding precision, linearity etc., and it appears comparable to analogous fluorometric assays for mastitis indicators in milk. From a local dairy herd, 825 milk samples were analysed for potential mastitis indicators, i.e. β-glucuronidase, lactate dehydrogenase (LDH), alkaline phosphatase (AP), and N-acetyl-β-d-glucosaminidase (NAGase) activity, and for somatic cell counts (SCC) and the variables were compared. Activity of β-glucuronidase was moderately but significantly correlated to SCC (r=0·21; n=768) as well as the other mentioned variables (r=0·25-0·43; n=825). Simple indices based on β-glucuronidase and LDH or NAGase activity were tested as indicators of mastitis (SCC), but were not found to improve the diagnostic value. Future studies may further verify whether β-glucuronidase can compete with well-established indicators of mastitis in cows such as LDH or NAGase as well as determine whether β-glucuronidase activity, in combination with other indicators of mastitis, has an advantage. Nineteen milk samples from subclinical and latent cases of mastitis (individual quarters) were identified for specific pathogens (PCR method) and measured for β-glucuronidase activity. The activity was tested at four different pH levels (5·5, 6·0, 6·5 and 7·0) in order to investigate the possibility of

  3. Development of enzyme immunoassays for 3,5,3'-triiodo-L-thyronine and L-thyroxine: time-course studies on the effect of food deprivation on plasma thyroid hormones in two marine teleosts, sea bass (Dicentrarchus labrax L.) and sea bream (Sparus aurata L.).

    PubMed

    Cerdá-Reverter, J M; Zanuy, S; Carrillo, M; Kah, O

    1996-09-01

    The effects of short-term food deprivation and photoperiod on plasma thyroid hormone levels of sea bass and sea bream were studied. Animals were acclimated under constant photoperiod regime (15L/9D) and feeding times (2 hr after light onset and 2 hr before light offset). Time-course studies involved monitoring plasma hormone levels every 4 hr throughout 1.5 24-hr cycles. Plasma 3,5, 3'-Triiodo-L-thyronine (T3) and L-thyroxine (T4) were assayed using a newly developed competitive enzyme immunoassay, utilizing acetylcholinesterase as a label of enzymatic tracers. Enzyme immunoassays had sensitivities of 1.25-0.02 and 62.5-0.2 ng/ml for T3 and T4, respectively, and reproducibilities of 3.7 and 5.6% intraassay variation for T3 and T4, respectively; interassay variations for T3 and T4 assays respectively were 1.6%, 11% and 6.6%, 8% for sea bass and sea bream plasma similar to RIA. In sea bass, 3 days of food deprivation resulted in depressed plasma T3 and T4, overriding significant diel variations seen during the second day of starvation. Sea bream displayed a slight decrease of T4 plasma levels while T3 levels remained constant for the whole sampling period. Both thyroidal systems responded to photoperiod with a significant increase in plasma T4 level at the time of light onset. In addition, sea bass also displayed increased T3 levels and decreases in both hormone levels coinciding with "lightoff." Data show different responses of the sea bass and sea bream thyroidal systems to both nutritional state and photoperiod in that the latter state is influenced by the former. Data suggest plasma thyroid levels can be used as a rapid indicator of nutritional status.

  4. Measurement of urinary histamine: comparison of fluorometric and radioisotopic-enzymatic assay procedures

    SciTech Connect

    Warren, K.; Dyer, J.; Merlin, S.; Kaliner, M.

    1982-02-01

    Assessment of urinary histamine may prove useful in determining the role of histamine in human health and disease. Urinary histamine may be accurately estimated by a modified fluorometric assay employing diamine oxidase (DAO) digestion and cation-exchange chromatography. However, the radioisotopic-enzymatic assay is less expensive, easier to perform, and possibly more sensitive. Therefore the two procedures were compared. The radioenzyme assay was found to be affected by factors in urine (possibly salt concentrations) requiring extraction of histamine from urine by butanol-heptane. Moreover, it was found to be necessary to compare DAO-digested samples with undigested samples to accurately estimate histamine levels and to run the standard curve of histamine in DAO-digested urine. Even with these modifications, the radioenzyme assay was not as accurate as the fluorometric assay for urine samples having histamine values about 60 ng/ml. Therefore we recommend utilization of the modified fluorometric assay for the measurement of urinary histamine levels.

  5. Fluorometric imaging methods for palladium and platinum and the use of palladium for imaging biomolecules.

    PubMed

    Tracey, Matthew P; Pham, Dianne; Koide, Kazunori

    2015-07-21

    Neither palladium nor platinum is an endogenous biological metal. Imaging palladium in biological samples, however, is becoming increasingly important because bioorthogonal organometallic chemistry involves palladium catalysis. In addition to being an imaging target, palladium has been used to fluorometrically image biomolecules. In these cases, palladium species are used as imaging-enabling reagents. This review article discusses these fluorometric methods. Platinum-based drugs are widely used as anticancer drugs, yet their mechanism of action remains largely unknown. We discuss fluorometric methods for imaging or quantifying platinum in cells or biofluids. These methods include the use of chemosensors to directly detect platinum, fluorescently tagging platinum-based drugs, and utilizing post-labeling to elucidate distribution and mode of action.

  6. Carbon Nanotubes with Enhanced Chemiluminescence Immunoassay for CCD-Based Detection of Staphylococcal Enterotoxin B in Food

    PubMed Central

    Yang, Minghui; Kostov, Yordan; Bruck, Hugh A.; Rasooly, Avraham

    2010-01-01

    Enhanced chemiluminescence (ECL) detection can significantly enhance the sensitivity of immunoassays but often requires expensive and complex detectors. The need for these detectors limits broader use of ECL in immunoassay applications. To make ECL more practical for immunoassays, we utilize a simple cooled charge-coupled device (CCD) detector combined with carbon nanotubes (CNTs) for primary antibody immobilization to develop a simple and portable point-of-care immunosensor. This combination of ECL, CNT, and CCD detector technologies is used to improve the detection of Staphylococcal enterotoxin B (SEB) in food. Anti-SEB primary antibodies were immobilized onto the CNT surface, and the antibody–nanotube mixture was immobilized onto a polycarbonate surface. SEB was then detected by an ELISA assay on the CNT–polycarbonate surface with an ECL assay. SEB in buffer, soy milk, apple juice, and meat baby food was assayed with a LOD of 0.01 ng/mL using our CCD detector, a level similar to the detection limit obtained with a fluorometric detector when using the CNTs. This level is far more sensitive than the conventional ELISA, which has a LOD of ~1 ng/mL. Our simple, versatile, and inexpensive point-of-care immunosensor combined with the CNT-ECL immunoassay method described in this work can also be used to simplify and increase sensitivity for many other types of diagnostics and detection assays. PMID:18855418

  7. Magnetic Beads-based Bioelectrochemical Immunoassay of Polycyclic Aromatic Hydrocarbons

    SciTech Connect

    Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.; Lin, Yuehe

    2007-07-01

    A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3, 3´, 5, 5´- tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.

  8. Fluorometric discrimination technique of phytoplankton population based on wavelet analysis

    NASA Astrophysics Data System (ADS)

    Zhang, Shanshan; Su, Rongguo; Duan, Yali; Zhang, Cui; Song, Zhijie; Wang, Xiulin

    2012-09-01

    The discrete excitation-emission-matrix fluorescence spectra (EEMS) at 12 excitation wavelengths (400, 430, 450, 460, 470, 490, 500, 510, 525, 550, 570, and 590 nm) and emission wavelengths ranging from 600-750 nm were determined for 43 phytoplankton species. A two-rank fluorescence spectra database was established by wavelet analysis and a fluorometric discrimination technique for determining phytoplankton population was developed. For laboratory simulatively mixed samples, the samples mixed from 43 algal species (the algae of one division accounted for 25%, 50%, 75%, 85%, and 100% of the gross biomass, respectively), the average discrimination rates at the level of division were 65.0%, 87.5%, 98.6%, 99.0%, and 99.1%, with average relative contents of 18.9%, 44.5%, 68.9%, 73.4%, and 82.9%, respectively; the samples mixed from 32 red tide algal species (the dominant species accounted for 60%, 70%, 80%, 90%, and 100% of the gross biomass, respectively), the average correct discrimination rates of the dominant species at the level of genus were 63.3%, 74.2%, 78.8%, 83.4%, and 79.4%, respectively. For the 81 laboratory mixed samples with the dominant species accounting for 75% of the gross biomass (chlorophyll), the discrimination rates of the dominant species were 95.1% and 72.8% at the level of division and genus, respectively. For the 12 samples collected from the mesocosm experiment in Maidao Bay of Qingdao in August 2007, the dominant species of the 11 samples were recognized at the division level and the dominant species of four of the five samples in which the dominant species accounted for more than 80% of the gross biomass were discriminated at the genus level; for the 12 samples obtained from Jiaozhou Bay in August 2007, the dominant species of all the 12 samples were recognized at the division level. The technique can be directly applied to fluorescence spectrophotometers and to the developing of an in situ algae fluorescence auto-analyzer for phytoplankton

  9. Fluorometric assessment of acetaminophen-induced toxicity in rat hepatocyte spheroids seeded on micro-space cell culture plates.

    PubMed

    Sanoh, Seigo; Santoh, Masataka; Takagi, Masashi; Kanayama, Tatsuya; Sugihara, Kazumi; Kotake, Yaichiro; Ejiri, Yoko; Horie, Toru; Kitamura, Shigeyuki; Ohta, Shigeru

    2014-09-01

    Hepatotoxicity induced by the metabolic activation of drugs is a major concern in drug discovery and development. Three-dimensional (3-D) cultures of hepatocyte spheroids may be superior to monolayer cultures for evaluating drug metabolism and toxicity because hepatocytes in spheroids maintain the expression of various metabolizing enzymes and transporters, such as cytochrome P450 (CYP). In this study, we examined the hepatotoxicity due to metabolic activation of acetaminophen (APAP) using fluorescent indicators of cell viability and intracellular levels of glutathione (GSH) in rat hepatocyte spheroids grown on micro-space cell culture plates. The mRNA expression levels of some drug-metabolizing enzymes were maintained during culture. Additionally, this culture system was compatible with microfluorometric imaging under confocal laser scanning microscopy. APAP induced a decrease in intracellular ATP at 10mM, which was blocked by the CYP inhibitor 1-aminobenzotriazole (ABT). APAP (10mM, 24h) decreased the levels of both intracellular ATP and GSH, and GSH-conjugated APAP (APAP-GSH) were formed. All three effects were blocked by ABT, confirming a contribution of APAP metabolic activation by CYP to spheroid toxicity. Fluorometric imaging of hepatocyte spheroids on micro-space cell culture plates may allow the screening of drug-induced hepatotoxicity during pharmaceutical development.

  10. Construction and Expression of Recombinant Streptolysin-O and Preevaluation of Its Use in Immunoassays

    PubMed Central

    Velázquez, Blanca; Massaldi, Hugo; Battistoni, Julio; Chabalgoity, José A.

    2005-01-01

    Commercially available immunoassays for assessment of anti-streptolysin-O antibodies use native streptolysin-O obtained by a complex process. We prepared a biologically active recombinant streptolysin-O with higher yield and a simpler purification process. An enzyme-linked immunosorbent assay developed with this recombinant showed good correlation with a commercial test, suggesting that it could be suitable for immunoassays. PMID:15879035

  11. ELEGANT ENVIRONMENTAL IMMUNOASSAYS

    EPA Science Inventory

    Immunochemical methods are based on selective antibodies directed to a particular target analyte. The specific binding between antibody and analyte can be used for detection and quantitation. Methods such as the enzyme-linked immunosorbent assay (ELISA) can provide a sensitiv...

  12. ELEGANT ENVIRONMENTAL IMMUNOASSAYS

    EPA Science Inventory

    Immunochemical methods are based on selective antibodies directed to a particular target analyte. The specific binding between antibody and analyte can be used for detection and quantitation. Methods such as the enzyme-linked immunosorbent assay (ELISA) can provide a sensitiv...

  13. Novel silver nanoparticle-enhanced fluorometric determination of trace tetracyclines in aqueous solutions.

    PubMed

    Wang, Ping; Wu, Tun-Hua; Zhang, Yong

    2016-01-01

    Metal-enhanced fluorescence (MEF) has exhibited promise for applications in fluorometric assays. The effects of silver nanoparticles (AgNP) on the fluorescence behaviours of tetracycline hydrochloride (TCH) and chlortetracycline hydrochloride (CTC) in aqueous solutions were investigated. The experimental results demonstrated that the fluorescence intensities of each tetracycline in water solutions were greatly enhanced by AgNP through the MEF effect. In addition, a novel silver nanoparticle-enhanced fluorometric method was established for the direct determination of TCH and CTC in aqueous solutions. Under optimum experimental conditions, the linear dynamic ranges for the determination of TCH and CTC in aqueous solutions varied from 0.10 to 6.0 mg L(-1) and 0.050 to 3.0 mg L(-1) with detection limits of 0.63 µg L(-1) and 0.19 µg L(-1), respectively, and with the relative standard deviation of less than 1.9% (n=9). The experimental recovery results for the determination of TCH and CTC in aqueous solutions ranged from 93-106% and 95-104%, respectively. Compared with the established method without the addition of AgNP, the limits of quantitation of the silver nanoparticle-enhanced fluorometric method were approximately 5-fold lower for TCH and 3-fold lower for CTC. Moreover, the newly established silver nanoparticle-enhanced fluorometric method was successfully applied to the direct determination of TCH and CTC in pharmaceutical preparations.

  14. Substrates and method for determining enzymes

    DOEpatents

    Smith, Robert E.; Bissell, Eugene R.

    1981-01-01

    A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate.

  15. Substrates and method for determining enzymes

    DOEpatents

    Smith, R.E.; Bissell, E.R.

    1981-10-13

    A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate. No Drawings

  16. Fluorescence Immunoassay for Cocaine Detection.

    PubMed

    Nakayama, Hiroshi; Kenjjou, Noriko; Shigetoh, Nobuyuki; Ito, Yuji

    2016-04-01

    A fluorescence immunoassay (FIA) has been developed for the detection of cocaine using norcocaine labeled with merocyanine dye and a monoclonal antibody specific to cocaine. Using this FIA, the detection range for cocaine was between 20.0 and 1700 μg/L with a limit of detection of 20.0 μg/L. Other cocaine derivatives did not interfere significantly with the detection when using this immunoassay technique with cross-reactivity values of less than 20%. Thus this FIA could be considered a useful tool for the detection of cocaine.

  17. Comparison of a commercial Varicella Zoster glycoprotein IgG enzyme immunoassay with a reference time resolved fluorescence immunoassay (VZV TRFIA) for measuring VZV IgG in sera from pregnant women, sera sent for confirmatory testing and pre and post vOka vaccination sera from healthcare workers.

    PubMed

    Maple, P A C; Breuer, J; Quinlivan, M; Kafatos, G; Brown, K E

    2012-03-01

    Recently, a commercial, standardised VZV IgG glycoprotein EIA, Binding Site VaccZyme™VZV glycoprotein IgG low level EIA (VaccZyme™EIA) has become available. The VaccZyme™EIA is more robust and user friendly than the reference VZV time-resolved fluorescence immunoassay (VZV TRFIA). To assess the usefulness of the VaccZyme™EIA in the diagnostic laboratory by comparing VZV IgG levels generated by both assays on serum panels representing, non-vaccinated, and vOka vaccinated populations. Sera from non-vaccinated individuals were tested; 248 from pregnant women, 117 from various patient groups referred to the Virus Reference Department for confirmatory VZV IgG testing and 102 from healthcare workers enrolled in a study (ROVE) of antibody/IgG response to vOka. From the ROVE study, 282 post vaccination sera were tested; 108 and 101 collected at six weeks post first and second doses of vOka, respectively, and 73 collected at 18 month follow-up. Sensitivities and specificities (equivocals treated as negatives) of the VaccZyme™EIA for sera from pregnant women were 97.8% (95% CI: [94.6%, 99.4%]) and 96.8% (95% CI: [89.0%, 99.6%]), respectively, and for sera referred for confirmatory testing were 81.2% (95% CI: [71.2%, 88.8%]) and 96.9% (95% CI: [83.8%, 99.9%]), respectively, and for ROVE baseline sera were 54.2% (95% CI: [32.8%, 74.4%]) and 100% (95% CI: [95.4%, 100.0%]), respectively. For the post vOka serum panels sensitivities of the VaccZyme™EIA ranged from 65.3% (95% CI: [50.4%, 78.3%]) to 80.4% (95% CI: [71.1%, 87.8%]). Specificities were all 100%. Correlation with VZV TRFIA was high and agreement varied between the serum panels tested. VaccZyme™EIA is recommended for detecting VZV IgG in sera from non-vaccinated populations; however, caution is advised when measuring post vOka VZV IgG levels. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Protein Adsorption in Microengraving Immunoassays

    PubMed Central

    Song, Qing

    2015-01-01

    Microengraving is a novel immunoassay forcharacterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales τD and τK determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when C0* is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 104–105 single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample. PMID:26501282

  19. Protein adsorption in microengraving immunoassays.

    PubMed

    Song, Qing

    2015-10-16

    Microengraving is a novel immunoassay for characterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales  and  determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when  is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 10⁴-10⁵ single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample.

  20. Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

    PubMed

    Chen, Jing; He, Qing-hua; Xu, Yang; Fu, Jin-heng; Li, Yan-ping; Tu, Zhui; Wang, Dan; Shu, Mei; Qiu, Yu-lou; Yang, Hong-wei; Liu, Yuan-yuan

    2016-01-15

    Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers.

  1. Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis.

    PubMed

    Teste, Bruno; Kanoufi, Frédéric; Descroix, Stéphanie; Poncet, Pascal; Georgelin, Thomas; Siaugue, Jean-Michel; Petr, Jan; Varenne, Anne; Hennion, Marie-Claire

    2011-07-01

    In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles' colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core-shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient's sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.

  2. Effect of Indian Ayurvedic medicine Ashwagandha on measurement of serum digoxin and 11 commonly monitored drugs using immunoassays: study of protein binding and interaction with Digibind.

    PubMed

    Dasgupta, Amitava; Peterson, Amanda; Wells, Alice; Actor, Jeffrey K

    2007-08-01

    Ashwagandha, a popular Ayurvedic medicine, is now available in the United States. Alkaloids found in this herb have structural similarity with digoxin. To study potential interference of Ashwagandha with serum digoxin measurement by immunoassays. Potential interference was also investigated with immunoassays for 11 other commonly monitored drugs. In addition, interaction of components of Ashwagandha with the Fab fragment of antidigoxin antibody (Digibind) was investigated. Two different brands of liquid extract and 1 dry powdered form of Ashwagandha were used for this investigation. Aliquots of drug-free serum were supplemented with various concentrations of Ashwagandha and apparent digoxin concentrations were measured by 3 digoxin immunoassays. Mice were fed with Ashwagandha and apparent digoxin concentrations were measured 1 and 3 hours after feeding. Potential interference of Ashwagandha with immunoassays of 11 other drugs was also investigated. Interaction of components of Ashwagandha with Digibind was studied in vitro. Significant apparent digoxin concentrations were observed both in vitro and in vivo using the fluorescence polarization immunoassay of digoxin, whereas the Beckman and the microparticle enzyme immunoassay digoxin assay demonstrated minimal interference. Immunoassays of 11 other drugs tested were unaffected. When Ashwagandha extract was added to a serum pool containing digoxin, falsely elevated digoxin value was observed with fluorescence polarization immunoassay, but values were falsely lowered when measured by the microparticle enzyme immunoassay. Digibind neutralized digoxin-like immunoreactive components of Ashwagandha in vitro. Components of Ashwagandha interfered with serum digoxin measurements using immunoassays. Digibind neutralized free digoxin-like immunoreactive components of Ashwagandha.

  3. A fluorometric assay for acetylcholinesterase activity and inhibitor detection based on DNA-templated copper/silver nanoclusters.

    PubMed

    Li, Wenhua; Li, Wang; Hu, Yufang; Xia, Yalin; Shen, Qinpeng; Nie, Zhou; Huang, Yan; Yao, Shouzhuo

    2013-09-15

    A novel label-free, rapid, cost-effective, and highly sensitive fluorometric sensor has been constructed for the detection of acetylcholinesterase (AChE) activity and its inhibitor based on the fluorescence quenching of DNA-templated copper/silver nanoclusters (DNA-Cu/AgNCs). In this assay, AChE catalyzes the hydrolysis of acetylthiocholine (ATCh) to form thiocholine which induces fluorescence quenching of DNA-Cu/AgNCs. The AChE activity could be detected as low as 0.05mU/mL and with a linear range from 0.05 to 2.0mU/mL. This assay offers a very convenient "mix and detect" approach for AChE activity. On the other hand, tacrine and organophosphorus pesticides (OPPs) were employed to inhibit the hydrolysis of ATCh, which could eliminate the fluorescence quenching of DNA-Cu/AgNCs. The IC50 of tacrine and methamidophos were estimated to be 16.9nM and 0.075mg/L, respectively. This method was also used to detect spiked OPPs in agricultural products successfully. The present work may expand the use of DNA-Cu/AgNCs to the field of enzyme sensors.

  4. Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.

    PubMed

    Hanson, Cynthia; Israelsen, Nathan D; Sieverts, Michael; Vargis, Elizabeth

    2016-11-10

    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.

  5. Automatic miniaturized fluorometric flow system for chemical and toxicological control of glibenclamide.

    PubMed

    Ribeiro, David S M; Prior, João A V; Taveira, Christian J M; Mendes, José M A F S; Santos, João L M

    2011-06-15

    In this work, and for the first time, it was developed an automatic and fast screening miniaturized flow system for the toxicological control of glibenclamide in beverages, with application in forensic laboratory investigations, and also, for the chemical control of commercially available pharmaceutical formulations. The automatic system exploited the multipumping flow (MPFS) concept and allowed the implementation of a new glibenclamide determination method based on the fluorometric monitoring of the drug in acidic medium (λ(ex)=301 nm; λ(em)=404 nm), in the presence of an anionic surfactant (SDS), promoting an organized micellar medium to enhance the fluorometric measurements. The developed approach assured good recoveries in the analysis of five spiked alcoholic beverages. Additionally, a good agreement was verified when comparing the results obtained in the determination of glibenclamide in five commercial pharmaceutical formulations by the proposed method and by the pharmacopoeia reference procedure.

  6. A biochemical fluorometric method for assessing the oxidative properties of HDL[S

    PubMed Central

    Kelesidis, Theodoros; Currier, Judith S.; Huynh, Diana; Meriwether, David; Charles-Schoeman, Christina; Reddy, Srinivasa T.; Fogelman, Alan M.; Navab, Mohamad; Yang, Otto O.

    2011-01-01

    Most current assays of HDL functional properties are cell-based. We have developed a fluorometric biochemical assay based on the oxidation of dihydrorhodamine 123 (DHR) by HDL. This cell-free assay assesses the intrinsic ability of HDL to be oxidized by measuring increasing fluorescence due to DHR oxidation over time. The assay distinguishes the oxidative potential of HDL taken from different persons, and the results are reproducible. Direct comparison of this measurement correlated well with results obtained using a validated cell-based assay (r2 = 0.62, P < 0.001). The assay can be scaled from a 96-well format to a 384-well format and, therefore, is suitable for high-throughput implementation. This new fluorometric method offers an inexpensive, accurate, and rapid means for determining the oxidative properties of HDL that is applicable to large-scale clinical studies. PMID:21957198

  7. Liquid chromatographic determination of amprolium in poultry feed and premixes using postcolumn chemistry with fluorometric detection.

    PubMed

    Vanderslice, J T; Huang, M H

    1987-01-01

    Two extraction and liquid chromatographic procedures are presented which separate amprolium from compounds in poultry feed or premixes that could interfere with its fluorometric determination. The procedures are based on earlier work on the determination of thiamine in food samples. Amprolium is extracted from feed with a hexane-aqueous sulfosalicylic acid mix, separated on a C18 column, and detected fluorometrically after postcolumn derivatization. For premixes, water extraction is used. Values for the amprolium content of poultry feed obtained with these procedures are in good agreement with those obtained with AOAC official methods. It is suggested that these methods with suitable modifications may be of use for routine analysis of amprolium in feeds. The overall methods are rapid and appear to give reasonable results.

  8. Dynamic modification of microorganisms by pyrenebutanoate for fluorometric detection in capillary zone electrophoresis.

    PubMed

    Horká, Marie; Růzicka, Filip; Holá, Veronika; Slais, Karel

    2005-02-01

    Pyrenebutanoate, a fluorescent amphiphilic probe, is suggested here as a capillary zone electrophoresis (CZE) buffer additive for dynamic modification and analysis of microbial cells. Mixed cultures of microorganisms Escherichia coli, Candida albicans, Enterococcus faecalis and Staphylococcus epidermidis were concentrated, resolved by CZE and detected. Using UV excitation for on-column fluorometric detection, a detection sensitivity for the microorganisms on the order of from one to tens of injected cells was achieved.

  9. Determination of beryllium in ores and rocks by a dilution-fluorometric method with morin

    USGS Publications Warehouse

    May, R.; Grimaldi, F.S.

    1961-01-01

    Beryllium in concentrations as little as a few parts per million is determined fluorometrically with morin in low grade ores by a dilution method without separations. A high sensitivity is obtained by the adoption of instrumental and reaction conditions that give a satisfactory ratio of beryllium to blank fluorescence and at the same time minimize iron interference. Data on the behavior of 47 ions are given. The method is applied to ores containing bertrandite and beryl as the beryllium minerals.

  10. Modeling metal binding by dissolved humic substance: a revisit to the fluorometric titration approach.

    PubMed

    Chen, Hao; Meng, Wei; Lei, Kun

    2014-01-01

    It is desirable to directly investigate metal cation binding by dissolved humic substance (HS) in environmental samples without isolation and purification of the HS. This is commonly achieved by the fluorometric titration approach, in which the variations of the HS components' fluorescence when titrated with metal cations, such as cupric ions (Cu(2+)), were commonly resolved by a well-established chemometric tool called parallel factor analysis and fit to a classical nonlin ear equation to obtain cation binding parameters. The nonlinear expression was derived based on the two assumptions that a given HS component (e.g., L) binds Cu(2+) with a 1:1 stoichiometry, forming only the complex LCu, and that other ligands competing with L for Cu(2+) are not explicitly considered. Given the deviations (e.g., the presence of multiple HS components competing for Cu(2+) and a likely 2:1 binding stoichiometry in addition to the 1:1 binding) from the assumptions, the fitting-derived binding parameters reported in past studies are questionable; those studies commonly reported high goodness-of-fit (R(2)) as a support of the validity of the assumptions. This study deconstructed the current equation and examined it with two organic ligand components in a simulated study to see what conditions could also yield a good fit. It turned out that high a R(2) value ranging between 0.9971 and 1.0 was observed despite the deviations from the above-mentioned assumptions. In addition, this study re-evaluated some published experimental data from these past studies and found that the fitting-derived parameters could not be accounted for based on the above-mentioned assumptions. The findings in this study therefore indicate that the current fluorometric titration approach is problematic when investigating HS component interactions with metal ions in situ. The combination of ion-selective electrode and fluorometric titration may be an alternative to the current fluorometric titration approach alone.

  11. Improved buprenorphine immunoassay performance after urine treatment with β-glucuronidase.

    PubMed

    Snyder, Marion L; Darragh, Alicia; Flood, James G; Jones, Jenny; Ropar, Kaitlin; Jarolim, Petr; Melanson, Stacy E F

    2014-01-01

    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: journals.permissions@oup.com.

  12. Determination of designer drug cross-reactivity on five commercial immunoassay screening kits.

    PubMed

    Regester, Laura E; Chmiel, Jeffrey D; Holler, Justin M; Vorce, Shawn P; Levine, Barry; Bosy, Thomas Z

    2015-03-01

    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.

  13. Flow method based on cloud point extraction for fluorometric determination of epinephrine in human urine.

    PubMed

    Davletbaeva, Polina; Falkova, Marina; Safonova, Evgenia; Moskvin, Leonid; Bulatov, Andrey

    2016-03-10

    A novel stepwise injection fluorometric method for the determination of epinephrine in human urine has been developed. In the current study, the stepwise injection analysis (SWIA) was successfully combined with on-line in-syringe cloud point extraction (CPE) and fluorometric detection. The procedure was based on the epinephrine derivatization in the presence of o-phenylenediamine followed by the preconcentration stage based on the CPE with the nonionic surfactant Triton X-114. After the phase separation into a syringe of the flow system, the micellar phase containing the epinephrine derivative was transported to a fluorometric detector. The excitation and emission wavelengths were set at 447 nm and 550 nm, respectively. The conditions of epinephrine derivatization and CPE have been studied. The calibration plot constructed using the developed procedure was linear in the range of 1·10(-11)-5·10(-7) mol L(-1). The limit of detection, calculated as 3 σ of a blank test (n = 10), was found to be 3·10(-12) mol L(-1). The proposed method was successfully applied for the determination of epinephrine in human urine samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification.

    PubMed

    Goumenou, Marina; Machera, Kyriaki

    2004-03-15

    The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye-ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N-nitroso-N-methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks.

  15. Measurement of urinary histamine: comparison of fluorometric and radioisotopic-enzymatic assay procedures

    SciTech Connect

    Warren, K.; Dyer, J.; Merlin, S.; Kaliner, M.

    1982-02-01

    Assessment of urinary histamine may prove useful in determining the role of histamine in human health and disease. Urinary histamine may be accurately estimated by a modified fluorometric assay employing diamine oxidase (DAO) digestion and cation-exchange chromatography. Normal urine histamine values obtained by this assay are: arithmetic means (+/- SEM), 8.6 +/- 0.6 ng/ml and 10.5 +/- 0.7 micrograms/24 hr; geometric means (+/- SEM), 6.2 +/- 1.1 ng/ml and 10.0 +/- 1.3 micrograms/24 hr. However, the radioisotopic-enzymatic assay is less expensive, easier to perform, and possibly more sensitive. Therefore the two procedures were compared. The radioenzyme assay was found to be affected by factors in urine (possibly salt concentrations) requiring extraction of histamine from urine by butanol-heptane. Moreover, it was found to be necessary to compare DAO-digested samples with undigested samples to accurately estimate histamine levels and to run the standard curve of histamine in DAO-digested urine. Even with these modifications, the radioenzyme assay was not as accurate as the fluorometric assay for urine samples having histamine values about 60 ng/ml. Therefore we recommend utilization of the modified fluorometric assay for the measurement of urinary histamine levels.

  16. Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

    SciTech Connect

    Yinfa, Ma.

    1990-12-10

    Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column'' detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal of this dissertation to present a new detection method for TLC -- indirect fluorometric detection method. This detection technique is universal sensitive, nondestructive, and simple. This will be described in detail from Sections 1 through Section 5. Section 1 and 3 describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. In Section 2, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. In Section 4, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages. A laser-based indirect fluorometric detection technique in TLC is developed in Section 5. Section 6 is totally different from Sections 1 through 5. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. 262 refs.

  17. Alpha-l-Fucosidase Immunoassay for Early Detection of Hepatocellular Carcinoma.

    PubMed

    Waidely, Eric; Al-Youbi, Abdulrahman O; Bashammakh, Abdulaziz S; El-Shahawi, Mohammad S; Leblanc, Roger M

    2017-09-05

    Detection of alpha-l-fucosidase has been shown to have relevance in diagnosing hepatocellular carcinoma. Few assays have been developed to measure this enzyme, with most relying on colorimetric techniques involving the enzyme's kinetics. While these assays are facile and quick, the sensitivity is not always sufficient for early tumor detection. To improve upon previous assays for alpha-l-fucosidase, a fluorescence based immunoassay was produced implementing an alpha-l-fucosidase specific antibody (FUCA2). The immobilization of the alpha-l-fucosidase-specific antibody onto a quartz slide was investigated with several bioconjugation approaches and an immunoassay for detection of alpha-l-fucosidase was produced. The immunoassay was utilized to produce calibration curves for quantifying alpha-l-fucosidase concentrations in both PBS and human blood serum. A detection limit of 10 nM was found using human blood serum, which is well below the diagnostic cutoff point of 80 nM.

  18. Enhancing immunoassay possibilities using magnetic carriers in biological fluids

    NASA Astrophysics Data System (ADS)

    Yu, Hao

    1997-05-01

    An antibody-based magnetic plate chemifluorescent immunoassay (MPFIA) for effective and rapid detection of bacteria and toxoid from biological fluids was developed. Streptavidin (SA)- magnetic particles and biotinylated antibody as a solid phase immunomagnetic carrier was used for antigen capture. An alkaline phosphatase-antibody conjugate as a secondary capture antibody to the antigen forms a sandwich with the primary antibody. The fluorgenic substrate, AttoPhos reacts with alkaline phosphatase that emits chemifluorescent intensities are proportional to captured antigens. Antigen separation and concentration from biological fluids using immunomagnetic carrier are the key step to reduce media interference for sensitive detection. Results of these efforts may actually enhance the immunoassay possibilities by concentration of specific antigen and reduction of background noise. Magnetic separation and chemifluorescent detection have been achieved by a multiple-well formatted magnetic plate separator and a fluorescent plate detector, respectively. Experiments were performed for virulent Escherichia coli cells, Staphylococcal enterotoxin B toxoid and Bacillus subtilus spore detection in biological fluids. In general, the fluorescent detection can be achieved at the same sensitivities as enzyme-linked immunoassay and the ECL detection is more sensitive than fluorescent assay. However, the unique features of MPFIA and MPECL are that the biological samples can be rapidly processed and detected on the same multiple sample formatted plate within one hour assay time.

  19. A microfluidic indirect competitive immunoassay for multiple and sensitive detection of testosterone in serum and urine.

    PubMed

    Wang, Huashan; Li, Juanjuan; Zhang, Xiaoqing; Hu, Binfeng; Liu, Yang; Zhang, Lin; Cha, Ruitao; Sun, Jiashu; Jiang, Xingyu

    2016-02-07

    We demonstrate a microfluidic-based indirect competitive chemiluminescence enzyme immunoassay (MIC) for multiple, sensitive, reliable and rapid detection of testosterone in human serum and urine samples. As MIC can detect biomarkers in a cost-effective and easy-to-operate manner, it may have great potential for clinical diagnosis and point-of-care testing (POCT).

  20. IMMUNOASSAY METHODS FOR MEASURING ATRAZINE AND 3,5,6-TRICHLORO-2-PYRIDINOL IN FOODS

    EPA Science Inventory

    This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) methods for the analysis of two potential environmental contaminants in food sample media, atrazine and 3,5,6-trichloro-2-pyridinol (3,5,6-TCP). Two different immunoassay formats are employed: a magnetic...

  1. Competitive chemiluminescent anzyme immunoassay for vitamin B12 analysis in human milk.

    USDA-ARS?s Scientific Manuscript database

    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 seru...

  2. IMMUNOASSAY METHODS FOR MEASURING ATRAZINE AND 3,5,6-TRICHLORO-2-PYRIDINOL IN FOODS

    EPA Science Inventory

    This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) methods for the analysis of two potential environmental contaminants in food sample media, atrazine and 3,5,6-trichloro-2-pyridinol (3,5,6-TCP). Two different immunoassay formats are employed: a magnetic...

  3. Evaluation of two immunoassay procedures for drug testing in hair samples.

    PubMed

    Musshoff, F; Kirschbaum, K M; Graumann, K; Herzfeld, C; Sachs, H; Madea, B

    2012-02-10

    A preliminary initial enzyme-linked immunosorbent assay (LUCIO-Direct ELISA kit) and a preliminary DRI enzyme immunoassay were evaluated for drug detection in head hair with respect to lowered cutoff values recommended in Germany for the control of abstinence in cases of re-granting of drivers' licences. Following drug classes were included: cannabinoids, opiates, cocaine like substances, amphetamine, methamphetamine (and methylenedioxyamphetamines), methadone, and benzodiazepines. 759 analyses were performed using LUCIO-Direct ELISA kits and 936 analyses using DRI enzyme immunoassay tests. Sample size for each drug group and immunoassay test reached from 74 to 178. The LUCIO-Direct ELISA kit revealed a sensitivity of 91% for amphetamine up to 98% for methadone (methamphetamine 92%, cocaine 94%, opiates 94%, benzodiazepines 96%) and values of specificity of 72% for methadone up to 89% for amphetamine and benzodiazepines. The test was not useful for a preliminary screening for tetrahydrocannabinol (sensitivity of 65%) in consideration of a suggested cutoff of 0.02 ng/mg. The DRI enzyme immunoassay test was only useful for morphine and cocaine testing at low recommended new cutoff values (0.1 ng/mg) revealing sensitivities of 94% and 99%, respectively.