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Sample records for igm enzyme immunoassay

  1. 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.

  2. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R. (Columbus, OH); Bednarski, Mark D. (Berkeley, CA); Gruber, Patrick R. (St. Paul, MN)

    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.

  3. Enzyme immunoassay system for panel testing.

    PubMed

    Donohue, J; Bailey, M; Gray, R; Holen, J; Huang, T M; Keevan, J; Mattimiro, C; Putterman, C; Stalder, A; Defreese, J

    1989-09-01

    An immunoassay system based on enzyme immunoassay technology has been developed for quantitative panel testing. The system includes test card disposables, reagents, and an instrument. Patients' samples are processed semiautomatically in the instrument with minimum user intervention. The test card has multiple test areas at individual locations on a membrane solid phase so that simultaneous determinations from a single specimen are possible. Each panel also includes positive and negative reagent procedural controls. Factory-determined calibration curves for each analyte are provided in barcode form with each test kit. The reagents include a specimen dilution buffer, enzyme conjugate, and precipitogenic substrate. Up to 10 test cards at a time can be processed in random-access and continuous-access modes, with automated agitation of sample and reagents over the solid phase, temperature-controlled incubation, and membrane washing and reading, data reduction, and printout of results. The optical reader measures diffuse reflectance and features source intensity and wavelength compensation. PMID:2673584

  4. Public Health Laboratory Service enzyme linked immunosorbent assay for detecting Toxoplasma specific IgM antibody.

    PubMed Central

    Payne, R A; Joynson, D H; Balfour, A H; Harford, J P; Fleck, D G; Mythen, M; Saunders, R J

    1987-01-01

    An enzyme linked immunosorbent assay (ELISA) based on the antibody class capture method for the detection of specific IgM against Toxoplasma gondii, using the microtitre plate format, was developed. Antigen binding was detected using a monoclonal antibody, CIE3, conjugated to horseradish peroxidase. Prior mixing of the conjugate and antigen improved the stability of these reagents as well as removing an incubation stage from the assay. The incubation time of less than four hours permits a rapid throughput of specimens. Using the assay, a total of 163 sera were examined in a three centre study and good agreement was found. Results were expressed as arbitrary enzyme immunoassay units (EIUs) against a freeze dried standard. Throughout the study the standard serum showed a coefficient of variation less than 10% across the microtitre plate. By measuring IgM titres in patients having toxoplasmic lymphadenopathy with a known date of onset, IgM class antibodies were shown to peak at two months, persisting for about six months. In addition, a case of laboratory acquired toxoplasmosis was monitored. Sera shown to contain rheumatoid factor and antinuclear factor did not give false positive results. This rapid, robust, and simplified assay is used by the Public Health Laboratory Service Toxoplasma Reference Units and will provide a standard with which other assays can be compared. PMID:3558860

  5. 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. PMID:2562487

  6. Multiplex Microsphere Immunoassays for the Detection of IgM and IgG to Arboviral Diseases

    PubMed Central

    Basile, Alison J.; Horiuchi, Kalanthe; Panella, Amanda J.; Laven, Janeen; Kosoy, Olga; Lanciotti, Robert S.; Venkateswaran, Neeraja; Biggerstaff, Brad J.

    2013-01-01

    Serodiagnosis of arthropod-borne viruses (arboviruses) at the Division of Vector-Borne Diseases, CDC, employs a combination of individual enzyme-linked immunosorbent assays and microsphere immunoassays (MIAs) to test for IgM and IgG, followed by confirmatory plaque-reduction neutralization tests. Based upon the geographic origin of a sample, it may be tested concurrently for multiple arboviruses, which can be a cumbersome task. The advent of multiplexing represents an opportunity to streamline these types of assays; however, because serologic cross-reactivity of the arboviral antigens often confounds results, it is of interest to employ data analysis methods that address this issue. Here, we constructed 13-virus multiplexed IgM and IgG MIAs that included internal and external controls, based upon the Luminex platform. Results from samples tested using these methods were analyzed using 8 different statistical schemes to identify the best way to classify the data. Geographic batteries were also devised to serve as a more practical diagnostic format, and further samples were tested using the abbreviated multiplexes. Comparative error rates for the classification schemes identified a specific boosting method based on logistic regression “Logitboost” as the classification method of choice. When the data from all samples tested were combined into one set, error rates from the multiplex IgM and IgG MIAs were <5% for all geographic batteries. This work represents both the most comprehensive, validated multiplexing method for arboviruses to date, and also the most systematic attempt to determine the most useful classification method for use with these types of serologic tests. PMID:24086608

  7. Detection of poliovirus antigen by enzyme immunoassay.

    PubMed Central

    Ukkonen, P; Huovilainen, A; Hovi, T

    1986-01-01

    A solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Rabbit and guinea pig antisera for the assay were raised against purified poliovirus type 3/Fin (strain 3/Fin/K) isolated from a fecal specimen from a meningitis patient during an outbreak of poliomyelitis in Finland in 1984. The EIA was highly specific for poliovirus type 3, and it was about 30 times more sensitive for strain 3/Fin/K than for strain 3/Saukett used in the inactivated poliovirus vaccine. The sensitivity of the EIA was 2 to 5 ng of purified strain 3/Fin/K per ml, whereas disrupted viruses and soluble viral proteins were almost undetectable by the assay. Only 5 of 51 (10%) stool specimens containing poliovirus type 3/Fin detected by virus isolation were positive by the EIA. Quantitation by the EIA, using purified poliovirus 3/Fin/K as a standard, revealed that concentrations of poliovirus type 3 in undiluted fecal specimens of patients with natural poliovirus infection were only 50 ng/ml or less. In conclusion, owing to the small amount of poliovirus in feces, the EIA is not suitable for the diagnosis of poliovirus infections directly from clinical specimens, but it can be used to detect, type, and quantitate poliovirus antigen in infected cells. PMID:3023440

  8. DETECTION OF ROTAVIRUS WITH A NEW POLYCLONAL ANTIBODY ENZYME IMMUNOASSAY (ROTAZYME 2) AND A COMMERCIAL LATEX AGGLUTINATION TEXT (ROTALEX): COMPARISON WITH A MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

    EPA Science Inventory

    A total of 176 human fecal specimens were examined for the presence of rotavirus using four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, Chicago, IL (Rotazyme I); a modification of...

  9. Microneutralization test for rabies virus based on an enzyme immunoassay.

    PubMed

    Mannen, K; Mifune, K; Reid-Sanden, F L; Smith, J S; Yager, P A; Sumner, J W; Fishbein, D B; Tong, T C; Baer, G M

    1987-12-01

    We have developed an enzyme immunoassay for rabies virus by using acetone-fixed infected cell cultures as the antigen. This test was used to demonstrate virus-neutralizing antibodies in human and animal sera and was as sensitive as and easier to perform than the rapid fluorescent-focus inhibition technique. PMID:3323234

  10. DETECTION OF NORWALK VIRUS IN STOOLS BY ENZYME IMMUNOASSAY

    EPA Science Inventory

    The development of a solid-phase microtiter enzyme immunoassay (EIA) for detection of Norwalk virus antigen in stool samples is described. The EIA was compared with a previously developed radioimmunoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 vol...

  11. Rapid determination of dioxin in water by enzyme immunoassay

    SciTech Connect

    Wang, H.; Wang, L.; George J.E. III; Ward, G.K.

    1996-10-01

    Dioxin in water, soil, sediments and other sample matrices is usually determined by the EPA method 1613 which was developed by the EPA Office of Science and Technology. This method however requires expensive instruments (high resolution gas chromatography/high resolution mass spectrometry) and a highly trained analyst. In order to reduce the cost and turn around time, a dioxin enzyme immunoassay (EIA) was developed to rapidly analyze trace levels of 2,3,7,8-tetra chlorinated dibenzodioxin (TCDD) in water samples. Water samples were extracted using a 47 mm, C18 Empore extraction disk (3M). Dioxin was eluted with dichloromethame. EnvironGard reagents and microwell strip reader (Millipore Corporation) were used to perform the dioxin enzyme immunoassay. The extraction efficiency was also tested by GC/MS with Varian`s large volume injector and Selected Ion Storage technique. The working range of the dioxin enzyme immunoassay was from 15 pg/L to 100 pg/L. The precision and accuracy of EIA was determined by performing five replicates of reagent water spiked at a concentration of 25 pg/L. The recovery of the dioxin assay ranged from 74% to 122%, and % CV for five replicates was less than 15%. In general, EIA provides a relatively easy and cost effective means for measuring trace levels of dioxin in water samples.

  12. Enzyme immunoassay for nomegestrol acetate in human plasma.

    PubMed

    Ezan, E; Benech, H; Bucourt, R; Ardouin, T; Tchernatinsky, C; Thomas, J L; Paris, J; Grognet, J M

    1993-10-01

    Currently available chromatographic assays of the progestative drug nomegestrol acetate in human plasma are not suitable for monitoring drug kinetics more than 24 h after clinical dosage. A specific and sensitive enzyme immunoassay was therefore developed. A 3(O-carboxymethyl)oxime derivative of nomegestrol acetate was synthesized and coupled to bovine serum albumin in order to raise polyclonal antibodies in rabbits. The enzymatic tracer was obtained by coupling the 3(O-carboxymethyl)oxime derivative to acetylcholinesterase (E.C.3.1.1.7.). HPLC fractionation of human plasma samples followed by enzyme immunoassay revealed the presence of cross-reacting metabolites. An automated procedure of metabolite separation was developed using silica bonded with diol groups (Diol Bakerbond column). This procedure ensured assay specificity. The quantification limit in human plasma was 0.1 ng/ml. Mean repeatability (intra-assay variation) and reproducibility (inter-assay variation) were 9 and 15%, respectively. The enzyme immunoassay allowed monitoring of the kinetics of nomegestrol acetate 144 h after oral administration of a single 5 mg dose. Values for human samples were in excellent agreement with those assayable by HPLC followed by u.v. detection. PMID:8217881

  13. 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

  14. Evaluation of enzyme immunoassay (Chlamydiazyme) for detecting Chlamydia trachomatis in genital tract specimens.

    PubMed Central

    Taylor-Robinson, D; Thomas, B J; Osborn, M F

    1987-01-01

    An enzyme immunoassay (Chlamydiazyme) for detecting Chlamydia trachomatis was evaluated on genital specimens from 96 men and 272 women attending a clinic for sexually transmitted diseases (STD clinic). Compared with a direct immunofluorescence test for chlamydial elementary bodies, the enzyme immunoassay had a sensitivity of 58% on specimens from men, a specificity of 90%, a positive predictive value of 93%, and a negative predictive value of 88%; the assay had a sensitivity of 67% on specimens from women, a specificity of 89%, a positive predictive value of 63% and a negative predictive value of 90%. Immunofluorescence provided the most stringent test for the performance of the enzyme immunoassay as values were improved a little when a cell culture procedure was used for comparison. Further evidence for the lack of sensitivity was the detection of elementary bodies, sometimes in large numbers, in the enzyme immunoassay buffer of 13 of 19 specimens that had given a negative enzyme immunoassay result and the finding in comparative titrations of four laboratory strains that the enzyme immunoassay was at least 100-fold less able to detect chlamydiae than either immunofluorescence or the cell culture procedure. Lack of specificity may be associated with the finding that the enzyme immunoassay antibody reacted with strains of Acinetobacter calcoaceticus, Escherichia coli, Gardnerella vaginalis, Neisseria gonorrhoeae and group B streptococci. The enzyme immunoassay was not considered to be sufficiently sensitive, specific, or reproducible for routine use. PMID:3546397

  15. Enzyme immunoassay validation for the detection of buprenorphine in urine.

    PubMed

    Cirimele, V; Kintz, P; Lohner, S; Ludes, B

    2003-03-01

    A solid-phase enzyme immunoassay involving microtiter plates was proposed by Microgenics to screen buprenorphine in urine. The intra-assay precision at 10 ng/mL was 7.7% (coefficient of variation). The immunoassay was determined to have no cross-reactivity with codeine, dihydrocodeine, morphine, ethylmorphine, 6-monoacetylmorphine, methadone, pholcodine, propoxyphene, dextromoramide, and dextromethorphan at 1 and 10 mg/L. A low cross-reactivity (3% at 1 ng/mL) was observed at low concentrations of norbuprenorphine. After comparing this new immunological test (Singlestep ELISA) for 76 urine specimens with our validated high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ES-MS) procedure, an optimum cutoff concentration of 2 ng/mL was determined for the kit. At this cutoff, the screening assay was able to determine more than 90% of true results with 43.4% true positives and 48.7% true negatives. Four positive urines (5.3%) were not confirmed by HPLC-ES-MS. In only one case, the negative urine test was confirmed as positive by HPLC-ES-MS (buprenorphine: 62.5 ng/mL). Buprenorphine concentrations determined by HPLC-ES-MS ranged from 1.2 to 1052 ng/mL. Of the four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that might be added to a positive urine specimen, none were able to cause a false-negative response by the immunoassay. The results of this study support the concept that the Singlestep ELISA for buprenorphine determination in urine should be considered as a new, valided screening procedure. PMID:12670004

  16. Enzyme immunoassay validation for qualitative detection of cocaine in sweat.

    PubMed

    Spiehler, V; Fay, J; Fogerson, R; Schoendorfer, D; Niedbala, R S

    1996-01-01

    A solid-phase enzyme immunoassay (EIA) involving microtiter plates was modified for analysis of cocaine in sweat. Sweat was collected with the PharmChek sweat patch and drugs were eluted from the collection pad of the patch. The sweat contained primarily parent cocaine. The assay was determined to have cross-reactivity for cocaine of 102% relative to 100% for the benzoylecgonine (BE) calibrators and for cocaethylene of 148%. The optimum cutoff concentration for this modified assay, determined by receiver-operating characteristic curve analysis, was 10 micrograms/L cocaine or BE equivalents. At this concentration the assay had 94.5% sensitivity and 99.1% specificity vs gas chromatography-mass spectrometry (GC-MS) as an acceptable indicator of the true clinical state. The positive predictive value at a prevalence of 50% was 99%. Threshold analysis for positives suggested that the 95% confidence interval for a positive result by the EIA was between 12.5 and 15 micrograms/L and that quality-control samples at 5 and 15 micrograms/L could be run with each batch to certify the precision around the cutoff. All positive samples must be confirmed by GC-MS. The sensitivity and specificity of the overall analysis system (immunoassay screen and GC-MS confirmation) was 86% and 97%, with known cocaine dosing of volunteers as the acceptable indicator of the true clinical state. PMID:8565229

  17. [Kinds and characteristics of solid-phase enzyme immunoassay].

    PubMed

    Sugiura, M; Mizuoka, K

    1995-09-01

    Technology with solid-phase enzyme immunoassay was markedly progressed. Kinds of solid-phase are as follows; (1) tube, (2) beads, (3) latex particle, (4) microtiter plate, (5) ferrite particle, (6) membrane. Recently, application of avidin/biotin system to the tube method was accomplished. As one of the B/F separation with beads method, spin washing method was also developed. Automated open system with the beads method was also developed. Microtiter plate using sponges as the solid-phase was routinely used for allergy tests (CAP-RAST). Multi-Access Open System (MAOS), applicable for various kinds of ELISA kit, will appear in Japan near future. Latex particle with ferrite plating and ferrite particle with glass coating were also used as the solid-phase of EIA. HCV-RIBA II and III systems using nitro-cellulose membrane as solid-phase were also described. PMID:7474379

  18. Chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine

    NASA Astrophysics Data System (ADS)

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

    2011-10-01

    Sulfamethoxydiazine (SMD), which is often used for animal disease treatment, is harmful to human health. No SMD residue should be detected in food in some countries, such as USA and Japan. Therefore, it is significant to develop a high-throughput, high-sensitivity and accurate method for the determination of the content of SMD in food. In this paper, chemiluminescence enzyme immunoassay (CLEIA) was developed for quantification of SMD. For this method, the limit of detection was 3.2 pg/ml, the linear range was from 10 to 2000 pg/ml, the within-day and inter-day precision were below 13% and below 18%, respectively, and the recovery was from 85% to 105%. Milk and egg were selected as samples to be examined with this method, and the result indicated that this CLEIA method was suitable for screening and quality control of food.

  19. Capillary electrophoretic enzyme immunoassay for digoxin in human serum.

    PubMed

    Liu, X; Xu, Y; Ip, M P

    1995-09-15

    The combined use of capillary electrophoretic (CE) separation and homogeneous enzyme immunoassay for analyzing drugs in hemolyzed, lipemic, or icteric serum samples was investigated. An FDA-approved EMIT assay kit for digoxin in human serum was used. After the enzyme immunoassay, the enzymatic reaction product (NADH) and remaining substrate (NAD+), together with internal standard (p-nitrophenol, NP), were electrokinetically injected into a polyacrylamide-coated electrophoresis capillary and separated under applied potential. Detection was made by monitoring the UV adsorption at wavelength of 260 nm. The digoxin level in human serum was determined by comparing the peak area ratio of NADH and NP to the ratios established by the known digoxin standards. In this study, the factors that influence the CE separation were also investigated. Under the optimum conditions, NADH, NAD+, and NP were separated at electric field strength of 438 V/cm in the coated capillary (100 microns x 57 cm) with 200 mM Tris-borate buffer (pH 7.9) containing 0.2% hydroxypropyl methylcellulose. CE analyses of serum samples spiked with NADH standards at concentrations of 100 and 400 microM resulted in detection variabilities of less than 2% and analytical recoveries of 98-102%. Both an internal calibration plot for NADH and a dose-response curve for digoxin in serum were constructed. Calibrator serum, patients' sera with hemolyzed, lipemic, and icteric interference factors, and other pigmented blood components (e.g., serum albumin, bilirubin, hemoglobin, uric acid, coproporphyrin, melanin, protoporphyrin IX, and uroprophyrin) demonstrated no interference in this method. The authors believed that this method is useful for analyzing digoxin in hemolyzed, lipemic, and icteric blood samples that are known to create problems in conventional EMIT assays and may be applicable to other EMIT-based assays for monitoring drugs in complex biological matrices. PMID:8686886

  20. 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

  1. Immunoassay using /sup 125/I- or enzyme-labeled protein A and antigen-coated tubes

    SciTech Connect

    Gee, A.P.; Langone, J.J.

    1981-09-15

    Antigen-coated plastic tubes were used with /sup 125/I- or enzyme-labeled stapylococcal protein A in a general immunoassay method for antigens and haptens. Protein A reacts with immunoglobulin G(IgG) regardless of antibody specificity at sites distal to the antigen combining site and does not inhibit the immune reaction. It therefore serves as a general tracer and its use eliminates the need to purify and to label individual components for each assay. Macromolecular antigens were bound to polystyrene or polypropylene tubes by direct passive absorption. Haptens with free carboxyl groups were bound covalently to poly-L-lysine and these conjugates passively absorbed to the tube surface. Optimal assay conditions were established for the quantitative determination of immunoglobulins and the folate derivatives, methotrexate and 5-methyltetrahydrofolate, using /sup 125/I-labeled protein A or protein A labeled with alkaline phosphatase. The method has been used to estimate levels of IgG, IgA, Igm, and IgE in serum in volumes up to 1 ml.

  2. Immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoassays are analytical methods that employ antibodies or molecules derived from antibodies for the essential binding reactions. The choice of immunoassay system for food safety analysis depends on the analyte, the matrix, and the requirements of the analysis (speed, throughput, sensitivity, spe...

  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. 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.

  5. 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.

  6. False-positive IgM serology in coccidioidomycosis.

    PubMed

    Kuberski, Tim; Herrig, Judith; Pappagianis, D

    2010-06-01

    The clinical observation has been made that there might be an unacceptable number of false-positive enzyme immunoassay (EIA) test results for IgM among persons suspected of having coccidioidomycosis. Patients with a positive result for IgM by EIA are thought to have a diagnosis of acute coccidioidomycosis. However, this study found that 82% of patients with an IgM-positive and IgG-negative EIA result did not have coccidioidomycosis. PMID:20357210

  7. Simple and specific enzyme immunoassay using monoclonal antibodies for serotyping human rotaviruses.

    PubMed Central

    Coulson, B S; Unicomb, L E; Pitson, G A; Bishop, R F

    1987-01-01

    An enzyme immunoassay for serotyping human rotaviruses in stools and in cell culture was developed. Hyperimmune rabbit antisera to rotaviruses were used as capture antibodies, and rotavirus-neutralizing mouse monoclonal antibodies specific for serotypes 1, 2, 3, and 4 were used as detection reagents. Partial purification of monoclonal antibodies and inclusion of skim milk powder in antibody diluents contributed to assay specificity. The sensitivity of this assay was greater than that of a direct enzyme immunoassay in which rotaviruses of the appropriate serotype were adsorbed directly to the solid phase. When fecal extracts were concentrated threefold, this serotyping enzyme immunoassay was of equal specificity and approached the sensitivity of electron microscopy for rotavirus detection. This assay is simple and rapid and is suitable for serotyping the large numbers of isolates obtained from epidemiological studies and vaccine trials. PMID:3033013

  8. Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides

    SciTech Connect

    Krogstad, D.J.; Granich, G.G.; Murray, P.R.; Pfaller, M.A.; Valdes, R.

    1981-07-01

    Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10/sup 5/ and greater than or equal to 3 X 10/sup 6/ USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase and kanamycin 6'-acetyltransferase enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10/sup 5/ and greater than or equal to10/sup 4/ USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. Heparin interference with these two assays and at concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes is described. (JMT)

  9. Development of a sensitive microarray immunoassay and comparison with standard enzyme-linked immunoassay for cytokine analysis.

    PubMed

    Knight, Paul R; Sreekumar, Arun; Siddiqui, Javed; Laxman, Bharathi; Copeland, Shannon; Chinnaiyan, Arul; Remick, Daniel G

    2004-01-01

    Cytokine and cytokine inhibitors represent important components of the inflammatory response in patients with trauma, shock, and sepsis. Many investigators wish to quantify cytokines and it would be advantageous to measure multiple cytokines in a multiplex manner to obtain an inflammatory profile rather than a single value. Using the well-accepted standard enzyme-linked immunoassay (ELISA) as a basis, a microarray immunoassay (MI) was designed to measure 16 different human cytokines simultaneously. The MI was performed by spotting antibodies on nitrocellulose pads affixed to glass slides. Detection of the mediators was performed with biotin-conjugated antibodies followed by fluorescently labeled streptavidin. All antibodies and other reagents were purchased commercially. The MI achieved a lower limit of detection that was generally similar to traditional ELISAs (approximately 4-12 pg/mL) and also had a similar coefficient of variation. In the multiplexed MI, there was no cross reactivity between mediators. To verify the utility of the MI, cytokines and cytokine inhibitors were measured in endotoxin stimulated human blood by both ELISA and MI. Virtually identical cytokine concentrations were measured by both methods. These results describe the development of a sensitive, specific and cost-effective multiplexed microarray immunoassay that produces values similar to traditional ELISAs. PMID:14676680

  10. Comparison of a Time-Resolved Fluorescence Immunoassay and an Enzyme-Linked Immunosorbent Assay for the Analysis of Atrazine

    E-print Network

    Hammock, Bruce D.

    Comparison of a Time-Resolved Fluorescence Immunoassay and an Enzyme-Linked Immunosorbent Assay of California, Davis, California 95616 Immunoassays for atrazine based on a time-resolved fluorescent label and an enzyme label were optimized and utilized to measure atrazine in water. The time-resolved fluorescent

  11. Validation of three commercially available immunoassays for quantification of IgA, IgG, and IgM in porcine saliva samples.

    PubMed

    Escribano, D; Gutiérrez, A M; Martínez Subiela, S; Tecles, F; Cerón, J J

    2012-10-01

    The objectives of this study were to perform the optimization and validation of three commercially available immunoassays for the measurement of IgA, IgG, and IgM (Igs) in porcine saliva samples and to determinate if their concentrations may be used to distinguish healthy from diseased animals. Intra and inter assay coefficients of variation were lower than 15% in all cases. All methods showed good linearity and recovery; and detection limits were low enough to detect Igs levels in healthy and diseased animals. The clinical validation showed an increase statistically significant (P<0.05) in the group of diseased animals versus healthy pigs. Therefore, these assays may be used in porcine saliva samples, in addition, the measurement of Igs in saliva could be a practical tool, simple and minimally invasive, to evaluate the humoral immune status of pigs. PMID:22019471

  12. 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 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

  13. 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

  14. 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 ?g/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.

  15. Automation of the enzyme immunoassay for the serodiagnosis of infectious diseases in cattle

    SciTech Connect

    Seawright, G.L.; Sanders, W.M.; Bryson, M.

    1980-01-01

    The enzyme immunoassay (EIA) is a versatile and highly sensitive new tool that can be used to detect a wide variety of infectious diseases or toxic agents and other low molecular weight compound. The paper describes the present state of development of the EIA-modified Technicon Autoanalyzer II instrumentation, standardization of quality control criteria for the automated EIA and standardization of the diagnostic decision process. (ACR)

  16. 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. PMID:10408118

  17. A sensitive surface-enhanced Raman scattering enzyme-catalyzed immunoassay of respiratory syncytial virus.

    PubMed

    Zhan, Lei; Zhen, Shu Jun; Wan, Xiao Yan; Gao, Peng Fei; Huang, Cheng Zhi

    2016-02-01

    Respiratory viruses have become a major global health challenge which would benefit from advances in screening methods for early diagnosis. Respiratory syncytial virus (RSV) is one of the most important pathogen causing severe lower respiratory tract infections. Here we present a novel surface-enhanced Raman scattering (SERS) enzyme-catalyzed immunoassay of RSV by employing peroxidase substrate 3, 3'-5, 5'-tetramethylbenzidine (TMB) as Raman molecule. Horseradish peroxidase (HRP) attached to the detection antibody in a novel sandwich immunoassay catalyzes the oxidation of TMB by H2O2 to give a radical cation (TMB(+)), which could be easily adsorbed on the negatively charged surface of silver nanoparticles (AgNPs) through electrostatic interaction, inducing the aggregation of AgNPs and thus giving a strong SERS signal. A linear relationship was obtained between the Raman intensity and the amount of RSV in the range from 0.5 to 20pg/mL, and the minimum detectable concentration of this SERS-based enzyme immunoassay was 0.05pg/mL, which was 20 times lower than that found in the colorimetric method. PMID:26653454

  18. Enzyme immunoassay and liquid chromatography-fluorescence detection for amikacin in raw milk.

    PubMed

    Chen, Yiqiang; Chen, Qian; He, Lidong; Shang, Bingru; Zhang, Liying

    2012-11-15

    An enzyme immunoassay and a liquid chromatography (LC) method for amikacin (AMK) in raw milk were developed in this study. Anti-AMK antibody was prepared by immunizing rabbits with AMK-BSA conjugate. The developed immunoassay exhibited an IC(50) value of 1.30 ng/mL and the spiked recoveries at 25-1000 ng/mL ranged from 69.8% to 93.3% with coefficients of variation (CVs) of 8.5-17.6%. For LC analysis, AMK was derivatized with 9-fluorenylmethyl chloroformate, which was followed by C8 column separation and fluorescence detection. By trichloroacetic acid extraction and MCX column purification, the recoveries at spiked concentrations of 500-5000 ng/mL were 80.7-91.3% with CVs less than 6.3%. The two methods can be selectively used for rapidly screening or quantitatively determining AMK in raw milk. PMID:22868103

  19. Determination of mycobacterial antigens in sputum by enzyme immunoassay.

    PubMed Central

    Yáñez, M A; Coppola, M P; Russo, D A; Delaha, E; Chaparas, S D; Yeager, H

    1986-01-01

    An enzyme-linked immunosorbent assay (ELISA) was examined for its usefulness in detecting mycobacterial antigens in sputum. A double-antibody sandwich procedure was set up by using a commercially available hyperimmune serum directed against Mycobacterium bovis, BCG. The ELISA was able to detect 10 ng of protein per ml of BCG sonic extract. The system also clearly distinguished Mycobacterium tuberculosis organisms from Mycobacterium avium and Mycobacterium kansasii organisms. A total of 68 unknown sputum specimens submitted to the clinical laboratories for examination for tuberculosis were tested by ELISA. Of the 20 specimens that were smear positive and culture positive, 12 (60%) were positive by ELISA; 6 of the 11 (55%) smear-positive culture-negative samples were positive by ELISA; 1 of 2 (50%) of the smear-negative culture-positive samples was positive by ELISA; and only 3 of 35 (9%) of the smear-negative culture-negative samples were positive by ELISA. This approach offers promise as an aid in the presumptive differentiation of nontuberculous mycobacteria from the M. tuberculosis complex. PMID:3086369

  20. 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.

  1. Enzyme immunoassay versus latex agglutination cryptococcal antigen assays in adults with non-HIV-related cryptococcosis.

    PubMed

    Panackal, Anil A; Dekker, John P; Proschan, Michael; Beri, Andrea; Williamson, Peter R

    2014-12-01

    We compared paired enzyme immunoassay (EIA) and latex agglutination (LA) assay results with 185 blood and 164 cerebrospinal fluid (CSF) samples from 44 and 33 non-HIV cryptococcosis patients, respectively. The LA assay cutoff of 1:256 in the blood and 1:32 in the CSF was most highly predictive of a positive EIA result. The EIA missed 18.4% detected by the LA assay in the blood samples and 7.8% detected by the LA assay in the CSF samples. We note here the improved sensitivity of the LA assay over the EIA in non-HIV patients. PMID:25253799

  2. 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-effective alternative to the current mass spectrometry based techniques for the quantitation of cocaine at forensically significant concentrations. PMID:25766738

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

    PubMed Central

    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

    1989-01-01

    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. PMID:2674196

  4. 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

    2015-08-01

    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. PMID:26320787

  5. Development of sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in plasma.

    PubMed

    Zehnacker, Laura; Nevers, Marie-Claire; Sinou, Véronique; Parzy, Dominique; Créminon, Christophe; Parzy, Daniel; Azoulay, Stéphane

    2015-10-01

    Despite significant progress in prevention and therapy, malaria is still one of the world's leading major diseases due to its high morbidity and mortality. Recommended treatments by the World Health Organization include the use of artemisinin and artemisinin derivative-based combination therapies. To allow efficient patient monitoring during antimalarial therapy without the use of expensive apparatus, we developed a sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in biological fluids. To produce specific antibodies against dihydroartemisinin (DHA), a synthetic DHA derivative was coupled to bovine serum albumin as the immunogen. In parallel, a new, rapid, and efficient procedure to covalently link glycoprotein to all amine-containing molecules has been established and the enzyme tracer was prepared by chemically coupling the DHA derivative in combination with SBP rather than the more commonly used HRP. It allowed us to develop, after optimization of the luminescent reagent, a sensitive and stable luminescent EIA, with a LLOQ of 90 pg mL(-1). This assay compares favorably with the most efficient HPLC methods previously reported with a LLOQ close to 1 ng mL(-1) and shows good precision and efficiency since recovery from human plasma spiked with DHA ranged between 91 and 103%, with coefficients of variation of <13%. To date, no immunoassay for DHA has been applied to plasma analysis and this EIA should be very useful in all clinical laboratories for rapid and cost-effective analysis. PMID:26280205

  6. Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay.

    PubMed

    Hashida, S; Ishikawa, E

    1990-12-01

    A novel and ultrasensitive enzyme immunoassay (immune complex transfer two-site enzyme immunoassay) for ferritin is described. Ferritin was reacted simultaneously with affinity-purified dinitrophenyl biotinyl anti-ferritin IgG and affinity-purified anti-ferritin Fab'-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto affinity-purified (anti-dinitrophenyl group) IgG-coated polystyrene balls. After eliminating excess conjugate by washing, the complex was eluted from the polystyrene balls with an excess of epsilon N-dinitrophenyl-L-lysine and transferred to streptavidin-coated polystyrene balls. The beta-D-galactosidase activity bound to streptavidin-coated polystyrene balls was assayed by fluorometry. Nonspecifically bound beta-D-galactosidase activity was remarkably lowered but there was much less decrease in specifically bound beta-D-galactosidase activity. As a result, the detection limit of ferritin was lowered to 1 milliattomole (1 x 10(-21) mol, 600 molecules as calculated from Avogadro's number). This technique will be useful for measuring, for example, antigens in single cells. PMID:2128491

  7. Plasma exchange to remove HIT antibodies: dissociation between enzyme-immunoassay and platelet activation test reactivities.

    PubMed

    Warkentin, Theodore E; Sheppard, Jo-Ann I; Chu, F Victor; Kapoor, Anil; Crowther, Mark A; Gangji, Azim

    2015-01-01

    Repeated therapeutic plasma exchange (TPE) has been advocated to remove heparin-induced thrombocytopenia (HIT) IgG antibodies before cardiac/vascular surgery in patients who have serologically-confirmed acute or subacute HIT; for this situation, a negative platelet activation assay (eg, platelet serotonin-release assay [SRA]) has been recommended as the target serological end point to permit safe surgery. We compared reactivities in the SRA and an anti-PF4/heparin IgG-specific enzyme immunoassay (EIA), testing serial serum samples in a patient with recent (subacute) HIT who underwent serial TPE precardiac surgery, as well as for 15 other serially-diluted HIT sera. We observed that post-TPE/diluted HIT sera-when first testing SRA-negative-continue to test strongly positive by EIA-IgG. This dissociation between the platelet activation assay and a PF4-dependent immunoassay for HIT antibodies indicates that patients with subacute HIT undergoing repeated TPE before heparin reexposure should be tested by serial platelet activation assays even when their EIAs remain strongly positive. PMID:25406354

  8. Improvement of an enzyme immunoassay for the determination of mercury (II)

    SciTech Connect

    Marx, A.; Kroetz, E.; Hock, B.

    1998-07-01

    Three systems were tested for the optimization of a heterogeneous noncompetitive enzyme immunoassay (EIA) for the determination of Hg (II). The sensitivity of the nonoptimized Hg-EIA with a detection limit of 2.1 {micro}g/L Hg (II) was improved by an avidin-biotin-complex (ABC) amplification system to a 2-fold lower detection limit (1.1 {micro}g/L Hg (II)). A conventional competitive EIA with the competition reaction between bound and free Hg (II) for antibody (ab) binding sites yielded a detection limit of 1.0 {micro}g/L Hg (II). Further improvement of sensitivity could be achieved by a competitive displacement EIA. In this case ab molecules bound to immobilized haptens are displaced in the next step by free Hg (II). The detection limit of the displacement approach is 0.4 {micro}g/L Hg (II).

  9. Noninvasive analysis of fecal reproductive hormone metabolites in female veiled chameleons (Chamaeleo calyptratus) by enzyme immunoassay.

    PubMed

    Kummrow, Maya S; Gilman, Christine; Mackie, Paula; Smith, Dale A; Mastromonaco, Gabriela F

    2011-01-01

    The noninvasive technique of gonadal steroid metabolite measurement in feces for evaluation of reproductive activity has proven an effective and important tool for population management in various captive species, but has not yet been validated and used in reptile species. In this study, enzyme immunoassays (EIAs) were validated for the analysis of fecal samples from female veiled chameleons (Chamaeleo calyptratus) for estrogen (E2), testosterone (T), and progesterone (P) and their metabolites. High performance liquid chromatography and physiological methods (GnRH stimulation) were used for the validation of the assays. Biological events, such as skin color changes indicative of ovarian activity and oviposition, correlated with the cyclical pattern of E2, T and P metabolites in feces over a period of two reproductive cycles. This is the first study to report frequent longitudinal measurements of fecal hormone levels by EIA in a reptile species. PMID:21319212

  10. Standardization of an enzyme immunoassay for human antibody to Haemophilus ducreyi.

    PubMed Central

    Desjardins, M; Thompson, C E; Filion, L G; Ndinya-Achola, J O; Plummer, F A; Ronald, A R; Piot, P; Cameron, D W

    1992-01-01

    We standardized a serologic enzyme immunoassay (EIA) for human immunoglobulin G and M antibodies against Haemophilus ducreyi. We evaluated the performance of this test with respect to the time from acute chancroid and coinfection with human immunodeficiency virus (HIV). Antibody to a crude, soluble bacterial antigen of one H. ducreyi strain was detected in a panel of serum samples from clinically and microbiologically confirmed cases of chancroid and from controls. Test interpretation was standardized for optimal sensitivity and specificity. Performance of the EIA was enhanced in the period of early convalescence from acute primary chancroid and was not diminished in the presence of HIV coinfection. The EIA performed adequately as a serologic screening test for field evaluation and epidemiologic application in conjunction with sexually transmitted disease and HIV detection and control efforts. PMID:1500508

  11. 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.

  12. 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. PMID:26431499

  13. 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.

  14. Detection of IgM antibodies against canine distemper virus in dog and mink sera employing enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Blixenkrone-Møller, M; Pedersen, I R; Appel, M J; Griot, C

    1991-01-01

    An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against canine distemper virus (CDV) in canine and mink serum is described. The diagnostic potential of this technique was evaluated by analyzing sera from natural or experimental infections in dog and mink and negative control sera. These results were compared with results obtained in the developed CDV IgG ELISA and in the virus neutralization test. The IgM test, which requires only a single serum specimen, is a useful method for diagnosing current or recent CDV infections in dog and mink. PMID:2039785

  15. Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin

    SciTech Connect

    Delaney, C.J.; Opheim, K.E.; Smith, A.L.; Plorde, J.J.

    1982-01-01

    We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay.

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

    PubMed Central

    Stenbaek, E I; De LaSalle, F; Gottschalk, M

    1997-01-01

    An inhibition enzyme immunoassay (EIA) for detection of antibodies against A. pleuropneumoniae serotype 5 (App-5) in pig sera, based on the inhibition of the binding of an App-5 specific monoclonal antibody was established. The monoclonal antibody (MAb 210-F11) was found to be directed against an epitope on the O-chain of App-5 LPS. In the inhibition EIA, highly purified App-5 LPS was used to coat microtitre plates. Serial dilutions of pig sera were added to the plates prior to the addition of the MAb 210-F11. The degree of binding of App-5 antibodies from pig sera was determined as the percentage inhibition of the MAb 210-F11. Pig serum from specific pathogen free (SPF) herds, from experimentally infected animals, and from acutely and chronically infected herds were tested. A serum dilution of 1/30 was found to be optimal, when using 50% inhibition as the discriminating inhibition percentage. No cross-reactivity was observed with serum from pigs infected with other App serotypes or bacteria isolated from the respiratory tract, such as A. suis and H. parasuis. The inhibition EIA will be used for surveillance of App-5 antibodies in SPF and conventional herds. Images Figure 1. PMID:9008793

  17. 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

  18. Evaluation of four commercially available enzyme immunoassays for laboratory diagnosis of Clostridium difficile-associated diseases.

    PubMed Central

    Whittier, S; Shapiro, D S; Kelly, W F; Walden, T P; Wait, K J; McMillon, L T; Gilligan, P H

    1993-01-01

    Four commercial enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxin A have recently been developed and marketed (Premier, Meridian Diagnostics, Cincinnati, Ohio; VIDAS, bioMerierux Vitek, Inc., Hazelwood, Mo.; Tox-A-Test, TechLab, Blacksburg, Va.; and Bartels, Baxter Diagnostics, McGaw Park, Ill.). The performances of these EIAs were compared with those of the tissue culture cytotoxicity assay and a definition of C. difficile-associated disease based on both laboratory and clinical criteria for 329 clinical specimens. Two EIAs (Premier and VIDAS) showed good overall agreement (96 and 95%, respectively) with the cytotoxicity assay. However, they were less sensitive (84 and 71%, respectively) than the Bartels (94%) or Tox-A-Test (93%) EIAs. The Bartels and Tox-A-Test assays were much less specific, resulting in poor positive predictive values (56%) of the two assays when compared with that of the cytotoxicity assay. Tox-A-Test had the added drawback of having a significant number of indeterminate results (6.4%). These data indicate that the four EIAs all have specific shortcomings. When using these EIAs, testing strategies that take these shortcomings into consideration should be developed. PMID:8263168

  19. 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. PMID:26218576

  20. Fieldable, real-time enzyme immunoassay kits for drugs on surfaces

    NASA Astrophysics Data System (ADS)

    Chiappini, Michele W.; Wendel, Gregory J.; Duquette, Peter H.; Hamilton, Martha J.; Chudzik, Stephen J.; Chappa, Ralph A.

    1994-03-01

    Immunoassays (e.g., RIA, EIA) have been demonstrated to be useful for rapid, convenient detection and semiquantitative analysis of drugs. Thermedics Detection, Inc. manufactures a rapid, sensitive, self-contained, disposable, EIA device, developed by Bio-Metric Systems, Inc., designed to allow untrained personnel to perform in field situations. This format has been developed for drugs in biological fluids and on surfaces. The analyte in the test sample competes with an enzyme-analyte conjugate for a limited number of immobilized antibody sites. The AccuPRESS Test format can detect analytes at 10 ppb in biological fluids, water, and soil, and on surfaces, such as suitcases, vehicles, tables and hands, with positive results indicated by clearly visible color development within 5 minutes. This format is designed to have all dry components and to have an ambient shelf life of greater than one year. The format is available for cocaine and opiate derivatives, including heroin, and is readily adaptable for use with numerous other drugs, explosives, and environmental pollutants.

  1. Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay using membrane antigens of Leishmania (Viannia) braziliensis.

    PubMed

    Skraba, Cissiara Manetti; Pedroso, Raíssa Bocchi; Fiorini, Adriana; Rosado, Fábio Rogério; Aristides, Sandra Mara Alessi; Lonardoni, Maria Valdrinez Campana; Teixeira, Jorge Juarez Vieira; Silveira, Thaís Gomes Verzignassi

    2014-04-01

    This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48-56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48-56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients. PMID:24485589

  2. Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by a commercial enzyme immunoassay.

    PubMed Central

    Swenson, P D; Kaplan, M H

    1986-01-01

    A commercial enzyme immunoassay (EIA) for the rapid detection of respiratory syncytial virus (RSV) in respiratory secretions was evaluated by comparison with both virus isolation in HEp-2 cells and indirect immunofluorescence (IFA) staining of exfoliated respiratory cells. Initial examination of 80 nasopharyngeal aspirates collected from infants with acute respiratory illness showed that the RSV EIA was positive for 21 of 24 specimens positive by virus isolation or IFA (87.5% sensitivity) and negative for 53 of 56 specimens negative by virus isolation and IFA (95% specificity). The EIA appears to be an acceptable and more rapid test than virus isolation for the detection of RSV, especially for laboratories in which prompt inoculation of specimens is not always possible. IFA staining with commercial bovine anti-RSV serum was found to be the most sensitive and rapid test for the detection of RSV. However, three of four specimens positive by IFA and negative by virus isolation were not cultured under optimal conditions. In addition, the IFA test requires a highly trained technologist to interpret the staining results. PMID:3514658

  3. 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. PMID:25437365

  4. 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. PMID:22204046

  5. Validation of an automated enzyme immunoassay for the measurement of serum total thyroxine in cats

    E-print Network

    Williams, Tim L.; Archer, Joy

    2015-05-18

    immunoassay (EIA) for use with feline serum and derivation of a TT4 reference interval (RI) for cats aged 9 years and older). Methods: Assay precision, reproducibility, and linearity were evaluated. Interference by hemolysis was also assessed. Method...

  6. Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae.

    PubMed Central

    Robison, B J; Pretzman, C I; Mattingly, J A

    1983-01-01

    An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed. PMID:6349525

  7. Environmental exposure to cockroach allergens: analysis with monoclonal antibody-based enzyme immunoassays.

    PubMed

    Pollart, S M; Smith, T F; Morris, E C; Gelber, L E; Platts-Mills, T A; Chapman, M D

    1991-02-01

    Quantitative two-site monoclonal antibody (MAb)-based enzyme-linked immunoassays for two cockroach (CR) allergens, Bla g I and Bla g II, have been developed and used to measure allergen levels in house-dust samples. Dust collected from the CR-infested homes of two patients with asthma from Charlottesville, Va., demonstrated wide variation in the levels of Bla g I, depending on the location of dust collection. Dust from kitchen floors and cabinets contained 50-fold more allergen (mean, 10,755 U/gm of dust) than dust from bedrooms and upholstered furniture (mean, 204 U/gm). One hundred forty-five dust samples were collected from the bedrooms and living rooms of 22 children with asthma and 16 control subjects without asthma living in Atlanta, Ga. Twenty-seven of the 38 homes (17/22 children with asthma; 10/16 control subjects) had detectable Bla g I (4 to 1340 U/gm of dust). Bla g II levels were assayed in 40 kitchen, bedroom, and living room samples from homes in Wilmington, Del. Highest levels of Bla g II were detected in kitchen-floor dust (300 U/gm of dust). Additionally, approximately 20% of homes with no visual evidence of CR infestation had significant levels of Bla g II in at least one dust sample (greater than 4 U/gm of dust). Our results demonstrate that CR may be an occult allergen in homes. The kitchen appears to be the primary site of CR-allergen accumulation, but significant CR-allergen levels can also be found at other sites in the home. The MAb-based assays can be used for quantitation of environmental exposure to CR allergens.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1993810

  8. 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. PMID:23811482

  9. Development and evaluation of an enzyme immunoassay for rapid diagnosis of rabies in humans and animals.

    PubMed

    Vasanth, Joel P; Madhusudana, S N; Abhilash, K V; Suja, M S; Muhamuda, K

    2004-10-01

    The presently advocated tests for rapid diagnosis of rabies such as fluorescent antibody test (FAT) is expensive and requires expertise to carry out and interpret the results. In this study we have developed and evaluated a simple enzyme immuno-assay (EIA) to detect rabies antigen in the brain specimens of animals and humans. We have also evaluated the utility of this test in ante mortem diagnosis of human rabies. The brain homogenates of suspected rabid animals (n=250), humans (n=16) and clinical samples like saliva (n=16) and cerebrospinal fluid (CSF, n=16) applied on to ELISA plates coated with rabies antinucleoprotein antibody and the absorbed rabies nucleoprotein antigen was detected using biotinylated anti-nucleoprotein antibody followed by treatment with streptavidin peroxidase conjugate and colour development with OPD. Rabies infected and normal mouse brain homogenates were used as positive and negative controls respectively. The results of this test was evaluated with fluorescent antibody technique (for brain samples) and mice inoculation test (for saliva and CSF samples). A distinct dark brown color was seen in positive control and all positive samples and there was no color development in negative control and samples. The concordance between FAT and EIA was 98.4%. With brain samples, 83.3% with saliva and 91.6% with CSF samples. The specificity of the test was found to be 100%. It can be concluded that the EIA described here is a sensitive, specific and rapid test for post mortem diagnosis of rabies in animals and humans. The utility of this test for ante mortem diagnosis of rabies needs to be further evaluated. PMID:16295401

  10. Glutathione S-transferases as biomarkers of organ damage: applications of rodent and canine GST enzyme immunoassays.

    PubMed

    Kilty, C; Doyle, S; Hassett, B; Manning, F

    1998-04-24

    The cytosolic glutathione S-transferase (GST) enzymes serve as ideal biomarkers of organ damage as they exhibit many of the required characteristics, i.e. specific localisation, high cytosolic concentration and relatively short half-life. The role of GSTs as early indicators of organ damage is applicable to both human and animal models. Because of the regio-specific localisation of the different isoforms of GST in liver and kidney, simultaneous monitoring of classes of GSTs in biological matrices permits the identification of specific areas of damage within a particular organ. Immunoassays have been developed which quantify canine alpha GST and roden microGST (Yb1). The immunoassays are solid phase EIAs, where GST in the sample or standard is captured by a specific anti-GST antibody coated onto the solid phase. After washing, a specific enzyme-labelled IgG conjugate is added which binds to the captured GST. After a further washing step, substrate is added and a colour developed. The absorbance is measured on an ELISA plate reader and is directly proportional to the amount of GST present in the sample. The assays are performed at room temperature and can be completed within 3 h. The immunoassays are specific for each GST and have a range of 0-100 micrograms/l. A range of assay parameters were investigated to validate the EIAs for GST detection. The assays are sensitive and reproducible. CV for inter- and intra-assay variation were below 9% for Yb1 assay and below 20% for the canine alpha GST EIA. Recovery of spiked GST over the standard curve range was 102 and 99%, respectively. No prozone effect was observed and samples exhibited linearity of dilution in both assays. Validation has shown that using these enzyme immunoassay, Yb1 and canine alpha GST can be measured accurately and precisely in biological matrices, tissue homogenates and cell lines and that changes in GST levels can be detected. The use of these assays have important applications in both in vitro and in vivo toxicity studies, where GST's serve as sensitive marker of hepatocellular and renal cell integrity. PMID:9679549

  11. 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

  12. The measurement of triclosan in water using a magnetic particle enzyme immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sensitive magnetic particle-based immunoassay to determine triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) in drinking water and wastewater was developed. Rabbit antiserum was produced by immunizing the rabbit with 6-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)hexanoic acid-keyhole limpet hemocya...

  13. 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.

  14. 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

  15. Evaluation of the Abbott TESTPACK RSV enzyme immunoassay for detection of respiratory syncytial virus in nasopharyngeal swab specimens.

    PubMed Central

    Swierkosz, E M; Flanders, R; Melvin, L; Miller, J D; Kline, M W

    1989-01-01

    The Abbott TESTPACK RSV assay (Abbott Laboratories, North Chicago, Ill.), a rapid (20-min) enzyme immunoassay, was compared with culture and direct immunofluorescence (DFA) of nasopharyngeal cells for the detection of respiratory syncytial virus (RSV) in nasopharyngeal swab specimens. Nasopharyngeal swab specimens, collected from 234 infants, were placed in viral transport medium. Portions of specimen in transport medium were used for each test. Of 234 specimens, 70 (30%) were culture positive, 103 (44%) were DFA positive, 107 (46%) were culture or DFA positive, and 112 (48%) were TESTPACK RSV positive. Of 19 specimens positive by TESTPACK RSV but negative by culture or DFA, 15 were positive by the blocking assay. A total of 122 specimens were culture, DFA, or blocking assay positive; TESTPACK RSV detected 108 specimens (sensitivity, 89%). The specificity, positive predictive value, and negative predictive value of TESTPACK RSV as compared with those of culture, DFA, and the blocking assay were 96, 96, and 89%, respectively. By comparison, the sensitivity, specificity, positive predictive value, and negative predictive value of combined culture and DFA were 88, 100, 100, and 88%, respectively. TESTPACK RSV is a rapid and reliable enzyme immunoassay for the direct detection of RSV antigen in nasopharyngeal swab specimens. Images PMID:2666434

  16. Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay.

    PubMed

    Lai, Guosong; Cheng, Hui; Xin, Dinghong; Zhang, Haili; Yu, Aimin

    2016-01-01

    A nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene (CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase (HRP) on the graphene oxide (GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles (Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis. PMID:26703270

  17. TRICLOSAN AND METHYL-TRICLOSAN MONITORING STUDY IN THE NORTHEAST OF SPAIN USING A MAGNETIC PARTICLE ENZYME IMMUNOASSAY AND CONFIRMATORY ANALYSIS BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. 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.

  19. 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. PMID:25793322

  20. 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. PMID:26067328

  1. Comparison of Antibody Titers Determined by Hemagglutination Inhibition and Enzyme Immunoassay for JC Virus and BK Virus

    PubMed Central

    Hamilton, R. S.; Gravell, M.; Major, E. O.

    2000-01-01

    A comparison of antibody titers to JC virus (JCV) or BK virus (BKV) was made by hemagglutination inhibition (HI) and enzyme immunoassay (EIA) with 114 human plasma samples. Antibody titers to JCV or BKV determined by HI were lower than those determined by EIA. Nevertheless, as HI titers increased so did EIA titers. When antibody data were compared by the Spearman rank correlation test, highly significant correlations were found between HI and EIA titers. Results obtained by plotting EIA antibody titers for JCV against those for BKV generally showed a reciprocal relationship, i.e., samples with high antibody titers to JCV had lower antibody titers to BKV and vice versa. Some samples, however, had antibody titers to both viruses. Of the samples tested, 25.4% (25 of 114) had HI and EIA antibody titers to JCV and BKV which were identical or closely related. This is not the scenario one would expect for cross-reactive epitopes shared by the two viruses, but one suggesting that these samples were from individuals who had experienced infections by both viruses. Adsorption with concentrated JCV or BKV antigen of sera with high antibody titers to both JCV and BKV and testing by JCV and BKV EIA gave results which support this conclusion. Although 52.6% (51 of 97) of the samples from the Japanese population tested had very high antibody titers (?40,960) to either JCV or BKV, none of the samples were found by a dot blot immunoassay to have antibodies which cross-reacted with simian virus 40. The results from this study, in agreement with those of others, suggest that humans infected by JCV or BKV produce antibodies to species-specific epitopes on their VP1 capsid protein, which is associated with hemagglutination and cellular binding. PMID:10618072

  2. 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 ?-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 ?-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. PMID:26255292

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. Diagnosis of herpes simplex virus infection in a clinical setting by a direct antigen detection enzyme immunoassay kit.

    PubMed Central

    Dascal, A; Chan-Thim, J; Morahan, M; Portnoy, J; Mendelson, J

    1989-01-01

    A commercial 4-h direct herpes simplex virus (HSV) antigen detection enzyme immunoassay (EIA) kit (Du Pont Herpchek) was evaluated by using 273 clinical specimens obtained in a hospital-based infectious disease practice. The EIA was compared with a standard culture method in which WI38 cells were inoculated within 20 min of sample collection. Cultures were observed for 2 weeks, and positive findings were confirmed by fluorescein-labeled monoclonal antibody (FA) staining. The values for the overall HSV detection rate were 40.7% by the standard culture method and 41.4% by EIA. In eight cases, the EIA was positive, while the culture method was negative; however, clinical data and confirmatory blocking EIA suggested that a true HSV infection was present. For six FA-confirmed, culture-positive samples, the direct EIA was negative; however, an EIA performed on the supernatants of these cultures was positive, suggesting that the failure of the EIA to detect these samples was not due to lack of strain specificity of the test. After confirmatory tests of standard culture and EIA discrepant results, the overall sensitivity of the test was 95.0% (113 of 119) and the specificity was 100% (154 of 154). PMID:2542362

  5. Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences.

    PubMed

    Felleisen, R S; Lambelet, N; Bachmann, P; Nicolet, J; Müller, N; Gottstein, B

    1998-02-01

    Tritrichomonas foetus is the causative agent of bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. Diagnosis is hampered by putative contamination of samples with intestinal or coprophilic trichomonadid protozoa which might be mistaken for T. foetus. Therefore, we developed a PCR test optimized for applicability in routine diagnosis. Amplification is based upon primers TFR3 and TFR4 directed to the rRNA gene units of T. foetus. In order to avoid potential carryover contamination by products of previous amplification reactions, conditions were adapted to the use of the uracil DNA glycosylase system. Furthermore, documentation and interpretation of results were facilitated by including a DNA enzyme immunoassay for the detection of amplification products. Specificity was confirmed with genomic material from different related trichomonadid protozoa. The high sensitivity of the test allowed the detection of a single T. foetus organism in diagnostic culture medium or about 50 parasites per ml of preputial washing fluid. The present methods are thus proposed as (i) confirmatory tests for microscopic diagnosis following diagnostic in vitro cultivation and (ii) a direct T. foetus screening test with diagnostic samples. PMID:9466768

  6. Performance of Clostridium difficile Toxin Enzyme Immunoassay and Nucleic Acid Amplification Tests Stratified by Patient Disease Severity

    PubMed Central

    Uslan, Daniel Z.; Rubin, Zachary

    2013-01-01

    Many clinical laboratories in the United States are transitioning from toxin enzyme immunoassays (EIA) to nucleic acid amplification tests (NAATs) as the primary diagnostic test for Clostridium difficile infection (CDI). While it is known that the analytical sensitivity of the toxin EIA is poor, there are limited clinical data on the performance of these assays for patients with mild or severe CDI. Two hundred ninety-six hospital inpatients with diarrhea and clinical suspicion for CDI were tested prospectively by toxin EIA, by C. difficile NAAT, and with a reference standard toxigenic culture. Following completion of laboratory testing, retrospective chart reviews were performed to stratify patients into mild and severe disease groups based on clinical criteria using a standard point-based system. One hundred forty-three patients with CDI confirmed by toxigenic culture were evaluated in this study. Among the patients with mild CDI, 49% tested positive by toxin EIA and 98% tested positive by NAAT. Among patients with severe CDI, 58% tested positive by toxin EIA and 98% tested positive by NAAT. Increased CDI disease severity was not associated with an increased sensitivity of EIA (P = 0.31). These data demonstrate that toxin EIA performs poorly both for patients with severe CDI and for those with mild CDI and support the routine use of NAAT for the diagnosis of CDI. The presence of stool toxin measured by EIA does not correlate with disease severity. PMID:23269736

  7. Enzyme immunoassay for the detection of porcine gelatine in edible bird's nests.

    PubMed

    Tukiran, Nur Azira; Ismail, Amin; Mustafa, Shuhaimi; Hamid, Muhajir

    2015-01-01

    Porcine gelatine is a common adulterant found in edible bird's nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g(-1), respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg(-1) (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry. PMID:25861981

  8. 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. PMID:26724762

  9. Comparison of two enzyme immunoassays for the detection of Haemonchus contortus infections in sheep.

    PubMed

    Schallig, H D; Hornok, S; Cornelissen, J B

    1995-04-01

    Two enzyme-linked immunosorbent assays (ELISA) using either excretory/secretory (ES) products or crude somatic antigens (CSA) of adult Haemonchus contortus were compared for their ability to detect antibodies against H. contortus in sheep. Serum samples obtained from a group of 32 H. contortus mono-infected sheep were tested in the two ELISAs and the obtained data were compared with the results of the faecal examinations of these sheep. The first sheep became patent 3 weeks post infection (p.i.) and all sheep had positive egg counts at week 5 p.i. The first antibodies against H. contortus were detected 1 week p.i. and all sheep were found positive by both ELISAs at week 4 p.i. Using sera from a large number of H. contortus-infected sheep a difference in sensitivity between the ES ELISA (97.7%) and the CSA ELISA (89.2%) was found. The specificity of each assay was determined by testing sera obtained from sheep with mono-infections of H. contortus, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Ostertagia circumcincta, Nematodirus battus, Cooperia curticei, Fasciola hepatica, Taenia ovis or Eimeria spp. The specificity of the ES ELISA was 87.2%, whereas the specificity of the CSA ELISA was 82.7%. PMID:7660570

  10. 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.

  11. 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

  12. 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. PMID:24452175

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

    PubMed Central

    Van Loock, Marnix; Verminnen, Kristel; Messmer, Trudy O; Volckaert, Guido; Goddeeris, Bruno M; Vanrompay, Daisy

    2005-01-01

    Background Laboratory diagnosis of Chlamydophila psittaci, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay (PCR-EIA) was developed to detect the Cp. psittaci outer membrane protein A (ompA) gene in pharyngeal swabs. Methods The fluorescein-biotin labelled PCR products were immobilized on streptavidin-coated microtiter plates and detected with anti-fluorescein peroxidase conjugate and a colorimetric substrate. An internal inhibition control was included to rule out the presence of inhibitors of DNA amplification. The diagnostic value of the ompA nested PCR-EIA in comparison to cell culture and a 16S-rRNA based nested PCR was assessed in pharyngeal turkey swabs from 10 different farms experiencing respiratory disease. Results The sensitivity of the nested PCR-EIA was established at 0.1 infection forming units (IFU). Specificity was 100%. The ompA nested PCR-EIA was more sensitive than the 16S-rRNA based nested PCR and isolation, revealing 105 out of 200 (52.5%) positives against 13 and 74 for the latter two tests, respectively. Twenty-nine (23.8%) out of 122 ompA PCR-EIA negatives showed the presence of inhibitors of DNA amplification, although 27 of them became positive after diluting (1/10) the specimens in PCR buffer or after phenol-chloroform extraction and subsequent ethanol precipitation. Conclusion The present study stresses the need for an internal control to confirm PCR true-negatives and demonstrates the high prevalence of chlamydiosis in Belgian turkeys and its potential zoonotic risk. The ompA nested PCR-EIA described here is a rapid, highly sensitive and specific diagnostic assay and will help to facilitate the diagnosis of Cp. psittaci infections in both poultry and man. PMID:16185353

  14. Low sensitivity of fecal toxin A/B enzyme immunoassay for diagnosis of Clostridium difficile infection in immunocompromised patients.

    PubMed

    Erb, S; Frei, R; Strandén, A M; Dangel, M; Tschudin-Sutter, S; Widmer, A F

    2015-11-01

    The optimal approach in laboratory diagnosis of Clostridium difficile infection (CDI) is still not well defined. Toxigenic culture (TC) or alternatively fecal toxin assay by cell cytotoxicity neutralization assay are considered to be the reference standard, but these methods are time-consuming and labor intensive. In many medical centers, diagnosis of CDI is therefore still based on fecal toxin A/B enzyme immunoassay (EIA) directly from stool alone, balancing cost and speed against limited diagnostic sensitivity. The aim of the study was to assess in which patient population the additional workload of TC is justified. All consecutive stool specimens submitted for diagnosis of suspected CDI between 2004 and 2011 at a tertiary-care center were examined by toxin EIA and TC. Clinical data of patients with established diagnosis of CDI were collected in a standardized case-report form. From 12?481 stool specimens submitted to the microbiologic laboratory, 480 (3.8%) fulfilled CDI criteria; 274 (57.1%) were diagnosed by toxin EIA; and an additional 206 (42.9%) were diagnosed by TC when toxin EIA was negative. Independent predictors for negative toxin EIA but positive TC were high-dose corticosteroids (odds ratio (OR) 2.97, 95% confidence interval (CI) 1.50-5.90, p 0.002), leukocytopenia <1000/?L (OR 2.52, 95% CI 1.22-5.23, p 0.013) and nonsevere CDI (OR 2.21, 95% CI 1.39-3.50, p 0.001). There was no difference in outcomes such as in-hospital mortality and recurrence between both groups. In conclusion, negative toxin EIA does not rule out CDI in immunocompromised patients in the setting of relevant clinical symptoms. Methods with improved sensitivity such as TC or PCR should be used, particularly in this patient population. PMID:26232535

  15. 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. PMID:23023352

  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. Validation of a New Recombinant Antibody Fragment (rFab)-Based Homogeneous Enzyme Immunoassay for the Highly Specific Detection of 6-Acetylmorphine in Urine.

    PubMed

    Wang, Guohong; Huynh, Kim; Barhate, Rekha; Liu, Jialin; Tam, Phillip; Rodrigues, Warren; Coulter, Cynthia; Moore, Christine; Soares, James

    2015-11-01

    The detection of 6-acetylmorphine (6-AM) in urine by immunoassay methods is challenging due to its short half-life and its similarity in structure to many commonly abused opiates that are often present at very high concentrations in urine. Current 6-AM homogeneous enzyme immunoassays use lyophilized reagents because of the instability of 6-AM in water or lack of the required specificity due to high cross-reactivity with morphine. A new 6-AM rFab-based homogeneous enzyme immunoassay (HEIA) has been developed with highly improved specificity. Using a cutoff concentration of 10 ng/mL, morphine or morphine glucuronides did not produce a positive signal up to 300,000 or 1,000,000 ng/mL, respectively. Assay imprecision (n = 80) was less than 1.5% using four replicates per day for 20 days over the range 0-20 ng/mL. Cross-reactivity with structurally related or non-related compounds was assessed at concentrations up to 1,000,000 ng/mL. Interferences from endogenous compounds at ±25% cutoff were also performed at the concentrations ranging from 100,000 to 500,000 ng/mL. The effect of varied pH values on assay performance at ±25% cutoff was investigated; no false-positive or false-negative results were observed between pH 4 and -11. Based on the analysis of 149 authentic urine samples, the accuracy of the 6-AM HEIA compared with LC-MS-MS was 100%. These results demonstrated that rFab can be suitable for traditional HEIA with desired detection sensitivity and stability. PMID:26311850

  18. DEVELOPMENT OF A MAGNETIC PARTICLE ENZYME IMMUNOASSAY AND ITS APPLICATION TO THE MEASUREMENT OF TRICLOSAN IN WATER AND WASTEWATER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sensitive magnetic particle-based immunoassay for triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) was developed. Rabbit antiserum was produced by immunizing the rabbit with 6-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)hexanoic acid-keyhole limpet hemocyanin. The triclosan ligand and horse radis...

  19. Evaluation of three different antigens in an indirect enzyme-linked immunoassay for the detection of antibodies against Brucella abortus SRB51 in vaccinated heifers.

    PubMed

    Robles, C A; Nielsen, K; Gall, D; Willems, P

    2009-01-15

    The live attenuated Brucella abortus SRB51 (SRB51) is a partial O-chain-deprived mutant. The relative lack of the polysaccharide prevents it from inducing antibodies detectable by most of the serological tests used for the diagnosis of bovine brucellosis. The performance of three antigens used in an indirect enzyme-linked immunoassay test for detecting SRB51 antibodies were evaluated. A homogeneous group of twenty-five 10-month-old Hereford heifers was used. The animals were bled on day 0 and then subcutaneously vaccinated with 2 ml of a commercially available SRB51 vaccine (Schering-Plough) containing 1x10(7) to 3.4x10(7) viable cells. Blood samples without anticoagulant for sera obtaining were then collected at days 30, 90, 210 and 360 post-vaccination. To detect the SRB51 antibodies, Brucella ovis hot saline extract, B. ovis RLPS (RLPS), and SRB51-RLPS were used. The buffered antigen plate agglutination test and an indirect enzyme-linked immunoassay (I-ELISA) using the smooth LPS (SLPS) antigen from B. abortus were used as control tests. All the sera samples were negative in the BPA test and in the standard I-ELISA using the SLPS. The SRB51-RLPS and the B. ovis RLPS antigens performed better than the B. ovis hot saline extract antigen. PMID:18980780

  20. Evaluation of a rapid membrane enzyme immunoassay for the simultaneous detection of glutamate dehydrogenase and toxin for the diagnosis of Clostridium difficile infection.

    PubMed

    Kim, Heejung; Kim, Wan Hee; Kim, Myungsook; Jeong, Seok Hoon; Lee, Kyungwon

    2014-05-01

    We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMérieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile. PMID:24790912

  1. 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. PMID:16722452

  2. Synthesis and characterization of mercapturic acid (N-acetyl-L-cysteine)-aflatoxin B1 adduct and its quantitation in rat urine by an enzyme immunoassay.

    PubMed

    Nayak, Sujatha; Tanuja, Penmatsa; Sashidhar, Rao Beedu

    2009-01-01

    A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-aflatoxin B1 (AFB1) adduct in rat urine is reported. A novel procedure was developed for in vitro synthesis of an immunogen, bovine serum albumen-glutathione-aflatoxin B1 (BSA-GSH-AFB1) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Sulphydryl group's analysis confirmed the conjugation of-SH groups to AFB1. Thin-layer chromatography and spectral analysis (absorption, fluorescence, and Fourier transform infrared) of the conjugates further confirmed the formation of the adducts. Polyclonal antibodies specific to mercapturic acid-AFB1 adduct were produced against BSA-GSH-AFB1. The assay was found to be linear in the range of 100 pg-100 ng of the analyte (y = a + bx). A 50% displacement of BSA-GSH-AFB1 antibodies was achieved at an inhibitory concentration (IC50) of 11.9 ng GSH-AFB1 (r2 = 0.98) and 1.22 ng N-acetyl-L-cysteine (NAC)-AFB1 (r2 = 0.98). Spiking 5 microg/mL of reference standard to the control rat urine showed a recovery of 98 +/- 2%. The immunoassay was validated in a rodent model exposed to a single oral dose of 1 mg/kg body mass of pure AFB1. The excretion of NAC-AFB1 adduct was quantitated at the end of 24 h. The concentration of the NAC-AFB1 adduct excreted in urine as determined by the immunoassay was found to be in the range of 3.22-5.97 microg/mg creatinine. The present method may find wide application as a biochemical tool in molecular epidemiological and intervention studies with respect to human exposure to dietary aflatoxins. PMID:19485208

  3. 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...

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

    PubMed

    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

  5. Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and its major metabolite 7-aminoflunitrazepam in hair.

    PubMed

    Negrusz, A; Moore, C; Deitermann, D; Lewis, D; Kaleciak, K; Kronstrand, R; Feeley, B; Niedbala, R S

    1999-10-01

    Flunitrazepam (Rohypnol) is a benzodiazepine used in the treatment of insomnia as a sedative hypnotic and as preanesthetic medication in European countries and Mexico. Although it has no medicinal purpose in the United States, the occurrence of its abuse is increasing. Sexual abuse of both men and women while under the influence of so-called "date-rape" drugs has been the focus of many investigations. Reported date-rape drugs include flunitrazepam (FN), clonazepam, diazepam, oxazepam, gamma-hydroxybutyrate, and many others. FN has been banned in the United States because of its alleged use in such situations. Unfortunately, the detection of FN or its metabolites 7-aminoflunitrazepam (7-AFN) and desmethylflunitrazepam in a single specimen such as urine or blood is difficult in criminal situations because of the likelihood of single-dose ingestion and the length of time since the alleged incident. Hair provides a solution to the second of these problems in that drugs tend to incorporate into hair and remain there for longer periods of time than either urine or blood. There are various techniques for the detection of FN in plasma, blood, and urine, but little work has been done with hair. Hair collection is a virtually noninvasive procedure that can supply information on drug use for several months preceding collection. The objective of this paper was to determine if a commercially available micro-plate enzyme immunoassay system was sufficiently sensitive for the routine screening of 7-AFN in hair by the development of extraction procedures and optimization of the immunoassay kit. Further, this study used the same solid-phase extraction to isolate FN and its major metabolite, 7-AFN, and gas chromatography-mass spectrometry with negative ion chemical ionization for confirmation. Two seven-point standard curves were established ranging from 0.5 pg/mg to 100 pg/mg for 7-AFN and 2.5 pg/mg to 200 pg/mg for FN with respective deuterated internal standards. A replicate analysis of controls was performed to establish inter- and intraday variabilities. Two suicide cases along with one alleged date-rape case and one case of an emergency room patient whose blood screened positive for benzodiazepines were analyzed. All the hair specimens screened positive for benzodiazepines using micro-plate enzyme immunoassay. Two cases, including the date-rape case, were negative for FN and 7-AFN, and two postmortem hair samples were confirmed positive for FN and its metabolite. PMID:10517547

  6. 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. PMID:17315457

  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. 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. PMID:23996355

  9. Development of a Reverse Transcription-PCR–DNA Enzyme Immunoassay for Detection of “Norwalk-Like” Viruses and Hepatitis A Virus in Stool and Shellfish

    PubMed Central

    Schwab, Kellogg J.; Neill, Frederick H.; Le Guyader, Françoise; Estes, Mary K.; Atmar, Robert L.

    2001-01-01

    Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly “Norwalk-like” viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR–oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Taq polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation. PMID:11157239

  10. Development of a reverse transcription-PCR-DNA enzyme immunoassay for detection of "Norwalk-like" viruses and hepatitis A virus in stool and shellfish.

    PubMed

    Schwab, K J; Neill, F H; Le Guyader, F; Estes, M K; Atmar, R L

    2001-02-01

    Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly "Norwalk-like" viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR-oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Taq polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation. PMID:11157239

  11. Plasma cortisol and 11-ketotestosterone enzyme immunoassay (EIA) kit validation for three fish species: the orange clownfish Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the blacktip reef shark Carcharhinus melanopterus.

    PubMed

    Mills, S C; Mourier, J; Galzin, R

    2010-08-01

    Commercially available enzyme immunoassay (EIA) kits were validated for measuring steroid hormone concentrations in blood plasma from three fish species: the orange clownfish Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the blacktip reef shark Carcharhinus melanopterus. A minimum of 5 microl plasma was required to estimate hormone concentrations with both kits. These EIA kits are a simple method requiring minimal equipment, for measuring hormone profiles under field conditions. PMID:20701653

  12. [Electro chemiluminescence immunoassay].

    PubMed

    Sano, M; Tatsumi, N

    1996-11-01

    Boehringer Mannheim is starting into a new age of immunoassay systems. A new immunoassay technology should allow the development of random access immunoassay analyzers with short incubation times. To satisfy the such matter, we have found a new technology ECL (Electro ChemiLuminescence). This technology has been licensed to Boehringer Mannheim by IGEN inc. (Maryland, USA). It's based on a microparticle solid phase coated streptavidin and a very promising complete new electro chemiluminescence technology. The ruthenium complex label used in the system will enables extremely stable reagents compared to enzyme conjugates. The test principle is the following : 1) pipette a sample, biotinylated antibodies, antibodies labeled ruthenium complex, and streptavidin coated microparticles into a reaction cup, 2) after incubation, the reaction mixture is aspirated into an electrochemical measuring cell, and unbound conjugate are washed away by tripropylamine (TPA) and a magnet, 3) by applying a potential to electrode, the ruthenium complex become to exciting stage, and the signal generation initiates. The analyzers using this technology have developed 2 type of fully automated instrument. The use of this new technology and these analyzers will make immunoassay analyses as convenient as clinical chemistry. PMID:8953939

  13. 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. PMID:25531426

  14. Enzyme immunoassay for rabies antibody in hybridoma culture fluids and its application to differentiation of street and laboratory strains of rabies virus.

    PubMed

    Smith, J S; Sumner, J W; Roumillat, L F

    1984-02-01

    A rapid and sensitive enzyme immunoassay is described for detecting rabies antibody in hybridoma culture fluids. Glass fiber filter disks were used to immobilize gamma-irradiated mouse neuroblastoma cells infected with street or laboratory strains of rabies virus. Bound rabies-specific antibody was detected by reaction with horseradish peroxidase-labeled goat anti-mouse immunoglobulin G. The assay was performed in a 96-well filtration device developed by Cleveland et al. (J. Clin. Microbiol. 15:402-407, 1982) for the typing of herpes simplex viruses. When partially disrupted cells were used, both internal and external viral antigens were available for reaction. The procedure is rapid (less than 4 h for completion) and requires only small amounts of fluid, and the gamma-irradiated antigen is noninfectious. When the procedure was used to screen 145 fluids from rabies-immune spleen-myeloma cell fusions, 132 were positive for rabies antibody. Other commonly used assays for the detection of rabies-specific antibody were less sensitive. Simultaneous analyses of many hybridoma fluids against a battery of street and laboratory strains of rabies virus are possible and allow rapid selection of useful monoclones. PMID:6365963

  15. Evaluation of three enzyme immunoassays and a loop-mediated isothermal amplification test for the laboratory diagnosis of Clostridium difficile infection.

    PubMed

    Bruins, M J; Verbeek, E; Wallinga, J A; Bruijnesteijn van Coppenraet, L E S; Kuijper, E J; Bloembergen, P

    2012-11-01

    The laboratory diagnosis of Clostridium difficile infection (CDI) consists of the detection of toxigenic Clostridium difficile, and/or its toxins A or B in stool preferably in a two-step algorithm. In a prospective study, we compared the performance of three toxin enzyme immunoassays (EIAs)-ImmunoCard Toxins A & B, Premier Toxins A & B and C. diff Quik Chek Complete, which combines a toxins test and a glutamate dehydrogenase (GDH) antigen EIA in one device -and the loop-mediated isothermal amplification assay Illumigene C. difficile. In total 986 stool samples were analyzed. Compared with toxigenic culture as the gold standard, sensitivities, specificities, PPV and NPV values of the toxin EIAs were 41.1-54.8 %, 98.9-100 %, 75.0-100 % and 95.5-96.5 % respectively, of the Illumigene assay 93.3 %, 99.7 %, 95.8 % and 99.5 %. Illumigene assays performed significantly better for non-014/020 PCR-ribotypes than for C. difficile isolates belonging to 014/020. Discrepant analysis of three culture-negative, but Illumigene-positive samples, revealed the presence of toxin genes using real-time PCRs. In addition to the GDH EIA (NPV of 99.8 %), the performance of Illumigene allows this test to be introduced as a first screening test for CDI- or as a confirmation test for GDH -positive samples, although the initial invalid Illumigene result of 4.4 % is a point of concern. PMID:22706512

  16. 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

  17. [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. PMID:25898736

  18. 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/or anti-Smith (Sm) antibodies. Reactivity against Sjögren's syndrome related antigens (SS)-A (including the Ro-60 and Ro-52 subcomponents), SS-B, histidyl tRNA synthetase (Jo-1), topoisomerase I antigen (Scl-70), polymyositis-scleroderma antigen (PM-Scl) and proliferating cell nuclear antigen (PCNA) was also noted in individual dogs. In conclusion, by using a commercial LIA and different ELISAs originally developed for detection of human ANA, we identified several specific ANA in serum samples from dogs sampled for IIF-ANA testing. Further, we found that the types of IIF-ANA pattern were associated with reactivity against some particular nuclear antigens. PMID:26547884

  19. 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. PMID:17263314

  20. A modified rapid enzyme immunoassay for the detection of rabies and rabies-related viruses: RREID-lyssa.

    PubMed

    Perrin, P; Gontier, C; Lecocq, E; Bourhy, H

    1992-03-01

    This paper presents a modification of the previously described Rapid Rabies Enzyme Immuno-Diagnosis test (RREID) by using biotinylated antibodies, streptavidin conjugate and a mixture of monospecific polyclonal antibodies against several lyssaviruses. In the modified technique (RREID-lyssa), microplates were sensitized with a mixture of purified antibodies against ribonucleoprotein (RNP) from Pasteur virus (Lyssavirus serotype 1), European Bat Lyssavirus (EBL, unclassified) and Mokola virus (Lyssavirus serotype 3). Bound RNP was detected by the same antibodies labelled with biotin and peroxidase-strepavidin conjugate. These techniques were used for the detection of RNP of different Lyssavirus serotypes (rabies and rabies-related viruses). For lyssavirus specimens of serotype 1, the threshold of detection of RREID and RREID-lyssa were similar. However, a smaller amount of labelled antibodies was needed when biotinylated antibodies were used. For specimens infected by rabies-related strains (serotypes 2, 3, 4 and EBL), the threshold of detection of the RREID-lyssa was between two and 512 times lower than with the RREID. The sensitivity and the specificity of the RREID-lyssa for rabies virus (serotype 1) when tested on a small field trial (53 specimens) were found to be identical to the RREID. Consequently, RREID-lyssa can be a useful tool for diagnostic laboratories that receive specimens infected by rabies-related viruses. PMID:1610558

  1. Comparison of commercial enzyme-linked immunosorbent assays and fluorescent microbead immunoassays for detection of antibodies against porcine reproductive and respiratory syndrome virus in boars.

    PubMed

    Gerber, Priscilla F; Giménez-Lirola, Luis G; Halbur, Patrick G; Zhou, Lei; Meng, Xiang-Jin; Opriessnig, Tanja

    2014-03-01

    The objective of this study was to compare the ability of two commercial enzyme-linked immunosorbent assays (ELISAs) and an in-house fluorescent microbead immunoassay (FMIA) to detect IgG antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) types 1 and 2 in serum and oral fluids from boars infected experimentally. Samples from uninfected control pigs and PRRSV-negative field samples were also used. Serum samples were tested by ELISAs (IDEXX Se, HIPRA Se) and an in-house FMIA-Se for detection of PRRSV types 1 and 2. Oral fluids were tested by ELISAs (IDEXX-SO, IDEXX-OF, HIPRA-OF) for detection of PRRSV types 1 and 2. Among the sera, IDEXX-Se and HIPRA-Se had similar sensitivity and specificity (p>0.05); however, IDEXX-Se detected positive animals earlier than HIPRA-Se (p<0.05). FMIA-Se had the highest false-positive rates in known negative field samples (1/205 for IDEXX-Se, 5/205 for HIPRA-Se, and 37/205 for FMIA-Se; p<0.01). Serum and oral fluid samples had similar detection rates and antibody kinetics using the IDEXX tests. There was a higher detection rate in serum than oral fluid using the HIPRA assays. In this study, the nucleocapsid protein utilized as antigen in the FMIAs yielded a low specificity. IDEXX-Se had the earliest detection and similar sensitivity and specificity to the HIPRA-Se. PMID:24361873

  2. 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

    Objective-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. Design-Prospective analytic study. Sample-Serum and urine from 70 dogs with and without blastomycosis. Procedures-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. Results-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. Conclusions and Clinical Relevance-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. PMID:26517616

  3. 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. PMID:23108105

  4. 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 11?-hydroxyetiocholanolone EIA as the most sensitive assay to reflect acute fGM elevations in the Eurasian lynx. HPLC immunograms demonstrated that the 11?-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 11?-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 11?-hydroxyetiocholanolone EIA is a reliable tool to assess fGM in both lynx species. PMID:25066418

  5. Survey of immunoassay techniques for biological analysis

    SciTech Connect

    Burtis, C.A.

    1986-10-01

    Immunoassay is a very specific, sensitive, and widely applicable analytical technique. Recent advances in genetic engineering have led to the development of monoclonal antibodies which further improves the specificity of immunoassays. Originally, radioisotopes were used to label the antigens and antibodies used in immunoassays. However, in the last decade, numerous types of immunoassays have been developed which utilize enzymes and fluorescent dyes as labels. Given the technical, safety, health, and disposal problems associated with using radioisotopes, immunoassays that utilize the enzyme and fluorescent labels are rapidly replacing those using radioisotope labels. These newer techniques are as sensitive, are easily automated, have stable reagents, and do not have a disposal problem. 6 refs., 1 fig., 2 tabs.

  6. The IGM Project

    NASA Astrophysics Data System (ADS)

    Martin, Christopher D.; Milliard, B.; Schiminovich, D.; Moore, A.; Chang, D.; Matuszewski, M.; Rahman, S.; Tuttle, S.; Deharveng, J.; Grange, R.; Frank, S.; Evrard, J.; FIREBALL Team; CWI Team; ISTOS Team; KCWI Team

    2010-01-01

    I discuss several experimental projects underway or proposed involving Caltech, NASA, Columbia, Laboratorie Astrophysique Marseille, CNES, and W. M. Keck Observatory, designed to discover and map emission from the IGM. The Cosmic Web Imager (CWI) is a ground-based high resolution spectrometer designed to detect low surface brightness emission from redshifted Lyman alpha, OVI and CIV at Palomar and Keck Observatories, over 2IGM emission in the 0.3IGM from 0.05IGM and Circum-Galactic Medium emission at levels predicted by three independent cosmological simulations.

  7. Rapid immunoenzymatic technique for titration of rabies antibodies IgG and IgM.

    PubMed

    Savy, V; Atanasiu, P

    1978-01-01

    This report is concerned with the application of the enzyme immunoassay to measure the antibodies in humans vaccinated against rabies or presenting symptoms of rabies without having been vaccinated. By the same technique we identify the IgG and IgM classes of antibodies. The antigen (5 microgram/ml), purified virus, is readily adsorbed into polystyrene tube by passive adsorption. The use of only one dilution for each serum assay (1/200) is particularly suitable for epidemiological studies. Antibody response of subjects in the course of rabies vaccination was an obvious application. After 5 inoculations of tissue culture vaccine the IgM response was poor and late; it was even negative in two cases. The IgG response appeared early on the 7th day. In the same way we tried to follow antibody response in three cases of rabies in man. Seroneutralisation (SN) antibody were not detected at the beginning of the illness. In case 1 antibodies were found on the 12th day, in case 2 on the 7th day and in case 3 on the 8th day. When we assayed the serum samples for immunoenzymatic test, we found that the sera became positive some days earlier: on the 5th for case 1, already on the 1st day for the two others. In each of these three cases the positivity of the test corresponded to the presence of IgM class globulins since IgG detection remained negative as did the SN test. Our results could have some clinical interest concerning future rabies treatment and early diagnosis. PMID:355018

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

    PubMed

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

    2013-11-01

    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

  9. 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

  10. Determination of plasma kisspeptin concentrations during reproductive cycle and different phases of pregnancy in crossbred cows using bovine specific enzyme immunoassay.

    PubMed

    Mondal, Mohan; Baruah, Kishore Kumar; Prakash, Bukkaraya Samudram

    2015-12-01

    Kisspeptin, a decapeptide and potent secretagogue of GnRH has been emerged recently as a master player in the regulation of reproduction in animals. Determination of kisspeptin in peripheral circulation is, therefore, very important for studying the control of its secretion and its role on reproduction in bovine species, the information on which is not available during any physiological state in this species, may probably be due to non-availability of simple assay procedure to measure the hormone. Therefore, the objective of this study was to develop and validate a simple and sufficiently sensitive enzyme immunoassay (EIA) for kisspeptin determination in bovine plasma using the biotin-streptavidin amplification system and second antibody coating technique. Biotin was coupled to kisspeptin and used to bridge between streptavidin-peroxidase and the immobilized kisspeptin antiserum in the competitive assay. The EIA was conducted directly in 100?l of unknown bovine plasma. Kisspeptin standards ranging from 0.01 to 25.6ng/100?l/well were prepared in hormone-free plasma. The lowest detection limit was 0.1ng/ml plasma. Plasma volumes for the EIA, viz., 50, 100 and 200?l 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 the endogenous bovine kisspeptin with kisspeptin standard used. It showed good parallelism with the kisspeptin standard curve. For the biological validation of the assay, plasma kisspeptin was measured in blood samples collected from six non-lactating cyclic cows during entire estrous cycle and from 18 pregnant cows during different stages of pregnancy. The mean plasma kisspeptin concentration during different days of the estrous cycle was different (P<0.001). Three peaks of kisspeptin were recorded, one on a day before appearance of preovulatory LH surge, second at day 6 and third one at day 18 of the estrous cycle. Plasma kisspeptin concentrations increased (P<0.001) from first through last trimester of pregnancy. Kisspeptin concentrations were also measured in different follicular, luteal and placental tissues. Follicular and placental kisspeptin levels increased (P<0.01) during follicular development and with the advancement of pregnancy, respectively. On the other hand, luteal concentrations of kisspeptin decreased (P<0.01) with its developmental process. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine plasma kisspeptin levels in bovine. A wide range of kisspeptin concentrations can be detected during different physiological stages in bovine using this kisspeptin-EIA procedure. PMID:26315389

  11. 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. PMID:26291091

  12. 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.

  13. 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 ...

  14. 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.; Petrovi?, 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 ?g/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).

  15. 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). PMID:22921803

  16. 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.

  17. Antibodies against cyclic citrullinated peptides of IgG, IgA and IgM isotype and rheumatoid factor of IgM and IgA isotype are increased in unaffected members of multicase rheumatoid arthritis families from northern Sweden

    PubMed Central

    Ärlestig, Lisbeth; Mullazehi, Mohammed; Kokkonen, Heidi; Rocklöv, Joacim; Rönnelid, Johan; Dahlqvist, Solbritt Rantapää

    2012-01-01

    Background Rheumatoid factors (RFs) and antibodies against cyclic citrullinated peptides (CCPs) of IgG, IgA and IgM isotype have been shown to precede disease onset by years. Objective To evaluate serological risk markers in first-degree relatives from multicase families in relation to genetic and environmental risk factors. Methods 51 multicase families consisting of 163 individuals with rheumatoid arthritis (RA) (mean±SD age, 60±14 years; disease duration 21 years; 71.8% female) and with 157 first-degree relatives unaffected by RA (54±17 years; 59.9% female) were recruited. Isotypes of antibodies against CCPs (IgG, IgA and IgM) and RFs (IgM and IgA) were determined using automated enzyme immunoassays. Cut-off levels were established using receiver operating characteristic curves based on values for 100 unrelated healthy controls. Results The concentrations and frequencies of all anti-CCP and RF isotypes were significantly increased in first-degree relatives and patients with RA compared with unrelated healthy controls. The relative distribution of IgA and IgM isotypes was higher than IgG in the relatives, whereas the IgG isotype dominated in patients with RA. The patients carried human leucocyte antigen-shared epitope (HLA-SE) significantly more often than the relatives (71.4% vs 53.9%, p=0.01), while the frequency of the PTPN22 T variant was similar. HLA-SE, combined with smoking, was significantly related to all combinations of anti-CCP and RF isotypes in patients with RA. No such relationships were found for the first-degree relatives. Conclusions All anti-CCP and RF isotypes analysed occurred more commonly in unaffected first-degree relatives from multicase families than in controls, but with different isotype distribution from patients with RA. PMID:22128080

  18. SITE EVALUATION OF FIELD PORTABLE PENTACHLOROPHENOL IMMUNOASSAYS

    EPA Science Inventory

    Four pentachlorophenol (PCP) enzyme immunoassays for environmental analysis have been evaluated through the U.S. EPA Superfund Innovative Technology Evaluation (SITE) program. Three assays were formatted for on-site field use and one assay could be used in a field laboratory sett...

  19. Evaluation of an enzyme immunoassay for the determination of cypermethrin in air samples with confirmation by gas chromatography/mass spectrometry 

    E-print Network

    Ball, Madalyn Eunice

    1993-01-01

    was initiated to investigate the applicability of a Competition Enzyme Linked Immunosorbent Assay to the analysis of trace levels of cypermethrin in air samples. The Occupational Safety and Health Administrations (OSHA) sampling procedure and a modified version...

  20. 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…

  1. Identification of a Novel Host-Specific IgM Protease in Streptococcus suis

    PubMed Central

    Seele, Jana; Singpiel, Alena; Spoerry, Christian; von Pawel-Rammingen, Ulrich; Valentin-Weigand, Peter

    2013-01-01

    Streptococcus suis serotype 2 is a highly invasive, extracellular pathogen in pigs with the capacity to cause severe infections in humans. This study was initiated by the finding that IgM degradation products are released after opsonization of S. suis. The objective of this work was to identify the bacterial factor responsible for IgM degradation. The results of this study showed that a member of the IdeS family, designated IdeSsuis (Immunoglobulin M-degrading enzyme of S. suis), is responsible and sufficient for IgM cleavage. Recombinant IdeSsuis was found to degrade only IgM but neither IgG nor IgA. Interestingly, Western blot analysis revealed that IdeSsuis is host specific, as it exclusively cleaves porcine IgM but not IgM from six other species, including a closely related member of the Suidae family. As demonstrated by flow cytometry and immunofluorescence microscopy, IdeSsuis modulates binding of IgM to the bacterial surface. IdeSsuis is the first prokaryotic IgM-specific protease described, indicating that this enzyme is involved in a so-far-unknown mechanism of host-pathogen interaction at an early stage of the host immune response. Furthermore, cleavage of porcine IgM by IdeSsuis is the first identified phenotype reflecting functional adaptation of S. suis to pigs as the main host. PMID:23243300

  2. 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. PMID:22734642

  3. 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...

  4. Field evaluation of four novel enzyme immunoassays for Chagas' disease in Venezuela blood banks: comparison of assays using fixed-epimastigotes, fixed-trypomastigotes or trypomastigote excreted-secreted antigens from two Trypanosoma cruzi strains.

    PubMed

    Berrizbeitia, M; Ndao, M; Bubis, J; Gottschalk, M; Aché, A; Lacouture, S; Medina, M; Ward, B J

    2006-12-01

    Many serological tests have been developed for the diagnosis of Chagas' disease, but few have been subjected to a rigorous field evaluation. We have recently described several novel enzyme immunoassays (EIAs) based on fixed-whole organisms or trypomastigote excretory-secretory antigens (TESA) from different Trypanosoma cruzi strains (Tulahuen or Brazil). This study evaluated the most promising of these novel assays (e.g. fixed-epimastigotes, fixed-trypomastigotes, TESA Brazil and TESA Tulahuen antigens) in a field study of Venezuelan blood bank specimens. The assays were tested in an operator-blinded fashion using 2038 blood bank samples obtained from low and high T.cruzi prevalence regions of Venezuela (n= 1050 and n= 988 from Bolivar and Portuguesa states, respectively). Based on National Laboratory for Chagas Immunodiagnosis (NLCI) 'gold standard' results, all novel EIAs were superior to the commercial kit currently used in Venezuela, achieving 100% sensitivity and >99% specificity at optimal cut-off values. The novel assays identified seven false-negative samples compared with the routine screening performed by the Venezuelan blood bank although two samples were also misclassified as positive. Minor differences in the performance of the four novel assays were observed at lower arbitrary cut-off values. This study confirms the potential utility of both the fixed-organism and the TESA-based assays in the diagnosis of T.cruzi infection. PMID:17163873

  5. 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. PMID:22259201

  6. High-voltage isoelectric focusing in ultrathin gels and enzyme-amplified immunoassay: a new method for analysis of cerebrospinal fluid proteins.

    PubMed

    Kjellin, K G; Hallander, L B

    1982-01-01

    A procedure using high-voltage isoelectric focusing (IF) in ultrathin (02. mm) gels and enzyme-amplified immuno-sandwich assay was elaborated to get optimal IF separation conditions, to avoid CSF concentration, e.g. by ultrafiltration preceding IF with the risk of unequal protein losses, to minimize the amounts of CSF and expensive reagents needed, especially antibodies and to shorten the analysis time, including the selective detection of proteins. The high voltage (2000-3000 V/10 cm) and efficient cooling during IF were obtained using ECPS 3000/150 and FBE 3000 (Pharmacia, Sweden). Ampholytes (Pharmalytes) of different pI intervals were used. The CSF and (diluted) serum samples were microdialysed in polyacrylamide gel before IF to minimize band curvature and to obtain optimal resolution. The IF separation was performed in about 1 h. Owing to the rapid fixation of ultrathin gels after IF, full use could be made of the high-voltage resolving capacity. The thin gels also made histochemical techniques applicable. Different immunological identification assays have been tested. An enzyme-amplified (alkaline phosphatase) immuno-sandwich method was found to be very sensitive and selective, and has so far given the best results. Many proteins in the same sample, applied as a line on the gel before IF, could be detected by overlaying antibody-soaked membrane strips. Furthermore, one specific protein could be examined in many samples simultaneously by overlaying or immersion of diluted antibody solutions. A few microlitres of unconcentrated CSF and diluted serum were used for the analysis performed within 1 day. The findings for albumin, transferrin and IgG in CSF and sera from patients with different neurological diseases, especially including cases with "normal" CSF, barrier damage, degenerative and demyelinating disorders, have been compared with the corresponding protein-stained (Coomassie R-250) patterns where the CSF had been concentrated by a special vacuum evaporation technique before IF. PMID:6184458

  7. Ultrasensitive enzyme-free electrochemical immunoassay for free thyroxine based on three dimensionally ordered macroporous chitosan-Au nanoparticles hybrid film.

    PubMed

    Zhang, Qi; Chen, Xiaojun; Tu, Fulai; Yao, Cheng

    2014-09-15

    The measurement of free thyroxine concentration in serum is considered to be an essential indicator of thyroid function. Here, a novel enzyme-free sandwich electrochemical immunosensor for the detection of FT4 antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous chitosan-Au nanoparticles hybrid (3DOM CS-AuNPs) film electrode, and magnetic multiwall carbon nanotubes (MMWCNTs) were used as label of secondary antibody (Ab2). The 3DOM CS-AuNPs film electrode was constructed by one-step electrodeposition of CS-AuNPs composite onto Au electrode with silica opal template. MMWCNTs were prepared by chemical co-precipitation of Fe(2+) and Fe(3+) salts on carboxylated MWCNTs. Ru(bpy)3(2+) labeled anti-FT4 (Ru(bpy)3(2+)-Ab2) was covalently attached to MMWCNTs through the formation of amide bond between the carboxylic groups of MWCNTs and the amine groups of antibody. Under the optimal conditions, FT4 was detected in a concentration range from 0.71 fg mL(-1) to 1.15 pg mL(-1) with a correlation coefficient of 0.998 and a detection limit of 0.20 fg mL(-1). Moreover, the immunosensor showed excellent selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method. PMID:24752149

  8. Clinical Performance of a New Soluble CD14-Subtype Immunochromatographic Test for Whole Blood Compared with Chemiluminescent Enzyme Immunoassay: Use of Quantitative Soluble CD14-Subtype Immunochromatographic Tests for the Diagnosis of Sepsis

    PubMed Central

    Sato, Masayuki; Takahashi, Gaku; Shibata, Shigehiro; Onodera, Makoto; Suzuki, Yasushi; Inoue, Yoshihiro; Endo, Shigeatsu

    2015-01-01

    We previously reported that a soluble CD14-subtype (sCD14-ST) immunochromatographic test (ICT) for plasma is more convenient than chemiluminescent enzyme immunoassay (CLEIA), but plasma separation makes bedside measurements difficult. We developed a new sCD14-ST ICT for whole blood and investigated whether quantitative determinations of sCD14-ST by ICT were useful for diagnosing sepsis and severe sepsis/septic shock. We studied 20 patients who fulfilled two or more systemic inflammatory response syndrome (SIRS) criteria and 32 patients who had been diagnosed with sepsis or severe sepsis/septic shock. Whole blood was collected on day 0 (on admission) and day 7, and the sCD14-ST concentration was quantitatively measured by CLEIA and ICT for whole blood. The patients’ Acute Physiology and Chronic Health Evaluation (APACHE) II, Sequential Organ Failure Assessment (SOFA), and Mortality in Emergency Department Sepsis (MEDS) scores were also calculated. The cut-off values obtained by the quantitative measurements made by ICT were 464.5 pg/mL for sepsis and 762.7 pg/mL for severe sepsis/septic shock (P < 0.0001). A Bland–Altman plot showed that no fixed bias or proportional bias was detected between CLEIA and quantitative ICT for whole blood. sCD14-ST concentrations were significantly correlated with APACHE II, SOFA, and MEDS scores (P < 0.0001). These results suggest that the new sCD14-ST ICT for whole blood may be a useful tool for the convenient, rapid bedside diagnosis and treatment of sepsis. PMID:26623644

  9. Application of an enzyme-immunoassay (EIA) for rapid screening of 5 alpha-pregnane-3,20-dione (DHP) in blood plasma of the Asian elephant, Elephas maximus.

    PubMed

    Dehnhard, M; Hildebrandt, T; Rohleder, M; Strauss, G; Meyer, H H; Göritz, F

    2001-01-01

    Populations of African (Loxodonta Africana) and Asian Elephants (Elephas maximus) in zoos and safari parks are at risk due to their low reproductive success. To extend the limited knowledge of their reproductive physiology, the development of easy and practical methods for the analysis of the relevant reproductive hormones is essential to support e.g. assisted reproduction. For the measurement of 5 alpha-pregnane-3,20-dione (DHP), the predominant ovarian gestagen in both species, an enzyme-immunoassay (EIA) based on commercial reagents was applied. Advantages of this EIA are the small volume of plasma needed for evaluation (5 microliters) and the possibility of direct processing without an extraction stage. The lower limit of detection was 0.16 ng/ml, the mean recovery was 101% and the mean coefficients of variation were 7.3% (intra-assay) and 9.9% (inter-assay). In Asian elephants, DHP levels reached 15 ng/ml during the luteal phase and up to 21 ng/ml during pregnancy. Estrous cycle lengths based on the lowest DHP concentrations varied from 12 to 20 weeks (mean 15.4 +/- 2.3). In two Asian elephant cows a calf was still-borne. Thereafter, the animals reassumed ovarian activity after approximately 8 and 13 weeks, respectively. In one animal estradiol implants for hormonal contraception caused a down regulation of the ovarian function as demonstrated by an irregular pattern of DHP secretion over a period of 48 weeks. We propose the direct DHP-EIA as a suitable method for reproductive monitoring in elephants, as it can be easily established in laboratories. PMID:11413705

  10. Detection of immunoglobulin M (IgM), IgA, and IgG Norwalk virus-specific antibodies by indirect enzyme-linked immunosorbent assay with baculovirus-expressed Norwalk virus capsid antigen in adult volunteers challenged with Norwalk virus.

    PubMed Central

    Gray, J J; Cunliffe, C; Ball, J; Graham, D Y; Desselberger, U; Estes, M K

    1994-01-01

    Pre- and postexposure sera collected from 17 adult volunteers challenged with Norwalk virus as described previously (D. Y. Graham, X. Jiang, T. Tanaka, A. Opekun, P. Madore, and M. K. Estes, J. Infect. Dis. 170:34-43, 1994) were examined for Norwalk virus-specific immunoglobulin M (IgM), IgA, and IgG by indirect enzyme-linked immunosorbent assays with recombinant Norwalk virus antigen bound to the solid phase. Sixteen of the 17 volunteers had evidence of past infection, all presenting with preexisting IgG antibody of high avidity; only one volunteer had no evidence of previous infection. Virus infection was detected in 14 of the 16 volunteers with evidence of past infection, and 9 of the infected volunteers had symptomatic illness. A significant rise in both virus-specific IgA and IgG titers was detected after challenge in all of the volunteers who became ill. Five of the asymptomatic volunteers who were infected had rising titers of virus-specific IgG, but only two of the five had a concomitant rise in their virus-specific IgA antibody titers. Antibody rises were detectable in eight of nine ill volunteers 8 to 11 days after challenge but in the asymptomatic volunteers only after more than 15 days had elapsed. Virus-specific IgM was detected after challenge in all 14 infected volunteers. Between symptomatic and asymptomatic volunteers there were no significant differences in titers of virus-specific IgG and IgA in serum before challenge; however, there were significantly higher titers in symptomatic volunteers between 8 and > 90 days after challenge for virus-specific IgG and 8 and 24 days after challenge for virus-specific IgA. PMID:7883902

  11. PCB immunoassay performance evaluation

    SciTech Connect

    Schreiber, J.A.; Pedersen, T.A.

    1995-12-31

    Field analytical technologies are becoming increasing relied upon in the performance of site investigations and remediation verification activities. Immunoassay screening methods provide a means to perform rapid analyses of soil, groundwater and waste samples in the field. When used in conjunction with a dynamic adaptive sampling/analyses approach immunoassay field screening data provides valuable information for decision making. Soil samples that were collected as part of site characterization and cleanup verification studies at an industrial facility in the northeast were analyzed using immunoassay kits to allow for expedited decisions on investigation and remediation approaches. The 161 samples collected were analyzed in the field using the Millipore EnviroGard{trademark} PCB and EnSys PCB RISc{trademark} immunoassay test methods. Confirmation analyses on all the samples were conducted in a laboratory using EPA Method 8080. Correlation of the field and laboratory results indicate that false negative and false positive rates vary depending on contaminant concentration and sample the matrix properties. Non-parametric statistical methods used to compare the quantified laboratory data to the immunoassay test range results show no significant difference at the 95 percent confidence intervals for some of the data sets although false positive and negative error in excess of 10 percent were observed in some cases. The results of the evaluation suggest that control of error rates can best be achieved by ensuring rigorous control of the field methodologies.

  12. 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.

  13. Updates in immunoassays: bacteriology.

    PubMed

    Josko, Deborah

    2012-01-01

    There are many immunoassays available that provide rapid, accurate and sensitive results. The intent of this article was to provide a brief overview of some of the products and methodologies available for clinical use and to discuss some of the principles behind the methodology and instrumentation. In the area of infectious disease, the use of immunoassays ensures rapid turnaround times that will result in the administration of prompt, accurate treatment for the patient. Ultimately, this will improve overall patient outcomes while possibly decreasing the costs associated with increased hospital stay. In conclusion, immunoassays are essentially easy to perform, cost-effective, produce highly sensitive and specific results, and allow the medical laboratory professional the ability to report accurate results in a timely manner. PMID:22953518

  14. Synthesis of optically pure chrysobactin and immunoassay development.

    PubMed

    Lu, C; Buyer, J S; Okonya, J F; Miller, M J

    1996-10-01

    Chrysobactin (alpha-N-(2,3-dihydroxybenzoyl)-D-lysyl-L-serine), a siderophore that is essential for systemic virulence by plant pathogenic Erwinia chrysanthemi, was synthesized with high diastereomeric purity. Chrysobactin was prepared by coupling the N-hydroxysuccinimide ester of alpha-N-(2,3-dibenzyloxybenzoyl)-epsilon-N-Cbz-D-lysine with L-serine benzyl ester followed by deprotection via hydrogenolysis. Optically pure chrysobactin was obtained with 98% overall yield. A monoclonal antibody to ferric chrysobactin was developed and characterized as IgM. The antibody reacts with chrysobactin, ferric chrysobactin and less strongly with ferric dihydroxybenzoic acid. The antibody reacts weakly with the siderophores ferrichrome, A, ferric pseudobactin and ferric rhodotorulic acid. This antibody was used in a competitive immunoassay to detect ferric chrysobactin at 10(-8) to 10(-10) mol. This immunoassay may provide a useful method for the detection of chrysobactin in plant samples. PMID:8837459

  15. Comparison of Common Immunoassay Kits for Effective Application in Workplace Drug Urinalysis.

    PubMed

    Liu, R H

    1994-06-01

    Workplace drug urinalysis protocols include an initial immunoassay followed by a confirmation gas chromatography/mass spectrometry (GC/MS) test of immunoassay-positive samples. (Drug categories that are commonly tested include: amphetamines, barbiturates, benzodiazepines, cannabis, cocaine, lysergic acid diethyl amide, methadone, methaqualone, opiates, phencyclidine, and propoxyphene. Not all drug categories are tested by all workplace drug urinalysis programs.) Only those samples that are tested positive by both the initial and the confirmatory procedures can be reported as positive. Thus, when adopting an immunoassay, one must have knowledge of the assay's cross-reacting characteristics and the assay's apparent analyze concentration that corresponds to a specific analyze concentration determined by the GC/MS procedure. The underlying principles of the commonly used radioimmunoassay, enzyme immunoassay, fluorescence polarization immunoassay, and particle immunoassay are outlined. Cross-reacting characteristics of these immunoassays as reported by the manufacturers and independent laboratories are tabulated. This information shown that commercial immunoassay kits for drug categories that are included in workplace drug urinalysis programs are generally more specific than those kits that are for clinical use only. Furthermore, recently manufactured immunoassay kits targeted for use in workplace drug urinalysis programs are more specific than those manufactured earlier. Reported effects of adulterants, such as salt, cleaning agents, etc., on commonly used immunoassays are summarized. Without more comprehensive and systematic studies, it is difficult to make general statements concerning the superiority of one methodology over the others. It is clear, however, that cannabinoid assesses are most susceptible to the influence of adulterants. Reported immunoassay-GC/MS correlation data are reviewed. Significant correlations exist in all cases. The immunoassay apparent analyze concentration corresponding to a specific GC/MS analyze concentration may be approximately based on the resulting regression equations. Since the corresponding immunoassay apparent analyze concentrations vary with the specificities of the reagent used, the immunoassay reagent manufacturers should carefully study specificity characteristics of each manufacturing batch and provide these correlation data for users' evaluation and adaptation. PMID:26270150

  16. Mass spectrometric immunoassay

    DOEpatents

    Nelson, Randall W (Phoenix, AZ); Williams, Peter (Phoenix, AZ); Krone, Jennifer Reeve (Granbury, TX)

    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.

  17. 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.

  18. 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.

  19. Immunoassay Techniques for Detection of the Herbicide Simazine Based on Use of Oppositely

    E-print Network

    Hammock, Bruce D.

    Immunoassay Techniques for Detection of the Herbicide Simazine Based on Use of Oppositely Charged to an assay for the herbicide simazine. Both enzyme-linked immu- nosorbent assay (ELISA) and dot blot formats

  20. Capillary Electrophoresis-Based Immunoassays: Principles & Quantitative Applications

    PubMed Central

    Moser, Annette C.; Hage, David S.

    2013-01-01

    The use of capillary electrophoresis as a tool to conduct immunoassays has been an area of increasing interest over the last decade. This approach combines the efficiency, small sample requirements, and relatively high speed of CE with the selectivity of antibodies as binding agents. This review examines the various assay formats and detection modes that have been reported for these assays, along with some representative applications. Most CE immunoassays in the past have employed homogeneous methods in which the sample and reagents are allowed to react in solution. These homogeneous methods have been conducted as both competitive binding immunoassays and as non-competitive binding immunoassays. Fluorescent labels are most commonly used for detection in these assays, but enzyme labels have also been utilized for such work. Some additional work has been performed in CE immunoassays with heterogeneous methods in which either antibodies or an analog of the analyte is immobilized to a solid support. These heterogeneous methods can be used for the selective isolation of analytes prior to their separation by CE or to remove a given species from a sample/reagent mixture prior to analysis by CE. These CE immunoassays can be used with a variety of detection modes, such as fluorescence, UV/visible absorbance, chemiluminescence, electrochemical measurements, mass spectrometry, and surface plasmon resonance. PMID:18646279

  1. Universal phosphorescence immunoassay.

    PubMed

    Mantrova EYu; Demcheva, M V; Savitsky, A P

    1994-05-15

    The aim of this study is to develop a universal phosphorescence immunoassay method using monoclonal antibodies to Pd-coproporphyrin (Pd-CP) and conjugates of various proteins with Pd-CP. Pd-CP and monoclonal antibodies obtained allow a convenient method for the determination of various antigens to be developed. The conditions for immunological reactions with Pd-CP were optimized with respect to the components affecting the nonspecific binding Pd-CP and Pd-CP conjugates. PMID:8059936

  2. 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.

  3. 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. PMID:22076332

  4. 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.

  5. Multiplex Electrochemical Immunoassay Using Gold Nanoparticle Probes and Immunochromatographic Strips

    SciTech Connect

    Mao, Xun; Baloda, Meenu; Gurung, Anant; Lin, Yuehe; Liu, Guodong

    2008-10-20

    We describe a multiplex electrochemical immunoassay based on the use of gold nanoparticle (Au-NP) probes and immunochromatographic strips (ISs). The approach takes advantage of the speed and low cost of the conventional IS tests and the high sensitivities of the nanoparticle-based electrochemical immunoassays. Rabbit IgG(R-IgG) and human IgM (H-IgM) were used as model targets for the demonstration of the proof of concept. The Au-NPs based sandwich immunoreactions were performed on the IS, and the captured gold nanoparticle labels on the test zones were determined by highly-sensitive stripping voltammetric measurement of the dissolved gold ions (III) with a carbon paste electrode. The detection limits are 1.0 and 1.5 ng/mL with the linear ranges of 2.5-250 ng/mL for quantitative detection of R-IgG and H-IgM, respectively. The total assay time is around 25 minutes. Such multiplex electrochemical immunoassay could be readily highly multiplexed to allow simultaneous parallel detection of numerous proteins and is expected to open new opportunities for protein diagnostics and biosecurity.

  6. 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

  7. A Wash-Free Homogeneous Colorimetric Immunoassay Method.

    PubMed

    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

  8. 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. PMID:8812399

  9. Enzyme-linked immunosorbent assays for the detection of antibody to Crimean-Congo haemorrhagic fever virus in the sera of livestock and wild vertebrates.

    PubMed Central

    Burt, F. J.; Swanepoel, R.; Braack, L. E.

    1993-01-01

    IgM antibody response to Crimean-Congo haemorrhagic fever (CCHF) virus was monitored in experimentally infected sheep and cattle by an IgM capture enzyme-linked immunoassay (ELISA). Specific binding of antigen was detected by a rabbit anti-CCHF horseradish peroxidase conjugate or a sandwich technique with hyperimmune mouse anti-CCHF ascitic fluid and commercially available anti-mouse immunoglobulin peroxidase conjugate. The persistence of IgM antibody activity was found to be of shorter duration than in humans, and this may be a function of the relative lack of susceptibility of these animals to infection with CCHF virus. IgG antibody responses in the sheep could be monitored by sandwich ELISA using commercially available anti-sheep immunoglobulin peroxidase conjugates. Total antibody activity in the sera of experimentally infected sheep, cattle and small mammals could be monitored in a competitive ELISA (CELISA) using rabbit anti-CCHF peroxidase conjugate. The CELISA was applied to the sera of 960 wild vertebrates from a nature reserve in South Africa, and the prevalence of antibody was found to be greatest in large mammals such as rhinoceros, giraffe and buffalo, which are known to be the preferred hosts of the adult tick (Hyalomma) vectors of the virus. PMID:8270014

  10. 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.

  11. 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.

  12. PAPAIN DIGESTION FRAGMENTS OF HUMAN IGM GLOBULINS

    PubMed Central

    Mihaesco, Constantin; Seligmann, Maxime

    1968-01-01

    Papain digestion of two Waldenström IgM globulins produced a high amount of small peptides and resulted in the formation of two end products, the Fabµ and Fcµ fragments. The Fcµ fragment is characterized by a fast electrophoretic mobility, a high content in carbohydrate, and a high molecular weight. It was demonstrated that this fragment is made of heavy chain pieces belonging to several disulfide-linked monomeric subunits, presumably representing the carboxy-terminal end of the µ-chains. Fc fragments from the two macroglobulins could not be distinguished immunologically. An appreciable proportion of IgM molecules apparently underwent degradation without the formation of a stable Fc fragment. An Fc-like fragment, analogous to the reduced Fc fragment, was obtained at early stages of papain digestion of the IgM subunits. The Fabµ fragment, with slow and individually distinct electrophoretic mobility, bears many physicochemical and immunological similarities to the Fab? fragment. It consists of one light chain and one Fd piece, both of which were isolated. The interaction of these two constituents was demonstrated by gel diffusion studies. Fab fragments of both IgM globulins were resolved into two subpopulations with different electric charges. In addition to these fragments, intermediary split products were observed at early stages of the degradation process, together with a high yield of small peptides mainly derived from the papain-sensitive region of the heavy chains. Immunologic data strongly suggested that this segment of µ-chains is situated between the Fd piece and the portion included in the Fc fragment. Several experiments indicated the importance of conformational antigenic specificity in both Fab and Fc regions of the IgM globulins. PMID:4169963

  13. Variability in Telavancin Cross-Reactivity among Vancomycin Immunoassays

    PubMed Central

    McConeghy, Kevin W.; Liao, Siyun; Clark, Douglas; Worboys, Philip; Barriere, Steven L.

    2014-01-01

    Telavancin is a semisynthetic lipoglycopeptide with a dual mechanism of action against Gram-positive pathogens. Two brief reports have suggested potential cross-reactivity of telavancin with the vancomycin particle-enhanced turbidometric immunoassay (PETIA). The purpose of this study was to evaluate several commercially available vancomycin immunoassays (fluorescence polarization [FPIA], enzyme-multiplied immunoassays [EMIT], PETIA, and chemiluminescent immunoassay [CMIA]) for cross-reactivity with telavancin. Seven sites were selected to analyze serum samples for vancomycin. Each site received a set of samples (n = 18) which combined drug-free serum with telavancin, 7-OH telavancin metabolite, or vancomycin. Immunoassays demonstrating potential cross-reactivity were further evaluated by sending a duplicate sample set to multiple laboratories. Cross-reactivity was defined as the percent theoretical concentration (reported concentration/theoretical concentration × 100). No cross-reactivity was seen with FPIA or EMIT. Within the theoretical concentration range of 5 to 120 ?g/ml of telavancin, the Synchron PETIA system reported vancomycin concentrations ranging from 4.7 to 54.2 ?g/ml compared to vancomycin concentrations from 1.1 to 5.6 ?g/ml for the Vista PETIA system. The Architect CMIA system reported vancomycin concentrations in the range of 0.27 to 0.97 ?g/ml, whereas Advia Centaur XP CMIA reported vancomycin concentrations between 1.6 and 31.6 ?g/ml. The Architect CMIA immunoassay had the lowest percent cross-reactivity (0.8 to 5.4%), while the Synchron PETIA immunoassay demonstrated the highest percent cross-reactivity (45.2 to 53.8%). Telavancin samples measured by liquid chromatography-mass spectroscopy were within 93.9 to 122% of theoretical concentrations. Vancomycin concentrations were not measured in any 7-OH telavancin-spiked sample. Vancomycin concentrations measured by liquid chromatography-mass spectroscopy were within 57.2 to 113% of theoretical concentrations. PETIA and CMIA measured vancomycin concentrations in telavancin-spiked samples. Significant variability in percent cross-reactivity was observed for each platform regardless of immunoassay method. PMID:25223996

  14. 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 0.48ngmL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ngmL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ngmL(-1), and the linear range was 0.01-10,000ngmL(-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. PMID:26592642

  15. 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?

  16. Evaluation of a stage-specific proteolytic enzyme of Schistosoma mansoni as a marker of exposure.

    PubMed

    Ramzy, R M; Faris, R; Bahgat, M; Helmy, H; Franklin, C; McKerrow, J H

    1997-06-01

    Cercarial elastase (CE) is one of the first proteins released in the host by invading schistosome cercariae. An enzyme-linked immunosorbent assay (ELISA)-formatted immunoassay has been developed to detect antibodies to the stage-specific CE antigen of Schistosoma mansoni as marker of exposure. We have evaluated this test system as an epidemiologic tool, using well-characterized sera collected from S. mansoni- and S. haematobium-infected subjects residing in endemic areas and from control subjects living in nonendemic areas in Egypt. Urine, stool specimens, and blood samples were collected from a sample of 272 endemic subjects randomly selected to represent different age groups in the range of 2-20 years of age. Of 47 S. mansoni-infected subjects, 41 (87.2%) had anti-CE IgG antibodies. Of 52 S. haematobium-infected cases, 38 (73.0%) had IgM antibodies to CE and 43 (82.7%) had IgG antibodies to CE. Of 173 egg-negative people in the endemic area, 84 (48.6%) were IgM positive and 99 (57.2%) were IgG positive. The mean IgM and IgG antibody levels were similar in the infected groups but were significantly lower in the egg-negative group (P = 0.001). All sera from young children (2-3 years of age) were uniformly ELISA negative. The prevalence of IgM and IgG antibodies to CE in children less than six years of age were significantly lower than in other age groups. There was no significant difference in prevalence rates of IgM and IgG anti-CE antibodies between subjects having other parasites present in the endemic area (Ascaris lumbricoides, Entrobius vermicularis, Hymenolepis nana, H. diminuta, Trichostrongylus spp., and Entamoeba histolytica) and those without any parasitic infection. All nonendemic sera (58), including those with other helminth infections, were uniformly ELISA negative for antibodies to CE. These findings suggest that antibodies to elastase indicate exposure, but not necessarily active schistosome infection. PMID:9230801

  17. 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...

  18. The IGM Project: Searching For IGM Emission Over 0

    NASA Astrophysics Data System (ADS)

    Martin, Christopher D.; Matuszewski, M.; Rahman, S.; Morrissey, P.; Moore, A.; Schiminovich, D.; Milliard, B.; Frank, S.; Deharveng, J.; Peroux, C.; CWI Team; FIREBALL Team; KCWI Team; ISTOS Team

    2011-01-01

    I discuss several experimental projects underway or proposed designed to discover and map emission from the IGM. The Cosmic Web Imager (CWI) is a ground-based high resolution spectrometer designed to detect low surface brightness emission from redshifted Lyman alpha, OVI and CIV at Palomar and Keck Observatories, over 2IGM emission in the 0.3IGM in the space UV. I will report on preliminary results from FIREBALL and CWI. This work is supported by NASA and NSF.

  19. The IGM Project: Searching For IGM Emission Over 0

    NASA Astrophysics Data System (ADS)

    Martin, Christopher D.; Matuszewski, M.; Morrissey, P.; Moore, A.; CWI Team

    2013-01-01

    I discuss several experimental projects underway or proposed designed to discover and map emission from the IGM. The Cosmic Web Imager (CWI) is a ground-based high resolution spectrometer designed to detect low surface brightness emission from redshifted Lyman alpha, OVI and CIV at Palomar and Keck Observatories, over 2IGM emission in the 0.3IGM in the space UV. I will report on preliminary results from FIREBALL and CWI. This work is supported by NASA and NSF.

  20. 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 ?D and ?K 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

  1. PCB detection by immunoassay -- A wipe test for surface contamination

    SciTech Connect

    Dautlick, J.X.; Teaney, G.B.; Hudak, R.T.; Melby, J.M.

    1995-12-31

    Immunoassay based field screening methods are gaining acceptance by the environmental diagnostics industry for on-site characterization and remediation monitoring. Polychlorinated biphenyls (PCBs), a family of molecules classified as potential carcinogens, can be easily detected on-site by immunoassay screening methods. This results in reduced project cost and improved onsite efficiency, since field screening immunoassays short cut the long turn around time of laboratory analysis while providing reliable results. On site wipe test technology for assessing PCB contamination on surfaces such as walls and floors of PCB storage facilities has been developed to supplement the D TECH{trademark} PCB soil assay. This sampling technique can also be used to monitor for transformer leaks, spills and to evaluate equipment decontamination processes. The D TECH PCB wipe test is quick, cost effective, highly specific and user friendly. The surface is sampled by wiping a 100 cm{sup 2} area with a 1 cm{sup 2} pad saturated with an extractant. The PCB is extracted from the sampling pad during a short extraction step. The sample is filtered, diluted, and run in the D TECH PCB field screening system. The components of the immunoassay include PCB specific antibodies (Ab) covalently linked to small latex particles, a PCB analog which is covalently linked to alkaline phosphatase, and the free PCB from the sample. The free PCB competes with the enzyme linked analog for the Ab binding sites. The latex particles are then collected on a filter device, washed, and an enzyme substrate is added. The amount of color produced is inversely proportional to the concentration of free PCB on the sample, and can be determined using a hand held reflectometer, or a color card.

  2. IgM in the kidney: a multiple personality disorder.

    PubMed

    Platt, Jeffrey L; Cascalho, Marilia

    2015-09-01

    IgM in the blood of normal individuals consists mainly of 'natural' polyreactive antibodies. Natural IgM is thought to provide an initial defense against infection and to promote the healing of wounded cells. Yet, as Panzer and colleagues show, these benefits can be eclipsed when the IgM binds to damaged cells of the glomerulus, activating complement. IgM in glomeruli thus signifies cellular damage and may warn that the pace of that damage exceeds the capacity for repair. PMID:26323070

  3. Hapten Synthesis and Antibody Development for Polychlorinated Dibenzo-p-dioxin Immunoassays

    E-print Network

    Hammock, Bruce D.

    Hapten Synthesis and Antibody Development for Polychlorinated Dibenzo-p-dioxin Immunoassays James R for the detection of dioxins such as TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) by ELISA (enzyme-linked immunosorbent assay). These novel haptens contain unsaturation between the halogenated dibenzo-p-dioxin ring

  4. Competitive chemiluminescent anzyme immunoassay for vitamin B12 analysis in human milk.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. 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...

  6. Study of IGM through high energy radiation from blazar

    NASA Astrophysics Data System (ADS)

    Medhi, Jayashri; Duorah, H. L.; Barua, A. G.; Duorah, K.

    2014-03-01

    The high energy gamma rays from blazar affects the intergalactic medium (IGM) to large distances at different redshifts. The blazar radiation has been taken as the result of synchrotron and Inverse Compton scattering of electrons. It is found that the intergalactic medium is clumpy. Our estimated values lie within the suggested limit of ?IGM ? 0.03 at redshift z = 3.

  7. Comparison of three automated immunoassay methods for the determination of Epstein-Barr virus-specific immunoglobulin M.

    PubMed

    Berth, Mario; Bosmans, Eugene

    2010-04-01

    In this study we compared the performances of three commercially available Epstein-Barr virus (EBV) immunoglobulin M (IgM) assays on highly automated immunoassay platforms: BioPlex 2200 (Bio-Rad Laboratories), Immulite 2000 (Siemens Healthcare Diagnostics), and Liaison (DiaSorin). As a confirmatory method, immunoblotting was performed. The specificity of the three EBV IgM assays was evaluated by testing 293 selected sera from patients with various infectious and noninfectious diseases. After the exclusion of 30 samples, the specificities were 96.2% for Liaison, 98.1% for Immulite, and 97.0% for BioPlex. For evaluation of the sensitivity, samples from 70 consecutive patients with a positive heterophile antibody test were examined, irrespective of clinical or biological findings. After the exclusion of six samples, the sensitivities were 89.1% for Liaison, 84.4% for Immulite, and 89.1% for BioPlex. Finally, in a prospective study performed with 500 samples obtained from consecutive patients and sent in by general practitioners, we also determined Epstein-Barr nuclear antigen IgG and viral capsid antigen IgG in a two-phase approach. Concordance of the EBV serologic status was 96.2% between Liaison and Immulite, 96.4% between Immulite and BioPlex, and 97.8% between BioPlex and Liaison. The three EBV IgM immunoassays that we evaluated have acceptable and comparable performances. PMID:20147496

  8. Pholcodine interference in the immunoassay for opiates in urine.

    PubMed

    Svenneby, G; Wedege, E; Karlsen, R L

    1983-01-01

    The excretion in urine after single oral therapeutic doses of morphine derivatives was analysed with radioimmunoassay (RIA) and homogeneous enzyme immunoassay (EMIT) for opiates. In contrast to the rapid excretion of ethylmorphine and codeine, pholcodine showed positive results for opiates 2-6 weeks after intake when the urines were analysed with the RIA-method. When analysed with the EMIT-method, positive results were obtained for pholcodine for approximately 10 days. As pholcodine is a common component in cough mixtures, its prolonged excretion could represent a hazard in interpreting the results from drug analyses of urines. PMID:6347841

  9. Isotope-labeled immunoassays without radiation waste

    E-print Network

    Hammock, Bruce D.

    Isotope-labeled immunoassays without radiation waste Guomin Shan*, Wei Huang*, Shirley J. Gee with radioactive materials, and (iii) short shelf-life of the labeled re- agents. The advantage of isotopic with ELISA or fluorescent detection systems. We developed a format for isotope label immunoassay

  10. Hydrophilic, bright CuInS2 quantum dots as Cd-free fluorescent labels in quantitative immunoassay.

    PubMed

    Speranskaya, Elena S; Beloglazova, Natalia V; Abé, Sofie; Aubert, Tangi; Smet, Philippe F; Poelman, Dirk; Goryacheva, Irina Y; De Saeger, Sarah; Hens, Zeger

    2014-07-01

    We report on the synthesis of core-shell CuInS2/ZnS quantum dots (QDs) in organic solution, their encapsulation with a PEG-containing amphiphilic polymer, and the application of the resulting water-soluble QDs as fluorescent label in quantitative immunoassay. By optimizing the methods for core synthesis and shell growth, CuInS2/ZnS QDs were obtained with a quantum yield of 50% on average after hydrophilization. After conjugation with an aflatoxin B1-protein derivative, the obtained QDs were used as fluorescent labels in microplate immunoassay for the quantitative determination of the mycotoxin aflatoxin B1. QDs-based immunoassay showed higher sensitivity compared to enzyme-based immunoassay. PMID:24892375

  11. Validation of a Microsphere Immunoassay for Serological Leptospirosis Diagnosis in Human Serum by Comparison to the Current Gold Standard

    PubMed Central

    Wynwood, Sarah J.; Burns, Mary-Anne A.; Graham, Glenn C.; Weier, Steven L.; McKay, David B.; Craig, Scott B.

    2015-01-01

    A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays. PMID:25807009

  12. Competitive Immunoassays Using Antigen Microarrays.

    PubMed

    Zhang, Zhaowei; Hu, Weihua; Zhang, Qi; Li, Peiwu; Li, Changming

    2016-01-01

    In this work, a non-fouling antigen competitive immunoassay microarray based on the polymer brush is reported to detect multiple mycotoxins. The detection is achieved by utilizing highly specific monoclonal antibodies produced in our laboratory. The polymer brush, poly[oligo(ethylene glycol) methacrylate-co-glycidyl methacrylate] (POEGMA-co-GMA), is synthesized via surface-initiated atom transfer radical polymerization (SI-ATRP) on standard glass slides. In the polymer brush, the epoxy groups of glycidyl methacrylate (GMA) residues provide covalent binding sites for spotted antigens. Moreover, the abundant poly(ethylene glycol) (PEG) side chains in the brush are able to ultimately suppress the nonspecific protein adsorption in solution (non-fouling). The polymer brush shows a high and uniform protein loading, along with a high resistance to nonspecific protein absorption that are both important to achieve a highly sensitive immunoassay. As a demonstration of a multiplex assay, aflatoxin B1 (AFB1), ochratoxin A (OTA), and zearalenone (ZEN) are selected as antigen targets for simultaneous detections using the microarray. PMID:26614080

  13. Numerical Simulations of Galactic Outflows and Evolution of the IGM

    E-print Network

    Martel, Hugo

    2011-01-01

    Galactic outflows play a major role in the evolution of galaxies and the intergalactic medium (IGM). The energy deposited into the interstellar medium by supernovae and active galactic nuclei can accelerate the gas past the escape velocity, and eject it into the IGM. This will affect the subsequent evolution of the galaxy, by reducing or eliminating star formation, and quenching the accretion of matter onto the central AGN. Galactic outflows is the main process by which energy and processed interstellar matter is transported into the IGM. This affects the subsequent formation of other galaxies. The energy carried by outflows can strip protogalactic halos of their gas, preventing galaxies from forming. Conversely, the metals carried by outflows can modify the composition and cooling rates of the gas in protogalactic halos, favoring the formation of galaxies. In this paper, I review the various techniques used to simulate galactic outflows and their impact on galaxy and IGM evolution.

  14. Varying results for immunoassay screening kits for cotinine level.

    PubMed

    Alterman, Arthur I; Gariti, Peter; Niedbala, R Sam

    2002-09-01

    Our earlier study found that although enzyme-linked immunosorbent analysis (ELISA) screening assays for urine cotinine indicated use in former smoking treatment patients who reported abstinence, this finding was sometimes incorrect when validated against gas chromatography/mass spectrometry (GC/ MS; P. Gariti, A. I. Alterman, R. Ehrmann, F. D. Mulvaney, & C. P. O'Brien, 2002). In the current validation study, separate urine samples of 71 of these same patients were reanalyzed by an independent laboratory in blinded fashion using a screening enzyme immunoassay (EIA) analysis and GC/MS confirmation. EIA results showed almost total agreement with confirmatory testing. The findings indicate that use of screening ELISA/EIA for urine cotinine can detect unreported cases of smoking in former patients, but that care is needed in selecting a laboratory for conducting these tests. PMID:12236461

  15. Diagnostic value of detecting specific IgA and IgM with recombinant Trypanosoma cruzi antigens in congenital Chagas' disease.

    PubMed

    Lorca, M; Veloso, C; Munoz, P; Bahamonde, M I; Garcia, A

    1995-06-01

    The present study compares the early diagnosis of congenital Chagas' disease with a DOT assay using recombinant antigens with immunofluorescence antibody testing (IFAT) and an enzyme-linked immunosorbent assay (ELISA). The studies were performed using cord blood and sera of 12 infected newborns (group I) and 12 uninfected ones born to Trypanosoma cruzi-infected mothers (group II). Conventional IFAT and ELISA showed positive results for IgG at high titers, in infants and mothers of both groups; IgA antibodies were detected by ELISA in four of the infected infants and IgM was detected in two of them. All sera of the uninfected infants were negative for IgA and IgM in the ELISA. Application of a DOT assay using eight recombinant T. cruzi antigens allowed detection of specific IgA in the cord blood of six of the infected cases and IgM in eight of them. Repetition of these serologic tests in samples obtained during a monthly follow-up gave positive results for IgA in two of the initially negative infants of group I and for IgM in four of them. This means that diagnosis of congenital T. cruzi infection was confirmed, through demonstration of specific IgM, in all infected infants, and of IgA in eight of them. The importance of late detection of IgM in siblings born of infected mothers is discussed. The detection of IgM and IgA in sera obtained after birth is believed to be due to a congenital transmission of the parasite that occurred late in pregnancy. No IgA or IgM antibodies could be detected by the DOT assay in the sera of the negative controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7611556

  16. Isolation and Characterization of IgM and IgY Antibodies from Plasma of Magellanic Penguins (Spheniscus magellanicus).

    PubMed

    Bizelli, Camila C; Silva, A Sandriana R; da Costa, Jessica D; Vanstreels, Ralph E T; Atzingen, Marina V; Santoro, Marcelo L; Fernandes, Irene; Catão-Dias, José L; Faquim-Mauro, Eliana L

    2015-03-01

    Infectious diseases such as aspergillosis, avian malaria, and viral infections are significant threats to the conservation of penguins, leading to morbidity and mortality of these birds both in captivity and in the wild. The immune response to such infectious diseases is dependent on different mechanisms mediated by cells and soluble components such as antibodies. Antibodies or immunoglobulins are glycoproteins that have many structural and functional features that mediate distinct effector immune functions. Three distinct classes of antibodies have been identified in birds: immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin Y (IgY). In this study we aim to establish an efficient laboratory method to obtain IgM and IgY antibodies from plasma samples of healthy adult Magellanic penguins (Spheniscus magellanicus). The protocol was developed combining plasma delipidation, sequential precipitation with caprylic acid and ammonium sulfate, and size-exclusion chromatography. The efficiency of the protocol and the identity of the purified IgM and IgY antibodies were confirmed through enzyme-linked immunosorbent assay, Western blotting, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, and lectin binding assay. Structural and physicochemical properties of IgM and IgY from Magellanic penguins were consistent with those of other avian species. This purification protocol will allow for more detailed studies on the humoral immunity of penguins and for the development of high specificity serologic assays to test Magellanic penguins for infectious pathogens. PMID:26292539

  17. [GoldMag particle-based chemiluminescence immunoassay for human high sensitive C-reactive protein].

    PubMed

    Guo, Boyang; Ma, Le; Zhang, Mengdan; Yang, Jiangcun; DU, Haiping; Ma, Ting; Yali, Cui

    2015-11-01

    To develop a sensitive, accurate detection method for high sensitive C-reactive protein (hsCRP). Methods With the GoldMag particle as the solid phase carrier, horseradish peroxidase (HRP)-lunimo-H2O2 as the chemiluminescence reaction system, we established a chemiluminescent immunoassay for hsCRP detection. Linear range, sensitivity, precision, accuracy and other indicators were evaluated. hsCRP level of 233 clinical human serum specimens were determined and compared by two different methods: the GoldMag particle-based magnetic chemiluminescence enzyme-linked immunoassay established in this study, and the commercialized scattering immunoturbidimetric assay from Germany SIEMENS. Results The chemiluminescent immunoassay for hsCRP based on GoldMag particle had a good linear relationship between 0.15 mg/L and 25 mg/L (R(2)=0.9937), with the detection limit of 0.076 mg/L. The intra-assay precision was less than 10.00% and the inter-assay precision was less than 15.00%. The average recovery rate for accuracy was 97.80%. In the contrast experiment of 233 clinical human serum specimens, the results obtained using the approach established in this study showed a high correlation and consistency with scattering immunoturbidimetric assay from Germany SIEMENS. Conclusion GoldMag particle-based magnetic chemiluminescence enzyme-linked immunoassay for hsCRP has been successfully developed. PMID:26522353

  18. Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

    PubMed

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe; Simon, Stéphanie

    2015-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

  19. Fast and Sensitive Detection of Enteropathogenic Yersinia by Immunoassays

    PubMed Central

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe

    2014-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 103 CFU/ml to 8.8 × 104 CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 105 CFU/ml to 106 CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

  20. Serological responses to experimental Norwalk virus infection measured using a quantitative duplex time-resolved fluorescence immunoassay.

    PubMed

    Kavanagh, Owen; Estes, Mary K; Reeck, Amanda; Raju, Ravikiran M; Opekun, Antone R; Gilger, Mark A; Graham, David Y; Atmar, Robert L

    2011-07-01

    A quantitative duplex time-resolved fluorescence assay, dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), was developed to measure Norwalk virus (NV)-specific IgA and IgG antibodies simultaneously. The duplex assay showed superior performance by detecting seroconversion following experimental NV infection at an earlier time point than a reference total immunoglobulin enzyme-linked immunosorbent assay (ELISA). PMID:21593238

  1. DEVELOPMENT OF A MAGNETIC PARTICLE IMMUNOASSAY FOR POLYBROMINATED DIPHENYL ETHERS AND APPLICATION TO ENVIRONMENTAL AND FOOD MATRICES.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sensitive magnetic particle enzyme-linked immunoassay (ELISA) was developed to analyze polybrominated diphenyl ethers (PBDEs) in water, milk, fish, and soil samples. The assay was rapid and can be used to analyze fifty samples in about one hour after sample cleanup. The assay has a limit of det...

  2. EVALUATION OF IMMUNOASSAY METHODS FOR DETERMINATION OF 3,5,6-TRICHLORO-2-PYRIDINOL IN MULTIPLE SAMPLE MEDIA

    EPA Science Inventory

    Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by the RaPID (TM) commercial immunoassay testing ...

  3. Recombinant Hepatitis A Virus Antigen: Improved Production and Utility in Diagnostic Immunoassays

    PubMed Central

    LaBrecque, F. D.; LaBrecque, D. R.; Klinzman, D.; Perlman, S.; Cederna, J. B.; Winokur, P. L.; Han, J.-Q.; Stapleton, J. T.

    1998-01-01

    Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies which were detected by commercial immunoassays. The present investigation compared the production of HAV antigen by standard tissue culture methods to the production of HAV antigen with the recombinant vaccinia virus system. In addition, HAV and rV-ORF antigens were assessed for their utility in diagnostic immunoassays. Serum or plasma samples from HAV antibody-positive and antibody-negative individuals were evaluated by immunoassay that used either HAV or rV-ORF antigen. All samples (86 of 86) in which HAV antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) also tested positive by the recombinant antigen-based immunoassay (VacRIA). Similarly, all samples (50 of 50) that were HAV antibody negative also tested negative by the VacRIA. The lower limit of detection of HAV antibody was similar among immunoassays with either HAV or rV-ORF antigen. Thus, in the population studied, the sensitivity and specificity of the VacRIA were equivalent to those of the commercial ELISA. Since production of recombinant antigen is faster and less expensive than production of traditional HAV antigen, the development of diagnostic HAV antibody tests with recombinant HAV antigen appears warranted. PMID:9650953

  4. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    PubMed

    Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Kumpoosiri, Mallika; Warin, Nuchnard; Oplatowska, Michalina; Gajanandana, Oraprapai; Grant, Irene R; Karoonuthaisiri, Nitsara; Elliott, Christopher T

    2013-01-01

    Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection. PMID:23638044

  5. Development of an Ultrasensitive Immunoassay for Detecting Tartrazine

    PubMed Central

    Li, Zhuokun; Song, Shanshan; Xu, Liguang; Kuang, Hua; Guo, Shidong; Xu, Chuanlai

    2013-01-01

    We have developed an ultrasensitive indirect competitive enzyme-linked immunosorbent assay for the determination of tartrazine. Two carboxylated analogues of tartrazine with different spacer lengths, and one derivative from commercial tartrazine after a little chemical modification, were synthesized as haptens in order to produce antibodies specific to tartrazine. The effect of sulfonic acid groups on the hapten structure of tartrazine was also studied carefully for the first time. A most specific monoclonal antibody against tartrazine was created and exhibited an IC50 value of 0.105 ng/mL and a limit of detection of 0.014 ng/mL, with no cross-reactivity to other structurally-related pigments. The established immunoassay was applied to the determination of tartrazine in fortified samples of orange juice and in real positive samples of carbonated beverages. PMID:23799494

  6. Silver deposition directed by self-assembled gold nanorods for amplified electrochemical immunoassay.

    PubMed

    Zhang, Hongfang; Ning, Danlei; Ma, Lina; Zheng, Jianbin

    2016-01-01

    A novel electrochemical immunoassay was developed based on the signal amplification strategy of silver deposition directed by gold nanorods (AuNRs), which was in-situ assembled on the sandwich immunocomplex. The superstructure formed by the self-assembly of AuNRs provided abundant active sites for the nucleation of silver nanoparticles. In this pathway, the stripping current of silver was greatly enhanced. Using human immunoglobulin G (HIgG) as a model analyte, the ultrasensitive immunoassay showed a wide linear range of six orders of magnitude from 0.1 fg mL(-1) to 100 pg mL(-1), with the low detection limit down to 0.08 fg mL(-1). The practicality of this electrochemical immunoassay for detection of HIgG in serum was validated with the average recovery of 93.9%. In addition, this enzyme-free immunoassay also has the advantages of acceptable reproducibility and specificity, and thus this immunosensing protocol can be extended to the detection of other low-abundant protein biomarkers. PMID:26703256

  7. A Reassessment of IgM Memory Subsets in Humans.

    PubMed

    Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2015-10-15

    From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD(+)CD27(+) ("marginal zone [MZ]"), IgD(-)CD27(+) ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD(-)CD27(-) cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and VH/JH usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The "IgM-only" subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27(+) switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities. PMID:26355154

  8. Monitoring human exposure to pesticides using immunoassay

    E-print Network

    Hammock, Bruce D.

    at the estimation of internal dose based on the fate of the compound in human body. Biological monitoring approachesMonitoring human exposure to pesticides using immunoassay Marja E. Koivunen1,2 , Shirley J. Gee1 of automated immunoanalyzers, immunosensors or microchips with flow-through systems. Biological monitoring

  9. Immunoassay on Free-standing Electrospun Membranes

    NASA Astrophysics Data System (ADS)

    Steckl, Andrew; Wu, Dapeng; Han, Daewoo

    2010-03-01

    For the purpose of immunoassay, electrospun membranes can be thought as the thread-like self-assembling of nano/microbeads. Non-woven membranes of electrospun poly(caprolactone) (PCL) fibers display excellent tenacity, flexibility and suitable surface energy. These PCL membranes exhibit easy handling in air, fast spreading and wetting in aqueous solution, and rapid adsorption of protein molecules by hydrophobic interaction. After a fold-and-press process, the membrane porosity was reduced from ˜ 75% to less than 10%, while the thickness increased from ˜5 to 300 ?m. The resulting fluorescence signal from adsorbed protein increased more than 120 times. With anti-HSA and HSA-FITC as an immunoassay model, a linear detection range from 500 ng/mL down to 1 ng/mL is obtained, with a detection of limit (LOD) of ˜ 0.08 ng/mL. By comparison, conventional nitrocellulose and thicker PCL fiber electrospun membrane displayed a much higher LOD of ˜100 ng/mL. Immunoassay on free-standing electrospun membrane successfully combines the low-cost and simplicity of conventional membrane immunoassay, with the fast reaction speed and high sensitivity characteristic of magnetic nano/microbeads bioassays.

  10. IMMUNOASSAY FOR P-NITROPHENOL IN URINE

    EPA Science Inventory

    Urinary excretion of nitrophenol metabolites is an important index of human exposure to organophosphate pesticides. In particular, p-nitrophenol, a major urinary metabolite of parathion, can be used as a biomarker of human exposure. Immunoassay methods have been recently describe...

  11. Immunoassay for Monitoring Environmental and Human Exposure

    E-print Network

    Hammock, Bruce D.

    Immunoassay for Monitoring Environmental and Human Exposure to the Polybrominated Diphenyl Ether) to monitor environmental and human exposure to polybrominated diphenyl ether BDE-47 that is used as a flame workers in electronic-recycling facilities that experience high PBDE exposure (11), a major route

  12. Liver Abscesses and Hyper IgM Syndrome

    PubMed Central

    Shah, Ira; Rahangdale, Aarti; Bhatnagar, Sushmita

    2013-01-01

    Hyper IgM (HIGM) syndrome is an immunodeficiency that can lead to liver disease in more than 80% of affected males by an age of 20 years. Hepatitis, sclerosing cholangitis, and hepatocellular malignancies are common among them. We encountered two cases in children of less than 12 years who presented with typical manifestations of liver abscess and were later detected to have a concomitant underlying HIGM syndrome. PMID:24479081

  13. Automated homogeneous immunoassay analysis of cotinine in urine.

    PubMed

    Niedbala, R Sam; Haley, Nancy; Kardos, Stephanie; Kardos, Keith

    2002-04-01

    A study was conducted to evaluate the performance comparison of a homogeneous enzyme immunoassay (EIA) designed to detect cotinine in urine and carbon monoxide (CO) breath measurements to determine smoking status. The clinical comparison was done using urine and breath specimens from 218 volunteers. Urine samples were analyzed by immunoassay and confirmed by gas chromatography-mass spectrometry (GC-MS). Breath carbon monoxide was determined by a commercial analyzer. Using cutoffs of 10 ppm for CO and 500 ng/mL for urinary cotinine, the relative sensitivity/specificity was 93.6%/74.0%. The positive predictive value was 86.8%, and the negative predictive value was 86.5%. However, comparison of the EIA to GC-MS showed a sensitivity/specificity of 96.2%/98.4% and a positive predictive value of 99.3%. The EIA was also evaluated non-clinically for precision, stability, recovery, and interferences. In addition, the non-clinical evaluation demonstrated coefficients of variation from 0.37 to 1.09% across cotinine concentrations ranging from 0 to 5000 ng/mL. The assay was found to be highly specific for cotinine and cross-reacted to a limited degree with 3-hydroxycotinine. Finally, multiple freeze-thaw cycles of urines containing cotinine showed no degradation of the drug in the specimen when tested in the EIA. Thus, the EIA tested is a rapid, lab-based test that can reliably determine cotinine levels and their relation to smoking status. PMID:11991533

  14. Industrial Fungal Enzymes: An Occupational Allergen Perspective

    PubMed Central

    Green, Brett J.; Beezhold, Donald H.

    2011-01-01

    Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes. PMID:21747869

  15. Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

    PubMed

    Woolley, Christine F; Hayes, Mark A; Mahanti, Prasun; Douglass Gilman, S; Taylor, Tom

    2015-11-01

    Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations. PMID:26342315

  16. A novel immunosensor for detecting toxoplasma gondii-specific IgM based on goldmag nanoparticles and graphene sheets.

    PubMed

    Jiang, Shuting; Hua, Erhui; Liang, Mo; Liu, Bei; Xie, Guoming

    2013-01-01

    A novel electrochemical immunosensor for detecting toxoplasma gondii-specific IgM (Tg-IgM) was constructed based on goldmag (Au-Fe(3)O(4)) nanoparticles and graphene sheets (GS). Thionine (Thi), as a mediator, was first electropolymerized on a nafion-GS (Nf-GS) modified electrode. Subsequently, gold nanoparticles (AuNPs) were attached onto the poly-thionine film through ?-stacking interactions, and then were used to immobilize toxoplasma gondii antigen (Tg-Ag) for immunosensor fabrication. A sandwich-type immunoassay for Tg-IgM was performed using Au-Fe(3)O(4) labeled anti-IgM-horseradish peroxidase (HRP) as trace label. Electrochemical detection was carried out in the presence of H(2)O(2) as HRP substrate. Using Au-Fe(3)O(4) provided a simple, non-chemical damaging method for regeneration, and enhanced the HRP reduction ability toward H(2)O(2). The AuNPs/Thi/Nf-GS nanocomposite also had good conductivity and biocompatibility, which effectively improved the immunosensor sensitivity. Under optimal conditions, the immunosensor can detect Tg-IgM in two linear ranges from 0.0375 to 1.2 AU mL(-1) and from 2.0 to 18 AU mL(-1) with a detection limit of 0.016 AU mL(-1) (S/N=3). The immunosensor exhibited good reproducibility, stability, and selectivity as well. PMID:23010058

  17. ABO (histo) blood group phenotype development and human reproduction as they relate to ancestral IgM formation: A hypothesis.

    PubMed

    Arend, Peter

    2016-01-01

    The formation of a histo (blood) group) ABO phenotype and the exclusion of an autoreactive IgM or isoagglutinin activity arise apparently in identical glycosylation of complementary domains on cell surfaces and plasma proteins. The fundamental O-glycan emptiness of the circulating IgM, which during the neonatal amino acid sequencing of the variable regions is exerting germline-specific O-GalNAc glycan-reactive serine/threonine residues that in the plasma of the adult human blood group O individuals apparently remain associated with the open glycosidic sites on the ABOH convertible red cell surface, must raise suggestions on a transient expression of developmental glycans, which have been "lost" over the course of maturation. In fact, while the mammalian non-somatic, embryogenic stem cell (ESC)- germ cell (GC) transformation is characterized by a transient and genetically as-yet-undefined trans-species-functional O-GalNAc glycan expression, in the C57BL/10 mouse such expression was potentially identified in growth-dependent, blood group A-like GalNAc glycan-bearing, ovarian glycolipids complementary with the syngeneic anti-A reactive IgM, which does not appear in early ovariectomized animals. This non-somatically encoded, polyreactive, ancestral IgM molecule has not undergone clonal selection and does primarily not differentiate between self and non-self and might, due to amino acid hydroxyl groups, highly suggest substrate competition with subsequent O-glycosylations in ongoing ESC-GC transformations and affecting GC maturation. However, the membrane-bound somatic N/O-glycotransferases, which initiate, after formation of the zygote, the complex construction of the human ABO phenotypes in the trans cisternae of the Golgi apparatus, are associated and/or completed with soluble enzyme versions exerting identical specificities in plasma and likely competing vice versa by glycosylation of neonatal IgM amino acids, where they suggest to accomplish the clearance of anti-A autoreactivity at germline serine and threonine residues. Sustaining the lineage-maintaining position of the classic A allele and the discovery of the OA hybrid alleles at the normal ABO locus and in heterozygous ESC lines have, together with clinical observations, raised discussions about a silent A-allelic support within blood group O reproduction. However, the question of whether a fictional "continued blood group O inbreeding" ultimately occurs without the A-allelic or somatic function remains unanswered because the genetic relationship between non-somatic O-GalNAc-glycosylations that operate before sperm-egg recognition and somatic O-GalNAc-glycosylations that arise after the formation of the zygote remains to be elucidated. PMID:26433867

  18. I. Studies on pyridine dinucleotide transhydrogenase in rat liver mitochondria. II. Identification of the tissue and cellular sites of catabolism of IgM in the rat

    SciTech Connect

    Weis, J.K.

    1988-01-01

    The orientation of the transmembrane enzyme, pyridine dinucleotide transhydrogenase, in the inner mitochondrial membrane of rat liver has been determined by evaluating effects of proteases on the integrity of the enzyme in mitoplasts and submitochondrial particles. Following treatment of these membranes with the non-specific protease, proteinase K, antigenic proteolytic products were detected by immunoblot analysis using polyclonal antibody prepared against purified bovine heart enzyme. Information from these proteolysis studies was used to construct a model of the orientation of transhydrogenase in the inner mitochondrial membrane. In this work, I have used the residualizing label, dilactitol-{sup 125}I-tyramine ({asterisk}I-DLT) to identify the tissue and cellular sites of catabolism of the immunoglobulin, IgM. Purified IgM was labeled conventionally with {sup 125}I or with the residualizing label, {asterisk}I-DLT. The circulating half-life of the protein, 2.7 {plus minus} 0.3 days, was the same when measured using either label, indicating that the residualizing label does not affect the kinetics of the protein's catabolism in vivo. At 2.4 or 5.1 days post injection, the liver contained the major fraction of catabolized protein compared to all the other organs in the body. Additionally, following collagenase digestion of the liver, the hepatocytes were shown to be 77% responsible for the catabolism of IgM by the liver. Autoradiography of the liver revealed that the remaining 23% of IgM catabolized by the liver was due to the Kupffer cells.

  19. COMPARISON OF IMMUNOASSAY AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY METHODS FOR MEASURING 3,5,6-TRICHLORO-2PYRIDINOL IN MULTIPLE SAMPLE MEDIA

    EPA Science Inventory

    Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by a commercial RaPID immunoassay testing kit. ...

  20. Laboratory diagnosis of syphilis with automated immunoassays.

    PubMed

    Marangoni, Antonella; Moroni, Alessandra; Accardo, Silvia; Cevenini, Roberto

    2009-01-01

    The serological detection of specific antibodies to Treponema pallidum is of particular importance in the diagnosis of syphilis. The purpose of this study was to evaluate diagnostic performances of automated immunoassays in comparison with T. pallidum hemagglutination test (TPHA) and Western Blot (WB). The retrospective study was performed with different panels of sera: 244 clinical and serological characterized syphilitic sera and 203 potentially interfering samples. All the sera were tested by Enzygnost Syphilis, ARCHITECT Syphilis TP, TPHA, and homemade WB. The diagnostic performances of the two assays were very similar: both Enzygnost Syphilis and ARCHITECT Syphilis TP performed with a sensitivity of 99.2%, whereas the specificity was 98.5 and 98.4%, respectively. Considering the suitability for automation, both immunoassays may represent a good choice as a screening test. However, the use of a confirmatory test, such as TPHA or WB, remains a must in order to avoid false-positive results. PMID:19140205

  1. Splenic extrafollicular reactions and BM plasma cells sustain IgM response associated with hypercholesterolemia.

    PubMed

    Khoo, Lawrence Han Boon; Thiam, Chung Hwee; Soh, Serena Ying; Angeli, Véronique

    2015-05-01

    Hypercholesterolemia associated with atherosclerotic disease is known to be associated with increased total and oxidized (ox) low-density lipoprotein (LDL)-specific IgM antibodies in circulation. However, the B-cell responses accounting for this increase remain to be elucidated. Here, we observed an association between total IgM and oxLDL-specific IgM autoantibodies with cholesterol in the plasma of hypercholesterolemic apolipoprotein E deficient (apoE(-/-)) mice. Our findings also indicated that oxLDL-specific IgM autoantibodies production was restricted to the spleen, but not the lymph nodes. Further examination of the spleen revealed that the extrafollicular responses, but not germinal center reactions, were the dominant antibody-producing pathway. A quiescent population of IgM(+) plasma cells including oxLDL-specific IgM antibody secreting cells in BM also sustained the elevated IgM antibodies response in circulation. We determined that IgM(+) plasma cells in the BM were, at least in part, splenic derived by depleting CD11c(+) DCs and plasmablasts to disrupt the humoral responses. In addition, lowering hypercholesterolemia reduced IgM response by interfering with extrafollicular and BM responses. By elucidating the mechanism underlying the elevated IgM response observed in hypercholesterolemia, this study provides insight into novel immunotherapeutic avenues. PMID:25639537

  2. Enzyme immunoassay by dynamic enhanced vibrational spectroscopy of the enzyme reaction product

    NASA Astrophysics Data System (ADS)

    Zhao, Haiying; Dou, Xiaoming

    2005-01-01

    This paper reports a kind of application of surface-enhanced Raman scattering (SERS) to immunology. In the proposed system, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibody labeled with peroxidase. If this immunocomplex is subjected to reaction with o-phenylenediamine and hydrogenperoxide at 37°C, azoaniline is generated. This azo compound is adsorbed on a silver colloid and only the azo compound gives a strong surface-enhanced resonance Raman (SERRS) spectrum. A linear relationship was observed between the peak intensity of the N=N stretching band and the concentration of antigen, revealing that one can determine the concentration of antigen by the SERRS measurement of the reaction product.

  3. Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling

    PubMed Central

    Shang, Jing; Zrazhevskiy, Pavel; Postupna, Nadia; Keene, C. Dirk; Montine, Thomas J.; Gao, Xiaohu

    2015-01-01

    In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes and technical complexity of alternative assay formats. Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. For a proof-of-principle, multiplexed detection of three model proteins was demonstrated on cultured HeLa cells, and two clinically-relevant markers of dementia, ?-amyloid and PHF-tau, were profiled in formalin-fixed paraffin embedded brain tissue sections, uncovering correlated increase in abundance of both markers in the “Alzheimer’s disease” cohort. Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics. PMID:26328896

  4. Analysis of drugs in human tissues by supercritical fluid extraction/immunoassay

    NASA Astrophysics Data System (ADS)

    Furton, Kenneth G.; Sabucedo, Alberta; Rein, Joseph; Hearn, W. L.

    1997-02-01

    A rapid, readily automated method has been developed for the quantitative analysis of phenobarbital from human liver tissues based on supercritical carbon dioxide extraction followed by fluorescence enzyme immunoassay. The method developed significantly reduces sample handling and utilizes the entire liver homogenate. The current method yields comparable recoveries and precision and does not require the use of an internal standard, although traditional GC/MS confirmation can still be performed on sample extracts. Additionally, the proposed method uses non-toxic, inexpensive carbon dioxide, thus eliminating the use of halogenated organic solvents.

  5. Magnetic Electrochemical Immunoassays with Quantum Dot Labels for Detection of Phosphorylated Acetylcholinesterase in Plasma

    SciTech Connect

    Wang, Hua; Wang, Jun; Timchalk, Charles; Lin, Yuehe

    2008-11-01

    A new magnetic electrochemical immunoassay has been developed as a tool for biomonitoring exposures to organophosphate (OP) compounds, e.g., insecticides and chemical nerve agents, by directly detecting organophosphorylated acetylcholinesterase (OP?AChE). This immunoassay uniquely incorporates highly efficient magnetic separation with ultrasensitive square wave voltammetry (SWV) analysis with quantum dots (QDs) as labels. A pair of antibodies was used to achieve the specific recognition of OP?AChE that was prepared with paraoxon as an OP model agent. Antiphosphoserine polyclonal antibodies were anchored on amorphous magnetic particles preferably chosen to capture OP?AChE from the sample matrixes by binding their phosphoserine moieties that were exposed through unfolding the protein adducts. This was validated by electrochemical examinations and enzyme-linked immunosorbent assays. Furthermore, antihuman AChE monoclonal antibodies were labeled with cadmium-source QDs to selectively recognize the captured OP?AChE, as characterized by transmission electron microscopy. The subsequent electrochemical SWV analysis of the cadmium component released by acid from the coupled QDs was conducted on disposable screen-printed electrodes. Experimental results indicated that the SWV-based immunoassays could yield a linear response over a broad concentration range of 0.3?300 ng/mL OP?AChE in human plasma with a detection limit of 0.15 ng/mL. Such a novel electrochemical immunoassay holds great promise as a simple, selective, sensitive, and field-deployable tool for the effective biomonitoring and diagnosis of potential exposures to nerve agents and pesticides.

  6. A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles.

    PubMed

    Sun, Qian; Zhao, Guangying; Dou, Wenchao

    2015-10-22

    A novel nonenzymatic optical immunoassay strategy was for the first time designed and utilized for sensitive detection of antibody to Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) in serum. The optical immunoassay strategy was based on blue silica nanoparticles (Blue-SiNps) and magnetic beads (MB). To construct such an optical immunoassay system, the Blue-SiNPs were first synthesized by inverse microemulsion method, characterized by SEM, Zeta potential and FTIR. Two nanostructures including Blue-SiNPs and MB were both functionalized with antibody against S. pullorum and S. gallinarum (anti-PG) without using enzyme labeled antibody. Anti-PG functionalized blue silica nanoparticles (IgG-Blue-SiNps) were used as signal transduction labels, while anti-PG functionalized magnetic beads (IgG-MB) were selected to separate and enrich the final sandwich immune complexes. In the process of detecting negative serum, a sandwich immunocomplex is formed between the IgG-MB and IgG-Blue-SiNPs. With the separation of the immunocomplex using an external magnetic field, the final plaque displayed bright blue color. While in the detection of infected serum, IgG-MB and anti-PG formed sandwich immunocomplexes, IgG-Blue-SiNPs were unable to bind to the limited sites of the antigen, and a light brown plaque was displayed in the bottom of microplate well. Stable results were obtained with an incubation time of 60 min at room temperature, and different colors corresponding to different results can be directly detected with naked eye. The reaction of IgG-Blue-SiNPs with S. pullorum was inhibited by 1:100 dilution of positive chicken serum. Such a simple immunoassay holds great potential as sensitive, selective and point-of-care (POC) tool for diagnosis of other biological molecules. PMID:26526916

  7. Statistical approaches to developing a multiplex immunoassay for determining human exposure to environmental pathogens.

    PubMed

    Augustine, Swinburne A J; Simmons, Kaneatra J; Eason, Tarsha N; Griffin, Shannon M; Curioso, Clarissa L; Wymer, Larry J; Shay Fout, G; Grimm, Ann C; Oshima, Kevin H; Dufour, Al

    2015-10-01

    There are numerous pathogens that can be transmitted through water. Identifying and understanding the routes and magnitude of exposure or infection to these microbial contaminants are critical to assessing and mitigating risk. Conventional approaches of studying immunological responses to exposure or infection such as Enzyme-Linked Immunosorbent Assays (ELISAs) and other monoplex antibody-based immunoassays can be very costly, laborious, and consume large quantities of patient sample. A major limitation of these approaches is that they can only be used to measure one analyte at a time. Multiplex immunoassays provide the ability to study multiple pathogens simultaneously in microliter volumes of samples. However, there are several challenges that must be addressed when developing these multiplex immunoassays such as selection of specific antigens and antibodies, cross-reactivity, calibration, protein-reagent interferences, and the need for rigorous optimization of protein concentrations. In this study, a Design of Experiments (DOE) approach was used to optimize reagent concentrations for coupling selected antigens to Luminex™ xMAP microspheres for use in an indirect capture, multiplex immunoassay to detect human exposure or infection from pathogens that are potentially transmitted through water. Results from Helicobacter pylori, Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella typhimurium singleplexes were used to determine the mean concentrations that would be applied to the multiplex assay. Cut-offs to differentiate between exposed and non-exposed individuals were determined using finite mixed modeling (FMM). The statistical approaches developed facilitated the detection of Immunoglobulin G (IgG) antibodies to H. pylori, C. jejuni, Toxoplasma gondii, hepatitis A virus, rotavirus and noroviruses (VA387 and Norwalk strains) in fifty-four diagnostically characterized plasma samples. Of the characterized samples, the detection rate was 87.5% for H. pylori, and 100% for T. gondii assays and 89% for HAV. Further, the optimized multiplex assay revealed exposure/infection to several other environmental pathogens previously uncharacterized in the samples. PMID:26070441

  8. On VI Absorption Lines and the IGM Baryon Content

    NASA Astrophysics Data System (ADS)

    Tripp, T. M.

    2005-12-01

    The Far Ultraviolet Spectroscopic Explorer has observed a large number of low-redshift QSOs and AGNs, and the spectra of these objects show an array of remarkable absorption lines ranging from cold, molecular gas (e.g., traced by C I and H2) to highly ionized and hot gas (e.g., traced by O VI and Ne VIII transitions). Many of the gas clouds detected in UV absorption cannot be studied by any other technique, especially in intergalactic regions where the densities are likely to be quite low. The O VI absorption lines detected in intervening gas clouds have particularly important implications regarding the chemical enrichment, physical conditions, and baryonic content of intergalactic gas in the nearby universe. For example, these absorbers can be used to search for the "warm-hot intergalactic medium", a shock-heated phase of the IGM that is theoretically predicted to be a major baryon reservoir at the present epoch. This talk will briefly review studies of the low-z IGM based on ultraviolet observations of O VI and related absorption lines, including findings on the metallicity, ionization, and cosmological mass of these systems as well as their relationships with galaxies. While the deployment of FUSE has led to substantial progress, the observations have also raised new questions. The talk will conclude with a few comments on outstanding questions and goals for future observations.

  9. Heavy element enrichment in the IGM at high redshift

    E-print Network

    S. Savaglio

    1997-09-16

    We present a detailed analysis of the ionisation state and heavy element abundances in the Intergalactic Medium (IGM). The CIV doublet is shown by 30 % of the 182 selected optically thin \\lya clouds in 10 QSO lines of sight. Direct metallicity calculations have been performed on individual systems with detected CIV and SiIV (10% of the sample) varying the UV photoionising source, cloud density and size and silicon relative abundance. The best solutions for carbon content in this subsample (redshift coverage $z=2.6 - 3.8$) span between 1/6 and 1/300 of the solar value with no evidence of redshift evolution in both the metallicity and the ionising source. Global properties of the whole sample indicate that the metallicity in \\lya clouds with CIV and SiIV is not typical of the IGM. The redshift evolution of the UVB is one of the possible sources of the observed SiIV/CIV trend presented by Cowie and collaborators during this meeting. Future detection of heavy elements in lower HI column density ($\\log N_{HI} < 14.5$) \\lya clouds relies on the presence of OVI and NV at $z=1-2.5$.

  10. Non-covalent association of IgM subunits produced by reduction and alkylation

    PubMed Central

    Parkhouse, R. M. E.

    1974-01-01

    Purified IgM isolated from the serum of mice bearing the transplantable plasmacytoma MOPC 104E was reduced and alkylated and then analysed by sucrose density gradient centrifugation and by sodium dodecyl sulphate—polyacrylamide gel electrophoresis. On partial reduction a mixture of IgM subunits was obtained in the absence of covalently linked 19S IgM. When examined under dissociating conditions this mixture was found to consist of disulphide-linked 7S subunits (IgMs), small amounts of HL subunits and oligomeric IgM of a size intermediate between monomeric and pentameric IgM. In the absence of a dissociating agent and on sucrose density gradient, however, the mixture resolved into a 19S and a 7S peak. The 19S peak consisted primarily of oligomeric IgM and IgMs with small amounts of HL subunits. Thus alkylated IgMs and HL subunits of IgM can associate through non-covalent forces to form a molecule sedimenting at 19S, providing oligomeric forms are present. In the absence of oligomeric forms, IgMs, HL subunits and heavy and light chains sediment at about 7S. The products of partial reduction which sediment at 7S and 19S could also be isolated by preparative polyacrylamide gel electrophoresis. When this was done J chain was absent in the former and present in the latter, raising the possibility that J chain does not disulphide bond to each of the five IgMs subunits, constituting an IgM molecule. Thus, within cells secreting IgM, J chain would be expected to mediate the formation of an oligomeric form of IgM. Once the oligomeric structure has been assembled, then non-covalent forces between this and IgMs subunits will cause the formation of a 19S structure, thereby facilitating the final assembly through disulphide bonds. ImagesFIG. 4FIG. 2FIG. 6 PMID:4217776

  11. [Evaluation of the anti-TP antibody latex agglutination immunoassay in routine testing and a clinical viewpoint].

    PubMed

    Fujimori, Chinami; Yukimasa, Nobuyasu; Miura, Keisuke; Mochizuki, Syofi; Yasuhara, Tsutomu; Takagi, Yasushi; Gomi, Kunihide

    2009-03-01

    We evaluated TP-LAIA (anti-TP latex agglutination immunoassay) and compared the results with those obtained using Serological Tests for Syphilis (STS), namely Venereal Disease Research Laboratory (VDRL) method and Rapid Plasma Reagin (RPR) test. We also examined early-stage antibody reaction using rabbits infected with active pathogen, and analyzed early-stage syphilis patient serum with IgM class anti-TP antibody. Based on routine test results and case history reviews for possible syphilis infection, TP-LAIA showed high specificity, 0.64% false positive results in comparison with 13.5% by VDRL method. Sensitivity was also significantly higher than TP-Hemagglutination Assay(TPHA). In the examination of TP early-stage infection, the fastest positive antibody reaction was observed with TP-LAIA, indicating its significance in the diagnosis of early-stage syphilis. TP-LAIA was confirmed to give a reliable reaction with IgM class anti-TP antibody. TP-LAIA results coincided with VDRL method results in the decrease in anti-treponemal antibody titers following medical treatment, suggesting that TP-LAIA will be a valuable tool for monitoring the effect of medical treatments. We concluded that not only the high sensitivity and specificity of TP-LAIA assay and its suitability for automation make it an ideal screening test, but also the assay performs sufficiently and satisfactorily for its use in monitoring medical treatments. PMID:19363990

  12. Microfluidic immunoassays as rapid saliva-based clinical diagnostics

    PubMed Central

    Herr, Amy E.; Hatch, Anson V.; Throckmorton, Daniel J.; Tran, Huu M.; Brennan, James S.; Giannobile, William V.; Singh, Anup K.

    2007-01-01

    At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip). Before anticipatory use of diagnostics for assessment of complex diseases becomes widespread, development of sophisticated bioassays capable of quantitatively measuring disease biomarkers is necessary. Successful translation of new bioassays into clinical settings demands the ability to monitor both the onset and progression of disease. Here we report on a clinical POC diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 ?l of saliva, we demonstrate rapid (<10 min) measurement of the collagen-cleaving enzyme matrix metalloproteinase-8 (MMP-8) in saliva from healthy and periodontally diseased subjects. In addition to physiologically measurable indicators of periodontal disease, conventional measurements of salivary MMP-8 were used to validate the microfluidic assays described in this proof-of-principle study. The microchip-based POC diagnostic demonstrated is applicable to rapid, reliable measurement of proteinaceous disease biomarkers in biological fluids. PMID:17374724

  13. Sensitive, Fast, and Specific Immunoassays for Methyltestosterone Detection

    PubMed Central

    Kong, Na; Song, Shanshan; Peng, Juan; Liu, Liqiang; Kuang, Hua; Xu, Chuanlai

    2015-01-01

    An indirect competitive enzyme-linked immunosorbent assay (icELISA) and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT) residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%–100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed. PMID:25938198

  14. Nanomaterial Labels in Electrochemical Immunosensors and Immunoassays

    SciTech Connect

    Liu, Guodong; Lin, Yuehe

    2007-12-15

    This article reviews recent advances in nanomaterial labels in electrochemical immunosensors and immunoassays. Various nanomaterial labels are discussed, including colloidal gold/silver, semiconductor nanoparticles, and markers loaded nanocarriers (carbon nanotubes, apoferritin, silica nanoparticles, and liposome beads). The enormous signal enhancement associated with the use of nanomaterial labels and with the formation of nanomaterial–antibody-antigen assemblies provides the basis for ultrasensitive electrochemical detection of disease-related protein biomarkers, biothreat agents, or infectious agents. In general, all endeavors cited here are geared to achieve one or more of the following goals: signal amplification by several orders of magnitude, lower detection limits, and detecting multiple targets.

  15. Gliadin Detection in Food by Immunoassay

    NASA Astrophysics Data System (ADS)

    Grant, Gordon; Sporns, Peter; Hsieh, Y.-H. Peggy

    Immunoassays are very sensitive and efficient tests that are commonly used to identify a specific protein. Examples of applications in the food industry include identification of proteins expressed in genetically modified foods, allergens, or proteins associated with a disease, including celiac disease. This genetic disease is associated with Europeans and affects about one in every 200 people in North America. These individuals react immunologically to wheat proteins, and consequently their own immune systems attack and damage their intestines. This disease can be managed if wheat proteins, specifically "gliadins," are avoided in foods.

  16. The contribution of naturally occurring IgM antibodies, IgM cross-reactivity and complement dependency in murine humoral responses to pneumococcal capsular polysaccharides.

    PubMed

    Jones, Hannah E; Taylor, Philip R; McGreal, Eamon; Zamze, Susanne; Wong, Simon Y C

    2009-09-25

    Immunogenicity of 12 capsular polysaccharides (CPS) from Streptococcus pneumoniae did not correlate with pre-existing levels of natural IgM anti-CPS antibodies in mice. Immunization of mice with individual CPS, with the exception of type 14 (the only neutral CPS tested), increased serum IgM that also bound other CPS serotypes independent of structural similarity or commonly known contaminants. Surprisingly only IgM response to type 4 (which has a small immunodominant epitope) was dependent on either complement C3 or complement receptors CD35/CD21. IgG anti-CPS responses were infrequently induced, but critically dependent on complement. Our results have clarified the role of complement in the induction of IgM and IgG anti-CPS antibody responses in mice and have implications for CPS vaccine development. PMID:19660585

  17. Fluorimetric Immunoassay for Multianalysis of Aflatoxins

    PubMed Central

    2013-01-01

    A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25–50?pg/mL. AFM1 as low as 1?pg/mL was detected by this method with assay volume 40??L. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling. PMID:24000318

  18. The Long Elusive IgM Fc Receptor, Fc?R

    PubMed Central

    Kubagawa, Hiromi; Oka, Satoshi; Kubagawa, Yoshiki; Torii, Ikuko; Takayama, Eiji; Kang, Dong-Won; Jones, Dewitt; Nishida, Naonori; Miyawaki, Toshio; Bertoli, Luigi F.; Sanders, Sheila K.; Honjo, Kazuhito

    2014-01-01

    IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both preimmune “natural” and antigen-induced “immune” IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (Fc?R). We have recently identified a bona fide Fc?R in both humans and mice. In this article we briefly review what we have learned so far about Fc?R. PMID:24793544

  19. IgM autoagglutinins in warm autoimmune hemolytic anemia: a poor prognostic feature.

    PubMed

    McCann, E L; Shirey, R S; Kickler, T S; Ness, P M

    1992-01-01

    The presence of both complete IgM autoagglutinins and IgG autoantibodies in warm autoimmune hemolytic anemia (AIHA) is an uncommon finding. Over a 6-year period, only 5 of 115 (4.1%) patients with AIHA had IgM and IgG autoantibodies. In 3 of the 5 cases, the complete IgM autoagglutinins reacted up to 30 degrees C and these patients responded well to corticosteroid or other therapies for warm AIHA. The 2 patients who had warm (37 degrees C) reactive IgM autoagglutinins, were refractory to corticosteroids, splenectomy and cytotoxic drugs, and died due to the complications of hemolytic anemia. The data in these 5 cases suggest that the thermal amplitude of the IgM antibody in these unusual AIHA cases may be predictive of refractoriness to therapy and poor clinical outcome. PMID:1466193

  20. Detection of IgM Antibrucella Antibody in the Absence of IgGs: A Challenge for the Clinical Interpretation of Brucella Serology

    PubMed Central

    Solís García del Pozo, Julián; Lorente Ortuño, Santiago; Navarro, Elena; Solera, Javier

    2014-01-01

    The use of enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibodies antibrucella has become widespread in the diagnosis of human brucellosis. IgM anti-Brucella antibodies are indicative of acute infection. Between 2009–2013, 5307 patients were evaluated for serologic diagnosis at the Microbiology Laboratory of the Albacete General Hospital. A ELISA IgM-positive, IgG-negative anti-Brucella antibody serology pattern was detected in 17 of those patients. Epidemiology data, symptoms, laboratory data, treatment and outcome from these patients were reviewed. Sixteen patients presented with musculoskeletal pain, fatigue and/or fever and 1 was asymptomatic. Five patients received treatment with doxycycline combined with rifampin, gentamycin or streptomycin during 6–12 weeks, with no improvement. None of the 17 patients were finally diagnosed with brucellosis. Our results indicate that anti-Brucella IgM positive serology, per se, is not enough to diagnose acute brucellosis and other methods should be used for confirmation. Brucella serology data should be interpreted taking into account the patient's clinical history and epidemiological context. PMID:25474572

  1. Anti-HDV IgM as a Marker of Disease Activity in Hepatitis Delta

    PubMed Central

    Wranke, Anika; Heidrich, Benjamin; Ernst, Stefanie; Calle Serrano, Beatriz; Caruntu, Florin Alexandru; Curescu, Manuela Gabriela; Yalcin, Kendal; Gürel, Selim; Zeuzem, Stefan; Erhardt, Andreas; Lüth, Stefan; Papatheodoridis, George V.; Bremer, Birgit; Stift, Judith; Grabowski, Jan; Kirschner, Janina; Port, Kerstin; Cornberg, Markus; Falk, Christine S.; Dienes, Hans-Peter; Hardtke, Svenja; Manns, Michael P.; Yurdaydin, Cihan; Wedemeyer, Heiner

    2014-01-01

    Background Hepatitis delta frequently leads to liver cirrhosis and hepatic decompensation. As treatment options are limited, there is a need for biomarkers to determine disease activity and to predict the risk of disease progression. We hypothesized that anti-HDV IgM could represent such a marker. Methods Samples of 120 HDV-infected patients recruited in an international multicenter treatment trial (HIDIT-2) were studied. Anti-HDV IgM testing was performed using ETI-DELTA-IGMK-2-assay (DiaSorin). In addition, fifty cytokines, chemokines and angiogenetic factors were measured using multiplex technology (Bio-Plex System). A second independent cohort of 78 patients was studied for the development of liver-related clinical endpoints (decompensation, HCC, liver transplantation or death; median follow up of 3.0 years, range 0.6–12). Results Anti-HDV IgM serum levels were negative in 18 (15%), low (OD<0.5) in 76 (63%), and high in 26 (22%) patients of the HIDIT-2 cohort. Anti-HDV IgM were significantly associated with histological inflammatory (p<0.01) and biochemical disease activity (ALT, AST p<0.01). HDV replication was independent from anti-HDV IgM, however, low HBV-DNA levels were observed in groups with higher anti-HDV IgM levels (p<0.01). While high IP-10 (CXCL10) levels were seen in greater groups of anti-HDV IgM levels, various other antiviral cytokines were negatively associated with anti-HDV IgM. Associations between anti-HDV IgM and ALT, AST, HBV-DNA were confirmed in the independent cohort. Clinical endpoints occurred in 26 anti-HDV IgM positive patients (39%) but in only one anti-HDV IgM negative individual (9%; p?=?0.05). Conclusions Serum anti-HDV IgM is a robust, easy-to-apply and relatively cheap marker to determine disease activity in hepatitis delta which has prognostic implications. High anti-HDV IgM levels may indicate an activated interferon system but exhausted antiviral immunity. PMID:25072849

  2. Immunoassay procedures for fiber optic sensors

    NASA Astrophysics Data System (ADS)

    Ligler, Frances S.

    1988-04-01

    There is an increasing need for the development of an ultrasensitive immunoassay for use with fiber optic sensors. These detection systems can be used for such applications as disease diagnosis, detection of chemical and biological warfare agents or drugs of abuse, pollution control, therapeutic monitoring, and explosive detection. This specific program is designed to produce generic chemistries for use with existing fiber optic-based sensors to detect pathogens of particular threat to Army personnel as determined by USAMRIID. The detection system under development involves the attachment of antibodies to an optical fiber at high density. In addition, the immobilization must be achieved in a way which retains the antibody's ability to bind antigen. The functionality of the antibody will be tested through the binding of a labelled antigen. In the future, this assay could incorporate the antibodies developed by the Army for pathogens of particularly military concern.

  3. Nanoparticles for Enhanced Sensitivity in Electrochemical Immunoassays

    SciTech Connect

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

    2008-10-12

    In this manuscript, we report on electrochemical biosensors based on various nanoparticles (NPs) as labels for sensitive detection of protein biomarkers. We used silica nanoparticle as a carrier to loading a large amount of electroactive species such as poly(guanine) for sensitive immunoassay of tumor necrosis factor-alpha (TNF-a). We took the advantages of the unique hollow structure and reconstruction properties of apoferritin to prepare Cd3(PO4)2 nanoparticles as labels for sensitive assay of TNF-a. A novel immunochromatographic/electro-chemical biosensor based on quantum dots as labels has also been developed for rapid and sensitive detection of prostate-specific antigen (PSA) in human serum. These biosensors are quite sensitive with the detection limit at pM level and these approaches based on nanoparticle labels offer a new avenue for sensitive detection of protein biomarkers.

  4. Age-related aspects of human IgM(+) B cell heterogeneity.

    PubMed

    Martin, Victoria; Wu, Yu-Chang; Kipling, David; Dunn-Walters, Deborah K

    2015-12-01

    The CD27(+) IgD(+) B cell population, known as IgM memory, reduces with age. It is thought that this population is responsible for pneumococcal polysaccharide T-independent responses, and that the age-related reduction might be partially responsible for the increased susceptibility of older people to bacterial pathogens. There are other IgM(+) B cell populations that do not express IgD. We compared the different IgM populations using high-throughput sequencing of the immunoglobulin (Ig) gene repertoire and multidimensional cell phenotyping and found that the different populations of IgM cells, defined by CD27 and IgD expression, have repertoire differences. Some of these differences are likely indicative of different selection pressures in an immune response, although the older individuals were found to have a changed repertoire in naive B cells, which may contribute to some of the changes seen in memory cells. In addition, even within the CD27(+) IgD(+) IgM memory population there are multiple cell types. We show that the level of IgM expression varies substantially and hypothesize that this distinguishes between T-dependent and T-independent types of IgM memory cells. Significant age-related changes in the relative proportions of these populations may exacerbate the reduction in T-independent responders in old age. PMID:26152370

  5. Seroprevalence of rubella-specific IgM and IgG antibodies among pregnant women seen in a tertiary hospital in Nigeria

    PubMed Central

    Olajide, Okikiola M; Aminu, Maryam; Randawa, Abdullahi J; Adejo, Daniel S

    2015-01-01

    Background Rubella is a contagious viral infection that in pregnant women leads to the infection of a developing fetus, causing fetal death or congenital rubella syndrome. Objective Pregnant women are not routinely screened for rubella in Nigeria. Epidemiological data on rubella is therefore necessary to create awareness and sensitize health care administrators and providers. Materials and methods A cross-sectional study was carried out at Ahmadu Bello University Teaching Hospital between June and August 2012 to determine the prevalence of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to rubella virus in pregnant women using enzyme-linked immunosorbent assay kits. Seroprevalence was compared among 160 pregnant women attending the antenatal clinic of Ahmadu Bello University Teaching Hospital and 20 nonpregnant women of childbearing age studying at Ahmadu Bello University. Prior to sample collection, questionnaires were administered to the women to obtain data on sociodemographics, awareness and knowledge of rubella, possible risk factors, and clinical symptoms associated with the viral infection. Results Of the 160 pregnant women, 149 (93.1%) and 62 (38.8%) were positive for anti-rubella IgM and IgG antibodies, respectively. Similarly, of the 20 nonpregnant women, 18 (90%) and eight (40%) were positive for rubella IgG and IgM antibodies, respectively. None of the possible risk factors studied were significantly associated with infection. Age and other sociodemographic factors were of little significance, and awareness of rubella was low. Conclusion The prevalence of rubella was high in both pregnant (93.1%) and nonpregnant women (90%), suggesting sustained transmission, which further suggests endemicity. The presence of rubella IgM and IgG antibodies in pregnant women predisposes babies to congenital rubella syndrome and emphasizes the need for the initiation of a national rubella vaccination program in Nigeria. PMID:25610003

  6. Development of immunoassays for human urokinase

    NASA Technical Reports Server (NTRS)

    Atassi, M. Zouhair

    1988-01-01

    Radioimmune assays (RIA) and enzyme linked immune assays for measurement of pro-urokinase and the two active forms of the enzyme were developed. Polyclonal and monoclonal antibodies, with desired specificities against preselected synthetic regions of urokinase (UK), were obtained by immunization with the respective synthetic peptides and used to develop RIA for zymogen and the two activated forms of UK.

  7. IgM nephropathy; can we still ignore it

    PubMed Central

    Vanikar, Aruna

    2013-01-01

    Context:IgM nephropathy (IgMN) is a relatively less recognized clinico-immunopathological entity in the domain of glomerulonephritis , often thought to be a bridge between minimal change disease and focal segmental glomerulosclerosis. Evidence Acquisitions: Directory of Open Access Journals (DOAJ), Google Scholar, Pubmed (NLM), LISTA (EBSCO) and Web of Science has been searched. Results: IgM nephropathy can present as nephritic syndrome or less commonly with subnephrotic proteinuria or rarely hematuria. About 30% patients respond to steroids whereas others are steroid dependent / resistant. They should be given a trial of Rituximab or stem cell therapy. Conclusions:IgM nephropathy (IgMN) is an important and rather neglected pathology responsible for renal morbidity in children and adults in developing countries as compared to developed nations with incidence of 2-18.5% of native biopsies. Abnormal T-cell function with hyperfunctioning suppressor T-cells are believed to be responsible for this disease entity. Approximately one third of the patients are steroid responders where as the remaining two thirds are steroid resistant or dependent. Therapeutic trials including cell therapies targeting suppressor T-cells are required. PMID:24475434

  8. IgM Myeloma or Waldenstrom's Macroglobulinemia Is the Big Question?

    PubMed Central

    BHATT, Vijaya Raj; MURUKUTLA, Srujitha; NAQI, Muniba; PANT, Shradha; KEDIA, Shiksha; TERJANIAN, Terenig

    2014-01-01

    Although critical from therapeutic and prognostic perspectives, differentiating IgM Myeloma (MM) from Waldenstrom's macroglobulinemia (WM) is fraught with failure. WM can usually be distinguished from IgM MM by the lymphoplasmacytic versus pure plasmacytic morphology, absent versus present lytic bone lesions, and immunophenotypic findings. However, all these features have their own limitations; hence, it requires constant vigilance and periodic re-evaluation. Here we describe a case of a 70-year-old woman initially diagnosed as smoldering IgM MM, who eventually turned out to have WM. PMID:25553130

  9. Suppressor T cells prevent in vitro expression of IgM rheumatoid factor in some healthy adults

    SciTech Connect

    Koopman, W.J.

    1981-12-01

    Peripheral blood mononuclear leukocytes (MNL) from 11 of 30 healthy adults elaborated detectable IgM RF when stimulated with pokeweed mitogen. The influence of T cells on IgM RF production by autologous B Cells prepared from donors whose unfractionated MNL synthesized IgM RF in response to PMW was investigated. Untreated T cells supported IgM RF production by autologous B cells with optimal synthesis observed at T:B cell ratios of 2:1 at higher T:B cell ratios a decline in IgM RF production occurred. In contrast, at higher T:B cell ratios irradiated T cells supported consistently higher levels of IgM RF production than untreated T cells suggesting the presence of radiosensitive suppressor T cells for IgM RF in these individuals. Irridiated T cells were compared to untreated T cells for capacity to support IgM RF production by autologous B cells from 12 randomly selected donors at T:B cell ratios of 3:1. Untreated T cells from 4 of 12 individuals were capable of cooperating in induction of IgM RF production by autologous B cells, whereas irradiated T cells supported IgM RF production in 6 of 12 individuals. Levels of IgM RF production in all 6 individuals were significantly higher with irradiated T cells than with untreated T cells; in 2 individuals IgM RF synthesis by autologous B cells was observed only in the presence of irradiated T cells. In 4 of 6 individuals increases in the ratio of IgM RF total IgM synthesis occured with irradiated T cells (when compared to untreated T cells), suggesting disproportionate suppression of RF production. These results indicate the presence of radiosensitive T cells capable of suppressing IgM RF production in a significant fraction of healthy adults and raise the possibility that these cells may regulate in vivo expression of RF.

  10. Detection of narcotics with an immunoassay film badge

    SciTech Connect

    Lukens, H.R.

    1993-12-31

    Efficient personnel performance, a major requirement for a safe nuclear industry, is jeopardized where personnel use narcotics. However, detection of narcotics at nuclear plants is a challenge. The unique specificity and sensitivity of an immunoassay has been implemented in the form of a small, dry immunoassay film badge (IFB) for the detection of vapors emitted by narcotics. The device is suitable as an area monitor, and its characteristics are suitable for use as a breath monitor for the detection of drug use.

  11. Clinical Applications of Capillary Electrophoresis-Based Immunoassays

    PubMed Central

    Moser, Annette C.; Willicott, Corey W.; Hage, David S.

    2014-01-01

    Immunoassays have long been an important set of tools in clinical laboratories for the detection, diagnosis and treatment of disease. Over the last two decades there has been growing interest in utilizing capillary electrophoresis (CE) as a means for conducting immunoassays with clinical samples. The resulting method is known as a CE immunoassay. This approach makes use of the selective and strong binding of antibodies for their targets, as is employed in a traditional immunoassay, and combines this with the speed, efficiency, and small sample requirements of CE. This review discusses the variety of ways in which CE immunoassays have been employed with clinical samples. An overview of the formats and detection modes that have been employed in these applications is first presented. A more detailed discussion is then given on the type of clinical targets and samples that have been measured or studied by using CE immunoassays. Particular attention is given to the use of this method in the fields of endocrinology, pharmaceutical measurements, protein and peptide analysis, immunology, infectious disease detection, and oncology. Representative applications in each of these areas are described, with these examples involving work with both traditional and microanalytical CE systems. PMID:24132682

  12. Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

    PubMed Central

    Hesketh, Emily E.; Dransfield, Ian; Kluth, David C.; Hughes, Jeremy

    2015-01-01

    Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)+PI (Propidium Iodide)+ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM+AnnV+PI+ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM-AnnV+PI+ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration. PMID:26121639

  13. Comparative Accuracy of the InBios Scrub Typhus Detect IgM Rapid Test for the Detection of IgM Antibodies by Using Conventional Serology.

    PubMed

    Kingston, Hugh W F; Blacksell, Stuart D; Tanganuchitcharnchai, Ampai; Laongnualpanich, Achara; Basnyat, Buddha; Day, Nicholas P J; Paris, Daniel H

    2015-10-01

    This study investigated the comparative accuracy of a recombinant 56-kDa type-specific antigen-based rapid diagnostic test (RDT) for scrub typhus for the detection of IgM antibodies by using conventional serology in well-characterized serum samples from undifferentiated febrile illness patients. The RDT showed high specificity and promising comparative accuracy, with 82% sensitivity and 98% specificity for samples defined positive at an IgM indirect immunofluorescence assay positivity cutoff titer of ?1:1,600 versus 92% and 95% at ?1:6,400, respectively. PMID:26291089

  14. Comparative Accuracy of the InBios Scrub Typhus Detect IgM Rapid Test for the Detection of IgM Antibodies by Using Conventional Serology

    PubMed Central

    Kingston, Hugh W. F.; Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Laongnualpanich, Achara; Basnyat, Buddha; Day, Nicholas P. J.

    2015-01-01

    This study investigated the comparative accuracy of a recombinant 56-kDa type-specific antigen-based rapid diagnostic test (RDT) for scrub typhus for the detection of IgM antibodies by using conventional serology in well-characterized serum samples from undifferentiated febrile illness patients. The RDT showed high specificity and promising comparative accuracy, with 82% sensitivity and 98% specificity for samples defined positive at an IgM indirect immunofluorescence assay positivity cutoff titer of ?1:1,600 versus 92% and 95% at ?1:6,400, respectively. PMID:26291089

  15. Genetics Home Reference: X-linked hyper IgM syndrome

    MedlinePLUS

    ... germs, marking them for destruction. There are several classes of antibodies, and each one has a different ... IgM syndrome have low levels of three other classes of antibodies: immunoglobulin G (IgG), immunoglobulin A (IgA), ...

  16. [Comparison of methods for the demonstration of Treponema-specific IgM].

    PubMed

    Panuccio, A; Borroni, G; Gelosa, L

    1989-01-01

    In this research 113 sera have been analysed with three methods for IgM treponema-specific determination: IgM-SPHA, IgM-EIA and 19S IgM FTA-ABS. Among these sera, 33 samples related to non-treated patients at different stages of infection, and 80 samples to treated patients. The results point out a light sensibility of the IgM-SPHA in primary lues. The IgM-EIA test has performed a good specificity, but displayed a sensibility lower to the 19S IgM FTA-ABS, which proved the best of tests. About the cases of treated lues at different stages, in 23 samples with VDRL negative has been found no positivity at three tests used, while in 49 samples with VDRL positive 8 are resulted positive at 19S IgM FTA-ABS. PMID:2518811

  17. Ground and Space-based Imaging of the IGM with CWI, FIREBALL, and ISTOS

    NASA Astrophysics Data System (ADS)

    Martin, Christopher

    I discuss several experimental projects underway or proposed designed to discover and map emission from the IGM. The Cosmic Web Imager (CWI) is a ground-based high resolution spectrometer designed to detect low surface brightness emission from redshifted Lyman alpha, OVI and CIV at Palomar and Keck Observatories, over 2 < z < 6. FIREBALL is a balloonborne telescope/spectrometer that will search for IGM emission in the 0.3 < z < 1.0 range in the UV balloon window at 2000 Angstroms. ISTOS (Imaging Spectroscopic Telescope for Origins Surveys) is a proposed SMEX mission to discover and map baryons in the IGM from 0.05 < z < 2 over the 1250-2800 Angstrom range. All three experiments are integral field spectrometers designed to detect low surface brightness emission and reject point and diffuse foregrounds, and all have sensitives required to detect IGM and Circum-Galactic Medium emission at levels predicted by three independent cosmological simulations.

  18. Vesiculovirus Neutralization by Natural IgM and Complement

    PubMed Central

    Tesfay, Mulu Z.; Ammayappan, Arun; Federspiel, Mark J.; Barber, Glen N.; Stojdl, David; Peng, Kah-Whye

    2014-01-01

    ABSTRACT Because of its very low human seroprevalence, vesicular stomatitis virus (VSV) has promise as a systemic oncolytic agent for human cancer therapy. However, as demonstrated in this report, the VSV infectious titer drops by 4 log units during the first hour of exposure to nonimmune human serum. This neutralization occurs relatively slowly and is mediated by the concerted actions of natural IgM and complement. Maraba virus, whose G protein is about 80% homologous to that of VSV, is relatively resistant to the neutralizing activity of nonimmune human serum. We therefore constructed and rescued a recombinant VSV whose G gene was replaced by the corresponding gene from Maraba virus. Comparison of the parental VSV and VSV with Maraba G substituted revealed nearly identical host range properties and replication kinetics on a panel of tumor cell lines. Moreover, in contrast to the parental VSV, the VSV with Maraba G substituted was resistant to nonimmune human serum. Overall, our data suggest that VSV with Maraba G substituted should be further investigated as a candidate for human systemic oncolytic virotherapy applications. IMPORTANCE Oncolytic virotherapy is a promising approach for the treatment of disseminated cancers, but antibody neutralization of circulating oncolytic virus particles remains a formidable barrier. In this work, we developed a pseudotyped vesicular stomatitis virus (VSV) with a glycoprotein of Maraba virus, a closely related but serologically distinct member of the family Rhabdoviridae, which demonstrated greatly diminished susceptibility to both nonimmune and VSV-immune serum neutralization. VSV with Maraba G substituted or lentiviral vectors should therefore be further investigated as candidates for human systemic oncolytic virotherapy and gene therapy applications. PMID:24648451

  19. Ultrasensitive on-chip immunoassays with a nanoparticle-assembled photonic crystal.

    PubMed

    Han, Jin-Hee; Sudheendra, L; Kim, Hee-Joo; Gee, Shirley J; Hammock, Bruce D; Kennedy, Ian M

    2012-10-23

    Electrophoretic particle entrapment system (EPES) is employed to generate 2D array of nanoparticles coated with biological molecules (i.e., antibodies). Phase matching of the excitation and the emission in the 2D arrays with particles produces a highly enhanced fluorescence signal that was shown to improve the limit of detection in immunoassays. The phase matching is achieved when the particle are in the sub-100 nm range. A comparison between different size particles shows that the sensitivity of an immunoassay is extended to a range that is difficult to achieve with standard technology (e.g., enzyme-linked immunosorbent assay-ELISA). The effectiveness of this novel configuration of particle-in-a-well was demonstrated with an assay for human epidermal growth factor receptor 2 (HER2; breast cancer biomarker), with a detection limit as low as 10 attomolar (aM) in less than 10 ?L of serum-based sample. The limit of detection of HER2 indicated far superior assay performance compared to the corresponding standard 96-well plate-based ELISA. The particle-based photonic platform reduces the reagent volume and the time for performing an assay in comparison to competing methods. The simplicity of operation and the level of sensitivity demonstrated here can be used for rapid and early stage detection of biomarkers. PMID:22957818

  20. Protein immunoassay methods for detection of biotech crops: applications, limitations, and practical considerations.

    PubMed

    Stave, James W

    2002-01-01

    Immunoassay methods are available for detection and quantitation of proteins expressed by most biotechnology-derived crops in commercial production. The 2 most common test formats are enzyme-linked immunosorbent assay (ELISA) and immunochromatographic (lateral flow) strip tests. Two ELISA methods, one for Roundup Ready soybeans and one for MON810 CrylAb corn, were the subject of large international collaborative studies and were demonstrated to quantitatively determine the concentrations of biotech crops in samples of ground grain. Quantitative ELISA methods are also useful for analysis of processed fractions of agricultural commodities such as soybean toasted meal or corn flour. Both strip tests and ELISAs for biotech crops are currently being used on a large scale in the United States to manage the sale and distribution of grain. In these applications, tests are used to determine if the concentration of biotech grain is above or below specified threshold limits. Using existing U.S. Department of Agriculture sampling techniques, the reliability of the threshold determination is expressed in terms of statistical confidence rather than analytical precision. Combining the use of protein immunoassays with Identity Preservation systems provides an effective means of characterizing the raw and processed agricultural inputs to the food production system in a way that allows food producers to comply with labeling laws. PMID:12083275

  1. Photoelectrochemical detection of enzymatically generated CdS nanoparticles: Application to development of immunoassay.

    PubMed

    Barroso, Javier; Saa, Laura; Grinyte, Ruta; Pavlov, Valeri

    2016-03-15

    We report an innovative photoelectrochemical process (PEC) based on graphite electrode modified with electroactive polyvinylpyridine bearing osmium complex (Os-PVP). The system relies on the in situ enzymatic generation of CdS quantum dots (QDs). Alkaline phosphatase (ALP) catalyzes the hydrolisis of sodium thiophosphate (TP) to hydrogen sulfide (H2S) which in the presence Cd(2+) ions yields CdS semiconductor nanoparticles (SNPs). Irradiation of SNPs with the standard laboratory UV-illuminator (wavelength of 365nm) results in photooxidation of 1-thioglycerol (TG) mediated by Os-PVP complex on the surface of graphite electrode at applied potential of 0.31V vs. Ag/AgCl. A novel immunoassay based on specific enzyme linked immunosorbent assay (ELISA) combined with the PEC methodology was developed. Having selected the affinity interaction between bovine serum albumine (BSA) with anti-BSA antibody (AB) as a model system, we built the PEC immunoassay for AB. The new assay displays a linear range up to 20ngmL(-1) and a detection limit (DL) of 2ngmL(-1) (S/N=3) which is lower 5 times that of the traditional chromogenic ELISA test employing p-nitro-phenyl phosphate (pNPP). PMID:26432195

  2. Clinical performance evaluation of four automated chemiluminescence immunoassays for hepatitis C virus antibody detection.

    PubMed

    Kim, Sinyoung; Kim, Jeong-Ho; Yoon, Seoyoung; Park, Youn-Hee; Kim, Hyon-Suk

    2008-12-01

    Various automated chemiluminescence immunoassay (CLIA) analyzers for the detection of antibodies to hepatitis C virus (HCV) are now commercially available in clinical laboratories and are replacing conventional enzyme immunoassays. We investigated the performance of four anti-HCV CLIAs (the Architect Anti-HCV assay on the Architect i2000 system, the Vitros Anti-HCV assay on the Vitros ECiQ Immunodiagnostic System, the Access HCV Ab PLUS assay on the UniCel DxI 800 analyzer, and the newly developed Elecsys Anti-HCV assay on the Cobas e 411 analyzer). The total percent coefficient of variation values of imprecision were 3.5 to 5.7% with positive control materials and 7.2 to 10.2% with negative control materials. The agreement between the results of the Elecsys, Architect, Vitros, and Access CLIAs ranged from 94.5 to 98.1%. The clinical sensitivity of all CLIAs was 100%. Each CLIA showed excellent reproducibility and clinical sensitivity. The Elecsys, Architect, Vitros, and Access CLIAs showed clinical specificities of 98.2, 98.8, 96.5, and 98.2%. PMID:18945839

  3. Ultrasensitive On-Chip Immunoassays with a Nanoparticle-Assembled Photonic Crystal

    PubMed Central

    Han, Jin-Hee; Sudheendra, L.; Kim, Hee-Joo; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2012-01-01

    Electrophoretic particle entrapment system (EPES) is employed to generate 2D array of nanoparticles coated with biological molecules (i.e. antibodies). Phase matching of the excitation and the emission in the 2D arrays with particles produces a highly enhanced fluorescence signal that was shown to improve the limit of detection in immunoassays. The phase matching is achieved when the particle are in the sub-100 nm range. A comparison between different size particles shows that the sensitivity of an immunoassay is extended to a range that is difficult to achieve with standard technology (e.g. Enzyme-linked immunosorbent assay-ELISA). The effectiveness of this novel configuration of particle-in-a well was demonstrated with an assay for human epidermal growth factor receptor 2 (HER2; breast cancer biomarker), with a detection limit as low as 10 aM in less than 10 ?l of serum-based sample. The limit of detection of HER2 indicated far superior assay performance compared to the corresponding standard 96-well plate-based ELISA. The particle-based photonic platform reduces the reagent volume and the time for performing an assay in comparison to competing methods. The simplicity of operation and the level of sensitivity demonstrated here can be used for rapid and early-stage detection of biomarkers. PMID:22957818

  4. Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.

    PubMed

    Liu, Yuan; Wu, Aihua; Hu, Jing; Lin, Manman; Wen, Mengtang; Zhang, Xiao; Xu, Chongxin; Hu, Xiaodan; Zhong, Jianfeng; Jiao, Lingxia; Xie, Yajing; Zhang, Cunzhen; Yu, Xiangyang; Liang, Ying; Liu, Xianjin

    2015-08-15

    3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA-bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I50 and I10 values of purified mAbs were 0.63 and 0.13 ?g/ml, respectively, with a dynamic range between 0.19 and 2.04 ?g/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 ?g/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples. PMID:25957127

  5. Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid*

    PubMed Central

    Planque, Stephanie A.; Nishiyama, Yasuhiro; Hara, Mariko; Sonoda, Sari; Murphy, Sarah K.; Watanabe, Kenji; Mitsuda, Yukie; Brown, Eric L.; Massey, Richard J.; Primmer, Stanley R.; O'Nuallain, Brian; Paul, Sudhir

    2014-01-01

    Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded ?-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid ? peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function. PMID:24648510

  6. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  7. Performance characteristics of eight estradiol immunoassays.

    PubMed

    Yang, David T; Owen, William E; Ramsay, Carol S; Xie, Hui; Roberts, William L

    2004-09-01

    Measurement of estradiol is useful in assisted reproduction, evaluation of infertility, menopause, and male feminization. The analytic performance of 8 estradiol immunoassays was evaluated. The imprecision and accuracy of the Access, ADVIA Centaur, ARCHITECT i2000, AutoDELFIA, Elecsys 2010, IMMULITE 2000, and Vitros ECi estradiol assays (see text for proprietary information) were evaluated by using an isotope dilution-gas chromatography-mass spectrometry (ID-GC-MS) reference method. The coefficient of variation (CV) ranged from 6.9% on the Elecsys 2010 to 42.6% on the ADVIA Centaur at an estradiol concentration of 18 pg/mL (66 pmol/L), with the ARCHITECT i2000 assay in development and the Vitros ECi having a CV below 10% at this estradiol concentration. Agreement between the automated assays and ID-GC-MS was variable, with slopes ranging from 0.87 to 1.20. The Access, ARCHITECT i2000 in development, and the IMMULITE 2000 were the most accurate, with slopes of 0.99, 0.98, and 1.03, respectively. These findings indicate that the ARCHITECT i2000 estradiol assay in development had the best precision and accuracy of the assays evaluated for measurement of serum estradiol concentrations. PMID:15362362

  8. Natural IgM is Produced by CD5? Plasma Cells that Occupy a Distinct Survival Niche in Bone Marrow

    PubMed Central

    Reynolds, Alexander E.; Kuraoka, Masayuki; Kelsoe, Garnett

    2014-01-01

    Natural IgM is constitutively present in the serum, where it aids in the early control of viral and bacterial expansion. Natural IgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. Nevertheless, the origins of natural IgM have not been precisely defined. Previous studies have focused on the role of CD5+ B1 cells in the production of natural IgM, but we show here that a discrete population of CD5? IgM plasmablasts and plasma cells in the BM produces the majority of serum IgM in resting mice. These ASC originate from peritoneal cavity-resident cells, as transfer of peritoneal cells completely restores serum IgM and the specific compartment of BM ASC in Rag1-deficient mice. We show that BM natural IgM ASC arise from a fetal-lineage progenitor that is neither B1a nor B1b, and that this IgM ASC compartment contains a substantial fraction of long-lived plasma cells that do not occupy the IgG plasma cell survival niche in the BM, but are instead supported by IL-5. In summary, we have identified the primary source of natural IgM, and shown that these ASC are maintained long-term in a unique survival niche within the BM. PMID:25429072

  9. Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels

    PubMed Central

    Tahara, Yusuke; Huang, Zhe; Kiritoshi, Tetsuro; Onodera, Takeshi; Toko, Kiyoshi

    2014-01-01

    The monitoring of salivary cortisol as a key biomarker of an individual’s stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38?ppt range with a measurement range of 10?ppt–100?ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8?min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R?=?0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels. PMID:25152888

  10. Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics

    PubMed Central

    Johlfs, Mary G.; Gorjala, Priyatham; Urasaki, Yasuyo; Le, Thuc T.; Fiscus, Ronald R.

    2015-01-01

    Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-I? and PKG-I? isoforms are present in preadipocytes, only PKG-I? isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems. PMID:26132171

  11. Survivability of immunoassay reagents exposed to the space radiation environment on board the ESA BIOPAN-6 platform as a prelude to performing immunoassays on Mars.

    PubMed

    Derveni, Mariliza; Allen, Marjorie; Sawakuchi, Gabriel O; Yukihara, Eduardo G; Richter, Lutz; Sims, Mark R; Cullen, David C

    2013-01-01

    The Life Marker Chip (LMC) instrument is an immunoassay-based sensor that will attempt to detect signatures of life in the subsurface of Mars. The molecular reagents at the core of the LMC have no heritage of interplanetary mission use; therefore, the design of such an instrument must take into account a number of risk factors, including the radiation environment that will be encountered during a mission to Mars. To study the effects of space radiation on immunoassay reagents, primarily antibodies, a space study was performed on the European Space Agency's 2007 BIOPAN-6 low-Earth orbit (LEO) space exposure platform to complement a set of ground-based radiation studies. Two antibodies were used in the study, which were lyophilized and packaged in the intended LMC format and loaded into a custom-made sample holder unit that was mounted on the BIOPAN-6 platform. The BIOPAN mission went into LEO for 12 days, after which all samples were recovered and the antibody binding performance was measured via enzyme-linked immunosorbent assays (ELISA). The factors expected to affect antibody performance were the physical conditions of a space mission and the exposure to space conditions, primarily the radiation environment in LEO. Both antibodies survived inactivation by these factors, as concluded from the comparison between the flight samples and a number of shipping and storage controls. This work, in combination with the ground-based radiation tests on representative LMC antibodies, has helped to reduce the risk of using antibodies in a planetary exploration mission context. PMID:23286207

  12. SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION (SITE) REPORT FOR THE WESTINGHOUSE BIOANALYTICAL SYSTEMS PENTACHLOROPHENOL (PCP) IMMUNOASSAYS

    EPA Science Inventory

    The results of the demonstration of two Westinghouse Bio-Analytic Systems (WBAS) immunoassay technologies are described in this report. The immunoassays measure parts per billion concentrations of pentachlorophenol in environmental water samples. The study was conducted under the...

  13. SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION PROGRAM DEMONSTRATION PLAN FOR WESTINGHOUSE BIO-ANALYTIC SYSTEMS PENTACHLOROPHENOL IMMUNOASSAYS

    EPA Science Inventory

    This plan provides a detailed design and description of the demonstration and evaluation program for the Westinghouse Bio-Analytic Systems immunoassay technologies specific for the analysis of pentachlorophenol. he immunoassays measure parts per billion concentrations of pentachl...

  14. The Effects of Sample Matrices on Immunoassays to Detect Microcystin-LR in Water

    EPA Science Inventory

    Abstract: Immunoassays are widely used biochemical techniques to detect microcystins in environmental samples. The use of immunoassays for the detection of microcystins is vulnerable to matrix components and other interferents. This study is an evaluation of the effects of interf...

  15. PRNP variants in goats reduce sensitivity of detection of PrPSc by immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoassays are extensively utilized in disease diagnostics with monoclonal antibodies serving as critical tools within the assay. Detection of scrapie in sheep and goats relies heavily on immunoassays including immunohistochemistry, western blotting, and ELISA. In the United States, regulatory tes...

  16. Functional characterization of IgM+ B cells and adaptive immunity in lumpfish (Cyclopterus lumpus L.).

    PubMed

    Rønneseth, Anita; Ghebretnsae, Dawit B; Wergeland, Heidrun I; Haugland, Gyri T

    2015-10-01

    The innate immune responses in lumpfish (Cyclopterus lumpus L.) have been shown to be functional, but little is currently known about the B cells, immunoglobulins or adaptive immune responses in this species. We have used anti-IgM antiserum to isolate B cells and compared them morphologically and functionally with other cell types. The fraction of IgM(+) cells among isolated peripheral blood leukocytes (PBL), head kidney leukocytes (HKL) and spleen leukocytes (SL) was in the range of 40%, 12% and 34%, respectively. The IgM(+) B cells had high phagocytic ability and were the predominant phagocytes in blood with higher capacity than IgM(+) B cells in HKL. Interestingly, among PBL, the most potent phagocytes were, in addition to monocytes, some small agranular uncharacterized IgM(-) cells. The IgM(+) B cells were positive for acid phosphatases (AcP), but negative for myeloperoxidase (MPO). Neutrophils were positive for MPO, while monocytes/macrophages and dendritic-like cells stained negatively. Monocytes/macrophages and the small, agranular IgM(-) cells stained most strongly positive for AcP corresponding to their high phagocytic capacity. Further, the ability to produce specific antibodies upon immunization verified adaptive immunity in the species. The high proportion of phagocytic IgM(+) B cells and their phagocytic ability indicate a significant role of phagocytic B cells in lumpfish innate immunity. The present analyses also give strong indications that vaccination and immunostimulation of farmed lumpfish can be used to prevent disease and mortality caused by pathogenic organisms. PMID:26021455

  17. Pancreatic Enzymes

    MedlinePLUS

    ... adequate nutrition and prevent weight loss. MCT (Medium Chain Triglyceride) oil may help control weight loss in ... enzyme products are regulated by the United States Food and Drug Administration (FDA) to ensure their effectiveness, ...

  18. Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays.

    PubMed

    Evangelista, R A; Pollak, A; Templeton, E F

    1991-08-15

    Enzyme-amplified lanthanide luminescence (EALL) is a new method which has been developed for enzymatically amplified signal detection in ultrasensitive bioanalytical assays where an enzyme is used as label or is itself the analyte of interest. Signal generation is performed by enzymatically transforming a substrate into a product which forms a luminescent lanthanide chelate; the product chelate can then be detected using time-resolved or normal fluorescence methods. Alkaline phosphatase substrates have been developed and demonstrated in a model immunoassay in microwell format. The method has also been demonstrated for detection of a variety of other hydrolytic and oxidative enzymes. Thus the EALL method shows promise for use in a wide variety of bioanalytical applications. PMID:1952068

  19. The Omentum Is a Site of Protective IgM Production during Intracellular Bacterial Infection

    PubMed Central

    Jones, Derek D.; Racine, Rachael; Wittmer, Susan T.; Harston, Louise; Papillion, Amber M.; Dishaw, Lisa M.; Randall, Troy D.; Woodland, David L.

    2015-01-01

    Infection of mice with the bacterium Ehrlichia muris elicits a protective T cell-independent (TI) IgM response mediated primarily by a population of CD11c-expressing plasmablasts in the spleen. Although splenic marginal zone (MZ) B cells are considered to be important for TI responses to blood-borne pathogens, MZ B cells were not responsible for generating plasmablasts in response to Ehrlichia muris. Moreover, antigen-specific serum IgM was decreased only modestly in splenectomized mice and in mice that lacked spleen, lymph nodes, and Peyer's patches (SLP mice). Both splenectomized and SLP mice were protected against lethal ehrlichial challenge infection. Moreover, we found a high frequency of Ehrlichia-specific plasmablasts in the omentum of both conventional and SLP mice. Omental plasmablasts elicited during Ehrlichia infection lacked expression of CD138 but expressed CD11c in a manner similar to that of their splenic counterparts. Selective ablation of CD11c-expressing B cells nearly eliminated the omental Ehrlichia-specific plasmablasts and reduced antigen-specific serum IgM, identifying the omental B cells as a source of IgM production in the SLP mice. Generation of the omental plasmablasts was route dependent, as they were detected following peritoneal infection but not following intravenous infection. Our data identify the omentum as an important auxiliary site of IgM production during intracellular bacterial infection. PMID:25776744

  20. Influenza Virus-Specific Neutralizing IgM Antibodies Persist for a Lifetime

    PubMed Central

    Skountzou, Ioanna; Satyabhama, Lakshmipriyadarshini; Stavropoulou, Anastasia; Ashraf, Zuhha; Esser, E. Stein; Vassilieva, Elena; Koutsonanos, Dimitrios; Compans, Richard

    2014-01-01

    Detection of immunoglobulin M (IgM) antibodies has long been used as an important diagnostic tool for identifying active viral infections, but their relevance in later stages has not been clearly defined in vivo. In this study, we followed the kinetics, longevity, and function of influenza virus-specific IgM antibodies for 2 years following sublethal infection of mice with live mouse-adapted A/PR/8/34 virus or immunization with formalin-inactivated virus. These groups mounted robust protective immune responses and survived lethal challenges with 50× 50% lethal dose (LD50) mouse-adapted A/PR/8/34 virus 600 days after the primary exposure. Surprisingly, the virus-specific IgM antibodies persisted along with IgG antibodies, and we found a significantly higher number of IgM-positive (IgM+) virus-specific plasma cells than IgG+ plasma cells that persisted for at least 9 months postexposure. The IgM antibodies were functional as they neutralized influenza virus in the presence of complement just as well as IgG antibodies did. PMID:25165027

  1. The omentum is a site of protective IgM production during intracellular bacterial infection.

    PubMed

    Jones, Derek D; Racine, Rachael; Wittmer, Susan T; Harston, Louise; Papillion, Amber M; Dishaw, Lisa M; Randall, Troy D; Woodland, David L; Winslow, Gary M

    2015-05-01

    Infection of mice with the bacterium Ehrlichia muris elicits a protective T cell-independent (TI) IgM response mediated primarily by a population of CD11c-expressing plasmablasts in the spleen. Although splenic marginal zone (MZ) B cells are considered to be important for TI responses to blood-borne pathogens, MZ B cells were not responsible for generating plasmablasts in response to Ehrlichia muris. Moreover, antigen-specific serum IgM was decreased only modestly in splenectomized mice and in mice that lacked spleen, lymph nodes, and Peyer's patches (SLP mice). Both splenectomized and SLP mice were protected against lethal ehrlichial challenge infection. Moreover, we found a high frequency of Ehrlichia-specific plasmablasts in the omentum of both conventional and SLP mice. Omental plasmablasts elicited during Ehrlichia infection lacked expression of CD138 but expressed CD11c in a manner similar to that of their splenic counterparts. Selective ablation of CD11c-expressing B cells nearly eliminated the omental Ehrlichia-specific plasmablasts and reduced antigen-specific serum IgM, identifying the omental B cells as a source of IgM production in the SLP mice. Generation of the omental plasmablasts was route dependent, as they were detected following peritoneal infection but not following intravenous infection. Our data identify the omentum as an important auxiliary site of IgM production during intracellular bacterial infection. PMID:25776744

  2. Natural IgM: Beneficial autoantibodies for the control of inflammatory and autoimmune disease?

    PubMed Central

    Grönwall, Caroline; Silverman, Gregg J.

    2014-01-01

    Natural IgM are highly represented in the circulation at birth, and these often autoreactive antibodies have been postulated to have innate-like properties and play crucial roles in apoptotic cell clearance, tissue homeostasis, and immune modulation. This review summarizes the known properties of these IgM autoantibodies, and the evidence that these anti-apoptotic cell IgM natural antibodies can regulate inflammatory responses through ancient pathways of the innate immune system that first arose long before the initial emergence of the adaptive immune system. While the regulatory contributions of these natural IgM autoantibodies are certainly not an essential and fundamental component of host defenses, these provide an additional layer to further protect the host. More importantly, these IgM antibody responses are highly inducible and their up-regulation can be a powerful means for the host to survive in a setting of chronic inflammation. The observed beneficial clinical associations for cardiovascular disease and autoimmunity, as well as opportunities for potential therapeutic implications are discussed. PMID:24691998

  3. West Nile virus IgM and IgG antibodies three years post- infection

    PubMed Central

    Papa, A; Anastasiadou, A; Delianidou, M

    2015-01-01

    Background: West Nile virus (WNV) causes to humans a variety of symptoms, from asymptomatic infection to severe neuroinvasive disease. In a previous study, it was shown that WNV IgM antibodies persisted in three of 26 (12%) patients, nine months after onset of the symptoms. The aim of the present study was to test 10 of these patients, three years post-infection for probable persistence of IgM antibodies and to investigate their IgG antibody patterns. Material and Methods: In summer 2013 serum samples were collected from 10 persons who were infected with WNV in 2010; 6 of them had a neuroinvasive disease. The three persons with detectable WNV IgM antibodies, nine months after onset of the symptoms, were included in the study. All samples were tested by ELISA in parallel with their stored paired samples taken in 2011. The positive results were confirmed by neutralization test. Results: WNV IgM antibodies were still detectable in the three persons, while high levels of WNV IgG and neutralizing antibodies were present in nine of the 10 persons, regardless the involvement of the nervous system. Conclusions: WNV IgM antibodies persist for more than three years in 12% of patients with WNV infection, while WNV IgG antibodies persist and even increase their levels, regardless the involvement of the nervous system, suggesting that the immune response in the symptomatic WNV infections is strong and long-lasting. Hippokratia 2015, 19 (1): 34-36. PMID:26435644

  4. Identification of the minimal binding region of a Plasmodium falciparum IgM binding PfEMP1 domain

    PubMed Central

    Semblat, Jean-Philippe; Ghumra, Ashfaq; Czajkowsky, Daniel M.; Wallis, Russell; Mitchell, Daniel A.; Raza, Ahmed; Rowe, J.Alexandra

    2015-01-01

    Binding of host immunoglobulin is a common immune evasion mechanism demonstrated by microbial pathogens. Previous work showed that the malaria parasite Plasmodium falciparum binds the Fc-region of human IgM molecules, resulting in a coating of IgM on the surface of infected erythrocytes. IgM binding is a property of P. falciparum strains showing virulence-related phenotypes such as erythrocyte rosetting. The parasite ligands for IgM binding are members of the diverse P. falciparum Erythrocyte Membrane Protein One (PfEMP1) family. However, little is known about the amino acid sequence requirements for IgM binding. Here we studied an IgM binding domain from a rosette-mediating PfEMP1 variant, DBL4? of TM284var1, and found that the minimal IgM binding region mapped to the central region of the DBL domain, comprising all of subdomain 2 and adjoining parts of subdomains 1 and 3. Site-directed mutagenesis of charged amino acids within subdomain 2, predicted by molecular modelling to form the IgM binding site, showed no marked effect on IgM binding properties. Overall, this study identifies the minimal IgM binding region of a PfEMP1 domain, and indicates that the existing homology model of PfEMP1-IgM interaction is incorrect. Further work is needed to identify the specific interaction site for IgM within the minimal binding region of PfEMP1. PMID:26094597

  5. Tyramine-based enzymatic conjugate repeats for ultrasensitive immunoassay accompanying tyramine signal amplification with enzymatic biocatalytic precipitation.

    PubMed

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

    2014-08-19

    A new impedimetric immunoassay protocol based on enzyme-triggered formation of tyramine-enzyme repeats on gold nanoparticle (AuNP) was designed for highly sensitive detection of carcinoembryonic antigen (CEA, as a model) by virtue of utilizing enzymatic biocatalytic precipitation toward 4-chloro-1-naphthol (4-CN) on anti-CEA antibody (Ab1)-modified immunosensor. Initially, AuNP was functionalized with horseradish peroxidase and detection antibody (HRP-AuNP-Ab2), and then HRP-tyramine conjugate was utilized for the formation of tyramine-HRP repeats through the triggering of the immobilized HRP on the AuNP with the aid of H2O2. In the presence of target CEA, the carried HRP-tyramine repeats accompanying the sandwiched immunocomplex catalyzed the 4-CN oxidation to produce an insoluble precipitation on the immunosensor, thus causing a local alteration of the conductivity. Three signal-transduction tags including HRP-Ab2, HRP-AuNP-Ab2, and HRP-AuNP-Ab2 with HRP-tyramine repeats were employed for target CEA evaluation, and improved analytical properties were achieved by HRP-AuNP-Ab2 with HRP-tyramine repeats. Using the unique signal-transduction tag, the analytical performance of the impedimetric immunoassay was studied in detail. Under the optimal conditions, the impedimetric immunosensor displayed a wide dynamic working range of between 0.5 pg mL(-1) and 40 ng mL(-1) with a detection limit (LOD) of 0.38 pg mL(-1) relative to target CEA. The coefficients of variation (CVs) were ?9.3% and 13.3% for the intra-assay and interassay, respectively. The levels of CEA in eight clinical serum specimens were measured by using the developed impedimetric immunosensor. The obtained results correlated well with those from the electrochemiluminescent (ECL)-based immunoassay with a correlation coefficient of 0.998. PMID:25088522

  6. Robust detection of peak signals for lateral flow immunoassays

    NASA Astrophysics Data System (ADS)

    Kim, Jongwon; Kim, Jong Dae; Nahm, Kie Bong; Choi, Eui Yul; Lee, Geumyoung

    2011-02-01

    Template matching method is presented to identify the peaks from the scanned signals of lateral flow immunoassay strips. The template is composed of two pulses separated by the distance of the control and the target ligand line in the assay, and is convolved with the scanned signal to deliver the maximum at the center of the two peaks. The peak regions were identified with the predefined distances from the center. Glycosylated haemoglobin immunoassay strips and fluorescent strip readers from Boditechmed Inc. were tested to estimate the lot and reader variations of the concentration measurands. The results showed the robustness of the propose method.

  7. Detection of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens

    PubMed Central

    Vojdani, Aristo

    2009-01-01

    Background Despite the first documented case of food allergy to cooked food in 1921 by Prausnitz and Kustner, all commercial food antigens are prepared from raw food. Furthermore, all IgE and IgG antibodies against dietary proteins offered by many clinical laboratories are measured against raw food antigens. Methods We developed an enzyme-linked immunosorbent assay for the measurement of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens. Sera with low or high reactivity to modified food antigens were subjected to myelin basic protein, oxidized low density lipoprotein, and advanced glycation end products (AGE) such as AGE-human serum albumin and AGE-hemoglobin. Results Compared to raw food antigens, IgE antibodies showed a 3–8-fold increase against processed food antigens in 31% of the patients. Similarly, IgG, IgA and IgM antibodies against modified food antigens overall were found at much higher levels than antibody reactions against raw food antigens. Almost every tested serum with high levels of antibodies against modified food antigens showed very high levels of antibodies against myelin basic protein, oxidized low density lipoprotein, AGE-human serum albumin and AGE-hemoglobin. Conclusion We conclude that the determination of food allergy, intolerance and sensitivity would be improved by testing IgE, IgG, IgA and IgM antibodies against both raw and processed food antigens. Antibodies against modified food antigens, by reacting with AGEs and tissue proteins, may cause perturbation in degenerative and autoimmune diseases such as diabetes, atherosclerosis, inflammation, autoimmunity, neurodegeneration and neuroautoimmunity. PMID:19435515

  8. Reversal of IgM deficiency following a gluten-free diet in seronegative celiac disease

    PubMed Central

    Montenegro, Lucia; Piscitelli, Domenico; Giorgio, Floriana; Covelli, Claudia; Fiore, Maria Grazia; Losurdo, Giuseppe; Iannone, Andrea; Ierardi, Enzo; Di Leo, Alfredo; Principi, Mariabeatrice

    2014-01-01

    Selective IgM deficiency (sIGMD) is very rare; it may be associated with celiac disease (CD). We present the case of an 18-year-old man with sIGMD masking seronegative CD. Symptoms included abdominal pain, diarrhea and weight loss. Laboratory tests showed reduced IgM, DQ2-HLA and negative anti-transglutaminase. Villous atrophy and diffuse immature lymphocytes were observed at histology. Tissue transglutaminase mRNA mucosal levels showed a 6-fold increase. The patient was treated with a gluten-free diet (GFD) and six months later the symptoms had disappeared, the villous architecture was restored and mucosal tissue transglutaminase mRNA was comparable to that of healthy subjects. After 1 year of GFD, a complete restoration of normal IgM values was observed and duodenal biopsy showed a reduction of immature lymphocytes and normal appearance of mature immune cells. PMID:25516687

  9. Diagnostic and prognostic significance of the IgM antibody to the Hepatitis delta virus

    SciTech Connect

    Farci, P.; Gerin, J.L.; Aragona, M.; Lindsey, I.; Crivelli, O.; Balestrieri, A.; Smedile, A.; Thomas, H.C.; Rizzetto, M.

    1986-03-21

    The IgM class antibody to the hepatitis delta virus (HDV) was determined in different clinical categories of hepatitis B surface antigen carriers infected by the HDV (positive in the test for total antibody to HDV). The IgM antibody was found at high titers in each 70 patients with inflammatory liver disease and at a low titer in one six patients with inactive cirrhosis; it was not found in eight carriers with normal liver histology. Testing for Igm antibody to HDV distinguishes hepatitis B surface antigen carriers who have underlying inflammatory HDV liver disease from those with past HDV infection and provides prognostic information on the course of chronic HDV hepatitis.

  10. Cold urticaria associated with C4 deficiency and elevated IgM.

    PubMed

    Stafford, C T; Jamieson, D M

    1986-04-01

    Various immunologic abnormalities have been implicated in cold urticaria. This is the first report of cold urticaria associated with C4 deficiency and elevated IgM. A 12-year-old male developed urticaria upon exposure to cold. He denied fever, purpura, hemoglobinuria, Raynaud's disease, or arthralgias. Family history was negative for cold urticaria. Immunologic studies revealed elevated IgM (186 mg/dL) as well as decreased CH100 and C4 (8.0 mg/dL). C1, C2, and C3 were normal. Ice cube skin test was positive, but passive transfer tests were negative. Biopsy was not diagnostic for vasculitis, although it revealed a few immunofluorescent deposits of IgM and C4. Complement genetic studies revealed deficiency of two half-null C4 haplotypes expressed as C4A*3QO and B*2QO. PMID:3963523

  11. Single-Digit Pathogen and Attomolar Detection with the Naked Eye Using Liposome-Amplified Plasmonic Immunoassay.

    PubMed

    Bui, Minh-Phuong Ngoc; Ahmed, Snober; Abbas, Abdennour

    2015-09-01

    We introduce an enzyme-free plasmonic immunoassay with a binary (all-or-none) response. The presence of a single pathogen in the sample results in a chemical cascade reaction leading to a large red to dark-blue colorimetric shift visible to the naked eye. The immediate and amplified response is initiated by a triggered breakdown of cysteine-loaded nanoliposomes and subsequent aggregation of plasmonic gold nanoparticles. Our approach enabled visual detection of a single-digit live pathogen of Salmonella, Listeria, and E. coli O157 in water and food samples. Furthermore, the assay allowed a naked-eye detection of target antibody concentrations as low as 6.7 attomolar (600 molecules in 150 ?L); six orders of magnitude lower than conventional enzyme-linked immunosorbent assay (ELISA). PMID:26308387

  12. Antagonism of cannabinoid receptor 2 pathway suppresses IL-6-induced immunoglobulin IgM secretion

    PubMed Central

    2014-01-01

    Background Cannabinoid receptor 2 (CB2) is expressed predominantly in the immune system, particularly in plasma cells, raising the possibility that targeting the CB2 pathway could yield an immunomodulatory effect. Although the role of CB2 in mediating immunoglobulin class switching has been reported, the effects of targeting the CB2 pathway on immunoglobulin secretion per se remain unclear. Methods Human B cell line SKW 6.4, which is capable of differentiating into IgM-secreting cells once treated with human IL-6, was employed as the cell model. SKW 6.4 cells were incubated for 4 days with CB2 ligands plus IL-6 (100 U/ml). The amount of secreted IgM was determined by an ELISA. Cell proliferation was determined by the 3H-Thymidine incorporation assay. Signal molecules involved in the modulation of IgM secretion were examined by real-time RT-PCR and Western blot analyses or by using their specific inhibitors. Results We demonstrated that CB2 inverse agonists SR144528 and AM630, but not CB2 agonist HU308 or CB1 antagonist SR141716, effectively inhibited IL-6-induced secretion of soluble IgM without affecting cell proliferation as measured by thymidine uptake. SR144528 alone had no effects on the basal levels of IgM in the resting cells. These effects were receptor mediated, as pretreatment with CB2 agonist abrogated SR144528-mediated inhibition of IL-6 stimulated IgM secretion. Transcription factors relevant to B cell differentiation, Bcl-6 and PAX5, as well as the protein kinase STAT3 pathway were involved in the inhibition of IL-6-induced IgM by SR144528. Conclusions These results uncover a novel function of CB2 antagonists and suggest that CB2 ligands may be potential modulators of immunoglobulin secretion. PMID:24913620

  13. Monitoring Pesticides and Personal Care Chemicals in Water by Immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Due to the increasing number and quantity of organic pollutants, regulatory authorities require implementation of rapid, reliable, and cost-effective technologies for monitoring of water quality. Immunoassays provide a simple, powerful and inexpensive method for monitoring organic contaminants in bo...

  14. A Compact Immunoassay Platform Based on a Multicapillary Glass Plate

    PubMed Central

    Xue, Shuhua; Zeng, Hulie; Yang, Jianmin; Nakajima, Hizuru; Uchiyama, Katsumi

    2014-01-01

    A highly sensitive, rapid immunoassay performed in the multi-channels of a micro-well array consisting of a multicapillary glass plate (MCP) and a polydimethylsiloxane (PDMS) slide is described. The micro-dimensions and large surface area of the MCP permitted the diffusion distance to be decreased and the reaction efficiency to be increased. To confirm the concept of the method, human immunoglobulin A (h-IgA) was measured using both the proposed immunoassay system and the traditional 96-well plate method. The proposed method resulted in a 1/5-fold decrease of immunoassay time, and a 1/56-fold cut in reagent consumption with a 0.05 ng/mL of limit of detection (LOD) for IgA. The method was also applied to saliva samples obtained from healthy volunteers. The results correlated well to those obtained by the 96-well plate method. The method has the potential for use in disease diagnostic or on-site immunoassays. PMID:24859022

  15. A fluorescence immunoassay for soluble antigens employing flow cytometric detection.

    PubMed

    Lisi, P J; Huang, C W; Hoffman, R A; Teipel, J W

    1982-04-01

    A "sandwich" fluorescence immunoassay is described which does not require the physical separation of bound from free label. Antibody coated microspheres, sample and fluorescent antibody are reacted together as in a conventional 'sandwich' immunoassay except that separation and washing steps are omitted. After the reaction is completed, the suspension is introduced directly into a flow cytometer equipped with a laser light source and both fluorescent and scattered light detection capabilities. By gating fluorescence light accumulation on scattered light pulses, particles associated fluorescence may be selectively measured. The system was evaluated in a model immunoassay for human immunoglobulin (hIgG), employing anti-hIgG coated microspheres (1--5 micrometer and 40--50 micrometer polyacrylamide beads and 30--40 micrometer dextran beads), fluorescein-labeled rabbit anti-hIgG and a Spectrum III flow cytometer. Sensitivities of 10 ng/ml and intra-assay precisions of 2--10% were achieved in a serum matrix. The approach potentially provides a general nonseparation immunoassay format for quantitatively measuring both small and large molecular weight soluble antigens, as well as cell surface antigens. PMID:7039872

  16. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  17. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  18. CLINICAL AND VACCINE IMMUNOLOGY, May 2011, p. 851859 Vol. 18, No. 5 1556-6811/11/$12.00 doi:10.1128/CVI.00409-10

    E-print Network

    Fan, Jianqing

    Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II

  19. Development of an immunoassay to detect benzene adducts in hemoglobin

    SciTech Connect

    Grassman, J.A.

    1993-01-01

    The purpose of this project was to develop an immunoassay to detect the adducts formed in hemoglobin after exposure to benzene, which is known to cause bone marrow degeneration and acute myelogenous leukemia. The use of benzene-adduct detection as a biological monitoring method would permit measurement of low exposures and exposures sustained weeks earlier. The reactivity of hydroquinone, an important benzene metabolite, with blood proteins and amino acids was investigated in order to decide which antigens and analytes were likely to be suitable for immunoassay development. The second section determined the combination of benzene-metabolite and antigen need to produce an immunoassay with the requisite low detection limit and specificity. The immunoassays with the best performance were tested on hemoglobin from benzene-exposed mice. In vitro studies showed that hydroquinone efficiently formed adducts with erythrocyte membranes and hemoglobin but not with albumin. Adduction efficiency was greater in incubations using purified hemoglobin than whole blood. Cysteine accounted for 15 to 27% of the adducts formed by hydroquinone. The site of the other adducts were not identified although there was evidence that the hemoglobin heme was adducted. Adducts were found on only 1 of the 2 globin chains. Tryptic digestion of the globin failed to associate the adducts with a specific peptide. Antigens made from hydroquinone-adducted hemoglobin but not hydroquinone-adducted cysteines coupled to carrier proteins effectively elicited adduct-specific antibodies. Interference due to reactivity to hemoglobin was controlled by using uniform quantities of hemoglobin in all wells. The mid-range of the best assays were approximately 12 pmoles HQ per well. Antibodies directed toward hemoglobin adducted with the benzene metabolites phenol, catechol and 1,2,4-trihydroxybenzene were also made. The performance of the anti-1,2,4-trihydroxybenzene were suitable for quantitative immunoassays.

  20. Detection of immunoglobulin M in cerebrospinal fluid from syphilis patients by enzyme-linked immunosorbent assay.

    PubMed Central

    Lee, J B; Farshy, C E; Hunter, E F; Hambie, E A; Wobig, G H; Larsen, S A

    1986-01-01

    Cerebrospinal fluid (CSF) samples were evaluated in an immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) for syphilis with sonic extracts of Treponema pallidum coated on polystyrene plates. The ELISA procedure was reproducible, and T. pallidum antigens were stable., A total of 15 CSF samples from patients with neurosyphilis, 18 CSF samples from patients with syphilis, 12 CSF samples from patients treated for syphilis, and 494 CSF samples from patients with neurologic or other systemic diseases were tested. The IgM ELISA gave reactive results in all of six symptomatic and congenital neurosyphilitic patients and none of nine asymptomatic neurosyphilitic patients. Of 524 CSF samples from nonneurosyphilitic individuals, 513 were nonreactive, resulting in 98% test specificity. The IgM ELISA in CSF should prove to be useful for confirmation of symptomatic neurosyphilis. PMID:3533984

  1. System-on-fluidics immunoassay device integrating wireless radio-frequency-identification sensor chips.

    PubMed

    Yazawa, Yoshiaki; Oonishi, Tadashi; Watanabe, Kazuki; Shiratori, Akiko; Funaoka, Sohei; Fukushima, Masao

    2014-09-01

    A simple and sensitive point-of-care-test (POCT) device for chemiluminescence (CL) immunoassay was devised and tested. The device consists of a plastic flow-channel reactor and two wireless-communication sensor chips, namely, a photo-sensor chip and a temperature-sensor chip. In the flow-channel reactor, a target antigen is captured by an antibody immobilized on the inner wall of the flow-channel and detected with enzyme labeled antibody by using CL substrate. The CL signal corresponding to the amount of antigen is measured by a newly developed radio-frequency-identification (RFID) sensor, which enables batteryless operation and wireless data communication with an external reader. As for the POCT device, its usage environment, especially temperature, varies for each measurement. Hence, temperature compensation is a key issue in regard to eliminating dark-signal fluctuation, which is a major factor in deterioration of the precision of the POCT device. A two-stage temperature-compensation scheme was adopted. As for the first stage, the signals of two photodiodes, one with an open window and one with a sealed window, integrated on the photo-sensor chip are differentiated to delete the dark signal. As for the second stage, the differentiated signal fluctuation caused by a temperature variation is compensated by using the other sensor chip (equipped with a temperature sensor). The dark-level fluctuation caused by temperature was reduced from 0.24 to 0.02 pA/°C. The POCT device was evaluated as a CL immunoassay of thyroid-stimulating hormone (TSH). The flow rate of the CL reagent in the flow channel was optimized. As a result, the detection limit of the POCT device was 0.08 ng/ml (i.e., 0.4 ?IU/ml). PMID:24735652

  2. Clinical value of multiplexed bead-based immunoassays for detection of autoantibodies to nuclear antigens.

    PubMed

    Avaniss-Aghajani, Erik; Berzon, Sophia; Sarkissian, Arlen

    2007-05-01

    The advent of multiplexed bead assays in recent years has introduced a new dimension of testing for complex diseases such as lupus, which can involve multiple autoantibodies. The ability to rapidly identify multiple autoantibodies, with high sensitivity and specificity in an automated fashion, is highly attractive. The aim of this study was to assess the performance and clinical value of multiplexed bead-based (AtheNA Multi-Lyte ANA-II test system) immunoassays both by comparing the results with those achieved by indirect fluorescent-antibody assay (IFA) or conventional enzyme immunoassays (EIAs) and by independent identification of autoantibodies in well-characterized samples. To achieve this goal, 984 samples were tested for seven analytes (SS/A, SS/B, Sm, RNP, Scl-70, double-stranded DNA [dsDNA], and centromere B) in both traditional and bead-based assays. The average concordance for the different analytes was 91%, ranging from 81% (dsDNA) to 97% (centromere B). The average relative specificity and sensitivity for the analytes were also high, 92% and 81%, respectively. An examination of 93 "normal controls" demonstrated a 7% false-positive rate, which was comparable to IFA. Percentages of different autoantibodies found in patients with a variety of disease conditions (34 with calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia; 41 with mixed connective tissue disease; 24 with scleroderma; and 35 with Sjogren's syndrome) were well within the range expected from each group. A scrutiny of results from AtheNA and EIA and Farr results for 185 systemic lupus erythematosus samples revealed comparable results by both methods, with the exception of SS/A and dsDNA, where AtheNA had a higher percentage of SS/A-positive results compared to EIA (51% versus 29%) and a lower percentage of dsDNA-positive results (18% versus 28% at a cutoff of 5 IU/ml). PMID:17376860

  3. IgM characterization directly performed in crude culture supernatants by a new simple electrophoretic method.

    PubMed

    Vorauer-Uhl, Karola; Wallner, Jakob; Lhota, Gabriele; Katinger, Hermann; Kunert, Renate

    2010-07-31

    A new electrophoretic technique for the qualitative and quantitative analyses of IgM isoforms and fragments has been developed. IgMs which are more complex than many other recombinantly expressed immunoglobulins are characterized by their high molecular weighted active forms and many additional isoforms and fragments in the molecular range between 25 and 1200kDa. To analyze the multimers, isoforms and fragments simultaneously a high-resolution method, which enables sufficient migration and separation is required. Furthermore, this method should be appropriate to analyze IgMs in crude culture supernatants as well as purified samples. Simple sample preparation avoiding unspecific protein loss has been established. Currently no standard method to analyze all of them accordingly is available. The IgM-SDS-PAGE investigated for this purpose includes all these aspects. The combination of simple sample preparation and the application of precast gels make this electrophoretic method suitable for research but also quality control. The selective quantification of the multimers and the relative isoform distribution were performed by sensitive Sypro Ruby staining obtaining reliable and reproducible data in clone screening and process development which has been demonstrated by recombinantly expressed IgMs with significantly different isoform pattern. PMID:20493871

  4. The Insertion Green Monster (iGM) Method for Expression of Multiple Exogenous Genes in Yeast

    E-print Network

    Roth, Frederick

    1 The Insertion Green Monster (iGM) Method for Expression of Multiple Exogenous Genes in Yeast to this work. 2 Corresponding authors. G3: Genes|Genomes|Genetics Early Online, published on April 28, 2014 from higher eukaryotes, including humans. However, studies of complex exogenous pathways using yeast

  5. Atypical IgM multiple myeloma with deletion of c-MAF.

    PubMed

    Juárez Salcedo, L M; López Rubio, M; Gil Fernández, J J; Garcia-Suarez, J; Magro, E; Arranz, E; Gutiérrez Jomarrón, I; Marcellini Antonio, S; Blasco, A; Burgaleta, C

    2015-10-01

    IgM multiple myeloma (MM) is a rare subtype of myeloma that shares clinical and pathological features with Waldenström's macroglobulinaemia. These are two separate entities that differ both in therapy and prognosis. We report a 57-year-old male, who presented with anaemia, hypercalcaemia, acute renal failure and several vertebral fractures that clinically suggested a multiple myeloma. Further investigations revealed a serum monoclonal component of IgM lambda type and a bone marrow infiltrated by small, lymphoplasmocytic cells. IgM MM was finally diagnosed by means of both inmunophenotypic and immunohistochemistry techniques, stressing the importance of inmunophenotypic evaluation when clinical and morphological features are discordant. Fluorescence in situ hybridization (FISH) studies disclosed a particular combination of deletion 13q14, t(11;14) and monoallelic deletion C-MAF without t(14;16). The clinical evolution after a Bortezomib-containing polychemotherapy and autologous stem cell transplantation (ASCT) conditioned with busulphan and melphalan is also presented. This very uncommon case highlights the impact of immunophenotyping on the differential diagnosis between IgM MM and WM, to choose the best treatment and establish an appropriate outcome. PMID:25996654

  6. Warm Dark Matter, the Temperature of the IGM, and the Ly-Forest

    E-print Network

    Maryland at College Park, University of

    Warm Dark Matter, the Temperature of the IGM, and the Ly- Forest Adam Lidz (CfA) DM_MCFP: April 2 dark matter and is a well-motivated particle DM candidate! #12;Sterile Neutrinos · One piece of beyond to explain atmospheric/solar oscillation data. · 3rd lightest one could be the dark matter! Want > keV mass

  7. Baryonic Content in the Warm-Hot IGM at Low Redshift

    NASA Technical Reports Server (NTRS)

    Sonneborn, George; Shull, M.; Danforth, C.; Moos, W.

    2007-01-01

    Baryons are 4.5% of the universe's mass/energy density; only 10% of these are in stars, galaxies, and clusters. At low-redshift 90% of baryons are in the IGM, 30% in Ly-alpha forest, but most are in hot gas (10(exp 5-7) K) produced by shocks during structure formation. O VI 1032-38 A are the best tracers of this gas. The distribution of O VI absorbers observed by FUSE rises as N(sup -2+/-0.2, down to 10(exp 13)/sq cm. Integrated to logN=13, 7% of baryons reside in the O VI-bearing IGM at 10% solar metallicity, T approx. 10(exp 5.5) K. At redshift z<0.1 metals have been transported less than 800/h kpc from L* galaxies and 200/h kpc from 0.1 L* galaxies. The steepness of dN/dz means that low-N absorbers contribute an equal mass of hot IGM as higher N gas. The total mass of O VI-bearing gas in the IGM depends on determining the turnover in dN/dz at low N(O VI). Future observations by FUSE are needed to reach lower N and to reduce the uncertainty in the dN/dz power law.

  8. Extracellular Enzymes Lab Biochemistry

    E-print Network

    Vallino, Joseph J.

    of the enzyme · The 3D enzyme structure and catalytic activity can be lost by exposing the enzyme to high temperatures, salinity, pH, and other extremes. These extremes "denature" the enzyme. · Many enzymes have) enzymes are affected by other compounds (modulators) that can either inhibit or activate an enzyme

  9. An enzyme-linked immunosorbent assay for the determination of dioxins in contaminated sediment and soil samples

    E-print Network

    Hammock, Bruce D.

    An enzyme-linked immunosorbent assay for the determination of dioxins in contaminated sediment-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin values and log- transformed GC/HRMS-derived TEQ values were. Keywords: PCDD; PCDF; 2,3,7,8-Tetrachlorodibenzo-p-dioxin; TCDD; GC/HRMS; Immunoassay 1. Introduction

  10. [Trial of a Soviet diagnostic kit for determining class M immunoglobulins to the hepatitis A virus by immunoenzyme analysis (an anti-HAV IgM test system)].

    PubMed

    Donets, M A; Nel'ga, I V; Grishina, G K; Kusov, Iu Iu; Balaian, M S

    1988-01-01

    The authors have studied the effectiveness of the first Soviet test system for the diagnosis of hepatitis A by means of the enzyme immunoassay (Diagn-A-Hep), developed at the Institute of Poliomyelitis and Viral Encephalitides, Moscow, under the conditions of different epidemic situations. In the process of this trial the high specificity and sensitivity of this test system, established earlier in the certification and commission trials, have been confirmed. Diagn-A-Hep has proved to be highly effective in the diagnosis of acute forms of hepatitis A and permitted its detection in patients during the incubation period, as well as in patients with anicteric and asymptomatic subclinical forms. Besides clinical diagnosis, the kit Diagn-A-Hep may be used in large-scale seroepidemiological surveys of the immune structure of the population, as well as in detection of HAV in different material under test. PMID:2834897

  11. Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

    PubMed Central

    Tak For Yu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

    2015-01-01

    Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45?min with a limit of detection down to 10?pg mL?1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications. PMID:26074253

  12. Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

    NASA Astrophysics Data System (ADS)

    TakYu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

    2015-06-01

    Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45?min with a limit of detection down to 10?pg mL-1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications.

  13. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  14. Lateral flow immunoassay using magnetoresistive sensors

    NASA Astrophysics Data System (ADS)

    Taton, Kristin; Johnson, Diane; Guire, Patrick; Lange, Erik; Tondra, Mark

    2009-05-01

    Magnetic particles have been adapted for use as labels in biochemical lateral flow strip tests. Standard gold particle lateral flow assays are generally qualitative; however, with magnetic particles, quantitative results can be obtained by using electronic detection systems with giant magnetoresistive (GMR) sensors. As described here, these small integrated sensor chips can detect the presence of magnetic labels in capture spots whose volume is approximately 150 ?m×150 ?m×150 ?m. The range of linear detection is better than two orders of magnitude; the total range is up to four orders of magnitude. The system was demonstrated with both indirect and sandwich enzyme-linked immunosorbent assays (ELISAs) for protein detection of rabbit IgG and interferon-?, respectively, achieving detection of 12 pg/ml protein. Ultimately, the goal is for the detector to be fully integrated into the lateral flow strip backing to form a single consumable item that is interrogated by a handheld electronic reader.

  15. Gaussia Luciferase as a Genetic Fusion Partner with Antibody Fragments for Sensitive Immunoassay Monitoring of Clinical Biomarkers.

    PubMed

    Oyama, Hiroyuki; Morita, Izumi; Kiguchi, Yuki; Miyake, Sayaka; Moriuchi, Ayaka; Akisada, Tatsuki; Niwa, Toshifumi; Kobayashi, Norihiro

    2015-12-15

    In this study, we show the utility of Gaussia luciferase (GLuc), which is much smaller than previously found luciferases, as the fusion partner with artificial antibody species for developing sensitive immunoassay systems. As an example, we constructed a bioluminescent enzyme-linked immunosorbent assay (BL-ELISA) system determining the major glucocorticoid cortisol. A monoclonal antibody was newly elicited against a cortisol-albumin conjugate, and the genes encoding its variable domains (VH and VL) were cloned and combined to encode a single-chain Fv fragment (scFv). scFv was then linked to the wild-type GLuc gene or that encoding GLuc mutants reported to show improved emission kinetics and expressed in the periplasmic space of several Escherichia coli strains. Notably, the wild-type GLuc fusion protein (scFv-wtGLuc) showed the most suitable luminescent properties for BL-ELISAs. In our system, scFv-wtGLuc was reacted competitively with the analyte and immobilized cortisol moieties, and the bound GLuc activity was monitored with coelenterazine as the substrate. Successful batch-type luminescence detection was achieved using a plate reader without built-in injectors. The midpoint and limit of detection in a typical dose-response curve were 4.1 and 0.26 pg/assay, respectively, thus exhibiting much more sensitivity than conventional cortisol immunoassays. Serum cortisol levels (as the sum with cortisone) for healthy subjects, determined without any pretreatment, were compatible with reported reference ranges. The scFv-wtGLuc probe was stable over a year under storage as periplasmic extracts at -30 °C or with repeated freeze-thawing. These results suggest that GLuc fusions with antibody fragments might serve as useful and highly sensitive immunoassay probes in various clinical settings. PMID:26625180

  16. Silver and gold enhancement methods for lateral flow immunoassays.

    PubMed

    Rodríguez, Myriam Oliveira; Covián, Lucía Blanco; García, Agustín Costa; Blanco-López, Maria Carmen

    2016-02-01

    Sensitivity is the main concern at the development of rapid test by lateral flow immunoassays. On the other hand, low limits of detection are often required at medical diagnostics and other field of analysis. To overcome this drawback, several enhancement protocols have been described. In this paper, we have selected different silver enhancement methods and one dual gold conjugation, and we critically compared the amplification produced when applied to a gold-nanoparticle based lateral flow immunoassay for the detection of prostate specific antigen (PSA). The highest amplification was obtained by using an immersion method based on a solution of silver nitrate and hydroquinone/citrate buffer in proportion 1:1. Under these conditions, the system is capable of detecting PSA within 20min at levels as low as 0.1ng/mL, with a 3-fold sensitivity improvement. PMID:26653449

  17. Enzyme immunoassay for semicarbazide--the nitrofuran metabolite and food contaminant.

    PubMed

    Cooper, Kevin M; Samsonova, Jeanne V; Plumpton, Laura; Elliott, Christopher T; Kennedy, D Glenn

    2007-05-29

    Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed--all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CCbeta) of 0.25 microg kg(-1) when a threshold of 0.21 microg kg(-1) is applied to the selection of samples for confirmation (lowest observed 0.25 microg kg(-1) fortified sample, n=20), thus satisfying the EU nitrofurans' minimum required performance limit of 1 microg kg(-1). NFZ-incurred muscles (12) containing SEM at 0.5-5.0 microg kg(-1) by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver. PMID:17499072

  18. COMPARISON OF SOIL PBDE LEVELS USING HRGC-HRMS AND MAGNETIC PARTICLE ENZYME IMMUNOASSAY.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polybrominated biphenyl ethers (PBDEs) are the major class of compounds used to prevent fires in furniture, textiles, and electronic equipment. Because they are structurally similar to dioxin and polychlorobiphenyl, PBDEs have been shown to be persistent environmental contaminants that can accumula...

  19. Application of enzyme immunoassay on infected cells (EIA-IC) for arboviruses.

    PubMed

    Xiao, Z S; Jia, L L; Qu, X S; Zhang, Y H

    1986-11-01

    Comparative titrations of alpha-, flavi- and Bunyamwera viruses were made by EIA-IC and according to cytopathic effect (CPE). Specific enzymatic reactions appeared earlier and in higher titres than CPE. The titres of dengue type 1, Mayaro, Powassan and Langat viruses measured by EIA-IC were comparable to those measured by intracerebral inoculation of mice. The cross-reactivity testing of EIA-IC among alphaviruses (Chikungunya, Sindbis and Mayaro), flaviviruses (Japanese encephalitis, Murray valley encephalitis, Kunjin, West Nile, yellow fever and louping ill, Powassan, Langat) and Bunyamwera arboviruses using polyclonal immune ascitic fluids confirmed the high specificity of EIA-IC. Homologous reactions mostly showed higher titres than heterologous ones. No cross-reactivity was seen between alpha-, flavi- and bunyaviruses, among the three alphaviruses, between mosquito-borne and tick-borne flaviviruses, or between JE complex and YF viruses. However, a cross-reactivity to different extent was observed among the four JE complex viruses and among louping ill, Powassan and Langat viruses. The results of EIA-IC cross tests showed that this method can distinguish togavirus group- or species-specific antigens, more precisely than conventional ELISA. PMID:2881468

  20. ENZYME IMMUNOASSAY WITH MONOCLONAL ANTIBODIES FOR THE DETECTION OF ROTAVIRUS IN STOOL SPECIMENS

    EPA Science Inventory

    Rotavirus infections are generally recognized as a major problem in young children; however, they have also been associated with severe gastroenteritis in adults and neonates. Infections in neonates are usually asymptomatic, although the incidence of infection may be high. Adult ...

  1. Benzene-RISc: The development and performance of an immunoassay to detect benzene in water

    SciTech Connect

    Friedman, S.B.; Withers, T.; Almond, R.; Stewart, T.; Allen, R.L.

    1994-12-31

    Immunoassay methods have become available for environmental applications. Their simplicity, reliability, and ability to provide information rapidly and on-site is enhancing the efficiency of many field and laboratory programs. Immunoassay methods rely upon antibody molecules to provide the sensitivity and specificity characteristics they exhibit, but many molecules are either insufficiently immunogenic or structurally unremarkable to induce an appropriate antibody response. Such compounds are usually considered to be incompatible with the development of an immunoassay method. An immunoassay method for the detection of benzene in water would have utility in detecting contamination from spills and leaking underground storage tanks. Benzene, however, is frequently considered to be in the class of compounds considered to be incompatible with antibody, and therefore immunoassay, development. The authors have developed an immunoassay for the detection of benzene in water by developing both sample processing and immunochemical procedures and reagents that overcome the technical limitations frequently encountered.

  2. Cooperative Immunoassays: Ultrasensitive Assays with Mixed Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Ehrlich, Paul H.; Moyle, William R.

    1983-07-01

    Mixtures of certain monoclonal antibodies appear to bind human chorionic gonadotropin in a ``cooperative'' fashion because they form circular complexes with the hormone. Experiments illustrate how this property might be exploited to develop very sensitive immunoassays for human chorionic gonadotropin or any other antigen. Since the assays are not based on competitive inhibition between radiolabeled and unlabeled antigen, they are much more sensitive than a traditional radioimmunoassay in which either one of the same antibodies is used alone.

  3. Seroprevalence of hepatitis B e antigen (HBe antigen) and B core antibodies (IgG anti-HBcore and IgM anti-HBcore) among hepatitis B surface antigen positive blood donors at a Tertiary Centre in Nigeria

    PubMed Central

    2012-01-01

    Background Hepatitis B virus (HBV) is a common cause of liver disease throughout the world. HBV is transmitted through blood and other body fluids, including semen and saliva. Chronic replication of HBV virons is characterized by persistence circulation of HBsAg, HBeAg and HBV DNA; usually with anti-HBc and occasionally with anti-HBs. Aim: To determine the prevalence of HBeAg, IgG anti-HBcore and IgM anti-HBcore amongst HBsAg positive blood donors. These parameters are reflective of transmissibility and active hepatitis B infection. A cross sectional study was carried out at the blood donor clinics of Lagos State University Teaching Hospital Ikeja and Lagos University Teaching Hospital Idiaraba. A total of 267 donors were recruited to determine HBe antigen, IgG and IgM anti-HBcore antibodies amongst hepatitis BsAg positive donors. Five milliliters of blood was collected from those who tested positive to HBsAg screen during donation. The sera were subjected to enzyme linked immunosorbent assay (ELISA). Pearson chi-squared test was used for the analytical assessment. Findings A total number of 267 HBsAg positive blood donors were studied. A seroprevalence of 8.2% (22 of 267) HBeAg was obtained, 4 of 267 (1.5%) were indeterminate while 241 (90.3%) tested negative. Only 27 out of 267 donors (10.1%) tested positive to IgM anti-HBcore, 234(87.6%) tested negative, while 6(2.2%) were indeterminate. A higher percentage of 60.7% (162 of 267) tested positive to IgG anti-HBcore, while 39.3% (105 of 267) tested negative. Conclusion There is a low seroprevalence rate of HBeAg-positive chronic hepatitis and relatively high IgG anti-HBcore and IgM anti-HBcore rates in South West Nigeria. PMID:22455501

  4. A Practical Guide to Immunoassay Method Validation.

    PubMed

    Andreasson, Ulf; Perret-Liaudet, Armand; van Waalwijk van Doorn, Linda J C; Blennow, Kaj; Chiasserini, Davide; Engelborghs, Sebastiaan; Fladby, Tormod; Genc, Sermin; Kruse, Niels; Kuiperij, H Bea; Kulic, Luka; Lewczuk, Piotr; Mollenhauer, Brit; Mroczko, Barbara; Parnetti, Lucilla; Vanmechelen, Eugeen; Verbeek, Marcel M; Winblad, Bengt; Zetterberg, Henrik; Koel-Simmelink, Marleen; Teunissen, Charlotte E

    2015-01-01

    Biochemical markers have a central position in the diagnosis and management of patients in clinical medicine, and also in clinical research and drug development, also for brain disorders, such as Alzheimer's disease. The enzyme-linked immunosorbent assay (ELISA) is frequently used for measurement of low-abundance biomarkers. However, the quality of ELISA methods varies, which may introduce both systematic and random errors. This urges the need for more rigorous control of assay performance, regardless of its use in a research setting, in clinical routine, or drug development. The aim of a method validation is to present objective evidence that a method fulfills the requirements for its intended use. Although much has been published on which parameters to investigate in a method validation, less is available on a detailed level on how to perform the corresponding experiments. To remedy this, standard operating procedures (SOPs) with step-by-step instructions for a number of different validation parameters is included in the present work together with a validation report template, which allow for a well-ordered presentation of the results. Even though the SOPs were developed with the intended use for immunochemical methods and to be used for multicenter evaluations, most of them are generic and can be used for other technologies as well. PMID:26347708

  5. A Practical Guide to Immunoassay Method Validation

    PubMed Central

    Andreasson, Ulf; Perret-Liaudet, Armand; van Waalwijk van Doorn, Linda J. C.; Blennow, Kaj; Chiasserini, Davide; Engelborghs, Sebastiaan; Fladby, Tormod; Genc, Sermin; Kruse, Niels; Kuiperij, H. Bea; Kulic, Luka; Lewczuk, Piotr; Mollenhauer, Brit; Mroczko, Barbara; Parnetti, Lucilla; Vanmechelen, Eugeen; Verbeek, Marcel M.; Winblad, Bengt; Zetterberg, Henrik; Koel-Simmelink, Marleen; Teunissen, Charlotte E.

    2015-01-01

    Biochemical markers have a central position in the diagnosis and management of patients in clinical medicine, and also in clinical research and drug development, also for brain disorders, such as Alzheimer’s disease. The enzyme-linked immunosorbent assay (ELISA) is frequently used for measurement of low-abundance biomarkers. However, the quality of ELISA methods varies, which may introduce both systematic and random errors. This urges the need for more rigorous control of assay performance, regardless of its use in a research setting, in clinical routine, or drug development. The aim of a method validation is to present objective evidence that a method fulfills the requirements for its intended use. Although much has been published on which parameters to investigate in a method validation, less is available on a detailed level on how to perform the corresponding experiments. To remedy this, standard operating procedures (SOPs) with step-by-step instructions for a number of different validation parameters is included in the present work together with a validation report template, which allow for a well-ordered presentation of the results. Even though the SOPs were developed with the intended use for immunochemical methods and to be used for multicenter evaluations, most of them are generic and can be used for other technologies as well. PMID:26347708

  6. Simple Patterned Nanofiber Scaffolds and Its Enhanced Performance in Immunoassay

    PubMed Central

    Lv, Xiao-guang; Song, Jia; Zhan, Na; Dong, Wei-guo; Huang, Wei-hua

    2013-01-01

    Cancer has become the leading cause of death worldwide; early diagnosis and treatment of cancers is critical for the survival of the patients. The concentration of cancer markers in easy-to-access biological fluids can provide great assistance in screening for occult primary cancers, distinguishing malignant from benign findings, determining prognosis and prediction for cancer patients. The multiplex detection technology of a panel of cancer markers can greatly increase the accuracy of disease diagnosis. Herein, we briefly fabricate a high-throughput micro-immunoassay based on the electrospun polystyrene (PS) substrates to improve detection sensitivity. The immunoassay was evaluated by analyzing three different cancer biomarkers (AFP, CEA, VEGF). For AFP, CEA, VEGF immunofluorescence assay, the LOD of assay conducted on electrospun PS substrates before or after plasma and the conventional PS substrates were 0.42, 0.10, 1.12 ng/mL, 0.57, 0.09, 1.24 ng/mL, and 159.75, 26.19, 385.59 pg/mL, respectively (P < 0.05). Due to the high porosity and large surface area-to-volume ratio which is the foremost merit of nanostructures, and the plasma treatment which make the hydrophobic PS nanofibers hydropholic, the nanofibers substrates showed sufficient retention of immunoassay functionality and high potential for capture molecules immobilization. Consequently, the immunofluorescence assay conducted on electrospun PS substrates could significantly enhance the sensitivity and limits of detection. PMID:24340065

  7. A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

    USGS Publications Warehouse

    Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

    2005-01-01

    Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Ku??tzing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L-1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method. ?? Springer 2005.

  8. Simple and Sensitive Detection of HBsAg by Using a Quantum Dots Nanobeads Based Dot-Blot Immunoassay

    PubMed Central

    Zhang, Pengfei; Lu, Huiqi; Chen, Jia; Han, Huanxing; Ma, Wei

    2014-01-01

    Simple and sensitive detection of infectious disease at an affordable cost is urgently needed in developing nations. In this regard, the dot blot immunoassay has been used as a common protein detection method for detection of disease markers. However, the traditional signal reporting systems, such as those using enzymes or gold nanoparticles lack sensitivity and thus restrict the application of these methods for disease detection. In this study, we report a simple and sensitive detection method for the detection of infectious disease markers that couples the dot-blot immunoassay with quantum dots nanobeads (QDNBs) as a reporter. First, the QDNBs were prepared by an oil-in-water emulsion-evaporation technique. Because of the encapsulation of several QDs in one particle, the fluorescent signal of reporter can be amplified with QDNBs in a one-step test and be read using a UV lamp obviating the need for complicated instruments. Detection of disease-associated markers in complex mixture is possible, which demonstrates the potential of developing QDNBs into a sensitive diagnostic kit. PMID:24505238

  9. Complex-specific immunoglobulin M antibody patterns in humans infected with alphaviruses.

    PubMed

    Calisher, C H; el-Kafrawi, A O; Al-Deen Mahmud, M I; Travassos da Rosa, A P; Bartz, C R; Brummer-Korvenkontio, M; Haksohusodo, S; Suharyono, W

    1986-01-01

    Sera from humans with serologically confirmed eastern equine encephalitis, western equine encephalitis, Pogosta (Ockelbo), Mayaro, Ross River, and chikungunya virus infections were tested by immunoglobulin M (IgM) antibody capture enzyme immunoassay. Diagnostically useful IgM antibody titers were detected, and selected sera with high IgM antibody titers were tested for IgM antibody with nine heterologous alphaviruses. The results provide evidence for the complex specificity of IgM antibody and indicate the usefulness of this test in both individual cases and epidemic situations. PMID:3009526

  10. An ImmunoChip prototype for simultaneous detection of antiepileptic drugs using an enhanced one-step homogeneous immunoassay1 2 3 4

    PubMed Central

    Yang, Xiaoyun; Janatova, Jarmila; Juenke, JoEtta M.; McMillin, Gwendolyn A.; Andrade, Joseph D.

    2007-01-01

    The development and characterization of a one-step homogeneous immunoassay-based multi-well ImmunoChip is reported for the simultaneous detection and quantitation of antiepileptic drugs (AEDs). The assay platform utilizes a Cloned Enzyme Donor Immunoassay (CEDIA), and a Beta-Glo assay system for generation of bioluminescent signal. Results of the one-step CEDIA for three AEDs (CBZ, carbamazepine; PHT, phenytoin; VPA, valproic acid), in the presence of serum, correlate well with the values determined by Fluorescence Polarization Immunoassay. CEDIA intra-assay and inter-assay coefficients of variation are lower than 10%. A microfabrication process, xurography, was utilized to produce the multi-well ImmunoChip. Assay reagents were dispensed, and lyophilized, in a three-layer pattern. The multi-well ImmunoChip prototype was used to detect and quantify AEDs in serum samples containing all three drugs. Luminescent signals generated from each well were recorded with a Charged Coupled Device (CCD) camera. The assays performed on an ImmunoChip were fast (5 min), requiring only small volumes of both the reagents (< 1 ?l per well) and the serum sample. The ImmunoChip assay platform described herein may be well suited for therapeutic monitoring of drugs and metabolites at the point-of-care. PMID:17448436

  11. Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark

    PubMed Central

    Rumfelt, Lynn L; Lohr, Rebecca L; Dooley, Helen; Flajnik, Martin F

    2004-01-01

    Background Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT) expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families. Results IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1) and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge) within a species. Conclusion As in ratfish, sandbar and horn sharks, most nurse shark IgM VH genes are from one family with multiple, heterogeneous loci. Their IgW VH genes have diversified, forming at least three families. The neonatal shark Ig VH CDR3 repertoire, diversified via N-region addition, is shorter than the adult VDJ junction, suggesting one means of postnatal repertoire diversification is expression of longer CDR3 junctions. PMID:15132758

  12. Multicenter Analytical Evaluation of the Automated Electrochemiluminescence Immunoassay for Cyclosporine

    PubMed Central

    Vogeser, Michael; Shipkova, Maria; Rigo-Bonnin, Raül; Wallemacq, Pierre; Orth, Matthias; Widmann, Monika

    2014-01-01

    Background: Cyclosporine A (CsA) is used as a posttransplantation immunosuppressant drug, and careful monitoring of CsA concentration in whole blood is essential. A new automated electrochemiluminescence immunoassay (ECLIA) for CsA measurement has been assessed in a multicenter evaluation. Methods: Residual EDTA whole blood samples from patients undergoing CsA therapy after organ transplant were used in assay evaluation at 5 clinical laboratories in Europe. Experiments included imprecision according to CLSI EP5-A2 (within-run and intermediate), lower limit of quantification, linearity according to CLSI EP6-A, and recovery of commercial external quality control samples. In addition, comparisons to liquid chromatography-tandem mass spectrometry methods in routine use at each investigational site and to commercial chemiluminescent microparticle immunoassay and antibody-conjugated magnetic immunoassay methods were performed. Results: Imprecision testing gave coefficients of variation of less than 9% in the 30–2000 mcg/L range for both within-run and intermediate imprecision. Lower limit of quantification of 6.8 mcg/L at one investigational site and 1.8 mcg/L at a second site at 20% coefficient of variation were observed. Linearity was measured over the concentration range 0–2000 mcg/L, yielding a deviation of less than ±12%. External quality control sample recovery by ECLIA was 93%–114% of LC-MS/MS sample recovery. Deming regression analysis of ECLIA method comparison to combined LC-MS/MS results yielded a slope of 1.04 [95% confidence interval (CI), 1.03–1.06] and intercept of 2.8 mcg/L (95% CI, 1.5–4.1 mcg/L). Comparison to chemiluminescent microparticle immunoassay yielded a slope of 0.87 (95% CI, 0.85–0.89) and intercept of 1.4 mcg/L (95% CI, ?0.89 to 3.7 mcg/L); comparison to antibody-conjugated magnetic immunoassay yielded a slope of 0.96 (95% CI, 0.93–0.98) and intercept of ?4.2 mcg/L (95% CI, ?7.1 to ?1.2 mcg/L). Conclusions: The data from this multicenter evaluation indicate that the new ECLIA-based cyclosporine assay is fit for its purpose, the therapeutic monitoring of CsA. PMID:24646730

  13. Application of an enzyme-linked immunosorbent assay (ELISA) to determine paraquat residues in milk, beef, and potatoes

    SciTech Connect

    Van Emon, J.; Seiber, J.; Hammock, B.

    1987-09-01

    The USDA Food Safety and Inspection Service (FSIS) has included paraquat on its list of compounds to be considered for monitoring in foods. However, present methods do not easily accommodate the processing of large numbers of samples, thus limiting routine monitoring of the compound. The conventional method, based on spectrophotometry of reduced paraquat solutions, requires time-consuming sample preparation. Although the advantages of immunoassays for pesticide residue analysis have been pointed out, the reported immunoassays for paraquat have only been applied to cases of clinical poisoning or human exposure assessment. In this study, spiked milk, potato, and beef were analyzed directly, without prior cleanup, by an enzyme-linked immunosorbent assay (ELISA).

  14. IGM Constraints from the SDSS-III/BOSS DR9 Ly? Forest Transmission Probability Distribution Function

    NASA Astrophysics Data System (ADS)

    Lee, Khee-Gan; Hennawi, Joseph F.; Spergel, David N.; Weinberg, David H.; Hogg, David W.; Viel, Matteo; Bolton, James S.; Bailey, Stephen; Pieri, Matthew M.; Carithers, William; Schlegel, David J.; Lundgren, Britt; Palanque-Delabrouille, Nathalie; Suzuki, Nao; Schneider, Donald P.; Yèche, Christophe

    2015-02-01

    The Ly? forest transmission probability distribution function (PDF) is an established probe of the intergalactic medium (IGM) astrophysics, especially the temperature-density relationship of the IGM. We measure the transmission PDF from 3393 Baryon Oscillations Spectroscopic Survey (BOSS) quasars from Sloan Digital Sky Survey Data Release 9, and compare with mock spectra that include careful modeling of the noise, continuum, and astrophysical uncertainties. The BOSS transmission PDFs, measured at langzrang = [2.3, 2.6, 3.0], are compared with PDFs created from mock spectra drawn from a suite of hydrodynamical simulations that sample the IGM temperature-density relationship, ?, and temperature at mean density, T 0, where T(?) = T 0?? - 1. We find that a significant population of partial Lyman-limit systems (LLSs) with a column-density distribution slope of ?pLLS ~ - 2 are required to explain the data at the low-transmission end of transmission PDF, while uncertainties in the mean Ly? forest transmission affect the high-transmission end. After modeling the LLSs and marginalizing over mean transmission uncertainties, we find that ? = 1.6 best describes the data over our entire redshift range, although constraints on T 0 are affected by systematic uncertainties. Within our model framework, isothermal or inverted temperature-density relationships (? <= 1) are disfavored at a significance of over 4?, although this could be somewhat weakened by cosmological and astrophysical uncertainties that we did not model.

  15. Constraints on the IGM Temperature-Density Relationship from BOSS Lyman-? Forest Data

    NASA Astrophysics Data System (ADS)

    Lee, Khee-Gan; Hennawi, J.; Spergel, D. N.; Hogg, D. W.; Viel, M.; Pieri, M.; Bolton, J.; Bailey, S. J.; Ge, J.; Schlegel, D. J.; Suzuki, N.; BOSS Collaboration

    2013-01-01

    The properties of the photoionized intergalactic medium (IGM) contain vital clues on the thermal history of the Universe, such as reionization events (both HI and HeII), quasar/AGN activity, and galaxy formation. We use 2541 quasar spectra from BOSS Data Release 9 to place constraints on the evolution of the IGM temperature-density relationship (usually parametrized as ?, where log T ? ? log ?) at 2.15IGM studies. We use a MCMC-based method to separate the photon-counting and CCD components of the spectral noise by differencing the individual exposures of each spectrum, while simultaneously providing accurate noise estimates. This allows us to create mock absorption spectra from detailed hydrodynamical simulations generated with different values of ?, with realistic noise properties tailored to each individual BOSS spectrum. For the continuum fitting, we use the mean-flux regulated PCA method, which allows accurate estimates 4% rms errors) of the underlying quasar continuum even in noisy spectra. Comparing the flux PDF from the mock spectra with the observed flux PDF from BOSS, we place constraints on the evolution of ? from z=3 to z=2 and discuss the results in the context of Helium reionization scenarios.

  16. IGM CONSTRAINTS FROM THE SDSS-III/BOSS DR9 Ly? FOREST TRANSMISSION PROBABILITY DISTRIBUTION FUNCTION

    SciTech Connect

    Lee, Khee-Gan; Hennawi, Joseph F.; Spergel, David N.; Weinberg, David H.; Hogg, David W.; Viel, Matteo; Bolton, James S.; Bailey, Stephen; Carithers, William; Schlegel, David J.; Pieri, Matthew M.; Lundgren, Britt; Palanque-Delabrouille, Nathalie; Yèche, Christophe; Schneider, Donald P.

    2015-02-01

    The Ly? forest transmission probability distribution function (PDF) is an established probe of the intergalactic medium (IGM) astrophysics, especially the temperature-density relationship of the IGM. We measure the transmission PDF from 3393 Baryon Oscillations Spectroscopic Survey (BOSS) quasars from Sloan Digital Sky Survey Data Release 9, and compare with mock spectra that include careful modeling of the noise, continuum, and astrophysical uncertainties. The BOSS transmission PDFs, measured at (z) = [2.3, 2.6, 3.0], are compared with PDFs created from mock spectra drawn from a suite of hydrodynamical simulations that sample the IGM temperature-density relationship, ?, and temperature at mean density, T {sub 0}, where T(?) = T {sub 0}?{sup ?} {sup –} {sup 1}. We find that a significant population of partial Lyman-limit systems (LLSs) with a column-density distribution slope of ?{sub pLLS} ? – 2 are required to explain the data at the low-transmission end of transmission PDF, while uncertainties in the mean Ly? forest transmission affect the high-transmission end. After modeling the LLSs and marginalizing over mean transmission uncertainties, we find that ? = 1.6 best describes the data over our entire redshift range, although constraints on T {sub 0} are affected by systematic uncertainties. Within our model framework, isothermal or inverted temperature-density relationships (? ? 1) are disfavored at a significance of over 4?, although this could be somewhat weakened by cosmological and astrophysical uncertainties that we did not model.

  17. Multi-allergen screening immunoassay for the detection of protein markers of peanut and four tree nuts in chocolate.

    PubMed

    Ben Rejeb, S; Abbott, M; Davies, D; Cléroux, C; Delahaut, P

    2005-08-01

    A multiresidue enzyme immunoassay was developed to check for the presence of markers of peanut, hazelnut, almond, cashew and Brazil nuts in a single run. The assay was designed under the competitive indirect format and adapted for screening purposes applied to chocolate samples. The limit of detection for this assay was below 1 microg g-1 protein for each allergenic food. In most cases, the high specificity of the antibodies used allowed the identification of each particular allergenic food with no possible confusion. This assay was proven to be useful as part of an analytical procedure involving the identification of the unknown allergenic food among peanut and other tree nuts in recalled samples before the application of a quantitative technique to determine the level of cross-contamination. PMID:16147426

  18. francois.marechal@epfl.chIndustrialEnergySystemsLaboratory-LENI-IGM-STI-EPFL2012 Prof. Franois Marechal

    E-print Network

    Ahrendt, Wolfgang

    .ch©IndustrialEnergySystemsLaboratory-LENI-IGM-STI-EPFL2012 The present system Raw materials Products& by-products Heat losses Food or agro process Waste Produces waste Waste management New products/services Energy Resources Recycling mass energy Production.ch©IndustrialEnergySystemsLaboratory-LENI-IGM-STI-EPFL2012 The integrated process Products and by-products Heat losses Waste Raw materials Electricity Heat

  19. HCR-stimulated formation of DNAzyme concatamers on gold nanoparticle for ultrasensitive impedimetric immunoassay.

    PubMed

    Hou, Li; Wu, Xiaoping; Chen, Guonan; Yang, Huanghao; Lu, Minghua; Tang, Dianping

    2015-06-15

    A novel signal-amplified impedimetric immunosensing strategy was successfully developed for ultrasensitive detection of low-abundance proteins (carcinoembryonic antigen, CEA, used as a model) based on hybridization chain reaction (HCR)-stimulated formation of DNAzyme concatamers on nanogold particle accompanying enzyme-triggered biocatalytic precipitation. The assay was carried out on capture antibody-modified electrode by using gold nanoparticle heavily functionalized with initiator strand and detection antibody as the signal-transduction tag with a sandwich-type immunosensing format. In the presence of target CEA, the formed immunocomplex underwent an unbiased strand-displacement reaction by the initiator strand on the gold nanoparticle between two auxiliary single-stranded DNA with the hemin aptamer. Upon hemin introduction, numerous DNAzyme molecules were formed on the concatamers and nanogold particle, which could catalyze 4-chloro-1-naphthol to produce an insoluble precipitation on the electrode, thereby resulting in the amplification of impedimetric signal. Under the optimal conditions, the immuno-HCR assay exhibited good impedimetric responses for the detection of target CEA in the working range from 1.0 pg mL(-1) to 20 ng mL(-1) with a detection limit of 0.42 pg mL(-1). In addition, the immuno-HCR assay was validated by measuring six human serum specimens for target CEA, receiving a highly matched correction between the obtained results by the immuno-HCR assay and the commercialized ELC-based immunoassay method. PMID:25636020

  20. An Immunoassay to Evaluate Human/Environmental Exposure to the Antimicrobial Triclocarban

    PubMed Central

    Ahn, Ki Chang; Kasagami, Takeo; Tsai, Hsing-Ju; Schebb, Nils Helge; Ogunyoku, Temitope; Gee, Shirley J.; Young, Thomas M.; Hammock, Bruce D.

    2011-01-01

    A sensitive, competitive indirect enzyme linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclocarban (TCC) was developed. The haptens were synthesized by derivatizing the para position of a phenyl moiety of TCC. The rabbit antisera were screened and the combination of antiserum #1648 and a heterologous competitive hapten containing a piperidine was further characterized. The IC50 and the detection range for TCC in buffer were 0.70 and 0.13–3.60 ng/mL, respectively. The assay was selective for TCC, providing only low cross-reactivity to TCC-related compounds and its major metabolites except for the closely related antimicrobial 3-trifluoromethyl-4,4?-dichlorocarbanilide. A liquid-liquid extraction for sample preparation of human body fluids resulted in an assay that measured low part per billion levels of TCC in small volumes of the samples. The limits of quantification of TCC were 5 ng/mL in blood/serum, and 10 ng/mL in urine, respectively. TCC in human urine was largely the N- or N?-glucuronide. TCC concentrations of biosolids measured by the ELISA were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid, inexpensive and convenient tool to aid researchers monitoring human/environmental exposure to TCC to better understand the health effects. PMID:22077920

  1. An immunoassay to evaluate human/environmental exposure to the antimicrobial triclocarban.

    PubMed

    Ahn, Ki Chang; Kasagami, Takeo; Tsai, Hsing-Ju; Schebb, Nils Helge; Ogunyoku, Temitope; Gee, Shirley J; Young, Thomas M; Hammock, Bruce D

    2012-01-01

    A sensitive, competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclocarban (TCC) was developed. The haptens were synthesized by derivatizing the para position of a phenyl moiety of TCC. The rabbit antisera were screened and the combination of antiserum 1648 and a heterologous competitive hapten containing a piperidine was further characterized. The IC(50) and detection range for TCC in buffer were 0.70 and 0.13-3.60 ng/mL, respectively. The assay was selective for TCC, providing only low cross-reactivity to TCC-related compounds and its major metabolites except for the closely related antimicrobial 3-trifluoromethyl-4,4'-dichlorocarbanilide. A liquid-liquid extraction for sample preparation of human body fluids resulted in an assay that measured low part per billion levels of TCC in small volumes of the samples. The limits of quantification of TCC were 5 ng/mL in blood/serum and 10 ng/mL in urine, respectively. TCC in human urine was largely the N- or N'-glucuronide. TCC concentrations of biosolids measured by the ELISA were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid, inexpensive, and convenient tool to aid researchers monitoring human/environmental exposure to TCC to better understand the health effects. PMID:22077920

  2. Development of an Immunoassay for the Detection of the Phenylpyrazole Insecticide Fipronil.

    PubMed

    Vasylieva, Natalia; Ahn, Ki Chang; Barnych, Bogdan; Gee, Shirley J; Hammock, Bruce D

    2015-08-18

    Phenylpyrazole insecticides such as fipronil have been used as replacements for organophosphates. The wide application of fipronil raises concern about environmental contamination and risk for fish, birds, and other nontargeted beings as well as human health. A sensitive, competitive indirect heterologous enzyme-linked immunosorbent assay (ELISA) was developed. Antibodies with different specificities to fipronil and its metabolites were produced. Two ELISAs having IC50 values of 0.58 ± 0.06 and 2.6 ± 0.4 ng/mL were developed. Design of different haptens and coating antigens resulted in two assays with distinct cross-reactivity patterns for structurally related compounds: 96, 38, and 101% versus 39, 1.4, and 25% for fipronil-sulfide, fipronil-detrifluoromethylsulfonyl, and fipronil-desulfinyl, respectively. Performance of the immunoassays was demonstrated by a recovery study from spiked water and human serum and urine matrices, giving recovery values in the range of 85-111% for different concentrations. The assays demonstrated good correlation in fipronil recovery with conventional LC-MS/MS analysis. The generic assay 2265 has the sensitivity to measure fipronil and its analogs in serum at levels relevant for exposure monitoring. The assays were used to analyze human urine samples obtained from exposure studies and serum samples from rats treated with a fipronil-containing diet. PMID:26196357

  3. A rapid sandwich immunoassay for human fetuin A using agarose-3-aminopropyltriethoxysilane modified microtiter plate.

    PubMed

    Vashist, Sandeep Kumar; Schneider, E Marion; Luong, John H T

    2015-07-01

    A rapid sandwich immunoassay (IA) with enhanced signal response for human fetuin A (HFA) was developed by modifying the surface of a KOH-treated polystyrene microtiter plate (MTP) with agarose and 3-aminopropyltriethoxysilane (APTES). The agarose-APTES complex binds covalently to the hydroxyl moiety of the MTP plate to serve as a binding platform for bioconjugation of EDC-activated anti-HFA antibody (Ab) via carbodiimide coupling. The one-step kinetics-based sandwich enzyme-linked immunosorbent assay (ELISA) enabled the detection of HFA in 30 min with a limit of detection (LOD) and a linear range of 0.02 ng mL(-1) and 1-243 ng mL(-1), respectively. It detected HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients with high precision similar to that of conventional ELISA. The anti-HFA Ab-bound agarose-functionalized MTPs retained their functional activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C. PMID:26088779

  4. A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses

    PubMed Central

    Beck, Cécile; Desprès, Philippe; Paulous, Sylvie; Vanhomwegen, Jessica; Lowenski, Steeve; Nowotny, Norbert; Durand, Benoit; Garnier, Annabelle; Blaise-Boisseau, Sandra; Guitton, Edouard; Yamanaka, Takashi; Zientara, Stéphan; Lecollinet, Sylvie

    2015-01-01

    West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using the Drosophila S2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases. PMID:26457301

  5. Development of ELISA and colloidal gold immunoassay for tetrodotoxin detetcion based on monoclonal antibody.

    PubMed

    Ling, Sumei; Chen, Qing-Ai; Zhang, Yuming; Wang, Rongzhi; Jin, Ni; Pang, Jie; Wang, Shihua

    2015-09-15

    A monoclonal hybridoma cell named 5B9 against tetrodotoxin (TTX) was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The 5B9 monoclonal antibody (McAb) with high affinity (about 2.55 × 10(9)) is specific to TTX, and this McAb belongs to the immunoglobulin G (IgG) isotype. Finally, an enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunoassay were established based on this McAb. The linear range of ELISA to detect TTX was 5-500 ng/mL, and the limit of detection (LOD) was 4.44 ng/mL. The average CV of intra- and inter-assay was less than 8%, with the samples recovery range of 70.93-99.99%. A competitive format colloidal gold strip was developed for detection of TTX in real samples, and the LOD for TTX is 20 ng/mL, and the assay time of the qualitative test can be finished in less than 10 min without any equipment. The result from test strip revealed that the test strip has a good agreement with those obtained from ELISA. PMID:25913446

  6. A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses.

    PubMed

    Beck, Cécile; Desprès, Philippe; Paulous, Sylvie; Vanhomwegen, Jessica; Lowenski, Steeve; Nowotny, Norbert; Durand, Benoit; Garnier, Annabelle; Blaise-Boisseau, Sandra; Guitton, Edouard; Yamanaka, Takashi; Zientara, Stéphan; Lecollinet, Sylvie

    2015-01-01

    West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using the Drosophila S2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases. PMID:26457301

  7. Immunoassay of red dyes based on the monoclonal antibody of ?-naphthol.

    PubMed

    Qi, Yong H; Zhang, Hui C; Xu, Rui T; Liu, Jin; Zhang, Lei; Wang, Jian P

    2015-01-01

    The objective of the present study was to develop a multi-analyte immunoassay for the determination of eight red dyes in food samples. Two dye intermediates (2-hydroxy-1-naphthoic acid and 1-amino-2-naphthol) were used as the haptens to produce the monoclonal antibodies. The obtained monoclonal antibodies recognized Sudan 1-4, Para red, Sudan red G, Sudan red B and Acid orange II simultaneously. After evaluation of different antibody/coating antigen combinations, a heterologous indirect competitive enzyme linked immunosorbent assay was developed to determine the eight red dyes in food samples (chili oil, chili powder, tomato sauce, hotpot seasoning). The crossreactivities to the eight analytes were in the range of 61%-79% (with ?-naphthol as 100%), and the limits of detection were in the range of 1.3-1.9 ng/mL. The recoveries of the eight analytes from the fortified blank samples were in the range of 84.2%-115% with coefficients of variation lower than 18.3%. Therefore, this method could be used as a rapid and simple tool to detect the residues of the eight red dyes in foods. PMID:26079338

  8. [IgM in the diagnosis of syphilis: results on the use of the IgM solid-phase hemadsorption test].

    PubMed

    Panuccio, A; Gelosa, L

    1985-01-01

    IgM-SPHA test was improved for IgM treponema-specific determination. The test can find routinary application because it does not require for its execution special equipments, and is very easy. With this test 160 sera of luetic subjects were examined 103 were treated and 57 were affected by lues at different stages and untreated. The test is rather sensitive in the secondary and latent lues and in the reinfections, while the results are paradoxically less satisfactory in the primary syphilis. The test can be a precious diagnostic tool since, beside allowing to decide the recovery from the disease from an immunological point, finds further applications in the connatal and neurological lues. PMID:4091975

  9. Micromotor-based lab-on-chip immunoassays

    NASA Astrophysics Data System (ADS)

    García, Miguel; Orozco, Jahir; Guix, Maria; Gao, Wei; Sattayasamitsathit, Sirilak; Escarpa, Alberto; Merkoçi, Arben; Wang, Joseph

    2013-01-01

    Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an `on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields.Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an `on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32400h

  10. Identification of serotype 9 human rotavirus by enzyme-linked immunosorbent assay with monoclonal antibodies.

    PubMed

    Midthun, K; Valdesuso, J; Kapikian, A Z; Hoshino, Y; Green, K Y

    1989-09-01

    Hybridomas producing monoclonal antibodies to VP7, a major neutralizing protein of serotype 9 rotavirus (strain W161), were prepared. One monoclonal antibody, W161-6A1, was shown to neutralize only serotype 9 rotavirus strains and reacted specifically with serotype 9 rotaviruses in an enzyme-linked immunosorbent assay. The development of an immunoassay for detection of serotype 9 rotaviruses should facilitate epidemiologic studies. PMID:2550520

  11. Identification of serotype 9 human rotavirus by enzyme-linked immunosorbent assay with monoclonal antibodies.

    PubMed Central

    Midthun, K; Valdesuso, J; Kapikian, A Z; Hoshino, Y; Green, K Y

    1989-01-01

    Hybridomas producing monoclonal antibodies to VP7, a major neutralizing protein of serotype 9 rotavirus (strain W161), were prepared. One monoclonal antibody, W161-6A1, was shown to neutralize only serotype 9 rotavirus strains and reacted specifically with serotype 9 rotaviruses in an enzyme-linked immunosorbent assay. The development of an immunoassay for detection of serotype 9 rotaviruses should facilitate epidemiologic studies. PMID:2550520

  12. ULTRASENSITIVE IMMUNOASSAYS BASED ON SURFACE-ENHANCED RAMAN SCATTERING BY IMMUNOGOLD LABELS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter describes recent advances in the use of surface-enhanced Raman Scattering (SERS) as a readout tool for chip-scale, sandwich-based immunoassays. It reviews progress made in developing SERS-based immunoassays for a wide range of biolytes, including proteins, viruses and bacteria. The st...

  13. Synthesis of Protein Conjugates and Development of Immunoassays for AAL Toxins

    E-print Network

    Hammock, Bruce D.

    Synthesis of Protein Conjugates and Development of Immunoassays for AAL Toxins Ferenc Szurdoki AAL toxins and fumonisins, produced by Alternaria alternata f. sp. lycopersici and Fusarium sensitive, inexpensive, and rapid immunoassays are needed to quantify these natural toxins in food products

  14. On-Chip Native Gel Electrophoresis-Based Immunoassays for Tetanus Antibody and Toxin

    E-print Network

    Herr, Amy E.

    On-Chip Native Gel Electrophoresis-Based Immunoassays for Tetanus Antibody and Toxin Amy E. Herr was developed for detection of tetanus antibodies in buffer as well as diluted serum samples. After an off was 0.68 nM. A competitive immunoassay was also developed for tetanus toxin C-fragment by allowing un

  15. Europium chelate labels in time-resolved fluorescence immunoassays and DNA hybridization assays

    SciTech Connect

    Diamandis, E.P.; Christopoulos, T.K. Univ. of Toronto, Ontario )

    1990-11-15

    Like many analytical methodologies, immunoassays and nucleic acid hybridization assays rely on the reaction between an analyte of interest and a specific reagent. The analyte concentration is then deduced by measuring either the amount of analyte-reagent complex formed (product) or the amount of residual reagent. The authors describe the application of fluorescent rare-earth chelates to immunoassay and DNA probing.

  16. Kinetics of protein binding in solid-phase immunoassays: Theory Konstantin V. Klenin

    E-print Network

    Langowski, Jörg

    Kinetics of protein binding in solid-phase immunoassays: Theory Konstantin V. Klenin Division 2005; accepted 13 April 2005; published online 7 June 2005 In a solid-phase immunoassay, binding in solution. We present a theory describing the kinetics of immunochemical reaction in such a system. A single

  17. A potent neutralizing IgM mAb targeting the N218 epitope on E2 protein protects against Chikungunya virus pathogenesis.

    PubMed

    Lam, Shirley; Nyo, Min; Phuektes, Patchara; Yew, Chow Wenn; Tan, Yee Joo; Chu, Justin Jang Hann

    2015-11-01

    Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore, this study aimed to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model. To evaluate their antigenicity and neutralization profile, indirect enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA) and a plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated, and reverse genetics techniques were then used to create an infectious CHIKV clone with a single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. The survival rate, CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralize CHIKV replication. Further analyses of 3E7b binding and neutralization of CHIKV single-mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In a pre-binding neutralization assay, 3E7b blocks CHIKV attachment to permissive cells, possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection, 3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively, these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design. PMID:26305993

  18. AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) TESTING OF THREE IMMUNOASSAY TEST KITS FOR ANTHRAX, BOTULINUM TOXIN AND RICIN

    EPA Science Inventory

    Immunoassay test kits are based on immunoassay methods, where specific antibodies are used to detect and measure the contaminants of interest. Immunoassay test kits rely on the reaction of a contaminant or antigen with a selective antibody to give a product that can be measures....

  19. Demonstration of four immunoassay formats using the array biosensor

    NASA Technical Reports Server (NTRS)

    Sapsford, Kim E.; Charles, Paul T.; Patterson, Charles H Jr; Ligler, Frances S.

    2002-01-01

    The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

  20. Redox-Magnetohydrodynamic Microfluidics Without Channels and Compatible with Electrochemical Detection Under Immunoassay Conditions

    PubMed Central

    Weston, Melissa C.; Nash, Christena K.; Fritsch, Ingrid

    2010-01-01

    A unique capability of redox-magnetohydrodynamics (redox-MHD) for handling liquids on a small scale was demonstrated. A 1.2-?L solution plug was pumped from an injection site to a detector without the need for a channel to direct the flow. The redox pumping species did not interfere with enzymatic activity in a solution compatible with enzyme-linked immunoassays. Alkaline phosphatase (AP), a common enzyme label, converted p-aminophenyl phosphate (PAPP) to p-aminophenol (PAPR) in the presence of 2.5 mM Ru(NH3)6Cl2 and 2.5 mM Ru(NH3)6 Cl3, in 0.1 M Tris buffer (pH=9). A solution plug containing PAPP (no AP) was pumped through the surrounding solution containing AP (no PAPP), and the enzymatically-generated PAPR was easily detected and distinguishable electrochemically from the pumping species with square wave voltammetry down to 0.1 mM concentrations. The test device consisted of a silicon chip containing individually-addressable microband electrodes, placed on a 0.5-T NdFeB permanent magnet with the field oriented perpendicular to the chip. A 8.0-mm wide × 15.5-mm long × 1.5-mm high volume of solution was contained by a poly(dimethylsiloxane) gasket and capped with a glass slide. A steady-state fluid velocity of ~30 ?m/s was generated in a reinforcing flow configuration between oppositely polarized sets of pumping electrodes with ~2.1 ?A. PMID:20681513

  1. Species Specific Bacterial Spore Detection Using Lateral-Flow Immunoassay with DPA-Triggered Tb Luminescence

    NASA Technical Reports Server (NTRS)

    Ponce, Adrian

    2003-01-01

    A method of detecting bacterial spores incorporates (1) A method of lateral-flow immunoassay in combination with (2) A method based on the luminescence of Tb3+ ions to which molecules of dipicolinic acid (DPA) released from the spores have become bound. The present combination of lateral-flow immunoassay and DPA-triggered Tb luminescence was developed as a superior alternative to a prior lateral-flow immunoassay method in which detection involves the visual observation and/or measurement of red light scattered from colloidal gold nanoparticles. The advantage of the present combination method is that it affords both (1) High selectivity for spores of the species of bacteria that one seeks to detect (a characteristic of lateral-flow immunoassay in general) and (2) Detection sensitivity much greater (by virtue of the use of DPA-triggered Tb luminescence instead of gold nanoparticles) than that of the prior lateral-flow immunoassay method

  2. J. Agric. Food Chem. 1993,41, 423-430 423 Development of an Enzyme-Linked Immunosorbent Assay for Carbaryl

    E-print Network

    Hammock, Bruce D.

    J. Agric. Food Chem. 1993,41, 423-430 423 Development of an Enzyme-Linked Immunosorbent Assay that this immunoassay can be used for the determination of carbaryl in water, soil, body fluids, and food samples of this insecticide, its metabolites, and its degradation products in food, feed, water, soil

  3. Production and characterization of monoclonal antibodies to IgM of Pacific herring (Clupea pallasii)

    USGS Publications Warehouse

    Purcell, Maureen K.; Bromage, Erin S.; Silva, Jessica; Hansen, John D.; Badil, Samantha M.; Woodson, James C.; Hershberger, Paul K.

    2012-01-01

    Pacific herring (Clupea pallasii) have a central role in the North Pacific ecosystem as a forage fish species and are natural reservoirs of several important finfish pathogens, including Viral hemorrhagic septicemia virus (VHSV). Here, we report the identification of the gene encoding the immunoglobulin mu (IgM) heavy chain, as well as the development and characterization of monoclonal antibodies (MAbs) that specifically react with Pacific herring IgM. Pacific herring immunoglobulin was purified and consisted of heavy and light chains of approximately 80 and 25 kDa. Three hybridoma clones were initially identified by ELISA as reactive with purified immunoglobulin but only one clone was able to detect an 80 kDa protein in Pacific and Atlantic herring (Clupea harengus) whole plasma by denaturing western blot. However, all three MAbs were able to precipitate an 80 kDa protein from Pacific herring and LCMS sequencing of peptide fragments derived from this protein matched the predicted amino acid sequence of the cloned, heavy chain gene. In addition, two of the MAbs stained cells within the putative lymphocyte gates for the spleen, anterior kidney and posterior kidney but were not reactive for myeloid/granulocyte gates, which is consistent with these MAbs reacting with surface IgM+ B-cells. To our knowledge, this is the first report of IgM-related gene sequences and anti-IgM monoclonal antibodies from any member of the family Clupeidae. The antibodies produced in this study are critical for achieving our long-term goal of conducting serological surveillance to assess pathogen exposure in natural populations of Pacific herring.

  4. Carbohydrate specificity of IgM autoantibodies to CD45 in systemic lupus erythematosus.

    PubMed

    Fernsten, P D; Czyzyk, J K; Mimura, T; Winfield, J B

    1994-01-01

    Patients with SLE develop IgM autoantibodies to different isoforms of CD45, the major surface membrane protein tyrosine phosphatase on lymphocytes and other nucleated hemopoietic cells. Because such autoantibodies could have a potential role in the development of immune dysfunction in this disorder, we performed a series of experiments to characterize their antigenic specificity further. Blots of recombinant E. coli fusion proteins encoded by exons 3-7 of the p220 and p180 isoforms were uniformly non-reactive with SLE IgM, suggesting that anti-CD45 autoantibodies in SLE are directed against conformational and/or carbohydrate epitopes, rather than linear polypeptide epitopes. This issue was examined further using chemically and enzymatically modified CD45 purified from T cells by lectin affinity chromatography as substrates. Treatment of CD45 with 25 mM sodium-m-periodate, sufficient to abrogate binding to various lectins, abolished the reactivity with SLE anti-CD45 autoantibodies. On the other hand, digestion of CD45 with neuraminidase enhanced the binding of anti-CD45 autoantibodies from some of the SLE sera. This result probably reflects decreased steric hindrance or charge repulsion because the binding of mouse monoclonal antibodies directed against linear polypeptide epitopes of CD45 was similarly enhanced. Digestion of CD45 with N-glycosidase F had no effect on autoantibody staining. Taken together, these data suggest that IgM anti-CD45 autoantibodies in SLE recognize non-sialylated carbohydrate determinants in the highly O-glycosylated polymorphic domains of CD45. PMID:7536298

  5. HOW TO SEARCH FOR ISLANDS OF NEUTRAL HYDROGEN IN THE z ? 5.5 IGM

    SciTech Connect

    Malloy, Matthew; Lidz, Adam

    2015-02-01

    Observations of the Lyman-alpha (Ly?) forest may allow reionization to complete as late as z ? 5.5, provided the ionization state of the intergalactic medium (IGM) is sufficiently inhomogeneous at these redshifts. In this case, significantly neutral islands may remain among highly ionized gas with the ionized regions allowing some transmission through the Ly? forest. This possibility has the important virtue that it is eminently testable with existing Ly? forest data. In particular, we describe three observable signatures of significantly neutral gas in the z ? 5.5 IGM. We use mock quasar spectra produced from numerical simulations of reionization to develop these tests. First, we quantify how the abundance and length of absorbed regions in the forest increase with the volume-averaged neutral fraction in our reionization model. Second, we consider stacking the transmission profile around highly absorbed regions in the forest. If and only if there is significantly neutral gas in the IGM, absorption in the damping wing of the Ly? line will cause the transmission to recover slowly as one moves from absorbed to transmitted portions of the spectrum. Third, the deuterium Ly? line should imprint a small but distinctive absorption feature slightly blueward of absorbed neutral regions in the Ly? forest. We show that these tests can be carried out with existing Keck HIRES spectra at z ? 5.5, with the damping wing being observable for ?x{sub H} {sub I}??0.05 and the deuterium feature observable with additional high-resolution spectra for ?x{sub H} {sub I}??0.2.

  6. Microsphere-Based Immunoassay for the Detection of Azaspiracids

    PubMed Central

    Rodríguez, Laura P.; Vilariño, Natalia; Louzao, M. Carmen; Dickerson, Tobin J.; Nicolaou, K. C.; Frederick, Michael O.; Botana, Luis M.

    2014-01-01

    Azaspiracids (AZAs) are a group of lipophilic toxins discovered in mussels from Ireland in 1995 following a human poisoning incident. Nowadays the regulatory limit for AZAs in many countries is set at 160 Fg of azaspiracid equivalents per kg of shellfish meat. In this work a microsphere-based immunoassay has been developed for the detection of AZAs using a Luminex system. This method is based on the competition between AZA-2 immobilized onto the surface of microspheres and free AZAs for the interaction with a monoclonal anti-azaspiracid antibody (mAb 8F4). In this inhibition immunoassay the amount of mAb 8F4 bound to AZA-2-microspheres was quantified using a phycoerythrin-labeled anti-mouse antibody, and the fluorescence was measured with a Luminex analyzer. Simple acetate/methanol or methanol extractions yielded final extracts with no matrix interferences and adequate recovery rates of 86.5% and 75.8%, respectively. In summary, this work presents, a sensitive and easily performed screening method capable of detecting AZAs at concentrations below the range of the European regulatory limit using a microsphere/flow cytometry system. PMID:24215909

  7. Competitive Homogeneous Immunoassay for Rapid Serodiagnosis of Hantavirus Disease.

    PubMed

    Hepojoki, Satu; Rusanen, Juuso; Hepojoki, Jussi; Nurmi, Visa; Vaheri, Antti; Lundkvist, Åke; Hedman, Klaus; Vapalahti, Olli

    2015-07-01

    In this study, we describe a competitive homogeneous immunoassay that makes use of Förster resonance energy transfer (FRET) in rapid detection of pathogen-specific antibodies. The assay principle is based on competition between a monoclonal antibody (MAb) and serum antibodies to a given antigen. In the assay, named competitive FRET immunoassay (CFRET-IA), the FRET signal is induced if MAb carrying a donor label binds to an acceptor-labeled antigen. Specific antibodies in serum compete for antigen binding, resulting in reduced FRET signal. The proof-of-principle for the assay was obtained using donor-labeled Puumala virus nucleocapsid protein (PUUV-N) and acceptor-labeled anti-PUUV-N MAb. The assay was evaluated by analyzing 329 clinical samples comprising 101 from individuals with acute PUUV infection, 42 from individuals with past infection, and 186 from individuals with PUUV-seronegative sera, and the results were compared to those of reference tests. The rapid serodiagnostic test we introduced herein performed with 100% sensitivity and 99% specificity for diagnosing acute hantavirus disease. PMID:25972427

  8. Competitive Homogeneous Immunoassay for Rapid Serodiagnosis of Hantavirus Disease

    PubMed Central

    Rusanen, Juuso; Hepojoki, Jussi; Nurmi, Visa; Vaheri, Antti; Lundkvist, Åke; Hedman, Klaus; Vapalahti, Olli

    2015-01-01

    In this study, we describe a competitive homogeneous immunoassay that makes use of Förster resonance energy transfer (FRET) in rapid detection of pathogen-specific antibodies. The assay principle is based on competition between a monoclonal antibody (MAb) and serum antibodies to a given antigen. In the assay, named competitive FRET immunoassay (CFRET-IA), the FRET signal is induced if MAb carrying a donor label binds to an acceptor-labeled antigen. Specific antibodies in serum compete for antigen binding, resulting in reduced FRET signal. The proof-of-principle for the assay was obtained using donor-labeled Puumala virus nucleocapsid protein (PUUV-N) and acceptor-labeled anti-PUUV-N MAb. The assay was evaluated by analyzing 329 clinical samples comprising 101 from individuals with acute PUUV infection, 42 from individuals with past infection, and 186 from individuals with PUUV-seronegative sera, and the results were compared to those of reference tests. The rapid serodiagnostic test we introduced herein performed with 100% sensitivity and 99% specificity for diagnosing acute hantavirus disease. PMID:25972427

  9. Finger-Actuated, Self-Contained Immunoassay Cassettes

    PubMed Central

    Qiu, Xianbo; Thompson, Jason A.; Chen, Zongyuan; Liu, Changchun; Chen, Dafeng; Ramprasad, Sudhir; Mauk, Michael G.; Ongagna, Serge; Barber, Cheryl; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L.A.M.; Bau, Haim H.

    2010-01-01

    The building blocks for an inexpensive, disposable, luminescence-based microfluidic immunoassay cassette are described, and their integration in a point-of-care diagnostic system is demonstrated. Fluid motion in the cassette is driven by depressing finger-actuated pouches. All reagents needed for the immunoassay can be stored in the cassette in liquid form. Prior to use, the cassette consists of two separate parts. A top storage component contains pouches, sealed storage chambers, a metering chamber, and needle seats. The bottom processing component contains connection needles, a mixing chamber, and a detection chamber with immobilized proteins. Subsequent to sample introduction, the storage and processing components are mated. The needles form hydraulic connections between the two parts and, in some cases, close valves. The pouches are then actuated sequentially to induce flow of various reagents and facilitate process operations. The cassette is compatible with different detection modalities. Both a cassette with immunochromatographic-based detection and a cassette with microbead-based detection were constructed and evaluated. The immunochromatographic cassette was used to detect antibodies to HIV in saliva samples. The bead-based cassette was used to detect the proinflammatory chemokine IL-8. The experimental data demonstrates good repeatability and reasonable sensitivity. PMID:19597994

  10. Sensitive Immunoassays of Nitrated Fibrinogen in Human Biofluids

    SciTech Connect

    Tang, Zhiwen; Wu, Hong; Du, Dan; Wang, Jun; Wang, Hua; Qian, Weijun; Bigelow, Diana J.; Pounds, Joel G.; Smith, Richard D.; Lin, Yuehe

    2010-05-05

    Three new sandwich immunoassays for detection of nitrated biomarker have been established with potential applications in biomedical studies and clinical practice. In this study, nitrated human fibrinogen, a potential oxidative stress biomarker for several pathologies, was chosen as the target. To improve the sensitivity and overcome the interference caused by the complexity of human biofluids, we developed three sandwich strategies using various combinations of primary antibody and secondary antibody. All three strategies demonstrated high sensitivity and selectivity towards nitrated forms of fibrinogen in buffer, but their performances were dramatically reduced when tested with human plasma and serum samples. Systematically optimizations were carried out to investigate the effects of numerous factors, including sampling, coating, blocking, and immunoreactions. Our final optimization results indicate that two of these strategies retain sufficient sensitivity and selectivity for use as assays in human physiological samples. Specifically, detection limits reached the pM level and the linear response ranges were up to nM level with a correlation coefficient > 0.99. To our best knowledge, this is the first example of using an electrochemical immunoassay for a nitrated biomarker in a physiological fluid. This novel approach provides a rapid, sensitive, selective, cost efficient and robust bioassay for detection of oxidative stress in pathology and for clinical applications. Moreover, the sandwich strategies developed in this paper can be readily used to establish effective methods targeting other nitration biomarkers.

  11. Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

    NASA Astrophysics Data System (ADS)

    Mirasoli, Mara; Michelini, Elisa; Deo, Sapna K.; Dikici, Emre; Roda, Aldo; Daunert, Sylvia

    2004-06-01

    The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

  12. Quantum-Dots Based Electrochemical Immunoassay of Interleukin-1?

    SciTech Connect

    Wu, Hong; Liu, Guodong; Wang, Jun; Lin, Yuehe

    2007-07-01

    We describe a quantum-dot (QD, CdSe@ZnS)-based electrochemical immunoassay to detect a protein biomarker, interleukin-1? (IL-1?). QD conjugated with anti-IL-1? antibody was used as a label in an immunorecognition event. After a complete sandwich immunoreaction among the primary IL-1? antibody (immobilized on the avidin-modified magnetic beads), IL-1?, and the QD-labeled secondary antibody, QD labels were attached to the magnetic-bead surface through the antibody-antigen immunocomplex. Electrochemical stripping analysis of the captured QDs was used to quantify the concentration of IL-1? after an acid-dissolution step. The streptavidin-modified magnetic beads and the magnetic separation platform were used to integrate a facile antibody immobilization (through a biotin/streptavidin interaction) with immunoreactions and the isolation of immunocomplexes from reaction solutions in the assay. The voltammetric response is highly linear over the range of 0.5 to 50 ng mL-1 IL 1?, and the limit of detection is estimated to be 0.3 ng mL-1 (18 pM). This QD-based electrochemical immunoassay shows great promise for rapid, simple, and cost-effective analysis of protein biomarkers.

  13. Enzyme-linked immunosorbent assays for the measurement of specific antibodies in experimentally induced ovine toxoplasmosis.

    PubMed Central

    Payne, R. A.; Joynson, D. H.; Wilsmore, A. J.

    1988-01-01

    Tachyzoites of the RH strain of Toxoplasma gondii were inoculated intravenously into sheep following which serum samples were collected at approximately weekly intervals for 9 months. The sera were examined by the toxoplasma dye test and two enzyme-linked immunosorbent assays (ELISA) specifically developed for investigations of ovine toxoplasmosis. One was an antibody class capture assay for the detection of anti-toxoplasma specific IgM, the other an indirect assay which detected anti-toxoplasma IgG. Some of the sheep had antibodies to toxoplasma prior to inoculation but none had specific IgM. Sera collected 17 days after inoculation showed that all had raised specific antibody levels but the only sheep that produced specific antitoxoplasma IgM were those that were initially without any antibody. Specific IgM could be detected in all these particular sheep for at least 1 month after infection and up to 3 months in some. Specific IgG persisted at high levels for at least 3 months and could still be detected at moderate levels for at least 9 months. The ELISA methods described are simple to perform and could clearly distinguish between previous infection and this experimental infection with Toxoplasma gondii. PMID:3356219

  14. The species specificity of the microimmunofluorescence antibody test and comparisons with a time resolved fluoroscopic immunoassay for measuring IgG antibodies against Chlamydia pneumoniae.

    PubMed Central

    Wong, Y K; Sueur, J M; Fall, C H; Orfila, J; Ward, M E

    1999-01-01

    AIMS: To examine the species specificity of the microimmunofluorescence test (MIF) and assess a time resolved fluoroscopic immunoassay (TRIA) for measuring IgG antibodies to C pneumoniae. METHODS: Sera from 1020 subjects were tested by MIF for IgG, IgM, and IgA antibodies to C pneumoniae, C trachomatis, and C psittaci; 501 serum samples were also tested by TRIA for IgG antibodies to C pneumoniae. RESULTS: C pneumoniae antibody titres as measured by MIF were correlated with those for C psittaci and trachomatis. It was estimated that on average, one third of the twofold dilution steps that make up the final C pneumoniae antibody titre may be due to cross reacting genus specific antibody. The results of TRIA correlated well with those of MIF. In 75% of cases, the TRIA result predicted a three titre range within which the actual MIF result would fall. CONCLUSIONS: MIF does not appear to be as species specific as claimed. TRIA is unlikely to be as specific but as it is completely objective, easier to perform, amenable to automation, and gives reproducible results, it is a rapid and useful method for comparing populations. PMID:10396235

  15. Preparation of a Magnetically Switchable Bioelectrocatalytic System Employing Cross-Linked Enzyme Aggregates in Magnetic Mesocellular Carbon Foam

    SciTech Connect

    Lee, Jinwoo; Lee, Dohun; Oh, Eunkeu; Kim, Jaeyun; Kim, Young-Pil; Jin, Sunmi; Kim, Hak Sung; Hwang, Yosun; Kwak, Ja Hun; Park, Je-Geun; Shin, Chae-Ho; Kim, Jungbae; Hyeon, Taeghwan

    2005-11-18

    Nanostructured magnetic materials (NMMs)[1] have attracted much attention recently because of their broad biotechnological applications including support matrices for enzyme immobilization,[2] immunoassays,[3] drug delivery,[4] and biosensors.[ 5] Specifically, the easy separation and controlled placement of NMMs by means of an external magnetic field enables their application in the development of immobilized enzyme processes[2] and the construction of magnetically controllable bio-electrocatalytic systems.[5, 6] Herein, we demonstrate the use of immobilized enzymes in NMMs for magnetically switchable bio-electrocatalysis.

  16. The etiology of Rubella IgM positivity in patients with rubella-like illness in Iran from 2011 to 2013.

    PubMed

    Khorrami, Seyed Mahmood Seyed; Mokhtari-Azad, Talat; Yavarian, Jila; Nasab, Gazal Sadat Fatemi; Naseri, Maryam; Jandaghi, Nazanin Zahra Shafiei

    2015-11-01

    Rubella is a mild self-limiting contagious viral disease caused by the rubella virus (RV). Although symptoms are often mild, the concern is centralized around the possible effect on a fetus growth and development in case of primary infection during early months of pregnancy. Recently acquired rubella is commonly confirmed by RV-specific IgM antibody detection in the serum. However, rubella primary infection is not always the only cause of IgM positivity. Other possible causes of rubella IgM positivity may include IgM persistence following vaccination or naturally acquired infection or even re-infection. Moreover, nonspecific IgM reactivity can cause false-positive results. There are few articles to differentiate the aetiology of rash in rubella-like illnesses. However, limited studies have been conducted on clarifying the source of IgM positivity in these cases. This article reports the study of 10,896 clinical cases demonstrating rubella-like illness between 2011 and 2013 in Iran. The rate of IgM positivity among these cases was 0.52% (57 cases). As predicted based on the high coverage of vaccination in Iran fewer than 16% of cases with ELISA IgM positive result, were due to current rubella primary infections. The greater part of the positive IgM reactions occurred in cross reactivity with other viruses (31.6%) or in prolonged IgM response post vaccination (24.6%). This research confirmed that the positive result of rubella IgM assay in vaccinated individuals is mainly caused by prolonged IgM production, rubella re-infection, and false positivity due to infection with other viruses, rather than the rubella primary infection itself. PMID:25950278

  17. Development and evaluation of a capture enzyme-linked immunosorbent assay for determination of rubella immunoglobulin M using monoclonal antibodies.

    PubMed Central

    Gerna, I; Zannino, M; Revello, M G; Petruzzelli, E; Dovis, M

    1987-01-01

    A capture enzyme-linked immunosorbent assay (ELISA) for detection of virus-specific immunoglobulin M (IgM) antibody was developed which used a panel of labeled monoclonal antibodies to rubella virus hemagglutinin. The rapidity of the test system was increased by using, after 1-h incubation of the test serum, a second 1-h incubation of the serum with a mixture of viral antigen and labeled monoclonal antibody. The new assay was tested for specificity on 371 human sera from people without any recent contact with rubella virus; of these, 66 were sera selected from people with rheumatoid factor or IgM antibody to human cytomegalovirus, Epstein-Barr virus, or other viruses. In parallel, the new assay was performed on 191 sera from patients having recent contact with rubella virus. Results were compared with those obtained by an indirect ELISA method on IgM serum fractions, using purified rubella virus as a solid phase. Of the 371 sera tested for specificity, 5 (1.3%) gave false-positive results with indirect ELISA (1 rheumatoid factor, 2 heterophil antibody, and 2 human cytomegalovirus sera positive for IgM), and none were false-positive with the capture assay. Two sera from a patient with primary cytomegalovirus infection, which were positive for rubella IgM antibody with both methods and were initially interpreted as false-positive, were finally considered to be true-positive, since they were reactive only in the presence of IgM antibody and viral antigen. Of the 191 sera from 92 patients (84 patients with acute rubella, four newborns from mothers with rubella during pregnancy, and four vaccinees), 136 (71.2%) were found to be positive for IgM by direct ELISA, and 128 (67.0%) were positive by capture ELISA; 12 sera drawn during the first 2 days of disease, or at least 40 days after onset (or after vaccination), were detected only by indirect ELISA, and 4 sera were detected only by capture ELISA. Thus, specificity and sensitivity, respectively, were 100 and 91.4% for capture ELISA and 98.6 and 97.1% for indirect ELISA. However, when the number of patients was considered, 86 were detected as IgM positive by indirect ELISA, and 87 were detected positive by capture ELISA. The overall agreement between the two assays was 96.2%. Capture ELISA using monoclonal antibody appears preferable over indirect ELISA on IgM serum fractions because of its higher specificity and shorter time for test performance; furthermore, there is no need for serum fractionation or virus purification for the capture ELISA. PMID:3597747

  18. 2D immunoblots show differential response of mouse IgG and IgM antibodies to antigens of mammary carcinoma 4 T1 cells

    PubMed Central

    2014-01-01

    Background Immunosuppression in breast cancer has been reported in women and in the highly metastatic mouse mammary tumor model 4 T1. The immunosuppressive environment complicates the use of the humoral response against the tumor as an immunodiagnostic tool. IgM has not been used in immunodiagnostic in part because its antitumor responses, both innate and adaptive, have not been studied in function of time in breast cancer. We show a new approach to analyzing the mouse humoral immune response, and compare the evolution with time of IgG and IgM responses against the antigens of 4 T1 cells. Methods The study is based on 2-dimensional immunoblotting detection of antigens from 4 T1 cells by the IgG and IgM antibodies in the serum of female mice injected with 4 T1 cells. Results There was a high variability in the intra-and inter-mouse response. Variability in the IgM response was manifested as a pattern of spots that could become a multibinomial variable of 0 and 1, which could represent a signature of the immune response. Different numbers of spots was found in the IgG and IgM responses from week 1 to 5. On average, the IgM had more but the IgG response decrease with the time. The natural IgM at t?=?0 responds stronger than w1; the adaptive response of both IgM and IgG were elicited where, with the former being stronger better than the latter. Antigens that are recognized by some female mice in the first week are also recognized by other female mice at time 0. Contamination of the natural IgM makes difficult use the adaptive IgM as a tool for immunodiagnostic. Conclusions IgM and IgG response varied with the time and individuals. Spot variation in 2D pattern for the natural IgM could be expressed as a binomial signature, which opens up the way to correlate a particular pattern with resistance or susceptibility. This uncovers a battery of IgMs for each individual to confront cancer or infections. The possibility to differentiate between adaptive IgM antibodies from the natural IgM will allow investigation of the adaptive IgM for early immunodiagnosis. PMID:24467921

  19. Development of a Disperse Dye Immunoassay Technique for Detection of Antibodies against Neospora caninum in Cattle

    PubMed Central

    Selahi, Fatemeh; Hosseini, Mohammad Hossein; Mansourian, Maryam; Tahamtan, Yahya

    2013-01-01

    In this study a disperse dye immunoassay method was standardized and evaluated for detection of antibodies against Neospora caninum in cattle. Sera from 150 cattle with a recent history of abortion were collected and tested by commercial ELISA kit and a standardized in-house dye immunoassay system. The positivity rate for the sera used in this study was 34.6% for the disperse dye immunoassay (DDIA) compared to 32% obtained by ELISA kit. This study showed no significant difference between DDIA and ELISA. The results indicated that the DDIA provide an economic, simple, rapid and robust test for detection of N. caninum infection in cattle. PMID:23467930

  20. Multiplexed immunoassays for biomonitoring of exposure to agrochemicals using quantum dots as fluorescent reporters

    NASA Astrophysics Data System (ADS)

    Nichkova, Mikaela; Dosev, Dosi; Davies, Alexander E.; Gee, Shirley J.; Kennedy, Ian M.; Hammock, Bruce D.

    2007-02-01

    The application of quantum dots (QDs) as labels in immunoassay microarrays for the multiplex detection of 3- phenoxybenzoic acid (PBA) and atrazine-mercapturate (AM) has been demonstrated. PBA and AM are biomarkers of exposure to the pyrethroid insecticides and to the herbicide atrazine, respectively. Microarrays were fabricated by microcontact printing of the coating antigens in line patterns onto glass substrates. Competitive immunoassays were successfully performed using quantum dots (QD560 and QD620) as reporters. The multiplexed immunoassays were characterized by fluorescence microscopy and SEM. The application of QD fluorophores facilitates multiplex assays and therefore can contribute to enhanced throughput in biomonitoring.

  1. Evaluation of recombinant line immunoblot for detection of Lyme disease in Slovakia: comparison with two other immunoassays.

    PubMed

    Lencáková, Daniela; Fingerle, Volker; Stefancíková, Astéria; Schulte-Spechtel, Ulrike; Petko, Branislav; Schréter, Ivan; Wilske, Bettina

    2008-06-01

    In the present study the sensitivity and the specificity of three serological tests (enzyme-linked immunosorbent assay [ELISA], indirect fluorescent antibody test [IFA], and recombinant line immunoblot) were compared by examining 74 sera from patients diagnosed with Lyme disease in Eastern Slovakia. In addition, the reactivity to each of the recombinant proteins in the immunoblot was examined in order to evaluate their diagnostic value. Generally, the immunoblot (93.2%) and the ELISA (90.5%) were significantly more sensitive than the IFA (64.9%; df = 1; p < or = 0.001). Correlation between results of the ELISA, IFA, and immunoblot for IgM or IgG, when two tests were always compared, one to the other, ranged from r(s) = 0.673 to r(s) = 0.905. In the immunoblot, the highest sensitivity was observed in DbpA and VlsE proteins (76.9% and 84.6%, respectively) in IgG testing of the sera from the patient group of Lyme arthritis. VlsE proteins, together with OspC proteins, were also shown to be useful for IgM antibody detection in erythema migrans patients (up to 44.4% and 53.7% sensitivity, respectively). Our results indicate that both the ELISA and the recombinant immunoblot test were more satisfactory for seroconfirmation of Lyme disease than IFA. Moreover, the reseach confirmed diagnostic value of the in-vivo expressed proteins (VlsE and DbpA), which might have the potential to play an important role in improving whole-cell antigen-based testing. PMID:18279004

  2. Competitive adsorption-desorption of IgM monomers-dimers on silica and modified silica surfaces.

    PubMed

    Guha, Suvajyoti; Wayment, Joshua; Rastogi, Vinayak; Li, Mingdong; Tarlov, Michael J; Zachariah, Michael R

    2013-07-15

    Understanding competitive adsorption-desorption of proteins onto surfaces is an important area of research in food processing and biomedical engineering. Here, we demonstrate, how electrospray-differential mobility analysis that has been traditionally used for characterizing bionanoparticles, can be used for quantifying complex competitive adsorption-desorption of oligomeric proteins or multiprotein systems using monomers and dimers of IgM as a model example onto silica and modified silica surfaces. Using ES-DMA, we show that IgM dimers show a preference to stay adsorbed to different surfaces although monomers adsorb more easily and desorption rates of monomers and dimers of IgM are surface-type-dependent and are not significantly affected by shear. We anticipate that this demonstration will make ES-DMA a popular "label-free" method for studying multicomponent multi-oligomeric protein adsorption to different surfaces in the future. PMID:23628202

  3. Sequential Push-Pull Pumping Mechanism for Washing and Evacuation of an Immunoassay Reaction Chamber on a Microfluidic CD Platform

    PubMed Central

    Thio, Tzer Hwai Gilbert; Ibrahim, Fatimah; Al-Faqheri, Wisam; Soin, Norhayati; Kahar Bador, Maria; Madou, Marc

    2015-01-01

    A centrifugal compact disc (CD) microfluidic platform with reservoirs, micro-channels, and valves can be employed for implementing a complete immunoassay. Detection or biosensor chambers are either coated for immuno-interaction or a biosensor chip is inserted in them. On microfluidic CDs featuring such multi-step chemical/biological processes, the biosensor chamber must be repeatedly filled with fluids such as enzymes solutions, buffers, and washing solutions. After each filling step, the biosensor chamber needs to be evacuated by a passive siphoning process to prepare it for the next step in the assay. However, rotational speed dependency and limited space on a CD are two big obstacles to performing such repetitive filling and siphoning steps. In this work, a unique thermo-pneumatic (TP) Push-Pull pumping method is employed to provide a superior alternative biosensor chamber filling and evacuation technique. The proposed technique is demonstrated on two CD designs. The first design features a simple two-step microfluidic process to demonstrate the evacuation technique, while the second design shows the filling and evacuation technique with an example sequence for an actual immunoassay. In addition, the performance of the filling and evacuation technique as a washing step is also evaluated quantitatively and compared to the conventional manual bench top washing method. The two designs and the performance evaluation demonstrate that the technique is simple to implement, reliable, easy to control, and allows for repeated push-pulls and thus filling and emptying of the biosensor chamber. Furthermore, by addressing the issue of rotational speed dependency and limited space concerns in implementing repetitive filling and evacuation steps, this newly introduced technique increases the flexibility of the microfluidic CD platform to perform multi-step biological and chemical processes. PMID:25853411

  4. Facilitating the Validation of Novel Protein Biomarkers for Dementia: An Optimal Workflow for the Development of Sandwich Immunoassays

    PubMed Central

    del Campo, Marta; Jongbloed, Wesley; Twaalfhoven, Harry A. M.; Veerhuis, Robert; Blankenstein, Marinus A.; Teunissen, Charlotte E.

    2015-01-01

    Different neurodegenerative disorders, such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD), lead to dementia syndromes. Dementia will pose a huge impact on society and thus it is essential to develop novel tools that are able to detect the earliest, most sensitive, discriminative, and dynamic biomarkers for each of the disorders. To date, the most common assays used in large-scale protein biomarker analysis are enzyme-linked immunosorbent assays (ELISA), such as the sandwich immunoassays, which are sensitive, practical, and easily implemented. However, due to the novelty of many candidate biomarkers identified during proteomics screening, such assays or the antibodies that specifically recognize the desired marker are often not available. The development and optimization of a new ELISA should be carried out with considerable caution since a poor planning can be costly, ineffective, time consuming, and it may lead to a misinterpretation of the findings. Previous guidelines described either the overall biomarker development in more general terms (i.e., the process from biomarker discovery to validation) or the specific steps of performing an ELISA procedure. However, a workflow describing and guiding the main issues in the development of a novel ELISA is missing. Here, we describe a specific and detailed workflow to develop and validate new ELISA for a successful and reliable validation of novel dementia biomarkers. The proposed workflow highlights the main issues in the development of an ELISA and covers several critical aspects, including production, screening, and selection of specific antibodies until optimal fine-tuning of the assay. Although these recommendations are designed to analyze novel biomarkers for dementia in cerebrospinal fluid, they are generally applicable for the development of immunoassays for biomarkers in other human body fluids or tissues. This workflow is designed to maximize the quality of the developed ELISA using a time- and cost-efficient strategy. This will facilitate the validation of the dementia biomarker candidates ultimately allowing accurate diagnostic conclusions. PMID:26483753

  5. IgE Epitope Mapping Using Peptide Microarray Immunoassay.

    PubMed

    Gimenez, Gustavo; Benedé, Sara; Lin, Jing

    2016-01-01

    IgE epitope mapping of food allergens contributes to a better understanding of the pathogenesis of food allergy and may become an additional tool for food allergy diagnosis/prognosis. Microarray platforms which allow for simultaneous screening of a large number of peptides corresponding to the sequences of food allergens are ideally suited for large-scale IgE epitope mapping. Here we describe the method of performing a reliable and sensitive peptide microarray immunoassay, which was developed in our lab and results in the identification of candidate IgE epitope biomarkers useful in determining allergic disease severity and prognosis, as well as in the prediction of treatment outcomes. A gastric digestion model that can be applied to prescreen peptides and reduce costs in the peptide microarray is also described in this chapter. PMID:26490481

  6. Biosensor immunoassay for traces of hazelnut protein in olive oil

    PubMed Central

    Smits, Nathalie G. E.; Haasnoot, Willem

    2009-01-01

    The fraudulent addition of hazelnut oil to more expensive olive oil not only causes economical loss but may also result in problems for allergic individuals as they may inadvertently be exposed to potentially allergenic hazelnut proteins. To improve consumer safety, a rapid and sensitive direct biosensor immunoassay, based on a highly specific monoclonal antibody, was developed to detect the presence of hazelnut proteins in olive oils. The sample preparation was easy (extraction with buffer); the assay time was fast (4.5 min only) and the limit of detection was low (0.08 ?g/g of hazelnut proteins in olive oil). Recoveries obtained with an olive oil mixed with different amounts of a hazelnut protein containing hazelnut oil varied between 93% and 109%. Electronic supplementary material The online version of this article (doi:10.1007/s00216-009-2720-1) contains supplementary material, which is available to authorized users. PMID:19263041

  7. Performance of immunoassay kits for site characterization and remediation

    SciTech Connect

    Waters, L.C.; Palausky, A.; Counts, R.W.; Jenkins, R.A.

    1995-12-31

    The US Department of Energy (DOE) is supporting efforts to identify, validate and implement the use of effective, low-cost alternatives to currently used analytical methods for environmental management. As part of that program, we have evaluated the performances of a number of immunoassay (IA) kits with specificities for environmental contaminants of concern to the DOE. The studies were done in the laboratory using both spiked and field test samples. The analyte specificity and manufacturers of the kits evaluated were the following: mercury, BioNebraska; polychlorinated biphenyls (PCBs), EnSys and Millipore; petroleum fuel hydrocarbons, Millipore and Ohmicron; and polyaromatic hydrocarbons (PAHs), Ohmicron and Millipore. The kits were used in either a semiquantitative or quantitative format according to the preference of the manufacturers.

  8. A fluorogenic heterogeneous immunoassay for cardiac muscle troponin cTnI on a digital microfluidic device.

    PubMed

    Tsaloglou, Maria-Nefeli; Jacobs, Adrian; Morgan, Hywel

    2014-09-01

    We describe a fluorogenic two-site noncompetitive heterogeneous immunoassay with magnetic beads on a low-voltage digital microfluidic platform using closed electrowetting-on-dielectric (EWOD). All the steps of an enzyme-linked immunosorbent assay (ELISA) were performed on the device using 9H-(1, 3-dichloro-9, 9-dimethylacridin-2-one-7-yl) phosphate as the fluorogenic substrate for the enzyme alkaline phosphatase. The performance of the system was demonstrated with cardiac marker Troponin I (cTnI) as a model analyte in phosphate-buffered saline samples. cTnI was detected within the diagnostically relevant range with a limit of detection of 2.0 ng/mL (CV?=?6.47 %). Washing of magnetic beads was achieved by movement through a narrow region of buffer bridging one drop to another with minimal fluid transfer. More than 90 % of the unbound reagents were removed after five washes. Further experiments testing human blood serum on the same platform demonstrated a sample-to-answer time at ?18.5 min detecting 6.79 ng/mL cTnI. PMID:25074544

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

    PubMed

    Kim, Myoung-Ho; Choi, Suk-Jung

    2015-04-15

    In this study, we devised a stationary liquid-phase lab-on-a-chip (SLP LOC), which was operated by moving solid-phase magnetic particles in the stationary liquid phase. The SLP LOC consisted of a sample chamber to which a sample and reactants were added, a detection chamber containing enzyme substrate solution, and a narrow channel connecting the two chambers and filled with buffer. As a model system, competitive immunoassays of saxitoxin (STX), a paralytic shellfish toxin, were conducted in the SLP LOC using protein G-coupled magnetic particles (G-MPs) as the solid phase. Anti-STX antibodies, STX-horseradish peroxidase conjugate, G-MPs, and a STX sample were added to the sample chamber and reacted by shaking. While liquids were in the stationary state, G-MPs were transported from the sample chamber to the detection chamber by moving a magnet below the LOC. After incubation to allow the enzymatic reaction to occur, the absorbance of the detection chamber solution was found to be reciprocally related to the STX concentration of the sample. Thus, the SLP LOC may represent a novel, simple format for point-of-care testing applications of enzyme-linked immunosorbent assays by eliminating complicated liquid handling steps. PMID:25460894

  10. Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 Variants per Genome Can Bind IgM via Its Fc Fragment Fc?.

    PubMed

    Jeppesen, Anine; Ditlev, Sisse Bolm; Soroka, Vladyslav; Stevenson, Liz; Turner, Louise; Dzikowski, Ron; Hviid, Lars; Barfod, Lea

    2015-10-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) adhesive proteins expressed on the surfaces of infected erythrocytes (IEs) are of key importance in the pathogenesis of P. falciparum malaria. Several structurally and functionally defined PfEMP1 types have been associated with severe clinical manifestations, such as cerebral malaria in children and placental malaria in pregnant women. PfEMP1 that can bind the Fc part of IgM (Fc?) characterizes one such type, although the functional significance of this IgM binding to PfEMP1 remains unclear. In this study, we report the identification and functional analysis of five IgM-binding PfEMP1 proteins encoded by P. falciparum NF54. In addition to the VAR2CSA-type PFL0030c protein, already known to bind Fc? and to mediate chondroitin sulfate A (CSA)-specific adhesion of IEs in the placenta, we found four PfEMP1 proteins not previously known to bind IgM this way. Although they all contained Duffy binding-like ? (DBL?) domains similar to those in VAR2CSA-type PfEMP1, they did not mediate IE adhesion to CSA, and IgM binding did not shield IEs from phagocytosis of IgG-opsonized IEs. In this way, these new IgM-binding PfEMP1 proteins resemble the rosette-mediating and IgM-binding PfEMP1 HB3VAR06, but none of them mediated formation of rosettes. We could map the capacity for Fc-specific IgM binding to DBL? domains near the C terminus for three of the four PfEMP1 proteins tested. Our study provides new evidence regarding Fc-dependent binding of IgM to PfEMP1, which appears to be a common and multifunctional phenotype. PMID:26216422

  11. The majority of human memory B cells recognizing RhD and tetanus resides in IgM+ B cells.

    PubMed

    Della Valle, Luciana; Dohmen, Serge E; Verhagen, Onno J H M; Berkowska, Magdalena A; Vidarsson, Gestur; Ellen van der Schoot, C

    2014-08-01

    B cell memory to T cell-dependent (TD) Ags are considered to largely reside in class-switched CD27(+) cells. However, we previously observed that anti-RhD (D) Igs cloned from two donors, hyperimmunized with D(+) erythrocytes, were predominantly of the IgM isotype. We therefore analyzed in this study the phenotype and frequency of D- and tetanus toxoid-specific B cells by culturing B cells in limiting dilution upon irradiated CD40L-expressing EL4.B5 cells and testing the culture supernatant. Most Ag-specific B cells for both TD Ags were found to reside in the IgM-expressing B cells, including CD27(-) B cells, in both hyperimmunized donors and nonhyperimmunized volunteers. Only shortly after immunization a sharp increase in Ag-specific CD27(+)IgG(+) B cells was observed. Next, B cells were enriched with D(+) erythrocyte ghosts and sorted as single cells. Sequencing of IGHV, IGLV, IGKV, and BCL6 genes from these D-specific B cell clones demonstrated that both CD27(-)IgM(+) and CD27(+)IgM(+) B cells harbored somatic mutations, documenting their Ag-selected nature. Furthermore, sequencing revealed a clonal relationship between the CD27(-)IgM(+), CD27(+)IgM(+), and CD27(+)IgG(+) B cell subsets. These data strongly support the recently described multiple layers of memory B cells to TD Ags in mice, where IgM(+) B cells represent a memory reservoir which can re-enter the germinal center and ensure replenishment of class-switched memory CD27(+) B cells from Ag-experienced precursors. PMID:24965774

  12. Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 Variants per Genome Can Bind IgM via Its Fc Fragment Fc?

    PubMed Central

    Jeppesen, Anine; Ditlev, Sisse Bolm; Soroka, Vladyslav; Stevenson, Liz; Turner, Louise; Dzikowski, Ron; Hviid, Lars

    2015-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) adhesive proteins expressed on the surfaces of infected erythrocytes (IEs) are of key importance in the pathogenesis of P. falciparum malaria. Several structurally and functionally defined PfEMP1 types have been associated with severe clinical manifestations, such as cerebral malaria in children and placental malaria in pregnant women. PfEMP1 that can bind the Fc part of IgM (Fc?) characterizes one such type, although the functional significance of this IgM binding to PfEMP1 remains unclear. In this study, we report the identification and functional analysis of five IgM-binding PfEMP1 proteins encoded by P. falciparum NF54. In addition to the VAR2CSA-type PFL0030c protein, already known to bind Fc? and to mediate chondroitin sulfate A (CSA)-specific adhesion of IEs in the placenta, we found four PfEMP1 proteins not previously known to bind IgM this way. Although they all contained Duffy binding-like ? (DBL?) domains similar to those in VAR2CSA-type PfEMP1, they did not mediate IE adhesion to CSA, and IgM binding did not shield IEs from phagocytosis of IgG-opsonized IEs. In this way, these new IgM-binding PfEMP1 proteins resemble the rosette-mediating and IgM-binding PfEMP1 HB3VAR06, but none of them mediated formation of rosettes. We could map the capacity for Fc-specific IgM binding to DBL? domains near the C terminus for three of the four PfEMP1 proteins tested. Our study provides new evidence regarding Fc-dependent binding of IgM to PfEMP1, which appears to be a common and multifunctional phenotype. PMID:26216422

  13. Insolubilization process increases enzyme stability

    NASA Technical Reports Server (NTRS)

    Billingham, J.; Lyn, J.

    1971-01-01

    Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes.

  14. Teleost Fish Mount Complex Clonal IgM and IgT Responses in Spleen upon Systemic Viral Infection

    PubMed Central

    Castro, Rosario; Jouneau, Luc; Pham, Hang-Phuong; Bouchez, Olivier; Giudicelli, Véronique; Lefranc, Marie-Paule; Quillet, Edwige; Benmansour, Abdenour; Cazals, Frédéric; Six, Adrien; Fillatreau, Simon; Sunyer, Oriol; Boudinot, Pierre

    2013-01-01

    Upon infection, B-lymphocytes expressing antibodies specific for the intruding pathogen develop clonal responses triggered by pathogen recognition via the B-cell receptor. The constant region of antibodies produced by such responding clones dictates their functional properties. In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT remain unknown. The expression of IgM and IgT are mutually exclusive, leading to the existence of two B-cell subsets expressing either both IgM and IgD or only IgT. Here, we undertook a comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout (Onchorhynchus mykiss) before, and after challenge with a rhabdovirus, the Viral Hemorrhagic Septicemia Virus (VHSV), using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, we observed distinct repertoires for IgM, IgD and IgT, respectively, with a few amplified ? and ? junctions, suggesting the presence of IgM- and IgT-secreting cells in the spleen. In infected animals, we detected complex and highly diverse IgM responses involving all VH subgroups, and dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal IgT, indicating that both IgM+ and IgT+ spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. Although fish IgD and IgT present different structural features and evolutionary origin compared to mammalian IgD and IgA, respectively, their implication in the B-cell response evokes these mouse and human counterparts. Thus, it appears that the general properties of antibody responses were already in place in common ancestors of fish and mammals, and were globally conserved during evolution with possible functional convergences. PMID:23326228

  15. Dark matter annihilation energy output and its effects on the high-z IGM

    NASA Astrophysics Data System (ADS)

    Araya, Ignacio J.; Padilla, Nelson D.

    2014-11-01

    We study the case of dark matter (DM) self-annihilation, in order to assess its importance as an energy injection mechanism, to the intergalactic medium (IGM) in general, and to the medium within particular DM haloes. We consider thermal relic WIMP (weakly interacting massive particle) particles with masses of 10 GeV and 1 TeV, and we analyse in detail the clustering properties of DM in a ? cold dark matter cosmology, on all hierarchy levels, from haloes and their mass function, to subhaloes and the DM density profiles within them, considering adiabatic contraction by the presence of a supermassive black hole. We then compute the corresponding energy output, concluding that DM annihilation does not constitute an important feedback mechanism. We also calculate the effects that DM annihilation has on the IGM temperature and ionization fraction, and we find that assuming maximal energy absorption, at z ˜ 10, for the case of a 1 TeV WIMP, the ionization fraction could be raised to 6 × 10-4 and the temperature to 10 K, and in the case of a 10 GeV WIMP, the IGM temperature could be raised to 200 K and the ionization fraction to 8 × 10-3. We conclude that DM annihilations cannot be regarded as an alternative reionization scenario. Regarding the detectability of the WIMP through the modifications to the 21 cm differential brightness temperature signal (?Tb), we conclude that a thermal relic WIMP with mass of 1 TeV is not likely to be detected from the global signal alone, except perhaps at the 1-3 mK level in the frequency range 30 < ? < 35 MHz corresponding to 40 < z < 46. However, a 10 GeV mass WIMP may be detectable at the 1-3 mK level in the frequency range 55 < ? < 119 MHz corresponding to 11 < z < 25, and at the 1-10 mK level in the frequency range 30 < ? < 40 MHz corresponding to 35 < z < 46.

  16. Multicenter evaluation of the Elecsys Toxo IgG and IgM tests for the diagnosis of infection with Toxoplasma gondii

    PubMed Central

    Meylan, Pascal; Paris, Luc; Liesenfeld, Oliver

    2015-01-01

    Detection of IgG and IgM antibodies is commonly performed for the diagnosis of infection with Toxoplasma gondii. We determined the accuracy of the Elecsys Toxo IgG and IgM test at four European laboratories compared to local reference methods. Coefficients of variation for reproducibility ranged from 1.0 to 6.5% for IgG and from 0.8 to 3.2% for IgM. Seroconversion panels revealed high overall concordance with the reference tests. The Elecsys test detected IgG antibodies earlier than the Cobas Core IgG test in 19 of 47 panels; persisting IgM antibodies were observed in the VIDAS but not the Elecsys test in five of 47 panels. In 31.4% of latent stage sera with persistent IgM antibodies (positive LIASON IgM), the Elecsys IgM test gave negative results indicating increased “clinical” specificity. Sensitivity and specificity of the Elecsys IgG assay ranged from 99.45 to 100% and 87.50–99.80%, respectively, and 91.11–95.74 and 98.45–99.79% for the Elecsys IgM assay, respectively. In conclusion, excellent reproducibility and accuracy make the Elecsys Toxo G and M tests highly suitable for the detection of anti-T. gondii IgG and IgM antibodies. The lower detection rates for persistent IgM in the Elecsys IgM test increase “clinical” specificity and decrease the need for follow-up testing. PMID:26185683

  17. An Inexpensive, Fast and Sensitive Quantitative Lateral Flow Magneto-Immunoassay for Total Prostate Specific Antigen

    PubMed Central

    Barnett, Jacqueline M.; Wraith, Patrick; Kiely, Janice; Persad, Raj; Hurley, Katrina; Hawkins, Peter; Luxton, Richard

    2014-01-01

    We describe the detection characteristics of a device the Resonant Coil Magnetometer (RCM) to quantify paramagnetic particles (PMPs) in immunochromatographic (lateral flow) assays. Lateral flow assays were developed using PMPs for the measurement of total prostate specific antigen (PSA) in serum samples. A detection limit of 0.8 ng/mL was achieved for total PSA using the RCM and is at clinically significant concentrations. Comparison of data obtained in a pilot study from the analysis of serum samples with commercially available immunoassays shows good agreement. The development of a quantitative magneto-immunoassay in lateral flow format for total PSA suggests the potential of the RCM to operate with many immunoassay formats. The RCM has the potential to be modified to quantify multiple analytes in this format. This research shows promise for the development of an inexpensive device capable of quantifying multiple analytes at the point-of-care using a magneto-immunoassay in lateral flow format. PMID:25587419

  18. Multiplexed immunoassays for proteins using magnetic luminescent nanoparticles for internal calibration

    E-print Network

    Hammock, Bruce D.

    Multiplexed immunoassays for proteins using magnetic luminescent nanoparticles for internalO3/luminescent Eu:Gd2O3 core/ shell nanoparticles (MLNPs) as a carrier. The magnetic properties. All rights reserved. Keywords: Luminescence; Nanoparticles; Lanthanide oxide; Magnetic; Multiplex

  19. Correction: Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene.

    PubMed

    Sanghavi, Bankim J; Varhue, Walter; Rohani, Ali; Liao, Kuo-Tang; Bazydlo, Lindsay A L; Chou, Chia-Fu; Swami, Nathan S

    2015-12-21

    Correction for 'Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene' by Bankim J. Sanghavi et al., Lab Chip, 2015, DOI: . PMID:26528632

  20. Development of an integrated capillary valve-based preconcentrator and surface-based immunoassay

    E-print Network

    Liu, Vincent Hok

    2009-01-01

    A new generation of integrated preconcentrator and immunoassay was developed. A novel, self-aligned method for patterning Nafion resin was developed and applied to create a preconcentrator. In a parallel effort, a surface-based ...

  1. Horseradish Peroxidase-Mediated, Iodide-Catalyzed Cascade Reaction for Plasmonic Immunoassays.

    PubMed

    Xianyu, Yunlei; Chen, Yiping; Jiang, Xingyu

    2015-11-01

    This report outlines an enzymatic cascade reaction for signal transduction and amplification for plasmonic immunoassays by using horseradish peroxidase (HRP)-mediated aggregation of gold nanoparticles (AuNPs). HRP-catalyzed oxidation of iodide and iodide-catalyzed oxidation of cysteine is employed to modulate the plasmonic signals of AuNPs. It agrees well with the current immunoassay platforms and allows naked-eye readout with enhanced sensitivity, which holds great promise for applications in resource-constrained settings. PMID:26460152

  2. Searching for fluctuations in the IGM temperature using the Lyman alpha forest

    E-print Network

    Matias Zaldarriaga

    2001-02-12

    We propose a statistical method to search for fluctuations in the temperature of the intergalactic medium (IGM) using the Lyman $\\alpha$ forest. The power on small scales ($\\sim 25 \\km/\\s$) is used as a thermometer and fluctuations of this power are constrained. The method is illustrated using Q1422+231. We see no evidence of temperature fluctuations. We show that in a model with two temperatures that occupy comparable fractions of the spectra, the ratio of small scale powers is constrained to be smaller than 3.5 (corresponding to a factor of 2.5 in temperature). We show that approximately ten quasars are needed constrain factors of two fluctuations in small scale power power.

  3. AGN activity and IGM heating in fossil cluster RX J1416.4+2315

    E-print Network

    Miraghaei, H; Sengupta, C; Raychaudhury, S; Jetha, N N; Abbassi, S

    2015-01-01

    We study Active Galactic Nucleus (AGN) activity in the fossil galaxy cluster, RX J1416.4+2315. Radio observations were carried out using Giant Metrewave Radio Telescope (GMRT) at two frequencies, 1420 MHz and 610 MHz. A weak radio lobe that extends from the central nucleus is detected in 610 MHz map. Assuming the radio lobe originated from the central AGN, we show the energy injection into the Inter Galactic Medium (IGM) is only sufficient to heat up the central 50 kpc within the cluster core, while the cooling radius is larger ( $\\sim$ 130 kpc). In the hardness ratio map, three low energy cavities have been identified. No radio emission is detected for these regions. We evaluated the power required to inflate the cavities and showed that the total energy budget is sufficient to offset the radiative cooling. We showed that the initial conditions would change the results remarkably. Furthermore, efficiency of Bondi accretion to power the AGN has been estimated.

  4. Identity of the elusive IgM Fc receptor (Fc?R) in humans

    PubMed Central

    Oka, Satoshi; Kubagawa, Yoshiki; Torii, Ikuko; Takayama, Eiji; Kang, Dong-Won; Gartland, G. Larry; Bertoli, Luigi F.; Mori, Hiromi; Takatsu, Hiroyuki; Kitamura, Toshio; Ohno, Hiroshi; Wang, Ji-Yang

    2009-01-01

    Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (Fc?R) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide Fc?R in human B-lineage cDNA libraries. Fc?R is defined as a transmembrane sialoglycoprotein of ?60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fc?/?R) but exhibits an exclusive Fc?-binding specificity. The cytoplasmic tail of Fc?R contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing Fc?R are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, Fc?R expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of Fc?R expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that Fc?R per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis. PMID:19858324

  5. Enzyme responsive acetaminophen hydrogels

    E-print Network

    Vemula, Praveen

    Utilization of enzyme catalysis as a tool to disassemble self-assembled hydrogels to control the release encapsulated drug provides an opportunity to design a wide range of enzyme-specific low-molecular-weight hydrogelators ...

  6. Detection of antibodies of the IgM class in sera of patients recently infected with influenza viruses.

    PubMed Central

    Buchner, Y I; Heath, R B; Collins, J V; Pattison, J R

    1976-01-01

    Sucrose density gradient ultracentrifugation can be used to detect specific 19S antibodies of the IgM class in the sera of patients recently infected with influenza A virus, provided steps are taken to remove non-specific inhibitors of haemagglutination. The usefulness of the procedure for the diagnosis of influenza requires further evaluation. PMID:945303

  7. Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

    PubMed

    Chromikova, Veronika; Mader, Alexander; Hofbauer, Stefan; Göbl, Christoph; Madl, Tobias; Gach, Johannes S; Bauernfried, Stefan; Furtmüller, Paul G; Forthal, Donald N; Mach, Lukas; Obinger, Christian; Kunert, Renate

    2015-10-01

    Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation. PMID:25748881

  8. Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM

    PubMed Central

    Chromikova, Veronika; Mader, Alexander; Hofbauer, Stefan; Göbl, Christoph; Madl, Tobias; Gach, Johannes S.; Bauernfried, Stefan; Furtmüller, Paul G.; Forthal, Donald N.; Mach, Lukas; Obinger, Christian; Kunert, Renate

    2015-01-01

    Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation. PMID:25748881

  9. Characterization of IgA and IgM binding and internalization by surface-expressed human Fc?/? receptor.

    PubMed

    Yoo, Esther M; Trinh, K Ryan; Lim, Hana; Wims, Letitia A; Morrison, Sherie L

    2011-09-01

    The Fc?/? receptor (Fc?/?R) is an unusual Fc receptor in that it binds to two different antibody isotypes, IgA and IgM. This receptor is of interest because it is thought to be involved in the capture of IgA- and IgM-immune complexes and antigen presentation. To further characterize this receptor, we were able to stably express human Fc?/?R on the surface of the 293T cell line. Using this system, we determined the affinity of the interactions of the receptor with IgA and IgM, which led to novel insights including the important finding that IgM polymers can bind to human Fc?/?R in the absence of J chain. This is in contrast to the polymeric immunoglobulin receptor (pIgR), which requires the presence of J chain to bind to polymeric IgA and IgM. The dissociation constants (K(d)) of all of the different human IgA isotypes and allotypes for human Fc?/?R were determined, and we show that the N-linked glycans on IgA1 are not required for binding to the receptor. In addition, we demonstrate that IgA can be rapidly internalized by human Fc?/?R in the presence of cross-linking antibody. PMID:21632111

  10. Simultaneous Detection of Forbidden Chemical Residues in Milk Using Dual-Label Time-Resolved Reverse Competitive Chemiluminescent Immunoassay Based on Amine Group Functionalized Surface

    PubMed Central

    Jiang, Haiyang; Wen, Kai; Shen, Jianzhong; Cao, Xingyuan

    2014-01-01

    In this study, a sensitive dual-label time-resolved reverse competitive chemiluminescent immunoassay was developed for simultaneous detection of chloramphenicol (CAP) and clenbuterol (CLE) in milk. The strategy was performed based on the distinction of the kinetic characteristics of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) in chemiluminesecence (CL) systems and different orders of magnitude in HRP CL value for CAP and ALP CL value for CLE in the chemiluminescent immunoassay. Capture antibodies were covalently bound to the amine group functionalized chemiluminescent microtiter plate (MTP) for efficient binding of detection antibodies for the enzymes labeled CAP (HRP-CAP) and CLE (ALP-CLE). The CL signals were recorded at different time points by the automatic luminometers with significant distinction in the dynamic curves. When we considered the ALP CL value (about 105) of CLE as background for HRP CL signal value (about 107) of CAP, there was no interaction from ALP CL background of CLE and the differentiation of CAP and CLE can be easily achieved. The 50% inhibition concentration (IC50) values of CAP and CLE in milk samples were 0.00501 µg L?1 and 0.0128 µg L?1, with the ranges from 0.0003 µg L?1 to 0.0912 µg L?1 and from 0.00385 µg L?1 to 0.125 µg L?1, respectively. The developed method is more sensitive and of less duration than the commercial ELISA kits, suitable for simultaneous screening of CAP and CLE. PMID:25313517

  11. Magnetic Particle-Based Immunoassay of Phosphorylated p53 Using Protein-Cage Templated Lead Phosphate and Carbon Nanospheres for Signal Amplification

    SciTech Connect

    Chen, Aiqiong; Bao, Yuanwu; Ge, Xiaoxiao; Shin, Yongsoon; Du, Dan; Lin, Yuehe

    2012-11-20

    Phosphorylated p53 at serin 15 (phospho-p53-15) is a potential biomarker of Gamma-radiation exposure. In this paper, we described a new magnetic particles (MPs)-based electrochemical immunoassay of human phospho-p53-15 using carbon nanospheres (CNS) and protein-cage templated lead phosphate nanoparticles for signal amplification. Greatly enhanced sensitivity was achieved by three aspects: 1) The protein-cage nanoparticle (PCN) and p53-15 signal antibody (p53-15 Ab2) are linked to CNS (PCNof each apoferritin; 3) MPs capture a large amount of primary antibodies. Using apoferritin templated metallic phosphate instead of enzyme as label has the advantage of eliminating the addition of mediator or immunoreagents and thus makes the immunoassay system simpler. The subsequent stripping voltammetric analysis of the released lead ions were detected on a disposable screen printed electrode. The response current was proportional to the phospho-p53-15 concentration in the range of 0.02 to 20 ng mL-1 with detection limit of 0.01 ng mL-1. This method shows a good stability, reproducibility and recovery.

  12. Investigating the function of Fc-specific binding of IgM to Plasmodium falciparum erythrocyte membrane protein 1 mediating erythrocyte rosetting.

    PubMed

    Stevenson, Liz; Huda, Pie; Jeppesen, Anine; Laursen, Erik; Rowe, J Alexandra; Craig, Alister; Streicher, Werner; Barfod, Lea; Hviid, Lars

    2015-06-01

    Acquired protection from Plasmodium falciparum malaria takes years to develop, probably reflecting the ability of the parasites to evade immunity. A recent example of this is the binding of the Fc region of IgM to VAR2CSA-type PfEMP1. This interferes with specific IgG recognition and phagocytosis of opsonized infected erythrocytes (IEs) without compromising the placental IE adhesion mediated by this PfEMP1 type. IgM also binds via Fc to several other PfEMP1 proteins, where it has been proposed to facilitate rosetting (binding of uninfected erythrocytes to a central IE). To further dissect the functional role of Fc -mediated IgM binding to PfEMP1, we studied the PfEMP1 protein HB3VAR06, which mediates rosetting and binds IgM. Binding of IgM to this PfEMP1 involved the Fc domains C?3-C?4 in IgM and the penultimate DBL domain (DBL?2) at the C-terminus of HB3VAR06. However, IgM binding did not inhibit specific IgG labelling of HB3VAR06 or shield IgG-opsonized IEs from phagocytosis. Instead, IgM was required for rosetting, and each pentameric IgM molecule could bind two HB3VAR06 molecules. Together, our data indicate that the primary function of Fc -mediated IgM binding in rosetting is not to shield IE from specific IgG recognition and phagocytosis as in VAR2CSA-type PfEMP1. Rather, the function appears to be strengthening of IE-erythrocyte interactions. In conclusion, our study provides new evidence on the molecular details and functional significance of rosetting, a long-recognized marker of parasites that cause severe P.?falciparum malaria. PMID:25482886

  13. Cross-reactions in IgM ELISA tests to Legionella pneumophila sg1 and Bordetella pertussis among children suspected of legionellosis; potential impact of vaccination against pertussis?

    PubMed Central

    2015-01-01

    The objective of this study was preliminary evaluation of IgM cross-reaction in sera collected from children hospitalized because of suspected legionellosis. Sera with positive IgM results to L. pneumophila sgs1-7, B. pertussis or with simultaneous detection of IgM antibodies to L. pneumophila sgs1-7 and B. pertussis, or IgM to L. pneumophila sgs1-7 and M. pneumoniae in routine tests, were selected. In total, an adapted pre-absorption test was used for the serological confirmation of legionellosis in the sera of 19 children suspected of legionellosis, and also in 3 adult persons with confirmed Legionnaires’ disease. Sera were pre-absorbed with antigens of L. pneumophila sg1, B. pertussis or both, and tested by ELISA tests. The reduction of IgM antibody level by pre-absorption with antigen/antigens was determined. Reduction of anti-Lpsgs1-7 IgM by pre-absorption with L.pneumophila sg1 antigen ranged from 1.5 to 80, and reduction of anti-Bp IgM by pre-absorption with B. pertussis ranged from 2.0 to 23.8. Reduction by both antigens varied depending on the age of the patients: among children <4 yrs.old, the reduction of anti-B. pertussis IgM by both antigens was higher than for B. pertussis antigen alone. Based on the high difference (? 2 times) between reduction by L.pneumophila sg1 and by B. pertussis antigen, legionellosis was confirmed in 8/19 children. The majority of them also indicated IgM positive/borderline results for B. pertussis or M.pneumoniae in routine ELISA tests. As a preliminary, we posed a hypothesis of a potential impact of an anti-pertussis vaccination on the results obtained in anti-L. pneumophila ELISA IgM tests among young children. PMID:26557032

  14. Clinical evaluation of dengue RNA, NS1, and IgM for diagnosis of dengue in Southern China.

    PubMed

    Chen, Xinliang; Chen, Rui; Gu, Wenshen; He, Jian; Cai, Weipeng; Li, Jiajia; Duan, Chaohui; Yan, Haiyan

    2016-01-01

    In 2014, a large outbreak of dengue occurred in Guangzhou, China. This outbreak prompted us to evaluate NS1 and RNA for the early diagnosis of acute dengue infection, in addition to the combination with IgM antibody. We aimed to find the differences of three assays about dengue diagnosis. This study was an evaluation of diagnosis test. Based on WHO criteria 2009, dengue RNA, NS1, and IgM/IgG were detected from 294 patients (180 dengue patients, 114 non-dengue patients) by three diagnostic kits made in China. The ?(2) test, sensitivity, and specificity were used in statistical analysis. The ratios of dengue patients with low platelet counts (<100?×?10(9) /L 32.2%) or white blood cell counts (<4.0?×?10(9) /L 58.9%) were significantly higher compared to non-dengue patients (P?IgM performed better at day 5 or more with 74.0% of sensitivity. The diagnostic rate using a combination of RNA and IgM was 97.8% and 96.7% using NS1 and IgM. A patient with low platelet and white blood cell counts needs additional tests for dengue during an epidemic. RNA and NS1 were most valuable for early diagnosis of dengue, whereas IgM was best suited as a supplementary method for patients at day 5 or more after illness onset. J. Med. Virol. 88:28-34, 2016. © 2015 Wiley Periodicals, Inc. PMID:26118588

  15. Circadian type, chronic fatigue, and serum IgM in the shift workers of an industrial organization

    PubMed Central

    Khaleghipour, Shahnaz; Masjedi, Mohsen; Kelishadi, Roya

    2015-01-01

    Background: Night shift workers are more vulnerable to immune-related diseases. Immunoglobulin M (IgM) is a potent activator of complement, and complement has a crucial role in defense against bacterial infections. Circadian type is known as an effective agent on vulnerability and adaptation with shift work due to non-compliance with shift stress. The objective of this study was to investigate the correlation of circadian type and chronic fatigue with the serum concentration of IgM in a group of shift workers. Materials and Methods: This cross-sectional study was performed in an industrial organization in Isfahan, Iran. The study population consisted of 221 male employees working at night shifts who were selected by random cluster sampling. The following questionnaires were used: composite morningness (Torsvall and Akerstedt), circadian type (Folkard), and chronic fatigue (Barton and colleagues). The serum concentration of IgM was measured by the nephelometric method. The data were analyzed with the Pearson coefficient correlation and the path analysis for finding the pattern of the structural equations to evaluate the direct and indirect relationships between variables, using the SPSS 15 and LISREL 8.5 statistical software. Results: Significant correlation was documented between morningness, flexibility, languidness, and chronic fatigue with the serum concentration of IgM (P < 0.01). Conclusion: The results showed that the shift workers with morningness and languidness experienced more problems during the working hours due to more tiredness, and had decreased serum concentration of IgM. Correct management of shift work may attenuate fatigue in workers and also improve many health issues experienced by the shift workers. PMID:25802830

  16. Diagnosis of recent hepatitis A infection: a comparison of two methods for detecting specific IgM.

    PubMed Central

    Mortimer, P. P.; Parry, J. V.; Appleton, H.

    1981-01-01

    Radioimmunoassay (RIA) tests for anti-hepatitis A virus (HAV) IgM were carried out on 728 sera: 283 were tested by both a method using an anti-mu serum bound to a solid phase and a method involving preliminary separation of igM by sucrose density gradient (SDG) centrifugation, 354 by the anti-mu method alone and two by the SDG method alone. Similar proportions of sera were found to be positive by each method (42.5%, 41.7%), but equivocal results were commoner by the SDG method (4.7% compared with 1.5%). There were 21 (5.5%) discrepant results from the sera tested by both methods, 20 of which could have been due to the higher sensitivity of the anti-mu method. The SDG method generally gave unequivocal results on sera collected within six weeks of the onset of jaundice. Separation of the IgM fraction by re-orientation centrifugation was quick, but otherwise offered no special advantage over separation on a swing-out rotor. The use of 2 mercaptoethanol (2 ME) reduction to assess the purity of the IgM fraction increased confidence in the specificity of the test. It led, however, to the exclusion of 16 reactive sera (4.2%), all of which were found to be positive in the anti-mu test. The anti-mu method gave better discrimination between positive and negative sera than the SDG method and detected IgM both earlier and later in infection. The results of tests designed to check the specificity of the anti-mu procedure were satisfactory. As it is potentially cheaper and easier to perform, the anti-mu method seems, in all respects, to be superior to the SDG method. PMID:6257778

  17. [Development of a Soviet diagnostic kit for determining class M immunoglobulins to the hepatitis A virus by immunoenzyme analysis (a test system for anti-HAV IgM)].

    PubMed

    Donets, M A; Nel'ga, I V; Grishina, G K; Korolev, M B; Kusov, Iu Iu

    1987-09-01

    A new test system Diagn-A-Hep for the laboratory diagnosis of hepatitis A (HA) by means of the enzyme immunoassay has been developed at the Institute of Poliomyelitis and Viral Encephalitides (Moscow). The sensitivity and specificity of the newly developed test system have proved to be similar to those of the well-known commercial diagnostic system HAVAB manufactured by Abbott Laboratories (USA). Diagn-A-Hep permits the diagnosis of HA with 96-100% effectiveness both in patients with the acute form of the disease and in patients with its anicteric or inapparent forms. This system is simple and convenient, it may be employed in inadequately equipped laboratories or even under field conditions. The rules for the selection of immunobiological preparations to be included in the test system have been worked out. PMID:2825452

  18. Ultrasensitive photoelectrochemical immunoassay through tag induced exciton trapping.

    PubMed

    Wen, Guangming; Ju, Huangxian

    2015-03-01

    The development of photoelectrochemical (PEC) sensors with novel principles is of significance in realizing sensitive and low-cost detection. This work uses CuO NPs labeled antibody to construct a simple and sensitive sandwich-type immunobiosensor for the detection of protein. The detection signal is produced by dissolving the CuO NPs to release copper ions, which are then added on a quantum dots (QDs) modified F-doped tin oxide to quench the photocurrent of QDs via copper ion-induced formation of exciton trapping. The formed exciton trapping blocks the escape of photoelectron and thus leads to a "signal off" PEC method for sensitive immunoassay. The proposed method shows a detectable range from 0.05 to 500 ng/mL for ?-fetoprotein (AFP) with a detection limit (LOD) of 0.038 ng/mL. This work further extends the application of exciton trapping-based PEC biosensing strategy in bioanalysis. The sensitive analytical performance of the designed route implies a promising potential of the PEC sensing in clinical diagnosis. PMID:25618699

  19. A Portable Analyzer for Pouch-Actuated, Immunoassay Cassettes

    PubMed Central

    Qiu, Xianbo; Liu, Changchun; Mauk, Michael G.; Hart, Robert W.; Chen, Dafeng; Qiu, Jing; Kientz, Terry; Fiene, Jonathan; Bau, Haim H.

    2011-01-01

    A portable, small footprint, light, general purpose analyzer (processor) to control the flow in immunoassay cassettes and to facilitate the detection of test results is described. The durable analyzer accepts disposable cassettes that contain pouches and reaction chambers for various unit operations such as hydration of dry reagents, stirring, and incubation. The analyzer includes individually controlled, linear actuators to compress the pouches in the cassette, which facilitates the pumping and mixing of sample and reagents, and to close diaphragm-based valves for flow control. The same types of actuators are used to compress pouches and actuate valves. The analyzer also houses a compact OEM scanner/reader to excite fluorescence and detect emission from labels. The analyzer is hydraulically isolated from the cassette, reducing the possibility of cross-contamination. The analyzer facilitates programmable, automated execution of a sequence of operations such as pumping and valving in a timely fashion, reducing the level of expertise required from the operator and the possibility for errors. The analyzer’s design is modular and expandable to accommodate cassettes of various complexities and additional functionalities. In this paper, the utility of the analyzer has been demonstrated with the execution of a simple, consecutive, lateral flow assay of a model biological system and the test results were detected with up converting phosphor labels that are excited at infrared frequencies and emit in the visible spectrum. PMID:22125359

  20. Backscattering particle immunoassays in wire-guide droplet manipulations

    PubMed Central

    Yoon, Jeong-Yeol; You, David J

    2008-01-01

    A simpler way for manipulating droplets on a flat surface was demonstrated, eliminating the complications in the existing methods of open-surface digital microfluidics. Programmed and motorized movements of 10 ?L droplets were demonstrated using stepper motors and microcontrollers, including merging, complicated movement along the programmed path, and rapid mixing. Latex immunoagglutination assays for mouse immunoglobulin G, bovine viral diarrhea virus and Escherichia coli were demonstrated by merging two droplets on a superhydrophobic surface (contact angle = 155 ± 2°) and using subsequent back light scattering detection, with detection limits of 50 pg mL-1, 2.5 TCID50 mL-1 and 85 CFU mL-1, respectively, all significantly lower than the other immunoassay demonstrations in conventional microfluidics (~1 ng mL-1 for proteins, ~100 TCID50 mL-1 for viruses and ~100 CFU mL-1 for bacteria). Advantages of this system over conventional microfluidics or microwell plate assays include: (1) minimized biofouling and repeated use (>100 times) of a platform; (2) possibility of nanoliter droplet manipulation; (3) reprogrammability with a computer or a game pad interface. PMID:19014703

  1. Analysis of cooked-food mutagens by HPLC/immunoassay

    SciTech Connect

    Watkins, B.E.; Vanderlaan, M.; Felton, J.S.

    1988-10-10

    Recognizing that an immunoassay for these aminoimidazoazaarenes (AIAs) could be an analytical method with higher sample throughput and would be less costly, we developed a set of monoclonal antibodies that selectively bind to each of the AIAs. We selected PhIP (6-phenyl-2-amino-1-methylimidazo(4,5-b)pyridine) for the initial assay development because it is the AIA that is most abundant by mass in cooked meat and it is the most genotoxic AIA in mammalian-cell short-term bioassays. It is, however, the least active AIA in the Ames Salmonella mutagenesis assay. We have recently developed a set of four monoclonals that bind to the PhIP. These antibodies were produced by standard methods and were derived from the immunogen described previously. The binding specificity of each of these antibodies has been well characterized. The most specific antibody, called PhIP-1, will bind PhIP with a 50% inhibition point (I/sub 50/) of 30 ng, and it will not bind to any of the other AIA mutagens, nor to any of a number of synthetically produced derivatives of PhIP, such as Iso-PhIP (6-phenyl-2-amino-3-methylimidazo(4,5-b)pyridine). PhIP-1 does bind with 2-deamino-PhIP and 2-deamino-2-nitro-PhIP with an I/sub 50/ of 13 and 16 ng, respectively. 9 refs., 3 figs.

  2. The microassay on a card: A rugged, portable immunoassay

    NASA Technical Reports Server (NTRS)

    Kidwell, David

    1991-01-01

    The Microassay on a Card (MAC) is a portable, hand-held, non-instrumental immunoassay that can test for the presence of a wide variety of substances in the environment. The MAC is a simple device to use. A drop of test solution is placed on one side of the card and within five minutes a color is developed on the other side in proportion to the amount of substance in the test solution, with sensitivity approaching 10 ng/ml. The MAC is self-contained and self-timed; no reagents or timing is necessary. The MAC may be configured with multiple wells to provide simultaneous testing for multiple species. As envisioned, the MAC will be employed first as an on-site screen for drugs of abuse in urine or saliva. If the MAC can be used as a screen of saliva for drugs of abuse, it could be applied to driving while intoxicated, use of drugs on the job, or testing of the identity of seized materials. With appropriate modifications, the MAC also could be used to test for environmental toxins or pollutants.

  3. Designing novel nano-immunoassays: antibody orientation versus sensitivity

    NASA Astrophysics Data System (ADS)

    Puertas, S.; Moros, M.; Fernández-Pacheco, R.; Ibarra, M. R.; Grazú, V.; de la Fuente, J. M.

    2010-12-01

    There is a growing interest in the use of magnetic nanoparticles (MNPs) for their application in quantitative and highly sensitive biosensors. Their use as labels of biological recognition events and their detection by means of some magnetic method constitute a very promising strategy for quantitative high-sensitive lateral-flow assays. In this paper, we report the importance of nanoparticle functionalization for the improvement of sensitivity for a lateral-flow immunoassay. More precisely, we have found that immobilization of IgG anti-hCG through its polysaccharide moieties on MNPs allows more successful recognition of the hCG hormone. Although we have used the detection of hCG as a model in this work, the strategy of binding antibodies to MNPs through its sugar chains reported here is applicable to other antibodies. It has huge potential as it will be very useful for the development of quantitative and high-sensitive lateral-flow assays for its use on human and veterinary, medicine, food and beverage manufacturing, pharmaceutical, medical biologics and personal care product production, environmental remediation, etc.

  4. Graphene oxide-based SPR biosensor chip for immunoassay applications

    NASA Astrophysics Data System (ADS)

    Chiu, Nan-Fu; Huang, Teng-Yi; Lai, Hsin-Chih; Liu, Kou-Chen

    2014-08-01

    This work develops a highly sensitive immunoassay sensor for use in graphene oxide sheet (GOS)-based surface plasmon resonance (SPR) chips. This sensing film, which is formed by chemically modifying a GOS surface, has covalent bonds that strongly interact with the bovine serum albumin (BSA), explaining why it has a higher sensitivity. This GOS film-based SPR chip has a BSA concentration detection limit that is 100 times higher than that of the conventional Au-film-based sensor. The affinity constants ( K A) on the GOS film-based SPR chip and the conventional SPR chip for 100 ?g/ml BSA are 80.82 × 106 M-1 and 15.67 × 106 M-1, respectively. Therefore, the affinity constant of the GOS film-based SPR chip is 5.2 times higher than that of the conventional chip. With respect to the protein-protein interaction, the SPR sensor capability to detect angle changes at a low concentration anti-BSA of 75.75 nM on the GOS film-based SPR chip and the conventional SPR chip is 36.1867 and 26.1759 mdeg, respectively. At a high concentration, anti-BSA of 378.78 nM on the GOS film-based SPR chip and the conventional SPR chip reveals two times increases in the SPR angle shift. Above results demonstrate that the GOS film is promising for highly sensitive clinical diagnostic applications.

  5. Comparison of different immunoassays for CA 19-9.

    PubMed

    Stern, P; Friedecky, B; Bartos, V; Bezdickova, D; Vavrova, J; Uhrova, J; Rozprimova, L; Zima, T; Palicka, V

    2001-12-01

    We compared six routinely employed immunoassay kits: Architect i2000 and AxSYM, Abbott Laboratories; Elecsys 2010, Roche Diagnostics; ELSA, CIS-BioInternational; Immulite 1, Diagnostic Products Corporation; and IRMA-mat, Byk-Sangtec Diagnostica. Using all analytical systems, we measured identical groups of clinical samples completed with selected control samples. The repeatability of measurements (coefficient of variation) ranged from 2.1% (Elecsys 2010) to 6.7% (ELSA). The parameters of Passing-Bablok regression show significant systematic differences among analytical systems. Data from a Bland-Altman diagram suggest that these differences project onto other, still more significant individual differences among individual samples. Though the cut-off values differ between various systems, no similar clinical efficacy appears to be attained. The behavior of individual systems is quite different for identical control materials and does not necessarily duplicate the calibration for biological samples. The results of determining CA 19-9 cannot be extrapolated from one analytical technique to another, even in cases where the same monoclonal antibody is used. Standardization of CA 19-9 measurement systems is necessary to allow use of the results for the purposes of evidence-based medicine. PMID:11798090

  6. System and method for a parallel immunoassay system

    DOEpatents

    Stevens, Fred J. (Naperville, IL)

    2002-01-01

    A method and system for detecting a target antigen using massively parallel immunoassay technology. In this system, high affinity antibodies of the antigen are covalently linked to small beads or particles. The beads are exposed to a solution containing DNA-oligomer-mimics of the antigen. The mimics which are reactive with the covalently attached antibody or antibodies will bind to the appropriate antibody molecule on the bead. The particles or beads are then washed to remove any unbound DNA-oligomer-mimics and are then immobilized or trapped. The bead-antibody complexes are then exposed to a test solution which may contain the targeted antigens. If the antigen is present it will replace the mimic since it has a greater affinity for the respective antibody. The particles are then removed from the solution leaving a residual solution. This residual solution is applied a DNA chip containing many samples of complimentary DNA. If the DNA tag from a mimic binds with its complimentary DNA, it indicates the presence of the target antigen. A flourescent tag can be used to more easily identify the bound DNA tag.

  7. Genome-wide scan identifies variant in TNFSF13 associated with serum IgM in a healthy Chinese male population.

    PubMed

    Yang, Ming; Wu, Yongming; Lu, Yanmei; Liu, Changyuan; Sun, Jielin; Liao, Ming; Qin, Min; Mo, Linjian; Gao, Yong; Lu, Zheng; Wu, Chunlei; Zhang, Youjie; Zhang, Haiying; Qin, Xue; Hu, Yanling; Zhang, Shijun; Li, Jianling; Dong, Min; Zheng, S Lilly; Xu, Jianfeng; Yang, Xiaobo; Tan, Aihua; Mo, Zengnan

    2012-01-01

    IgM provides a first line of defense during microbial infections. Serum IgM levels are detected routinely in clinical practice. And IgM is a genetically complex trait. We conducted a two-stage genome-wide association study (GWAS) to identify genetic variants affecting serum IgM levels in a Chinese population of 3495, including 1999 unrelated subjects in the first stage and 1496 independent individuals in the second stage. Our data show that a common single nucleotide polymorphism (SNP), rs11552708 located in the TNFSF13 gene was significantly associated with IgM levels (p?=?5.00×10(-7) in first stage, p?=?1.34×10(-3) in second stage, and p?=?4.22×10(-9) when combined). Besides, smoking was identified to be associated with IgM levels in both stages (P<0.05), but there was no significant interaction between smoking and the identified SNP (P>0.05). It is suggested that TNFSF13 may be a susceptibility gene affecting serum IgM levels in Chinese male population. PMID:23118916

  8. Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene.

    PubMed

    Sanghavi, Bankim J; Varhue, Walter; Rohani, Ali; Liao, Kuo-Tang; Bazydlo, Lindsay A L; Chou, Chia-Fu; Swami, Nathan S

    2015-12-21

    Heterogeneous immunoassays usually require long incubation times to promote specific target binding and several wash steps to eliminate non-specific binding. Hence, signal saturation is rarely achieved at detection limit levels of analyte, leading to significant errors in analyte quantification due to extreme sensitivity of the signals to incubation time and methodology. The poor binding kinetics of immunoassays at detection limit levels can be alleviated through creating an enriched analyte plug in the vicinity of immobilized capture probes to enable signal saturation at higher levels and at earlier times, due to higher analyte association and its faster replenishment at the binding surface. Herein, we achieve this by coupling frequency-selective dielectrophoretic molecular dam enrichment of the target biomarker in physiological media to capture probes immobilized on graphene-modified surfaces in a nanoslit to enable ultrafast immunoassays with near-instantaneous (<2 minutes) signal saturation at dilute biomarker levels (picomolar) within ultra-low sample volumes (picoliters). This methodology is applied to the detection of Prostate Specific Antigen (PSA) diluted in serum samples, followed by validation against a standard two-step immunoassay using three de-identified patient samples. Based on the ability of dielectrophoretic molecular dam analyte enrichment methods to enable the detection of PSA at 1-5 pg mL(-1) levels within a minute, and the relative insensitivity of the signals to incubation time after the first two minutes, we envision its application for improving the sensitivity of immunoassays and their accuracy at detection limit levels. PMID:26496877

  9. 25OHD analogues and vacuum blood collection tubes dramatically affect the accuracy of automated immunoassays

    PubMed Central

    Yu, Songlin; Cheng, Xinqi; Fang, Huiling; Zhang, Ruiping; Han, Jianhua; Qin, Xuzhen; Cheng, Qian; Su, Wei; Hou, Li’an; Xia, Liangyu; Qiu, Ling

    2015-01-01

    Variations in vitamin D quantification methods are large, and influences of vitamin D analogues and blood collection methods have not been systematically examined. We evaluated the effects of vitamin D analogues 25OHD2 and 3-epi 25OHD3 and blood collection methods on vitamin D measurement, using five immunoassay systems and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum samples (332) were selected from routine vitamin D assay requests, including samples with or without 25OHD2 or 3-epi 25OHD3, and analysed using various immunoassay systems. In samples with no 25OHD2 or 3-epi 25OHD3, all immunoassays correlated well with LC-MS/MS. However, the Siemens system produced a large positive mean bias of 12.5?ng/mL and a poor Kappa value when using tubes with clot activator and gel separator. When 25OHD2 or 3-epi 25OHD3 was present, correlations and clinical agreement decreased for all immunoassays. Serum 25OHD in VACUETTE tubes with gel and clot activator, as measured by the Siemens system, produced significantly higher values than did samples collected in VACUETTE tubes with no additives. Bias decreased and clinical agreement improved significantly when using tubes with no additives. In conclusion, most automated immunoassays showed acceptable correlation and agreement with LC-MS/MS; however, 25OHD analogues and blood collection tubes dramatically affected accuracy. PMID:26420221

  10. Superfund innovative technology evaluation (site) program evaluation report for antox BTX water screen (BTX immunoassay)

    SciTech Connect

    Gerlach, R.W.; White, R.J.; O'Leary, N.F.; Van Emon, J.M.

    1993-06-01

    The results of a demonstration of a portable immunoassay for the detection of benzene, toluene, and xylene(s) (BTX) are described in the report. Seventy-nine field samples were obtained from monitoring wells at several sites with gasoline contaminated ground water. Sample splits were analyzed on-site by the BTX immunoassay and in the laboratory by gas chromatography (GC) using EPA Method 8020. The BTX immunoassay was rapid and simple to use. It performed well in identifying high level contamination and gasoline contaminated samples having BTX concentrations greater than 100 ppb. It did not fully meet the claims of the developer of identifying contamination levels down to 25 ppb BTX. Two field samples determined by GC to have between 25 and 100 ppb BTX failed to be classified correctly by the immunoassay. Results from quality assurance samples with BTX concentrations of 2.5, 25, and 100 ppb also showed that false negative results would be expected at higher than a 5 percent rate when BTX contamination levels were between 25 and 100 ppb. However, for samples with higher BTX levels, the immunoassay gave excellent results. Two field samples yielded false positive results compared to GC values, but these samples showed signs of low-level gasoline contamination.

  11. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.; Gottesfeld, S.

    1992-12-31

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid, polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  12. Catalyzed enzyme electrodes

    DOEpatents

    Zawodzinski, Thomas A. (Los Alamos, NM); Wilson, Mahlon S. (Los Alamos, NM); Rishpon, Judith (Ramat-Aviv, IL); Gottesfeld, Shimshon (Los Alamos, NM)

    1993-01-01

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  13. Magnetically responsive enzyme powders

    NASA Astrophysics Data System (ADS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (-20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties.

  14. Rational enzyme redesign

    SciTech Connect

    Ornstein, R.L.

    1994-05-01

    Protein engineering is first a means of elucidating structure-function relations in an enzyme, and second, a means of changing a protein to make it serve a different, but generally related, purpose. In principle, one may change the functional characteristics of an enzyme by altering its substrate specificity, kinetics, optimum range of activity, and chemical mechanism. Obviously one cannot make all possible combinations of amino acid changes for even the smallest enzyme, so the essential question is which changes to make. The intent of rational protein/enzyme redesign is to alter a protein/enzyme in a timely and premeditated fashion. This article provides an outline of the process of rational enzyme redesign.

  15. Evaluation of screen-printed gold on low-temperature co-fired ceramic as a substrate for the immobilization of electrochemical immunoassays.

    PubMed

    Fakunle, Eyitayo S; Aguilar, Zoraida P; Shultz, John L; Toland, Alan D; Fritsch, Ingrid

    2006-12-01

    Screen-printed gold (SPG, Dupont gold conductor 5734) on low-temperature co-fired ceramic (LTCC) materials (Dupont dielectric tape 951, mostly composed of alumina and silica) has been demonstrated to be a substrate for electrochemical enzyme-linked immunosorbant assays. The effect of two different cleaning treatments and the extent of nonspecific adsorption on the SPG/LTCC and plain LTCC surfaces were also evaluated. LTCC materials hold promise for constructing a new generation of devices for microelectrochemical sensing and assays. Facile fabrication in three dimensions with integrated conducting elements makes them attractive. A standard sandwich immunoassay for a model analyte, mouse IgG, was used to evaluate the LTCC materials. After the assembly of components onto chips of SPG/LTCC and plain LTCC, p-aminophenol that was generated enzymatically by the enzyme label was detected electrochemically with a separate glassy carbon electrode. Cleaning SPG/LTCC with a piranha solution (7:1 vol/vol of concentrated H2SO4/30% H2O2), traditionally used for other gold surfaces prior to SAM assembly, resulted in a notable decrease in assay signal and an increase in nonspecific adsorption when compared to cleaning with water alone. Assay components assemble specifically on plain LTCC, with only a small percent attributed to NSA. Environmental scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy reveal the tremendous chemical heterogeneity and complexity of both SPG/LTCC and plain LTCC surfaces and aid in the explanation of assay results. A 10% acetate Tween bovine serum albumin solution and an ethanolic solution of 4 mM 1-butanol eliminate assay signals originating from plain LTCC. The outcomes of these studies can now be used to achieve miniaturized electrochemical immunoassays on LTCC materials where both plain and SPG surfaces are present. PMID:17129069

  16. Late Reheating of the IGM by Quasars: A Radiation Hydrodynamical Simulation of Helium II Reionization

    E-print Network

    Paschos, Pascal; Bordner, James O; Harkness, Robert

    2007-01-01

    We study the ionization and thermal evolution of the intergalactic medium during the epoch of \\heii reionization by means of radiation hydrodynamical cosmological simulations. We post-process baryonic density fields from a standard optically-thin IGM simulation with a homogeneous galaxy-dominated UV background (UVB) which reionizes \\hi and \\hei at z=6.5 but does not have any contribution to the ionization of \\heii. Quasars with luminosities proportional to the mass of the host halos are then introduced as point sources throughout the 100 Mpc simulation volume consistent with the Pei luminosity function. We evolve the spatial distribution of the \\heii ionizing radiation field using a time-implicit variable tensor Eddington factor radiative transfer scheme. Simultaneously, we also solve for the local ionization of \\heii to \\heii and the associated photoheating of the gas. We find that the percolation of the \\heiii regions is essentially complete by z=2.5. When comparing to a self-consistent optically thin simul...

  17. Nanostructures for enzyme stabilization

    SciTech Connect

    Kim, Jungbae; Grate, Jay W.; Wang, Ping

    2006-02-02

    The last decade has witnessed notable breakthroughs in nanotechnology with development of various nanostructured materials such as mesoporous materials and nanoparticles. These nanostructures have been used as a host for enzyme immobilization via various approaches, such as enzyme adsorption, covalent attachment, enzyme encapsulation, and sophisticated combinations of methods. This review discusses the stabilization mechanisms behind these diverse approaches; such as confinement, pore size and volume, charge interaction, hydrophobic interaction, and multipoint attachment. In addition, we will introduce recent rigorous approaches to improve the enzyme stability in these nanostructures or develop new nanostructures for the enzyme stabilization. Especially, we will introduce our recent invention of a nanostructure, called single enzyme nanoparticles (SENs). In the form of SENs, each enzyme molecule is surrounded with a nanometer scale network, resulting in stabilization of enzyme activity without any serious limitation for the substrate transfer from solution to the active site. SENs can be further immobilized into mesoporous silica with a large surface area, providing a hierarchical approach for stable, immobilized enzyme systems for various applications, such as bioconversion, bioremediation, and biosensors.

  18. Food and feed enzymes.

    PubMed

    Fraatz, Marco Alexander; Rühl, Martin; Zorn, Holger

    2014-01-01

    Humans have benefited from the unique catalytic properties of enzymes, in particular for food production, for thousands of years. Prominent examples include the production of fermented alcoholic beverages, such as beer and wine, as well as bakery and dairy products. The chapter reviews the historic background of the development of modern enzyme technology and provides an overview of the industrial food and feed enzymes currently available on the world market. The chapter highlights enzyme applications for the improvement of resource efficiency, the biopreservation of food, and the treatment of food intolerances. Further topics address the improvement of food safety and food quality. PMID:23873095

  19. Enzymes on material surfaces.

    PubMed

    Talbert, Joey N; Goddard, Julie M

    2012-05-01

    Enzyme interactions with material surfaces are of interest for industrial food and pharmaceutical transformations, biosensors, artificial cells, cell free reactions, drug and nutrition delivery technologies, and imaging. When in contact with a material surface, an enzyme may lose or appear to lose activity due to the nature of the enzyme, the nature of the material, and/or the nature of the interface between the enzyme, material, and substrate environment. The purpose of this review is to survey recent advances that have been made towards the preservation, optimization, and enhancement of enzyme activity on material surfaces within the context of well-known concepts that describe the loss of activity after immobilization. This review breaks down the immobilized enzyme system to look at the individual components of the system-namely the enzyme, the material, and the interface. For each piece, possible causes for the loss of enzyme activity are described as well as strategies that have been applied to limit the affect. At the conclusion we identify areas of future research needed to overcome limitations in the current state-of-the art for immobilized enzyme systems. PMID:22269888

  20. Replacing antibodies with aptamers in lateral flow immunoassay.

    PubMed

    Chen, Ailiang; Yang, Shuming

    2015-09-15

    Aptamers have been identified against various targets as a type of chemical or nucleic acid ligand by systematic evolution of ligands by exponential enrichment (SELEX) with high sensitivity and specificity. Aptamers show remarkable advantages over antibodies due to the nucleic acid nature and target-induced structure-switching properties and are widely used to design various fluorescent, electrochemical, or colorimetric biosensors. However, the practical applications of aptamer-based sensing and diagnostics are still lagging behind those of antibody-based tests. Lateral flow immunoassay (LFIA) represents a well established and appropriate technology among rapid assays because of its low cost and user-friendliness. The antibody-based platform is utilized to detect numerous targets, but it is always hampered by the antibody preparation time, antibody stability, and effect of modification on the antibody. Seeking alternatives to antibodies is an area of active research and is of tremendous importance. Aptamers are receiving increasing attention in lateral flow applications because of a number of important potential performance advantages. We speculate that aptamer-based LFIA may be one of the first platforms for commercial use of aptamer-based diagnosis. This review first gives an introduction to aptamer including the selection process SELEX with its focus on aptamer advantages over antibodies, and then depicts LFIA with its focus on aptamer opportunities in LFIA over antibodies. Furthermore, we summarize the recent advances in the development of aptamer-based lateral flow biosensing assays with the aim to provide a general guide for the design of aptamer-based lateral flow biosensing assays. PMID:25912679

  1. Scheme for the selection of measurement uncertainty models in blood establishments' screening immunoassays.

    PubMed

    Pereira, Paulo; Westgard, James O; Encarnação, Pedro; Seghatchian, Jerard; de Sousa, Gracinda

    2015-02-01

    Blood establishments routinely perform screening immunoassays to assess safety of the blood components. As with any other screening test, results have an inherent uncertainty. In blood establishments the major concern is the chance of false negatives, due to its possible impact on patients' health. This article briefly reviews GUM and diagnostic accuracy models for screening immunoassays, recommending a scheme to support the screening laboratories' staffs on the selection of a model considering the intended use of the screening results (i.e., post-transfusion safety). The discussion is grounded on a "risk-based thinking", risk being considered from the blood donor selection to the screening immunoassays. A combination of GUM and diagnostic accuracy models to evaluate measurement uncertainty in blood establishments is recommended. PMID:25620757

  2. A SERS-based microfluidic immunoassay using an in-situ synthesized gold substrate

    NASA Astrophysics Data System (ADS)

    Fan, Kequan; Wang, Zhuyuan; Wu, Lei; Zong, Shenfei; Cui, Yiping

    2015-05-01

    A sensitive SERS (surface-enhanced Raman scattering)-based immunoassay in microfluidic system has been developed with in-situ synthesis of gold substrate and immune reporter named as 4MBA (4-Mercaptobenzoic acid)-labeled immuno-Ag aggregates. The gold substrate was fabricated simply by introducing the hydrogen tetrachloroaurate (III) trihydrate (HAuCl4) solution to microchannels using a microfluidic pump. It was found that the obtained deposited gold nanoparticles were uniform in size and shape. Then the sandwich immunoassays were performed using the gold substrates based on SERS signals. In the immunoassay, the gold nanoparticles decorated surface was modified with certain antibodies to recognize the specific kind of antigen, which was flowed through the microfluidic channel afterwards. Then 4MBA-labeled immuno-Ag aggregates were employed as the SERS probes to quantitatively detect the antigen. The experimental results showed a good specificity and limit of detection (LOD) about 1 ng/mL.

  3. Association of IgM monoclonal gammopathy with progressive muscular atrophy and multifocal motor neuropathy: a case-control study.

    PubMed

    Vlam, Lotte; Piepers, Sanne; Sutedja, Nadia A; Jacobs, Bart C; Tio-Gillen, Anne P; Stam, Marloes; Franssen, Hessel; Veldink, Jan H; Cats, Elisabeth A; Notermans, Nicolette C; Bloem, Andries C; Wadman, Renske I; van der Pol, W-Ludo; van den Berg, Leonard H

    2015-03-01

    Monoclonal gammopathy in patients with amyotrophic lateral sclerosis (ALS) and related disorders has been reported in small studies but the validity of the reported associations remains uncertain. Presence of monoclonal gammopathy may indicate specific pathogenic pathways and may facilitate the development of novel treatment strategies. The objective of this large case-control study was to determine the prevalence of monoclonal gammopathy in motor neuron diseases (MND) and multifocal motor neuropathy (MMN). Monoclonal gammopathy was determined by immunoelectrophoresis and immunofixation in serum from 445 patients with ALS, 158 patients with progressive muscular atrophy (PMA), 60 patients with primary lateral sclerosis (PLS), 88 patients with MMN and in 430 matched healthy controls. Anti-ganglioside antibody titers were determined in sera from patients with MMN and PMA, and in ALS and PLS patients with monoclonal gammopathy. Logistic regression analysis was used to investigate associations of monoclonal gammopathy with motor neuron diseases and clinical characteristics. Neither ALS nor PLS was associated with monoclonal gammopathy. IgM monoclonal gammopathy was more frequent in patients with PMA (8 %) (OR = 4.2; p = 0.001) and MMN (7 %) (OR = 5.8; p = 0.002) than in controls (2 %). High titers of anti-GM1 IgM antibodies were present in 43 % of MMN patients and 7 % of PMA patients. Patients with PMA and IgM monoclonal gammopathy or anti-GM1 antibodies had a higher age at onset, more often weakness of upper legs and more severe outcome than patients with MMN. PMA and MMN, but not ALS and PLS, are significantly associated with IgM monoclonal gammopathy and anti-GM1 antibodies. These results may indicate that a subset of patients presenting with PMA share pathogenic mechanisms with MMN. PMID:25549972

  4. Natural and adaptive IgM antibodies in the recognition of tumor-associated antigens of breast cancer (Review)

    PubMed Central

    DÍAZ-ZARAGOZA, MARIANA; HERNÁNDEZ-ÁVILA, RICARDO; VIEDMA-RODRÍGUEZ, RUBÍ; ARENAS-ARANDA, DIEGO; OSTOA-SALOMA, PEDRO

    2015-01-01

    For early detection of cancer, education and screening are important, but the most critical factor is the development of early diagnostic tools. Methods that recognize the warning signs of cancer and take prompt action lead to an early diagnosis; simple tests can identify individuals in a healthy population who have the disease but have not developed symptoms. Early detection of cancer is significant and is one of the most promising approaches by which to reduce the growing cancer burden and guide curative treatment. The early diagnosis of patients with breast cancer is challenging, since it is the most common cancer in women worldwide. Despite the advent of mammography in screening for breast cancer, low-resource, low-cost alternative tools must be implemented to complement mammography findings. IgM is part of the first line of defense of an organism and is responsible for recognizing and eliminating infectious particles and removing transformed cells. Most studies on breast cancer have focused on the development of IgG-like molecules as biomarkers or as a treatment for the advanced stages of cancer, but autoantibodies (IgM) and tumor-associated antigens (proteins or carbohydrates with aberrant structures) have not been examined as early diagnostic tools for breast cancer. The present review summarizes the function of natural and adaptive IgM in eliminating cancer cells in the early stages of pathology and their value as early diagnostic tools. IgM, as a component of the immune system, is being used to identify tumor-associated antigens and tumor-associated carbohydrate antigens. PMID:26133558

  5. DC-SIGN–expressing macrophages trigger activation of mannosylated IgM B-cell receptor in follicular lymphoma

    PubMed Central

    Amin, Rada; Mourcin, Frédéric; Uhel, Fabrice; Pangault, Céline; Ruminy, Philippe; Dupré, Loic; Guirriec, Marion; Marchand, Tony; Fest, Thierry; Lamy, Thierry

    2015-01-01

    Follicular lymphoma (FL) results from the accumulation of malignant germinal center (GC) B cells leading to the development of an indolent and largely incurable disease. FL cells remain highly dependent on B-cell receptor (BCR) signaling and on a specific cell microenvironment, including T cells, macrophages, and stromal cells. Importantly, FL BCR is characterized by a selective pressure to retain surface immunoglobulin M (IgM) BCR despite an active class-switch recombination process, and by the introduction, in BCR variable regions, of N-glycosylation acceptor sites harboring unusual high-mannose oligosaccharides. However, the relevance of these 2 FL BCR features for lymphomagenesis remains unclear. In this study, we demonstrated that IgM+ FL B cells activated a stronger BCR signaling network than IgG+ FL B cells and normal GC B cells. BCR expression level and phosphatase activity could both contribute to such heterogeneity. Moreover, we underlined that a subset of IgM+ FL samples, displaying highly mannosylated BCR, efficiently bound dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN), which could in turn trigger delayed but long-lasting BCR aggregation and activation. Interestingly, DC-SIGN was found within the FL cell niche in situ. Finally, M2 macrophages induced a DC-SIGN–dependent adhesion of highly mannosylated IgM+ FL B cells and triggered BCR-associated kinase activation. Interestingly, pharmacologic BCR inhibitors abolished such crosstalk between macrophages and FL B cells. Altogether, our data support an important role for DC-SIGN–expressing infiltrating cells in the biology of FL and suggest that they could represent interesting therapeutic targets. PMID:26272216

  6. Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody

    PubMed Central

    Ramsland, Paul A.; Terzyan, Simon S.; Cloud, Gwendolyn; Bourne, Christina R.; Farrugia, William; Tribbick, Gordon; Geysen, H. Mario; Moomaw, Carolyn R.; Slaughter, Clive A.; Edmundson, Allen B.

    2006-01-01

    The 2.6 Å (1 Å=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenström's macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611–39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024–14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds. PMID:16422668

  7. Increase in Anti-Gal IgM Level is Associated With Early Graft Failure in Intraportal Porcine Islet Xenotransplantation

    PubMed Central

    Lee, Haneulnari; Park, Eun Mi; Kim, Jong Min; Shin, Jun-Seop; Kim, Jung-Sik; Park, Chung-Gyu; Kim, Sang Joon

    2015-01-01

    Background Anti-Gal is a major antibody induced in non-human primates (NHPs) after xenotransplantation. To understand the mechanism of graft rejection, we investigated the association between anti-Gal responses and graft failure in NHP recipients of porcine islet transplantation (PITx). Methods Intraportal PITx was performed in 35 diabetic NHPs, and graft function was monitored. Early graft failure (EGF) was defined as loss of graft function within a month after PITx. Seven, 19, nine NHPs received immunosuppression (IS) without CD40 pathway blockade (Group I), with anti-CD154 (Group II), and with anti-CD40 (Group III), respectively. The anti-Gal levels on day 0 and day 7 of PITx were measured by ELISA. Results The frequency of EGF was significantly lower in Group II (26.3%) than in Group I (100%, P=0.0012) and Group III (77.8%, P=0.0166). While levels of anti-Gal IgG in Group I and anti-Gal IgM in Group III increased on day 7 compared with day 0 (P=0.0156 and 0.0273), there was no increase in either on day 7 in Group II. The ratio of anti-Gal IgM or IgG level on day 7 to that on day 0 (Ratio7/0) was significantly higher in recipients with EGF than without EGF (P=0.0009 and 0.0027). ROC curve analysis of anti-Gal IgM Ratio7/0 revealed an area under the curve of 0.789 (P=0.0003). Conclusions IS with anti-CD154 suppressed anti-Gal responses and prevented EGF in PITx. Anti-Gal IgM Ratio7/0, being associated with EGF, is a predictive marker for EGF. PMID:26354349

  8. Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody.

    PubMed

    Ramsland, Paul A; Terzyan, Simon S; Cloud, Gwendolyn; Bourne, Christina R; Farrugia, William; Tribbick, Gordon; Geysen, H Mario; Moomaw, Carolyn R; Slaughter, Clive A; Edmundson, Allen B

    2006-05-01

    The 2.6 A (1 A=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenström's macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611-39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024-14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds. PMID:16422668

  9. Structural property of soybean lunasin and development of a method to quantify lunasin in plasma using an optimized immunoassay protocol.

    PubMed

    Dia, Vermont P; Frankland-Searby, Sarah; del Hierro, Francisco Laso; Garcia, Guadalupe; de Mejia, Elvira Gonzalez

    2013-05-01

    Lunasin is a 43-amino acid naturally occurring chemopreventive peptide with demonstrated anti-cancer and anti-inflammatory properties. The objectives of this study were to determine the effect of temperature on the secondary structure of lunasin, to develop a method of isolating lunasin from human plasma using an ion-exchange microspin column and to quantify the amount of lunasin using an optimized enzyme-linked immunosorbent assay. Lunasin was purified using a combination of ion-exchange chromatography, ultrafiltration and gel filtration chromatography. Circular dichroism showed that increased in temperature from 25 to 100 °C resulted in changes on the secondary structure of lunasin and its capability to interact with rabbit polyclonal antibody. Enzyme linked immunosorbent assay showed that lunasin rabbit polyclonal antibody has a titer of 250 and a specific activity of 0.05 mL/?g. A linear response was detected between 16 to 48 ng lunasin per mL (y=0.03x-0.38, R(2)=0.96). The use of diethylaminoethyl microspin column to isolate spiked lunasin in human plasma showed that most lunasin (37.8-46.5%) bound to the column eluted with Tris-HCl buffer, pH 7.5 with a yield up to 76.6%. In conclusion, lunasin can be isolated from human plasma by a simple DEAE microspin column technique and can be quantified using a validated and optimized immunoassay procedure. This method can be used directly to quantify lunasin from plasma in different human and animal studies aiming to determine its bioavailability. PMID:23265496

  10. Using cheminformatics to predict cross reactivity of “designer drugs” to their currently available immunoassays

    PubMed Central

    2014-01-01

    Background A challenge for drug of abuse testing is presented by ‘designer drugs’, compounds typically discovered by modifications of existing clinical drug classes such as amphetamines and cannabinoids. Drug of abuse screening immunoassays directed at amphetamine or methamphetamine only detect a small subset of designer amphetamine-like drugs, and those immunoassays designed for tetrahydrocannabinol metabolites generally do not cross-react with synthetic cannabinoids lacking the classic cannabinoid chemical backbone. This suggests complexity in understanding how to detect and identify whether a patient has taken a molecule of one class or another, impacting clinical care. Methods Cross-reactivity data from immunoassays specifically targeting designer amphetamine-like and synthetic cannabinoid drugs was collected from multiple published sources, and virtual chemical libraries for molecular similarity analysis were built. The virtual library for synthetic cannabinoid analysis contained a total of 169 structures, while the virtual library for amphetamine-type stimulants contained 288 compounds. Two-dimensional (2D) similarity for each test compound was compared to the target molecule of the immunoassay undergoing analysis. Results 2D similarity differentiated between cross-reactive and non-cross-reactive compounds for immunoassays targeting mephedrone/methcathinone, 3,4-methylenedioxypyrovalerone, benzylpiperazine, mephentermine, and synthetic cannabinoids. Conclusions In this study, we applied 2D molecular similarity analysis to the designer amphetamine-type stimulants and synthetic cannabinoids. Similarity calculations can be used to more efficiently decide which drugs and metabolites should be tested in cross-reactivity studies, as well as to design experiments and potentially predict antigens that would lead to immunoassays with cross reactivity for a broader array of designer drugs. PMID:24851137

  11. Discrepancy between Vitamin D Total Immunoassays due to Various Cross-reactivities

    PubMed Central

    Lee, Jun Hyung; Choi, Jee-Hye; Kweon, Oh Joo

    2015-01-01

    Background The purpose of this study was to find out the cause of discrepancy between various automated immunoassays for 25-hydroxy-vitamin D (25-[OH]D). Methods National Institute of Standards & Technology Standard Reference Material (SRM) 972a is SRM for 25-(OH)D and consists of 4 vials of frozen serum with different concentrations of 25-(OH)D. Each concentration was measured 6 times in 3 different immunoassays: ADVIA Vitamin D Total assay (Siemens Healthcare, Erlangen, Germany), ARCHITECT 25-(OH)D (Abbott Laboratories, Abbott Park, IL, USA), and COBAS Vitamin D Total assay (Roche Diagnostics, Basel, Switzerland). Results When using the certified reference values of SRM 972a as it is, discarding the cross-reactivity of each immunoassay, for ADVIA, the coefficient of determination (R2) as a score of regression analysis was 0.8995 and maximal difference between measured value and certified reference value was 3.6 ng/mL in level 3. The R2 and maximal differences of ARCHITECT were 0.5377 and 6.9 ng/mL, respectively, in level 4. Those of COBAS were 0.3674 and 22.3 ng/mL, respectively, in level 4. When considering cross-reactivities of each immunoassays to various 25-(OH)D metabolites, the ADVIA had R2 and maximal difference of 0.9254 and 3.3 ng/mL, respectively, in level 3. For ARCHITECT, the R2 and maximal differences were 0.7602 and 5.1 ng/mL, respectively, in level 1. Those of COBAS were 0.9284 and 4.9 ng/mL, respectively, in level 1. Conclusions The cause of discrepancies between vitamin D immunoassays was mainly on the difference in cross-reactivities to various vitamin D metabolites. The discrepancies can be considerably decreased by considering cross-reactivities of each immunoassay. PMID:26389085

  12. A research standard for human serum immunoglobulins IgG, IgA and IgM.

    PubMed

    Rowe, D S; Anderson, S G; Grab, B

    1970-01-01

    A pooled human serum, partly diluted, has been distributed into ampoules and freeze-dried in several batches. The freeze-dried material has been examined in an international collaborative assay and certain properties have also been estimated in individual laboratories.On the basis of these tests this material was considered to be suitable for use as a standard for the estimation of IgG, IgA and IgM for clinical purposes using the single-radial-diffusion or similar techniques. Greater uniformity of results than is obtained at present should be achieved if this material were in general use.Estimates of immunoglobulins from different laboratories using this material as a standard showed small but significant variability. This variability was probably related to the heterogeneity of immunoglobulins and of antisera, and it limits the precision of immunoglobulin estimations by techniques at present in use.Batches of this material have been distributed to various centres. 67/68 has been established as the British research standard for human serum immunoglobulins IgG, IgA and IgM for which the unit of potency is defined as the activity present in 0.8147 mg of dry powder. The average activity per ampoule of 67/86 is 100 units of IgG, IgA and IgM. The average activities of other related preparations have been estimated. PMID:4194813

  13. A Research Standard for Human Serum Immunoglobulins IgG, IgA and IgM

    PubMed Central

    Rowe, D. S.; Anderson, S. G.; Grab, B.

    1970-01-01

    A pooled human serum, partly diluted, has been distributed into ampoules and freeze-dried in several batches. The freeze-dried material has been examined in an international collaborative assay and certain properties have also been estimated in individual laboratories. On the basis of these tests this material was considered to be suitable for use as a standard for the estimation of IgG, IgA and IgM for clinical purposes using the single-radial-diffusion or similar techniques. Greater uniformity of results than is obtained at present should be achieved if this material were in general use. Estimates of immunoglobulins from different laboratories using this material as a standard showed small but significant variability. This variability was probably related to the heterogeneity of immunoglobulins and of antisera, and it limits the precision of immunoglobulin estimations by techniques at present in use. Batches of this material have been distributed to various centres. 67/68 has been established as the British research standard for human serum immunoglobulins IgG, IgA and IgM for which the unit of potency is defined as the activity present in 0.8147 mg of dry powder. The average activity per ampoule of 67/86 is 100 units of IgG, IgA and IgM. The average activities of other related preparations have been estimated. ImagesFIG. 2 PMID:4194813

  14. The infrared fingerprint signals of silica nanoparticles and its application in immunoassay

    NASA Astrophysics Data System (ADS)

    Ding, Yadan; Chu, Xueying; Hong, Xia; Zou, Peng; Liu, Yichun

    2012-01-01

    Infrared absorption properties of silica nanoparticles were studied. The transverse optical and the longitudinal optical phonon modes from the silica were proved to be the characteristic spectroscopic fingerprint signals. Based on this, a sandwich-structured immunoassay was performed, and the detection of the analyte (human IgG) was achieved by using biofunctional silica nanoparticles as infrared probes. The immunoassay based on Fourier transform infrared reflection absorption spectroscopy of silica nanoparticles shows significant value for potential applications in many areas, such as biomedicine, food safety, and waste treatment.

  15. A Microfluidic Device for Immunoassay-Based Protein Analysis of Single E. coli Bacteria.

    PubMed

    Stratz, Simone; Dittrich, Petra S

    2015-01-01

    We present a method suitable for quantitative analysis of intracellular proteins, metabolites and secondary messengers of single bacterial cells. The method integrates the concept of immunoassays on a microfluidic device that facilitates single cell trapping and isolating in a small volume of a few tens of picoliters. Combination of the benefits of microfluidic systems for single cell analysis with the high analytical selectivity and sensitivity of immunoassays enables the detection of even low abundant intracellular analytes which occur only at a few hundred copies per bacterium. PMID:26542712

  16. IgM-class rheumatoid factor interference in the solid-phase radioimmunoassay of rubella-specific IgM antibodies.

    PubMed Central

    Meurman, O H; Ziola, B R

    1978-01-01

    The interference of IgM-class rheumatoid factor (RF) in the solid-phase radioimmunoassay (RIA) of rubella virus IgM antibodies was studied. Acute rubella infections did not significantly activate RF. False-positive rubella antibody results were obtained, however, when patients with raised RF levels were tested. If a low rubella IgG antibody titre was present, a high level of RF was required to cause a false-positive IgM result; conversely, in sera with high IgG titres, only a low level of RF was required for interference. Although the false-positive IgM titres obtained were generally low, thet did show a positive correlation to both RF levels and rubella IgG titres. False-positive results were successfully avoided by removing the RF by absorption with heat-aggregated human gamma globulin. The absorption procedure did not affect true rubella IgM antibody titres. PMID:77280

  17. Enzymes in cleaning products: an overview of toxicological properties and risk assessment/management.

    PubMed

    Basketter, David; Berg, Ninna; Broekhuizen, Cees; Fieldsend, Mark; Kirkwood, Sheila; Kluin, Cornelia; Mathieu, Sophie; Rodriguez, Carlos

    2012-10-01

    Enzymes used in cleaning products have an excellent safety profile, with little ability to cause adverse responses in humans. For acute toxicity, genotoxicity, sub-acute and repeated dose toxicity, enzymes are unremarkable. Reproductive toxicity and carcinogenicity are also not endpoints of concern. Exceptions are the ability of some proteases to produce irritating effects at high concentrations and more importantly, the intrinsic potential of these bacterial/fungal proteins to act as respiratory sensitizers. It is a reasonable assumption that the majority of enzyme proteins possess this hazard. However, methods for characterising the respiratory sensitisation hazard of enzymes are lacking and the information required for risk assessment and risk management, although sufficient, remains limited. Previously, most data was generated in animal models and in in vitro immunoassays that assess immunological cross-reactivity. Nevertheless, by the establishment of strict limits on airborne exposure (based on a defined minimal effect limit of 60ng active enzyme protein/m(3)) and air and health monitoring, occupational safety can be assured. Similarly, by ensuring that airborne exposure is kept similarly low, coupled with knowledge of the fate of these enzymes on skin and fabrics, it has proven possible to establish a long history of safe consumer use of enzyme containing products. PMID:22743221

  18. Enhancement by ascorbic acid 2-glucoside or repeated additions of ascorbate of mitogen-induced IgM and IgG productions by human peripheral blood lymphocytes.

    PubMed

    Tanaka, M; Muto, N; Gohda, E; Yamamoto, I

    1994-12-01

    In this study, the effect of ascorbic acid 2-glucoside (AA-2G), a stable derivative of ascorbic acid (AsA), or repeated additions of ascorbate on antibody productions by human peripheral blood lymphocytes (PBLs) was examined, and the physiological function of AsA was evaluated. When human PBLs were stimulated with Staphylococcus aureus Cowan I or pokeweed mitogen, AA-2G remarkably increased the numbers of IgM- and IgG-secreting cells which were detected by enzyme-linked immunospot assay. Although a single addition of ascorbate was without effect, the effect of AA-2G was remarkably inhibited by the addition of castanospermine, an alpha-glucosidase inhibitor; and moreover, repeated additions of AsA to the culture medium during the culture period enhanced the response to the same level as did a single addition of AA-2G. These results indicate that AsA has the ability to stimulate the immunoglobulin productions by AA-2G. The phytohemagglutinin-induced proliferative response of PBLs was also stimulated by AA-2G. The intracellular AsA content in PBLs cultured with AA-2G was maintained at relatively high levels during the culture period, whereas the content with a single dose of AsA reached nearly zero by the end of the experiment. These in vitro findings suggest that AA-2G and AsA function as potent immunostimulators of antibody production in humans and that the intracellular AsA content is a key parameter for establishing the immune response of PBLs. PMID:7723222

  19. Simultaneous Binding of the Anti-Cancer IgM Monoclonal Antibody PAT-SM6 to Low Density Lipoproteins and GRP78

    PubMed Central

    Rosenes, Zachary; Mok, Yee-Foong; Yang, Shuo; Griffin, Michael D. W.; Mulhern, Terrence D.; Hatters, Danny M.; Hensel, Frank; Howlett, Geoffrey J.

    2013-01-01

    The tumour-derived monoclonal IgM antibody PAT-SM6 specifically kills malignant cells by an apoptotic mechanism linked to the excessive uptake of plasma lipids. The mechanism is postulated to occur via the multi-point attachment of PAT-SM6 to the unfolded protein response regulator GRP78, located on the surface of tumour cells, coupled to the simultaneous binding of plasma low density lipoprotein (LDL). We prepared and characterised LDL and oxidized LDL using sedimentation velocity and small-angle X-ray scattering (SAXS) analysis. Enzyme-linked immunosorbent (ELISA) techniques indicated apparent dissociation constants of approximately 20 nM for the binding of LDL or oxidized LDL to PAT-SM6. ELISA experiments showed cross competition with LDL inhibiting PAT-SM6 binding to immobilised GRP78, while, in the reverse experiment, GRP78 inhibited PAT-SM6 binding to immobilized LDL. In contrast to the results of the ELISA experiments, sedimentation velocity experiments indicated relatively weak interactions between LDL and PAT-SM6, suggesting immunoabsorbance to the microtiter plate is driven by an avidity-based binding mechanism. The importance of avidity and the multipoint attachment of antigens to PAT-SM6 was further investigated using antigen-coated polystyrene beads. Absorption of GRP78 or LDL to polystyrene microspheres led to an increase in the inhibition of PAT-SM6 binding to microtiter plates coated with GRP78 or LDL, respectively. These results support the hypothesis that the biological action of PAT-SM6 in tumour cell apoptosis depends on the multivalent nature of PAT-SM6 and the ability to interact simultaneously with LDL and multiple GRP78 molecules clustered on the tumour cell surface. PMID:23620733

  20. Fetal lambs are depleted of IgM+ cells following a single injection of an anti-IgM antibody early in gestation.

    PubMed Central

    Press, C M; Reynolds, J D; McClure, S J; Simpson-Morgan, M W; Landsverk, T

    1996-01-01

    B-cell depleted fetal sheep were created following a single injection of an anti-IgM monoclonal antibody early in gestation. Six sheep fetuses were given a single intraperitoneal injection of a monoclonal antibody directed against IgM at 63 days of gestation (gestation in sheep = 150 days). The fetuses were killed at 138-142 days of gestation and lymphoid tissues were collected for subsequent light microscopy and immunohistochemical examination. The ileal and jejunal Peyer's patch (PP) follicles in four of the six injected fetuses were markedly reduced in size. Cells in the rudimentary follicles of the ileal PP of these animals showed no reactivity for IgM and most were negative for CD45. The dome regions contained many T cells, which were predominantly CD8+ cells and included gamma delta T cells. The interfollicular areas of the PP of the markedly affected fetuses contained large populations of T cells. The spleen and lymph nodes were also markedly depleted of IgM+ cells and these tissues contained only a small, scattered population of weakly IgM+ cells. Follicular accumulations of IgM+ cells were absent. Large populations of T cells were present in the white pulp of the spleen and cortex of the lymph nodes. The liver did not contain IgM+ cells and the medulla of the thymus was depleted of IgM+ cells. The results of this study suggest that a surface IgM+ B-cell population is present in the sheep fetus at 63 days of gestation, which is essential for the colonization of the ileal PP and subsequent B-cell development. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8707346

  1. Bead-based microfluidic immunoassay for diagnosis of Johne's disease

    SciTech Connect

    Wadhwa, Ashutosh; Foote, Robert; Shaw, Robert W; Eda, Shigetoshi

    2012-01-01

    Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne s disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types of SAB, and types of magnetic beads) were optimized for a great degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested by commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads by laser-induced fluorescence. Antigen-coated magnetic beads treated with bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples obtained with the system were compared to those obtained with the flow cytometer, a high level of correlation (linear regression, r2 = 0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD. Key Words: Mycobacterium avium ssp. paratuberculosis, Johne s disease, microfluidics, lab-on-a-chip.

  2. Multiplex serum cytokine immunoassay using nanoplasmonic biosensor microarrays.

    PubMed

    Chen, Pengyu; Chung, Meng Ting; McHugh, Walker; Nidetz, Robert; Li, Yuwei; Fu, Jianping; Cornell, Timothy T; Shanley, Thomas P; Kurabayashi, Katsuo

    2015-04-28

    Precise monitoring of the rapidly changing immune status during the course of a disease requires multiplex analysis of cytokines from frequently sampled human blood. However, the current lack of rapid, multiplex, and low volume assays makes immune monitoring for clinical decision-making (e.g., critically ill patients) impractical. Without such assays, immune monitoring is even virtually impossible for infants and neonates with infectious diseases and/or immune mediated disorders as access to their blood in large quantities is prohibited. Localized surface plasmon resonance (LSPR)-based microfluidic optical biosensing is a promising approach to fill this technical gap as it could potentially permit real-time refractometric detection of biomolecular binding on a metallic nanoparticle surface and sensor miniaturization, both leading to rapid and sample-sparing analyte analysis. Despite this promise, practical implementation of such a microfluidic assay for cytokine biomarker detection in serum samples has not been established primarily due to the limited sensitivity of LSPR biosensing. Here, we developed a high-throughput, label-free, multiarrayed LSPR optical biosensor device with 480 nanoplasmonic sensing spots in microfluidic channel arrays and demonstrated parallel multiplex immunoassays of six cytokines in a complex serum matrix on a single device chip while overcoming technical limitations. The device was fabricated using easy-to-implement, one-step microfluidic patterning and antibody conjugation of gold nanorods (AuNRs). When scanning the scattering light intensity across the microarrays of AuNR ensembles with dark-field imaging optics, our LSPR biosensing technique allowed for high-sensitivity quantitative cytokine measurements at concentrations down to 5-20 pg/mL from a 1 ?L serum sample. Using the nanoplasmonic biosensor microarray device, we demonstrated the ability to monitor the inflammatory responses of infants following cardiopulmonary bypass (CPB) surgery through tracking the time-course variations of their serum cytokines. The whole parallel on-chip assays, which involved the loading, incubation, and washing of samples and reagents, and 10-fold replicated multianalyte detection for each sample using the entire biosensor arrays, were completed within 40 min. PMID:25790830

  3. Commercial production of microbial enzymes

    SciTech Connect

    Munro, I.G.

    1985-01-01

    The advantages and uses of industrially produced microbial enzymes are described. The processes involved in the production of these enzymes, cultivation techniques, enzyme extraction, enzyme purification and immobilization are outlined. Both the history of enzyme technology and its future development are discussed.

  4. Seroepidemiology of toxoplasma gondii infection in human adults. From three rural communities in Derango State, Mexico

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is scarce information concerning the epidemiology of Toxoplasma gondii infection in people of rural Mexico. Anti-T. Gondii IgG and IgM antibodies were sought in 462 adult inhabitants from 3 rural communities of Durango State, Mexico, using enzyme-linked immunoassays. In total, 110 (23.8% of ...

  5. Pregnancy Outcomes of Mothers with Detectable CMV-Specific IgM Antibodies: A Three-Year Review in a Large Irish Tertiary Referral Maternity Hospital

    PubMed Central

    Drew, Richard J.; Stapleton, Patrick; Abu, Hala; Healy, Eibhlín; Ferguson, Wendy; De Gascun, Cillian; O'Gorman, Joanne; Eogan, Maeve

    2015-01-01

    A retrospective audit was performed for all obstetric patients who had positive CMV IgM results between January 2012 and December 2014 in the Rotunda Hospital, Ireland. In total, 622 CMV IgM positive tests were performed on samples from 572 patients. Thirty-seven patients had a positive CMV IgM result (5.9%) on the Architect system as part of the initial screening. Three patients were excluded as they were not obstetric patients. Of the 34 pregnant women with CMV IgM positive results on initial screening, 16 (47%) had CMV IgM positivity confirmed on the second platform (VIDAS) and 18 (53%) did not. In the 16 patients with confirmed positive CMV IgM results, four (25%) had acute infection, two (12.5%) had infection of uncertain timing, and ten (62.5%) had infection more than three months prior to sampling as determined by the CMV IgG avidity index. Two of the four neonates of women with low avidity IgG had CMV DNA detected in urine. Both these cases had severe neurological damage and the indication for testing their mothers was because the biparietal diameter (BPD) was less than the 5th centile at the routine 20-week gestation anomaly scan. PMID:26696757

  6. Application of an enzyme immunoassay for the quantitative determination of azo dye (Orange II) in food products.

    PubMed

    Xue, Huyin; Xing, Yue; Yin, Yongmei; Zhang, Taichang; Zhang, Bo; Zhang, Yu; Song, Pei; Tian, Xi; Xu, Yinghui; Wang, Peng; Meng, Meng; Xi, Rimo

    2012-01-01

    This paper reports the preparation of polyclonal antibodies against a synthetic azo dye, Orange II, and the development of an indirect ELISA to detect Orange II in foods. The sulfonic group of Orange II was modified and linked with carrier protein to synthesise an artificial antigen. Based on the checkerboard titration, the method showed excellent sensitivity (IC?? = 0.61 ng g?¹) to Orange II in the linear range of 0.05-10 ng g?¹. The antibody had little cross-reactivity with Chromotrope FB, Gardenia Yellow, Ponceau 4R, Sunset Yellow and Sudan dyes. The ELISA had limits of detection (LOD) of 0.22, 0.97 and 0.74 ng g?¹ in chilli powder, chilli oil and braised pork, respectively. The limits of quantification (LOQ) of the assay were 0.91 ng g?¹ in chilli powder, 1.48 ng g?¹ in chilli oil and 1.10 ng g?¹ in braised pork. For food products fortified with 1-10 ng g?¹ Orange II, the inter- and intra-assay variations were all less than 24.0% and 18.0%, respectively. Therefore, the proposed test could be used as a rapid screening method for Orange II detection in food samples. PMID:22889210

  7. STABILIZATION OF A RACTOPAMINE ENZYME CONJUGATE IN AQUEOUS SOLUTION, A RAPID AND CONVENIENT IMMUNOASSAY METHOD FOR THE DETECTION OF RACTOPAMINE.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is an increasing demand for a sensitive screening method for ractopamine because of the zero tolerance policy in many countries. Most of the commercially available ractopamine ELISA kits require concentrated conjugate to be diluted prior to use. We have observed that the highly diluted racto...

  8. 2220 J. Agric. Food Chem. 1993, 41, 2220-2227 An Enzyme Immunoassay for the Environmental Monitoring of the

    E-print Network

    Hammock, Bruce D.

    , and Bruce D. Hammock' Departments of Entomology and Environmental Toxicology,University of California, 1991; Jaynes, 1991; Reddy et al., 1992)because of its wide application and recent concerns about health for EcologicalHealth Research CR819047-01-0,and NIEHS Center for Health Effects of Agrochemicals1P30ES05707. H

  9. J. Agric. Food Chem. 1992, 40, 1459-1465 1459 Synthesis of Haptens and Conjugates for an Enzyme Immunoassay for

    E-print Network

    Hammock, Bruce D.

    ,t*llMaria-Pilar Marco,*~~Marvin H. Goodrow,t and Bruce D. Hammock'J Departments of Entomology and Environmental environmental and health hazards due to the contami- nation ofgroundwater andsoilbybromacil(Valencia,1981; Rao; California Code of Regulations 1991;Allender, 1991). The Lifetime Health AdvisoryLevel in drinking water

  10. A Semester-Long Student-Directed Research Project Involving Enzyme Immunoassay: Appropriate for Immunology, Endocrinology, or Neuroscience Courses

    ERIC Educational Resources Information Center

    Goyette, Sharon Ramos; 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…

  11. Development of an enzyme immunoassay using recombinant invariant surface glycoprotein (rISG) 75 for serodiagnosis of bovine trypanosomosis.

    PubMed

    Rudramurthy, G R; Sengupta, P P; Metilda, B; Balamurugan, V; Prabhudas, K; Rahman, H

    2015-01-01

    Trypanosomosis or surra is caused by the haemoflagellate parasite, Trypanosoma evansi and is an important disease of animals, including domestic and wild herbivores and carnivores, in tropical countries. The invariant surface glycoproteins (ISGs) are blood stream stage specific and are uniformly distributed over the entire surface of the trypanosomes. In the present study, the extracellular domain (ED) region of ISG-75 from T. evansi, consisting of 1320 nt, encoding a polypeptide of 440 amino acids, has been heterologously expressed in Escherichia coli. Further, the immunoreactivity of recombinant ISG-75 (rISG-75) was characterized in immunoblot and ELISA using T. evansi hyper immune sera raised in experimental animals. The protein was found immunoreactive when compared with a panel of antigens (VSG RoTat 1.2 and whole cell lysate) using bovine serum samples from field. The diagnostic potential of rISG-75 was evaluated in ELISA with large number of bovine field serum samples. The optimum sensitivity and specificity were 98.47 and 99.1, respectively. The present finding showed that the expressed protein has potential use in the serodiagnosis of trypanosomosis. PMID:25675706

  12. Terbuthylazine and deethylterbuthylazine in rain and surface water - Determination by enzyme immunoassay and gas chromatography/mass spectrometry

    USGS Publications Warehouse

    Dankwardt, A.; Thurman, E.M.; Hock, B.

    1997-01-01

    Rain and surface water samples from Southern Germany were investigated from 1991 to 1995 for terbuthylazine and one of its major metabolites, deethylterbuthylazine. The concentrations observed were compared to the concentrations found for atrazine and deethylatrazine in the same water samples. Concentrations ranged from <0.02 ??g/L to 0.7 ??g/L for terbuthylazine and from <0.02 ??g/L to 0.6 ??g/L for deethylterbuthylazine, compared to concentrations of <0.02 ??g/L to 3 ??g/L and <0.02 ??g/L to 0.5 ??g/L for atrazine and deethylatrazine, respectively. The ratios of metabolite concentrations to parent compound concentrations were calculated for deethylterbuthylazine to terbuthylazine (DTR) and deethylatrazine to atrazine (DAR). In rain water, DTR of 0.8...3.0 and DAR of 0.3...1.9 were determined with mean values of 0.9...1.7 for DTR and 0.6...0.9 for DAR in the different years. The ratios increased during summer periods. The highest ratios were observed in samples from forest stands, showing that degradation of the herbicide has occurred during transport between the source and the sampling site. The DTR in rain water were about 50...100% higher than the DAR. This indicates a higher degradation rate of terbuthylazine during atmospheric transport. In surface water, DTR of 0.3...1.2 with mean values of 0.5...0.8 and DAR of 0.2...2.2 with mean values of 0.2...1.3 were observed. The ratios increased from June to September.

  13. Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry.

    PubMed

    Miller, Robert; Plessow, Franziska; Rauh, Manfred; Gröschl, Michael; Kirschbaum, Clemens

    2013-01-01

    Assessing the amount of bioavailable cortisol in saliva with immunoassays and thus sampling an endocrine marker of hypothalamus-pituitary-adrenal axis activity is of major interest in both research and clinical practice. However, absolute cortisol concentrations obtained with different immunoassays (IAs) are barely comparable precluding direct comparison between studies or individuals whenever cortisol analyses were not based on the same IA. The present technical report aims to solve this problem by evaluating the validity of, as well as agreement between the most commonly used immunoassays in psychoneuroendocrinological research (i.e., IBL, DRG, Salimetrics, DSL, and DELFIA) and a reference method (LC-MS/MS) in a sample of 195 saliva specimen covering the whole range of cortisol concentrations in adults. A structural equation modelling framework is applied to decompose systematic assay variance and estimate cortisol reference values, which are adjusted for measurement error and interference of salivary cortisone. Our findings reveal nonlinear relations between IAs and LC-MS/MS, which are discussed in terms of IA cross-reactivity with saliva matrix components. Finally guidelines for converting cortisol concentrations being obtained by these immunoassays into comparable reference values are proposed by providing conversion functions, a conversion table, and an online conversion tool. PMID:22641005

  14. Determination of deoxynivalenol in wheat bran and whole-wheat flour by fluorescence polarization immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A rapid and accurate fluorescence polarization (FP) immunoassay has been optimized for the determination of deoxynivalenol (DON) in bran and whole-wheat flour. A preliminary treatment with activated charcoal was used to eliminate the strong matrix effect due to highly colored interfering compounds p...

  15. Metallic nanocrystallites-incorporated ordered mesoporous carbon as labels for a sensitive simultaneous multianalyte electrochemical immunoassay.

    PubMed

    Fang, Yishan; Huang, Xinjian; Zeng, Qiang; Wang, Lishi

    2015-11-15

    This work reports on a facile, novel multianalyte electrochemical immunoassay for simultaneous detection of a-fetoprotein (AFP) and human epidermal growth factor receptor type-2 (HER-2) using metal-containing nanomaterials confined in the ordered mesoporous carbon matrix (OMC-M) as labels. Well-dispersed uniform metallic nanocrystallites incorporated OMC materials were fabricated through a simple, economical, and green preparative strategy toward phenolic resol as a carbon source and metal nitrate as metal sources. The large amount of metallic nanocrystallites loading on the OMC nanomaterials, greatly amplified the detection signals, and the good biocompatibility of carbon nanotubes-chitosan retained excellent stability for the sandwich-type immunoassay. Under optimal experimental conditions, the proposed immunoassay exhibited high sensitivity and selectivity for the detection of analytes, providing a better linear response range from 0.001 to 150 ng/mL for AFP and for HER-2, with a lower limit of detectionof 0.6p g/mL and 0.35 pg/mL (S/N=3), respectively. The immunosensor exhibited convenience, low cost, rapidity, good specificity, acceptable stability and reproducibility. Moreover, satisfactory results were obtained for the determination of AFP and HER-2 in real human serum samples, indicating that the developed immunoassay has the potential to find application in clinical detection of AFP and HER-2 and other tumor markers as an alternative approach. PMID:26046316

  16. Immunoassays for the cancer biomarker CA125 based on a large-birefringence nematic liquid-

    E-print Network

    Wu, Shin-Tson

    for gastric cancer patients," J. Cancer Res. Clin. Oncol. 133(7), 471­476 (2007). 7. B. Zhang, F. F. CaiImmunoassays for the cancer biomarker CA125 based on a large-birefringence nematic liquid- crystal for the immunodetection. Experiments were performed, targeting at the cancer biomarker CA125. We showed that the larger

  17. Enhanced Sensitive Immunoassay: Noncompetitive Phage Anti-Immune Complex Assay for the Determination of Malachite Green

    E-print Network

    Hammock, Bruce D.

    for the Determination of Malachite Green and Leucomalachite Green Jie-Xian Dong,, Chao Xu, Hong Wang,*, Zhi-Li Xiao ABSTRACT: To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG for monitoring food safety. KEYWORDS: malachite green, leucomalachite Green, phage anti-immunocomplex assay

  18. Electrophoretic build-up of multi nanoparticle array for a highly sensitive immunoassay

    E-print Network

    Hammock, Bruce D.

    Electrophoretic build-up of multi nanoparticle array for a highly sensitive immunoassay Jin-Hee Han design of the nanoarray, electrophoretic trapping of a nanoparticle was modeled numerically. The trapping facilitated many breakthroughs in the life sciences by identifying specific gene sequences or protein ana

  19. Magnetic colorimetric immunoassay for human interleukin-6 based on the oxidase activity of ceria spheres.

    PubMed

    Peng, Juan; Guan, Jufang; Yao, Huiqin; Jin, Xiaoyong

    2016-01-01

    A novel magnetic colorimetric immunoassay strategy was designed for sensitive detection of human interleukin-6 (IL-6) using ceria spheres as labels. Ceria spheres showed excellent oxidase activity, which can directly catalyze the oxidation of substrate o-phenylenediamine (OPD) to a stable yellow product, 2,3-diaminophenazine (oxOPD). The absorbance of oxOPD was recorded to reflect the level of IL-6. The relatively mild conditions made the immunoassay strategy more robust, reliable, and easy. A linear relationship between absorbance intensity and the logarithm of IL-6 concentrations was obtained in the range of 0.0001-10 ng mL(-1) with a detection limit of 0.04 pg mL(-1) (S/N = 3). The colorimetric immunoassay exhibited high sensitivity and specificity for the detection of IL-6. This immunoassay has been successfully applied in the detection of IL-6 in serum samples and can be readily extended toward the on-site monitoring of cancer biomarkers in serum samples. PMID:26416691

  20. Fluorescence polarization immunoassay using IgY antibodies for detection of valnemulin in swine tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassay for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence p...

  1. ON-SITE MERCURY ANALYSIS OF SOIL AT HAZARDOUS WASTE SITES BY IMMUNOASSAY AND ASV

    EPA Science Inventory

    Two field methods for Hg, immunoassay and anodic stripping voltammetry (ASV), that can provide onsite results for quick decisions at hazardous waste sites were evaluated. Each method was applied to samples from two Superfund sites that contain high levels of Hg; Sulphur Bank Me...

  2. Rapid Detection of Nivalenol and Deoxynivalenol in Wheat Using Surface Plasmon Resonance Immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Surface plasmon resonance immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A DON-immobilized sensor chip having high sensitivity and stability was prepared, and an SPR detection procedure was developed. The competitiv...

  3. Microarray Immunoassay for Phenoxybenzoic Acid Using Polymer Encapsulated Eu:Gd2O3

    E-print Network

    Hammock, Bruce D.

    Microarray Immunoassay for Phenoxybenzoic Acid Using Polymer Encapsulated Eu:Gd2O3 Nanoparticles red emission, large Stokes shift, photostable laser-induced fluorescence with a long lifetime (1 ms nanostructures (polymer beads, fluorophore-doped nanoparticles, semiconductor quantum dots, inorganic and organic

  4. Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The extensive use of organophosphorus pesticides (OPs) in agriculture and domestic settings can result in widespread water contamination. The development of easy-to-use and rapid-screening immunoassay methods in a class-selective manner is a topic of considerable environmental interest. In this wo...

  5. Immunoassay on Free-Standing Electrospun Dapeng Wu, Daewoo Han, and Andrew J. Steckl*

    E-print Network

    Cincinnati, University of

    -HSA and HSA-FITC as an immunoassay model, a linear detection range from 500 ng/mL down to 1 ng/mL is obtained. Electrospinning is an efficient and simple tool to spray micro/nano polymer fibers from viscous polymer solutions

  6. DEVELOPMENT OF A FLUORESCENT LATEX IMMUNOASSAY FOR DETECTION OF SPECTINOMYCIN ANTIOBIOTIC

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spectinomycin is an antimicrobial agent used to treat infections caused by Gram negative and positive microorganisms in poultry, swine and non-lactating cattle. There is a need to develop a rapid and sensitive method to detect spectinomycin residues in animal tissues. A latex fluorescent immunoassay...

  7. IMMUNOASSAY METHOD FOR THE DETERMINATION OF PENTACHLOROPHENOL IN SOIL AND SEDIMENT

    EPA Science Inventory

    The journal article describes the use of a prototype immunoassay method for the determination of pentacholorphenol (PCP) in soil and sediment. PCP was used as a pesticide and wood preservative and is not currently available to the general public. The paper stresses the importan...

  8. AUTOMATED FLOW FLUORESCENT IMMUNOASSAY FOR PART PER TRILLION DETECTION OF THE NEONICOTINOID INSECTICIDE THIAMETHOXAM.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ultra sensitive automated flow fluorescent immunoassay was developed using the KinExATM 3000 system for quantitative analysis of the neonicotinoid insecticide thiamethoxam. Five monoclonal antibodies were obtained and screened with a competitive ELISA. One monoclonal antibody designated as E6VI ...

  9. A commercial rapid optical immunoassay detects Streptococcus agalactiae from aquatic cultures and clinical specimens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The BioStar STREPT B Optical ImmunoAssay (OIA) (BioStar® OIA® Strep B Assay Kit; Biostar Incorporation; Louisville, CO, USA) was used to identify 32 known group B streptococcus (GBS) isolates of fish, dolphin, bovine, and human origin. Thirteen non-GBS isolates from fish and other animals were test...

  10. Analytica Chimica Acta 444 (2001) 169178 Highly sensitive dioxin immunoassay and its

    E-print Network

    Hammock, Bruce D.

    2001-01-01

    Analytica Chimica Acta 444 (2001) 169­178 Highly sensitive dioxin immunoassay and its application Jonesc, Daniel P.Y. Changb, Bruce D. Hammocka, a UCD Cancer Research Center, Department of Entomology Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known highly toxic compound that is present in nearly all components

  11. An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads.

    PubMed

    Tao, Xiaoqi; Jiang, Haiyang; Yu, Xuezhi; Zhu, Jinghui; Wang, Xia; Wang, Zhanhui; Niu, Lanlan; Wu, Xiaoping; Shen, Jianzhong

    2013-05-01

    A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC50 values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) were 0.017 µg L(-1) for extract method I and 0.17 µg L(-1) for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7-98.0% (extract method I) and 80.0-103.0% (extract method II), and the coefficients of variation (CVs) were all <15%. The satisfactory recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis. PMID:23512826

  12. Fluorescence polarization immunoassays for rapid, accurate and sensitive determination of mycotoxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fluorescence polarization immunoassay (FPIA) is a type of homogeneous assay. For low molecular weight antigens, such as mycotoxins, it is based on the competition between an unlabeled antigen and its fluorescent-labeled derivative (tracer) for an antigen-specific antibody. The antigen content is det...

  13. Surface-Enhanced Raman Scattering (SERS) for Detection in Immunoassays: applications, fundamentals, and optimization

    SciTech Connect

    Jeremy Daniel Driskell

    2006-08-09

    Immunoassays have been utilized for the detection of biological analytes for several decades. Many formats and detection strategies have been explored, each having unique advantages and disadvantages. More recently, surface-enhanced Raman scattering (SERS) has been introduced as a readout method for immunoassays, and has shown great potential to meet many key analytical figures of merit. This technology is in its infancy and this dissertation explores the diversity of this method as well as the mechanism responsible for surface enhancement. Approaches to reduce assay times are also investigated. Implementing the knowledge gained from these studies will lead to a more sensitive immunoassay requiring less time than its predecessors. This dissertation is organized into six sections. The first section includes a literature review of the previous work that led to this dissertation. A general overview of the different approaches to immunoassays is given, outlining the strengths and weaknesses of each. Included is a detailed review of binding kinetics, which is central for decreasing assay times. Next, the theoretical underpinnings of SERS is reviewed at its current level of understanding. Past work has argued that surface plasmon resonance (SPR) of the enhancing substrate influences the SERS signal; therefore, the SPR of the extrinsic Raman labels (ERLs) utilized in our SERS-based immunoassay is discussed. Four original research chapters follow the Introduction, each presented as separate manuscripts. Chapter 2 modifies a SERS-based immunoassay previously developed in our group, extending it to the low-level detection of viral pathogens and demonstrating its versatility in terms of analyte type, Chapter 3 investigates the influence of ERL size, material composition, and separation distance between the ERLs and capture substrate on the SERS signal. This chapter links SPR with SERS enhancement factors and is consistent with many of the results from theoretical treatments of SPR and SERS. Chapter 4 introduces a novel method of reducing sample incubation time via capture substrate rotation. Moreover, this work led to a method of virus quantification without the use of standards. Chapter 5 extends the methodology developed in Chapter 4 to both the antigen and ERL labeling step to perform assays with improved analytical performance in less time than can be accomplished in diffusion controlled assays. This dissertation concludes with a general summary and speculates on the future of this exciting approach to carrying out immunoassays.

  14. RNA as an Enzyme.

    ERIC Educational Resources Information Center

    Cech, Thomas R.

    1986-01-01

    Reviews current findings that explain RNA's function as an enzyme in addition to being an informational molecule. Highlights recent research efforts and notes changes in the information base on RNA activity. Includes models and diagrams of RNA activity. (ML)

  15. Overproduction of ligninolytic enzymes

    SciTech Connect

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  16. Functionalized gold nanoclusters as fluorescent labels for immunoassays: Application to human serum immunoglobulin E determination.

    PubMed

    Alonso, María Cruz; Trapiella-Alfonso, Laura; Fernández, José M Costa; Pereiro, Rosario; Sanz-Medel, Alfredo

    2016-03-15

    A quantitative immunoassay for the determination of immunoglobulin E (IgE) in human serum using gold nanoclusters (AuNCs) as fluorescent label was developed. Water soluble AuNCs were synthesized using lipoic acid and then thoroughly characterized. The obtained AuNCs have a particle size of 2.7±0.1nm and maximum fluorescence emission at 710nm. The synthesized AuNCs showed very good stability of the fluorescent signal with light exposure and at neutral and slightly basic media. A covalent bioconjugation of these AuNCs with the desired antibody was carried out by the carbodiimide reaction. After due optimization of such bioconjugation reaction, a molar ratio 1:3 (antibody:AuNCs) was selected. The bioconjugate maintained an intense luminescence emission, slightly red-shifted as compared to the free AuNCs. Two typical immunoassay configurations, competitive and sandwich, were assayed and their performance for IgE determination critically compared. After the different immunoassay steps were accomplished, the fluorescence emission of the bioconjugate was measured. While the sandwich format provided a detection limit (DL) of 10ng/mL and a linear range between 25 and 565ng/mL of IgE, the competitive format revealed a DL of 0.2ng/mL with a linear range between 0.3 and 7.1ng/mL The applicability of the more sensitive competitive fluorescent immunoassay was assessed by successful analysis of the IgE in human serum and comparison of results with those from a commercial kit. The main advantages of the proposed AuNCs-based fluorimetric method include a low DL and a simple immunoassay protocol involving few reagents. PMID:26547433

  17. Significantly improved accuracy of diagnosis of early Lyme disease by peptide enzyme-linked immunosorbent assay based on the borreliacidal antibody epitope of Borrelia burgdorferi OspC.

    PubMed

    Jobe, Dean A; Lovrich, Steven D; Asp, Krista E; Mathiason, Michelle A; Albrecht, Stephanie E; Schell, Ronald F; Callister, Steven M

    2008-06-01

    Highly specific borreliacidal antibodies are induced by infection with Borrelia burgdorferi, and the immunodominant response during early Lyme disease is specific for an epitope within the 7 amino acids nearest the C terminus of OspC. We evaluated the ability of an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (OspC7) that matched the region to detect the response and compared the sensitivity during early Lyme disease to that for an FDA-approved Western blot. When the optical density value was adjusted to 98% specificity based on the results from testing normal or uncharacterized sera (n = 236) or sera from patients with blood factors or illnesses that commonly produce antibodies that cross-react with B. burgdorferi antigens (n = 77), 115 (73%) of 157 sera from patients likely to have early Lyme disease were positive for immunoglobulin M (IgM) antibodies and 17 (11%) also had IgG antibodies. In addition, the IgM ELISA reactivities and the titers of antibodies detected by a flow cytometric borreliacidal antibody test correlated closely (r = 0.646). Moreover, the IgM ELISA was significantly more sensitive (P < 0.001) than the Western blot procedure. The findings therefore confirmed that the peptide IgM ELISA detected OspC borreliacidal antibodies and provided strong evidence that the test can eliminate the necessity for confirming early Lyme disease by a supplementary test such as Western blotting. PMID:18417666

  18. Magnetic bead droplet immunoassay of oligomer amyloid ? for the diagnosis of Alzheimer's disease using micro-pillars to enhance the stability of the oil-water interface.

    PubMed

    Kim, Jeong Ah; Kim, Moojong; Kang, Sung Min; Lim, Kun Taek; Kim, Tae Song; Kang, Ji Yoon

    2015-05-15

    Despite scientific progress in the study of Alzheimer's disease (AD), it is still challenging to develop a robust and sensitive methodology for the early diagnosis of AD due to the lack of a decisive biomarker in blood. Recent reports on the oligomer amyloid ? (A?) as a biomarker demonstrated its possibility for identifying early onset of AD in patients, but its low concentration in blood requires highly reliable detection techniques. To overcome the low reliability and labor-intensive procedures of conventional enzyme-linked immunosorbent assay (ELISA), we present a magnetic bead-droplet immunoassay platform for simple and highly sensitive detection of oligomer A? for the diagnosis of AD. This microchip consists of chambers that contain water-based reagents or oil for consecutive assay procedures, and there are arrays of micro-pillars fabricated between the two adjacent chambers to form robust water-oil interfaces. With the aid of these micro-pillars, magnetic beads can stably pass through each chamber by linearly actuating a magnet along the microchip. The robust water-oil interface and simple procedures of the assay make it possible to obtain reliable results from this microchip. The intensity of the fluorescence at the read-out chamber increased quantitatively and linearly, depending on the amount of serially-diluted standard A? solution. The results of the assay indicated that the limit of detection was about 10 pg/mL even though it was done with manual manipulation of the magnet. This platform simplified the complicated ELISA procedure and achieved high sensitivity that was no lower than that of the conventional magnetic bead immunoassay. The magnetic bead-droplet platform reduced the assay time to 45 min, and it also reduced the amount of antibody usage in a single diagnosis significantly (10-30 ng of antibody per single assay). Consequently, this microfluidic chip has strong potential as a feasible system for use in the diagnosis of AD with a fast and easy immunoassay process, since the suggested platform can be automated with ease for point-of-care testing as well as high-throughput diagnostic equipment. PMID:25459055

  19. An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets.

    PubMed

    Sánchez Guiu, I M; Martínez-Martinez, I; Martínez, C; Navarro-Fernandez, J; García-Candel, F; Ferrer-Marín, F; Vicente, V; Watson, S P; Andrews, R K; Gardiner, E E; Lozano, M L; Rivera, J

    2015-08-01

    Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin ?IIb?3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and ?IIb?3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (Fc?RIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding. PMID:25994029

  20. Generation and Characterization of Antibodies against Asian Elephant (Elephas maximus) IgG, IgM, and IgA

    PubMed Central

    Humphreys, Alan F.; Tan, Jie; Peng, RongSheng; Benton, Susan M.; Qin, Xiang; Worley, Kim C.; Mikulski, Rose L.; Chow, Dar-Chone; Palzkill, Timothy G.; Ling, Paul D.

    2015-01-01

    Asian elephant (Elephas maximus) immunity is poorly characterized and understood. This gap in knowledge is particularly concerning as Asian elephants are an endangered species threatened by a newly discovered herpesvirus known as elephant endotheliotropic herpesvirus (EEHV), which is the leading cause of death for captive Asian elephants born after 1980 in North America. While reliable diagnostic assays have been developed to detect EEHV DNA, serological assays to evaluate elephant anti-EEHV antibody responses are lacking and will be needed for surveillance and epidemiological studies and also for evaluating potential treatments or vaccines against lethal EEHV infection. Previous studies have shown that Asian elephants produce IgG in serum, but they failed to detect IgM and IgA, further hampering development of informative serological assays for this species. To begin to address this issue, we determined the constant region genomic sequence of Asian elephant IgM and obtained some limited protein sequence information for putative serum IgA. The information was used to generate or identify specific commercial antisera reactive against IgM and IgA isotypes. In addition, we generated a monoclonal antibody against Asian elephant IgG. These three reagents were used to demonstrate that all three immunoglobulin isotypes are found in Asian elephant serum and milk and to detect antibody responses following tetanus toxoid booster vaccination or antibodies against a putative EEHV structural protein. The results indicate that these new reagents will be useful for developing sensitive and specific assays to detect and characterize elephant antibody responses for any pathogen or vaccine, including EEHV. PMID:25658336